View
75
Download
0
Category
Preview:
Citation preview
i
Ameliorative anti - hepatotoxic potential of various herbal
combinations
By
(M. Phil. UAF)
A thesis submitted in partial fulfillment of the requirements for the
degree of
DOCTOR OF PHILOSOPHY
IN
BIOCHEMISTRY
DEPARTMENT OF BIOCHEMISTRY
FACULTY OF SCIENCES,
UNIVERSITY OF AGRICULTURE,
FAISALABAD
PAKISTAN
2020
i
i
To
My Beloved Parents
Who taught me The first word to speak, The first word to write,
And the first step to take
Acknowledgements
First and the foremost, I would like to give my humble thanks and praise to the Almighty Allah for
His grace and blessings throughout my entire life and in the completion of this dissertation particularly.
Without the Blessings of Almighty Allah and His teachings taught by Prophet Muhammad (PBUH), my
life is nothing.
During the completion of this thesis, there were many kinds of supports I have got. I would like to
express my deepest thanks and gratitude to my supervisor Prof. Dr. Khalil-ur-Rehman, (Department of
Biochemistry University of Agriculture, Faisalabad), for the valuable guidance, assistance and constructive
advice throughout the entire project. I would like to make my sincere appreciation and pleasure and
sincerest thanks to my committee members Dr. Muhammad Shahid (Department of Biochemistry
University of Agriculture Faisalabad) and Dr. Nazish Jahan (Department of Chemistry, University of
Agriculture, Faisalabad), for valuable assistance and guidance. I also pay homage to all teachers goal of
my academic carrier with light of knowledge and enable me to touch a section in my life.
Very special thanks to ORIC (University of Agriculture Faisalabad) for its moral & financial support,
without its assistance this dissertation was merely a dream.
I am unable to express my deepest appreciation towards my affectionate parents. I am most
earnestly obliged to my Dear father, Muhammad Akram for the strenuous efforts done by him in
enabling me to join the higher ideas of life and to my Loving Mother, Nasira Perveen for her
patience and for the prayers she had made for my success. The names of my parents will always be in
front of my eyes, as I will look on the cover of my life. I feel my immense pleasure to express my deepest
gratitude and sincere thanks to my brother Sajeel Akram, sisters Abeeda Akram and Deeba Akram and
sister -in-law Masooma Sajeel for their encouraging and inspiring cooperation all the time. I have no
words to express my sweet sensation to my beloved cousins and my all family for their moral boost,
encouragements and countless prayers for me to achieve higher ideal of life.
I have no words to express my sweet sensations to my loving friends Zara Jabeen, Zirwa Rizwan,
Nighat Zia and Fatima Yousaf who are near and dear to me. I deeply obliged of my seniors Dr. Abid
Ali, Dr. Saman Hina, Dr. Nadia Afsheen and Dr. Allah Rakhha for support and guidance. May
Allah’s blessings be upon all these people with His countless favors and I would like to pray for their happy
and peaceful lives (Ameen)!
UMM-E-HABIBA
LIST OF TABLES
Table No. Title
Page No.
2.1 Experimentally reported hepatoprotective medicinal plants 10
2.2 Reported different hepatoprotective herbal combinations 12
3.1 Voucher numbers of the selected medicinal plants with their parts
used for evaluation of hepatoprotective potential 19
3.2 Krebs – Ringer – Hepes medium composition (g/ Liter) 28
3.3 Experimental layout for liver slice culture assay 30
3.4 Central Composite Design suggested herbal combinations 34
3.5 Curative and preventive groups for in vivo hepatoprotective
actitivity 37
4.1 Total phenolic and flavonoid Content in medicinal plants 47
4.2 Percentage inhibition of DPPH at different concentration of
medicinal plants 49
4.3 Percentage of hydroxyl radical scavanging activity of selected
plants at different concentrations. 51
4.4 FRAP values (Fe (II) mg/mL) of plants at different concentrations 53
4.5 Percentage hemolysis in selected plants at different concentrations 55
4.6 Percentage of Clot lysis in medicinal plants at different
concentrations 57
4.7 Percentage inhibition of Acetylcholinesterase of medicinal plants
at different concentrations. 59
4.8 Percentage Cytotoxicity of medicinal plants at different
concentrations against heaptotoxicant CCl4 62
4.9
RSM model0020under Central Composite Design (CCD) for
determination of optimum concentrations as hepatoprotective
potential of medicinal plants
85
4.10 Analysis of variance (ANOVA) of data for RSM model to
determine hepatoprotective potential of medicinal plants 87
4.11 ANOVA for Response Surface Quadratic model to determine
hepatoprotective potential of medicinal plants 87
4.12 Hepatoprotective effects of herbal combination on MDA, GSH and
SOD levels 110
4.13 Preventive hepatoprotective effects of herbal combination on
MDA, GSH and SOD levels 128
LIST OF FIGURES
Figure No. Title
Page No.
2.1 Difference in lactate production in normal and cancer cells
14
2.2 Cell proliferation and apoptosis through Acetylcholine and its
receptors 15
2.3 Hepatotoxicity inducing mechanism of CCl4 17
3.1 Standard curve of Gallic acid to measure total phenolic content 22
3.2 Standard curve of Catechin to measure total flavonoid content
23
3.3 Standard curve of FeSO4 to calculate ferric reducing antioxidant
potential of medicinal plants 25
3.4 Liver lobes cut into thin slices. B: Treatment of liver slices with
medicinal planst at different concentrations. 30
3.5 Rats divided into groups, B: observation of body weight of rats
for dose calculation, C: Dose addministration to rat 38
3.6 A: Blood collection, B: Blood in gel activator tubes, C:
Separtion of serum from blood 39
3.7 Standard curve of MDA (nmol/mL) to measure MDA level in
terms of lipidperoxidation 41
3.8 Standard curve of GSH (µg/mg) to calculate gluthathione
reductase level in liver homogenate 42
4.1 Total phenolic content in indigenous medicinal plants 47
4.2 Total flavonoid content in indigenous medicinal plants 48
4.3 Percentage inhibition of DPPH of medicinal plants 50
4.4 Percentage of hydroxyl radical scavanging activity of plants 52
4.5 FRAP values (Fe (II) mg/mL) of indigenous medicinal plants 53
4.6 Percentage of haemolysis of medicinal plants at three
concentrations 55
4.7 Percentage of clot lysis through medicinal plants of three
different concentrations 57
4.8 Percentage inhibition of Acetylcholinesterase in medicinal
plants 59
4.9 Presented percentage cytotoxicity in high control, low control
and standard groups 61
4.10 Trend of percentage Cytotoxicity of medicinal plants at
different concentrations in terms of LDH released 62
4.11 Full mass spectrum of S. marianum 66
4.12 Mass spectrum indicating the presence of Caffeic acid and
Chlorogenic acid 66
4.13 Mass spectrum of S. marianum showing Sweroside at 381.25
m/z 66
4.14 Mass spectrum indicating the presence of Taxifolin and
Luteolin 7-O- glucoside 67
4.15 MS/MS of S. marianum peak 303 at CID (20.00) showing
Luteolin 7-O- glucoside at 285 m/z 67
4.16 Mass spectrum of S. marianum showing Silybin at 481.25 m/z 68
4.17 MS/MS of S. marianum peak 481.25 at CID (22.00) indicating
the presence of Silybin 68
4.18 Full mass spectrum of T. officinale 69
4.19 Mass spectrum indicating the presence of Sweroside (381.25
m/z) and Isoquercetin (465.33 m/z) 69
4.20 Mass spectrum of T. officinale showed Quercetin and Myricetin
at 303.25 and 319.33 m/z. 70
4.21 MS/MS with CID (25.00) of peak 303.17 m/z indicating the
presence of Quercetin 70
4.22 MS2 with CID (25.00) of peak 319.25 m/z shown Myricetin in
T. officinale 71
4.23 Mass spectrum of T. officinale indicating the presence of Silybin
A and B at 481.33 m/z. 71
4.24 Mass spectrum of T. officinale showed Chlorogenic acid at
377.25 m/z 72
4.25 Full mass spectrum of P. viviparum 72
4.26 MS of P. viviparum showed Caffeic acid at 180.08 m/z and
Sweroside at 381.25 m/z 73
4.27 Mass spectrum of P. viviparum shown Morroniside at 428.25
m/z 73
4.28 Mass spectrum of P. viviparum shown Gallic acid at 169.08 m/z
and Quinic acid at 191.08 m/z 74
4.29 Mass spectrum of P. viviparum shown Chlorogenic acid at
353.25 m/z 74
4.30 MS- MS at 353.17 m/z by CID 20:00 indicated the confirm
presence of Chlorogenic acid 75
4.31 MS-MS of P. viviparum shown Protocatechuic acid at 154 and
109.83 m/z 75
4.32 MS-MS of P. viviparum shown Catechin at 290.17 and 272.08
m/z 76
4.33 Full mass spectrum of F. arabica 76
4.34 Mass spectrum indicated the presence of quercetin and
Isorhamnetin at 301.33 and 317.33 m/z in F. arabica 77
4.35 Mass spectrum at 381.25 and 397.42 m/z indicated the presence
of Sweroside and Swertiamarin in F. arabica 77
4.36 Mass spectrum at 413.42 m/z indicated the presence of Loganin
in F. rabica 78
4.37 MS-MS with CID (29:00) indicated the presence of Loganin
(413.33 m/z) and Quercetin (301.17 m/z) in F. arabica 78
4.38 Mass spectrum shown the presence of 5 hydroxymethylfurfural
at 127 m/z and Isorhamnetin at 315 m/z in F. arabica 79
4.39 Mass spectrum shown the presence of Chlorogenic acid at
377.25 m/z in F. arabica 80
4.40 Full mass spectrum of M. annua 80
4.41 Mass spectrum indicated Gallic acid at 170 m/z and Myricetin
at 319.33 m/z in M. annua 81
4.42 MS-MS indicated Gallic acid at 170 m/z CID (27:00) 81
4.43 Mass spectrum shown Ferulic acid, Chlorogenic acid and
Swertiamarin in M. annua 82
4.44 Mass spectrum indicated Hydroxymethylfurfural and Myricetin
correspondingly at 127 and 317.42 m/z in M. annua 82
4.45 Mass spectrum showed Loganin in M. annua at 413.42 m/z 83
4.46 MS-MS of peak at 153 m/z with CID 27:00 indicated
Protocatechuic acid in M. annua 83
4.47 Mass spectrum showed Echinacoside in M. annua at 785.33 m/z 84
4.48 Presented quadratic model with normal probability plot of the
studentized residuals as hepatoprotective potential 88
4.49 Presented quadratic model run number verses internally
studentized residuals plot for hepatoprotective potential 89
4.50 Shown quadratic model correlation between predicted and
actual values of herbal combinations 90
4.51 Box-Cox plot of Lambda and Ln residuals SS in quadratic
model 90
4.52 Response surface plot of interaction in S. marianum with P.
viviparum for the hepatoprotective activity 91
4.53 Response surface plot of interaction between S. marianum and
T. officinale for the hepatoprotective activity 92
4.54 Response surface plot of interaction in S. marianum with F.
arabica for the hepatoprotective activity 93
4.55 Response surface plot of interaction between S. marianum and
M. annua for the hepatoprotective activity 93
4.56 Response surface plot of interaction in T. officinale and P.
viviparum for the hepatoprotective activity 94
4.57 Response surface plot of interaction in P. viviparum with F.
arabica for the hepatoprotective activity 95
4.58 Response surface plot of interaction in P. viviparum with M.
annua for the hepatoprotective activity 95
4.59 Response surface plot of interaction in T. officinale with F.
arabica for the hepatoprotective activity 96
4.60 Response surface plot of interaction in T. officinale with M.
annua for the hepatoprotective activity 97
4.61 Response surface plot of interaction between F. arabica and M.
annua for the hepatoprotective activity 97
4.62 Curative effect of herbal combination against hepatotoxicity
induced by CCl4 on body weights of rats 99
4.63 Shown curative hepatoprotective effects of herbal combination
on ALT (U/L) level at different days 101
4.64 Graphical presentation of curative hepatoprotective effects of
herbal combination on AST (U/L) level at different days 102
4.65 Presented curative hepatoprotective effects of herbal
combination on ALP (U/L) level at different days 103
4.66 Shown curative hepatoprotective effects of herbal combination
on ƔGT (U/L) level at different days 104
4.67 Presented curative hepatoprotective effects of herbal
combination on total bilirubin (mg/dL) level at different days 105
4.68 Presented curative hepatoprotective effects of herbal
combination on total protein (g/dL) level at different days 106
4.69 Presented curative hepatoprotective effects of herbal
combination on Albumin (g/dL) level at different days 107
4.70 Presented curative hepatoprotective effects of herbal
combination on globulin (g/dL) level at different days 108
4.71
Presented curative hepatoprotective effects of herbal
combination on AChE activity (µM of ATC hydrolyzed/min/g)
in different groups 109
4.72 Presented curative hepatoprotective effects of herbal
combination on MDA (nmol/g tissue) in different groups 111
4.73 Presented curative hepatoprotective effects of herbal
combination on GSH (µg/mg) in different groups 111
4.74 Presented curative hepatoprotective effects of herbal
combination on SOD (U/mg) in different groups 112
4.75
Photomicrograph (X400) shown normal architecture of the
Hepatic cell (H), with nucleolus and prominent chromatin (→),
Sinusoidal spaces between hepatocytes (S and circles) and
Kupffer cells (K)
114
4.76
Photomicrograph (X400) shown necrosis of hepatic cell (→),
inflammation in the hepatic cells and sinusoidal spaces (circles)
and dilation of sinusoidal spaces (←) in curative positive control
group
114
4.77
Photomicrograph (X400) shown mild degree of inflammation of
hepatic cell (←), binucleated cells (↑) and dilation of sinusoidal
spaces (→) in liver of curative negative control group 115
4.78
Photomicrograph (X400) displayed mild degree of necrosis of
hepatic cell (→), vacuolar degeneration (↑), diffused kupffer
cells (↓) and inflammation of hepatic cells (←) in liver of
curative treatment group
116
4.79
Photomicrograph (X400) shown mild degree of necrosis of
hepatic cell (→), binucleated cells (↑) and dilation of sinusoidal
spaces (←) in liver of curative standard group 116
4.80 Preventive effect of herbal combination against hepatotoxicity
induced by CCl4 on body weights of rats 118
4.81 Shown preventive hepatoprotective effects of herbal
combination on ALT (U/L) level at different days 119
4.82 Presented preventive hepatoprotective effects of herbal
combination on AST (U/L) level at different days 120
4.83 Shown preventive hepatoprotective effects of herbal
combination on ALP (U/L) level at different days 121
4.84 . Presented preventive hepatoprotective effects of herbal
combination on ƔGT (U/L) level at different days 122
4.85 Presented preventive hepatoprotective effects of herbal
combination on total bilirubin (mg/dL) level at different days 123
4.86 Shown preventive hepatoprotective effects of herbal
combination on total protein (g/dL) levels at different days 124
4.87 Presented preventive hepatoprotective effects of herbal
combination on total albumin (g/dL) levels at different days 125
4.88 Presented preventive hepatoprotective effects of herbal
combination on total globulin (g/dL) levels at different days 125
4.89 Presented preventive hepatoprotective effects of herbal
combination on AChE activity (U/mg) in different groups 127
4.90 Presented preventive hepatoprotective effects of herbal
combination on MDA level (nmol/g tissue) in different groups 128
4.91 Presented preventive hepatoprotective effects of herbal
combination on GSH levels (µg/mg) in different groups 129
4.92 Presented preventive hepatoprotective effects of herbal
combination on SOD levels (U/mg) in different groups 130
4.93
Photomicrograph (X400) shown normal architecture of the
Hepatic cell (H), with nucleolus and prominent chromatin (←),
Sinusoidal spaces between hepatocytes (S), Kupffer cells (K)
and binucleated cells in normal control group
131
4.94
Photomicrograph (X400) shown inflammation of hepatic cell
(←), necrosis of the hepatic cells (→) and dilation of sinusoidal
spaces (↑) leakage of blood cells through portal vein (↓) and
diffusing of hepatocytes and kupffer cells in liver of the
preventive positive control group
132
4.95
Photomicrograph (X400) presented hepatic cell (→), sinusoidal
spaces (↑), blood cells (↓), endothelial cells (←), hepatic vein
(HV), hepatic artery (HA) and hepatic bile duct (BD) in liver of
preventive negative control group
132
4.96
Photomicrograph (X400) shown mild degree of necrosis of
hepatic cell (→), vacuolar degeneration (↓), inflammation of
hepatic cells (←) and dilation of sinusoidal spaces (↑) in liver
of preventive treatment group
133
4.97
Photomicrograph (X400) shown mild degree of necrosis of
hepatic cell (→), binucleated cells (←) and dilation of
sinusoidal spaces (→) in liver of preventive standard group 134
ABSTRACT
The increasing prevalence of hepatic disorders has become a major challenge for the scientists.
Medicinal plants have remarkable potential to cure liver diseases by reducing reactive oxygen
species that induce oxidative stress to the cells. Therefore, the present study was planned to prepare
a herbal formulation that may prevent and manage several liver disorders and its related
complications. This study was accomplished in three phases. In phase I, in vitro screening of
indigenous medicinal plants was done on the basis of their antioxidant, toxicological and
hepatoprotective potential. The total phenolic and flavonoid content were found in the range of
67.02 to 267.43 GAE and 73.26 to 131.86 CE respectively. The antioxidant potential of medicinal
plants was explored through ferric reducing antioxidant power assay, DPPH and hydroxyl radical
scavenging activities. The obtained results showed 33.04 to 84.73% DPPH inhibition, 32 to 94%
hydroxyl radical scavenging activity and 10.14 to 44.49 ferric reducing antioxidant power at 100,
300 and 500 µg/mL concentrations. The antioxidant potential of studied medicinal plants increased
in dose dependent manner. For toxicity evaluation of medicinal plants, hemolytic and thrombolytic
activities were performed in terms of percentage hemolysis and clot lysis respectively. Medicinal
plants that showed minimum percentage hemolysis and clot lysis were considered best and safe.
The hepatoprotective potential of medicinal plants was exhibited through acetylcholinesterase
(AChE) inhibition assay and Liver Slice culture (LSC) assay. Medicinal plants that showed
maximum hepatoprotective potential in terms of minimum inhibition of AChE activity and
minimum percentage cytotoxicity were selected for phase II. In phase II, biochemical profiling of
selected plants was done through LC-MS to detect and confirm the presence of hepatoprotective
bioactive compounds. Moreover, different herbal combinations of selected plants suggested by
Response surface methodology (RSM) were prepared and analyzed through in vitro Liver Slice
Culture assay in phase II. The herbal combination that showed minimum percentage cytotoxicity
in terms of maximum hepatoprotective potential was selected for phase III, in vivo studies. In this
phase, rats were used as animal model to evaluate the ameliorative effects of selected herbal
combination against hepatotoxicity. The heaptotoxicant CCl4 with the dose of 1 mL/kg b.wt was
used to induce hepatotoxicity and herbal combination with the dose of 200 mg/kg b.wt was used
as curative and preventive mode of treatments for thirty days. Biochemical parameters including
ALT, AST, ALP, ƔGT, total bilirubin, total protein, albumin and globulin were measured in blood
serum of all groups. Percentage of acetylcholinesterase enzyme activity, malondialdehyde (MDA)
in terms of lipid peroxidation and antioxidant enzymes SOD and GSH were measured in liver
homogenate of rats. Liver histopathological analysis was done to see any alteration in hepatic cells
due to exposure of heaptotoxicant CCl4 and then healed with herbal combination in curative and
preventive mode of treatments. The significant decrease in ALT, AST, ALP, ƔGT, total bilirubin,
MDA and increase of total protein, albumin, globulin, AChE activity SOD and GSH levels in both
curative and preventive treatment groups as compared to positive control groups indicated
significant ameliorative effects of herbal formulation. Likewise histopathological examination
with normal architecture of hepatic cells in curative and preventive treatment groups endorsed the
hepatoprotective potential of herbal combination. Therefore, this herbal combination might be
used as an alternative medicines against various liver disorders and its related complications.
1
CHAPTER 1
INTRODUCTION
Hepatitis has become a serious health issue throughout the world and is becoming more
severe in some countries including Pakistan (Ashraf and Ahmed, 2015). This health menace,
particularly in its chronic form is leading to high level of morbidity and mortality (Averhoff et al.,
2012). A number of efforts are being made to get rid of this threatened disease from Pakistan
(Umer and Iqbal, 2016). The preventive and curative measures taken by the Pakistani government,
including public health awareness programs, blood screening, free treatment of poor patients and
vaccinations of high risk population and new-borns are insufficient to overcome chronic liver
disorders (Ahmed et al., 2016). Owing to high prevalence and inadequate measures, it is necessary
to take immediate and innovative actions to manage hepatic disorders (Qureshi et al., 2010; Ullah
et al., 2017).
Liver is second largest organ of the body and hepatocytes, sinusoidal endothelial and
kupffer cells are its main components (Ozougwu and Eyo, 2014; Jevas, 2017). The core function
of the liver is to maintain homeostasis and metabolism of the body (Ishibashi et al., 2009). It also
plays a fundamental role in nutrient supply, energy provision, excretion of waste (drugs,
xenobiotics and metabolites) and fight against diseases (Raj et al., 2013). Therefore, healthy liver
is important for the overall health of an individual (Kumar et al., 2012; Kumar, 2012). Hepatitis is
the inflammation of hepatocytes and instigated by numerous infectious and non-infectious factors
(Chuang et al., 2009; Secretan et al., 2016).
Infectious hepatitis caused by bacteria and viruses. Viral hepatitis is more prevalent then
bacteria and categorised as hepatitis A, B, C, D and E (Jacobsen et al., 2010). Among them
hepatitis B and C are the 8th highest cause of mortality worldwide (Belyhun et al., 2016). Around
240 million population is suffering from hepatitis C and about 130-150 million people are infected
from hepatitis B virus globally (WHO 2013). Pakistan has the second highest number in prevalence
of the hepatitis C after Egypt (Hwang and Cheung, 2011). Infection of hepatitis C virus is a serious
health issue in medical and public sectors globally (Murphy et al., 2000; Haley and Fischer, 2001).
The seroprevalence of hepatitis C virus among the adult population of Pakistan is 6.8 percent
(Umer and Iqbal, 2016). Around 55-85 percent of hepatitis C patients are at high risk of developing
liver cirrhosis, hepatocellular injury, carcinoma and liver failure (Bisceglie, 2000; Zhang et al.,
2
2013). Acutely infected people of hepatitis C virus are rare to be diagnosed because they are mostly
asymptomatic (Orland et al., 2001; Nomura et al., 2004).
Hepatitis is also caused by several non- infectious factors including toxicants,
chemotherapeutic agents, excessive use of alcohol and pollutants (Navarro and Senior, 2006; Perz
et al., 2006). Moreover autoimmune hepatitis is a type of hepatitis in which antibodies are
produced against liver tissues (Harsha and Latha, 2011). The other liver diseases are classified as
hepatosis, steatosis, acute on chronic liver failure (ACLF) and cirrhosis (Samir, 2001; Ross and
Wilson, 2005). Hepatosis (non- inflammatory) and steatosis (fatty liver) are associated with non-
alcoholic fatty liver diseases (NAFLD), caused by stress related metabolic liver disorders (Fan et
al., 2009; Abdul et al., 2017). Consequently, NAFLD is under full consideration of healthcare
departments in Asia because of high obesity and metabolic complications in people (Misra et al.,
2013; Ashtari et al., 2015). Acute on chronic liver failure (ACLF) is a recently documented disease
that arises with or without previously diagnosed cirrhosis of the liver. ACLF is characterized by
acute hepatic decomposition and its mortality rate is increased (30-40 percent) within three months
(Jalan et al., 2014).
Cirrhosis is a progression of hepatic fibrosis with distortion of the hepatic architecture and
regenerative nodule formation (Lefton et al., 2009). Different complications including hepatic
encephalopathy, spontaneous bacterial peritonitis, ascites, portal hypertension and hepatorenal
syndrome are also associated with liver cirrhosis (Heidelbaugh and Bruderly, 2006).
Hepatocellular carcinoma (HCC) is the fifth most common cancer in the world and the third most
common cause of death worldwide (Bosch et al., 2004). Numerous factors like excessive use of
alcohol, chemical toxicants and viral infections are the major reasons of hepatic injury and liver
metabolic dysfunctions (Fernandez and Kaplowitz, 2005; Srivastava and Shivanandappa, 2010).
Hepatocellular carcinoma occurred in result of transient elevation of hepatic enzymes, hepatic
fibrosis and liver cirrhosis (Harsha and Latha, 2011; Hartmut et al., 2012).
A number of therapeutic and preventive drugs are being used to treat liver injuries (Muriel
and Rivera-Espinoza, 2008). In allopathic treatment of chronic hepatitis C, a combination of peg
interferon and ribavirin is commonly used (Hutchison et al., 2000; Reddy et al., 2001; Zoulim and
Perrillo, 2008). Interferons (IFNs) are cytokines, possessing a variety of biological properties
3
including antiviral, immune modulatory, anti-proliferative and anti angiogenic effects (Wang et
al., 2003). IFNs are also effective in suppressing the replication of HBV and it was the first agent
that was approved for the treatment of chronic HBV infection (Lo et al., 2007). IFN has also been
used for HCV infection and has effectively decreased progression to liver cirrhosis and
development of HCC (Lin et al., 2007). The combination of interferon alfa with ribavirin increased
sustained virological response rates from 10 to > 40% (Frird, 2002). A triple therapy (telaprevir
with peg interferon and ribavirin) is also used for the treatment of hepatitis C (Hezode et al., 2009;
Kumada et al., 2012). Moreover a combination of sofosbuvir (Sovaldi) and daclatasvir is used to
treat hepatitis C infection and is effective to suppress viral rate of hepatitis C genotypes 1-4 up to
97 percent (Ven et al., 2015).
Excessive use of IFN alfa and peg interferon induces severe psychiatric side effects such
as depression, anxiety, irritability and disturbance in sleep cycle (Schaefer et al., 2003). Interferon
also persuades pancytopenia, through its bone marrow depressing activity (Manns et al., 2001;
Fried et al., 2002). Moreover, the use of triple therapy causes adverse effects, such as the rapid
progression of anaemia, severe rash and even renal dysfunctions (Hayashi et al., 2012; Mauss et
al., 2014). On the other side these modern synthetic medicines are not hundred percent effective
to stimulate liver functions and to regenerate hepatic cells (Wang et al., 2003; Orman et al., 2013).
Likewise in healthcare departments of underdeveloped countries, the number of patients suffering
from liver diseases are increasing due to the high cost of these drugs (Hill and Cooke, 2014).
Pakistan is an under developed country with dense population and majority of people cannot afford
the cost of these treatments to manage liver diseases. Therefore, the requirement of current
situations is to search and explore alternative solutions to form new effective medicine for the
treatment of various liver disorders (Ashraf and Ahmad, 2015).
Medicinal plants are highly valuable source of pharmaceutical drugs (Palanisamy et al.,
2014). Herbal remedies possess many therapeutic effects and are used for the treatment of various
diseases from ancient times (Samudram et al., 2008). Different medicinal plants can also play an
imperative role to cure acute and chronic hepatic diseases (Verpoorte and Alfermann, 2002;
Saroopa et al., 2016). Phytochemicals are the active constituents of medicinal plant that protect
the hepatic cells by delaying fibrogenesis, inhibition of oxidative stress and suppression of
tumorigenesis process (Dhiman et al., 2012; Del et al., 2012). Many in vitro and in vivo studies
4
have been conducted on several medicinal plants naming S. marianum, M. olifera, A. absinthium,
G. glabra, S. nigrum, C. intybus, C. fistula, T. officinale, and S. aromaticum to reveal the
hepatoprotective potential against hepatotoxicity induced by chemicals and drugs (Kannampalli et
al., 2007; Adewusi and Afolayan, 2010; Ali et al., 2015; Hina et al., 2017).
Silybum marianum (Milk thistle) is a member of Asteraceae family and has been used from
2000 decades for the treatment of liver and gall bladder diseases (Kren and Walterova 2005;
Abenavoli et al., 2010). S. marianum showed many pharmacological activities including anti-
inflammatory (Kren and Walterova, 2005), antioxidative, anti-tumor (Amani et al., 2011),
antifibrotic, liver regenerating mechanism membrane stabilizing (Radko and Cybulski, 2007)
immunomodulatory, anti-atherosclerotic, and anti-diabetic effects (Pradhan and Girish, 2006). The
active component of S. marianum is Silymarin, which has been used as a complementary
alternative medicine to treat liver disorders (Hawke et al., 2010; Loguercio and Festi, 2011). S.
marianum also contains flavonoid taxifolin and many falvolignans which are good
hepatoprotectors through their antioxidative properties (Vargas et al., 2014).
Taraxacum officinale a perineal herb, commonly known as Dandelion belongs to family
Asteraceae (You et al., 2010; Martinez et al., 2015). Owing to its antidiabetic, antioxidant, diuretic,
antirhematic, anti-allergic and choleretic properties, dandelion is traditionally used as a herbal
medicine for the treatment of liver disease, digestive complaints, gallbladder disorders, rheumatic
and arthritic diseases (Schütz et al.,2006; Koh et al.,2010). Recent studies on T. officinale depicted
that it has potential to reduce the risk of inflammation and tumour (Kim et al., 2007; Sigstedt et
al., 2008). Leaves of T. officinale used as diuretic and bitter digestive stimulant, while roots of this
plant are beneficial for digestion, improvement of liver functions and gastrointestinal remedies
(Yarnell et al., 2009).
Martynia annua (herbaceous annual plant), belongs to family Martyniaceae and commonly
known as Devil’s claw (Nagda et al., 2009). Leaves and seeds of M.annua are commonly used for
the treatment of inflammation, epilepsy and tuberculosis (Hosamani et al., 2002; Babu et al.,
2010). Seed oil of M. annua is used for skin affections, itching and applied on abscesses, while
fruit is used as a local sedative agent (Khare, 2007; Kenwat et al., 2013). The leaves, fruits and
seeds extracts of M. annua have exhibited antioxidant (Sermakkani and Thangapandian, 2010),
5
antibacterial, antifungal (Dhruti et al., 2009), anticonvulsant and wound healing properties (Babu
et al., 2010; Lodhi and Singhai, 2011; Vishal et al., 2012).
Medicinal plants are natural resources of antioxidants that are potent to reduce oxidative
stress that is arisen due to the imbalance of reactive oxygen species (ROS) (Videla et al., 2004).
Homeostasis of the body and antioxidant defence system are also disrupted by excessive
production of ROS (Zhu et al., 2012; Kabiri et al., 2013). As a result they damage cellular
components such as cell membrane and DNA through oxidation process (Abdollahi et al., 2004;
Birben et al., 2012). Normally free radicals are generated through several biochemical and
metabolic reactions which are mostly taken place in hepatic cells (Cichoż-Lach and Michalak,
2014). While excessive production of these free radicals induced various liver diseases and other
ailments including cancer, stroke, atherosclerosis, arthritis, asthma and age related diseases
(Jaeschke, 2011).
Both infectious and non- infectious liver diseases are also progressed through ethanol
mediated reactive oxygen species (Jayant et al., 2012). In subcellular compartment of liver, alcohol
is metabolized in cytosol, peroxisomes and endoplasmic reticulum with the help of alcohol
dehydrogenase, catalase and microsomal ethanol oxidizing system respectively (Xu et al., 2003;
Caro and Cederbaum, 2004). In a result of alcohol metabolism ROS including superoxide,
hydrogen peroxide and hydroxal radical are generated (Artee, 2003; Wu and Cederbaum, 2003).
The overproduction of ROS is responsible for damaging of the hepatic cells through lipid
peroxidation, necrosis and apoptosis (Moquin and Chan, 2010; Challa and Chan, 2010).
Pakistan is rich in biodiversity of medicinal plants and different herbal formulations are
available in the Pakistan market for the treatment of liver disease (Bashir et al., 2011). But these
herbal medicine are not experimentally tested and verified, so it is necessary to investigate the
beneficial and toxic effects of medicinal plants to improve efficacy and safety of herbal
formulations (Kanwal and Ali, 2017). Therefore, in vitro liver slice culture method is a unique and
sensitive method to evaluate the cytotoxic effects of medicinal plants on liver cells (Inge et al.,
2014).
In liver slice culture assay, level of Lactate dehydrogenase (LDH) enzyme (released from
the hepatic cells) is measured and expressed in terms of percentage cytotoxicity of medicinal plants
(Moquin and Chan, 2010). Lactate dehydrogenase enzyme is present in many organs including
6
liver, kidney and cardiac tissues (Thomas et al., 2015). Lactate dehydrogenase catalyses the
interconversion pyruvate to lactate and vice versa in glycolytic pathways (Shi and Pinto, 2014). In
necrosis or damaging of the cell, LDH is released by rupturing of the plasma membrane into
extracellular fluid and through its concentration hepatoprotective effects of herbal medicines
against heaptotoxicant can be measured easily (Suresh et al., 2015; Yingjuan et al., 2015).
On the other hand acetylcholinesterase inhibition assay is also a valuable and convenient
method for the screening of hepatoprotective medicinal plants (Salud et al., 2006). Acetylcholine
is a neurotransmitter that is involved in cell differentiation and proliferation during S phase of cell
cycle (Johnson et al., 2004). Acetylcholinesterase (AChE) converts acetylcholine into acetate and
choline. When acetylcholinesterase enzyme is absent in liver cells then acetylcholine level
increased that ultimately induced more differentiation and proliferation of cells and as a result
hepatocellular carcinoma occurred (Benjamin et al., 2015; Uzunhisarcikli and Kalender, 2011;
Uzun and Kalender, 2013). Different previous studies depicted the significance inhibitory activity
of acetylcholinesterase in medicinal plants including P. a crucis, L. pedunculata, M. suaveolens
and H. undulatum (Ferreira et al., 2006; Saroopa et al., 2016).
Therefore, the present study was planned to develop an auspicious herbal formulation of
medicinal plants to protect and manage hepatic disorders and its related complications.
AIMS AND OBJECTIVES
The aim of this study was to prepare a hepatoprotective herbal formulation to cure and
prevent numerous liver disorders and their side effects. Following were the main objectives of
present study:
I. In vitro screening of indigenous medicinal plants on the basis of antioxidant and
hepatoprotective potential through qualitative and quantitative analysis
II. In vitro and in vivo assessment of ameliorative effects of herbal combinations of
selected plant against hepatotoxicity
7
CHAPTER 2
REVIEW OF LITERATURE 2.1 The Liver
Liver is the second largest organ of the body and situated on the top of stomach at right
corner side of the abdomen. Liver carries out more than 500 dynamic metabolic functions (Ahsan
et al., 2009; Hazem and Hassan, 2012).
2.1.1 Functions of liver
Liver metabolized nutrients such as carbohydrates, lipids and proteins and also synthesized
plasma proteins, glucose through glycogenesis, different blood clotting factors and urea
(Saukkonen et al., 2006). Liver also plays critical role in homeostasis and regulation of enzymes
(Kumar, 2012). Different products like fat soluble vitamins and glycogen are stored in the liver
parenchyma tissues. Bile acid also produced by liver, is involved in the removal of toxic and
harmful substances from the body (Pradhan and Girish, 2006; Naruse et al., 2007). Smooth
endoplasmic reticulum of liver cells is a metabolic clearing house for endogenous substances
including cholesterol, proteins, steroid hormones and fatty acid and exogenous chemicals like
alcohol and drugs. Main function of the liver is metabolism and clearance of toxic substances
(Singh et al., 2011; Colin et al., 2018).
2.1.2 Liver diseases and its related complications
Hepatitis is the swelling of the hepatocytes and mainly caused by the viral infections
(Locarnini et al., 2016). There are eight types A, B, C, D, E, F, G and H of viral hepatitis from
which HBV and HCV are more prevalent (Satsangi and Chawla, 2016). Hepatitis A and E are
transmitted through faecal-oral rout and epidemics of acute viral hepatitis (Verma and Khanna,
2012). On the other side hepatitis B and C are spread through parenteral rout and caused chronic
hepatitis that leads to liver cirrhosis and hepatocellular carcinoma (Puri, 2014). Hepatitis D virus
is a satellite virus and it depend upon the hepatitis B virus for production of its envelop protein
(Rizzetto, 2016).
Obesity, insulin resistance diabetes, hypertriglyceridemia, metabolic health complications,
unhealthy and sedentary life style are the major risk factors of non-alcoholic fatty liver diseases
(NAFLD) (Veena et al., 2014; Hannah et al., 2016). In Western countries NAFLD is the major
health issue because if it remains untreated, it could progress to severe hepatic complications and
liver cirrhosis (Farrel et al., 2013). Cirrhosis mainly occurred due to prolonged non-alcoholic fatty
8
liver diseases, excessive alcohol consumption and chronic viral hepatitis infections (Gildea et al.,
2004; Schuppan et al., 2008).
2.1.3 Prevalence of hepatitis and other liver diseases globally and in Pakistan
High prevalence of liver disorders are the serious health issue in under developed countries.
About 200 million people are infected with HCV worldwide, which covers about 3.3% of the
world’s population (Wands, 2004). Pakistan has the second highest number of hepatitis C virus
(HCV) infection in the world after Egypt. In Pakistan with high morbidity and mortality, more
than 10 million people are living with Hepatitis C (Waheed et al., 2009). Among the adult
population of Pakistan, the seroprevalence of hepatitis C is 6.8% with genotype 3a (Muhammad
and Iqbal 2016).Patients suffering from hepatitis C have 75-85% chance to develop chronic
hepatitis and 15-20% among of these may develop liver cirrhosis above a period of 10-20 years
(Zhang et al., 2013). Hepatitis D is also prevalent and associated with the most severe form of viral
hepatitis (Alves et al., 2013). Likewise hepatitis E is one of the leading causes of acute viral
hepatitis worldwide, especially in under developed countries (Kim et al., 2014).
The prevalence rate of non-alcoholic fatty liver disease (NAFLD) is 25.24% globally and
it ranges between 15-45% in Asian countries (Younossi et al., 2016). The prevalence rate of
NAFLD is 18% in general Pakistani population (Niaz et al., 2011; Afzal et al., 2016). The
estimated prevalence rate of NAFLD in US is 19-46% (Williams et al., 2011; Lazo et al., 2013;
Doycheva et al., 2016). The evidence from recent studies has suggested that NAFLD may play an
imperative role in the progression of cardiovascular disease and chronic kidney disease (Targher
et al., 2010). Therefore, early detection of NAFLD is necessary to halt the disease progression and
its extrahepatic manifestations (Qurrat-ul-Ain et al., 2017).
Globally the prevalence of acute chronic liver failure (ACLF) is high. According to recent
studied seven million people hospitalized a year in the United States for cirrhosis and among of
these 32,335 people are suffered from ACLF (Allen et al., 2016). According to a survey in
Pakistan, 30 percent hepatitis B and 52 percent hepatitis C cases are reported every year (Ali et
al., 2015). In United States 29,000 deaths occurred annually due to liver cirrhosis (Bhurgri et al.,
2006). Hepatitis C disease is the single largest cause of hepatocellular carcinoma (HCC) in
Pakistan. The prevalence of HCC is 7.6% in males and 2.8% in females of Pakistan (Butt et al.,
2012).
9
2.2 Medicinal plants
Medicinal plants have been used from ancient times as therapeutic agents for treatment of
various human diseases. Around 80% traditional herbal remedies are used globally to cure several
diseases (Harish and Shivanandappa, 2006). Recently, the appreciation of traditional and green
medicine as a substitute health care by CAM practitioners have gained much attention in the
research area to explore the biological activities of medicinal plants (Tumah, 2005; Gurib-Fakim,
2006). About 70 % population of Pakistan depends upon folk and traditional system of medicines
that are obtained from natural recourses. The high cost and increasing side effects of allopathic
medicines, reinvigorated the people to use herbal remedies for treatment of numerous diseases
(Zaidi et al., 2012).
2.2.1 Importance of medicinal plants
In pharmaceutical, food and cosmetic industries the increasing demand of medicinal plant
proved that green herbs are very dynamic due to the presence of active constituents (Nostro et al.,
2000). In under developed countries, prevalence of severe infections caused by pathogenic
microorganisms has been increasing rapidly (Reddipalli, 2010). Owing to the increase of resistance
against pathogenic microbes and existing antibiotics, natural herbal remedies are used for the
treatment of several diseases (Kumar et al., 2011). Drug resistance against pathogenic
microorganisms is frequently increasing, therefore medicinal plants are under full consideration of
researchers for the treatment of pathogenic diseases (Pretorius and Watt, 2001; Doughari et al.,
2007). Medicinal plants have many antioxidant compounds that helping to cure and prevent several
diseases (Rajesh and Latha, 2004; Reddipalli, 2010).
2.2.2 Hepatoprotective medicinal plants
Liver is important organ of the body and involved in different metabolic activities and
regulation of physiological processes (Kumar et al., 2011). For that reason, any damage to hepatic
cells can result in transient elevation of liver enzymes, life threatening liver cirrhosis and hepatic
failure (Bera et al., 2012). In spite of remarkable advances in modern medicine, hepatic diseases
remains a health problem worldwide (Nithianantham et al., 2011). These liver diseases and its
complications can be managed through herbal remedies. Different experimentally reported
hepatoprotective plants are listed below:
10
Table: 2.1. Experimentally reported hepatoprotective medicinal plants
Plants name
and
used parts
Solvents
used
Effects on biochemical
markers and hepatocytes
Findings and potent
effects
Reference
Abrus mollis
Dry aerial
parts
Ethanol Significantly decreased
liver index enzymes ALT,
AST, ALP, total bilirubin
and hydroxyproline
contents. Increased
albumin and globulin level
Against hepatotoxicity
CCl4 and D-glactosmaine
in rats and mice
respectively
Abrus mollis has
hepatoprotective and
anti-inflammatory
agent to cure hepatitis,
hepatic fibrosis and
inflammation.
Mi et al., 2014
Sphallerocarp
us gracilis
Stems and
leaves
Ethanol Significantly reduced
serum AST, ALT, ALP
total cholesterol,
triacylglycerols total
bilirubin and lipid
peroxidation and also
increased levels of GSH,
SOD and catalase in plant
treated rats as compared to
CCl4 treated.
Ethanolic extracts of
Sphallerocarpus
gracilis could be used
as a preventive
hepatoprotective agent
and as a therapeutic
functional food
Ma et al., 2015
Clitoria
ternatea L.
Flower
Methanol Significant decrease in
ALT, AST and total
bilirubin against
acetaminophen. Protective
effect on histopathological
alterations and restored
glutathione levels
Clitoria ternatea L.
flower extract useful
hepatoprotectents
against paracetamol
Nithianant-
ham et al.,
2011
Urtica dioica
L.
Whole plant
Water,
alcohol,
petroleum
ether, ethyl
acetate,
n-butanol
and
aqueous
fractions of
extracts
Ethyl acetate fractions
(EAF) significantly
attenuated the increased
liver enzyme activities
induced by CCl4 and also
significantly decrease the
oxidative parameters in
liver tissues.
Ferulic acid obtained
through column
chromatography of
ethyl acetate fractions
of Urtica dioica L. is
responsible for
hepatoprotective
potential.
Chandra et al.,
2015
Nigella sativa
seeds
Water and
n-hexane
Pre-treatment of both
aqueous and n-hexane
extracts significantly
Nigella sativa has
hepatoprotective
properties by reducing
Yasmin et al.,
2013
11
decreased serum ALT and
MDA levels and increased
GSH level in hepatic cells
oxidative stress and
liver damage
Swertia
Chirayita
Whole plant
Methanol Plant extract significantly
acts as a hepatoprotectent
against intoxication of
CCL4 by decreasing serum
enzyme ALT, ALP and
AST levels
Possesses
hepatoprotective and
antioxidant activities
by reducing
biochemical
parameters
Mahmood et
al., 2014
Artemisia
absinthium L.
Arial parts
Methanol Level of AST and ALT
decreased in plant treated
group. Total thiol group
(TTG) also decreased in
plant treated group and
antioxidant power
increased
Ameliorate liver
toxicity by reducing
oxidative damage and
liver enzymes.
Mohammadia
n et al., 2016
Ficus
religiosa L.
Latex
Methanol Significant reduction in
liver enzymes and
improvement antioxidant
and hepatocytes in
preventive plant treated
group
Ficus religiosa L. has
hepatoprotective and
antioxidant effects
against cisplatin
hepatotoxic inducing
agent
Yogesh and
Yadav, 2015
Ocmuim
basilicum
Leaves
Water Reduction in AST and
ALT and improvement in
immune histoch-emical
and histological changes in
both plant and dianzinon
(insecticide) administrated
rats.
Basil leaves have
hepatoprotective and
antioxidant properties
Faiza et al.,
2015
Taraxacum
officinale
Roots
water Significant reduction in
LDH, AST, ALT and ALP
and increase in GSH and
catalase treated group as
compared to alcohol alone
treated group
T. officinale has
hepatoprotective and
antioxidant potential
by decreasing lipid
peroxidation
You et al.,
2010
Ziziphus
jujuba M.
Dry fruit
Water Polysaccharide plant
extract lower the AST &
ALT and restoration of
antioxidant enzymes in
both plant and CCl4
intoxicated mice
Hepatoprotectiv-e
potential
Yue et al.,
2014
12
2.2.3. Use of herbal combination as a hepatoprotectors
Plants contain bioactive components and wide variety of pharmacological properties such
as anti- inflammatory, anti- proliferative and anti- carcinogenic. Antioxidant properties of plants
prevent the oxidative stress related diseases by reducing reactive oxygen species (Dey and
Lakshmanan, 2013; Chowdhury et al., 2015). A single medicinal plant cannot be effective for all
type of severe liver diseases. Therefore, herbal formulations are developed that enhance
bioavailability and antioxidant potential of plants due to synergism. Some experimentally reported
herbal formulations are listed below:
Table 2.2. Reported different hepatoprotective herbal combinations
Herbal
formulation
Plants Biochemical parameter
evaluation
Findings Reference
Rohitaka
ghrita
Tecomella undulate,
Zinziber officinalis,
Piperm longum,
Plumbago zeylanica
and Piper
retrofractum
Remarkably reverse the
effect of paracetamol by
reducing AST, ALP,
ALT and bilirubin. Also
improves the antioxidant
catalase and glutathione
Significant
hepatoprotective
potential and
recommended to use
in liver
complications such
as jaundice,
cholestasis and
cirrhosis
Goyal et al.,
2012
Herbal drug Trichosanthes
cucumerina L. and
Coriandrum sativum
Significant effect on liver
function test and total
protein globulin and
albumin
Herbal combination
has hepatoprotective
potential against
hepatotoxicity
induced by
paracetamol
Palanisamy
et al., 2014
Jian-Gan-Bao
Folk medicen
Coriolus versicolor,
Salvia miltiorrhiza
and Schisandra
chinensis
Significant effect on the
liver function enzymes
and antioxidants
Jian-Gan-Bao has
preventive potential
to cure alcoholic
liver diseases and
NAFLD
Li et al.,
2018
13
SAL herbal
combination
Schisandra chinensis,
Artemisia capillaris,
and Aloe barbadensis,
Significant reduction in
serum ALT and
antioxidant enzymes in
both SAL and CCl4
administrated group
SAL has synergistic
potential to against
hepatic
detoxification agents
Yimam et
al., 2016
Bi herbal
extract
Eclipta alba and
Piper longum
Substantially high liver
marker enzymes, LDH,
ƔGT and 5’nucleotidase
reverse to normalization
Strongly
hepatoprotective
against CCl4
induced toxicity
Samudram
et al., 2008
Ginseng
essence
(GE)
Panax ginseng Panax
uinquefolius,
Nelumbo nucifera and
Lilium longiflorum.
Significantly ameliorate
the high level of ALT,
AST and antioxidants
SOD, GSH and catalase
GE improves liver
inflammation and
fibrosis by
attenuation of
oxidative stress
Lu et al.,
2017
2.2.4. Starring role of phytochemicals as a antioxidants
About 80% of the world population is using green medicines to cure different diseases.
Medicines prepared from plants extracts are firstly evaluated through biological, chemical and
physical parameters (Veena et al., 2014). For chemical analysis, different chromatographic and
spectroscopic techniques are used to study the phytochemical constituents of the plants. LC-MS,
GC-MS and HPLC are most commonly used for the phytochemical study of plants (Demiray et
al., 2009; Talreja et al., 2017). In metabolic pathways production of free radicals is a normal
mechanism but uncontrolled and continues production induced DNA damage and protein
deterioration that cause cancer, cardiac and liver diseases (Sen et al., 2010). Many phytochemical
compounds such as tannins, phenolic acids, flavonoids has been present in medicinal plants
prevent the cells from oxidative damage by inhibiting reactive oxygen species (ROS) (Valko et
al., 2004; Govindarajan et al., 2005; Pandey, 2011).
2.3. In vitro techniques for identification of antioxidant and hepatoprotective potential of
medicinal plants
Different in vitro techniques including ferric reducing antioxidant power (FRAP) DPPH
and hydroxyl radical scavenging activity have been used to explore the antioxidant potential of
medicinal plants (Rashid et al., 2016). In vitro study of medicinal plants is easier than in vivo
studies in which animal models are used to perform experiments. For that reason liver models
14
such as Liver Slice Culture (LSC), precision cut liver slice and liver cell lines have been developed
to study the hepatoprotective potential of different medicinal plants (Farkas and Tannenbaum,
2005; Lemaire et al., 2011). In vitro Liver Slice Culture (LSC) assay is easy and sensitive method
to study the hepatoprotective potential of different plants against hepatotoxicity induced by
chemicals and drugs. Fresh liver of animal models are used in Liver Slice Culture assay under
controlled physiological conditions for the evolution of medicinal plants (Liu et al., 2015).
2.3.1 Role of lactate dehydrogenase in LSC assay
Normal function of the LDH is to convert pyruvate to lactate and vice versa but in case of
cancer and necrotic cells, cells membrane ruptured and lactate released from the cells (Moquin and
Chan, 2010). Liver Slice Culture assay is used for the study of hepatoprotective potential of
medicinal plants against toxicants such as carbon tetrachloride (CCl4). Liver cell become swelled
and necrotic when treated with CCl4 (Vatakuti et al., 2015). As a result of plasma membrane
breakdown, intracellular contents like LDH released into extracellular milieu in cancerous cells
(Figure 2.1). Lactate dehydrogenase (LDH) enzyme is also released in necrosis of hepatic cells
(Chan et al., 2013). The potent effects of medicinal plants against hepatotoxicity induced by
chemicals and drugs can be measured through amount of LDH released in terms of percentage
cytotoxicity (Rajopadhye and Upadhye, 2011).
Figure 2.1. Difference in lactate production in normal and cancer cells
(Adopted from Suely and Marie, 2011)
15
2.3.2 Role of Acetylcholinesterase in cell cycle
Acetylcholinesterase (AChE) is an enzyme that hydrolysis the acetylcholine (ACh) in
cholinergic synapse and convert it into acetate and choline. AChE also participates in cell
differentiation and development of different tissues (Vidyasagar et al., 2004; Sharma et al., 2005).
Acetylcholinesterase also present in the hematopoietic cells including megakaryocytes and
erythrocytes. Its overexpression can lead to abnormal mega karyo cytopoiesis (Zhang et al., 2002).
Acetylcholine is present in prokaryotic and eukaryotic cells and act as signalling molecule
(Kawashima and Fujii, 2000; Horiuchi et al., 2003).
Acetylcholine has two receptors muscarinic and nicotinic that promotes differentiation,
proliferation, apoptosis and cytoskeleton organization of the cell during phases of cell cycle (Tobin
and Budd, 2003; Resende et al., 2008). Hyper production of ACh intriguingly increases the Ca2+
influx and diverse activation of molecules such as Ras-mitogen activated protein kinase,
phosphatidylinositol 3-kinase-Akt, protein kinase C and c-Src through muscarinic and nicotinic
receptors and in result cell proliferation and apoptosis occurred (Resende and Adhikari, 2009). In
case of acute intoxication of pesticides and chemicals irreversible inhibition of
acetylcholinesterase activity is happened in blood, hepatic cells and nervous system, as a result,
ACh accumulated and may lead to proliferation and apoptosis of the cells (Nazam et al.,
2015)(Figure 2.2).
Figure 2.2. Cell proliferation and apoptosis through Acetylcholine and its receptors
(Adopted from Schuller, 2009)
16
2.3.4 Acetylcholinesterase inhibition assay: for screening of hepatoprotective plants
Hepatitis, acute and chronic liver diseases, liver cirrhosis and hepatocellular carcinoma are
more prevalent in East Asia and South Africa and cure of these diseases is still challenge for the
scientist (El-Serag and Rudolph, 2007; Gish et al., 2007). Different experimental studies
performed on the hepatocellular carcinoma cells and Huh-7 and HepG2 cell lines, it was revealed
that AChE plays an important role in cell proliferation suppression by the inhibition of
acetylcholine catalysis and degradation (Zhao et al., 2011; Perez-Aguilar et al., 2015). In vitro
many studies were performed on the medicinal plants to find out AChE inhibition activity.
According to experimental findings different medicinal plants such as Ziziphus jujube, Acorus
gramineus, Erodium malacoides and Asphodelus microcarpus have lower potential to inhibit
AChE (Oha et al., 2004). In present research work different medicinal plant were taken and
secerned out on the basis of AChE inhibition activity and then further used in combination to find
out their hepatoprotective activity.
2.4. In vivo evolution of hepatoprotective medicinal plants by chemically inducing
hepatotoxicity
Liver plays and important role in transportation and excretion of harmful chemicals from
the body but dysfunction of the liver caused hepatotoxicity (Singh et al., 2011). On the basis of in
vitro studies, in vivo studies are performed to find out the effects of hepatotoxic agents such as
acetaminophen, amodiaquine, allyl alcohol, carbon tetrachloride, cisplatin, isoniazid and halothane
on experimental animals (Lee, 2003; Navarro and Senior, 2006; Shimizu et al., 2009). An in vivo
system fully reflects the exposing profile and the cellular function as the compounds are exposed
in the successive manner through absorption from the first exposed site followed by metabolism,
distribution and elimination. They may be used to elucidate basic mechanism of xenobiotic
activities which will be useful in understanding their impact on human health (Dambach et al.,
2005).
2.4.1 Mechanism of carbon tetra chloride (CCl4) intoxication
Carbon tetrachloride (CCl4) is an organic chemical and mostly used as a cleaning agent
and precursor to refrigerants. It is a hepatotoxic agent and mostly used in experimental studies on
animals to find out the hepatoprotective potential of medicinal plants against hepatotoxicity
(Ritesh et al., 2015). CCl4 becomes active into highly reactive trichloro methyl radical (CCl3•)
after metabolization in endoplasmic reticulum through cytochrome p450 enzyme (Figure 2.3). This
CCl3• rapidly reacts with oxygen and form trichloromethyl peroxyl radical (CCl3OO•) a free
17
radical, which acts as a reactive oxygen species (ROS) and formed lipid peroxidation products
(Weber and Boll, 2003; Risal et al., 2012; Dey et al., 2016).
Figure 2.3. Hepatotoxicity inducing mechanism of CCl4 (Adopted from Dey et al., 2016)
Many experimental studies were performed on experimental animals to explore the effects
of medicinal plants as a hepatoprotectents against carbon tetra chloride. The results of plants
including Cinnamomum zeylanicum, Tapinanthus bangwensis, Abrus mollis, Silybum marianum,
Zizyphus spina-christi and many other plants have shown hepatoprotective potential by reduction
in liver enzymes and restoring of antioxidant enzymes (Patrick-Iwuanyanwu et al., 2010; Ezz El-
Din et al., 2011; Eidi et al., 2012; Mi et al., 2014).
18
CHAPTER 3
MATERIALS AND METHODS
The main objective of present study was to formulate an auspicious herbal combination of
medicinal plants to protect and manage hepatic disorders and its related complications. In order to
accomplish this target, research work was divided into three phases. In first phase (Phase-I), the
collected medicinal plants were screened out through in vitro antioxidant, acetylcholinesterase and
hepatoprotective activities. Toxicity evaluation through haemolytic and thrombolytic assay was
also the part of this phase. In second phase (Phase-II) detailed biochemical profiling of screened
plants was performed. Moreover, combinations of screened plants were formulated through RSM
and analyzed through liver slice culture (LSC) assay. At the end of phase-II, herbal combination
with least percentage cytotoxicity was selected for in vivo studies. In third and last phase (Phase-
III), in vivo trials were performed to evaluate the hepatoprotective effects of selected herbal
formulation on biochemical parameters and pathological changings on liver cells. All the research
work was conducted in Clinico-Medical Biochemistry Laboratory, Department of Biochemistry
of the University of Agriculture Faisalabad. Following methods and protocols were followed:
3.1 Phase I: In vitro screening of indigenous medicinal plants
In vitro screening of indigenous medicinal plants was executed through different
antioxidant activities, toxicological evaluation and hepatoprotective potential of selected
medicinal plants.
3.1.1 Collection of medicinal plants
For the development of promising hepatoprotective formulation, ten medicinal plants were
selected on the basis of already investigated and reported data of these plants. Some of the plants
were collected from the botanical garden, University of Agriculture Faisalabad and others were
procured from local herbal stores. The selected plants were made identified by Dr. Mansoor
Hameed, Associate Professor of the Botany Department, University of Agriculture Faisalabad.
Voucher specimens of medicinal plants were prepared and put down in the Herbarium, Department
of Botany, University of Agriculture Faisalabad. Detail of selected medicinal plants, their parts
used in presented research work and issue voucher numbers are given in the table 3.1.
19
Table 3.1: Voucher numbers of the selected medicinal plants with their parts used for
evaluation of hepatoprotective potential
Sr.
No
Botanical name Family Common
name
Selected parts of
plants
Voucher
number
1 Silybum marianum Asteraceae Milk
thistle
Seeds
804-1-17
2 Ficus religiosa Moraceae Peepul
tree
Leaves
804-2-17
3 Cassia fistula Fabaceae Amaltas
Leaves
804-3-17
4 Taraxacum officinale Asteraceae Dandelion
Roots
804-4-17
5 Polygonum
viviparum
Polygonaceae Anjbar
Rhizomes
53-1-18
6 Ziziphus jujuba Rhamnaceae Unaab
Fruit
804-5-17
20
7 Phyllanthus emblica Phyllanthaceae Aamla
Fruit
804-6-17
8 Fagonia arabica Zygophyllaceae Dhamasa
Shoots and leaves
804-7-17
9 Ocimum basilicum Lamiaceae Niazboo
Seeds
804-8-17
10 Martynia annua Martyniaceae Bichu phal
Fruits and seeds
53-2-18
3.1.2 Preparation of plant extracts
The selected parts of the plants were washed, dried and grinded into fine powder. The
powdered plants (25 gram of each) were soaked in 250 mL of methanol and kept in orbital shaker
for 7 days. After 7 days, supernatant was taken out through filtration, Whatman’s filter papers
No.1 were used to filter the macerate of each plant and methanol. The filtrate of each plant was
concentrated by rotary evaporator under reduced pressure (Rotavapor, Buchi R-215, Switzerland).
These concentrated plants extracts were dried completely and stored in air tight containers at 4°C
for further use (Sheng et al., 2014).
21
3.1.3 In vitro antioxidant activities of indegenous medicinal plants
In vitro total phnolic contnts (TPC), total flavonoid contents (TFC), DPPH redical
scavanging activity, hydroxyl redical scavanging (HRS) activity and feric reducing antioxidant
power (FRAP) assay were performed to explore antioxidant potential of slelected medicinal planst.
3.1.3.1 Total Phenolic Content (TPC)
Total phenolic content of indeginous medicinal plants were measured by following the
protocol of Lallianrawna et al. (2013).
3.1.3.1.2 Principle
Folin-Ciocalteu, a reducing reagent, is used to measure polyphenolic compounds in
medicinal plants. Folin-Ciocalteu reagent consists of two components phosphomolybdate and
phosphotungstate. When Folin-Ciocalteu reacts with phenolic contents prsent in plants, a reduced
chromophore blue color complex is generated. The absorbance of this blue colour product was
noted through spectrophotometer at 765 nm.
3.1.3.1.3 Procedure
The methanolic extracts of each plant (0.5 mg) were taken, dissolved in 1mL of Dimethyl
sulfoxide (DMSO) and used to measure total phnolic content. Subsequently 20 µL of each plant
and 100 µL of Folin –Ciocalteau reagent were taken in microwell plate and incubated for 3 minuts
in dark. After incubation, 100 µL of 10% Na2CO3 solution was added in the mixture of plant
extracts and Folin –Ciocalteau reagent and again incubated in the dark for one hour and then the
absobance was noted at 765 nm by using ELISA plate reader (µ Quant). Different concentrations
( 100, 200, 300, 400, 500 and 600 µg/mL) of Gallic acid was used as a standard to calculate TPC
in selected plants (Figure 3.1). The results of TPC were expressed as mean ± SD Gallic acid
equilants (GAE µg/g) (Nathia-Nevesa et al., 2017).
22
Figure 3.1. Standard curve of Gallic acid to measure total phenolic content
3.1.3.2 Total Flavonoid Content (TFC)
Aluminimum cloride (AlCl3) colorimetric method was used for the determination of total
flavonoid content of indejinous medicinal plants (Medini et al., 2017).
3.1.3.2.1 Principle
In Aluminimum cloride (AlCl3) colorimetric method, AlCl3 reacts with C4 keto groups
and either C3 or C5 hydroxal groups of falovonoids (present in plants) and forms acid stable
complexes. Moreover, AlCl3 also forms acid labile complexes with the orthodihydroxyl groups of
A and B rings of flavonoids and in result yellow colour product is generated that absorbance is
read spectrophotometrically at 510 nm (Pal et al., 2009).
3.1.3.2.2 Procedure
Total flavonoid content in methanolic extracts (0.5mg) of each plant were measured in
triplicates . For this purpose, 1mL of each plant extract (prepared in DMSO) and 300 µL of 5 %
NaNO2 solution were taken in test tubes and incubated at room temperature for 5 minutes. After
incubation, 300 µL solution of 10 % AlCl3.6H2O was added in the mixture of each test tube and
kept at room temperature for 5 minutes. After that 1 mL of 1M NaOH solution was added and
made up total volume up to 3 mL of each test tube by adding deionized water. The absobance of
the resulting mixture was taken at 510 nm through a double beam (UV- VIS) spectrophotometer
(Halo DB-20S, Dynamics instrument, UK). Catechin used as standard and its stanadard curve was
prepared by using different concentrations (100, 200, 400, 600, 800 and 1000 µg/mL) to measure
y = 0.0035x + 0.368R² = 0.9908
0
0.5
1
1.5
2
2.5
3
0 100 200 300 400 500 600 700
Ab
sorb
an
ce 7
65
nm
Concentrations of Gallic acid (µg/mL)
23
total flavonoid contents (Figure 3.2). The resrults of total flavonoid contents were presented as
mean ± SD of Catechin equivalants (CE µg/g plant) (Medini et al., 2014).
Figure 3.2. Standard curve of Catechin to measure total flavonoid content
3.1.3.3 DPPH radical scavanging activity
The DPPH radical scavanging activity of selected plants at different concentartions (100,
300 and 500 µg/mL) were determined by following the method of Sasikumar and Kalaisezhiyen,
(2014). This method is highly sensitive and extensively used for the detection of bioactive
constituents (antioxidants) in medicinal plants. The percentage inhibition of DPPH due to
antioxidants was taken at 517 nm and Ascorbic acid (1000 µg/mL) was used as a reference
standard.
3.1.3.3.1 Principle
DPPH (1, 1 Diphenyl 2- Picryl Hydrazyl) is a free radical stable compound. Antioxidants
of plants reduce DPPH into DPPH-H as a result the purplish / reddish colour of DPPH is changed
into yellow and absorbance is decreased. The change in colour and decrease in absorbance indicate
the scavenging potential of plants against free radicals (Sannigrahi et al., 2009).
3.1.3.3.2 Procedure
DPPH radical scavenging activity of plants extracts at 100, 300 and 500 µg/mL
concentrations were measured in triplicates. Firslty the fersh solution of 100 µM of DPPH in 95%
methanol was prepared. After that 195 µL of DPPH soution and 5 µL of each plant extract
y = 0.0019x + 0.6157R² = 0.9933
0
0.5
1
1.5
2
2.5
3
0 2 0 0 4 0 0 6 0 0 8 0 0 1 0 0 0 1 2 0 0
Ab
sorb
an
ce 5
10
nm
Catechin concentation (µg/mL)
24
concentration were taken in 96 well microplate and incubated in dark for 30 minutes. After
incubation absorbance was noted at 517 nm. The percentage inhibition of DPPH was calculated
by the follwing equation:
% Inhibition of DPPH = {𝟏 − (𝐀𝐬/𝐀𝐜)}× 100
Where “As” represents the absorbane of DPPH with plants extracts and “Ac” is the
absorbance of DPPH without plants extracts.
3.1.3.4 Hydroxyl radical scavanging (HRS) activity
Hydroxyl (OH) radical is a highly reactive oxygen specie and causes cell damage by
reacting with the phospholoipids of polyunsaturated fatty acids in the cell membrane.
3.1.3.4.1 Principle
In HRS assay hydroxyl radicals are measured by using hydrogen peroxide, FeSO4 and
Sodium salicylate along with plants extractas. The hydroxyl radicals produced by hydrogen
peroxide react with antioxidants of plants in the presence of sodium salicylate and a complex of
hydoxylated salicylate is genrated that absorbance is noted at 562 nm (Sharma et al., 2013).
3.1.3.4.2 Procedure
For hydroxyl radical scavenging activity, different concentrations (100, 300 and 500
µg/mL) of methnolic extratced slected plants were prepared in DMSO. The fersh solutions of 1.5
mM FeSO4, 6 mM of hydrogen peroxide (H2O2) and 20 mM sodium salicylate were prepared in
distil water. Reaction mixture was prepared in three ways. Mixture one was denoted as “A0” and
contained 1 mL FeSO4, 700 µL hydrogen peroxide and 300 µL of sodium salicylate. Mixture two
(A1) was prepared by adding 1 mL FeSO4, 700 µL hydrogen peroxide, 300 µL of sodium salicylate
and 1mL of each plant extract concentration. Whereas mixture three (A2) contained all the reagents
and plant samples except sodium salicylate solution. Incubated these reaction mixtures for 1 hour
and then absorbance of these mixtures were taken at 562 nm. Follwing equation was used to
calculate the hydroxyl radical scavanging activity of selected plants:
Scavanging activity (%) = {𝟏 − (𝑨𝟏 − 𝑨𝟐)/𝐀𝟎} × 100
Where “A0” presents the absorbance of the control samples without plants extracts, “A1”
is the absorbance in the presence of plants extracts and “A2” represents the absorbance without
sodium salicylate.
25
3.1.3.5 Ferric reducing antioxidant power assay
Ferric reducing antioxidant power (FRAP) is a powerful assay used to measure the
antioxidant potential of medicinal plants. Antioxidants act as a reducing and reduced reactive
oxygen species (ROS) that are harmful for DNA and cell membrane (Rabeta and Faraniza, 2013).
3.1.3.5.1 Principle
Ferric reducing antioxidant power (FRAP) assay is extensively used for the detection of
reductants in redox linked colorimetric reaction, where ferric (Fe3+) tripyridyltriazine complex
reduced into ferrous (Fe2+) 2, 4, 6 – tripyridyl -S- triazine and as a result blue color product is
formed that absorbance is noted at 593 nm through spectrophotometrically (Kumar and Seasotiya,
2011).
3.1.3.5.2 Procedure
Different concentrations (100, 300 and 500 µg/mL) of plants were prepared in DMSO and
used for ferric reducing antioxidant power assay. FRAP working reagent was prepared by adding
300 mM acetate buffer of pH 3.6, 20 mM FeCl3. H2O and 10 mM 2, 4, 6 – tripyridyl -S- triazine
(TPTZ) (dissolved in 40 mM of HCl) in a ratio of 10:1:1. After that 200 µL of each plant
concentration and 3 mL of FRAP reagent were taken in test tubes and incubated in water bath for
30 minutes. The change in absorbance of the incubated mixture was measured at 593 nm against
FRAP reagent used as a blank. FeSO4 .7H2O used as standard to measure the antioxidant contents.
A standard curve was prepared by using different concentrations (100, 200, 400, 600, 800 and
1000 µg/mL) of FeSO4 .7H2O (Figure 3.3). The values obtained from standard curve were
expressed as ferrous (Fe2+) equivalent µg/mL plant.
Figure 3.3. Standard curve of FeSO4 to calculate ferric reducing antioxidant potential of
medicinal plants
y = 0.0001x + 0.0391R² = 0.9852
0
0.02
0.04
0.06
0.08
0.1
0.12
0.14
0.16
0 200 400 600 800 1000 1200
Ab
sorb
ance
(5
93
nm
)
FeSO4 concentrations (µg/mL)
26
3.1.4 In vitro toxicological evaluation of medicinal plants
For toxicological evaluation, hemolytic and thrombolytic activities of selected plants were
performed in Bioassay Section, Medicinal Biochemistry Lab, Department of Biochemistry,
University of Agriculture Faislabad.
3.1.4.1 Hemolytic activity
Hemolytic assay is the primary assesment tool for the evaluation of toxicological
interaction of red blood cells with bioactive compounds present in medicinal plants. Hemolytic
activity of medicinal plants at 100, 300 and 500 µg/mL concentrations were measured by following
the method of Kumar et al. (2016).
3.1.4.1.1 Principle
In hemolytic assay generally human red blood cells (RBCs) are used. The free rasdicals,
present in plants, induce oxidative stress and lipid peroxidation of RBCs and in result destruction
of red blood cells occured. Therefore, hymolysis is dependent on antioxidant potential and
efficiency of plants (Vinjamur et al., 2015).
3.1.4.1.2 Preparation of erythrocytes suspension
Fresh blood was taken from the healthy persons and centrifuged at 2000 rpm for 3 minutes.
After centrifugation pallet was taken and washed three times with chilled (4°C) phosphate buffer
saline of pH 7.4 through centrifugation. The washed red blood cells (RBCs) were suspended in 20
mL chilled phosphate buffer saline and kept on ice to perform hemolytic activity (Kumar et al.,
2016).
3.1.4.1.3 Procedure
Different concentrations (100, 300 and 500 µg/mL) of metanolic extracted plants were
prepared in phosphate buffer saline. In micro centrifuge tubes 20 µL of each plant sample and 180
µL of diluted blood cell suspension were taken and incubated at 37 °C for 30 minutes with
agitation (80 rev/min). After incubation immediately placed these samples on ice for 5 minutes
and then centrifuged at 3000 rpm for 5 minutes. After centrifugation 100 µL supernatant of each
sample was taken in microcentrifuge tubes and added 900 µL of chilled phosphate buffer saline
for dilution. From these diluted samples took 180 µL of each sample in microplate and absorbance
was measured through ELISA reader at 576 nm. Triton X-100 (100 % blood lyses) was used as a
positive control and phosphate buffer saline (0% blood lyses) as a negative control. Hemolytic
27
assay of each plant at different concentrations were performed in triplicates and results were
expressed as Mean ± S.D in terms of percentage hemolysis.
Hemolysis (%) = (Absorbance of sample – Absorbance of negative control ) × 100
Absorbance of positive control
3.1.4.2 Thrombolytic activity
Thrombolytic activity of methanolic extracted plants were performed by follwing the
method of Chowdhury et al. (2015).
3.1.4.2.1 Blood clots preparation
In pre weighted micro centrifuge tubes, 500 µL of blood was taken for the preparation of
blood clot. These blood containing micro centrifuge tubes were incubated in heat controlled
incubator for 45 minutes at 37°C. After incubation, serum was removed cautiously from each tube
without disturbing the clot. Weighed the tubes with blood clots and used for thrombolytic activity.
3.1.4.2.2 Procedure
Prepared the fresh concentrations (100, 300 and 500 µg/mL) of each methanolic extracted
plant and then 100 µL of each plant sample was taken in micro-centrifuge tube with blood clot.
Distilled water and streptokinase were used correspondingly as a negative and positive control.
Incubated the all micro centrifuge tubes at 37 0C for 90 minutes. After incubation dissolved clots
were removed carefully from the tubes and weighed again. The difference in weight of the tubes
before and after clot lysis was calculated and expressed in terms of % clot lysis. The ability of
extracts and synthetic compounds to dissolve blood clot were compared with standard and blank.
All the experiment was repeated three times to decrease the chance of error and results were
presented as mean ± SD of percentage clot lysis.
3.1.5 In vitro hepatoprotective potential of medicinal plants
In vitro secreening of medicinal plants was done on he basis of hepatoprotoective potential
in which acetylcholinesterase inhibition and liver slice culture assay were performed.
3.1.5.1 Acetylcholineterase (AChE) inhibition assay
In vitro acetylcholinesterase inhibitory activity of medicinal plants was measured through
Ellman et al. (1961) method, which was modified by Rahman and Choudhary (2001).
3.1.5.1.1 Principle
Acetylcholinesterase is a crucial enzyme which degrades the neurotransmitter
acetylcholine into acetate and choline. Acetylthiocholine (ATCI) is used in this assay that reactes
28
with DTNB (5,5′-Dithiobis 2-nitrobenzoic acid) and formed a yellow color, 5-thio-2-nitrobenzoate
anion complex. Absorbance of this coloured product was noted at 412 nm.
3.1.5.1.2 Procedure
Different concentrations of all selected plants (100, 300 and 500 µg/mL) were prepared in
DMSO to perform acetylcholinesterase inhibition assay. Firstly sodium phosphate (0.1 M) buffer
of pH 7.8 was prepared. Then 10 mM solution of DTNB and 14 mM solution of acetylthiocholine
(ATCI) were perpared in sodium phosphate buffer and distill water respectively. After preparing
the solutions, 120 µL sodium phosphate buffer, 20 µL of each plant concentration and 20 µL of
enzyme solution (0.1 units/ mL) were taken in microplate and incubated at 25° C for 15 minutes.
Subsequently, 10 µL of DTNB and 10 µL of ATCI solutions were added in this mixture and again
incubated for 10 minutes and then absorbance of the coloured product was taken at 412 nm through
ELISA reader. Galantamine (10 µM) was used as a positive control. The percentage inhibition of
AChE was calculated by following formula and result were expressed in terms of mean ± SD of
AChE activity.
AChE Inhibition (%) = (1 – Abs. of sample/Abs. of control) × 100
3.1.5.2 In vitro hepatoportective activity through liver slice culture assay
In vitro Liver slice culture (LSC) assay was used for the evaluation of hepatoprotective
activity of methanolic extracted plants at different concentrations (50, 100, 300, 500, 700 and 1000
µg/mL). Wormser et al., (1990) protocol was used to perform liver slice culture assay after the
slight modification.
3.1.5.2.1 Reagents of liver slice culture assay
Krebs – Ringer – Hepes (KRH) medium of pH 7.4 was used LCS assay for washing and
incubation of liver slices. Composition of the Krebs – Ringer - Hepes medium is given in table
3.2.
Table 3.2: Krebs – Ringer – Hepes medium composition (g/ Liter)
Sr.
No.
Compunds Name Concentrations
(mM)
Amount
(g/L)
1 KCl 2.85 0.212
2 NaCl 118 6.895
3 KH2PO4 1.15 0.156
4 CaCl2 2.5 0.277
5 MgSO4.7H2O 1.18 0.290
29
6 β Hydroxy butyrate 5 0.499mL/L
7 Glucose 4 0.720
8 HEPES(4-(2-hydoxyethyl)-1-piperazine
ethanesulfide acid)
2.5 0.5957
3.1.5.2.2. Liver slice culture prepration
Healthy Albino rats were taken from animal house of University of Agriculture Faislabad.
These rats were dissected, removed their liver and transferred these liver lobs into pre warmed
Krebs – Ringer – Hepes (KRH) medium. The liver lobes were washed with KRH medium and
then cut into thin slices using sharp scalpe blades (Figure 3.4: A). After cutting transferred these
slices into Capped Eelemeyer flask containing 30 mL of KRH medium and incubated at 37°C on
a shaker water bath for 1 hour. In one hour time period, after every 10 minutes liver slices were
washed with 10 mL of new KRH medium.
3.1.5.2.3 Experimental procedure
Different concenrtations (50, 100, 300, 500, 700 and 1000 µg/mL) of methanolic extracted
plants were prepared in DMSO to eveluate hepatoprotective potential against hepatotoxcity
induced by CCl4 (Table 3.3). All experimental procedure of liver slice culture assay was run in
triplicates to minimize the chance of errors. From freshly prpared liver slice culture 20-22 liver
slices, of 100-120 mg were taken in 15 mL capped falcon tubes that already contain 2 mL KRH
media and incubated at 37°C for 30 minutes on a shaker water bath. After incubation, replaced
the KRH medium with fresh 2 mL KRH medium which contained different plants concentrations
(50, 100, 300, 500, 700 and 1000 µg/mL) (Figure 3.4: B). Ascorbic acid (10 mM) was used as a
reference standard. Incubated these liver slices with plants for 1 hour at 37°C and then 1 mL of
CCl4 (40 mM) was added in each falcon tube to induce hepatotoxcity. After adding CCl4 incubated
these slices again for 2 hours on shaker water bath at 37°C. In two hours time period, aerated the
liver slices after every 10 minutes with oxygen by removing the caps of falcon tubes. At the end
culture medium of each sample was collected after centirfugation to measure the Latate
dehydrogenase (LDH) level, which was the cytotoxic marker of liver injury.
30
Figure 3.4. A: Liver lobes cut into thin slices. B: Treatment of liver slices with
medicinal planst at different concentrations.
3.1.5.2.4 Estimation of lactate dehyrogenase (LDH)
A commerical LDH cytotoxicity assay kit II Abcam (ab65393) was used to measure the
lactate dehydrogenase in a culture medium. For this purpose, 100 µL of culture medium and 100
µL of raction mixture (WST substrate mix and LDH buffer) were taken in clear micro well plate.
The reaction mixture was incubated in dark at room temperature for 30 minutes and then absorbane
was noted at 490 nm by using micro plate reader. Firslty substratced the background control value
from all the other values to measure the percentage cytotoxicity. Following formula was used to
calcutae the percentage cytotoxicity of LDH released from liver slices:
Cytotoxicity (%) = Plant extract Abs – Low control Abs × 100
Low control Abs – High control Abs
After calulations, those plants were selected that showed minimum percentage cytotoxicity
againts hepatotoxicant CCl4.
Table 3.3: Experimental layout for Liver Slice Culture (LSC) assay
Sr.
No.
Experimental layout Treatments
1 Backgroung control KRH medium only
2 Low control KRH medium + liver slices
3 High control KRH medium + liver slices + 40 mM CCl4
4 Reference standard Liver slices + Ascorbic acid (10mM) + 40 mM CCl4
5 Silybum marianum
Liver slices + S. marianum (50 µg/ mL) + 40 mM CCl4
Liver slices + S. marianum (100 µg/ mL) + 40 mM CCl4
Liver slices + S. marianum (300 µg/ mL) + 40 mM CCl4
31
Liver slices + S. marianum (500 µg/ mL) + 40 mM CCl4
Liver slices + S. marianum (700 µg/ mL) + 40 mM CCl4
Liver slices + S. marianum (1000 µg/ mL) + 40 mM CCl4
6 Ficus religiosa
Liver slices + F. religiosa (50 µg/ mL) + 40 mM CCl4
Liver slices + F. religiosa (100 µg/ mL) + 40 mM CCl4
Liver slices + F. religiosa (300 µg/ mL) + 40 mM CCl4
Liver slices + F. religiosa (500 µg/ mL) + 40 mM CCl4
Liver slices + F. religiosa (700 µg/ mL) + 40 mM CCl4
Liver slices + F. religiosa (1000 µg/ mL) + 40 mM CCl4
7 Cassia fistula
Liver slices + C. fistula (50 µg/ mL) + 40 mM CCl4
Liver slices + C. fistula (100 µg/ mL) + 40 mM CCl4
Liver slices + C. fistula (300 µg/ mL) + 40 mM CCl4
Liver slices + C. fistula (500 µg/ mL) + 40 mM CCl4
Liver slices + C. fistula (700 µg/ mL) + 40 mM CCl4
Liver slices + C. fistula (1000 µg/ mL) + 40 mM CCl4
8 Taraxacum officinale
Liver slices + T. officinale (50 µg/ mL) + 40 mM CCl4
Liver slices + T. officinale (100 µg/ mL) + 40 mM CCl4
Liver slices + T. officinale (300 µg/ mL) + 40 mM CCl4
Liver slices + T. officinale (500 µg/ mL) + 40 mM CCl4
Liver slices + T. officinale (700 µg/ mL) + 40 mM CCl4
Liver slices + T. officinale (1000 µg/ mL) + 40 mM CCl4
9 Polygonum viviparum
Liver slices + P. viviparum (50 µg/ mL) + 40 mM CCl4
Liver slices + P. viviparum (100 µg/ mL) + 40 mM CCl4
Liver slices + P. viviparum (300 µg/ mL) + 40 mM CCl4
Liver slices + P. viviparum (500 µg/ mL) + 40 mM CCl4
Liver slices + P. viviparum (700 µg/ mL) + 40 mM CCl4
Liver slices + P. viviparum (1000 µg/ mL) + 40 mM CCl4
10 Ziziphus jujuba Liver slices + Z. jujuba (50 µg/ mL) + 40 mM CCl4
Liver slices + Z. jujuba (100 µg/ mL) + 40 mM CCl4
Liver slices + Z. jujuba (300 µg/ mL) + 40 mM CCl4
Liver slices + Z. jujuba (500 µg/ mL) + 40 mM CCl4
Liver slices + Z. jujuba (700 µg/ mL) + 40 mM CCl4
Liver slices + Z. jujuba (1000 µg/ mL) + 40 mM CCl4
11 Phyllanthus emblica
Liver slices + P. emblica (50 µg/ mL) + 40 mM CCl4
Liver slices + P. emblica (100 µg/ mL) + 40 mM CCl4
Liver slices + P. emblica (300 µg/ mL) + 40 mM CCl4
Liver slices + P. emblica (500 µg/ mL) + 40 mM CCl4
Liver slices + P. emblica (700 µg/ mL) + 40 mM CCl4
Liver slices + P. emblica (1000 µg/ mL) + 40 mM CCl4
12 Fagonia arabica
Liver slices + F. arabica (50 µg/ mL) + 40 mM CCl4
Liver slices + F. arabica (100 µg/ mL) + 40 mM CCl4
Liver slices + F. arabica (300 µg/ mL) + 40 mM CCl4
32
Liver slices + F. arabica (500 µg/ mL) + 40 mM CCl4
Liver slices + F. arabica (700 µg/ mL) + 40 mM CCl4
Liver slices + F. arabica (1000 µg/ mL) + 40 mM CCl4
13 Ocimum basilicum
Liver slices + O. basilicum (50 µg/ mL) + 40 mM CCl4
Liver slices + O. basilicum (100 µg/ mL) + 40 mM CCl4
Liver slices + O. basilicum (300 µg/ mL) + 40 mM CCl4
Liver slices + O. basilicum (500 µg/ mL) + 40 mM CCl4
Liver slices + O. basilicum (700 µg/ mL) + 40 mM CCl4
Liver slices + O. basilicum (1000 µg/ mL) + 40 mM CCl4
14 Martynia annua
Liver slices + M. annua (50 µg/ mL) + 40 mM CCl4
Liver slices + M. annua (100 µg/ mL) + 40 mM CCl4
Liver slices + M. annua (300 µg/ mL) + 40 mM CCl4
Liver slices + M. annua (500 µg/ mL) + 40 mM CCl4
Liver slices + M. annua (700 µg/ mL) + 40 mM CCl4
Liver slices + M. annua (1000 µg/ mL) + 40 mM CCl4
On the basis of in vitro screening results of indigenous ten medicinal plants, five plants that
showing maximum antioxidant and hepatoprotective potential were finally selected and further
analysed in phase II.
3.2 Phase II: Biochemical profiling of selected plants and their synergistic
hepatoprotective potential in different combinations Biochemical profiling of selected medicinal plants was performed through Liquid
chromatography mass spectrometry (LC-MS). The in vitro hepatoprotective potential of herbal
combinations (selected plants) was determined through Liver Slice Culture (LSC) assay.
3.2.1 Biochemical profiling of selected plants through LC-MS
S. marianum, T. officinale, P. viviparum, F. arabica and M. annua were analysed by using
Liquid chromatography combined with Electron Spray Ionization Mass spectrometry (LC-ESI-
MS) from National Institute of Biotechnology and Genetic Engineering (NIBGE) Faisalabad,
Pakistan. The extracts of selected medicinal plants were prepared in HPLC grade methanol. The
separation of the phytoconstituents was performed on HPLC Surveyor plus System equipped with
Surveyor auto (Thermo Scientific, San Jose, CA, USA). An equipped pump of Luna RP C-18
analytical column with 4.6 × 150 mm length and 3.0 µm particle size was used for HPLC analysis.
For the elution, two solvents were used including LCMS grade methanol (mobile phase A) and
acidified water 0.5% formic acid v/v (mobile phase B). On the base of gradient system solvent,
elution was executed with 0.3 mL/ min of flow rate. The gradient elution was encoded as follow:
from 10% A in 5 minutes, form 20 % B in 20 minutes and maintained it till the end of analysis.
33
The injection volume was 5.0 µL and temperature of the column was maintained at 25 °C. The
effluent from HPLC column was fixed for electron spray ionization mass spectrometer (LTQ XLTM
linear ion trap Thermo Scientific River Oaks Parkway, USA). Negative ion mode with spectra
acquired over a mass range m/z 200 to 2000 were used for LCMS analysis. The optimum values
of ESI-MS factors were sheath gas and auxiliary gas with 45 and 5 units/min respectively, spray
voltage +4.0 kV, Capillary temperature 320 °C, capillary voltage -20 V and tube lens -66.51 V.
For the interpretation of mass spectra data of the molecular ions X- caliber software (Thermo
Fisher Scientific Inc, Waltham, MA, USA) was used (Jiao and Zuo, 2009).
3.2.2 In vitro hepatoprotective potential of herbal combinations
Herbal combinations may have more medicinal potential against many diseases due to
synergistic effects of various components that increase bioavailability, specific activity and
antioxidant potential of medicinal plants (Eman et al., 2017, Li et al., 2018). Therefore, after
determination of hepatoprotective potential in terms of percentage cytotoxicity of indigenous
medicinal plants individually at various concentrations, best plants that showed minimum
percentage cytotoxicity were selected. Different herbal combinations of selected plants were
prepared by using central composite design (CCD) in response surface methodology (RSM) and
evaluated through liver slice culture assay (Myers et al., 2009).
3.2.2.1 Preparation of herbal combinations
Five medicinal plants S. marianum (150-250 mg), T. officinale (200-300 mg), P. viviparum
(25-100 mg), F. arabica (200-400 mg) and M. annua (20-100 mg) were selected to formulate
different herbal combinations through RSM with low and high dose levels. A statistical model was
developed by using various concentration of these medicinal plants through central composite
design (CCD). Design Expert 10.0 trial version (State-Ease Inc., Minneapolis, USA) was used to
design the experiment. Hepatoprotective potential of herbal combinations, suggested by the model
were then evaluated through Liver Slice Culture assay.
Central composite design suggested fifty (50) experimental runs of medicinal plants with
different concentrations and independent variables name as S. marianum (A), P. viviparum (B), T.
officinale (C), F. arabica (D) and M. annua (E). Each experimental run correspond to a different
herbal combination. In table 3.4 different herbal combinations with different concentrations of
medicinal plants were shown.
34
Table 3.4: Central Composite Design suggested herbal combinations
Run S. marianum
(mg)
(A)
P. viviparum
(mg)
(B)
T. officinale
(mg)
(C)
F. arabica
(mg)
(D)
M. annua
(mg)
(E)
1 221 47 271 258 77
2 179 47 271 342 43
3 179 78 229 258 43
4 200 63 300 300 60
5 200 63 250 300 60
6 221 47 229 342 77
7 221 78 229 258 77
8 200 63 250 200 60
9 179 78 229 342 43
10 179 78 271 258 43
11 200 63 250 400 60
12 200 63 250 300 60
13 221 47 229 342 43
14 221 47 271 342 77
15 200 63 250 300 60
16 221 47 271 258 43
17 200 25 250 300 60
18 200 100 250 300 60
19 179 47 229 258 77
20 200 63 250 300 20
21 200 63 250 300 60
22 179 78 229 342 77
23 221 78 229 342 43
24 221 78 271 342 43
25 179 78 271 342 77
26 200 63 200 300 60
27 179 47 271 258 77
28 200 63 250 300 60
29 179 78 229 258 77
30 221 78 271 258 43
31 150 63 250 300 60
32 179 78 271 258 77
33 200 63 250 300 60
34 221 47 229 258 43
35 250 63 250 300 60
36 179 78 271 342 43
37 221 47 271 342 43
38 221 78 229 258 43
35
39 179 47 271 258 43
40 221 78 229 342 77
41 200 63 250 300 60
42 179 47 229 258 43
43 179 47 229 342 77
44 200 63 250 300 100
45 221 78 271 342 77
46 221 47 229 258 77
47 221 78 271 258 77
48 200 63 250 300 60
49 179 47 271 342 77
50 179 47 229 342 43
3.2.2.2 In vitro Liver Slice Culture (LSC) assay of herbal combinations
Fifty combinations of selected plants suggested by central composite design were
formulated and evaluated for in vitro hepatoprotective potential through Liver Slice Culture assay
(Liu et al., 2015; Wormser et al., 1990; Rajoapdhye and Upadhye, 2011) as described in section
3.7 for individual medicinal plants. A brief protocol of Liver Slice Culture assay is given below.
For in vitro hepatoprotective activity fifty different concentrations of each plant were
prepared to form fifty herbal combinations. After disection of experimental rats livers were
removed and transferred into pre warmed Krebs – Ringer – Hepes medium (Table 3.2). Liver lobes
were washed with KRH medium and then cut into thin slices using sharp scalpe blades. After
cutting shifted these liver slices into Capped Eelemeyer flask containing 30 mL KRH medium and
incubated at 37°C on a shaker water bath for 1 hour. After incubation weighing liver slices (100-
120 mg) were taken in test tubes containing 2 mL KRH medium and incubated again at 37°C for
30 minutes on a shaker water bath. After that KRH medium was replaced with fresh 2 mL KRH
medium which contained different herbal combinations of selected plants and incubated for 1 hour
at 37°C Ascorbic acid (10 mM) was used as a reference standard. Incubated these slices and then
1 mL of CCl4 (40 mM) was added in each test tube to induce the heaptotoxcity and incubated again
for 2 hours on shaker water bath at 37°C. In this period of 2 hours aerated these liver slices after
every 10 minutes with oxygen by removing the caps of test tubes. After the final incubation culture
medium was collected to measure the Latate dehydrogenase (LDH) level using a commercial LDH
cytotoxcity kit by Abcam. Following formula was used to calcutae the percentage cytotoxicity in
terms of LDH released from liver slices:
36
Cytotoxicity (%) = Test sample Abs – Low control Abs × 100
Low control Abs – High control Abs
Here, “Test samples Abs” indicated liver slice culture medium of herbal combinations and 40 mM
CCl4, “High control Abs” showed absorbance of liver slice culture medium incubated with only 40
mM CCl4 and “Low control Abs” desiginated as culture medium of liver slices incubated with only
KRH medium.
All the concentrations of herbal combinations were recoded as optimum concentrations for
the hepatoprotective potential. The obtained values of percentage cytotoxcity from liver slice
culture assay were added in Design Expert software to calculate regression, analysis of varience
(ANOVA) and interactions between two variables (plants) by holding three variables constant.
After the measurememt of LDH levels and calulation of percentage cytotoxcity of herbal
combinations against CCl4, herbal combination that showed least percentage cytotoxcity was
considered as a best hepatoprotective herbal combination.
3.3 Phase III: In vivo Hepatoprotective potential of herbal combination
Herbal combination that showed least percentage cytotoxicity (in liver slice culture assay)
was selected to evalute in vivo hepatoprotective potential againts CCl4 induced hepatotoxcity. Both
curative and preventive mode of treatments were used for in vivo studie of herbal combination.
The dose of 200 mg/kg b. wt of herbal combination was used for in vivo analysis. As a standard
50 mg/kg b. wt of Silliver (Abboet labotries, Pakistan) was used. In rats Liver intoxication was
induced by 1 mL/Kg b. wt of CCl4 administration with olive oil in the ratio 1:1 v/v.
3.3.1 Ethical Approval
The ethical approval for animal trials was taken from Institutional biosafety and bioethical
Committee, University of Agriculture, Faisalabad. (Appendix 1)
3.3.2 Experimental animals
Albino (Sprague-Dawley) male rats (200-250g) were taken and placed in the animal house
of Clinical Medicine and Surgery (CMS) Department, University of Agriculture Faisalabad,
Pakistan. All animals were divided into two main groups comprising of curative and preventive
groups. Cutaive group was divided into five sub groups naming normal control, curative positive
control, curative negative control, cutaive treatment and curative standard group. Similarly
preventive group was divided into five sub group normal control, preventive positive control,
37
preventive negative control, preventive treatment and preventive standard group. Rats fed with
normal pallet diet. Rats were acclimatized for one week before start of experiment.
3.3.3 Power Calculation Analysis
Power calculation was used to calculate the minimum sample size for number of animals
per group. G* Power version 3.1.9.2. calculator software was used to measure the sample size in
this study. Sample size was calculated by inserting the values of alpha (α) and beta (1-β) error of
probabilty as 0.10 and 0.6 respectively. Total sample size of fifty (50) animals for ten (10) groups
were suggested by the power calculator was five that means each group was contained five
animals.
3.3.4 Hepatoprotective potential in Curative and preventive groups
Fifty male rats were divided into two main curative and preventive groups which were
further divided into ten sub groups (n=5) (Figure 3.5: A). Normal control group of curative and
preventive were received only normal diet for one month. Nagative control groups of both curative
and preventive were addministrated with herbal combination (200 mg/kg b. wt) for 30th days.
Curative positive control group rats were treated with 1mL/kg b. wt CCl4 for twice a week for the
period of thirty days, while curative treatment group was addministrated with CCl4 1mL/kg b. wt
twice a week and herbal combination 200 mg/kg b. wt daily for one month. Curative standard
group was received silliver 50 mg/kg b. wt daily and CCl4 1mL/kg b. wt twice a week for thirty
days (Table 3.5). In preventive mode of treatment, preventive positive control group was treated
with 1mL/kg b. wt of CCl4 at 28th and 29th day. Preventive treatment group was received herbal
combination 200 mg/kg b. wt daily for 27 days and at 28th and 29th day administrated with CCl4
1mL/kg b. wt. Preventive standard group was firslty treated with silliver 50 mg/kg b. wt for 27
days and then treated with 1mL/kg b. wt of CCl4 at 28th and 29th day in a period of one month
(Table 3.5).
Table 3.5: Curative and preventive groups for in vivo hepatoprotective actitivity
Curative
Groups
Dose addminitration Preventive
groups
Dose addminitration
Normal control Normal diet (30 days) Normal control Normal diet (30 days)
Curative
Negative
control
Herbal combination daily
(200 mg/kg b.wt) for 30
days
Preventive
Negative
control
Herbal combination daily
(200 mg/kg b.wt) for 30
days
38
Curative
Positive control
CCl4 (1 mL/kg b.wt)
twice a week for 30 days Preventive
Positive control
CCl4 (1 mL/kg b.wt) at
28th and 29th day
Curative
treatment
group
Herbal combination (200
mg/kg b.wt) daily and
CCl4 (1 mL/kg b.wt)
twice a week for 30 days
Preventive
treatment
group
Herbal combination daily
(200 mg/kg b.wt) for 27
days and CCl4 (1 mL/kg
b.wt) at 28th and 29th day
Curative
standard group
Silliver (50 mg/kg b.wt)
daily and CCl4 (1 mL/kg
b.wt) twice a week for 30
days
Preventive
standard group
Silliver (50 mg/kg b.wt)
for 27 days and CCl4 (1
mL/kg b.wt) at 28th and
29th day
3.3.5 Observation of body weight of rats
Body weight of rats were firstly measured before started the experiment to find out the
dose of herbal combination, hepatotoxicant CCl4 and silliver (Figure 3.5: B and C). At 7th, 14th,
21st and 28th day the body weight of rats were recorded for the dose calculation and administration.
Figure 3.5. A: Rats divided into groups, B: observation of body weight of rats for
dose calculation, C: Dose addministration to rat
3.3.6 Collection of blood of rats at different days
Blood samples of all curative groups were collected at 0th 2nd and 3rd, 10th, 20th and 30th
day into gel activator tubes to compare the changes occurred between these groups. Moreover,
blood samples all preventive groups were collected on 0th, 15th, 29th and 30th day to see the
difference in biochemical parameters between groups (Figure 3.6: A, B and C).
A B C
39
Figure 3.6. A: Blood collection, B: Blood in gel activator tubes, C: Separtion of
serum from blood
3.3.7 Measurement of Biochemical Parameters
The collected blood in gel activator tubes at different days were centrifuged at 3000 rpm
and serum was seprated in eppendrofs. These serum samples were used for the analysis of heaptic
markers, Alanine aminotranferase (ALT), Aspartate aminotranferase (AST), Alkaline
phosphatase, (ALP), Gamma glutamyl tranferase (GGT), Total bilirubin (TB), direct and indirect
bilirubin, Total protein (TP), Albumin (Alb), Globulin (Glb) and albumin globulin ratio (A/G).
These biochemical parameters were measured by using standard kits through Clinilab 200 semi
automated chemistry analyzer (Eidi et al., 2012; Cui et al., 2017).
3.3.8 Determination of acetylcholinesterase enzyme, Malondialdehyde (MDA) and
antioxidant enzymes in liver homogenate samples
3.3.8.1 Prepration of liver homogenate
Rats of all groups were secrificed to remove the liver and washed with phosphate buffer
saline (pH, 7.4), at the end of the experiment to see the ameliorative effects of herbal cpmbination
on acetylcholinesterase enzyme (AChE), malondialdehyde (MDA) and antioxidant enzymes
including Superoxide dismutase (SOD) and glutathione reductase (GSH). After washing liver
samples were homogenized (10% w/v) in phosphate buffer saline with pH 7.4. These liver samples
were then centrifuged for 10 minutes at 10,000 rpm and 4°C. After centifugation supernatents were
seprated and stored at -20 °C (Yimam et al., 2016).
A C B
40
3.3.8.2 In vivo acetylcholinesterase assay
In vitro acetylcholinesterase inhibitory activity of medicinal plants was measured through
Ellman et al., (1961) method with slight modifications suggested by Srikumar et al., 2004.
3.3.8.2.1 Procedure
Firstly sodium phosphate (0.1 M) buffer of pH 7.8 was prepared. Then 10 mM solution of
DTNB and 14 mM solution of acetylthiocholine (ATCI) were perpared in sodium phosphate buffer
and distill water respectively. After preparing the solutions, 120 µL sodium phosphate buffer, 20
µL of liver homogentae of each group and 20 µL of DTNB were taken in microplate and incubated
at 25° C for 15 minutes. Reaction was started by adding 20 µL of ATCI solutions in this mixture
and change in absorbance of was taken at 412 nm through ELISA reader for a period of 10 min
with 2 min. interval. The AChE activity was expressed in terms of µM of substrate
hydrolyzed/min/g tissue.
3.3.8.3 Estimation of Malondialdehyde (MDA)
A spectrophotometric method described by Okhawa et al. (1979) was used for the
determination of MDA level in liver homogenate samples.
3.3.8.3.1 Principle
This method is based on the condensation of two molecules of TBA with one molecule of
MDA, as a result two water molecules are eliminated and a coloured TBA pigment is formed that
gives absorbance at 532 nm.
3.3.8.3.2 Procedure
Liver homogenate samples (0.2mL) of each group were added into the reaction mixture
comprising of 0.2 mL of 8.1% SDS, 1.5 mL of 0.8% aqueous solution of TBA and 1.5 mL of 20%
acetic acid solution with adjusted pH 3.5. In this mixture, 0.6 mL of distilled water was added to
make the total volume of 4 mL and then heated for 60 minutes on water bath at 95°C. These
samples were cooled with tap water after heating and then added 5 mL mixture of n-butanol,
pyridine in a ratio of 15:1 v/v and 1 mL of distilled water. These samples were centrifuged at 4000
rpm for 10 min, separated the organic layer of each sample carefully and absorbance was noted at
532 nm. A stanadard curve (Figure 3.7) was prepared by using 100, 200, 300, 400, 500 and 600
nmol/mL concentraions of stanadrd MDA (Sigma). The values obtained from standard curve were
expressed as MDA nmol/mL in liver tissue homogenate samples.
41
Figure 3.7. Standard curve of MDA (nmol/mL) to measure MDA level in terms of
lipidperoxidation
3.3.8.4 Estimation of superoxide dismutase (SOD)
In liver homogenate samples superoxide dismutase activity was excuted by following the
method of Rajinder et al. (1981). SOD activity was measured by photoreduction and inhibition of
Nitro Blue tetrazol (NBT).
3.3.8.4.1 Procedure
All chemical reagents of SOD were prepared firstly which were consist of 67mM
phosphate buffer (pH, 7.8), 100 mM EDTA, 63 µM NBT, 13 mM methionine and 1.3 µM of
riboflavin. In micro well plate 10 µL of liver homogenate sample of each group, 204 µL of
phosphate buffer and 6 µL of riboflavin were taken and incubated in light box for 12 minutes.
After incubation 16 µL of EDTA, 8 µL of NBT and 6 µL of methionine were added in this mixture
and incubated again for 20 seconds. Absorbance was taken after each 20 second at 560 nm up to
120 seconds. All the experiments was run in triplicate to reduce the error and phosphate buffer was
used as a blank. SOD activity was measured in terms of percentage inhibition of NBT. Following
formula was used to measure the % inhibition and specific activity of enzyme 1U/mL/min/mg of
protein.
Inhibition (%) = Blank Abs- Sample Abs × 100
Blank Abs
Specific activity = Enzyme activity × mg of protein
y = 8E-05x + 0.0406R² = 0.9848
0
0.01
0.02
0.03
0.04
0.05
0.06
0.07
0.08
0.09
0.1
0 100 200 300 400 500 600 700
Ab
sorb
an
ce a
t 5
32
nm
Concentration of MDA nmol/mL
42
3.3.8.5 Estimation of Glutathione reductase (GSH)
The activity of GSH was measured by its capability to reduce the dithiobisnitro benzoate
(DTNB) (Razzaq and Malik, 2014).
3.3.8.5.1 Procedure
Reagents used in this assay were 0.1 M phosphate buffer saline (pH, 7), 0.5mM of DTNB
and 5 % TCA solution. For reaction mixture 250 µL of liver homogenate and 8.9 mL of phosphate
buffer saline were taken in test tubes and then 5% TCA solution was added to precipitate the
protein. After precipitation, centrifuged the reaction mixture at 3000 rpm for 10 minutes and
supernatant was taken in separate test tubes. Then 50 µL of DTNB was added in the supernatant
and incubated for one hour at 37°C. After incubation absorbance was of the coloured product was
taken at 412 nm. All the experiment was run in triplicates to minimize the chance of error.
Glutathione standard curve was prepared through GSH standard of different concentrations (20,
40, 60, 80, 100, 120, 140, 160, 180 and 200 mg/ml) at 412 nm. GSH µg /mg in liver tissue
homogenate were measured through the standard curve of GSH (Figure 3.8).
Figure 3.8. Standard curve of GSH (µg/mg) to calculate gluthathione reductase level
in liver homogenate
3.3.9 Histopathological studies
At the end of animal trial at 30th day, for histopathological examination all the rats were
sacrificed and livers were removed. These livers were washed to remove blood and preserved
y = 0.0037x + 0.2073
R² = 0.9939
0
0.2
0.4
0.6
0.8
1
1.2
0 50 100 150 200 250
Ab
sorb
ance
at
41
2 n
m
Concentration of GSH µg /mg
43
immediately in 10 % buffered formalin (pH, 7.2) (Chandra et al., 2015). Following steps were
used to process for histopathological examination.
3.3.9.1 Tissue preparation and sectioning of liver
For tissue preparation and sectioning of liver all the samples of liver were collected from rat
of different groups were labelled with India ink. The labelled samples were then subjected to tissue
processing which involved dehydration, clearing, embedding, casting and blocking.
3.3.9.1.1 Dehydration
After fixation in aqueous fixatives delicate liver samples were put in increasing strengths
of alcohols for dehydration. For this purpose the liver samples were first put in 70% ethanol for 1
hour, then in 90% ethanol for 2 hours and finally in 100% ethanol for 2 hours in order to replace
water in the tissue with alcohol.
3.3.9.1.2 Clearing (to remove alcohol)
Clearing of the tissue was achieved by immersing the tissue in Xylene in order to remove
alcohol from tissue.
3.3.9.1.3 Wax impregnation and embedding
For wax impregnation and embedding of tissue, the tissue was embedded in paraffin wax
for 2 hours in order to provide hard consistency to tissue so that thin section of 3-4 micron thickness
can be cut. The wax was melted at 60° C prior to impregnation.
3.3.9.1.4 Casting and blocking
Embedded tissues were place in a mold, which was made up of metal with their label and
then fresh melted wax was pour in it and was allow to settle and solidify. When the block became
cool it was immersed in cold water in order to prevent crystallization of wax. After the block was
completely cooled it was cut in to individual blocks and each was trimmed and individual blocks
were then labelled.
3.3.9.2 Staining of sections of liver
Haematoxylin and eosin staining was performed in order to stain thin section of liver. This
stain is commonly used for routine histopathology and in diagnostic cytology. It particular value
lies in its ability of imparting proper differentiation to distinguish between different types of
connective tissue fibbers’ and matrices, by staining them different shades of red and pink.
44
3.3.9.2.1 Principle
First the tissues are cleared of all wax and then are dehydrated to facilitate the entry of
dyes. The tissue sections and then sequentially exposed to basic dye e.g. Harris’s Haematoxylin
and an acid dye e.g. eosin. These stains bith acid are basic components of the tissue.
3.3.9.2.2 Reagents
a) Harris’s haematoxylin
Haematoxylin crystals 5.0 g
Alcohol 95% 50 mL
Ammonium or potassium Alum 100 g
Mercuric oxide 2.5 g
Distilled water 1L
Glacial acetic acid 40 mL
b) Acid Alcohol
Alcohol 70% 500 mL
HCL concentrated 5 mL
c) Ammonia water
Ammonia 2 mL
Tap water 1 L
d) Alcoholic eosin solution
Eosin (water soluble) 2 g
Distilled water 160 mL
Alcohol 95% 640 mL
e) Other reagents
Xylol, Absolute Alcohol, rectified spirit and methylated spirit.
3.3.9.2.3 Staining procedure
Liver sections were fixed on a glass slide and were kept for 3 minutes in xylol and then
transferred to absolute alcohol for 3 minutes. After that slides were transferred to rectified spirit
(80% alcohol) for 2 minutes. Slides were washed in running water for 1 minute and then put in
Harris’s haematoxylin for 5 minutes. After dyeing, all slides were washed in running water in order
to wash the excess dye in 1% acid alcohol by continuous agitation for 15 minutes. Then all the
slides were dipped in ammonia water solution until the tissues attained a blue colour. Slides were
then washed in running water and were counter stained with eosin for 3 minutes and washed again
in running tap water for 30 seconds. All the slides were then subjected to dehydration by keeping
in increasing concentration of alcohol (2-3 dips in 70%, 95% and absolute alcohol). After the
completion of staining procedure, slides were then cleared by xylol, examined and photographed
through Labomed LX 600, equipped with iVu 3100 (3 Megapixel) digital camera (Labo America
Inc.).
45
3.4 Statistical analysis
All the values were expressed as mean ± SD. Graph Pad prism (version 7.0) was used for
the statistical analysis. Two way ANOVA followed by Turkey’s multiple comparison test was
performed for the statistical difference in mean and P< 0.05 value was considered as statistically
significant (Montgomery, 2017).
46
CHAPTER 4
RESULTS AND DISCUSSION
The present research work was designed to formulate a herbal combination of indigenous
medicinal plants to prevent and manage numerous hepatic diseases and its related complications.
To achieve the objectives, the research work was divided into three phases and all the experimental
work was conducted in Clinico-Medical Biochemistry Laboratory, Department of Biochemistry,
University of Agriculture Faisalabad, Pakistan.
In phase I, ten indigenous medicinal plants naming S. marianum, F. religiosa, C. fistula,
T. officinale, P. viviparum, Z. jujuba, P. emblica, F. arabica, O. basilicum and M. annua were
collected on the basis of already investigated and reported data of these plants for in vitro
screening. On the basis of this in vitro screening in terms of antioxidant activities, toxicological
evaluation and hepatoprotective potential of these plants, five plants including S. marianum, T.
officinale, P. viviparum, F. arabica and M. annua were selected and used for further study. In
phase II, biochemical profiling of five selected plants was preformed through LC-MS and their
synergistic hepatoprotective potential in different combinations was determined through Liver
Slice Culture (LSC) assay in terms of percentage cytotoxicity. In phase III, the herbal combination
showing least percentage cytotoxicity was selected for in vivo studies to evaluate ameliorative
effects by following both curative and preventive mode of treatments.
4.1 Phase I: In vitro screening of medicinal plants
In phase one the in vitro screening of medicinal plants was executed. The results obtained
have been described and discussed under following headings.
4.1.1 Antioxidant activities of medicinal plants
Total phenolic and flavonoid content, DPPH radical scavenging activity, hydroxal radical
scavenging activity and ferric reducing antioxidant power (FRAP) assay were performed to
explore the antioxidant potential of studied medicinal plants.
4.1.1.1 Total phenolic and flavonoid content in medicinal plants
Total phenolic content of all the studied methanolic extracts of medicinal plants were
measured and expressed in terms of Gallic acid equivalents microgram per gram (GAE µg/g). The
highest total phenolic content (267.43 ± 20.06 GAE µg/g) was recorded in P. viviparum, while P.
emblica showed least amount of total phenolic content (67.02 ± 8.3 GAE µg/g) (Figure 4.1).
47
Whereas, M. annua and S. marianum contain correspondingly 236.62 ± 9.74 and 167.70 ± 21.02
GAE µg/g of total phenolic content (Table 4.1).
The total flavonoid content in all studied plants (methanolic extracts) were measured in
terms of Catechin equivalents (CE µg/g) and results were expressed as mean ± SD. The range of
total flavonoid content from 73.26 ± 1.56 to 131.86 ± 1.13 CE µg/g was observed among all
medicinal plants (Table 4.1). In M. annua, maximum flavonoid content (131.86 ± 1.13 CE µg/g)
was observed, whereas C. fistula was shown least quantity (73.26 ± 0.56 CE µg/g) of total
flavonoid content (Figure 4.2). Moreover, P. viviparum and T. officinale showed 130.46 ± 2.13
CE µg/g and 118.36 ± 2.12 CE µg/g flavonoid content respectively.
Table 4.1: Total phenolic and flavonoid Content in medicinal plants
Medicinal plants Total phenolic contents
(GAE µg/g plant)
Total Flavonoid Contents
(Catechin µg/g plant)
S. marianum 167.70 ± 21.02 116.86 ± 2.13
F. religiosa 130.94 ± 19.11 97.16 ± 1.41
C. fistula 76.35 ± 13.33 73.26 ± 1.56
T. officinale 140.67 ± 21.06 118.36 ± 2.12
P. viviparum 267.43 ± 20.06 130.46 ± 2.13
Z. jujuba 127.29 ± 28.22 98.76 ± 4.10
P. emblica 67.02 ± 8.3 112.86 ± 1.69
F. Arabica 95.21 ± 17.18 102.26 ± 1.84
O. basilicum 146.21 ± 15.28 110.86 ± 1.28
M. annua 236.62 ± 9.74 131.86 ± 1.13
Figure 4.1. Total phenolic content in indigenous medicinal plants
48
Figure 4.2. Total flavonoid content in indigenous medicinal plants
Natural bioactive compounds such as phenolic and flavonoids of plants are antioxidants and
are used as a therapeutic agent to control many diseases (Ghasemzadeh and Ghasemzadeh, 2011).
Many types of studies were conducted by researchers to find out the presence of phenolic and
flavonoid contents in different medicinal plants. The range of total phenolic content from 4.21 ±
0.01 to 11.88 ± 0.02 and total flavonoid content from 12.98 ± 0.03 to 26.42 ± 0.01 were observed
in different types of O. basilicum extracts (Kaurinovic et al., 2011). In another experimental study,
the ranges of total phenolic content from 81.5 ± 3.7 to 111.1 ± 2.7 mg/g GAE and total flavonoids
from 20.3 ± 1.6 to 38.7 ± 3.6 mg/g QE were recorded in P. emblica from different regions of China
(Liu et al., 2008). Lodhi et al. (2015) measured total flavonoid contents in ethanolic extracts of M.
annua which were 126.2 ± 4.69 mg quercetin equivalent. In another study 8.017 ± 0.130 mg
Chlorogenic acid equivalent phenolic content were recorded in T. officinale (Domitrovic et al.,
2010).
Both phenolic and flavonoids secondary metabolites present in medicinal plants, fruits and
vegetables have shown powerful anti-oxidative properties. Phenolics are strong reducing agents,
due to electron donating property that inhibit many free radical reaction, which induce oxidative
stress (John and Shahidi, 2010; Davaatseren et al., 2013; Angelis et al., 2018). These phenolic
contents are universally disseminated in plants and essential for plant growth and development
(Elzaawely et al., 2007; Dai and Mumper, 2010).
Several diseases including cancer, inflammation, neurodegenerative, cardiovascular and liver
infections are associated with increase of oxidative stress that damages DNA and cell membrane
49
(Babbar et al., 2011; Rodrigo et al., 2011). Plants are rich source of antioxidant in the form of
phenolic and flavonoids. These antioxidants have significant potential to inhibit the process of
oxidation and generation of reactive oxygen species that are harmful for human health (Lucio et
al., 2009). The presence of these secondary metabolites (Phenolics and flavonoids) show the strong
antioxidant potential and pharmacological actions against many diseases (Yogesh and Yadav,
2015; Preethi et al., 2016).
4.1.1.2 DPPH radical scavenging activity
PPH redical scavanging activity of medicinal plants of metanolic extracts at different
concentartions were determined through spectrophotometric method. This method is highly
sensitive and extensively used for the detection of bioactive antioxidants in medicinal plants
(Sasikumar and Kalaisezhiyen, 2014).
The DPPH free radical scavenging activity of medicinal plants at 100, 300 and 500 µg/mL
concentrations were performed in terms of percentage inhibition. The maximum percentage
inhibition of DPPH, at 100, 300 and 500 µg/mL concentrations of P. viviparum was 79.93 ± 3.53,
83.04 ± 3.52 and 84.73 ± 2.65 respectively (Table 4.2). On the other hand F. religiosa showed
least percentage inhibition of DPPH (33.04 ± 1.76, 39.34 ± 6.17 and 44.76 ± 3.53) at studied
concentration of 100, 300 and 500 µg/mL. Moreover, M. annua, T. officinale and F. arabica
showed 83.17 ± 7.05, 77.62 ± 2.65 and 82.54 ± 1.76 percentage inhibition of DPPH respectively
at 500 µg/mL.
Table 4.2: Percentage inhibition of DPPH at different concentration of medicinal plants
Medicinal plants Percentage inhibition of DPPH
100 µg/mL 300 µg/mL 500 µg/mL
S. marianum 49.63 ± 5.29 59.1 ± 1.76 65.96 ± 3.53
F. religiosa 33.04 ± 1.76 39.34 ± 6.17 44.76 ± 3.53
C. fistula 43.52 ± 2.47 49.31 ± 4.41 67.27 ± 1.58
T. officinale 52.12 ± 2.12 68.58 ± 1.76 77.62 ± 2.65
P. viviparum 79.93 ± 3.53 83.04 ± 3.5 84.73 ± 2.65
Z. jujuba 54.68 ± 2.65 70.01 ± 2.65 77.49 ± 2.65
P. emblica 65.9 ± 8.82 68.2 ± 3.53 77.31 ± 1.76
F. arabica 64.4 ± 2.65 70.14 ± 2.65 82.54 ± 1.76
O. basilicum 47.44 ± 1.15 74.44 ± 1.76 81.48 ± 2.65
M. annua 54.18 ± 1.68 77.87 ± 7.94 83.17 ± 7.05
50
Graphical presentation (Figure 4.3) of selected medicinal plants at three different
concentration showed that percentage inhibition of DPPH radicals increased significantly (p<
0.0001) in dose dependent manner with the increase in the concentration of medicinal plants.
Figure 4.3. Percentage inhibition of DPPH of medicinal plants
The spectophotometric DPPH radical sacavging assay is an inexpensive and accurate,
used widley for the determination of antioxidant activity in medicinal herbs, fruits and vegetables
(Salamone et al., 2011; Salamone et al., 2012). It is an appropriate method to qunatify the
antioxidative potential of medicinal planst againt free redical due to its reducing ability (Schaich
et al., 2015). DPPH radicals are reduced by aceptance of electrons and change their color from
blue to yellow in the presence of antioxidants (Dehpour et al., 2009).
Mostly, antioxidative properties of medicinal plants is due to the presence of flavonoids
and phenolics. Several human disorders including heart diseases, cancer, diabetes, Alzheimer’s,
Parkinson’s diseases and ageing are caused due to the over production of free radicals through
oxidation process which can be trapped by antioxidants (Young, 2001; Embuscado, 2015).
Different findings of previously studied plants naming F. religiosa, M. annua, Z. jujuba and P.
emblica supported the present data as percentage inhibition of DPPH activity is increased in dose
dependent manner due to the increase of antioxidant potential of these plants (Kaurinovic et al.,
2011; Yue et al., 2014; Arshad et al., 2017).
51
4.1.1.3 Hydroxyl radical scavanging activity
Hydroxyl (OH) radical is the highly reactive oxygen specie and causes cell damage by
reacting with the phospholoipids of polyunsaturated fatty acids in the cell membrane (Birben et
al., 2012). In hydroxyl radical scvanging activity, spectrophomrtricaly measured the hydroxyl
radicals in medicinal plants of methanol extracts at different (100, 300 and 500 µg/mL)
concentartions.
According to results it was revelead that percentage of hydroxyl radical scavanging activity
increased with the increase of plant concentrations (Table 4.3). At 100 and 300 µg/mL, P.
viviparum showed maximum hydroxal radical scavenging activity 59 ± 0.54 and 82 ± 0.25
respectively. Whereas at 500 µg/mL, high hydroxyl radical scavenging activity was recorded in S.
marianum (94 ± 0.17) and T. officinale (94 ± 0.11). On the other hand, Z. jujuba showed 32 ± 0.14,
74 ± 0.11 and 89 ± 0.20 percentage of hydroxyl radical scavenging activity at 100, 300 and 500
µg/mL concentrations respectively (Table 4.3). Results of hydroxyl radical scavenging activity
also depicted that M. annua, F. arabica and O. basilicum have good tendency to reduce free
radicals generated by hydrogen peroxide.
Table 4.3: Percentage of hydroxyl radical scavanging activity of selected plants at different
concentrations.
Medicinal plants
% Hydroxyl radical scavanging activity
100 µg/mL 300 µg/mL 500 µg/mL
S. marianum 54 ± 0.2 77 ± 0.12 94 ± 0.17
F. religiosa 52 ± 0.5 80 ± 0.20 91 ± 0.34
C. fistula 45 ± 0.3 65 ± 0.68 92 ± 0.57
T. officinale 58 ± 0.28 72 ± 0.62 94 ± 0.11
P. viviparum 59 ± 0.54 82 ± 0.25 93 ± 0.20
Z. jujuba 32 ± 0.14 74 ± 0.11 89 ± 0.20
P. emblica 56 ± 0.8 64 ± 0.22 86 ± 0.8
F. arabica 49 ± 0.22 76 ± 0.91 92 ± 0.14
O. basilicum 41 ± 0.45 65 ± 0.2 93 ± 0.40
M. annua 58 ± 0.5 78 ± 0.14 93 ± 0.28
Graphical presentation (Figure 4.4) of medicinal plants at three different concentration
clearly showed that hydroxyl radical scavenging activity significantly (p<0.0001) increased in
dose dependent manner.
52
Figure 4.4. Percentage of hydroxyl radical scavanging activity of plants.
Hydroxyl radical scavenging activity is used for the detection of antioxidant potential of
medicinal plants. Reactive oxygen species are highly reactive anions that are produced in oxidative
reactions which are taken place in mitochondria (Herraiz and Galisteo, 2015). The excessive
production of these ROS is harmful and caused many diseases like cancer, inflammation,
cardiovascular and hepatic disorders. ROS damages the biomolecules proteins and lipids and also
induced oxidative DNA damage, lipid peroxidation and apoptosis of the cells (Giesea et al., 2015).
The free radical scavenging activity in green hull of Juglans regia was studied and results of this
study showed antioxidant potential at different ethanolic concentrations (Sherma et al., 2013).
Medicinal plants are vulnerable sources of antioxidants that inhibit or reduced the process of
oxidation. In hydroxyl radical scavenging activity free hydroxyl radicals are present that are
reduced by antioxidant compounds of medicinal plants (Adjimani and Asare, 2015).
4.1.1.4 Ferric reducing antioxidant power assay
Ferric reducing antioxidant power (FRAP) is a powerful assay to measure the antioxidant
abilities of plants extracts (Birben et al., 2012). For FRAP assay methanolic extracts of plants at
100, 300 and 500 µg/mL concentration were used. The results of FRAP assay (Table 4.4) showed
that ferric reducing antioxidant potential increased with the increase in the concentration of plants.
53
In P. viviparum at 100, 300 and 500 µg/mL concetrations maximum ferric reducing
antioxidant power 164.79 ± 2.82, 215.24 ± 24.74 and 240.54 ± 17.67 were recorded respectively
(Table 4.4). Whereas minimum ferric reducing antioxidant power was observed in F. religiosa
10.14 ± 2.12, 21.79 ± 2.82 and 44.49 ± 8.48 at all studied concetrations. Moreover, S. marianum,
F. arabica and M. annua also showed good ferric reducing antioxidant potential as compare to C.
fistula, T. officinale, and Z. jujuba at 100, 300 and 500 µg/mL concentrations (Table 4.4).
Table 4.4: FRAP values (Fe (II) mg/mL) of plants at different concentrations.
Medicinal plants
Ferrous (Fe2+) equivalent mg/g plant
100 µg/mL 300 µg/mL 500 µg/mL
S. marianum 51.44 ± 2.12 93.24 ± 0.70 111.84 ± 2.12
F. religiosa 10.14 ± 2.12 21.79 ± 2.82 44.49 ± 8.48
C. fistula 35.49 ± 1.41 45.74 ± 6.36 47.24 ± 2.12
T. officinale 16.04 ± 3.53 37.19 ± 1.41 51.04 ± 6.36
P. viviparum 164.79 ± 2.82 215.24 ± 24.74 240.54 ± 17.67
Z. jujuba 40.94 ± 0.70 70.19 ± 2.82 83.24 ± 28.99
P. emblica 37.84 ± 2.12 105.54 ± 19.09 185.59 ± 26.87
F. arabica 35.89 ± 2.82 42.69 ± 2.82 127.19 ± 41.01
O. basilicum 73.64 ± 6.36 104.84 ± 0.70 174.94 ± 4.94
M. annua 75.49 ± 11.31 102.94 ± 13.43 237.54 ± 2.12
Ferric reducing antioxidant power (FRAP) results of selected medicinal plants were
graphically presented in Figure 4.5. It was clear from results that ferric reducing antioxidant power
potential was directly proportional to concentration of medicinal plants.
Figure 4.5. FRAP values (Fe (II) mg/mL) of indigenous medicinal plants
54
The ferric reducing antioxidant power assay is extensively used to find out antioxidant
potential of medicinal plants (Vijayalakshmi and Ruckmani, 2016). The antioxidant potential of
the bioactive compounds in medicinal plants detected through reduction of Fe3+ in to Fe2+, a blue
colour product (Gulcin, 2012; Benzie and Choi, 2014). Previously, various studies were done to
evaluate the ferric reducing ability of the antioxidant compounds of plants and their findings are
in a line with the results of present study (Kasture et al., 2014: Angelica et al., 2015).
Arshad et al. (2017) studied the antioxidant potential of M. annua fruit extracts through
ferric reducing antioxidant power assay. Results of his study showed that phytoconstituents of M.
annua significantly reduced Fe3+ into Fe2+ with the increase in plant concentration. In another study
FRAP assay was performed to determine the antioxidants in Japanese Lichens. Similar results were
observed in which samples exhibited ferric reducing antioxidants in a dose dependent manner by
reducing TPTZ - Fe3+ into TPTZ-Fe2+ (Eman et al., 2017). Phytochemicals of medicinal plants
are beneficial for human health due to their antioxidant ability to improve immune system of the
body by regulation of innate and adaptive immunity (Kumar et al., 2012; Estrada et al., 2013;
Patel, 2013).
4.1.2 Toxicological evaluation of medicinal plants
Toxicological evaluation was done by performing haemolytic and thrombolytic activities.
4..1.2.1 Hemolytic activity
Hemolytic activity of selected medicinal plants at different concentrations was performed
by follwing the method of Kumar et al. (2016). Haemolytic activity was performed at 100, 300
and 500 µg/mL concentrations of medicinal plants of metanolic extracts. Phosphate buffer saline
and triton X was used as a negative control and positive control respectively.
Results were presented in form of percentage hemolysis in table 4.5. The percentage
haemolysis increased with the increase of plant concentrations. At 100 µg/mL minimum
percentage of haemolysis was observed in C. fistula (2 ± 0.14), T. officinale (2 ± 0.82), Z. jujuba
(2 ± 0.77) and M. annua (2 ± 0.77) plants, whereas maximum percentage of haemolysis 14 ± 0.42
was recorded in F. arabica at same concentration. At 300 and 500 µg/mL concentrations C. fistula
showed least percentage of haemolysis 6 ± 0.71 and 12 ± 0.56 respectively. Among all the plants,
maximum percentage haemolysis (41 ± 0.7 and 50 ± 0.35) was found in P. emblica at 300 and 500
µg/mL concentrations (Table 4.5).
55
Table 4.5: Percentage haemolysis in selected plants at different concentrations.
Medicinal plants
Haemolysis (%)
100 µg/mL 300 µg/mL 500 µg/mL
S. marianum 3 ± 0.53 17 ± 1.6 39 ± 2.6
F. religiosa 4 ± 0.36 19 ± 0.707 34 ± 1.5
C. fistula 2 ± 0.14 6 ± 0.71 12 ± 0.56
T. officinale 2 ± 0.82 14 ± 0.42 21 ± 1.4
P. viviparum 4 ± 0.43 9 ± 1.4 23 ± 0.31
Z. jujuba 2 ± 0.77 9 ± 0.24 16 ± 1.06
P. emblica 4 ± 0.91 41 ± 0.7 50 ± 0.35
F. arabica 14 ± 0.42 20 ± 1.41 40 ± 0.49
O. basilicum 12 ± 0.7 23 ± 0.98 34 ± 0.45
M. annua 2 ± 0.77 9 ± 2.1 21 ± 1.5
Negative control 1.8 ± 0.24 (PBS)
Positive control 83 ± 0.83 (0.1 % Triton X)
It was elucidate from graphical representation (Figure 4.6) of medicinal plants at three
different concentration that the percentage haemolysis was significantly increased (p<0.0001) in
dose dependent manner with the increase in the concentration of medicinal plants from 100 to 500
µg/mL.
Figure 4.6. Percentage haemolysis of medicinal plants at three concentrations
Medicinal plants are used as alternative source of drugs to cure many diseases including
cardiovascular disorders, cancer, inflammation, liver and kidney related diseases due to their
antioxidant and therapeutic properties (Zohra and Fawzia, 2014). The natural and active
56
compounds such as flavonoids, phenolics, terpenes, alkaloids and steroids of medicinal plants have
pharmacological and biological properties against different diseases (Nakachi et al., 2000). But
before any treatment with the use of medicinal plants, it is necessary to find out its efficacy and
toxic effects (Kumar et al., 2016). Therefore to evaluate the toxic effects of medicinal plants,
haemolytic activity is performed on human erythrocytes that are used as a model due to same
membrane structure like other cells (Malagoli, 2007; De et al., 2008).
In present study for the screening of hepatoprotective medicinal plants, ten plants are used
and their haemolytic activity was performed to evaluate toxic effects of these plants. The depicted
results of present study are consistent with previous different studies in which haemolytic activity
was performed on different plants including Bridellia ferruginea benth, Solanum nigrum,
Passiflora species and Tragia involucrate. Results indicated that these plants significantly
inhibited the haemolysis of erythrocytes and percentage of haemolysis increased with the increase
in concentrations of these plants (Vinjamuri et al., 2015; Kumar et al., 2016; Cotos et al., 2017).
4.1.2.2 Thrombolytic activity of medicinal plants
Thrombolytic activity of medicinal plants (metnaolic extracts) at different concentrations
(100, 300 and 500 µg/mL) was performed by follwing the method of Chowdhury et al. (2015).
Thrombolytic activity was performed to assess the plants toxicity on blood clotting time period
and expressed in terms of percentage of clot lysis. Distilled water and streptokinase were used as
a negative and positive control respectively.
T. officinale showed maximum percentage of clot lysis 16 ± 5.73 at 100 µg/mL, whereas
minimum percentage of clot lysis 1 ± 0.76 was observed in C. fistula at same concentration (Table
4.6). At 300 and 500 µg/mL high percentage of clot lysis correspondingly 16 ± 4.39 and 30 ± 0.57
was observed in O. basilicum. The lowest percetage clot lysis was found in F. religiosa (7 ± 1.14)
and F. arabica (7 ± 1.14) at 300 µg/mL concentration. On the other hand At 500 µg/mL C. fistula
showed (3 ± 5.73) minimum percentage of clot lysis (Table.4.6). Moreover, S. marianum, T.
officinale, P. viviparum and M. annua were possessed against thrombolytic activity in terms of
percentage of clot lysis at all studied different concentrations.
57
Table 4.6: Percentage of Clot lysis in medicinal plants at different concentrations. Medicinal plants
Clot lysis (%)
100 µg/mL 300 µg/mL 500 µg/mL
S. marianum 6 ± 0.76 12 ± 3.64 27 ± 0.57
F. religiosa 8 ± 0.95 7 ± 1.14 4 ± 1.33
C. fistula 1 ± 0.76 10 ± 0.38 3 ± 5.73
T. officinale 16 ± 5.73 13 ± 0.84 11 ± 1.33
P. viviparum 6 ± 5.39 10 ± 0.38 22 ± 3.63
Z. jujuba 2 ± 0.93 8 ± 2.86 9 ± 0.38
P. emblica 2 ± 0.95 9 ± 0.57 10 ± 3.82
F. arabica 3 ± 0.87 7 ± 1.14 8 ± 2.86
O. basilicum 15 ± 6.32 16 ± 4.39 30 ± 0.57
M. annua 7 ± 2.10 14 ± 2.67 17 ± 1.33
Positive control 99 ± 14.56 (Streptokinase)
Negative control 8 ± 0.58 (Distilled water)
Figure 4.7 presented the percentage of clot lysis of medicinal planst at different
concentration. It was depicted from the results that thrombolytic acitivty was not increased in a
concentration dependent manner. S. marianum, P. vivparum, O. bacilicum and M. annua showed
signifcant (p>0.0001) change in percentage of clot lysis at all concentrations as comapre to other
studied plants.
Figure 4.7. Percentage of clot lysis through medicinal plants at three different
concentrations
Acute and chronic hepatitis and its associated liver diseases are the major health problem
now a days (Lisman et al., 2010; Kopec et al., 2016). Haemostasis is the blood clotting system and
it is also linked with liver disease. The component of haemostatic system are altered in patients of
liver diseases (Charles, 2008; Fontana et al., 2015). Liver is involved in the synthesis of many
58
plasma proteins including thrombopoietin, antifibrinolytic factors, pro and anti-coagulants factors
(Lisman et al., 2017). Thrombocytopenia a decrease in platelet count is also associated with
chronic liver cirrhosis (Tsao and Bosch, 2010). Medicinal plants play an important role in world
care health systems (Pandey and Mishra, 2010). Many pharmaceutical drugs are prepared from
medicinal plants and they have lesser side effect and less toxic as compared to synthetic drugs
(Annapurna et al., 2001).
Therefore to overcome these effect, in the present study screening of medicinal plants was
also performed on the basis of their thrombolytic activity. Results of thrombolytic activity of
studied plants are a line with other experimental works. Ahmad et al. (2015) evaluated the
thrombolytic activity of Macaranga denticulata bark. He observed that Macaranga denticulata
bark has significant thrombolytic activity with the percentage of clot lysis 31.59. In another study
thrombolytic activity of Asteraceae family plants was conducted and results showed the significant
percentage of clot lysis in all plants (Tabassum et al., 2017).
4.1.3 In vitro hepatoprotective potential of medicinal plants
In vitro hepatoprotective potential of medicinal plants was determined through
acetylcholinesterase inhibition assay and liver slice culture assay. The obtained results are as
followed
4.1.3.1 Acetylcholineterase (AChE) inhibition assay
Acetylcholinesterase is a crucial enzyme which degrades the neurotransmitter
acetylcholine into choline and acetate. In the absence of acetylcholinesterase enzyme cell
proliferation mechanism is increased that lead to more proliferation of the hepatocellular cells and
results in hepatocellular carcinoma (Johnson et al., 2004; Perez-Aguilar et al., 2015). In vitro
acetylcholinesterase inhibitory activity of medicinal plants (methanolic extracts) at 100, 300 and
500 µg/mL concentrations were measured through Ellman et al.,(1961) method which was
modified by Rahman and Choudhary (2001). Galantamine 10 µM concentration was used as a
positive control. The results of percentage inhibition of acetylcholinesterase enzyme are expressed
as mean ± SD.
Maximum percentage inhibition of AChE 52 ± 0.13 was recorded in P. emblica at 100
µg/mL, while C. fistula showed minimum percentage of AChE inhibition 26 ± 0.24 at same
concentration. At 300 and 500 µg/mL, P. emblica showed maximum percentage inhibition of
acetylcholinesterase 60 ± 0.22 and 86 ± 0.28 respectively (Table 4.7). On the other hand, F.
59
religiosa showed least inhibition of AChE 39 ± 0.20 and 42 ± 0.24 correspondingly at 300 and
500 µg/mL concentrations. Moreover, P. viviparum, P. emblica, F. arabica and M. annua have
also good potential to inhibit acetylcholinesterase enzyme at all studied concentrations.
Table 4.7 Percentage inhibition of AChE of medicinal plants at different
concentrations.
Medicinal plants
Percentage inhibition of Acetylcholinesterase
(AChE)
100 µg/mL 300 µg/mL 500 µg/mL
S. marianum 49 ± 0.14 56 ± 0.41 57 ± 0.39
F. religiosa 28 ± 0.28 39 ± 0.20 42 ± 0.24
C. fistula 26 ± 0.24 39 ± 0.11 58 ± 0.29
T. officinale 49 ± 0.22 50 ± 0.33 57 ± 0.46
P. viviparum 33 ± 0.21 47 ± 0.33 64 ± 0.27
Z. jujuba 46 ± 0.13 50 ± 0.15 57 ± 0.19
P. emblica 52 ± 0.13 60 ± 0.22 86 ± 0.28
F. arabica 44 ± 0.18 48 ± 0.13 75 ± 0.26
O. basilicum 51 ± 0.39 57 ± 0.34 63 ± 0.22
M. annua 42 ± 0.21 54 ± 0.03 78 ± 0.25
Galantamine + ve control 95 ± 0.23
Graphical presentation (Figure 4.8) of medicinal plants at three different concentration
clearly depicted that the percentage inhibition of acetylcholinesterase was significantly increased
(<0.0001) in a dose dependent manner.
Figure 4.8. Percentage inhibition of Acetylcholinesterase in medicinal plants
60
Acetylcholinesterase (AChE) is a hydrolytic enzyme that splits acetylcholine into acetate
and choline. Acetylcholine is a key regulator for cell survival, differentiation and proliferation
(Shen et al., 2013). Excessive production of acetylcholine in the absence of AChE, increased the
cell proliferation by downregulation of p27 and cyclin proteins that are main component of cell
cycle (Catassi et al., 2008; Pérez-Aguilar et al., 2015). Visceral organs including liver, heart,
kidney, placenta, intestines and pancreas contained variable amount of AChE (Soreq and Seidman,
2001). In the absence of AChE, acetylcholine is accumulated in the haptic cells and induced
hepatotoxicity and hepatocellular carcinoma by increasing cell proliferation mechanism (Forner et
al., 2012; Bruix and Colombo, 2014).
Present study was designed to evaluate the hepatoprotective potential of medicinal plants.
Therefore plants that showed minimum percentage inhibition of AChE are beneficial for the
hepatic cell. Findings of the present study are comparable with different previous studies in which
acetylcholinesterase percentage inhibition was measured in different medicinal plants and
galantamine was used a positive control (Soreq and Seidman, 2001; Moniruzzaman et al., 2015).
4.1.3.2 In vitro hepatoportective activity through liver slice culture assay
In vitro liver slice culture assay was performed to measure release of Lactate
dehydrogenase (LDH) from liver cells in terms of percentage cytotoxicity at different
concentrations (50, 100, 300, 500, 700 and 1000 µg/mL) of medicinal plants (methanolic extracts).
For liver slice culure assay Womser et al., (1990) protocol was used with slight modification by
Rajoapdhye and Upadhye (2011). Results of percentage cytotoxicity of medicinal plants were
presented in terms of mean ± SD (Table. 4.8).
Persentage cytotoxcicity was measured in high control, low control and stanadard groups.
In high control group, liver slices were treated with hepatotoxin 40 mM CCl4, while low control
group was not received any treatment. Stanadrd group was treated with 10 mM ascorbic acid and
40 mM CCl4. High control group was shown maximum percentage cytotoxicity (72%), whereas
minimum percentage cytotoxicity 4% was observed in low control group (Figure 4.9). These
findings inndicated that more LDH is releasd from the hepatic cells when treated with hepatotoxin
CCl4 as compare to the non treated liver slices. In standard group 17% cytotoxcity was observed
as compare to high control group (72%) that means acorbic acid protectes the liver cells from
hepatotoxin CCl4.
61
Figure 4.9. Presented percentage cytotoxicity in high control, low control and
standard groups
Liver slices treated with plants at different concentrations and hepatotoxicant CCl4 showed
decrease in percentage cytotoxicity (LDH) with the increase of plants concentrations (Figure 4.10).
The range from 50 ± 0.55 to 6 ± 0.01of percentage cytotoxicity was found from 50 to 1000 µg/mL
concentrations of among studied medicinal plants.
Z. jujuba was showed high percentage cytotoxicity 50 ± 0.55 at 50 µg/mL, while minimum
percentage cytotoxicity 29 ± 0.39 was observed in M. annua at same concentration. Similarly at
100 µg/mL Z. jujuba and M. anuua showed maximum (38 ± 0.27) and minimum (26 ± 0.11)
percentage cytotoxicity respectively. F. arabica showed lowest percentage cytotoxicity 19 ± 0.39
and 13 ± 0.12 correspondingly at 300 and 500 µg/mL concentrations. M. annua was shown lowest
percentage cytotoxicity 8 ± 0.50 at 700 µg/mL and 6 ± 0.18 at 1000 µg/mL, while maximum
percentage cytotoxicity 33 ± 0.70, 31 ± 0.88, 24 ± 0.104 and 14 ± 0.49 were observed in F.
religiosa at 300, 500, 700 and 1000 µg/mL concentrations respectively. Moreover, it was proven
from the results that S. marianum, C. fistula, P. viviparum, T. officinale, F. arabica and M. annua
have hepatoprotective potential against CCl4 at all studied concentrations (Figure 4.10).
62
Table: 4.8: Percentage Cytotoxicity of medicinal plants at different concentrations against
hepatotoxicant CCl4
Medicinal
plants
% Cytotoxicity in liver slices against CCl4
50 µg/mL 100 µg/mL 300 µg/mL 500 µg/mL 700 µg/mL 1000 µg/mL
S. marianum 32 ± 0.22 30 ± 0.4 26 ± 0.45 21 ± 0.3 13 ± 0.66 8 ± 0.30
F. religiosa 46 ± 0.077 35 ± 0.5 33 ± 0.70 31 ± 0.88 24 ± 0.104 14 ± 0.49
C. fistula 30 ± 0.89 28 ± 0.6 22 ± 0.41 16 ± 0.21 10 ± 0.38 7 ± 0.01
T. officinale 34 ± 0.38 32 ± 0.59 24 ± 0.64 19 ± 0.08 13 ± 0.44 10 ± 0.33
P. viviparum 34 ± 0.33 28 ± 0.12 24 ± 0.31 19 ± 0.16 15 ± 0.50 9 ± 0.21
Z. jujuba 50 ± 0.55 38 ± 0.27 26 ± 0.4 21 ± 0.29 17 ± 0.39 14 ± 0.74
P. emblica 44 ± 0.15 36 ± 0.93 30 ± 1.03 23 ± 0.06 21 ± 0.54 12 ± 0.30
F. arabica 38 ± 0.62 31 ± 0.2 19 ± 0.39 13 ± 0.12 11 ± 0.56 8 ± 0.602
O. basilicum 39 ± 0.18 30 ± 0.51 23 ± 0.61 14 ± 0.32 9 ± 0.86 8 ± 0.204
M. annua 29 ± 0.39 26 ± 0.11 28 ± 0.68 18 ± 0.49 8 ± 0.50 6 ± 0.18
It was depicted from the graphical presentation that percentage cytotoxicity in terms of
LDH released decreased with the increase of plants concentrations (Figure 4.10).
Figure 4.10. Trend of percentage Cytotoxicity of medicinal plants at different concentrations
in terms of LDH released
63
Liver slice culture assay is in vitro technique that provide a suitable environment to liver
cells and valuable approach for the screening of hepatoprotective medicinal plants (Wormser and
Ben, 1990; Rajopadhye and Upadhye, 2011). In this assay Krebs – Ringer – Hepes (KRH) medium
was used for the maintenance of actual environment of the liver cells (Naik et al., 2004). Different
drug and chemicals are used as a hepatotoxicant including paracetamol, cisplatin, carbon
tetrachloride and ethanol to induce oxidative stress in the liver cells through generation of free
radicals (ROS) (Liu et al., 2015; Vatakuti et al., 2015).
These free radicals or supper anions that react with biological molecules like
carbohydrates, lipids and proteins in cells and in result lipid peroxidation of the cell membrane
and oxidative DNA damage occurred (Lim et al., 2007; Loguidice et al., 2011). Lactate
dehydrogenase (LDH) is cytosolic enzyme that convert lactate into pyruvate and wise versa. In
cell cytotoxicity and lipid peroxidation LDH released in the culture medium. Plants exhibited
cytotoxic effects against hepatotoxicant CCl4 by decreasing the LDH release. Plants are used along
with heaptotoxicants in liver slice culture assay and amount of LDH released in the medium
elucidate the hepatoprotective potential of the plants (Kim, 2002; Shi et al., 2006). Medicinal
plants have therapeutic potential to cure many diseases which are associated with oxidative stress
due to their anti-oxidative properties (Matsuda et al., 2009; Huang et al., 2010; Kumar et al., 2011).
In the present research work liver slice culture assay was used for the screening of ten
medicinal on the basis of their hepatoprotective potential against hepatotoxicant CCl4. The
outcomes of present study were consistent with previous studies. Gevrenova et al. (2015)
determined the antioxidant effects through DPPH, ABTS method and hepatoprotective potential
of three Bupleurum species on rats liver cells. Hepatotoxicity was induced by carbon tetrachloride
and tert-butyl hydroperoxide and measured the level of lactate dehydrogenase and
malondialdehyde released from hepatic cells. It was revealed from his study that Bupleurum
species have hepatoprotective potential by inhibiting the production of free radicals.
In another study similar type of results were observed in which in vitro hepatoprotective
activity was performed through liver slice culture model to assess the hepatoprotective and
antioxidant potential of Rungia repens whole plant against CCl4. Lipid peroxidation in terms of
thiobarbituric acid reactive substances (TBARS) and LDH released from the liver cells were
measured. Results of this study showed the significant decrease in LDH and lipid peroxidation in
that group which was treated with both plant and CCl4 as compared to only CCl4 treated group
64
(Rajopadhye and Upadhye, 2011). The present study results were in line with another research
work in which in vitro hepatoprotective activity of S. aromaticum was performed at different
concentrations through liver slice culture assay. The range of percentage cytotoxicity from 7.35 to
16.16% was observed in S. aromaticum at different concentration in comparison with only CCl4
treated group in which 75.58 % cytotoxicity was observed (Hina et al., 2017).
Medicinal plants are important for human health due their pharmacological and biological
properties and used to cure many diseases which are originated from oxidative stress (Amudha
and Komala, 2014). Antioxidants including phenolic, flavonoids and glycosides are the secondary
metabolites of plants which inhibited the process of oxidation and generation of free radicals (Cui
et al., 2014). Almost all the biochemical reactions of drug metabolism are taken place in the liver
and many drug induced harmful side effects on hepatic cells (Hong et al., 2015; Yimam et al.,
2016). Therefore to overcome these side effects green medicine seek the attention of the scientist
to treat different diseases. To find out the therapeutic potential of plants, in vitro models like human
hepatic cell lines, neuronal PC-12 cells and rat’s hepatocytes models are used (Pramyothin et al.,
2007; Choi et al., 2015). These models are beneficial to screen the medicinal plants on the basis
of their lesser side effects and more therapeutic potential against diseases (Asad et al., 2012).
Therefore, on the basis of obtained results of antioxidant activities, toxicological evaluation
and in vitro hepatoprotective potential five plants S. marianum, T. officinale, P. viviparum, F.
arabica and M. annua were selected and further analyzed in Phase II.
4.2 Phase II: Biochemical profiling of selected plants and their synergistic
hepatoprotective potential in different combinations
In phase II, biochemical profiling of the finally selected medicinal plants was performed
through LC-MS. Moreover, fifty combinations of selected medicinal plants (suggested by RSM)
were prepared and analysed for hepatoprotective potential through Liver Slice Culture (LSC)
assay.
4.2.1 Biochemical profiling of medicinal plants
Biochemical profiling of five selected plants naming S. marianum, T. officinale, P.
viviparum, F. arabica and M. annua was done through LC-MS. Methanolic extracts of these plants
were used for LC-MS analysis. Different bioactive compounds including phenolics, flavonoids,
and alkaloids were identified through liquid chromatography (LC) and structural elucidation of
specific compounds was determined by peak fragmentation through Collision Induced
65
Dissociation (CID) in mass spectroscopy (MS) technique. LC-MS analysis was performed in
National Institute of Biotechnology and Genetic Engineering (NIBGE), Pakistan.
4.2.1.1 Biochemical profiling of S. marianum
LC-MS analysis of S. marianum was performed to determine the phytoconstituents. The
full mass spectrum showed the high peaks at 130.08, 335.33, 365.33, 381.25.428.25 and 521.25
m/z (Figure 4.11). The peaks at m/z of 179 and 377.17 depicted the presence of Caffeic acid
(C9H8O4) and Chlorogenic acid (C16H18O9) respectively (Figure 4.12). Previous studies on other
medicinal plants also idetenfied chlogenic acid on this peak (Michael et al., 2003). The mass
spectrum obtained from LC-MS analysis in figure 4.13 of S. marianum showed the highest peak
at 381.25 m/z that indicated the presence of sweroside with molecular formula C16H22O9.
Sweroside is an iridoid glycoside which exhibits both pharmacological and biological effects
including anti hepatitis, anti- inflammatory, anti-allergic and anti- fungal activities (Sun et al.,
2013; Yang et al., 2016). It was also revealed from different studies that sweroside ameliorates
liver injury by decreasing oxidative damage of hepatic cells (Jeong et al., 2015).
66
Figure 4.11. Full mass spectrum of S. marianum
Figure 4.12. Mass spectrum indicating the presence of Caffeic acid and Chlorogenic acid.
Figure 4.13. Mass spectrum of S. marianum showing Sweroside at 381.25 m/z
67
The peaks at 304.08 and 448.25 m/z indicated the presence of flavonoids Taxifolin
(dihydroquercetin) and Luteolin 7-O-glucoside (Cynaroside) respectively (Figure 4.14). Luteolin
7-O-glucoside (C21H20O11) presence in figure 4.15 was further confirmed by MS-MS using CID
(20:00) at 285 m/z. Similar peaks of Luteolin 7-O-glucoside were observed in characterization of
danlion (Katrin et al., 2005; ). It was revealed from different studies of plants that both of these
flavonoids, Taxifolin and Luteolin 7-O-glucoside possess antioxidant a nd anticancer potential
(Tudorel et al., 2015; Thi et al., 2016).
Figure 4.14. Mass spectrum indicating the presence of Taxifolin and Luteolin 7-O-
glucoside
Figure 4.15. MS/MS of S. marianum peak 303 at CID (20.00) showing Luteolin 7-O-
glucoside at 285 m/z
The highest peak 481.25 m/z presented in figure 4.16 revealed the presence of Silybin A
and B in S. marianum. The MS-MS of peak 481.25 into 257.08, 301.08 and 453.25 m/z by CID
68
(22:00) indicated the confirm presence of Silybin (Figure. 4.17). It is an active phytoconstituents
and main component of silymarin present in S. marianum. Silymarin has a strong antioxidant
potential and ability to promote liver cell regeneration and reduction in blood cholesterol (Lee et
al., 2006). Silybin is a major hepatoprotective flavonolignan and have pharmacological activities
including anti-inflammatory and anti-tumour effects (Kren and Walterva, 2005).
Figure 4.16. Mass spectrum of S. marianum showing Silybin at 481.25 m/z
Figure 4.17. MS/MS of S. marianum peak 481.25 m/z at CID (22.00) indicating the presence
of Silybin.
4.2.1.2 Biochemical profiling of T. officinale
The LC-MS analysis of T. officinale was executed to find out the presence of
phytochemicals that are helpfull to cure heaptic infections. Figure 4.18 depicted full mass spectrum
of T. officinale and different peaks were found at 203.08, 219.08, 381.25 and 428.25 m/z. The
peaks at m/z 381.25 and 465.33 were indicated the presence of bioactive compounds Swerosid
69
(C16H22O9) and Isoquercetin (C21H20O12) respectively (Figure 4.19). Sweroside is an irioid, active
secondary metabolite involved in different metabolic process. It is a natural bioactive compund
present in Swertia pseudochinensis that is used as therapeutic agent to treat diarrhea and juandice
(Han et al., 2014). Sweroside has potential to cure hepatobiliary disorders and possess antioxidant,
cell neuritogenic and wound healing properties (Wei et al., 2012; Mihailovic et al., 2013).
Isoquercetin a bioactive compound present in T. officinale, also found in Apocynum venetum L.a
hepatoprotective plant (Xie et al., 2016). Isoquercetin extensively present in different fruits and
vegetables and it shows different biological including free radical scavenging potential,
neuroprotective, diuretic, antihypertensive and anti-inflammatory effects (Huang et al., 2014;
Chen et al., 2015).
Figure 4.18. Full mass spectrum of T. officinale
Figure 4.19. Mass spectrum indicating the presence of Sweroside (381.25 m/z) and
Isoquercetin (465.33 m/z)
70
The mass spectrum of T. officinale illustrated the presence of Quercetin and Myricetin
correspondingly at 303.25 and 319.33 m/z correspondingly (Figure 4.20). Presence of these two
bioactive compounds were further confirmed by MS-MS. The MS-MS of peak 303.17 m/z (CID
25:00) into 271.08 and 285.08 m/z with positive ESI directed the confirmation of Quercetin in T.
officinale (Figure 4.21). The MS-MS of peak 319.25 m/z by CID (25:00) with + ESI confirmed
the presence of Myricetin (Figure 4.22). Myricetin is well known flavonoid present in teas, fruits
and vegetables. Myricetin is a powerful antioxidant compound that exhibit scavenging activity
towards a number of radicals and ions (Chang et al., 2015). It was observed from different studies
that Myricetin plays a crucial role against diabetes, cancer, inflammation, hepatic and
neurodegenerative disorders (Shimmyo et al., 2008; Matic et al., 2013; Grenier et al., 2015).
Figure 4.20. Mass spectrum of T. officinale showed Quercetin and Myricetin at 303.25 and
319.33 m/z.
Figure 4.21. MS/MS with CID (25.00) of peak 303.17 m/z indicating the presence of
Quercetin
71
Figure 4.22. MS2 with CID (25.00) of peak 319.25 m/z shown Myricetin in T. officinale
The mass spectrum of T. officinale illustrated the presence of Silybin A and B at peak
481.33 m/z (Figure 4.23). Silybin is a hepatoprotective compound also found in S. marianum,
decreases inflammatory lipid peroxidation through antioxidants in chronic liver diseases
(Moustafa et al., 2012; Grattagliano et al., 2013). Chlorogenic acid a polyphenolic compound
showed the presence in T. officinale at 377.25 m/z (Figure 4.24). A phenolic compound
Chlorogenic acid found in fruits, vegetables and coffee, antioxidant, anti- carcinogenic, anti-
diabetic, anti-inflammatory, anti-lipidemic and anti-microbial properties (Yun et al., 2012; Ji et
al., 2013; Ma et al., 2015). It was also revealed from different studies that Chlorogenic acid
inhibited the vascular NADPH in hypertensive rats and reduced liver fibrosis in rats treated with
CCl4 (Suzuki et al., 2006; Shi et al., 2013).
Figure 4.23. Mass spectrum of T. officinale indicating the presence of Silybin A and B at
481.33 m/z.
72
Figure 4.24. Mass spectrum of T. officinale showed Chlorogenic acid at 377.25 m/z
4.2.1.3 Biochemical profiling of P. viviparum
LC-MS analysis of rhizomes extract of P. viviparum was executed to assess the
phytochemicals such as alkaloids, flavonoids and phenolics. In figure 4.25 full mass spectrum of
P. viviparum was presented at high peaks 130.08, 219.08, 266.25, 337.33 381.25 and 402.33 m/z.
The mass spectrum of positive mode of electron spray ionization (ESI) depicted the presence of a
polyphenolic compound Caffeic acid (C9H9O4) and natural iridoid sweroside (C16H22O9) at 180.08
and 381.25 m/z respectively (Figure 4.26).
Figure 4.25. Full mass spectrum of P. viviparum
73
Figure 4.26. MS of P. viviparum showed Caffeic acid at 180.08 m/z and Sweroside at 381.25
m/z
The figure 4.27 showed the presence of a glycoside Morroniside (C17H26O11) at 428.25 m/z
with positive mode of ESI. Morroniside is an iridoid glycoside, used as a vegetable drug in china
and found in Cornus officinalis (Zhao et al., 2016). It was revealed from previous studies that
morroniside have different biological properties like tissue regeneration and protecting cells
against apoptosis (Hu et al., 2013; Meunier et al., 2015).
Figure 4.27. Mass spectrum of P. viviparum shown Morroniside at 428.25 m/z
The LC-MS analysis of P. viviparum revealed the presence of Gallic acid (C7H6O5) at
169.08 m/z with negative mode of electron spray ionization (Figure 4.28). Gallic acid a naturally
occurring low molecular weight antioxidant polyphenolic compound, derivative of many
medicines. Gallic acid has several biological and pharmacological properties like anti-microbial,
anti-ulcer and anticancer by interfering with cell signalling pathways and apoptosis of cancer cells
74
(Badhani et al., 2015; Tasi et al., 2018). In hepatotoxicity which is associated with oxidative stress
can be reduced through Gallic acid by reducing lipid peroxidation mechanism of cell membrane
(Shahrzad et al., 2001; Zarei et al., 2013). The peak of negative mode ESI at 191.08 m/z depicted
the presence of Quinic acid with molecular formula C7H12O6 in P. viviparum (Figure 4.28).
Figure 4.28. Mass spectrum of P. viviparum shown Gallic acid at 169.08 m/z and Quinic
acid at 191.08 m/z
The mass spectrum of P. viviparum executed by LC-MS analysis illustrated the presence
of Chlorogenic acid (C16H18O9) at 353.25 m/z by negative mode of electron spray ionization
(Figure 4.29). The MS–MS of peak 353.17 m/z with CID 20:00 into three peaks 173, 179 and 191
m/z indicated the confirm presence of Chlorogenic acid in P. viviparum (Figure 4.30). Chlorogenic
acid is a strong polyphenolic compound present in different plants, fruits and vegetables (Xu et
al., 2010; Heitman and Ingram, 2014). According to different in vitro and in vivo studies,
Chlorogenic acid acts as a strong antioxidant agent by increasing the activity of antioxidant
enzymes catalase and superoxide dismutase and inhibiting lipid peroxidation in kidney and liver
cells (Feng et al., 2016).
Figure 4.29. Mass spectrum of P. viviparum shown Chlorogenic acid at 353.25 m/z
75
Figure 4.30. MS- MS at 353.17 m/z by CID 20:00 indicated the confirm presence of
Chlorogenic acid
The MS-MS of peak 154 m/z by CID 27:00 in negative mode ESI into 109.83 and 123.75
m/z indicated the presence of Protocatechuic acid (Figure 4.31). Protocatechuic acid is a phenolic
compound, extensively present in edible plants and folk medicines (Liu, 2004). Different in vitro
and in vivo studies on Protocatechuic acid revealed that it has numerous pharmacological potentials
against diseases including diabetes, cancer and cardiovascular diseases (Kakkar and Bais, 2014).
It acts as a strong antioxidant and hepatoprotective by reducing oxidative stress (Liu et al., 2002;
Lee at al., 2009). The MS-MS of peak at 290.17 m/z with collision ion dissociation (CID) 25:00
was shown the presence of Catechin at 272.08 and 290.17 m/z (Figure 4.32). Catechin a phenolic
compound mainly found in green tea (Liu et al., 2015). According to reported data on previous
studies on Catechin it has several pharmacological effects comprising of antioxidant, anti-
inflammatory, anti-carcinogenic, anti-mutagenic, anti-diabetic and anti-microbial activities
(Jullian et al., 2007).
Figure 4.31. MS-MS of P. viviparum shown Protocatechuic acid at 154 and 109.83 m/z
76
Figure 4.32. MS-MS of P. viviparum shown Catechin at 290.17 and 272.08 m/z
4.2.1.4 Biochemical profiling of F. arabica
F.arabica commonly known as Dhamasa and used mostly in ayurvedic medicines to cure
neurological, haematological and inflammatory disorders (Saeed and Wahid, 2003; Veena et al.,
2014). In the present study full mass spectrum obtained by LC-MS analysis of F. arabica shown
the high peaks at 115, 239.25, 317.33, 365.33, 381.25 and 413.42 m/z (Figure 4.33).
The peaks at 301.33 and 317.33 m/z indicated the presence of Quercetin (C15H10O7) and
Isorhamnetin (C16H12O7) respectively (Figure 4.34). Isorhamnetin a flavonoid compound present
in many fruits and vegetables (Hu et al., 2015). It has many biological functions comprising of
antioxidant, anti- tumor and anti-inflammatory effects (Lua et al., 2018). According to in vivo
studies on Isorhamnetin, it was observed that isorhamnetin effectively protects the hepatic cells
against oxidative stress via transforming growth factor β and exhibit breast cancer which are
mediated by Akt and MEK signaling pathway (Yang et al., 2016: Wu et al., 2017).
Figure 4.33. Full mass spectrum of F. arabica
77
Figure 4.34. Mass spectrum indicated the presence of quercetin and Isorhamnetin at 301.33
and 317.33 m/z in F. arabica
The mass spectrum of F. arabica showed the presence of Sweroside and Swertiamarin at
381.25 and 397.42 m/z respectively (Figure 4.35). Swertiamarin an iridoid glycoside, found in
different therapeutic plants (Patel et al., 2013). It has a number of biological and pharmacological
effects like antidiabetic, hepatoprotective, anti edematogenic, anti arrithritic and anticholinergic
(Vaidya et al., 2009; Vaijanathappa et al., 2009; Saravanan et al., 2014). A case study on diabetic
rats showed that Swertiamarin improves the insulin sensitivity by altered the gene expression of
glucose metabolism in liver and also lowers the surplus cholesterol by inhibiting HMG-CoA
reductase activity (Kumar and Jairaj 2018).
Figure 4.35. Mass spectrum at 381.25 and 397.42 m/z indicated the presence of Sweroside
and Swertiamarin in F. arabica
78
The LC-MS analysis of F. arabica shown the occurrence of Loganin at 413.42 m/z electron
spray ionization (Figure 4.36). The MS-MS of the peak 413.33 m/z by collision ion dissociation
(29:00) confirmed the presence of Loganin (Figure 4.37). The peak found at 301.17 m/z with
positive mode ESI indicated the presence of Quercetin. Loganin is an iridoid glycoside, used as a
traditional medicine in China and Japan (Kwon et al., 2011). It was reported from the case study
that Loganin has a potential to decrease glucose level in plasma and regulate immune function in
rats. Loganin also showed pharmacological effects including neuroprotective, anti-shock and anti-
inflammatory effects (Yamabe et al., 2007).
Figure 4.36. Mass spectrum at 413.42 m/z indicated the presence of Loganin in F. arabica
Figure 4.37. MS-MS with CID (29:00) indicated the presence of Loganin (413.33 m/z) and
Quercetin (301.17 m/z) in F. arabica
79
The mass spectrum of LC-MS analysis of F. arabica showed the presence of
Hydroxymethylfurfural (C6H6O3) at 127 m/z and isorahmentin at 315.42 m/z with negative mode
of ESI (Figure 4.38). 5-Hydroxymethylfurfural is an organic compound and found in Alpinia
oxyphylla. It has multiple biological activities such antioxidative, anti-allergic, anti-inflammatory,
anti-hypoxic, anti-sickling, and anti-hyperuricemic effects (Kagami et al., 2008; Mahfuza et al.,
2018). It was also revealed from a clinical study on rats that 5 hydroxymethylfurfural reverse the
level of MDA and SOD by inhibiting oxidative stress against hepatotoxicant CCl4 (Liu et al.,
2014).
The mass spectrum in figure 4.39 was shown the presence of Chlorogenic acid (C16H18O9)
at 377.25 m/z with negative mode of electron spray ionization in F. arabica. In the present study
Chlorogenic acid presence was also observed in S. marianum, T officinale and P. viviparum.
Chlorogenic acid present in fruits and vegetables is a phenolic compound that exhibited
pharmacological effects against many diseases (Shi et al., 2013; Lou et al., 2011; Ong et al., 2013).
Chlorogenic acid also showed potent effects on liver fibrosis in vitro and in vivo through
suppression of oxidative stress in rats which that induced by carbon tetrachloride (Shi et al., 2016).
Figure 4.38. Mass spectrum shown the presence of 5 hydroxymethylfurfural at 127 m/z and
Isorhamnetin at 315 m/z in F. arabica
80
Figure 4.39. Mass spectrum shown the presence of Chlorogenic acid at 377.25 m/z in F.
arabica
4.2.1.5 Biochemical profiling of Martynia annua
M. annua fruit extracts are used from ancient times to cure epilepsy, inflammation and
tuberculosis (Kenwat, 2013). The phytochemical analysis of M. annua was performed through LC-
MS and the full mass spectrum was shown in figure 4.40. The high peaks at 319.33, 337.42, 377.42
and 393.42 m/z were observed with positive mode ESI.
Figure 4.40. Full mass spectrum of M. annua
The mass spectrum of M. annua from 150 to 400 m/z indicated the presence of Gallic acid
and Myricetin at 170 and 319.33 m/z respectively (Figure 4.41). The MS-MS of peak at 170 m/z
into 127.83 and 169. 92 m/z CID (27:00) with positive mode of ESI indicated the confirmed
presence of Gallic acid (Figure 4.42). Gallic acid is a low molecular triphenolic compound also
found in methanol extracts of P. viviparum. It is a powerful antioxidant agent and has a potential
to inhibit apoptosis and lipid peroxidation in cancer cells (Badhani et al., 2015).
81
Figure 4.41. Mass spectrum indicated Gallic acid at 170 m/z and Myricetin at 319.33 m/z in
M. annua
Figure 4.42. MS-MS indicated Gallic acid at 170 m/z CID (27:00)
The mass spectrum in figure 4.43 depicted the presence of Ferulic acid, Chlorogenic acid
and Swertiamarin at 194.17, 377.42 and 397.42 m/z respectively with positive mode of electron
spray ionization. In presented study presence of both Chlorogenic acid a phenolic compound and
Swertiamarin an iridoid glycoside were also observed in F. arabica (Figure 4.35 and 4.39).
Chlorogenic acid and Swertiamarin have a therapeutic potential to cure various diseases by
controlling oxidative stress and lipid peroxidation through radical scavenging activity (Shi et al.,
2016: Kumar and Jairaj, 2018).
The mass spectrum of M. annua also showed the presence of Ferulic acid a phenolic
compound at 194.17 m/z (Figure 4.43). Ferulic acid widely distributed antioxidant and found in
many plants with lots of pharmacological effects to cure cancer, cardiovascular and hepatic
82
diseases, diabetes, respiratory and neurological disorders. It has also photoprotective effects and
biological effects including antioxidant, anti-inflammatory and anti-microbial activities (Brenelli
et al., 2013). According to a research study on Ferulic acid extracted from root of Scrophularia
buergeriana, has a significant potential to cure acute liver injuries by inhibition of oxidative stress
and inflammatory signalling pathway (Kim et al., 2011).
Figure 4.43. Mass spectrum shown Ferulic acid, Chlorogenic acid and Swertiamarin in M.
annua
Figure 4.44 of mass spectrum of M. annua was showed the presence of
Hydroxymethylfurfural (C6H6O3) and Myricetin (C15H10O8) at 127 and 317.42 m/z respectively
with negative mode of ESI. The mass spectrum also showed the presence of Loganin at 413.42
m/z with negative mode of ESI in M. annua (Figure 4.45).
Figure 4.44. Mass spectrum indicated Hydroxymethylfurfural and Myricetin
correspondingly at 127 and 317.42 m/z in M. annua
83
Figure 4.45. Mass spectrum showed Loganin in M. annua at 413.42 m/z
The MS-MS of peak at 153 m/z into 108.83 and 152.83 m/z depicted the presence of
Protocatechuic acid (C7H6O4) at CID 27:00 with negative mode of ESI (Figure 4.46). The mass
spectrum of M. annua indicated the presence of Echinacoside (C35H46O20) at 785.33 with negative
mode of ESI (Figure. 4.47). Both Protocatechuic acid and Echinacoside are phenolic compounds
and found in different plants. These compounds have many in vitro and in vivo pharmacological
effects comprising of anti-inflammatory, cardio and hepato protective activities (Semaming et al.,
2015; Liu et al., 2018).
Figure 4.46. MS-MS of peak at 153 m/z with CID 27:00 indicated Protocatechuic acid in
M. annua
84
Figure 4.47. Mass spectrum showed Echinacoside in M. annua at 785.33 m/z
LC-MS of selected medicinal plants S. marianum, T. officinale, P. viviparum, F. arabica
and M. annua exhibited the presence of several biologically active compounds that showed
antioxidant and hepatoprotective activities (Jaishree and Badami, 2010; Mihailovic et al., 2013;
Serviddio et al., 2014; Yeon et al., 2017). Hence occurrence of these bioactive compounds in
selected plants support their potential uses as antioxidant and hepatoprotective agents. Therefore
increase in antioxidant potential of selected medicinal plants may be effective in the treatment and
overcome of the side effects of liver diseases.
4.2.2 Optimization of in vitro hepatoprotective potential of herbal combinations through
Liver Slice culture assay
The five medicinal plants (S. marianum, T. officinale, P. viviparum, F. arabica and M.
annua) those showed minimum percentage of cytotoxicity in liver slice culture assay were selected
for preparation of herbal combinations by following the tools of response surface methodology
(RSM). Hepatoprotective potential of these herbal combinations were evaluated through liver
slice culture assay against CCl4 and the released LDH was measured and expressed in terms of
percentage cytotoxicity (Table 4.9). Through Central Composite Design (CCD) of response
surface methodology (RSM), a total of 50 experiments were conducted with five factors (plants)
at two levels with eight replicates at centre point for constructing quadratic model. Herbal
combination (run # 45) that showed minimum percentage cytotoxicity was considered the best
one. The concentrations of medicinal plants in this combination (run # 45) were considered as
optimum concentration for synergic hepatoprotective activity.
85
Table. 4.9: RSM model under Central Composite Design (CCD) for determination of
optimum concentrations as hepatoprotective potential of medicinal plants
Run
#
S. marianum
(mg)
P. viviparum
(mg)
T. officinale
(mg)
F. arabica
(mg)
M. annua
(mg)
%
Cytotoxicity
A B C D E
1 221 47 271 258 77 4.91
2 179 47 271 342 43 4.69
3 179 78 229 258 43 5.02
4 200 63 300 300 60 4.49
5 200 63 250 300 60 5.09
6 221 47 229 342 77 5.73
7 221 78 229 258 77 2.65
8 200 63 250 200 60 5.26
9 179 78 229 342 43 4.43
10 179 78 271 258 43 4.29
11 200 63 250 400 60 4.68
12 200 63 250 300 60 5.19
13 221 47 229 342 43 4.85
14 221 47 271 342 77 3.6
15 200 63 250 300 60 3.9
16 221 47 271 258 43 4.05
17 200 25 250 300 60 4.05
18 200 100 250 300 60 2.39
19 179 47 229 258 77 4.78
20 200 63 250 300 20 5.16
21 200 63 250 300 60 3.72
22 179 78 229 342 77 3.09
23 221 78 229 342 43 4.57
24 221 78 271 342 43 5.08
25 179 78 271 342 77 3.29
26 200 63 200 300 60 4.36
27 179 47 271 258 77 5.13
28 200 63 250 300 60 4.47
29 179 78 229 258 77 4.34
30 221 78 271 258 43 4.35
31 150 63 250 300 60 5.13
32 179 78 271 258 77 4.09
33 200 63 250 300 60 4.58
34 221 47 229 258 43 4.94
35 250 63 250 300 60 8.65
36 179 78 271 342 43 4.89
86
37 221 47 271 342 43 4.12
38 221 78 229 258 43 4.21
39 179 47 271 258 43 5.05
40 221 78 229 342 77 2.85
41 200 63 250 300 60 3.06
42 179 47 229 258 43 1.63
43 179 47 229 342 77 1.02
44 200 63 250 300 100 1.52
45 221 78 271 342 77 0.82
46 221 47 229 258 77 1.13
47 221 78 271 258 77 1.21
48 200 63 250 300 60 3.2
49 179 47 271 342 77 1.57
50 179 47 229 342 43 1.26
Results of in vitro liver slice culture assay of herbal combinations revealed that run # 45
showed minimum percentage cytotoxicity (0.82%) against CCl4 (Table 4.9). This least percentage
cytotoxicity herbal combination comprised of 221 mg of S. marianum, 78 mg of P. viviparum,
271mg of T. officinale, 342 mg of F. arabica and 77 mg of M. annua. Design Expert trial version
10.0 was used for the analysis, interpretation, construction of three dimensional (3D) response
surface graphs, ANOVA table, effect of regression coefficient of linear interactions and square
terms. The overall regression equation providing the analysis of data is as follow:
R = 3.70 - 0.33A – 0.76B – 0.33C – 0.42D -0.9E - 0.45AB – 0.15AC + 0.15AD -0.13 AE
– 0.11BC + 0.22BD + 0.28 BE – 0.17 CD – 0.088 CE – 0.30 DE + 0.24A2 – 0.14B2 + 0.10 C2 +
0.061 D2 – 0.018E2
Here, “R” indicated the response variable of percentage cytotoxicity, A, B, C, D and E
were the linear terms, “A” was the S. marianum, “B” was the P. viviparum, “C” was the T.
officinale, “D” was the F. arabica and “E” was the M. annua. AB, AC, AD, AE, BC, BD, BE, CD,
CE and DE were the interaction coefficients and A2, B2, C2, D2 and E2 were the square coefficients.
4.2.2.1 Validation of herbal combinations in quadratic model
Standard deviation showed the strong resemblance between the model and predicted
response. In present work 0.35 value of standard deviation and 3.97 mean value were observed
(Table 4.10). The coefficient of variation (C.V) was 8.78% that indicated better degree of precision
and reliability of experiment. R2 (coefficient of determination) generally used to measure the
percentage of variation and fitness relationship between estimated model and experimental data.
87
Its value lies between 0 and 1 that indicated good correlation between experimental and predicted
values. The value of R2 (coefficient of determination) was 0.9659. The adjusted and predicted R2
values were 0.9425 and 0.9132 respectively, those were close to R2 (0.9659). Adequate precision
measures the signal to noise ratio and 26.492 adequate precision was noted (Table 4.10). A signal
to noise ratio greater than 4 was desirable that means this model can be used to navigate design
space (Ohale et al., 2017; Ragavendran et al., 2017).
Table 4.10: Analysis of variance (ANOVA) of data for RSM model to determine
hepatoprotective potential of medicinal plants
Standard deviation 0.35 R-Squared (R2) 0.9659
Mean 3.97 Adjusted R-Squared 0.9425
C.V 8.78% Predicted R-Squared 0.9132
PRESS 8.97 Adequate Precision 26.492
ANOVA of the quadratic model depicted that the selected plants S. marianum, P.
viviparum, T. officinale, F. arabica and M. annua of herbal combinations were shown synergic
hepatoprotective effects against hepatotoxic CCl4. F and p values of the ANOVA of quadratic
model suggested the significant level of the each coefficient. The F-value of 41.12 with low
probability advocated the high significance of the quadratic model. In this model A, B, C, D, E,
AB, AC, AD, AE, BD, BE, CD, DE, A2, B2, C2 were significant (p <0.05) terms, whereas BC, CE,
D2 and E2 were non-significant (Table 4.11). In this model the Lack of Fit (F-value) was 0.37 that
was not significant relative to the pure error. The model with non-significant lack of fit was
considered a good (Tan et al., 2017; Wang et al., 2018).
Table 4.11: ANOVA for Response Surface Quadratic model to determine hepatoprotective
potential of medicinal plants
Source Sum of
Squares
df Mean
Square
F
Value
p-value
Prob > F
Significance
Model 99.86 20 4.99 41.12 < 0.0001 Significant
A-S. marianum 4.99 1 4.99 41.07 < 0.0001 Significant
B-P. viviparum 25.68 1 25.68 211.48 < 0.0001 Significant
C-T. officinale 4.78 1 4.78 39.39 < 0.0001 Significant
D-F. arabica 7.85 1 7.85 64.69 < 0.0001 Significant
E-M. annua 33.98 1 33.98 279.89 < 0.0001 Significant
AB 6.90 1 6.90 56.84 < 0.0001 Significant
AC 0.74 1 0.74 6.12 0.0194 Significant
AD 0.81 1 0.81 6.65 0.0153 Significant
88
AE 0.58 1 0.58 4.74 0.0377 Significant
BC 0.40 1 0.40 3.26 0.0813 Not significant
BD 1.64 1 1.64 13.47 0.0010 Significant
BE 2.46 1 2.46 20.24 0.0001 Significant
CD 1.03 1 1.03 8.48 0.0069 Significant
CE 0.25 1 0.25 2.06 0.1619 Not significant
DE 2.76 1 2.76 22.72 < 0.0001 Significant
A2 3.15 1 3.15 25.92 < 0.0001 Significant
B2 1.12 1 1.12 9.21 0.0050 Significant
C2 0.58 1 0.58 4.75 0.0375 Significant
D2 0.21 1 0.21 1.70 0.2029 Not significant
E2 0.018 1 0.018 0.14 0.7066 Not significant
Residual 3.52 29 0.12
Lack of Fit 1.90 22 0.086 0.37 0.9644 Not significant
Pure Error 1.62 7 0.23
Cor Total 103.39 49
4.2.2.2 Normal % probability plot of herbal combinations as hepatoprotectents
The pattern of least square fit model of the residuals directed the accuracy of the model
(Figure 4.48). The straight line of the normal plot of residuals confirmed the hepatoprotective
potential of the studied herbal combinations.
Figure 4.48. Presented quadratic model with normal probability plot of the studentized
residuals as hepatoprotective potential
89
4.2.2.3 Independency of the herbal combinations on percentage cytotoxicity
Independency of the data of the quadratic model was verified by plotting a graph between
residuals and run order (Xio et al., 2012). The residual plots of medicinal plants and their
hepatoprotective potential were shown in figure 4.49. Fifty runs of selected plants were formulated
to find out the hepatoprotective potential against hepatotoxicity induced by CCl4. The range of -3
to +3 of all residuals indicated that predictable pattern of this model was accurate (Figure 4.49).
Figure 4.49. Presented quadratic model run number verses internally studentized residuals
plot for hepatoprotective potential
4.2.2.4 Correlation between predicted and actual values of herbal combinations
The actual and predicted percentage cytotoxicity of herbal combinations were shown in
Figure 4.50. A significant correlation was observed between actual and predicted values. The
values of R2 and adjusted R2 were 0.9659 and 0.9425 respectively (Table 4.10). These values
showed the strong correlation and validation of the model between predicted and actual values.
The standard deviation of the model was 0.35 that indicated the significant correlation between
predicted and actual values of response.
90
Figure 4.50. Shown quadratic model correlation between predicted and actual values of
herbal combinations
4.2.2.5 Box-Cox plot of herbal combinations for power transformation
This plot is used to find out the normality of the data. It was observed that natural logarithm
(ln) of the residuals sum of squares (SS) suddenly decreased with the lowest value of 0.57 against
lambda (λ) as presenting in figure 4.51 The best value of residual against Lambda (λ was 0.88 and
high value was 1.24. The current value of confidence interval with lambda was very close to
optimum value of data, therefore data of herbal combinations of medicinal plants did not need any
transformation.
Figure 4.51. Box-Cox plot of Lambda and Ln medicinal plants SS in quadratic model.
91
4.2.2.6 Response surface analysis of three dimensional plots of medicinal plants
The three dimensional plots were constructed to analyse the experimental data for the
hepatoprotective potential of herbal combinations. The three dimensional plots were used to
illustrate the significance relationship between independent (medicinal plants) and dependent
(percentage cytotoxicity) variables. To find out the hepatoprotective potential of variables,
interaction between two variables in 3D curve plots of response surface were observed by keeping
the other three variables constant at their central values (Belwal et al., 2016). The interactions
among the variables S. marianum and P. viviparum, S. marianum and T. officinale, S. marianum
and F. arabica, S. marianum and M. annua, P. viviparum and T. officinale, P. viviparum and F.
arabica, P. viviparum and M. annua, T. officinale and F. arabica, T. officinale and M. annua and
F. arabica and M. annua have been presented in figure 4.52 to 4.61 These 3D plots of medicinal
plants with optimum values showed minimum percentage of cytotoxicity in terms of maximum
hepatoprotective potential.
4.2.2.7 Interaction of S. marianum with P. viviparum
Figure 4.52 showed the interaction between S. marianum and P. viviparum variables,
whereas other variables (T. officinale, F. arabica and M. annua) were constant at their centre
values. It was revealed from the response graph that with the increase in concentration of S.
marianum from 150 to 250 mg and P. viviparum from 25 to 80 mg percentage cytotoxicity was
decreased. Significant interaction was observed at 221 mg of S. marianum and 78 mg of P.
viviparum with minimum percentage cytotoxicity (0.82%) and p value less than 0.05 as presenting
in ANOVA (Table 4.11).
Figure 4.52. Response surface plot of interaction in S. marianum with P. viviparum for the
hepatoprotective activity
92
4.2.2.8 Interaction of S. marianum with T. officinale
The interaction between variables S. marianum and T. officinale was shown in the Figure
4.53. Here other three variables (P. viviparum, F. arabica and M. annua) were constant. Response
surface plot of S. marianum and T. officinale showed significant interaction between them with F
and p value 6.12 and 0.0194 respectively (Table 4.11). Inverse relation was found between
independent variables (S. marianum and T. officinale) and dependant variable (% cytotoxicity).
When S. marianum concentration increased from 150 to 250 then decrease in percentage
cytotoxicity was observed. On the other side, percentage cytotoxicity was decrease when T.
officinale concentration increased from 200 to 271 mg then slightly increased with the increase in
concentration of T. officinale from 272 to 300 mg (Figure 4.53)
Figure 4.53. Response surface plot of interaction between S. marianum and T. officinale for
the hepatoprotective activity
4.2.2.9 Interaction of S. marianum and F. arabica
The response surface interaction plot of S. marianum and F. arabica was presenting in
figure 4.54, while other three variables (P. viviparum, T. officinale and M. annua) were constant.
It was revealed from the ANOVA and 3D response surface plot that significant relation was present
between S. marianum and F. arabica. When concentrations of S. marianum and F. arabica were
increased then decreased in the dependent variable percentage cytotoxicity was observed.
Minimum percentage cytotoxicity 0.82 was found at 221 mg of S. marianum and 342 mg of F.
arabica.
93
Figure 4.54. Response surface plot of interaction in S. marianum with F. arabica for the
hepatoprotective activity
4.2.2.10 Interaction between S. marianum and M. annua
The response surface interaction plot of S. marianum and M. annua have been presented
in figure 4.65. Significant relation between these two variables (S. marianum and M. annua) was
observed with the p value < 0.05, while other three variables (P. viviparum, T. officinale and F.
arabica) were constant. It was revealed from the 3D plot that at certain concentrations of S.
marianum and M. annua percentage cytotoxicity was decreased and then increased with the
increase of plants concentrations.
Figure 4.55. Response surface plot of interaction between S. marianum and M. annua for
the hepatoprotective activity
94
4.2.2.11 Interaction of T. officinale and P. viviparum
Figure 4.56 showed interaction between T. officinale and P. viviparum variables, whereas
other variables (S. marianum, F. arabica and M. annua) were constant at their centre values.
According to ANOVA and 3D response surface plot non-significant relation was observed
between these two variable T. officinale and P. viviparum because p value was > 0.05. It was also
observed from the plot that at certain level percentage cytotoxicity was decreased with the increase
in the plants concentrations.
Figure 4.56. Response surface plot of interaction in T. officinale and P. viviparum for the
hepatoprotective activity
4.2.2.12 Interaction of P. viviparum with F. arabica
The 3D response surface plot (Figure 4.57) presenting the interaction between P. viviparum
and F. arabica variables, while other three variables (S. marianum, T. officinale and M. annua)
were constant at their centre values. Significant interaction between these two variables was found
as p value < 0.05. It was also revealed from the 3D response surface that with the increase in the
plants concentrations P. viviparum 25 to 80 mg and F. arabica 200 to 350 mg, percentage
cytotoxicity was decreased. However percentage cytotoxicity was increased, when concentrations
of P. viviparum 80 to 100 mg and F. arabica 350 to 400 mg were increased.
95
Figure 4.57. Response surface plot of interaction in P. viviparum with F. arabica for the
hepatoprotective activity
4.2.2.13 Interaction of P. viviparum and M. annua
The response surface interaction plot of P. viviparum and M. annua was shown in figure
4.58. Significant relation between these two variables (P. viviparum and M. annua) was noted with
the p value < 0.05, while other three variables (S. marianum, T. officinale and F. arabica) were
constant at their centre values. It was revealed from the 3D plot that at certain concentrations of P.
viviparum and M. annua percentage cytotoxicity was increased and then decreased with the
increase in concentrations of these plants.
Figure 4.58. Response surface plot of interaction in P. viviparum with M. annua for the
hepatoprotective activity
96
4.2.2.14 Interaction between T. officinale and F. arabica
The interaction between variables T. officinale and F. arabica was shown in the Figure
4.59, while other three variables (S. marianum, P. viviparum and M. annua) were constant.
Response surface plot of T. officinale and F. arabica was showed the significant interaction
between them with F and p values 8.48 and 0.0069 respectively (Table 4.11). Inverse relation was
found between independent variables (T. officinale and F. arabica) and dependant variable (%
cytotoxicity). When concentrations of plants T. officinale increased from 200 to 300 and F. arabica
from 200 to 400 mg, decrease in percentage cytotoxicity was observed.
Figure 4.59. Response surface plot of interaction in T. officinale with F. arabica for the
hepatoprotective activity
4.2.2.15 Interaction of T. officinale and M. annua
Figure 4.60 depicted the interaction between T. officinale and M. annua variables, whereas
other variables (S. marianum, P. viviparum and F. arabica) were constant at their centre values.
According to ANOVA and 3D response surface plot non-significant relation was present between
these two variable T. officinale and M. annua because p value was > 0.05. It was also observed
from the plot that at certain level percentage cytotoxicity was decreased with the increased in the
plants concentrations and then increased with the increase of plants concentrations.
97
Figure 4.60. Response surface plot of interaction in T. officinale with M. annua for the
hepatoprotective activity
4.2.2.16 Interaction between F. arabica and M. annua
The 3D response surface plot (Figure 4.61) presented the interaction between P. F. arabica
and M. annua variables, while other three variables (S. marianum, T. officinale and P. viviparum)
were constant at their centre values. Significant interaction between these two variables was found
as p value < 0.05 (Table 4.11). It was also revealed from the 3D response surface that with the
increase of plants concentrations F. arabica from 200 to 400 mg and M. annua from 20 to 100 mg
percentage cytotoxicity was decreased.
Figure 4.61. Response surface plot of interaction between F. arabica and M. annua for
hepatoprotective activity
It was elucidated from these results that quadratic model was useful to predict optimum
concentrations of medicinal plants that showed maximum hepatoprotective potential. Therefore
98
RSM based central composite design model appeared to be accurate and reliable for the dose
optimization of herbal combinations of medicinal plants.
Response surface methodology (RSM) is a most commonly used to obtain optimal
parameters and consist of mathematical and statistical techniques (Gu et al., 2012; Kuo et al.,
2013). RSM estimates the problem with design variables of different quantities, using several
analytical or experimental results. The predicted values from RSM can be compared with
experimental values and they are very close to experimental values and showed excellent fit of
model (Amin et al., 2011; Barbosa et al., 2010). There are two main experimental tools of RSM
Box-Behnken and central composite design (CCD) are used in different fields for the optimization
of samples and experimental runs (Zhao et al., 2012; Shannon and Abu-Ghannam, 2017). RSM
has been extensively used in food companied and herbal medicines for the optimization of
chemical and biochemical process and the relation between dependent and independent variables.
(Granato et al., 2010, Liu et al., 2012, Delgado et al., 2012, Ellendersen et al., 2012).
Similar type of RSM study was done by Kalagatur et al. (2018) to evaluate the inhibitory
effects of H. spicatum essential oil on production and growth rate of maize grains. A total 13
experiments were designed by central composite design through Stat Ease Design Expert.
According to obtain results of this study, quadratic models was well fitted an all the parameters
including larger F value, smaller p - value, adequate precision and regression coefficients (R2)
were statistically significant. Also appropriate normal plot residuals, Box-Cox, and actual vs.
predicted plots were observed in this study that confirmed the accuracy of optimized design. In
another study, related findings with present study like antioxidant potential of Berberis asiatica
fruit with optimum concentrations were observed by using tools of response surface methodology.
In this experimental study Central Composite Design was used with three factors and two levels
and well fitted quadratic model with non-significant lack of fit was observed (Belwal et al., 2016).
Therefore, in the present study fifty different herbal combinations of five selected plants
were analyzed for their hepatoprotective potential in terms of percentage cytotoxicity. On the basis
of obtained results and model suggested by RSM, herbal combination that showing minimum
percentage cytotoxicity was finally selected for in vivo studies.
4.3 Phase III: In vivo hepatoprotective potential of selected herbal combination
Both curative and preventive mode of treatments were used for evaluation of ameliorative
effects of selected herbal combinations on Albino rats against hepatotoxicity induced by carbon
tetrachloride (CCl4).
99
4.3.1 Curative effects of herbal combination
In curative mode of treatment, Albino rats were divided into five group. The first group
was normal control that received only normal diet. The second curative positive control group
which was administrated with 1mL/kg b.wt of CCl4 twice a week for one month, while curative
negative control (third group) was treated with and 200 mg/kg b.wt of herbal combination daily
for thirty days. On the other hand, fourth curative treatment group received both 1 mL/kg b.wt of
CCl4 twice a week and 200 mg/kg b.wt of herbal combination daily for one month. Curative
standard (fifth group) was administrated with both 1 mL/kg b.wt of CCl4 twice a week and 50
mg/kg b.wt of Siliver (standard drug) for thirty days. The obtained results of curative mode of
treatment were described and discussed under following headings.
4.3.1.1 Curative effect on body weight of rats.
Body weights of all the curative group were measured at zero, 7th, 14th, 21st and 28th day to
see hepatoprotective effect of herbal combination in CCl4 toxicity induced rats. In control group,
a significant increase of weight was observed at 21st and 28th day, while in curative positive control
elevation of weight were seen at 28th day (Figure 4.62). The significant increase of weight of rats
at 14th, 21st and 28th day was exhibited in curative negative control group, whereas there were no
significant change in weights of rats were observed in curative treatment group at all days. The
curative standard group showed a significant increase in weight at 21st and 28th day (Figure 4.62).
Figure 4.62. Curative effect of herbal combination against hepatotoxicity induced by CCl4
on body weights of rats.
100
4.3.1.2 Curative effects of herbal combination on Biochemical parameters
Blood samples of all curative group were taken at zero, 2nd, 3rd, 10th, 20th and 30th day for
the evaluation of hepatoprotective effects of herbal combination on biochemical parameters of
liver against hepatotoxicity induced by CCl4. The liver biochemical parameters including ALT,
AST, ALP, ƔGT, total bilirubin, total protein, albumin and globulin were determined to find out
ameliorative effects of herbal combination.
4.3.1.2.1 Curative effects on Alanine aminotransferase (ALT)
The level of ALT (U/L) were measured in all curative groups as compared to normal
control group at Zero, 2nd, 3rd, 10th, 20th and 30th day (Figure.4.63). The results of ALT (U/L)
enzymes in all curative groups were presented in mean ± SD. The significant or non-significant
change in enzyme level between groups as compared to normal control was analyzed through two-
way ANOVA, Tukey’s multiple comparison test (Figure 4.63).
At zero day (before starting experiment), no significant change in ALT enzyme (U/L) was
observed in all curative groups i.e, curative positive control (43 ± 7.32), curative negative control
(46 ± 3.21), curative treatment (47 ± 7.54) and curative standard group (42 ± 4.01) as compared to
normal control group (44 ± 3.60). At 2nd day (after 24 hours of CCl4 intoxication) in curative
positive control (81.33 ± 14.18), curative treatment (67 ± 12.05) and curative standard (63 ± 6.8)
groups significant (p-value = 0.05) increase in ALT level was observed as compared to normal
control (45.66 ± 2.08) group, while there was no significant change found in ALT level of negative
curative group (44 ± 4.03) as compared to normal control. Correspondingly a significant elevation
of ALT level 95 ± 17.05, 78 ±15.82 and 74 ± 9.71 was observed in curative positive control,
curative treatment and curative standard groups as compared to normal control group 43 ± 3.05 at
3rd day. The high level of ALT was the indication of hepatotoxicity in liver cells of rats due to
CCl4 that disrupted the normal functions of liver.
The highly significant (p>0.0001) increase of ALT level 71.66 ± 7.76, 87.66 ± 11.50 and
96.02 ± 4.01 was seen at 10th, 20th and 30th day respectively in curative positive control group as
compared to normal control (45 ± 4.00, 48.56 ± 14.40 and 47.38 ± 9.84) at the same days. On the
other hand, no significant alteration in ALT levels (56 ± 9.01, 59.33 ± 9.45 and 63.67 ± 3.21) were
recorded in negative control group as compared to normal control at 10th, 20th and 30th day. The
curative treatment and curative standard group showed significant increase in ALT levels as
compare to normal control (45 ± 4.00) at 10th day, while at 20th and 30th day curative treatment
101
(65.66 ± 6.65 and 66 ± 5.29) and curative standard group (63.67 ± 5.13 and 54.33 ± 4.11) exhibited
non-significant change in ALT levels as compared to normal control (48.56 ± 14.40 and 47.38 ±
9.84). The normal levels of ALT in these groups indicated the hepatoprotective potential of herbal
combination and standard drug Siliver against CCl4 intoxication.
Figure 4.63. Shown curative hepatoprotective effects of herbal combination on ALT (U/L)
level at different days.
4.3.1.2.2 Curative effects on Aspartate aminotransferase (AST)
In all curative groups i.e, curative negative control (173 ± 22.51), curative positive control
(142 ± 28.63), curative treatment (174.33 ± 31.73) and curative standard group (115 ± 22.91) non-
significant alteration in AST (U/L) level was observed as compared to normal control (124 ±
26.51) at zero day, that means liver of all rats was in proper functioning prior to administration of
hepatotoxicant CCl4 (Figure 4.64). At 2nd day (after 24 hours of CCl4 administration), there was a
significant (p-value = 0.05) elevation of AST (U/L) level in curative positive control (209.33 ±
40.52) and curative treatment group (203.66 ± 36.43) with comparison of normal control (133.66
± 29.67) group. On the other hand, curative negative control (175.33 ± 17.47) and curative standard
group (178.66 ± 38.88) showed non-significant change in AST (U/L) level at 2nd day.
The highly significant (p<0.0001) increase in AST (U/L) levels were seen in curative
positive control (271.66 ± 25.83), curative treatment (287 ± 47.69) and curative standard group
(278 ± 37.1) at 3rd day (after 48 hours CCl4 administration) as compared to normal control (131 ±
102
20.07) at the same day. The increase in AST (U/L) level indicated disturbance in normal
functioning of the hepatocytes. The curative negative control (treated with only herbal
combination) was shown no significant alteration in AST (U/L) at 3rd day. At 10th, 20th and 30th
day gradual (significant) increase in AST (U/L) was seen from 312 ± 19.15 to 363.33 ± 21.19 in
curative positive control group (treated with CCl4 twice a week) as compared to normal control
(Figure 4.64).
The curative treatment group (treated with herbal combination daily and CCl4 twice a
week) was shown ameliorative hepatoprotective effect of herbal combination by decreasing of
AST (U/L) level from 235 ± 38.21 to 218.33 ± 28.61 as compared to curative positive control. AT
10th, 20th and 30th day, in curative negative control (114.33 ± 11.67, 103.66 ± 26.35 and 117.66 ±
29.70) and curative standard group (127 ± 33.28, 148.66 ± 20.03 and 173.33 ± 30.58) non-
significant (p<0.0001) change in AST (U/L) was recorded as compared with normal control and
curative positive control group. The decrease of AST (U/L) levels in curative treatment group
advocated the effectiveness of herbal combination to restore the normal functions of the liver.
Figure 4.64. Graphical presentation of curative hepatoprotective effects of herbal
combination on AST (U/L) level at different days.
4.3.1.2.3 Curative effects on Alkaline phosphatase (ALP)
ALP enzyme is present in liver and bone cells and in case of injury it comes out from the
cells. Therefore, in liver disorders ALP level is increased due to destruction of hepatocytes. The
103
graphical presentation in figure 4.65 depicted that at zero day (before any treatment) no significant
change in ALP (U/L) was observed in all curative groups as compare to normal control.
At 2nd and 3rd day after the administration of CCl4, a significant elevation of ALP (U/L)
was found in curative positive control (445 ± 28.05 and 639 ± 80.29), curative treatment (467.66
± 46.09 and 564.33 ± 84.08) and curative standard (479.33 ± 53.92 and 721.33 ± 147.57) groups
as compared to control group (274.66 ± 38.79 and 256.66 ± 30.98), whereas no significant
alteration of ALP (U/L) was observed in curative negative control (210 ± 13.11 and 207.66 ±
8.02) in comparison with normal control at the same days (Figure 4.65).
On the other side, at 10th day a significant (p< 0.0001) increase in ALP (U/L) was seen in
curative positive control (511.66 ± 30.53), curative treatment (553.3 ± 26.38) and curative standard
(642 ± 90.02) groups in comparison with normal control (208 ± 33.15) and negative control (238
± 2.01) group. From 20th to 30th day, a significant increase of ALP (U/L) level (478 ± 77.38 to
522.66 ± 42.44) was observed in curative positive control group, while significant decrease in ALP
(U/L) levels were recorded in curative treatment (502 ± 62 to 447.33 ± 92.22) and curative standard
(632.66 ± 65.73 to 520 ± 68.55) groups at the same days. The ALP (U/L) levels were in normal
range in normal control (211.66 ± 26.76 and 244 ± 20.84) and curative negative control (282.33 ±
22.50 and 333.33 ± 50.29) group at 20th and 30th day. The decrease in ALP (U/L) level in curative
treatment group showing the ameliorative effect of herbal combination by restoring the normal
functions of liver cells.
Figure 4.65. Presented curative hepatoprotective effects of herbal combination on ALP (U/L)
level at different days.
104
4.3.1.2.4 Curative effect on Gamma-glutamyltransferase (ƔGT)
The graphical presentation of ƔGT (U/L) depicted that all curative groups exhibited normal
ƔGT (U/L) level at zero day (Figure.4.66). Likewise no significant (p>0.0001) variation in ƔGT
(U/L) level was observed in curative positive control (10.33 ± 1.52), curative negative (8.66 ±
0.57) and curative treatment (10.33 ± 2.08) groups as compared to normal control (8.33 ± 0.57) at
2nd day, while curative standard group showed significant (p<0.0001) increase in ƔGT (U/L) level
at the same day.
At 3rd day, a significant (p<0.0001) increase in ƔGT (U/L) level was found in curative
positive control (13.3 ± 1.15), curative treatment (14.66 ± 1.52) and curative standard (14.41 ±
2.65) groups as compared to normal control (7.21 ± 0.57), while no significant change in ƔGT
(U/L) level (9.05 ± 2.08) was observed in curative negative control at same day. On the other hand,
from 10th to 30th day in curative positive control a significant (p<0.0001) increase of ƔGT (U/L)
level (12.87 ± 1.22 to 14 ± 2.02) was observed from, whereas curative treatment (13.33 ± 1.15,
12.56 ± 1.15 and11.66 ± 0.57) and curative standard (10 ± 2.02, 9.33 ± 2.30 and 8.75 ± 0.57)
groups showed gradual decrease in ƔGT (U/L) levels at 10th, 20th and 30th day (Figure.4.66). The
curative negative control group exhibited normal level ƔGT (U/L) 9 ± 1.13, 8.33 ± 1.52 and 7.33
± 0.57 correspondingly at 10th, 20th and 30th day as compared to normal control (8 ± 1.01, 9 ± 1.73
and 9.66 ± 1.15) at the same days.
Figure 4.66. Shown curative hepatoprotective effects of herbal combination on ƔGT (U/L)
level at different days.
4.3.1.2.5 Curative effect on Total bilirubin
The graphical presentation of total bilirubin (mg/dL) exhibited a non-significant
(p<0.0001) change in curative negative control (0.043 ± 0.026), curative positive control (0.18 ±
105
0.035), curative treatment (0.076 ± 0.014) and curative standard (0.14 ± 0.06) groups as compared
to normal control (0.106 ± 0.023) at zero day (Figure 4.67). At 2nd day a significant (p>0.0001)
increase in total bilirubin (mg/dL) level 0.27 ± 0.055 and 0.23 ± 0.079 was seen in curative positive
control and curative standard group respectively as compared to normal control (0.12 ± 0.02).
Whereas curative negative control and curative treatment group showed non-significant increase
(0.092 ± 0.024 and 0.18 ± 0.030) in total bilirubin (mg/dL) at the same day as compared to normal
control (0.12 ± 0.02).
The significantly increase in total bilirubin (mg/dL) level was observed in curative positive
control (0.39 ± 0.041), curative treatment (0.28 ± 0.051) and curative standard (0.40 ± 0.119) group
in comparison with normal control (0.15 ± 0.030) at 3rd day, while at the same day no significant
increase of total bilirubin (mg/dL) was seen in curative negative control (0.12 ± 0.02) and curative
positive control (0.39 ± 0.041). At 10th, 20th and 30th day a significant (p>0.0001) increase in total
bilirubin (mg/dL) levels correspondingly 0.52 ± 0.072, 0.56 ± 0.056 and 0.58 ± 0.064 were
recorded in curative positive control group. In this group high level of total bilirubin (mg/dL)
indicated the abnormal functioning of liver cells due to CCl4 intoxication. Similarly a significant
increase of total bilirubin (mg/dL) level was found in curative treatment (0.35 ± 0.053, 0.44 ±
0.053 and 0.49 ± 0.065) and curative standard (0.51 ± 0.052, 0.56 ± 0.02 and 0.58 ± 0.02) groups
regarding to normal control (0.18 ± 0.02, 0.17 ± 0.023 and 0.16 ± 0.04) at 10th, 20th and 30th day
(Figure 4.67).
Figure 4.67. Presented curative hepatoprotective effects of herbal combination on total
bilirubin (mg/dL) level at different days.
106
4.3.1.2.6 Curative effect on Total Protein
The total protein (g/dL) contents were normal in all curative groups at zero day (Figure
4.68). At 2nd, 3rd and 10th day, significant decrease of total protein (g/dL) contents (5.03 ± 0.68, 4.5
± 0.63 and 4.4 ± 0.21) were observed in curative treatment group (treated with both herbal
combination and CCL4). There was no significant change of total protein (g/dL) exhibited in both
curative positive control (6.1 ± 0.50 and 5.8 ± 0.97) and curative negative control (6 ± 0.72 and
6.0 ± 0.50) at 2nd and 3rd day as compared to normal control (6.3 ± 0.50 and 6.2 ± 0.59). Whereas
in curative positive control a significant (p< 0.0001) decrease in total protein (g/dL) from 5.03 ±
0.60 to 4.3 ± 0.30 was detected from 20th to 30th day. On the other side, curative treatment and
curative standard groups showed total protein (g/dL) in normal range 5.4 ± 0.15 and 5.5 ± 0.94
respectively as compared to normal control (6.2 ± 0.55) at 30th day.
Figure 4.68 Presented curative hepatoprotective effects of herbal combination on total
protein (g/dL) level at different days
4.3.1.2.7 Curative effect on Albumin levels The normal albumin (g/dL) level was observed in curative positive control (3.26 ± 0.23),
curative negative control (2.96 ± 0.60), curative treatment (2.93 ± 0.25) and curative standard (2.7
± 0.75) groups as compared to normal control (2.73 ± 0.46) at zero day. Similarly at 2nd and 3rd
day, no significant change in albumin (g/dL) level was detected in curative positive control (2.86
± 0.11 and 2.73 ± 0.11), curative negative control (3.06 ± 0.50 and 3.2 ± 0.6) and curative standard
107
(2.56 ± 0.20 and 2.5 ± 0.1) groups as compared to normal control. While a significant decrease in
albumin (g/dL) level from 2.6 ± 0.2 to 2.23 ± 0.20 was noted from 2nd to 3rd day in curative
treatment group (Figure 4.69)
At 10th, 20th and 30th day a significant (p<0.0001) decrease in albumin (g/dL)
correspondingly 2.53 ± 0.61, 2.2 ± 0.34 and 1.6 ± 0.40 was recorded in curative positive control
as compared to normal control (3.73 ± 0.31, 4 ± 0.4 and 4.2 ± 0.52) on the same days. On the other
side, curative treatment and curative standard groups showed an increase in albumin (g/dL) level
2.53 ± 0.76 and 3.36 ± 0.35 respectively at 30th day as compared to 1curative positive control (1.6
± 0.40). The increase of albumin (g/dL) level in curative treatment group advocated the
hepatoprotective potential of herbal combination against hepatotoxicity induced by CCl4.
Figure 4.69. Presented curative hepatoprotective effects of herbal combination on Albumin
(g/dL) level at different days.
4.3.1.2.7 Curative effect on globulin level
There was no significant change in globulin (g/dL) levels observed at zero day and 2nd day
in all curative groups as compared to normal control (Figure 4.70). The curative positive control
showed a significant decrease in globulin (g/dL) level 2.3 ± 0.25, 1.96 ± 0.20, 1.63 ± 0.32 and 1.36
± 0.35 at 3rd, 10th, 20th and 30th day respectively in comparison with normal control (3.22 ± 0.43,
3.06 ± 0.55, 2.98 ± 0.60 and 2.9 ± 0.60). However, no significant change in globulin (g/dL) levels
were observed in curative negative control, curative treatment and curative standard groups at 3rd,
108
10th, 20th and 30th day (Figure 4.70). The non-significant trend of globulin (g/dL) in curative
treatment group indicated the effectiveness of herbal combination against hepatotoxicant CCl4.
Figure 4.70. Presented curative hepatoprotective effects of herbal combination on globulin
(g/dL) level at different days.
Medicinal plants are storehouse of many bioactive compounds that are effective to control
various diseases through antioxidative properties (Weber et al., 2003; Subba et al., 2017).
Different studies on medicinal plants supported the present study results. Such as protective effect
of polyherbal formulation of two plants naming T. cordifolia and C. orchioides were evaluated by
measuring SGPT, SGOT, creatinine, total protein and albumin levels as biomarkers against
hepatotoxicity induced by ethanol. Results of this study indicated that rats treated with 200 and
400 mg/kg body weight of polyherbal formulation significantly inhibited the elevated levels of
SGPT, SGOT creatinine and albumin levels. It was also revealed from the results that the
combination of T. cordifolia and C. orchioides due to their phytoconstituents is more useful for
the protection of the liver cells (Sreshta and Ravindra, 2018).
The hepatoprotective potential of Ginseng essence (GE) prepared from four edible
medicinal plants including P. ginseng, P. quinquefolius, N. nucifera and L. longiflorum were
evaluated against liver injury induced through carbon tetrachloride (CCl4). Results of serum
biochemical analysis showed that the use of GE herbal formulation with hepatotoxicant CCl4,
109
ameliorate and reverse the increased levels of ALT, AST and bilirubin (Lu et al., 2017). Similarly
in another study the hepatoprotective effects of individual and polyherbal formulation of C.
procera, G. sylvestre and L. inermis in methanolic aqueous extracts were evaluated and Silymarin
was used as a standard drug. Results indicated the significant potential of polyherbal formulation
by controlling elevated serum enzymes SGOT, SGPT, total bilirubin and cholesterol level at
normal range in albino rats. It was revealed that polyherbal formulation exhibited excellent
hepatoprotective potential as compare to individual plants due to synergistic effects of
phytochemical of medicinal plants (Yogi and Mishra, 2016).
4.3.1.3 Curative hepatoprotective effect on Acetylcholinesterase activity
Liver of three rats from each curative group were removed. Liver homogenate was
prepared and used to measure acetylcholinesterase (AChE) activity. The level of
acetylcholinesterase enzyme activity in liver homogenate of all curative groups was measured and
expressed in terms of mean ± SD (µM of ATC hydrolyzed/min/g). The first group, normal control
group showed 4.2 ± 1.06 of AChE activity, while curative positive control group exhibited a
significant (p>0.0001) decrease 1.4 ± 0.70 in AChE activity (Figure 4.71). On the other side, no
significant change in AChE activity was observed in curative negative control (3.8 ± 0.61),
curative treatment (3.1 ± 1.16) and curative standard (3.5 ± 0.60) group as compared to normal
control.
Figure 4.71. Presented curative hepatoprotective effects of herbal combination on AChE
activity (µM of ATC hydrolyzed/min/g) in different groups.
Acetylcholinesterase (AChE) is an enzyme that hydrolysed acetylcholine, a
neurotransmitter into acetyl and choline. AChE can be used as a biomarker for the identification
110
of xenobiotic, pesticides, herbicides and toxic effects of chemical on liver and kidney of Wister
rats (Yilmaz et al., 2015). An experimental study performed to see the toxic effect of Malathion
on live. It was revealed from the results that rats treated with Malathion significantly decreased
the acetylcholinesterase activity as compared to control group (Akhgari et al., 2003; Rezg et al.,
2008). Similar findings were observed in present study in which rats treated with CCl4 were shown
a significant decrease in AChE as compare to normal control group. In another study similar results
were observed in which etoxazole was used to induce toxicity in hepatocytes. Results of this
study exhibited the significant reduction of AChE and increase of MDA levels due to the toxic
effects of etoxazole on liver cells (Yilmaz et al., 2017).
4.3.1.4 Curative hepatoprotective effects of herbal combination on lipid peroxidation and
antioxidant enzymes
Liver homogenate of rats from each group were prepared and used to measure
malondialdehyde (MDA) and antioxidants (SOD and GSH) activities. Curative hepatoprotective
effects of herbal combination were measured on lipid peroxidation in terms of nmol/g tissue of
malondialdehyde (MDA), antioxidant enzymes glutathione reductase (GSH) µg/mg and
superoxide dismutase (SOD) U/mg in liver homogenates of rats (Table 4.12).
Table 4.12 Curative hepatoprotective effects of herbal combination on MDA, GSH and
SOD levels
Curative Groups MDA (nmol/g tissue) GSH (µg/mg) SOD (U/mg)
Normal Control 74.5 ± 4.41 42.34 ± 1.66 18.44 ± 0.37
Curative positive control 205.5 ± 4.49 17.83 ± 2.46 7.47 ± 0.41
Curative negative control 61.5 ± 2.65 49.82 ± 4.27 16.24 ± 0.31
Curative treatment group 100.5 ± 7.9 45.76 ± 1.86 13.32 ± 0.45
Curative standard group 99.5 ± 4.4 45.76 ± 1.86 14.71 ± 0.8
The normal control showed 74.5 ± 4.4 MDA (nmol/g tissue) level, while was significantly
high (205.5 ± 4.49) MDA levels was observed in curative positive control group that was treated
with only CCl4 twice a week for a period of one month (Table 12). The high MDA level indicated
the extreme lipid peroxidation of hepatic cells due to the exposure of CCl4 and generation of
reactive oxygen species. Likewise curative negative control which was treated with only herbal
combination showed a normal level of MDA as compared to normal control. In curative treatment
group (treated with both herbal combination and CCl4) significant decrease of MDA level 100.5 ±
7.9 nmol/g tissue was observed as compared to curative positive control (205.5 ± 4.49) that
indicated the ameliorative anti- hepatotoxic effect of herbal combination. Similarly curative
111
standard group (treated with both Siliver and CCl4) was exhibited 99.5 ± 4.4 nmol/g MDA level
in liver homogenate as compared to normal control group (Figure 4.72).
Figure 4.72. Presented curative hepatoprotective effects of herbal combination on MDA
(nmol/g tissue) in different groups
The curative negative, curative treatment and curative standard group exhibited 49.82 ±
4.27, 45.76 ± 1.86 and 45.76 ± 1.86 µg/mg GSH levels respectively as compared to normal control
42.34 ± 1.66 µg/mg (Table 4.12). On the other side, significant decrease of GSH level 17.83 ±
2.46 µg/mg was observed in curative positive control as compared to normal control (Figure 4.73).
Figure 4.73. Presented curative hepatoprotective effects of herbal combination on GSH
(µg/mg) in different groups
The significantly low superoxide dismutase (SOD) (U/mg) levels 16.24 ± 0.31, 13.32 ±
0.45 and 14.71 ± 0.8 were observed correspondingly in curative negative control, curative
treatment and curative standard groups as compared to normal control group (18.44 ± 0.37) (Table
112
4.12). The curative positive control group showed a significant decrease in SOD level (U/mg) 7.47
± 0.41 as compare to normal control (Figure 4.74). The normal level of SOD and GSH in curative
treatment group as compared to curative positive control advocated the effectiveness of herbal
combination due to synergism of bioactive compounds.
Figure 4.74. Presented curative hepatoprotective effects of herbal combination on SOD
(U/mg) in different groups
The finding of present study were co-related with the study in which combination of R.
verniciflua and E. ulmoides was assessed against hepatotoxicity induced by CCl4 in HepG2 cells.
The HepG2 cells treated with plant combination showed significant results by inhibiting lipid
peroxidation and restoration of antioxidant enzyme SOD (Young et al., 2018). Similar findings
were observed in another study in which self-micro emulsifying formulation of L. speciose, leaves
extract were used to evaluate the hepatoprotective potential against chemically induced
hepatotoxicity. Both plant and toxicant (CCl4) treated group was exhibited a significant antioxidant
potential of plant by increasing of GSH, SOD and CAT contents and decreasing of lipid
peroxidation. On the other hand in CCl4 treated group a significant depletion of GSH, SOD and
CAT and increase of lipid peroxidation was observed as compare to control group (Amresh et al.,
2018).
In another study, an ayurvedic formulation Rhhitaka ghrita (Rg) was evaluated against
hepatotoxicity induced through paracetamol in rats. The results of this study depicted that use of
paracetamol significantly impaired the antioxidant system by increase of lipid peroxidation and
decrease of catalase and glutathione levels. The use of Rg formulation remarkably reverse the toxic
113
effect of paracetamol by restoring of catalase, glutathione and inhibiting of lipid peroxidation
process (Goyal et al., 2012). Oxidative stress also disrupted the signal transduction pathway that
are responsible for induction and activation of antioxidant enzymes (Reddy and Urooj, 2017).
Therefore, natural products that have ROS-scavenging properties may use as promising
therapeutic agents to treat and manage oxidative stress related diseases (Hu and Kong 2004).
4.3.1.5 Curative effects of herbal combination on liver Histopathology
Liver of rats from each group were removed at 30th day and used to see the
histopathological investigation on hepatocytes. Histopathological examination of normal control
group liver cells (Figure 4.75) exhibited the normal hepatocellular architecture with well –distinct
hepatic cells, kupffer cells and sinusoidal spaces. Nucleus of the hepatic cells was in normal shape
with clear nucleolus and prominent chromatin material. Hepatic cells with cytoplasm and
parenchyma were regular in shape and arranged in a plate of hepatocytes. Likewise no any vacuolar
necrotic damage and pathological abnormality were observed in liver of normal control rats. On
the other side, histopathological examination of curative positive control group rats treated with
CCl4 twice a week for one month showed abrupt changes in liver cells (Figure. 4.76). Sever
necrosis and inflammation were seen in hepatic parenchyma of curative positive control group.
Likewise dilation of sinusoidal spaces due to inflammation, microvascular steatosis and ballooning
was noted in curative positive control group. These unusual changes indicated hepatotoxic effect
of CCl4 that disrupted the hepatic cells and their functions.
114
Figure 4.75. Photomicrograph (X400) shown normal architecture of the Hepatic cell (H),
with nucleolus and prominent chromatin (→), Sinusoidal spaces between hepatocytes (S and
circles) and Kupffer cells (K).
Figure 4.76. Photomicrograph (X400) shown necrosis of hepatic cell (→), inflammation in
the hepatic cells and sinusoidal spaces (circles) and dilation of sinusoidal spaces (←) in
curative positive control group
115
Histopathological evaluation of curative negative control group (treated with only herbal
combination) showed a clear hepatocytes with parenchyma and nucleolus with prominent
chromatin. Similarly mild inflammation, dilation of sinusoidal spaces and binucleated nucleus
were observed in curative negative control group (Figure 4.77). The normal architecture of hepatic
cells confirmed the potential of herbal combination with no cytotoxicity in the liver cells.
Figure 4.77. Photomicrograph (X400) shown mild degree of inflammation of hepatic cell
(←), binucleated cells (↑) and dilation of sinusoidal spaces (→) in liver of curative negative
control group
The liver histology of the curative treatment group (treated with herbal combination daily
and CCl4 twice a week for a period of one month) showed ameliorative effects of herbal
combination (Figure 4.78). A mild degree of narcosis, inflammation and vacuolar degeneration of
hepatic cells were observed in liver sections of curative treatment group. Likewise visible
parenchyma and nucleolus with prominent chromatin of hepatic cells were seen that indicated the
recovery of these cells due to anti-hepatotoxic effect of herbal combination.
116
Figure 4.78. Photomicrograph (X400) displayed mild degree of necrosis of hepatic cell (→),
vacuolar degeneration (↑), diffused kupffer cells (↓) and inflammation of hepatic cells (←) in
liver of curative treatment group
The histopathological examination of curative standard group (treated with Siliver daily
and CCl4 twice a week for one month) depicted a minor changes in the liver cells (Figure 4.79). A
mild degree of narcosis and binucleated hepatocytes with clear parenchyma were observed in the
liver cells of this group. These minor histopathological changes showed the potent effects of Siliver
against hepatotoxicant CCl4.
Figure 4.79. Photomicrograph (X400) shown mild degree of necrosis of hepatic cell (→),
binucleated cells (↑) and dilation of sinusoidal spaces (←) in liver of curative standard group
117
Numerous threating side effects have been appeared on hepatic cells due to allopathic
medications, if the dose and course of the medication is not followed wisely (Vliegenthart, 2014).
Drug-induced liver injury is the major challenge for researches in clinical medicine and drug
development. Medicinal herbs have unified power of healing though their versatile biologically
active components such as Flavonoids, phenolics, alkaloids, Terpenoids, etc (Vinayak et al., 2018).
An experimental study was performed to evaluate synergistic effect of G. galbra and S. marianum
in combination against hepatotoxicity induced by CCl4 in Wister albino rats. The histopathological
finding of this study showed abnormalities in liver architecture such as dilated hepatic sinusoids,
cellular necrosis and apoptotic bodies, diffused kupffer cells and degenerated hepatocytes in CCl4
treated group. On the other side rat treated with plant combination showed a significant
hepatoprotective potential by healing and repairing of hepatocytes and necroinflammatory lesion
induced (Rasool et al., 2014).
Similar changes in liver histology were seen another study in which a combination of T.
cucumerina and C. sativum (150mg/kg body weight) was given as a hepatoprotective agent against
paracetamol. In paracetamol intoxicated group degeneration of hepatic plates, ballooning, necrosis
and loos of cellular boundaries were noted as compare to control group. However group treated
with plant combination showed minimal inflammation with normal hepatic cord that indicating
the significant protective potential of T. cucumerina and C. sativum against toxic effects of
paracetamol on liver cells (Palanisamy et al., 2017).
4.3.2 Preventive hepatoprotective effect of herbal combination
Five preventive groups including normal control, preventive negative control, preventive
positive control, preventive treatment and preventive standard group were made to see preventive
hepatoprotective effect of herbal combination. Normal control group received normal diet and
preventive negative control treated with only herbal combination (200 mg/kg b. wt) for thirty days,
while preventive positive control was on normal diet for 27 days and at 28th and 29th day
administrated with hepatotoxicant CCl4 (1mL/kg b. wt). The preventive treatment group was
received herbal combination (200mg/kg b. wt) daily for 27 days and at 28th and 29th day treated
with hepatotoxicant CCl4 (1mL/kg b. wt). In preventive standard group rats were treated with
Siliver (50 mg/kg b.wt) for a period of twenty seven days and at 28th and 29th day administrated
with hepatotoxicant CCl4 (1mL/kg b. wt).
118
4.3.2.1 Preventive effect of herbal combination on body weights of rats
Body weights among all the preventive groups were measured at zero, 7th, 14th, 21st and
28th day to see effect of toxicant (CCl4) and herbal combination on rats. The body weight of rat
were measured in grams (g) and presented in figure 4.80. It was depicted from the graph that
preventive positive control and preventive standard group showed a significant elevation of weight
of rats from 21st to 28th day. Similarly a significant increase of weight of rats in preventive negative
control and preventive treatment groups were observed at 14th, 21st and 28th day (Figure 4.80)
Figure 4.80. Preventive effect of herbal combination against hepatotoxicity induced by CCl4
on body weights of rats.
4.3.2.2 Preventive effect of herbal combination on Biochemical parameters
The blood samples of all preventive groups were taken to measure liver function test at
zero day, 15th, 29th (after 24 hours CCl4 administration) and 30th day (after 48 hours CCl4
administration). Biochemical parameters of livernincluding ALT, AST, ALP, ƔGT, total bilirubin,
total protein, albumin and globulin were measured at zero, 15th, 29th and 30th day.
4.3.2.2.1 Preventive effect on Alanine aminotransferase (ALT)
The level of ALT (U/L) was measured in all preventive groups as compared to normal
control group at Zero, 15th, 29th and 30th day. The results of ALT (U/L) enzymes in all preventive
groups were presented as mean ± SD and significant change in enzyme level between groups as
compared to normal control and within groups were analyzed through two-way ANOVA, Tukey’s
119
multiple comparison test. There were no significant change in ALT enzyme (U/L) in all preventive
groups at zero and 15th day (Figure 4.81).
At 29th day, preventive positive control and preventive standard group were shown a
significant (p<0.0001) increase of ALT (U/L) level 82.67 ± 4.51 and 68.67 ± 3.06 respectively as
compared to normal control 45.24 ± 12.53, while no significant change was noted in preventive
negative control 49.67 ± 3.51 at the same day. Similarly preventive treatment group (54 ± 7.81)
showed a non-significant change in ALT (U/L) level as compared to normal control at 29th day.
A significant (p>0.0001) elevation of ALT (U/L) level was observed in preventive positive control
(96.67 ± 8.02), preventive treatment (65.67 ± 10.21) and preventive standard (71.43 ± 6.24) as
compared to normal control (46.33 ± 6.11), whereas preventive negative control group was shown
no change in ALT (U/L) 47 ± 2.65 at 30th day. The low level of ALT (U/L) in preventive treatment
group as compared to preventive positive control advocated the potent effect of herbal combination
of selected medicinal plants against CCl4.
Figure 4.81. Shown preventive hepatoprotective effects of herbal combination on ALT (U/L)
level at different days.
120
4.3.2.2.2 Preventive effect on Aspartate aminotransferase (AST)
All preventive groups comprising of preventive negative control (95 ± 21and 102.33 ±
4.73), preventive positive control (103.67 ± 26.08 and 131.67 ± 25.74), preventive treatment
(109.33 ± 17.01and 105.33 ± 16.04) and preventive standard (119.67 ± 17.9 and 148.33 ± 54.2)
were shown non- significant alteration in AST (U/L) level as compared to normal control (81.33
± 9.07 and 96.33 ± 8.14) and at zero and 15th day (Figure 4.82).
There was a significant (p>0.0001) increase of AST (U/L) level 261 ± 34.09, 172.67 ±
20.11 and 208.67 ± 35.26 observed correspondingly in preventive positive control, preventive
treatment and preventive standard group in comparison with normal control 112.67 ± 15.53, while
preventive negative control again showed (138.33 ± 14.01) a non-significant change in AST (U/L)
at 29th day. The significant (p<0.0001) increase of AST (U/L) level 369.33 ± 39.88 was observed
in preventive positive control group at 30th day in comparison with normal control (116.33 ±
14.98), preventive negative control (162 ± 18.08), preventive treatment (265 ± 18.25) and
preventive standard (291.67 ± 5.69) group. Similarly preventive treatment and preventive standard
were shown increase in AST (U/L) level 265 ± 18.25 and 291.67 ± 5.69 respectively as compare
to normal control 116.33 ± 14.98 at 30th day. The comparison of AST (U/L) level between groups
at 30th day advocated that herbal combination of medicinal plants was effective in preventive mode
of treatment by controlling liver enzymes.
Figure 4.82. Presented preventive hepatoprotective effects of herbal combination on AST
(U/L) level at different days.
121
4.3.2.2.3 Preventive effect on Alkaline phosphatase (ALP)
ALP enzyme is present in liver and bone cells and in case of injury it comes out from the
cells. Therefore, in liver disorders ALP level is increased due to destruction of hepatic cells. The
graphical presentation figure 4.83 depicted that at zero and 15th day (before the administration of
hepatotoxicant CCl4) correspondingly a non- significant change in ALP (U/L) was observed in
preventive positive control (198.67 ± 22.03 and 217 ± 21.79), preventive negative control (160.67
± 21.78 and 184.67 ± 17.9), preventive treatment (207.67 ± 39.68 and 249.67 ± 17.95) and
preventive standard (142.33 ± 21.59 and 226.33 ± 22.85) group as compared to normal control
(165 ± 31.61 and 208.67 ± 23.71).
After the administration of CCl4, at 29th and 30th day a significant (p>0.0001) elevation of
ALP (U/L) was noted in preventive positive control (302 ± 53.25 and 366.33 ± 58.79) and
preventive treatment (298.45 ± 31.66 and 314.67 ± 37.17) groups as compared to control group
(229.67 ± 18.77and 245 ± 27.22). On the other side, preventive standard showed a non-significant
change at 29th day (264.67 ± 21.96) and significant elevation of ALP (U/L) (303.67 ± 4.04) at 30th
day. There were no significant alteration in preventive negative control (199.67 ± 17.39 and 222
± 5.29) in comparison with normal control at 29th and 30th day. Therefore, low level of ALP (U/L)
in preventive treatment group 314.67 ± 37.17as compared to preventive positive control 366.33 ±
58.79, supported the ameliorative effects of herbal combination.
Figure 4.83. Shown preventive hepatoprotective effects of herbal combination on AST (U/L)
level at different days.
122
4.3.2.2.4 Preventive effect on Gamma-glutamyltransferase (ƔGT)
It was depicted from graph (Figure 4.84) of ƔGT (U/L) at zero and 15th day that preventive
negative control (11 ± 1.05 and 9.33 ± 1.15), preventive positive control (8.33 ± 1.53 and 7.33 ±
0.58), preventive treatment (10.33 ± 1.53 and 9.33 ± 2.31) and preventive standard (10.67 ± 1.53
and 7 ± 2.02) groups showed non-significant change in ƔGT (U/L) level as compared to normal
control (8.33 ± 1.15 and7.33 ± 0.58). In preventive positive control a significant (p>0.0001)
increase in ƔGT (U/L) level 14 ± 2.03 and 15.33 ± 2.31 was seen at 29th and 30th day respectively
in contrast to normal control (9.67 ± 1.53 and 10.33 ± 0.58) at same days. The preventive treatment
(10.67 ± 1.15 and 12 ± 2.06) and preventive standard (8.33 ± 2.08 and 8 ± 1.73) groups exhibited
a non- significant increase in ƔGT (U/L) level at 29th and 30th day as compared to normal control
(9.67 ± 1.53 and 10.33 ± 0.58). Similarly in preventive negative control group, a non-significant
alteration in ƔGT (U/L) level was noted 8 ± 1.73 and 9.33 ± 1.53 correspondingly at 29th and 30th
day.
Figure 4.84. Presented preventive hepatoprotective effects of herbal combination on ƔGT
(U/L) level at different days.
4.3.2.2.5 Preventive effect on Total bilirubin
The graphical presentation of total bilirubin (mg/dL) exhibited a non-significant
(p<0.0001) alteration in preventive negative control (0.2 ± 0.05 and 0.28 ± 0.01), preventive
positive control (0.18 ± 0.03 and 0.26 ± 0.02), preventive treatment (0.17 ± 0.02 and 0.28 ± 0.03)
123
and preventive standard (0.16 ± 0.05 and 0.25 ± 0.05) groups as compared to normal control (0.15
± 0.06 and 0.21 ± 0.05) at zero and 15th day (Figure 4.85). In curative positive control a significant
(p>0.0001) increase in total bilirubin (mg/dL) 0.5 ± 0.11 and 0.57 ± 0.06 was observed at 29th and
30th day respectively. Similarly elevated total bilirubin (mg/dL) level was noted in preventive
treatment (0.34 ± 0.02 to 0.44 ± 0.02) and preventive standard (0.35 ± 0.03 to 0.47 ± 0.07) at 29th
to 30th day as compared to normal control (0.22 ± 0.03 and 0.22 ± 0.01). However no significant
increase of total bilirubin (mg/dL) was observed in preventive treatment and preventive standard
groups as compared to preventive positive control due to the persuasive effects of herbal
combination and standard Siliver drug in respective groups.
Figure 4.85. Presented preventive hepatoprotective effects of herbal combination on total
bilirubin (mg/dL) level at different days.
4.3.2.2.6 Preventive effect on Total protein
The total protein (g/dL) contents were found to be normal in preventive positive control
(5.79 ± 0.40 and 6.06 ± 0.25), preventive negative control (6.06 ± 0.50 and 6.2 ± 0.34), preventive
treatment (5.33 ± 0.41and 5.13 ± 0.47) and preventive standard (5.33 ± 0.64 and 5.6 ± 0.6) groups
as compared to normal control (5.85 ± 0.87 and 5.56 ± 0.50) group at zero and 15th day (Figure
4.86). Similarly at 29th day, a non-significant change in total protein (g/dL) 5.25 ± 0.36, 6.26 ±
0.57 and 5 ±0.7 were observed in preventive positive control, preventive negative control and
preventive standard groups, while preventive treatment group showed a significant depletion of
124
total protein (g/dL) 4.86 ± 0.37 at 29th day as compared to normal control 5.8 ± 0.58 group at same
day. At 30th day (after 48 hours of CCl4 administration) preventive treatment (4.34 ± 0.27),
preventive standard (4.73 ± 0.70) and preventive positive (4.02 ± 0.15) control groups exhibited a
significant decrease of total protein in comparison with preventive negative control (6.06 ± 0.50)
and normal control (5.85 ± 0.35) group.
Figure 4.86. Shown preventive hepatoprotective effects of herbal combination on total
protein (g/dL) levels at different days.
4.3.2.2.7 Preventive effect on albumin
The preventive positive control (2.86 ± 0.11, 2.73 ± 0.11and 2.53 ± 0.61), preventive
negative control (3.06 ± 0.50, 3.2 ± 0.6 and 3.56 ± 0.56) and preventive standard (2.63 ± 0.42, 2.7
± 0.75 and 2.56 ± 0.21) groups showed a non-significant change in albumin (g/dL) at zero, 15th
and 29th day as compared to normal control group (3.05 ± 0.31, 3.12 ± 0.4 and 3.16 ± 0.25). On
the other side, at 29th day in preventive treatment group a significant decrease in albumin (g/dL)
2.23 ± 0.21 was noted. At 30th day only preventive positive control group showed a significant
decrease of albumin (g/dL) (2.2 ± 0.34) as compared to normal control (3.2 ± 0.2) (Figure 4.87).
125
Figure 4.87. Presented preventive hepatoprotective effects of herbal combination on total
albumin (g/dL) levels at different days.
4.3.2.2.8 Preventive effect on globulin protein
At zero, 15th and 29th day all preventive groups were exhibited a non-significant change in
globulin protein (g/dL) as compared to normal control group (Figure 4.88). At 30th day in
preventive positive control group a significant decrease in globulin (g/dL) 1.96 ± 0.21 was
observed in comparison with normal control group 2.9 ± 0.61. The preventive treatment (3.23 ±
0.25), preventive standard (2.78 ± 0.22) and preventive negative control (3.16 ± 0.57) showed non-
significant depletion of globulin (g/dL) at 30th day in comparison with normal control group.
Figure 4.88. Presented preventive hepatoprotective effects of herbal combination on total
globulin (g/dL) levels at different days.
126
Medicinal plants are used as alternative source of drugs to cure many diseases including
cardiovascular disorders, cancer, inflammation, liver and kidney related diseases due to their
antioxidant and therapeutic properties (Zohra and Fawzia, 2014). The natural and active
compounds such as flavonoids, phenolics, terpenes, alkaloids and steroids of medicinal plants have
pharmacological and biological properties against different diseases (Nakachi et al., 2000).
Various studies on hepatoprotective medicinal plants supported the present research
findings. Like the preventive hepatoprotective activity of polyherbal tablets of 50mg/kg and
100mg/kg, prepared by three plants B. monosperma, B. variegata and O. gratissimum was assessed
in rats against hepatotoxicity induced by paracetamol. The high levels of SGPT, SGOT, ALP and
total bilirubin were reversed due to the ameliorate effect of polyherbal tablet of 100mg/kg
significantly (Gupta et al., 2013). Similar finding were observed in another study in which the
synergistic hepatoprotective potential of a combination (SAL) of three plants S. chinensis, A.
capillaris and A. barbadensis was evaluated. Hepatotoxicity was induced by acetaminophen and
carbon tetrachloride (CCl4) that increased the ALT, AST, ALP, total bilirubin, total protein and
bile acid levels. However pre-treatment with SAL 250 to 400 mg/kg body weight significantly
inhibited the over production of these enzymes ALT, AST and bile acid (Yimam et al., 2016).
4.3.2.3 Preventive effect of herbal combination on acetylcholinesterase activity
Liver of three rats from each group were removed at 30th day (end of animal trial). Liver
homogenate was prepared to measure acetylcholinesterase activity in all preventive groups and
expressed in terms of mean ± SD (µM of ATC hydrolyzed/min/g). In normal control group 4.6 ±
1.06 µM of ATC hydrolyzed/min/g of AChE levels was observed, while preventive positive
control group showed a significant (p>0.0001) decrease in AChE activity (2.3 ± 0.71 µM of ATC
hydrolyzed/min/g) as compared normal control group (Figure 4.89). On the other side in
preventive negative control (4 ± 0.61), preventive treatment (3.2 ± 0.84) and preventive standard
(3.5 ± 0.58) groups no significant change in AChE activity was observed as compared to normal
control (Figure 4.89).
127
Figure 4.89. Presented preventive hepatoprotective effects of herbal combination on AChE
activity (U/mg) in different groups.
Acetylcholinesterase (AChE) is a hydrolytic enzyme that splits acetylcholine into acetate
and choline. Acetylcholine is a key regulator for cell survival, differentiation and proliferation
(Shen et al., 2013). Excessive production of acetylcholine in the absence of AChE, increased the
cell proliferation by downregulation of p27 and cyclin proteins that are main component of cell
cycle (Pérez-Aguilar et al., 2015). Visceral organs including liver, heart, kidney, placenta,
intestines and pancreas contained variable amount of AChE (Soreq and Seidman, 2001). In case
of liver injury and chemically induced hepatotoxicity, AChE level is decreased that stimulate more
proliferation of the liver cells (Catassi et al., 2008). Similar finding were observed in present study
in which CCl4 intoxication decrease the AChE activity in hepatocytes. However, use of medicinal
plants in combinations protect the liver cells and increased the production of AChE activity
(Yilmaz et al., 2017).
4.3.2.4 Preventive effects of herbal combination on lipid peroxidation and antioxidant
enzymes
At 30th day liver of three rats from each preventive group were removed and liver
homogenate was prepared. Lipid peroxidation in terms of nmol/g tissue of malondialdehyde
(MDA) and antioxidant enzymes glutathione reductase (GSH) µg/mg and superoxide dismutase
(SOD) U/mg were measure in liver homogenate (Table 4.13).
128
Table 4.13: Preventive hepatoprotective effects of herbal combination on MDA, GSH and
SOD levels
Preventive Groups MDA (nmol/g tissue) GSH (µg/mg) SOD (U/mg)
Normal Control 74.5 ± 4.41 30.9 ± 1.66 18.44 ± 0.17
Preventive positive control 199.5 ± 3.86 15.04 ± 3.69 7.2 ± 0.465
Preventive negative control 61.5 ± 2.65 49.82 ± 4.27 16.24 ± 0.21
Preventive treatment group 112 ± 4.4 30.9 ± 5.7 15.85 ± 0.49
Preventive standard group 118 ± 4.7 34.32 ± 5.88 19.4 ± 0.45
Normal control showed 74.5 ± 4.41 MDA (nmol/g tissue) level, while MDA levels was
significantly high (199.5 ± 3.86) in preventive positive control group (received CCl4 at 28th and
29th day) as compared to normal control group (Figure 4.90). The high level of MDA, indicated
the increase of lipid peroxidation in hepatic cells due to the exposure of CCl4. Likewise preventive
negative control which was treated with only herbal combination showed a normal level of MDA.
In preventive treatment group (treated with herbal combination for 28 days and CCl4 for last two
days in a period of one month), a non-significant increase in MDA level 112 ± 4.4 nmol/g tissue
was observed that specified the ameliorative preventive effect of herbal combination against
hepatotoxicity. Similarly non-significantly high MDA level nmol/g tissue 118 ± 4.7 was observed
in preventive standard group as compared to normal control group (Figure 4.90).
Figure 4.90. Presented preventive hepatoprotective effects of herbal combination on MDA
level (nmol/g tissue) in different groups.
129
In preventive negative, preventive treatment and preventive standard groups GSH levels
49.82 ± 4.27, 30.9 ± 5.7 and 34.32 ± 5.88 µg/mg were observed respectively as compared to normal
control 30.9 ± 1.66 µg/mg (Table 4.13). On the other side in preventive positive control a
significant decrease in GSH level 15.04 ± 3.69 µg/mg was noted as compared to normal control
(Figure 4.91).
Figure 4.91. Presented preventive hepatoprotective effects of herbal combination on GSH
levels (µg/mg) in different groups
The antioxidant SOD level (U/mg) was found normal in preventive negative control (16.24
± 0.21), preventive treatment (15.85 ± 0.49) and preventive standard (19.4 ± 0.45) groups as
compared to normal control group (18.44 ± 0.17). The preventive positive control group showed
a significant decrease in SOD levels (U/mg) 7.2 ± 0.465 as compared to all group (Figure 4.92).
The normal level in preventive treatment group as compared to preventive negative control was
the evidence of potent antioxidant effect of herbal combination.
130
Figure 4.92. Presented preventive hepatoprotective effects of herbal combination on SOD
levels (U/mg) in different groups.
Medicinal plants are used from ancient times to treat several disease due to their
antioxidative properties that reduced the reactive oxygen species (Embuscado, 2015). Reactive
oxygen species are highly reactive anions that are produced in oxidative reactions which are taken
place in mitochondria (Herraiz and Galisteo, 2015). Different experimental studies on medicinal
plants supported the present research work such as Chinese herbal medicine Jian-Gan-Bao (JGB)
preventive hepatoprotective effects were studied on acute and chronic alcoholic liver disease
(ALD) and non-alcoholic fatty liver disease (NAFLD). In Jian-Gan-Bao formulation three
medicinal plants C. versicolor, S. miltiorrhiza and S. chinensis were used. In preventive mode of
treatment JGB significantly reduced the malondialdehyde, the end product of lipid peroxidation
against alcohol induced liver damage in mice. Moreover JGB had no preventive effects on SOD
and catalase activities, while significant increase in activity of glutathione peroxidase was
observed at high dose treatment with JGB (Li et al., 2018).
A similar study was done to assess the hepatoprotective potential of SAL consisting of
three medicinal plants S. chinensis, A. capillaris and A. barbadensis against acute liver toxicity
induced by acetaminophen and carbon tertachrloide (CCl4). The author evaluated glutathione and
superoxide dismutase levels in liver homogenate. It was revealed from the results that SAL
significantly replenished depleted SOD and hepatic glutathione levels by reducing oxidative stress
and synergistic antioxidative properties (Yimam et al., 2016). Therefore, herbal combination of
131
medicinal plants have synergistic effect due to presences of more secondary metabolites (Phenolics
and flavonoids) that enhanced the pharmacological actions against many diseases (Yogesh and
Yadav, 2015; Preethi et al., 2016).
4.3.2.5 Preventive effect of herbal combination on liver histopathology
Liver of rats from each preventive group were removed and preserved for histopathological
examination at the end of trial. Histopathological analysis of normal control group showed normal
architecture of liver cells (Figure 4.93). The polygonal shape and well preserved nucleus of
hepatocytes was observed in normal control group. Similarly parenchyma of the hepatic cells,
nuclei of the hepatic cells with prominent chromatin and nucleolus were seen in the liver of normal
control group rats. However in preventive positive control group inflammation and necrosis of
hepatic cells were observed that was the evidence of receiving hepatotoxicity of CCl4.
Histopathological examination also showed dilation of sinusoidal spaces and diffusing of kupffer
cells and hepatocytes (Figure 4.94).
Figure 4.93. Photomicrograph (X400) shown normal architecture of the Hepatic cell (H),
with nucleolus and prominent chromatin (←), Sinusoidal spaces between hepatocytes (S),
Kupffer cells (K) and binucleated cells in normal control group
132
Figure 4.94. Photomicrograph (X400) shown inflammation of hepatic cell (←), necrosis of
the hepatic cells (→) and dilation of sinusoidal spaces (↑) leakage of blood cells through portal
vein (↓) and diffusing of hepatocytes and kupffer cells in liver of the preventive positive
control group
Histopathological analysis of preventive negative control group treated with only herbal
combination showed a clear hepatocytes with parenchyma and nucleolus with prominent
chromatin. Similarly normal sinusoidal spaces, endothelial cells, hepatic vein with blood cells,
hepatic artery and hepatic bile duct were seen in preventive negative control group (Figure 4.95).
The normal architecture of hepatic cells confirmed the potential of herbal combination with no
cytotoxicity in the liver cells.
Figure 4.95. Photomicrograph (X400) presented hepatic cell (→), sinusoidal spaces (↑), blood
cells (↓), endothelial cells (←), hepatic vein (HV), hepatic artery (HA) and hepatic bile duct
(BD) in liver of preventive negative control group
133
The liver histology of the preventive treatment group (treated with herbal combination
daily for 28 days and hepatotoxicant CCl4 for last two days for a period of one month) showed
ameliorative effects of herbal combination (Figure 4.96). A mild degree narcosis, inflammation
and vacuolar degeneration of hepatic cells were observed in liver sections of preventive treatment
rats. The recovery of these cells against hepatotoxicity induced by CCl4 proved the ameliorative
effects of herbal combination.
Figure 4.96. Photomicrograph (X400) shown mild degree of necrosis of hepatic cell (→),
vacuolar degeneration (↓), inflammation of hepatic cells (←) and dilation of sinusoidal spaces
(↑) in liver of preventive treatment group
The preventive standard group (treated with Siliver daily for 28 days and then
administrated with CCl4 for last two days in one month) exhibited a minor changes in the liver
cells of histopathological examination (Figure 4.97). A mild degree of narcosis, dilation of
sinusoidal spaces and binucleated hepatocytes with clear parenchyma were observed in the liver
cells of this group. These minor histopathological changes supported the potent effect of Siliver
against hepatotoxicant CCl4.
134
Figure 4.97. Photomicrograph (X400) shown mild degree of necrosis of hepatic cell (→),
binucleated cells (←) and dilation of sinusoidal spaces (→) in liver of preventive standard
group
Similar changes were noted in another study in which hepatoprotective potential of ginseng
formulation against liver injury induced by carbon tetrachloride (CCl4) was evaluated through
histopathological examination of hepatic cells. The ginseng formulation was contained four
medicinal plants P. ginseng, P. quinquefolius, N. nucifera, and L. longiflorum. Histopathological
results indicated that pre-treatment of ginseng formulation significantly reduced the toxic effects
of CCl4 by inhibiting inflammation and fibrosis (Lu et al., 2017). Another study supported the
findings of present study in which poly herbal formulation assessed against paracetamol
intoxication. Histopathological findings depicted that rat’s treated with 50 mg/kg and 100 mg/kg
of polyherbal formulation significantly exhibited the injury of hepatic cells. On the other hand rats
administrated with paracetamol showed a damage of hepatic cells with necrosis, vascular swelling,
and congestion in central vein and proliferation of kupffer cells (Gupta et al., 2013).
It is elucidated from the present study that selected herbal combination, comprising of S.
marianum, T. officinale, P. viviparum, F. arabica and M. annua have synergic hepatoprotective
potential against hepatotoxicity induced by CCl4. The restoration of liver enzymes, antioxidant
enzymes and histopathological alterations in both curative and preventive mode of treatments are
the evidenced of ameliorative anti-hepatotoxic potential of herbal combination. Therefore, this
herbal combination might be used as a therapeutic agent to manage and control of various liver
diseases and its related complications.
135
CHAPTER 5
SUMMARY
Hepatitis has become a serious health issue throughout the world and is becoming more severe in
Asian countries including Pakistan. This health menace, particularly in its chronic form is leading
to high level of morbidity and mortality. The increasing prevalence of hepatic disorders and its
related complications are major challenges for the scientists to overcome these hitches by
developing safer and green alternatives. Medicinal plants have remarkable potential to cure many
diseases through antioxidative properties but individually they are not sufficient to combat against
liver diseases, while in combinations form they have more medicinal potential against many
diseases due to synergistic effects of various components that increase bioavailability, specific
activity and antioxidant potential of medicinal plants. Therefore, in present study a combination
of medicinal plants was formulated through in vitro and in vivo studies to prevent and manage
numerous hepatic diseases and its related complications. To achieve the objectives of this study, it
was divided into three phases and experimental work was conducted in Clinico-Medical
Biochemistry Laboratory, Department of Biochemistry, University of Agriculture Faisalabad,
Pakistan. In phase I, in vitro screening of indigenous medicinal plants naming S. marianum, F.
religiosa, C. fistula, T. officinale, P. viviparum, Z. jujuba, P. emblica, F. arabica, O. basilicum
and M. annua was executed through antioxidant, toxicological and hepatoprotective potential.
Among all the plants, the maximum phenolic content (267.43 ± 20.06 GAE µg/g) were found in
P. viviparum and total flavonoid content (131.86 ± 1.13 CE µg/g) were observed in M. annua. The
antioxidant potential of medicinal plants was determined through ferric reducing antioxidant
power (FRAP) assay, DPPH and hydroxyl radical scavenging (HRS) activities on different
concentrations (100, 300 and 500 µg/mL). The antioxidant potential in the ranges from 33.04 to
84.73% as DPPH inhibition, from 32 to 94 % as scavenging of hydroxal radical and from 10.14 to
44.49 as ferric reducing power were observed from 100 to 500 µg/mL concentrations of the studied
medicinal plants. These findings indicated that the antioxidative properties increased with the
increase of plant concentrations. Toxicological evaluation of medicinal plants at different
concentrations was executed by hemolytic and thrombolytic activities in terms of percentage
hemolysis and percentage of clot lysis respectively. The medicinal plants that showed minimum
toxic effects on erythrocytes as compared to positive control in hemolytic and thrombolytic
activities were considered best and safe. Moreover, in phase I, hepatoprotective potential was
evaluated through acetylcholinesterase (AChE) inhibition assay and Liver Slice Culture (LSC)
assay. Plants that showed minimum percentage inhibition of AChE activity were considered as
136
best hepatoprotective plants. Liver Slice Culture assay was the main assay to explore the
hepatoprotective potential of medicinal plants in terms of percentage cytotoxicity. In this assay
fresh liver slices of rats under controlled physiological conditions were used to measure
hepatoprotective potential of plants at various concentrations against hepatotoxicity induced by
CCl4. Among all the plants S. marianum, T. officinale, P. viviparum, F. arabica and M. annua
showed maximum hepatoprotective potential in terms of least percentage cytotoxicity against
CCl4. Therefore, these plants were used for phase II studies in which biochemical profiling of
selected plants was performed through Liquid chromatography mass spectrometry (LC-MS). The
presence of different hepatoprotective phytoconstituents like Sweroside, Chlorogenic acid,
Quercetin, Myricetin, Isorhamnetin, Caffeic acid, Swertiamarin, 5-Hydroxymethylfurfural and
Ferulic acid were confirmed in selected plants. Subsequently, fifty herbal combinations of selected
plants suggested by Central Composite Design (CCD) of response surface methodology (RSM)
were prepared and analyzed through Liver Slice Culture assay in phase II. The herbal combination
that showed maximum hepatoprotective potential in terms of minimum percentage cytotoxicity,
comprised of 221 mg of S. marianum, 78 mg of P. viviparum, 271 mg of T. officinale, 342 mg of
F. arabica and 77 mg of M. annua was selected for in vivo studies. In phase III, this herbal was
used for in vivo study in which rats were used as animal model. Both curative and preventive mode
of treatments were used to evaluate the ameliorative effects of selected herbal combination against
hepatotoxicity induced by CCl4. For this purpose a dose of 1 mL/kg b.wt of CCl4 was used to
induce hepatotoxicity and a dose of 200 mg/kg b.wt of herbal combination was used for both
curative and preventive mode of treatments. Biochemical parameters including ALT, AST, ALP,
ƔGT, total bilirubin, total protein, albumin and globulin were measured in blood serum of all
curative and preventive groups. Acetylcholinesterase (AChE) enzyme activity, malondialdehyde
(MDA) in terms of lipid peroxidation and antioxidant enzymes superoxide dismutase (SOD) and
glutathione reductase (GSH) were also measured in liver homogenate of rats of all groups.
Likewise liver histopathological analysis was done to see any alteration in hepatic cells due to
exposure of hepatotoxicant and treatment with herbal combination. It was clarify from the obtained
results that this studied herbal combination have remarkable ameliorative anti-hepatotoxic
potential by restoration of increased liver enzymes, MDA, decreased antioxidants, AChE activity
and histopathological alterations in both curative and preventive mode of treatment groups.
Therefore, this herbal combination would be an excellent alternative remedy to manage and control
various liver disorders and its related complications.
137
LITERATURE CITED
Abdollahi, M., A. Ranjbar, S. Shadnia, S. Nikfar and A. Rezaiee. 2004. Pesticides and oxidative stress:
a review. Med. Sci. Rev. 10:141-147.
Abdul, R.G., M. Saqlain, M. Mobeen, Z. J. Shagufta, S. M.N. Saqlan and G. R. Kaukab. 2017.
Identification of Metabolic risk phenotypes predisposing to Non-Alcoholic Fatty Liver Disease
in a Pakistani Cohort. Pak. J. Med. Sci. 33:121-126.
Abenavoli, L., R. Capasso, N. Milic and F. Capasso. 2010. Milk thistle in liver diseases: past, present,
future. Phytother. Res. 24:1423-1432.
Adewusi, E.A and A.J. Afolayan. 2010. A review of natural products with hepatoprotective activity.
JMPR. 4:1318-1334. Adjimani, J.P and P. Asare. 2015. Antioxidant and free radical scavenging activity of iron chelators.
Toxicol. Reports. 2:721-728.
Afzal, M.Y., H.S. Anjum, U.N. Siddiqui and S. Shahid. 2016. Nonalcoholic fatty liver disease
(NAFLD) frequency in Diabetes Mellitus (DM) type–II patients. FUUAST J. Biol. 6:7-10.
Ahmad T.C., A.T.M.M. Kamal, K.A. Ahmed, A. Jahan, M.S. Hossain, A. Mamur, M. Hasan and J.
Hossain. 2015. Cytotoxic & thrombolytic activity of methanolic extract of Macaranga
denticulata Bark. The Pharma. Innov. J. 4:36-39.
Ahmed, T., Rahool, N.K. Nasir, F. Khan, Shahabudddin and M. K. Saleem. 2016. Prevalence of
Hepatitis B virus, Hepatitis C virus and HIV in blood donors of different areas of Khyber
Pukhtoonkhwa, Pakistan. J. Biodiversity Environ. Sci. 9:304-309.
Ahsan, R., K.M. Islam, Musaddik and E. Haque. 2009. Hepatoprotective Activity of Methanol Extract
of Some Medicinal Plants against Carbon Tetrachloride Induced Hepatotoxicity in Albino
Rats. Global J. Pharmacol. 3:116-122.
Akhgari, M., M. Abdollahi, A. Kebryaeezadeh, R. Hosseini and O. Sabzevari. 2003. Biochemical
evidence for free radical-induced lipid peroxidation as a mechanism for subchronic toxicity of
Malathion in blood and liver of rats. Hum. Exp. Toxicol. 4:205–211.
Ali, S.M.S., S.M. Aimen, M.Y. Faizan, A. Ghauri, R. Ejaz, H. Alshalabi, I.K. Khan and M. Umar.
2015. Hepatic Cirrhosis - Disease Burden. J. Rawalpindi Med. College.19:17-20.
Allen, A.M., W.R. Kim, J.P. Moriarty, N.D. Shah, J.J. Larson, P.S. Kamath. 2016. Time trends in the
health care burden and mortality of acute on chronic liver failure in the United States. Hepatol.
64:2165-2172.
Alves, C., C. Branco and C. Cunha. 2013. Hepatitis Delta Virus: A Peculiar Virus. Adv. Virol.
2013:1–11.
Amani, N. S., M. M. Khodeir, A.G. Nadia and E. Mohamed. 2011. Improved antifibrotic effect of a
combination of verapamil and silymarin in rat-induced liver fibrosis. Arab J.
Gastroenterol.12:143-149.
Amin, F., H.N. Bhatti and S. Rehman. 2011. Optimization of growth parameters for lipase production
by Ganoderma lucidum using response surface methodology. Afr. J. Biotechnol. 10:5514-
5523.
Amresh, G., V.A. Kumar and C.R. Venkateswara. 2018. Rao Self microemulsifying formulation of
Lagerstroemia speciosa against chemically induced hepatotoxicity. J. Traditional Complemen.
Med. 8:164-169.
Amudha and M. Komala. 2014. Evaluation of hepatoprotective activity of nanosuspension containing
Coriander sativum extracts. Inter. J. Phytopharmacol. 5:463-465.
138
Angelica, A.S., B. Torre, T. Henderson, P.N. Singh and R.K. Owusu-Apenten. 2015. A universally
calibrated microplate ferric reducing antioxidant power (FRAP) assay for foods and
applications to Manuka honey. Food Chem. 174: 119-123.
Angelis, J.C.B., D.A.N. Laise, A.R. Cristina, R.S. Kelly, D.G. Lopes, K.T.M. Rachel, J. Almeida-
Lima, R.G.C. Barros, N.S. Bezerra and H.O.R. Alexandre. 2018. In vivo evaluation of the
antioxidant activity and protective action of the seaweed Gracilaria birdiae. Oxidative Med.
Cellular Longevity. 2018:1-12.
Arshad, Z., S. Saied and S. Naz. 2017. Antioxidant Activities and Phytochemical Screening of
Martynia annua Fruit Extract. Biosci. Biotech. Res. Asia. 14:1363-1369.
Artee, G.E. 2003. Oxidants and antioxidants in alcohol-induced liver disease. Gastroenterol. 124:778-
790.
Asad, S. B., M.M. Iqbal, M. Kiranmai and M. Ibrahim. 2012. Hepatoprotective activity of Phyllanthus
amarus seeds extracts in CCl4 treated rats: In vitro & In Vivo. Global J. Medical Res. 12:39-
49.
Ashraf, S and A. Ahmad. 2015. Viral hepatitis in Pakistan: challenges and priorities. Asian Pac. J.
Trop. Biomed. 5:190-191.
Ashtari, S., M.A. Pourhoseingholi and M.R. Zali. 2015. Non-alcohol fatty liver disease in Asia:
Prevention and planning. World J. Hepatol. 7:1788-1796.
Averhoff, F.M., N. Glass and D. Holtzman. 2012. Global burden of hepatitis C: considerations for
healthcare providers in the United States. Clinic. Infect. Dis. 55:10-15.
Babbar, N., H.S. Oberoi, D.S. Uppal and R.T. Patil. 2011. Total phenolic content and antioxidant
capacity of extracts obtained from six important fruit residues. Food Res. Inter. 44:391-396.
Babu, H.B., L.S Mohana and A.K. Saravana. 2010. Studies on phytochemical and anticonvulsant
property of Martynia annua Linn. Int. J. Phytopharmacol. 1:82-86.
Badhani, B., N. Sharma and R. Kakkar. 2015. Gallic acid: a versatile antioxidant with promising therapeutic
and industrial applications. R.SC Adv. 5:27540-27557.
Barbosa, A.M., E.C. Giese, R.F.H. Dekker, D. Borsato and A.I.B. Perez. 2010. Extracellular β-
glucosidase production by the yeast Debaryomyces pseudopolymorphus UCLM-NS7A:
optimization using response surface methodology. New Biotechnol. 27:374-381.
Bashir, S., R. Memon and A.H. Gilani. 2011. Antispasmodic and Antidiarrheal Activities of Valeriana
hardwickii Wall. Rhizome Are Putatively Mediated through Calcium Channel Blockade. Evid.
Based. Comple. Alternat. Med. 2011:1-6.
Belwal. T., P. Dhyani, I.D. Bhatt, R.S. Rawal and V. Pande. 2016. Optimization extraction conditions
for improving phenolic content and antioxidant activity in Berberis asiatica fruits using
response surface methodology (RSM). Food Chem. 207:115-124.
Belyhun, Y., M. Maier, A. Mulu, E. Diro and U. L. Gerd. 2016. Hepatitis viruses in Ethiopia: a
systematic review and meta-analysis. BMC Infect. Dis. 16:761.
Benzie, I.F and S.W. Choi. 2014. Antioxidants in food: Content, measurement, significance, action,
cautions, caveats, and research needs. Adv. Food Nutrition Res. 71:1–53.
Bera, T.k., K. Chatterjee, K. Jana, K.M. Ali, D. De and S. Maiti. 2012. Antihepatotoxic effect of
“Livshis,” a polyherbal formulation against carbon tetrachloride-induced hepatotoxicity in
male albino rat. J. Nat. Pharm. 3:17-24.
Bhurgri, Y., S. Pervez, N. Kayani, A. Bhurgri, A. Usman and I. Bashir. 2006. Cancer profile of
Larkana, Pakistan (2000-2002). Asian Pac. J. Cancer Prev. 7:518-521.
139
Birben, E., U.M. Sahiner, C. Sackesen, S, Erzurum and O. Kalayci. 2012. Oxidative stress and
antioxidant defense. World Allergy Organ. J. 5:9-19.
Bisceglie, D.A. M. Natural history of hepatitis C: its impact on clinical management. Hepatol.
31:1014-1018.
Bosch, F.X., J. Ribes, M. Diaz and R. Cleries.2004. Primary liver cancer: worldwide incidence and
trends. Gastroenterol. 127:5-16.
Brenelli, L.P., R. Goldbeck, W.S. Dantas and F.S. Marcio. 2013. Ferulic acid and derivatives:
molecules with potential application in the pharmaceutical field. Brazilian J. Pharmaceut. Sci.
49:396-411.
Bruix, J and M. Colombo. 2014. Hepatocellular carcinoma: current state of the art in diagnosis and
treatment. Best Pract. Res. Clin. Gastroenterol. 28:751.
Butt, A.S., Z. Abbas and W. Jafri. 2012. Hepatocellular carcinoma in Pakistan: where do we stand?
Hepat. Mon. 12:6023.
Caro, A.A and A.I. Cederbaum. 2004. Oxidative stress, toxicology, and pharmacology of CYP2E1.
Annu. Rev. Pharmacol. Toxicol. 44:27-42.
Catassi, A., D. Servent, L. Paleari, A. Cesario and P. Russo. 2008. Multiple roles of nicotine on cell
proliferation and inhibition of apoptosis: implications on lung carcinogenesis. Mutat. Res.
659:221-231.
Challa S. and F.K. Chan. 2010. Going up in flames: necrotic cell injury and inflammatory diseases.
Cell. Mol. Life Sci. 67:3241-53.
Chan, F.K., K. Moriwaki and M. D.R. Jose. 2013. Detection of Necrosis by Release of Lactate
Dehydrogenase (LDH) Activity. Methods Mol. Biol. 979:65-70.
Chandra, B. J., A. Prakashb, N. Ajudhia and Kaliaa. 2015. Hepatoprotective potential of antioxidant
potent fraction from Urtica dioica Linn. (Whole plant) in CCl4 challenged rats. Toxicol.
Reports. 2:1101–1110.
Chang, Y., C.Y. Chang, S.J. Wang, S.K. Huang. 2015. Myricetin inhibits the release of glutamate in
rat cerebrocortical nerve terminals. J. Med. Food. 18:516-523.
Charles, D. 2008. Thrombosis and Anticoagulation in Liver Disease. Hepatol. 47:1384-1393.
Chen, Q., P. Li, P. Li, Y. Xu, Y. Li and B. Tang. 2015. Isoquercitrin inhibits the progression of
pancreatic cancer in vivo and in vitro by regulating opioid receptors and the mitogen activated
protein kinase signaling pathway. Oncol. Rep. 33:840-848.
Choi, J., X. An, B.L. Hang, J.L. Suk, H.H. Jin, T. Kim, J. Ahn and D. Kim. 2015. Protective Effects
of Bioactive Phenolics from Jujube (Ziziphus jujuba) Seeds against H2O2–induced Oxidative
Stress in Neuronal PC-12 Cells. Food Sci. Biotechnol. 24: 2219-2227.
Chowdhury, M.R., M.A. Sagor, N. Tabassum, M.A. Potol, H. Hossain, M.A. Alam. 2015.
Supplementation of Citrus maxima peel powder prevented oxidative stress, fibrosis, and
hepatic damage in carbon tetrachloride (CCl4) treated rats. Evid. Based Complement Alternat.
Med. 2015:1-10.
Chuang, S.C., L. C. Vecchia and P. Boffetta. 2009. Liver cancer: descriptive epidemiology and risk
factors other than HBV and HCV infection. Cancer Lett. 286:9-14.
Cichoż-Lach, H and A. Michalak. 2014. Oxidative stress as a crucial factor in liver diseases. World J.
Gastroenterol. 20:8082-8091.
Colin, H., B. Amanda, M. Clarka, S.D. Wheelera, L.T. Donna, B. Stolzcd, L. Griffithg and A.
Wellsabd. 2018. Liver organ on a chip. Expt. Cell Res. 363: 15-25.
140
Cotos, C., M. Rosario, M. H. Yahya and I. Hadi. 2017. Antimicrobial, Antioxidant, Hemolytic, Anti-
anxiety, and Antihypertensive activity of Passiflora species. Research J. Pharm. and Tech.
10:4079-4084.
Cui, Y., Y. Cheng, Y. Guo, Y. Xie, W. Yao, W. Zhang and H. Qian. 2017. Evaluating the
hepatoprotective efficacy of Aloe vera polysaccharides against subchronic exposure of
aflatoxins. J. Taiwan Inst. Chem. Eng.76:10–17.
Dai, J and R. Mumper. 2010. Plant phenolics: extraction, analysis and their antioxidant and anticancer
properties. Molecules. 15:7313-7352.
Dambach, D.M., B.A. Andrews and F. Moulin. 2005. New technologies and screening strategies for
hepatotoxicity: use of in vitro models. Toxicol. Pathol. 33:17-26.
Davaatseren, M., H.H. Jeon, H.Y. Jeong, J.T. Hwang, J. P. Ho, H.J. Kim, M.K. Jung, D.K. Young and
M.S. Jeong. 2013. Taraxacum official (dandelion) leaf extract alleviates high-fat diet-induced
nonalcoholic fatty liver. Food Chem. Toxicol. 58:30-36.
De, M.V.F., R.C. Netto, J.C. Da, T.M.S. De, J.O. Costa, C.B. Firmino and N. Penha-Silva. 2008.
Influence of aqueous crude extracts of medicinal plants on the osmotic stability of human
erythrocytes. Toxicol. In Vitro. 22:219-24.
Dehpour, A.A., M.A. Ebrahimzadeh, S.F. Nabavi and S.M. Nabavi. 2009. Antioxidant activity of
methanol extract of Ferula assafoetida and its essential oil composition. Grasas. Aceites.
60:405-412.
Del P. A., A. Scalera, M.D. Iadevaia, A. Miranda, C. Zulli, L. Gaeta, C. Tuccillo, A. Federico and C.
Loguercio. 2012. Herbal products: Benefits, limits, and applications in chronic liver disease.
Evid. Based Complemen. Alter. Med. 2012: 837-856.
Delgado, D.A., A.S. Sant’ana, D. Granato and P.R. Massaguer. 2012. Inactivation of Neosartorya
fischeri and Paecilomyces variotii on paperboard packaging material by hydrogen peroxide
and heat. Food Control. 23:165-170.
Demiray, S., M.E. Pintado and P.M.L. Castro. 2009. Evaluation of phenolic profiles and antioxidant
activities of Turkish medicinal plants: Tilia argentea, Crataegi folium leaves and Polygonum
bistorta roots. Inter. J. Med. Health Biomed. Bioengine. Pharmaceu. Engg. 54:312-317.
Dey, A and J. Lakshmanan. 2013. The role of antioxidants and other agents in alleviating
hyperglycemia mediated oxidative stress and injury in liver. Food Funct. 4:1148-1184.
Dey, P., S. Dutta, A. Biswas-Raha and T.C. Kumar. 2016. Haloalkane induced hepatic insult in murine
model: Amelioration by Oleander through antioxidant and anti-inflammatory activities, an in
vitro and in vivo study. BMC. Complementary and Alternative Medicine. 6:1260- 86.
Dhiman, A., A. Nanda and S. Ahmad. 2012. A recent update in research on the anti-hepatotoxic
potential of medicinal plants. Chin. J. Integr. Med. 10:117-127.
Dhruti, N., S. Ajay and N. Chirag. 2009. Antioxidant activities of methanolic and aqueous extracts
from leaves of Martynia annua Linn. Phcog. J. 1:291-96.
Domitrovi´c, R., H. Jakovac, Z. Romic, D. Rahelic and Z. Tadic. 2010. Antifibrotic activity of
Taraxacum officinale root in carbon tetrachloride-induced liver damage in mice.J.
Ethnopharmacol.130: 569–577.
Doughari, J.H., A.M.E. Mahmood and S. Manzara. 2007. Studies on the antibacterial activity of root
extracts of Carica papaya L. Afri. AJMR. 1:037-041.
Doycheva, I., J. Cui and P. Nguyen. 2016. Non-invasive screening of diabetics in primary care for
NAFLD and advanced fibrosis by MRI and MRE. Aliment Pharmacol. Ther. 43:83-95.
Eidi, A., P. Mortazavi, M. Bazargan and J. Zaringhalam. 2012. Hepatoprotective activity of Cinnamon
ethanolic extract against ccl4-induced liver injury in rats. Excli J. 11:495-507.
141
Ellendersen, L.S.N., D. Granato, K.B. Guergoletto, and G. Wosiacki. 2012. Development and sensory
profile of a probiotic beverage from apple fermented with Lactobacillus casei. Eng. Life Sci.
12:475-485.
Ellman, G.L., K. D. Courtney, V. Andres and R. M. Featherstone. 1961. A new and rapid colorimetric
determination of acetylcholinesterase activity. Biochem. Pharmacol. 7:88-95.
El-Serag, H.B and K.L. Rudolph. Hepatocellular carcinoma: epidemiology and molecular
carcinogenesis. Gastroenterol. 132:2557-2576.
Elzaawely, A.A., T.D. Xuanand S. Tawata. 2007. Essential oils, kava pyrones and phenolic
compounds from leaves and rhizomess of Alpinia zerumbet and their antioxidant activity. Food
Chem. 103:486-494.
Eman, F. A., A.E. Waill, A.A. Hanan, E.R. Mostafa and W. Fayad. 2017. Biological capacity and
chemical composition of secondary metabolites from representatives Japanese Lichens. J.
Applied Pharma. Sci. 7:098-103.
Estrada, M.J., C.V. Contreras and A.G. Escobar. 2013. In vitro antioxidant and anti-proliferative
activities of plants of the ethno pharmacopeia from northwest of Mexico. BMC. Complemen.
Alter. Med. 2013:12.
Ezz El- Din H.Y, Abeer A. K. Mohamed and Z. Mahran. 2011. Hepatoprotective activity and
antioxidant effects of El Nabka (Zizyphus spina-christi) fruits on rats hepatotoxicity induced
by carbon tetrachloride. Nat. and Sci. 9:1-7.
Faiza, A., Mahboub and M.A Samah. 2015. Hepatoprotective effect of Ocimum basilicum extract
against the toxicity of diazinon in albino rats: Histopathological and immune histochemical
evaluation. World J. Pharm. Sci. 3:790-799.
Fan, J.G., C. Geoffrey and Farrell. 2009. Epidemiology of non-alcoholic fatty liver disease in China.
J. Heptol. 50:204-210.
Farkas, D and S.R. Tannenbaum. 2005. In vitro methods to study chemically-induced hepatotoxicity:
a literature review. Curr. Drug Metab. 6:111–125.
Farrell, G.C., V.W. Wong and S. Chitturi. 2013. NAFLD in Asia—as common and important as in the
West. Nature Reviews Gastroenterol. Hepatol. 10:307-318.
Feng, Y., Y.H. Yu, S.T. Wang, J. Ren, D. Camer, Y.Z. Hua, Q. Zhang, J. Huang, D. L. Xue, X. F.
Zhang, X.F. Huang and Y. Liu. 2016. Chlorogenic acid protects d-galactose-induced liver and
kidney injury via antioxidation and antiinflammation effects in mice. Pharmaceutic. Bio.
54:1027–1034.
Fernandez, C.J.C. and N. Kaplowitz. 2005. Hepatic mitochondrial glutathione: transport and role in
disease and toxicity. Toxicol. Appl. Pharmacol. 204:263-73.
Ferreira, C., Proenc, M.L.M. Serralheiro and M.E.M. Ara´ujo. 2006. The in vitro screening for
acetylcholinesterase inhibition and antioxidant activity of medicinal plants from Portugal. J.
Ethnopharmacol. 108:31-37.
Fontana, R.J., C. Ellerbe, V.E. Durkalski, A. Rangnekar, R.K. Reddy, T. Stravitz, B. McGuire, T.
Davern, A. Reuben, I. Liou, O. Fix, D.R. Ganger, R.T. Chung, M. Schilsky, S. Han, L.S.
Hynan, C. Sanders, W.M. Lee. 2015. Acute Liver Failure Study Group. Two-year outcomes in
initial survivors with acute liver failure: results from a prospective, multicentre study. Liver
Int. 35:370-380.
Forner, A., J.M. Llovet and J. Bruix. 2012. Hepatocellular carcinoma. Lancet. 379:1245-1255.
Fried, M.W., M.L. Shiffman, K.R. Reddy, C. Smith., G. Marinos, F.L. Gonçales, D. Häussinger, M.
Diago, G. Carosi, D. Dhumeaux, A. Craxi, A. Lin, J. Hoffman and J. Yu. 2002. Peginterferon
alfa-2a plus ribavirin for chronic hepatitis C virus infection. N. Engl. J. Med. 347:975-982.
142
Gevrenova, R., M. Kondeva-Burdina, N. Denkov and D. Zheleva-Dimitrova. 2015. Flavonoid
profiles of three Bupleurum species and in vitro hepatoprotective activity of Bupleurum
flavum Forsk. Pharmacognosy, magazine. 11:14-23
Ghasemzadeh, A and N. Ghasemzadeh. 2011. Flavonoids and phenolic acids: Role and biochemical
activity in plants and human. J. Med. Plants Res. 5:6697-6703.
Giesea, E.C., J. Gascona, G. Anzelmo, A.M. Barbosaa, M.A. Alves and R.F.H. Dekker. 2015. A Free-
radical scavenging properties and antioxidant activities of botryosphaeran and some other -D-
glucans. Inter J. Biol Macromolecules. 72:125-130.
Gildea, T.R., W.C. Cook, D.R. Nelson and A. Aggarwal. 2004. Predictors of long-term mortality in
patients with cirrhosis of the liver. Am. Col. Chest Physicians. 126:1598-1603.
Gish, R.G., C. Porta, L. Lazar, P. Ruff, R. Feld and A. Croitoru. 2007. Phase III randomized controlled
trial comparing the survival of patients with unrespectable hepatocellular carcinoma treated
with nolatrexed or doxorubicin. J. Clin. Oncol. 25:3069-3075.
Govindarajan, R., M. Vijayakumar and P. Pushpangadan. 2005. Antioxidant approach to disease
management and the role of ‘Rasayana’ herbs of ayurveda. J. Ethnopharm. 99:165-178.
Goyal, R., B. Ravishankar, V.J. Shukla and M. Singh. 2012. Hepatoprotective activity of Rohitaka
ghirita against paracetamol induce liver injury in rat. Pharmacologia. 3: 227-232.
Granato, D., J.C.B. Ribeiro, I.A. Castro and M.L. Masson. 2010. Sensory evaluation and
physicochemical optimisation of soy-based desserts using response surface methodology.
Food Chem. 121:899-906.
Grattagliano, I., C.V. Diogo, M. Mastrodonato, O. deBari, M. Persichella, D.Q. Wang, A. Liquori, D.
Ferri, M.R. Carratu, P.J. Oliveira, P.A. Portincasa. 2013. Silybin–phospholipids complex
counteracts rat fatty liver degeneration and mitochondrial oxidative changes. World J.
Gastroenterol. 19:3007-3017.
Grenier, D., H. Chen, A.B. Lagha, J. Fournier-Larente and M.P. Morin. 2015. Dual action of myricetin
on Porphyromonas gingivalis and the inflammatory response of host cells: A promising
therapeutic molecule for periodontal diseases. PLoS ONE. 10:1-14.
Gu, F., F. Xu, L. Tan, H. Wu, Z. Chu and Q. Wang. 2012. Optimization of enzymatic process for
vanillin extraction using response surface methodology. Molecules. 17:8753–8761.
Gulcin, I. 2012. Antioxidant activity of food constituents: An overview. Archives Toxicol. 86:345-
391.
Gupta, A., R.S. Navin, S. Pandey, D.R. Shah and J.S. Yadav. 2013. Design and evaluation of herbal
hepatoprotective formulation against paracetamol induced liver toxicity. J. Young
Pharmacists. 5:180-187.
Gurib-Fakim, F. 2006. Medicinal plants: Traditions of yesterday and drugs of tomorrow. Molecular
Aspect. Med. 27:1-93.
Haley, R.W and R.P. Fischer. 2001. Commercial tattooing as a potentially important source of
hepatitis C infection. Medicine. 80:134-151.
Han, H., W. Zeng, C. He, S. W.A. Bligh, Q. Liu, L. Yanga and Z. Wanga. 2014. Characterization of
metabolites of sweroside in rat urine using ultra-high-performance liquid chromatography
combined with electrospray ionization quadrupole time-of-flight tandem mass spectrometry
and NMR spectroscopy. J. Mass Spectrom. 49:1108-1116.
Hannah, J.W.N and S.A. Harrison. 2016. Lifestyle and Dietary Interventions in the management of
Nonalcoholic Fatty Liver Disease. Dig. Dis. Sci. 61:1365-1374.
Harish, R and T. Shivanandappa. 2006. Antioxidant Activity and Hepatoprotective Potential of
Phyllanthus Niruri. Food Chem. 95:180-185.
143
Harsha, S.N and B.V. Latha. 2011. In vitro antioxidant and in vitro anti-inflammatory activity of Ruta
graveolens methanol extract. Asian J. Pharm. Clin. Res. 5:32-35.
Hartmut, J., R. Mitchell, McGill and R. Anup. 2012. Oxidant stress, mitochondria and cell death
mechanism in drug- induced liver injury: Lessons learned from acetaminophen hepatotoxicity.
Drug Metabol. Rev. 44:88-106.
Hawke, R.L., S.J. Schrieber, T.A. Soule, Z. Wen, P.C. Smith, K.R. Reddy, A.S. Wahed, S.H. Belle,
N.H. Afdhal, V.J. Navarro, J. Berman, Q.Y. Liu, E. Doo and M.W. Fried. 2010. Silymarin
ascending multiple oral dosing phase I study in non-cirrhotic patients with chronic hepatitis C.
J. Clin. Pharmacol. 50:434-449.
Hayashi, N., T. Okanoue, H. Tsubouchi, J. Toyota, K. Chayama and H. Kumada. 2012. Efficacy and
safety of telaprevir, a new protease inhibitor, for difficultto- treat patients with genotype 1
chronic hepatitis C. J. Viral Hepat. 19:134-142.
Hazem, M and M. Hassan. 2012. Hepatoprotective Effect of Red Grape Seed Extracts Against
Ethanol-Induced Cytotoxicity. Global J. Biotech. Biochem. 7:30-37.
He, Y.M., S. Zhu, Y.W. Ge, K. Kazuma, K. Zou and S.Q. Cai. 2015. The anti-inflammatory
secoiridoid glycosides from Gentianae Scabrae Radix: the root and rhizome of Gentiana
scabra. J Nat. Med. 69:303-312.
Heidelbaugh, J.J., M. Bruderly. 2006. Cirrhosis and chronic liver failure. Part I: Diagnosis and
evaluation. Am Fam Physician. 74:756-762.
Heitman, E and D.K. Ingram. 2014. Cognitive and neuroprotective effects of Chlorogenic acid. Nutr.
Neurosci. 20:32-39.
Herraiz, T and J. Galisteo. 2015. Hydroxyl radical reactions and the radical scavenging activity of b-
carboline alkaloids. Food Chem. 172:640-649.
Hezode, C., N. Forestier and G. Dusheiko. 2009. Telaprevir and peginterferon with or without ribavirin
for chronic HCV infection. N. Engl. J. Med. 360:839-1850.
Hill, A and G. Cooke. 2014. Hepatitis C can be cured globally, but at what cost? Sci. 345:141-142.
Hina, S., K. Rehman, M. Shahid and N. Jahan. 2017. In vitro antioxidant, hepatoprotective potential
and chemical profiling of Syzygium aromaticum using HPLC and GC‐MS. Pak. J. Pharm. Sci.
30:1031-1039.
Hong, M., S. Li, H.T. Yue, N. Wang, S. Tsao and Y. Feng. 2015. Current status of herbal medicines
in chronic liver disease therapy: the biological effects, molecular targets and future prospects.
Int. J. Mol. Sci. 16:28705-28745.
Horiuchi, Y., R. Kimura, N. Kato, T. Fujii, M. Seki, T. Endo, T. Kato and K. Kawashima. 2003.
Evolutional study on acetylcholine expression. Life Sci. 72:1745-1756.
Hosamani, K.M., R.M. Sattigeri and K.B. Patil. 2002. Studies on chemical compounds of Martynia
annua syn. M. diandra seed oil. J. Med. Aromatic Plant Sci. 24:12.
Hu, N., S. Ren, W. Li, T. Zhang and C. Zhao. 2013. Morroniside promotes bone marrow mesenchymal
stem cell proliferation in rats. Mol. Med. Rep. 7:1565-1570.
Hu, R and A.N. Kong. 2004. Activation of MAP kinases, apoptosis and nutrigenomics of gene
expression elicited by dietary cancer-prevention compounds. Nutrition 20:83-88.
Hu, S., L. Huang, L. Meng, H. Sun, W. Zhang and Y. Xu. 2015. Isorhamnetin inhibits cell proliferation
and induces apoptosis in breast cancer via Akt and mitogen-activated protein kinase kinase
signaling pathways. Molecular Med. Rep.12:6745-6751.
Huang, B., X. Ban, J. He, J. Tong, J. Tian and Y. Wang. 2010. Hepatoprotective and antioxidant
activity of ethanolic extracts of edible lotus (Nelumbo Nucifera Gaertn.) Leaves. Food Chem.
120:873-878.
144
Huang, G., B. Tang, K. Tang, X. Dong, J. Deng, L. Liao, Z. Liao, H. Yang and S. He. 2014.
Isoquercitrin inhibits the progression of liver cancer in vivo and in vitro via the MAPK
signalling pathway. Oncol. Rep. 31:2377-2384.
Hutchison, M.J.G., T. Poynard, S. Pianko, S.C. Gordon, A.E. Reid, J. Dienstag and T. Morgan. The
impact of interferon plus ribavirin on response to therapy in black patients with chronic
hepatitis C. The International Hepatitis Interventional Therapy Group. Gastroenterol.
119:1317-1323.
Hwang, E.W and R. Cheung. 2011. Global epidemiology of hepatitis B virus (HBV) infection. N Am.
J. Med. Sci. 4:7-13.
Inge, M.W., O. Dorenda, M.M. Geny, Groothuis and O. Peter. 2014. Precision-cut liver slices as a
model for the early onset of liver fibrosis to test anti-fibrotic drugs. Toxicol. Appl. Pharm.
274:328-338.
Ishibashi, H., M. Nakamura, A. Komori, K. Migita and S. Shimoda. 2009. Liver architecture, cell
function, and disease. Springer Semin. Immunopathol. 31:399-409.
Jacobsen, K.H and S.T. Wiersma. 2010. Hepatitis A virus seroprevalence by age and world region,
1990 and 2005. Vaccine. 28:6653-6657.
Jaeschke, H., R. Mitchell, M. Gill and A. Ramachandran. 2012. Oxidant stress, mitochondria, and
cell death mechanisms in drug-induced liver injury: Lessons learned from acetaminophen
hepatotoxicity. Drug Metabol. Rev. 44:88-106.
Jaishree, V and S. Badami. 2010. Antioxidant and hepatoprotective effect of swertiamarin from
Enicostemmaaxillare against d-galactosamine induced acute liver damage in rats. J.
Ethnopharmacol.130:103-106.
Jalan, R., C. Yurdaydin, J.S. Bajaj, S.K. Acharya, V. Arroyo and H.C. Lin. 2014. Toward an improved
definition of acute-on-chronic liver failure. Gastroenterol. 147:4-10.
Jayant, S.L., P.A. Thomas, Devasagayam, L.Y. Foo, P. Shastry and S.S. Ghaskadbi. 2012. Geraniin
and amariin, ellagitannins from Phyllanthus amarus, protect liver cells against ethanol induced
cytotoxicity. Fitoterapia. 83:1562-1568.
Jeong, Y.T., S.C. Jeong, J.S. Hwang and J.H. Kim. 2015. Modulation effects of sweroside isolated
from the Lonicera japonica on melanin synthesis. Chem. Biol. Interact. 238:33-39.
Jevas, O.C. 2017. Physiology of the liver. Int. J. Res. Pharma. Bio. Sci. 4:13-24.
Ji, L., P. Jiang, B. Lu, Y. Sheng, X. Wang and Z. Wang Z. 2013. Chlorogenic acid, a dietary
polyphenol, protects acetaminophen-induced liver injury and its mechanism. J Nutr Biochem.
24:1911- 9.
Jiao, Y., Y. Zuo. 2009. Ultrasonic extraction and HPLC determination of anthraquinones, aloe-
emodine, emodine, rheine, chrysophanol and physcione, in roots of Polygoni multiflori.
Phytochem. Anal. 20:272-278.
John, J.A and F. Shahidi. 2010. Phenolic compounds and antioxidant activity of Brazil nut
(Bertholletia excelsa). J. Func. Foods. 2:196-209.
Johnson, G and S.W. Moore. 2004. Identification of a structural site on acetylcholinesterase that
promotes neurite outgrowth and binds laminin-1 and collagen IV. Biochem. Biophys. Res.
Commun. 319:448-455.
Jullian, C., S. Miranda, G. Zapata-Torres, F. Mendizabal and C. Olea-Azar. 2007. Studies of inclusion
complexes of natural and modified cyclodextrin with (+) catechin by NMR and molecular
modeling. Bio. org. Med. Chem. 15:3217-3224.
145
Kabiri, N., M. Ahangar-Darabi, M. Setorki and M. Rafieian- Kopaei. 2013. The effect of silymarin on
liver injury induced by thioacetamide in rats. J. Herb.med. Pharmacol. 2:29-33.
Kagami, K., K. Onda, K. Oka and T. Hirano. 2008. Suppression of blood lipid concentrations by
volatile Maillard reaction products. Nutrition. 24:1159-1166.
Kakkar, S and S. Bais. 2014. A Review on Protocatechuic Acid and Its Pharmacological Potential.
ISRN Pharmacol. 2014:1-10.
Kalagatur, N.K., R.K. Jalarama, C. Siddaiah, V.K. Gupta, K. Krishna and V. Mudili. 2018.
Combinational Inhibitory Action of Hedychium spicatum L. Essential Oil and g-Radiation on
Growth Rate and Mycotoxins Content of Fusarium graminearum in Maize: Response Surface
Methodology. Front Microbiol. 9:1-15.
Kannampalli, P., V.R. Chandrasekaran., G. Kuppannan and K. Sivanesan. 2007. Effect of Cassia
fistula Linn. leaf extract on diethylnitrosamine induced hepatic injury in rats. Chem. Biol.
Interact. 167:12-18. Kanwal, S and B. S. Ali. 2017. Herbal medicine: Trend of practice, perspective, and limitations in
Pakistan. Asian Pacific J. Health Sci. 4:6-8.
Kasture, S.V., S.A. Gosavi, J.B. Kolpe and S.G. Deshapande. 2014. Phytochemical and biological
evaluation of Fagonia species: a review. World J. pharmacy and pharma. Sci. 3: 1206-1217.
Katrin, S., Dietmar, R. Kammerer, Reinhold, C and Andreas, S. 2005. Characterization of phenolic
acids and flavonoids indandelion (Taraxacum officinale WEB. ex WIGG.) root and herb by
high-performance liquid chromatography/electrospray ionization mass spectrometry. Rapid
Commun. Mass Spectrom. 19:179-186.
Kaurinovic, B., M. Popovic, S. Vlaisavljevic and S. Trivic. 2011. Antioxidant Capacity of Ocimum
basilicum L. and Origanum vulgare L. Extracts. Molecules. 16:7401-7414.
Kawashima, K and T. Fujii. 2000. Extraneuronal cholinergic system in lymphocytes. Pharmacol. Ther.
86:29-48.
Kenwat, P. 2013. Martynia annua. UK J. Pharmaceut. Biosci. 1:7-10.
Kenwat, R., P. Prasad, T. Satapathy and A. Roy. Martynia annua: An Overview .UK J. Pharma.
Biosci. 1:7-10.
Khare, C.P. 2007. Indian Medicinal Plants An illustrated Dictionary. Springer publica.. 2007: 399-
400.
Khurelbat, D., M. Purevkhuu, B. Luvsansharav, S. Bandi, D. Tseveen, T. Sanjjav, E. Dorjbal and A.
Miegombo. 2014. The hepatoprotective activity of the herbal preparation Salivin against
carbon tetrachloride (Ccl4) induced hepatotoxicity in rabbits. Curr. Issues Pharm. Med. Sci.
27:263-266.
Kim, H.S. 2002. Effects of the Zizyphus jujuba seed extract on the lipid components in hyperlipidemic
rats. J. Food Sci. Nutr. 7:72-77.
Kim, H.Y., J. Park, K.H. Lee, D.U. Lee, J.H. Kwak, Y. S. Kim, S.M. Lee. 2011. Ferulic acid protects
against carbon tetrachloride-induced liver injury in mice. Toxicol. 282: 104-111.
Kim, J., K. Nelson and U.A. Panzner.2014. A systematic review of the epidemiology of hepatitis E
virus in Africa. BMC Infect. Dis. 14:308.
Kim, J., K. Noh, M. Cho, J. Jang and Y. Song. 2007. Anti-oxidative, anti-inflammatory and anti-
atherogenic effects of dandelion (Taraxacum officinale) extracts in C57BL/6 mice fed
atherogenic diet. FASEB J. 21:862-867.
146
Koh, Y.J., D.S. Cha, J.S. Ko, H.J. Park and H.D. Choi. 2010. Anti-inflammatory effect of Taraxacum
officinale leaves on lipopolysaccharide-induced inflammatory responses in RAW 264.7 cells.
J. Med. Food. 13:870-878.
Kopec, K., N. Joshi and J.P. Luyendyk. 2016. Role of hemostatic factors in hepatic injury and disease:
animal models de-liver. J. Thromb. Haemost. 14:1337-1349.
Kren, V and D. Walterova. 2005. Silybin and silymarin – new effects and applications. Biomed. Pap.
149:29-41.
Kumada, H., J. Toyota, T. Okanoue, K. Chayama, H. Tsubouchi and N. Hayashi. 2012. Telaprevir
with peginterferon and ribavirin for treatment-naive patients chronically infected with HCV of
genotype 1 in Japan. J. Hepatol. 56:78–84.
Kumar, A. 2012. A review on hepatoprotective herbal drugs. Int. J. Res. Pharmacy. Chem. 2:92-102.
Kumar, H.C., A. Ramesh, J.N.S Kumar and B.M. Ishaq. 2011. A review on hepatoprotective activity
of medicinal plants. Inter. J. Pharma. Sci. Res. 2:501-515.
Kumar, R.S and L. Seasotiya. 2011. Free radical scavenging activity, phenolic contents and
phytochemical evaluation of different extracts of stem bark of Butea monosperma (Lam.)
Kuntze. Frontiers Life Sci. 5:107–116.
Kumar, S and V. Jairaj. 2018. An Effective Method for Isolation of Pure Swertiamarin from
Enicostemma littorale Blume. Indo. Global J. Pharmaceut. Sci. 8:1-8.
Kumar, S.R.A., R.J. Hariprasanth, M.P. Siddharth, M. Gobinath and C. Rajukutty. 2016. Evaluation
of the Antioxidant, Antimicrobial, Antidiabetic and Hemolytic Activity of Organically Grown
Solanum nigrum and Solanum xanthocarpum. Int. J. Curr. Pharma. Rev. Res. 7:296-299.
Kumar, U.A., C. Manjunath, T. Thaminzhmani, Y. R. Kiran and Y. Brahmaiah. 2012. A review on
immunomodulatory activity plants. Indian J. Novel Drug Delivery. 4:93-103.
Kumar, V., T. Sanjeev, S. Ajay, S.P. Kumar and S. Anil. 2012. A Review on Hepatoprotective Activity
of Medicinal Plants. Int. J. Adv. Res. Pharma. Biosci. 2:31-38.
Kuo, C.H., F.W. Hsiao, J.H. Chen, C.W. Hsieh, Y.C. Liu and C.J. Shieh. 2013. Kinetic aspects of
ultrasound-accelerated lipase catalyzed acetylation and optimal synthesis of 4’-
acetoxyresveratrol. Ultrason. Sonochem. 20:546-552.
Kwon, S.H., J.A. Kim, S.I. Hong, Y.H. Jung, H.C. Kim and S.Y. Lee. 2011 Loganin protects against
hydrogen peroxide-induced apoptosis by inhibiting phosphorylation of JNK, p38, and ERK
1/2 MAPKs in SH-SY5Y cells. Neurochem Int. 58:533-541.
Lallianrawna, S., R. Muthukumaran, V. Ralte, G. Gurusubramanian and N.S. Kumar. 2013.
Determination of total phenolic content, total flavonoid content and total antioxidant capacity
of Ageratina adenophora (Spreng.) King & H. Rob. Sci. Vis. 13:149-156.
Lazo, M., R. Hernaez and M.S. Eberhardt. 2013. Prevalence of non-alcoholic fatty liver disease in
the United States: the Third National Health and Nutrition Examination Survey, 1988–1994.
Am. J. Epidemiol. 178:38-45.
Lee, J.H., H.J. Lee and H.J. Lee. 2009. Rhus verniciflua Stokes prevents cisplatin-induced cytotoxicity
and reactive oxygen species production in MDCK-I renal cells and intact mice. Phytomedicine.
16:188-197.
Lee, J.I., B.H. Hsu, D. Wu and J.S. Barrett. 2006. Separation and characterization of silybin, isosilybin,
silydianin and silychristin in milk thistle extract by liquid chromatography–electrospray
tandem mass spectrometry. J. Chromatogr. A. 1116:57-68.
Lee, W.M. 2003. Drug-induced hepatotoxicity. N Engl. J. Med. 349:474-485.
Lefton, H., A. Rosa and M. Cohen. 2009. Diagnosis and Epidemiology of Cirrhosis. Med. Clini. North
America. 93:787-799.
147
Lemaire, B., M. Beck, M. Jaspart, C. Debier, P.B. Calderon, J.P. Thomé and J.F. Rees. 2011.
Precision-cut liver slices of Salmo salar as a tool to investigate the oxidative impact of CYP1A-
mediated PCB 126 and 3-methylcholanthrene metabolism. Toxicol. In Vitro. 25:335–342.
Li, S., N. Wang, M. Hong, H.Y. Tan, G. Pan and Y. Feng. 2018. Hepatoprotective Effects of a
Functional Formula of Three Chinese Medicinal Herbs: Experimental Evidence and Network
Pharmacology-Based Identification of Mechanism of Action and Potential Bioactive
Components. Molecules. 23:352-369.
Lim, P.L., W. Tan. C. Latchoumycandane, W.C. Mok, Y.M. Khoo and H.S. Lee. 2007. Molecular and
functional characterization of drug-metabolizing enzymes and transporter expression in the
novel spontaneously immortalized human hepatocyte line HC-04. Toxicol. In Vitro. 21:1390-
1401.
Lin, S.M., M.L. Yu and C.M. Lee. 2007. Interferon therapy in HBeAg positive chronic hepatitis
reduces progression to cirrhosis and hepatocellular carcinoma. J. Hepatol. 46:45-52.
Lisman, T., J. Robert and M.D. Porte. 2017. Pathogenesis, prevention, and anagement of bleeding and
thrombosis in patients with liver diseases. Res. Pract. Thromb. Haemost. 1:150-161.
Lisman, T., S.H. Caldwell, A.K. Burroughs, P.G. Northup, M. Senzolo, R.T. Stravitz, A. Tripodi, J.F
Trotter, D.C. Valla and R.J. Porte. 2010. Coagulation in liver disease study group hemostasis
and thrombosis in patients with liver disease: the ups and downs. J. Hepatol. 53:362-371.
Liu C, J. Yu, J. Wang, Z. Liu and Q. Wang. 2012. Application of response surface methodology to
optimise supercritical carbon dioxide extraction of oil from rapeseed (Brassica napus L.). Int.
J. Food Sci. Technol. 47:1115-1121.
Liu, A., X. Zhao, H. Li, Z. Liu, B. Liu, X. Mao, L. Guo, K. Bi and Y. Jia. 2014. 5-
Hydroxymethylfurfural, an antioxidant agent from Alpinia oxyphylla Miq. improves cognitive
impairment in Aβ1–42mouse model of Alzheimer's disease. Inter. Immunopharmacol. 23:719-
725.
Liu, C.L., J.M. Wang, C.Y. Chu, M.T. Cheng and T.H. Tseng. 2002. In vivo protective effect of
protocatechuic acid on tert-butyl ISRN Pharmacology 9 hydroperoxide-induced rat
hepatotoxicity. Food Chemical Toxicol. 40:635-641.
Liu, J., J. Lu, X. Wen, J. Kan and C. Jin. 2015. Antioxidant and protective effect of inulin and catechin
grafted inulin against CCl4-induced liver injury. Inter. J Bio. Macromolecules. 72: 1479-1484.
Liu, J., L. Yang, Y. Dong, B. Zhang and X. Ma. 2018. Echinacoside, an Inestimable Natural Product
in Treatment of Neurological and other Disorders. Molecules. 23:1213.
Liu, R.H. 2004. Potential synergy of phytochemicals in cancer prevention: mechanism of action. J.
Nutr. 134:3479-3485.
Liu, S., W. Wei, K. Shi, X. Cao, Zhou and Z. Liu. 2014. In vitro and in vivo anti-hepatitis B virus
activities of the lignan niranthin isolated from Phyllanthus niruri L. J. Ethnopharmacol.
55:1061-1067.
Liu, X., M. Zhao, J. Wanga, B. Yangb and Y. Jiang. 2008. Antioxidant activity of methanolic extract
of emblica fruit (Phyllanthus emblica L.) from six regions in China. J. Food Composition
Anal. 21:219-228.
Liu, Y., L. Cao, J. Du, R. Jia, J. Wang, P. Xu and G. Yin. 2015. Protective effects of Lycium barbarum
polysaccharides against carbon tetrachloride-induced hepatotoxicity in precision-cut liver
slices In vitro and in vivo in common carp (Cyprinus carpio L.) Compara. Biochem.
Physio.169:65-72.
Lo, C.M., C.L. Liu and S.C. Chan. 2007. A randomized, controlled trial of postoperative adjuvant
interferon therapy after resection of hepatocellular carcinoma. Ann. Surg. 245:831-842.
148
Locarnini, S., D.S. Chen and K. Shibuya. 2016. No more excuses: viral hepatitis can be eliminated.
Lancet. 387:1703-1704.
Lodhi, S and A.K. Singhai. 2011. Preliminary pharmacological evaluation of Martynia annua Linn
leaves for wound healing. Asian Pacific J. Tropical Biomed. 1:421-427.
Lodhi, S., A. Jain, A.J. Pal, R.P. Singh and A. S. Kumar. 2016. Effects of flavonoids from Martynia
annua and Tephrosia purpurea on cutaneous wound healing. Avicenna J. Phytomed. 6:578-
591.
Loguercio, C and D. Festi. 2011. Silybin and the liver: from basic research to clinical practice. World.
J. Gastroenterol. 17:2288-2301.
Loguidice, A and U.A. Boelsterli. 2011. Acetaminophen overdose induced liver injury in mice is
mediated by peroxynitrite independently of the cyclophilin D-regulated permeability
transition. Hepatol. 54:969-978.
Lou, Z., H. Wang, S. Zhu, C. Ma and Z. Wang. 2011. Antibacterial activity and mechanism of action
of chlorogenic acid. J. Food Sci. 76:398-403.
Lu, K.H., C. Weng, W.C. Chen and L.Y. Sheen. 2017. Ginseng essence, a medicinal and edible herbal
formulation, ameliorates carbon tetrachloride-induced oxidative stress and liver injury in rats.
J. Ginseng Res. 41:316-325.
Lua, X., T. Liua, K. Chena, Y. Xiaa, W. Daib, S. Xuc, L. Xud, F. Wange, L. Wua, J. Lia, S. Lia, W.
Wanga, Q. Yuc, J. Fenga, X. Fanf, Y. Zhoua, P. Niug and C. Guoa. 2018. Isorhamnetin: A
hepatoprotective flavonoid inhibits apoptosis and autophagy via P38/PPAR-α pathway in
mice. Biomed. Pharmacotherapy. 103:800-811.
Lucio, M., C. Nunes, D. Gaspar, H. Ferreira, J.L.F.C. Lima and S. Reis. 2009. Antioxidant activity of
vitamin E and Trolox: understanding of the factors that govern lipid peroxidation studies in
vitro. Food Biophy. 4:312-320.
Ma, T., X. Sun, C. Tian, Y. Zheng, C. Zheng, J. Zhan. 2015. Chemical composition and
hepatoprotective effects of polyphenols extracted from the stems and leaves of
Sphallerocarpus gracilis. J. Funct. Food. 18:673-683.
Ma, Y., M. Gao and D. Liu. 2015. Chlorogenic acid improves high fat diet-induced hepatic steatosis
and insulin resistance in mice. Pharma. Res. 32:1200-1209.
Mahfuza, U.S., M. Solayman, N. Alam, M.I. Khalil and S.H. Gan. 2018. 5-Hydroxymethylfurfural
(HMF) levels in honey and other food products: effects on bees and human health. Chem.
Central J. 12:35.
Mahmood, S., S. Hussain, S. Tabassum, F. Malik and H. Riaz. 2014. Comparative phytochemical,
hepatoprotective and antioxidant activities of various samples of Swertia Chirayita collected
from various cities of Pakistan. Pak. J. Pharm. Sci. 27:1975-1983.
Malagoli, M. 2007. A full-length protocol to test hemolytic activity of palytoxin on human
erythrocytes. Inter. Surgery J. 4:92-104.
Manns, M.P., M.J.G. Hutchison, S.C. Gordon, V.K. Rustgi, M. Shiffman, R. Reindollar, Z.D.
Goodman, K. Koury, M. Ling and J.K. Albrecht. 2001. Peginterferon alfa-2b plus ribavirin
compared with interferon alfa-2b plus ribavirin for initial treatment of chronic hepatitis C: a
randomised trial. Lancet. 358:958-965.
Martinez, M., P. Poirrier, R. Chamy, D. Prüfer, C. Schulze-Gronover, L. Jorquera and G. Ruiz. 2015.
Taraxacum officinale and related species-An ethno pharmacological review and its potential
as a commercial medicinal plant. J. Ethnopharmacol.169:244-262.
149
Mathew, M and S. Subramanian. 2014. In Vitro Screening for Anti-Cholinesterase and Antioxidant
Activity of Methanolic Extracts of Ayurvedic Medicinal Plants Used for Cognitive Disorders.
PLoS ONE. 9:1-7.
Matic, S., S. Stanic, D. Bogojevic, M. Vidakovic, N. Grdovic, S. Dinic, S. Solujic, M. Mladenovic, N.
Stankovic, M. Mihailovic. 2013. Methanol extract from the stem of Cotinus coggygria Scop
and its major bioactive phytochemical constituent myricetin modulate pyrogallol-induced
DNA damage and liver injury. Mutat. Res. 755:81-89.
Matsuda, H.A., K. Ninomiya, T. Morikawa, D. Yasuda, I. Yamaguchi and M. Yoshikawa. 2009.
Hepatoprotective Amide Constituents From The Fruit Of Piper Chaba: Structural
Requirements, Mode Of Action, And New Amides. Bioorganic Med Chem. 17: 7313-7323.
Mauss, S., D. Hueppe and U. Alshuth. 2014. Renal impairment is frequent in chronic hepatitis C
patients under triple therapy with telaprevir or boceprevir. Hepatolo. 59:46–48.
Medini, F., H. Fellah, R. Ksouri and C. Abdelly. 2014. Total phenolic, flavonoid and tannin contents
and antioxidant and antimicrobial activities of organic extracts of shoots of the plant Limonium
delicatulum. J. Taibah Uni. Sci. 8:216–224.
Meunier, F. J., J. Mondejar-Fernandez, F. Goussard, G. Clement and M. Herbin. 2015. Presence of
plicidentine in the oral teeth of the coelacanth Latimeria chalumnae Smith 1939 (Sarcopterygii;
Actinistia). J. Struct. Biol. 190:31-37.
Mi, C., W. Tao, J. Zhen-Zhou, S. Chun, W. Hao, W.U. Mei-Juan, Z. Shuang, Z. Yun, Z. Lu-Yong.
2014. Anti-inflammatory and hepatoprotective effects of total flavonoid C-glycosides from
Abrus mollis extracts. Chinese J. Natural Med. 12:0590-0598.
Michael, N. C., Johnston, K.L. Knight, S and Kuhnert, N. 2003. Hierarchical Scheme for LC-MSn
Identification of Chlorogenic Acids. J. Agric. Food. Chem. 51:2900-11.
Mihailovic, V., M. Mihailovic, A. Uskokovic, J. Arambasic, D. Misic, V. Stankovic, J. Katanic, M.
Mladenovic, S. Solujic and S. Matic. 2013. Hepatoprotective effects of Gentiana asclepiadea
L. extracts against carbon tetrachloride induced liver injury in rats. Food Chem. Toxicol.
52:83-90.
Misra, A and U. Shrivastava. 2013. Obesity and dyslipidemia in South Asians. Nutr. 5:2708-2033.
Mohammadian, A., S. Moradkhani, S. Ataei, T.S. Heidary, M. Sedaghat, N. Kheiripour, A. Ranjbar.
2016. Antioxidative and hepatoprotective effects of hydroalcoholic extract of Artemisia
absinthium L. in rat. J. Herb. Med. Pharmacol. 5:29-32.
Moniruzzaman, M., M. Asaduzzaman, M.S. Hossain, J. Sarker, S.M. A. Rahman, M. Rashid and
M.M. Rahman. 2015. In vitro antioxidant and cholinesterase inhibitory activities of
methanolic fruit extract of Phyllanthus acidus. BMC Complement. Alter. Med. 15:403.
Montgomery, D.C. 2017. Design and Analysis of Experiments, 9th Edition, John Wiley and Sons,
Inc., New-York, USA. Moquin, D and F.K. Chan. 2010. The molecular regulation of programmed necrotic cell injury. Trends
Biochem. Sci. 35:434-41.
Moustafa,T., P. Fickert, C. Magnes, C. Guelly, A. Thueringer, S. Frank, Kratky, W. Sattler, H.
Reicher, F. Sinner, J. Gumhold, D. Silbert, G. Fauler, G. Hofler, A. Lass, R. Zechner and M.
Trauner. 2012. Alterations in lipid metabolism mediate inflammation, fibrosis, and
proliferation in a mouse model of chronic cholestatic liver injury. Gastroenterol. 142:140-151.
Muriel, P. and Y. Rivera-Espinoza. 2008. Beneficial drugs for liver diseases. J. Appl. Toxicol. 28: 93-
103.
150
Murphy, E.L., S.M. Bryzman, S.A. Glynn, D.I. Ameti, R.A. Thomson, A.E. Williams, C.C. Nass,
H.E. Ownby, G.B. Schreiber, F. Kong, K.R. Neal and G.J. Nemo. 2000. Risk factors for
hepatitis C virus infection in United States blood donors. Hepatol. 31:756-762.
Myers, R.H., D.C. Montgomery and C.M. Anderson-Cook. 2009. Response Surface Methodology:
Process and Product Optimization Using Designed Experiments, 4th Ed., John Wiley and Sons,
Inc., New-York, USA.
Nagda, D., A. Saluja and C. Nagda. 2009. Antioxidant activities of methanolic and aqueous extract
from leaves of Martynia annua Linn. J. pharmacognosy. 1:288-297.
Naik, R.S., A.M. Mujumdar and S.S. Ghaskadbi. 2004. Protection of liver cells from ethanol
cytotoxicity by curcumin in liver slice culture in vitro. J. Ethnopharmacol. 95:31-37.
Nakachi, K., S. Matsuyama, S. Miyake, M. Suganuma and K. Imai. 2000. Preventive effects of
drinking green tea on cancer and cardiovascular disease: epidemiological evidence for multiple
targeting prevention. Biofactors. 13:49-54.
Naruse K, W. Tang and M. Makuuchi. 2007. Artificial and bioartificial liver support: A review of
perfusion treatment for hepatic failure patients. World J. Gastroenterol. 13:1516-1521.
Nathia-Nevesa, G., A.G. Taroneb, M.M Tosic, M.R.M. Juniorb, M. Angela and A. Meireles. 2017.
Extraction of bioactive compounds from genipap (Genipa americana L.) by pressurized
ethanol: Iridoids, phenolic content and antioxidant activity. Food Res. Int. 102:595–604.
Navarro, V.J and J.R. Senior. 2006. Drug-related hepatotoxicity. N. Engl. J. Med. 354:731-739.
Nazam, N., S. Shaikh, M. L. Iqbal, M. Sharma and W. Ahmad. 2015. Combined in silico and in vivo
studies shed insights into the acute acetylcholinesterase response in rat and human brain.
Biotechnol. Appl. Biochem. 62:407-415.
Niaz, A., Z. Ali, S. Nayyar and N. Fatima. 2011. Prevalence of NAFLD in Healthy and Young Male
Individuals. ISRN Gastroenterol. 2011:1-4.
Nithianantham, K., M. Shyamala, Y. Chen, L.Y. Latha, S.L. Jothy and S. Sasidharan. 2011.
Hepatoprotective potential of Clitoria ternatea leaf extract against paracetamol induced
damage in mice. Molecules. 16:10134-10145.
Nomura, H., S. Sou, H. Tanimoto, T. Nagahama, Y. Kimura, J. Hayashi, H. Ishibashi and S.
Kashiwagi. 2004. Short-term interferon-alfa therapy for acute hepatitis C: a randomized
controlled trial. Hepatol. 39:1213-1219.
Nostro, A., M.P. Germano, V. D’angelo, A. Marino and M.A. Cannatelli. 2000. Extraction methods
and bio autography for evaluation of medicinal plant antimicrobial activity. Lett. Appl.
Microbiol. 30:379-384.
Oha, M.H., P.J. Houghtona, W.K. Whangc and J.H. Chob. 2004. Screening of Korean herbal
medicines used to improve cognitive function for anti-cholinesterase activity. Phytomedicine.
11:544-548.
Ohale, P., C.F. Uzoh and O.D. Onukwuli. 2017. Optimal factor evaluation for the dissolution of
alumina from azaraegbelu clay in acid solution using RSM and ANN comparative analysis. S.
Afr. J. Chem. Eng. 24:43-54.
Okhawa, H., N. Ohishi and K. Yagi. 1979. Assay for lipid peroxide in animal tissue by thiobarbituric
acid reaction. Anal. Biochem. 95:351-53.
Ong, K.W., A. Hsu and B.K. Tan. 2013. Anti-diabetic and anti-lipidemic effects of Chlorogenic acid
are mediated by AMPK activation. Biochem. Pharmacol. 85:1341-1351.
Orland, J.R., T.L. Wright and S. Cooper. 2001. Acute hepatitis C. Hepatol. 33:321-327.
Orman, E.S., G. Odena and R. Bataller. 2013. Alcoholic liver disease: Pathogenesis, management, and
novel targets for therapy. J. Gastroenterol. Hepatol. 28:77-84.
151
Ozougwu, J.C and J.E. Eyo. 2014. Hepatoprotective effects of Allium cepa extracts on paracetamol-
induced liver damage in rat. African J. Biotech. 13:2679 -2688.
Pal, D., S. Sannigrahi and U. Mazumder. 2009. Analgesic and anticonvulsant effects of saponin
isolated from the leaves of Clerodendrum infortunatum Linn. in mice. Indian J. Expt. Biol.
47:743-747.
Palanisamy, V., S. Shanmugam and S. Balakrishnan. 2014. An Improvement in Hepatoprotective
Activity by Herbal Drug Combination. Middle-East J Sci. Res. 211454-1459.
Pandey, G. 2011. Medicinal plants against liver diseases. Inter. Res. J. Pharmacy. 2:115-121
Pandey, R and A. Mishra. 2010. Antibacterial activities of crude extract of Aloe barbadensis to
clinically isolated bacterial pathogens. Appl. Biochem. Biotechnol. 160:1356-1361.
Patel, R.M. 2013. Ferrous ion chelating activity (FICA)- a comparative antioxidant activity evaluation
of extracts of eleven naturally growing plants of Gujarat, India. Inter. J. Sci. Res. 2:426-428.
Patel, T.P., S. Soni, P. Parikh, J. Gosai, R. Chruvattil and S. Gupta. 2013. Swertiamarin: An Active
Lead from Enicostemma littorale Regulates Hepatic and Adipose Tissue Gene Expression by
Targeting PPAR-𝛾 and Improves Insulin Sensitivity in Experimental NIDDM Rat Model.
Evid. Based Complemen. Alternat. Med. 2013:1-11.
Patrick-Iwuanyanwu, K.C., N. Eugene, Onyeike and O. W. Mathew. 2010. Hepatoprotective effects
of methanolic extract and fractions of African mistletoe Tapinanthus bangwensis (engl. & k.
krause) from Nigeria. EXCLI J. 9:187-194.
Perez-Aguilar, B., C J. Vidal, G. Palomec, F. García-Dolores, M.C. Gutierrez-Ruiz, L. Bucio, J.L.
Gomez-Olivares and L.E. Gomez-Quiroz. 2015. Acetylcholinesterase is associated with a
decrease in cell proliferation of hepatocellular carcinoma cells. Biochimica. Biophy. Acta.
1852:1380-1387.
Perz, J.F., G.L. Armstrong, L.A. Farrington, Y.J. Hutin and B.P. Bell. 2006. The contributions of
hepatitis B virus and hepatitis C virus infections to cirrhosis and primary liver cancer
worldwide. World J. Hepatol. 45:529-538
Pradhan, S.C and C. Girish. 2006. Hepatoprotective herbal drug, silymarin from experimental
pharmacology to clinical medicine. Indian J. Med. Res. 124:491-504.
Pradhan, S.C and C. Girish. 2006. Hepatoprotective herbal drug, silymarin from experimental
pharmacology to clinical medicine. Indian J. Med. Res. 124: 491-504.
Pramyothin, P., C. Ngamtin, S. Poungshompoo and C. Chaichantipyuth. 2007. Hepatoprotective
activity of Phyllanthus amarus Schum. et. Thonn. extract in ethanol treated rats: In vitro and
in vivo studies. J. Ethnopharmacol. 114:169-173.
Preethi, J., K. Vennila, S. Penislusshiyan and S. Velvizhi. 2016. Hepatoprotective and antioxidant role
of Ziziphus jujuba leaves on paracetamol induced hepatic damage in rats. J. diseases med.
Plants. 2:1-10.
Pretorius, C.J. and E. Watt. 2001. Purification and identification of active components of Carpobrotus
edulis L. J. Ethnopharmarcol. 76:87-91.
Puri, P. 2014. Tackling the Hepatitis B disease burden in India. J. Clin. Exp. Hepatol. 4:312-319.
Qureshi, H., K.M. Bile, R. Jooma, S.E. Alam and H.U. Afridi. 2010. Prevalence of hepatitis B and C
viral infections in Pakistan: findings of a national survey appealing for effective prevention
and control measures. East. Mediterr. Health J. 16:15-23.
Qurrat-ul-Ain., A. Latif, S.J. Raza and M. Saleem. 2017. Validation of fatty liver index for
nonalcoholic fatty liver disease in Pakistani adults. Pakistan J. Pathol. 28:21-27.
152
Rabeta, M.S and R.N. Faraniza. 2013. Total phenolic content and ferric reducing antioxidant power
of the leaves and fruits of Garcinia atrovirdis and Cynometra cauliflora. Int. Food Res. J.
20:1691-1696.
Radko, L and W. Cybulski. 2007. Application of silymarin in human and animal medicine. J. of Pre-
Clinic. Clinical Res.1:022-026.
Ragavendran, M., N. Chandrasekhar, R. Ravikumar, R. Saxena, M. Vasudevan and A. Bhaduri. 2017.
Optimization of hybrid lasere TIG welding of 316LN steel using response surface
methodology (RSM). Optic Laser. Eng. 94: 27-36.
Rahman, A.U and M.I. Choudhary. 2001. Bioactive natural products as a potential source of new
pharmacophores a theory of memory. Pure Appl. Chem. 73:555-560.
Raj, B., S.D.J. Singh, V.J. Samual, S. John and A. Siddiqua. 2013. Hepatoprotective and antioxidant
activity of Cassytha filiformis against CCl4-induced hepatic damage in rats. J. Pharm. Res.
7:15-19.
Rajesh, M.G. and M.S. Latha. 2004. Protective Activityof Glycyrrhiza Glabra Linn. On
Carbantetrachloride-Induced Peroxidative Damage. Indian J. Pharmacol. 38:284-287.
Rajinder, S.D., P.D. Plumb and T.A. Trevor. 1981. Leaf Senescence: Correlated with Increased Levels
of Membrane Permeability and Lipid Peroxidation, and Decreased Levels of Superoxide
Dismutase and Catalase. J. Exp. Botany. 32:93-101.
Rajopadhye, A.A and A.S. Upadhye. 2011. In vitro Antioxidant and Hepatoprotective Effect of the
Whole Plant of Rungia repens (L.) Nees, Against CCl4 Induced Oxidative Stress in Liver Slice
Culture Model. Int. J. Phytomed. 3:540-548.
Rashid, U., M. K. Rashid and M. Sajid. 2016. Hepatoprotective potential of Fagonia olivieri DC.
against acetaminophen induced toxicity in rat. Complemen. Alter. Med. 16: 445-1463.
Rasool, M., J. Iqbal, A. Malik, H.R. Sobia, M.Q. Saeed, M. Asif, M.Q. Husain, M.K. Amjad, A.A.
Gulzar, M.A. Hussain, S.G. Hua and S. Karim. 2014. Hepatoprotective Effects of Silybum
marianum (Silymarin) and Glycyrrhiza glabra (Glycyrrhizin) in Combination: A Possible
Synergy. Evid. Based Complemen. Alternat. Med. 2014:1-9.
Razzaq, Z and A. Malik. 2014. Viral load is associatedwith abnormal serumlevels of micronutrients
and glutathione and glutathione-dependent enzymes in genotype 3 HCV patients. BBA
Clinical. 2::72–78.
Reddipalli, H. 2010. Anti-Hepatotoxic and Anti-Oxidant Defense Potential of Tridax Procumbens.
IJGP. 3:164-169.
Reddy, K.R., T.L. Wright, P.J. Pockros, M. Shiffman, G. Everson, R. Reindollar, and M.W. Fried.
2001. Efficacy and safety of pegylated (40-kd) interferon alpha-2a compared with interferon
alpha-2a in noncirrhotic patients with chronic hepatitis C. Heptol. 33:433- 438.
Reddy, V.P and A. Urooj. 2017. Evaluation of hepatoprotective activity of Morus indica linn. against
toxicity induced by carbon tetrachloride in rats. Int. J. Pharma. Sci. Res. 8:845-851.
Resende, R and A. Adhikari. 2009. Cholinergic receptor pathways involved in apoptosis, cell
proliferation and neuronal differentiation. Cell Commun. & Signaling. 7:7-20.
Resende, R.R., A.S. Alves, L.R. Britto and H. Ulrich. 2008. Role of acetylcholine receptors in
proliferation and differentiation of P19 embryonal carcinoma cells. Exp. Cell. Res. 314:1429-
1443.
Rezg, R., B. Mornagui, S. El-Fazaa and N. Gharbi. 2008. Biochemical evaluation of hepatic damage
in subchronic exposure to Malathion in rats: effect on superoxide dismutase and catalase
activities using native PAGE. Comptes Rendus Biologies. 331:655-662.
153
Risal, P., P.H. Hwang, B.S. Yun, H.K. Yi, B.H. Cho, K.Y. and Y.J. Jeong. 2012. Hispidin analogue
davallialactone attenuates carbontetrachloride induced hepatotoxicity in mice. J. Nat. Prod.
75:1683-1689.
Ritesh , K,R., A. Suganya, H.V. Dileepkumar, Y. Rajashekar and T. Shivanandappa. 2015. A single
acute hepatotoxic dose of CCl4causes oxidative stress in therat brain. Toxicol. Reports.
2:891–895.
Rizzetto, M. 2016. The adventure of delta. Liver Int. 36: 135-140.
Rodrigo, R., A. Miranda and L. Vergara. 2011. Modulation of endogenous antioxidant system by wine
polyphenols in human disease. Clinica. Chimica. Acta. 412:410-424.
Ross and Wilson. 2005. Anatomy and Physiology in Health and illness. Ninth Edition, published by
Elsevier Ltd. 307-310 and 333-336.
Saeed, M.A and S.A. Wahid. 2003. Effects of Fagonia cretica L constituent on various hematological
parameter in rabbits, J. Ethopharmacol. 85:195-200.
Salamone, M., G.A.L. Di and M. Bietti. 2011. Hydrogen atom abstraction reactions from tertiary
amines by benzyloxyl and cumyloxyl radicals: Influence of structure on the rate determining
formation of a hydrogen-bonded pre reaction complex. J. Organic Chem.76:6264-6270.
Salamone, M., R. Martella and M. Bietti. 2012. Hydrogen abstraction from cyclic amines by the
cumyloxyl and benzyloxyl radicals. The role of stereo electronic effects and of
substrate/radical hydrogen bonding. J. Organic Chem. 77:8556-8561.
Salud, M. G., M. X. Silveyra, A. Candela, A. Compa, J. Claria, R. Jover, M. Perez-Mateo, V. Felipo,
S. Martınez, J. Galceran and J. Saez-Valero. 2006. Changes in liver and plasma
acetylcholinesterase in rats with cirrhosis induced by bile duct ligation. Hepatol. 43:444-453.
Samir, S. 2001. Hepatoprotective Natural Products. Int. J. Adv. Res. Pharm. Biosci. 2: 110-111.
Samudram, P., R. Hari, R. Vasuki, A. Geetha and S. Moorthi. 2008. Hepatoprotective activity of Bi -
herbal ethanolic extract on CCl4 induced hepatic damage in rats. African J. Biochem. Res.
2:061-065.
Sannigrahi, S., U.K. Mazumder, D. Pal and Mishra. 2009. Hepatoprotective potential of methanol
extract of Clerodendrum infortunatum Linn. against CCl4 induced hepatotoxicity in rats.
Indian J. Expt. Biol. 5:394-399.
Saravanan, S., H. Islam, P. Babu. 2014. Swertiamarin attenuates inflammation mediators via
modulating NF- ĸB/Iĸb and JAK2/STAT3 transcription factors in adjuvant induced arthritis.
Eur J Pharm Sci. 56:70-86.
Saroopa, P., Samaradivakaraa, R. Samarasekeraa, M. Shiroma, O.V. D. S. Handunnettib and J.
Weerasena. 2016. Cholinesterase, protease inhibitory and antioxidant capacities of SriLankan
medicinal plants. Ind. Crops and Prod. 83:227-234.
Sasikumar. V and P. Kalaisezhiyen. 2014. Evaluation of free radical scavenging activity of various
leaf extracts from Kedrostis foetidissima (Jacq.) Biochem. Analyt. Biochem. 3:1-7.
Satsangi, S and Y.K. Chawla. 2016. Viral hepatitis: Indian scenario. Med. J. Armed Forces India.72:
204-210.
Saukkonen, J.J., D.L. Cohn, R.M. Jasmer, S. Schenker and J.A. Jereb. 2006. An Official ATS
Statement: Hepatotoxicity of anti-tuberculosis therapy. Am. J. Respir Crit. Care Med. 174:935-
952.
Schaefer, M., F. Schmidt, C. Folwaczny, R. Lorenz, G. Martin, N. Schindlbeck, W. Heldwein, M.
Soyka, H. Grunze, A. Koenig and K. L. Adherence. 2003. Mental side effects during Hepatitis
C treatment with Interferon Alfa and Ribavirin in Psychiatric risk groups. Hepaol. 37:443-451.
154
Schaich, K.M., X. Tian and J. Xie. 2015. Hurdles and pitfalls in measuring antioxidant efficacy: A
critical evaluation of ABTS, DPPH, and ORAC assays. J. Funct. Food. 14:111-125.
Schuller, H. 2009. Is cancer triggered by altered signalling of nicotinic acetylcholine receptors? Nat.
Rev. Cancer. 9:195-205.
Schuppan, D and N. Afdhal. 2008. Liver Cirrhosis. Lancet. 371:838-851.
Schutz, K., R. Carle and A. Sxhieber. 2006. Taraxacum - a review on its phytochemical and
pharmacological profile. J. Ethnopharmacol. 107:313-323.
Secretan, L.B., C. Scoccianti, D. Loomis, Y.Grosse, F. Bianchini and K. Straif. 2016. International
agency for research on cancer hand book working group body fatness and cancer-viewpoint of
the Iarc working group. N. Engl. J. Med. 375:794-798.
Semaming, Y., P. Pannengpetch, S.C. Chattipakorn and N. Chattipakorn. 2015. Pharmacological
Properties of Protocatechuic Acid and Its Potential Roles as Complementary Medicine. Evid.
Based Complemen. Alter. Med. 2015:1-12.
Sen, S., R. Chakraborty, C. Sridhar, Y.S.R. Reddy and D. Biplab. 2010. Free radicals, antioxidants,
diseases and phytomedicines: Current status and future prospect. Int. J. Pharm. Sci. Rev. Res.
3:91-100.
Sermakkani, M and V. Thangapandian. Phytochemical and antibacterial activity of Martynia annua
L. against the different pathogenic bacteria. J. Herbal Med. Toxicol. 4:221- 224.
Serviddio, G., F. Bellanti, E. Stanca, P. Lunetti, M. Blonda, R. Tamborra, L. Siculella, G. Vendemiale,
L. Capobianco and A.M. Giudetti. 2014. Silybin exerts antioxidant effects and induces
mitochondrial biogenesis in liver of rat with secondary biliary cirrhosis. Free Radical Bio.
Med. 73:117-126.
Shahrzad, S., K. Aoyagi, A. Winter, A. Koyama and I. Bitsch. 2001. Pharmacokinetics of gallic acid
and its relative bioavailability from tea in healthy humans. J. Nutr. 131:1207-10.
Shannon, E and N. Abu-Ghannam. 2017. Optimisation of fucoxanthin extraction from Irish seaweeds
by response surface methodology. J. Appl. Phycol. 29:1027-1036.
Sharma, P., G. Ravikumar, M. Kalaiselvi, D. Gomathi and C. Uman. 2013. In vitro antibacterial and
freer radical scavenging activity of green hull of Juglans regia. J. Pharma. Anal. 3:298-302.
Sharma, Y., S. Bashir, M. Irshad, Gupta, S. D., and Dogra, T. D. 2005. Effects of acute dimethoate
administration on antioxidant status of liver and brain of experimental rats. Toxicol. 6:49-57.
Shen, J.X., D. Qin, H. Wang, C. Wu, F.D. Shi and J. Wu. 2013. Roles of nicotinic acetylcholine
receptors in stem cell survival/apoptosis, proliferation and differentiation. Curr. Mol. Med.
13:1455-1464.
Shi, G.F., L.J. An, B. Jiang, S. Guan and Y.M. Bao. 2006. Alpinia protocatechuic acid protects against
oxidative damage in vitro and reduces oxidative stress in vivo. Neurosci. Lett. 403:206-210.
Shi, H., A. Shi, L. Dong, X. Lu, Y. Wang, J. Zhao, F. Da and X. Guo. 2016. Chlorogenic acid protects
against liver fibrosis in vivo and in vitro through inhibition of oxidative stress. Clinical
Nutrition. 35:1366-1373.
Shi, H., L. Dong, J. Jiang, J. Zhao, G. Zhao and X. Dang. 2013. Chlorogenic acid reduces liver
inflammation and fibrosis through inhibition of toll-like receptor 4 Signaling pathway. Toxicol.
303:107-114.
Shi, H., L. Dong, X. Dang, Y. Liu, J. Jiang and Y. Wang. 2013. Effect of chlorogenic acid on LPS-
induced pro inflammatory signaling in hepatic stellate cells. Inflamm. Res.62:581-587.
Shi, Y and B. M. Pinto. 2014. Human Lactate Dehydrogenase A Inhibitors: A Molecular Dynamics
Investigation. PLoS ONE. 9:86365.
155
Shimizu, S., R. Atsami, K. Itokawa, M, Iwasaki and T. Aoki. 2009. Metabolism dependent
hepatotoxicity of amodiaquine in glutathione-depleted mice. Arch. Toxicol. 83:701-701.
Shimmyo, Y., T. Kihara, A. Akaike, T. Niidome and H. Sugimoto. 2008. Multifunction of myricetin
on Aβ: Neuroprotection via a conformational change of Aβ and reduction of Aβ via the
interference of secretases. J. Neurosci. Res. 86:368-377.
Sigstedt, S.C., C.J. Hooten, M.C. Callewaert, A.R. Jenkins, A.E. Romero, M.J. Pullin, A. Kornienko,
T.K. Lowrey, S.V. Slambrouch, W.F. Steelant. 2008. Evaluation of aqueous extracts of
Taraxacum officinale on growth and invasion of breast and prostate cancer cells. Int. J. Oncol.
32:1085-1890.
Singh, A., T.K. Bhat and O.P. Sharma. 2011 Clinical Biochemistry of hepatotoxicity. J. Clinic.
Toxicol. 4:2-19.
Soreq, H and S. Seidman. 2001. Acetylcholinesterase—new roles for an old actor, nature reviews.
Neurosci. 2:294-302.
Sreshta, B and S.B. Ravindra. 2018. Evaluation of hepatoprotective activity of polyherbal formulation
against ethanol induced hepatotoxicity in rats. World J Pharm. Pharmaceutic. Sci. 7: 1390-
1399.
Srikumar, B. N., K. Ramkumar, T.R. Raju and R.B.S. Shankarnarayana. 2004. Assay of
Acetylcholinesterase Activity in the Brain. National Inst. Mental Health neurosci. 2004:142-
144.
Srivastava, A. and T. Shivanandappa. 2010. Hepatoprotective effect of the root extract of Decalepis
hamiltonii against carbon tetrachloride-induced oxidative stress in rats. Food Chem. 18:411-
417.
Subba, A., B. Dutta, R.S. Kumar and P. Mandal. 2017. Antioxidant, anti-inflammatory, and
hepatoprotective activity of Fraxinus floribunda bark and the influence of extraction process
on their bioactivity. J. Pharma. Res. 11:983-990.
Sun, H., L. Li, A. Zhang, N. Zhang, H. Lv, W. Sun and X. Wang. 2013. Protective effects of sweroside
on human MG-63 cells and rat osteoblasts. Fitoterapia. 84:174-179.
Suresh, V., G. E.Willem, J. Schoonen, L. G. Marieke, G. Elferink, M. M Geny, M. Groothuis and O.
Peter. 2015. Acute toxicity of CCl4 but not of paracetamol induces a transcriptomic signature
of fibrosis in precision-cut liver slices. Toxicology in Vitro. 29: 012-1020.
Suzuki, A., N. Yamamoto, H. Jokura, M. Yamamoto, A. Fujii and I. Tokimitsu. 2006. Chlorogenic
acid attenuates hypertension and improves endothelial function in spontaneously hypertensive
rats. J. Hypertens. 24:1065-73.
Talreja, T., M. Kumar, A. Goswami, G. Gahlot, S. J. Kumar and T. Sharma. 2017. HPLC analysis of
saponins in Achyranthes aspera and Cissus quadrangularis. J. Pharmacognos. Phytochem.
6:89-92.
Tan, Y.H., M.O. Abdullah, C. Nolasco-Hipolito and N.S.A. Zauzi. 2017. Application of RSM and
Taguchi methods for optimizing the transesterification of waste cooking oil catalyzed by solid
ostrich and chicken-eggshell derived CaO. Renew. Energy 114:437-447.
Targher, G., C.P. Day and E. Bonora. 2010. Risk of cardiovascular disease in patients with
nonalcoholic fatty liver disease. N. Engl. J. Med. 363:1341-50.
Thi, B.P.T., N.T.A. Nhung, T. Duong, P.V. Trung, N. M. Quang, H.T.K. Dung and P.V. Tat. 2016.
Prediction of anticancer activities of cynaroside and quercetin in leaf of plants Cynara
scolymus L and Artocarpus incisa L using structure–activity relationship. Cogent Chem. 2:1-
12.
156
Thomas, M.G., M. R. Marwood, A.E. Parsons and R.B. Parsons. 2015. The effect of foetal bovine
serum supplementation upon the lactate dehydrogenase cytotoxicity assay: Important
considerations for in vitro toxicity analysis. Toxicol. In Vitro. 30:300–308.
Tobin, A. B and D.C. Budd. 2003. The anti-apoptotic response of the Gq/11- coupled muscarinic
receptor family. Biochem. Soc. Trans. 31:1182-1185.
Tsai, C.H., Y.M. Chiu, T.Y. HO, C.T. Hsieh, D.C. Hieh, Y.J. Lee, G.J. Tsay and Y.Y. Wu. 2018.
Gallic Acid Induces Apoptosis in Human Gastric Adenocarcinoma Cells Anticancer Res.
38:42057-42067.
Tsao, G and J. Bosch. 2010. Management of varices and variceal hemorrhage in cirrhosis. N. Engl. J.
Med. 362:823-32.
Tudorel, O.O., G.M. Niţulescu, A. Orțan and C.E. Dinu-Pirvu. 2015. Ethnomedicinal, Phytochemical
and Pharmacological Profile of Anthriscus sylvestris as an Alternative Source for Anticancer
Lignans. Molecules. 20:15003-15022.
Tumah, H. 2005. Fourth-generation cephalosporins: In vitro activity against nosocomial gram-
negative bacilli compared with β-lactam antibiotics and ciprofloxacin. Chemotherapy. 51: 80-
85.
Ullah, Q., K. Khan, K. Saeed, H. U. Rehman, M. Arif, S. Khattak, M. Zaman, F. N. Falak, R. Ahmad,
I. Ali, A. Ahmad and A. Hezbullah. 2015. Prevalence of Hepatitis C and B in MURCY Hospital
Peshawar, KP, Pakistan. J. Entomol. Zool. Studies. 5:1081-1084.
Umer, M and M. Iqbal. 2016. Hepatitis C virus prevalence and genotype distribution in Pakistan:
Comprehensive review of recent data. World J. Gastroenterol. 22:1684-1700.
Uzun, F.G and Y. Kalender. 2013. Chlorpyrifos induced hepatotoxic and hematologic changes in rats:
the role of quercetin and catechin. Food Chem. Toxicol. 55:549-556.
Uzunhisarcikli, M and Y. Kalender. 2011. Protective effects of vitamins C and E against
hepatotoxicity induced by methyl parathion in rats. Ecotoxicol. Environ. Saf. 74:2112-2118.
Vaidya, H., M. Rajani, V. Sudarsanam, H. Padh and R. Goyal. 2009. Antihyperlipidaemic activity of
swertiamarin, a secoiridoid glycoside in poloxamer-407-induced hyperlipidaemic rats. J Nat
Med. 63:437-442.
Vaijanathappa, J and S. Badami. 2009. Antiedematogenic and free radical scavenging activity of
swertiamarin isolated from Enicostemma axillare. Planta Med. 75:12-17.
Valko, M., M. Izakovic, M. Mazur, C.J. Rhodes and J. Telser. 2004. Role of oxygen radicals in DNA
damage and cancer incidence. Mole. Cell Biochem. 266:37-56.
Vargas, N.M., E. S. Madrigal, A. G. Morales, J. S. Esquivel, C. Esquivel, M. García-Luna, G. Rubio,
J.A. Gayosso-de-Lucio, J.A. Morales-González. 2014 Hepatoprotective effect of silymarin.
World J. Hepatol. 6:144-149.
Vatakuti, S., G.E.J. Willem, Schoonen, L. Marieke, Elferink, M.M. Geny, Groothuis and P. Olinga.
2015. Acute toxicity of CCl4 but not of paracetamol induces a transcriptomic signature of
fibrosis in precision-cut liver slices. Toxicolo. In Vitro. 29:1012-1020.
Veena, S.K., A.S. Gosavi, J.B. Kolpe and S.G. Deshapande. 2014. Phytochemical and biological
evaluation of Fagonia species: a review. World J. Pharma. Pharmaceut. Sci. 3:1206-1217.
Velu, V and H. Malipeddi. 2017. In vitro anti-arthritic and hemolytic activity of leaf extracts of tragia
involucrate. Inter. J. Pharm Tech Res. 8:46-50.
Ven, V.D.N., J. Fortunak and B. Simmons. 2015. Minimum target prices for production of direct-
acting antivirals and associated diagnostics to combat hepatitis C virus. Hepatol. 61:1174-
1182.
157
Verma, R and P. Khanna. 2012. Hepatitis A vaccine should receive priority in National Immunization
Schedule in India. Hum Vaccin Immunother. 8:1132-1134.
Verpoorte, R. and A.W. Alfermann. 2000. Metabolic Engineering of Plant Secondary Metabolism.
Curr. Opin. Biotechnol. 9:323-343.
Videla, L.A., R. Rodrigo and M. Orellana. 2004. Oxidative stress-related parameters in the liver of
non-alcoholic fatty liver disease patients. Clin. Sci. 106:261-268.
Vidyasagar, J., N. Karunakar, M.S. Reddy, K. Rajnarayana, T. Surender and D.R. Krishna. 2004.
Oxidative stress and antioxidant status in acute organop hosphorous insecticide poisoning.
Indian J. Pharmacol. 36:76-79.
Vijayalakshmi, M and K. Ruckmani. 2016. Ferric reducing anti-oxidant power assay in plant extract.
Bangladesh J. Pharmacol. 11:570-572.
Vinayak, S., R. Gayatri, S. Sivakumar, D. Sivaraman, A. Sundaresan andV. Banumathi. 2018.
Evaluation of hepatoprotective Potential of Classical Siddha Distillate Sanjeevi Theeneer
against Paracetamol Induced Hepatotoxicity in Zebrafish (Danio rerio) Model. Int. J. Curr.
Res. Chem. Pharm. Sci. 5:44-49.
Vinjamuri, S., D. Shanker, R. Shreesha, Ramesh and S. Nagarajan. 2015. In vitro evaluation of
hemolytic activity and cell viability assay of hexanoic extracts of bridelia ferruginea benth.
World J. Pharmacy Pharma. Sci. 4:1263-1268.
Vishal, R., K. S. Bazegah, and N.Qureshi. 2012. Screening of CSDPs for AM Fungal Association
from Arnala and Kalamb Beach Maharashtra. J. Pharmacy Bio. Sci. 2:44-47.
Vliegenthart, B.A D. 2014. Zebrafish as model organisms for studying drug-induced liver injury. J.
Clin. Pharmacol. 78:1217-1227.
Waheed, Y., T. Shafi, S.Zaman and I. Qadri. 2009. Hepatitis C virus in Pakistan: A systematic review
of prevalence, genotypes and risk factors. World J. Gastroenterol.15:5647-5653.
Wands, J.R. 2004. Prevention of hepatocellular carcinoma. N. Engl. J. Med. 351: 1567-1570.
Wang, L., W.Z. Wu and H.C. Sun. 2003. Mechanism of interferon alpha on inhibition of metastasis
and angiogenesis of hepatocellular carcinoma after curative resection in nude mice. J.
Gastrointest. Surg. 7:587-594.
Wang, W., G. Huang, C. An, S. Zhao, X. Chen and P. Zhang. 2018. Adsorption of anionic azo dyes
from aqueous solution on cationic gemini surfactant-modified flax shives: synchrotron
infrared, optimization and modeling studies. J. Clean. Prod. 172:1986-1997.
Weber, L.W., M. Boll and A. Stamp. 2003. Hepatotoxicity and mechanism of action ofhaloalkanes:
carbon tetrachloride as a toxicological model, CRC Crit. Rev. Toxicol. 33:105-136.
Weber, L.W., M. Boll and A. Stampfl. 2003. Hepatotoxicity and mechanism of action of haloalkanes:
Carbon tetrachloride as a toxicological model. Crit Rev Toxicol. 33:105-136.
Wei, S., G. Chen, W. He, H. Chi, H. Abe, K. Yamashita, M. Yokoyama and H. Kodama. 2012.
Inhibitory effects of secoiridoids from the roots of Gentiana straminea on stimulus-induced
superoxide generation, phosphorylation and translocation of cytosolic compounds to plasma
membrane in human neutrophils. Phytother. Res. 26:168.
WHO. 2013. Global policy report on the prevention and control of viral hepatitis. WC: WHO member
states. 536.
Williams, C.D., J. Stengel and M.I. Asike. 2011. Prevalence of non-alcoholic fatty liver disease and
nonalcoholic steatohepatitis among a largely middle-aged population utilizing ultrasound and
liver biopsy: a prospective study. Gastroenterol. 140:124-131.
Wormser, U and Z.S. Ben. 1990. The liver slice system-an in vitro acute toxicity test for assessment
of hepatotoxins and their antidotes. Toxicol. In Vitro. 4:449-451.
158
Wu, D and A.I Cederbaum. 2003. Alcohol, oxidative stress, and free radical damage. Alcohol Res.
Health. 27:274-284.
Wu, H., G. Zhang, L. Huang, H. Pang, N. Zhang, Y. Chen and G. Wang. 2017. Hepatoprotective effect
of polyphenol-enriched fraction from folium microcos on oxidative stress and apoptosis in
acetaminophen-induced liver injury in mice. Oxid. Med. Cell. Longevity. 2018:1-14.
Xie, W., M. Wang, C. Chen, X. Zhang and M.F. Melzig. 2016. Hepatoprotective effect of isoquercitrin
against acetaminophen-induced liver injury. Life Sci.152:180-189.
Xu, Y., J. Chen, X. Yu, W. Tao, F. Jiang, Z. Yin and C. Liu. 2010. Protective effects of chlorogenic
acid on acute hepatotoxicity induced by lipopolysaccharide in mice. Inflamm. Res. 59:871-
877.
Xu, Y., M.A. Leo and C.S. Lieber. 2003. Lycopene attenuates alcoholic apoptosis in HepG2 cells
expressing CYP2E1. Biochem. Biophys. Res. Commun. 308:614-618.
Yamabe, N., K.S. Kang, E. Goto, T. Tanaka and T. Yokozawa. 2007. Beneficial effect of Corni
Fructus, a constituent of Hachimi-jio-gan, on advanced glycation end-product-mediated renal
injury in Streptozotocin-treated diabetic rats. Biol. Pharm. Bull. 30:520-526.
Yang, J.H., S.C. Kim, K.M. Kim, C.H. Jang, S.S. Cho, S.J. Kim, S.K. Ku, I.J. Cho and S.H. Ki. 2016.
Isorhamnetin attenuates liver fibrosis by inhibiting TGF-beta/ Smad signalling and relieving
oxidative stress, Eur. J. Pharmacol. 783:92-102.
Yang, Q., F. Yang, J. Gong, X. Tang, G. Wang, Z. Wang, L. Yang. 2016. Sweroside ameliorates α-
naphthyl isothiocyanate induced cholestatic liver injury in mice by regulating bile acids and
suppressing pro-inflammatory responses. Acta. Pharmacologica Sinica.37:1218-1228.
Yarnell, E., N.D. RH (AHG) and K. Abascal. 2009. Dandelion (Taraxacum officinale and T
mongolicum). Integrative Med. 8: 35-38.
Yeon, S.P., G. Ahnb, J. U. Hyung, E. H. Jeong, C.B. Ahnc, N.Y. Young and J.J Young. 2017.
Hepatoprotective effect of chitosan-caffeic acid conjugate against ethanol treated mice. Expt.
Toxicol. Pathology. 69:618-624.
Yesmin, F., Z. Rahman, J.D. Fouzia, A.H. Mazid, N. A. Rahman, A.G. Alattraqchi, A. Ahmed, R.
Yousuf, A. Salam and M. Haque. Hepato-protective role of the aqueous and n-hexane extracts
of nigella sativa linn. in experimental liver damage in rats. Asian J. Pharm. Clin. Res. 6:205-
209.
Yilmaz, M., E. Rencuzogullari and M. Canli. 2015. The effects of cyfluthrin on some biomarkers in
the liver and kidney of Wistar rats. Environ Sci Pollut Res. 22:4747–4752.
Yilmaz, M., E. Rencuzogullari and M. Canli. 2017. Investigations on the effects of etoxazole in the
liver and kidney of Wistar rats. Environ Sci Pollut Res. 24:19635-19639.
Yimam, M., P. Jiao, B. Moore, M. Hong, S. Cleveland, M. Chu, Q. Jia, Y.C. Lee, H. J. Kim, J. B.
Nam, M.R. Kim, E.J. Hyun, G. Jung and S. G. Do. 2016. Hepatoprotective Activity of Herbal
Composition SAL, a Standardize Blend Comprised of Schisandra chinensis, Artemisia
capillaris, and Aloe barbadensis. J. Nutr. Metabol. 4:1-10.
Yingjuan, L., C. Liping, D. Jinliang, J. Rui, W. Jiahao, X. Pao and Y. Guojun. 2015. Protective effects
of Lycium barbarum polysaccharides against carbon tetrachloride-induced hepatotoxicity in
precision-cut liver slices in vitro and in vivo in common carp. Comp. Biochem. Physio. 169:65-
72.
Yogesh, C and Yadav. 2015. Hepatoprotective effect of Ficus religiosa latex on cisplatin induced liver
injury in Wistar rats. Brazilian J. Pharmacognos.. 25:278–283.
159
Yogi, B and A. Mishra. 2017. Hepatoprotective effects of polyherbal formulation against carbon
tetrachloride-induced hepatic injury in albino rats: a toxicity screening approach. Asian J
Pharm Clin Res. 10:192-195.
You, Y., Y. Soonam, H. Yoon, J. Park, Y.H. Lee, S. Kim, K.T. Oh, J. Lee, H.Y. Cho and W. Jun.
2010. In vitro and in vivo hepatoprotective effects of the aqueous extract from Taraxacum
officinale (dandelion) root against alcohol-induced oxidative stress. Food Chem. Toxicol.
48:1632-1637.
Young, H.L., Y. Yoon and H.C. Jung. 2018. The effects of herbal extracts on CCl4‑induced ROS
accumulation and cell death in hepatocytes. Oriental Pharma. Expt. Med. 18:257-264.
Young, I.S. 2001.Woodside Antioxidant in health and diseases. Clinical. Pathology. 54:176- 186.
Younossi, Z.M., A.B. Koenig, D. Abdelatif, Y. Fazel, L. Henry and M. Wymer. 2016. Global
Epidemiology of Non-Alcoholic Fatty Liver Disease-Meta-Analytic Assessment of
Prevalence, Incidence and Outcomes. Hepatol. 64:73-84.
Yue, Y., S. Wu, H. Zhang, X. Zhang, Y. Niu, X. Cao, F. Huang and H. Ding. 2014. Characterization
and hepatoprotective effect of polysaccharides from Ziziphus jujuba Mill. var. spinosa (Bunge)
Hu ex H. F. Chou sarcocarp. Food Chem. Toxicol. 74:76-84.
Yun, N., J.W. Kang and S.M. Lee. 2012. Protective effects of chlorogenic acid against
ischemia/reperfusion injury in rat liver: molecular evidence of its antioxidant and anti-
inflammatory properties. J. Nutr. Biochem. 23:1249-55.
Zaidi, S.F., J.S. Muhammad, S. Shahryar, K. Usmanghani, A.H. Gilani, W. Jafri and T. Sugiyama.
2012. Anti-inflammatory and cytoprotective effects of selected Pakistani medicinal plants in
Helicobacter pylori-infected gastric epithelial cells. J. Ethn. pharmacol. 14:403-410.
Zarei, M and T. Shivanandappa. 2013. Amelioration of cyclophosphamide-induced hepatotoxicity by
the root extract of Decalepis hamiltonii in mice. Food Chem. Toxicol. 57:179-84.
Zhang, A., H. Sun and X. Wang. 2013. Recent advances in natural products from plants for treatment
of liver diseases. Eur J Med Chem. 63:570-77.
Zhang, X.J., L. Yang, Q. Zhao, J.P. Caen, H.Y. He, Q.H. Jin, L.H. Guo, M. Alemany, L.Y. Zhang and
Y.F. Shi. 2002. Induction of acetylcholinesterase expression during apoptosis in various cell
types. Cell Death and Different. 9:790-800.
Zhao, L.C., Y. He, X. Deng, G.L. Yang, W. Li, J. Liang and Q. L. Tang. 2012. Response surface
modeling and optimization of accelerated solvent extraction of four lignans from Fructus
schisandrae. Molecules. 17:3618-3629.
Zhao, M., J. Tao, D. Qian, P. Liu, E.X. Shang, S. Jiang, J. Guo, S.L. Su, J.A. Duan and L. Du. 2016.
Simultaneous determination of loganin, morroniside, catalpol and acteoside in normal and
chronic kidney disease rat plasma by UPLC-MS for investigating the pharmacokinetics of
Rehmannia glutinosa and Cornus ofcinalis Sieb drug pair extract. J. Chromatogr. B. Analyt.
Technol. Biomed. Life. Sci. 1009–1010:122–129.
Zhao, Y., X. Wang, T. Wang, X. Hu, X. Hui, M. Yan, Q. Gao, T. Chen, J. Li, M. Yao, D. Wan, J.
Gu, J. Fan and X. He. 2011. Acetylcholinesterase, a Key Prognostic Predictor for
Hepatocellular Carcinoma, Suppresses Cell Growth and Induces Chemosensitization. Hepatol.
53:494-505.
Zhu, R., Y. Wang, L. Zhang and Q. Guo. 2012. Oxidative stress and liver disease. Hepatol. Res.
42:741-749.
Zohra, M and A. Fawzia. 2014. Hemolytic activity of different herbal extracts used in Algeria. Inter.
J Pharma Sci. Res. 5:495-500.
160
Zoulim, F and R. Perrillo. 2008. Hepatitis B: reflections on the current approach to antiviral therapy.
J. Hepatol. 48:2-19.
Appendix I
Recommended