BIOCHEMISTRY DEPARTMENT OF BIOCHEMISTRY FACULTY OF

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.


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.


4.60 Response surface plot of interaction in T. officinale with M.


4.61 Response surface plot of interaction between F. arabica and M.


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


combination on total bilirubin (mg/dL) level at different days 105


combination on total protein (g/dL) level at different days 106


combination on Albumin (g/dL) level at different days 107


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


combination on MDA (nmol/g tissue) in different groups 111


combination on GSH (µg/mg) in different groups 111


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


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


combination on total bilirubin (mg/dL) level at different days 123


combination on total protein (g/dL) levels at different days 124


combination on total albumin (g/dL) levels at different days 125


combination on total globulin (g/dL) levels at different days 125


combination on AChE activity (U/mg) in different groups 127


combination on MDA level (nmol/g tissue) in different groups 128


combination on GSH levels (µg/mg) in different groups 129


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


hepatic cell (→), vacuolar degeneration (↓), inflammation of

hepatic cells (←) and dilation of sinusoidal spaces (↑) in liver

of preventive treatment group

133

4.97


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


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


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


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


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)

https://www.researchgate.net/profile/Hildegard_Schuller

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

https://www.herbal-supplement-resource.com/dandelion-root.html

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



31




6 Ficus religiosa

Liver slices + F. religiosa (50 µg/ mL) + 40 mM CCl4






7 Cassia fistula

Liver slices + C. fistula (50 µg/ mL) + 40 mM CCl4






8 Taraxacum officinale

Liver slices + T. officinale (50 µg/ mL) + 40 mM CCl4






9 Polygonum viviparum

Liver slices + P. viviparum (50 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





11 Phyllanthus emblica

Liver slices + P. emblica (50 µg/ mL) + 40 mM CCl4






12 Fagonia arabica

Liver slices + F. arabica (50 µg/ mL) + 40 mM CCl4



32




13 Ocimum basilicum

Liver slices + O. basilicum (50 µg/ mL) + 40 mM CCl4






14 Martynia annua

Liver slices + M. annua (50 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



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



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


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


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

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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).

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Table 4.5: Percentage haemolysis in selected plants at different concentrations.

Medicinal plants

Haemolysis (%)


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.

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Table 4.6: Percentage of Clot lysis in medicinal plants at different concentrations. Medicinal plants

Clot lysis (%)


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)


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

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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.

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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).

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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

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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

https://www.ncbi.nlm.nih.gov/pubmed/?term=Gevrenova%20R%5BAuthor%5D&cauthor=true&cauthor_uid=25709205

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(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 antifungal 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).

https://pubmed.ncbi.nlm.nih.gov/?term=Clifford+MN&cauthor_id=12720369

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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

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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

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(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)

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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

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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

https://pubs.rsc.org/en/results?searchtext=Author%3ABharti%20Badhani

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

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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

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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

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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

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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

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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).

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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

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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.

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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

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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.

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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

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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.

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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.

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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

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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.

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Figure 4.54. Response surface plot of interaction in S. marianum with F. arabica for the


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

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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


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.

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Figure 4.57. Response surface plot of interaction in P. viviparum with F. arabica for the


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


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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


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.

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Figure 4.60. Response surface plot of interaction in T. officinale with M. annua for the


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


It was elucidated from these results that quadratic model was useful to predict optimum

concentrations of medicinal plants that showed maximum hepatoprotective potential. Therefore

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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).

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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.

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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

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(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 ±

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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

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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)


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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)


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 ±

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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.

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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

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(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,

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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,

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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

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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

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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

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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

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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.

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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

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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.

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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

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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).

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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

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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)


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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.

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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)


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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)

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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

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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


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.


globulin (g/dL) levels at different days.

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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).

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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.

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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.


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


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

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Appendix I

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