prr.hec.gov.pkprr.hec.gov.pk/jspui/bitstream/123456789/7659/1/Abid Ali full final thesis.pdf · 20 Declaration I hereby declare that the contents of the thesis, “Biochemical Profiling

19

Biochemical Profiling and Synergistic Potential of

Medicinal Plants as Hepatoprotective

By

Abid Ali M. Phil (Biochemistry)

A dissertation submitted in partial fulfillment of the requirement for the

Degree of

DOCTOR OF PHILOSOPHY

In

BIOCHEMISTRY

DEPARTMENT OF BIOCHEMISTRY

FCAULTY OF SCIENCES

UNIVERSITY OF AGRICULTURE FAISALABAD,

PAKISTAN

(2015)

20

D ec l ar at i o n

I h e r e b y d e c l a r e t h a t t h e c on t en t s o f t h e t h e s i s , “ Biochemical

Profiling and Synergistic potential of medicinal plants as

Hepatoprotective” are product of my own research and no part has been copied

from any published sources (except the references, standard mathematical or

genetic models/ equations/ formulate/ protocols etc.). I further declare that this

work has not been submitted for award of any other diploma/ degree. The

university may take action if the information provided is found inaccurate at any

stage. (In case of any default the scholar will be preceded as per HEC

plagiarism policy).

A b i d A l i

R eg . N o . 2 0 0 4 - ag -

2 1 2

T o

21

The Controller of Examinations,

University of Agriculture,

Faisalabad.

“We, the Supervisory Committee, certify that the contents and form of thesis

submitted by Mr. Abid Ali, Reg. No. 2004-ag-212, have been found satisfactory and

recommend that it may be processed for evaluation, by the External Examiner(s) for the

award of degree” .

Supervisory Committee

1. Chairman __________________________

Prof. Dr. Khalil-Ur-Rahman

2. Member __________________________

Dr. Muhammad Shahid

3. Member __________________________

Prof. Dr. Muhammad Arshad

22

To

My Affectionate Parents

My Brother Muhammad Majid

&

My caring wife

Their faith in and support of me has been constant during times

When all seemed impossible

23

ACKNOWLEDGMENT In the name of Allah, the merciful, the beneficent

All praises (belong) to Allah alone, the cherisher and Sustainer of the world. He is

the first, he is the last, he is the hidden, and he knows about everything. He brings the night

into the day and brings the day into the night, and he knows the thoughts of hearts (Al-

Quran)

I have the pearls of my eyes to admire countless blessings of Allah Almighty because

the words are bound, knowledge is limited and time of life is too short to express his

dignity. It is the one of his infinite benedictions that he bestowed upon me with the

potential and ability to complete the present research program and to make a meek

contribution to the deep oceans of knowledge already existing.

Then the trembling lips and wet eyes praise the greatest man of Universe, the last

messenger of ALLLAH, HAZRAT MUHAMMAD (PBUH) whom ALLAH has sent as

mercy for worlds, the illuminating torch, the blessing for the literate, illiterate, rich, poor,

powerful, weaker, able and disable. Whose life and sayings are ultimate source of guidance

and way of "NIJAT" for the mankind, who enlighten our conscious with the essence of faith

on ALLAH, merging all his kindness and mercy upon us.

I deem it my utmost pleasure to avail an opportunity to express my heartiest

gratitude and deep sense of obligation to a very hardworking and personalized man. My

honorable supervisor, Prof. Dr. Khalil-Ur-Rahman, Department of Chemistry &

Biochemistry, University of Agriculture, Faisalabad for his kind behavior, generous

transfer of knowledge, moral support, constructive criticism and enlightened supervision

during the whole study period. His available words will always serve as a beacon of light

throughout my life.

I express my deep sense of gratitude to member of my supervisory committee, Dr.

Muhammad Shahid, (Associate professor, Department of Chemistry and Biochemistry) for

gidence and support during research work. I am also thankful to Prof. Dr. Muhammad

Arshad (Institute of Microbiology, University of Agriculture, Faisalabad) for his kind

behavior and critical review in my research.

Above all I am very thankful to Dr. Nazish Jahan (Assistant professor, Department

of Chemistry), Prof. Dr. Amer Jamil Department of Biochemistry, Dr. Muhammad Atiq

(Assistant professor, Department of Plant Pathology), Dr. Muhammad Kashif Saleemi

(Assistant professor, Department of veterinary Pathology), Dr. Muhammad Yousaf

Hussain (Professor of Physics) and Dr. Mazhar Iqbal (Principal Scientist and group leader

NIBGE) for their guidance during my research.

24

I am also very thankful to all of my fellows for their sincere co-operation and

appreciation, throughout the study period, especially Sumaira Sharif, Aisha Tahir, Sobia

Aleem, Hira Munir, Sadia Noreen & Aisa Atta PhD scholars, Department of Chemistry

and Biochemistry UAF and my research fellows, Nadia Afsheen, Saman Hina and Umme

Habiba.

True friends, the asset of my life, who always helped me and never make me feel that

anything is difficult or impossible, include a list of small in number but great and sincere

people Atiq-Ur-Rehman, Mazhar Abbas, Ghulam Mustafa,Umar Farooq, Rana Mudassar

Hussain and Muhammad Usman . May ALLAH bless them with success and happiness for

their future.

At last but not the least I pay my cordial thanks to those who live in my mind and

soul, whose love will never mitigate, whose prayers will never die, who are nearest, deepest

and dearest to me, my parents especially my Mother and my Grandfather. I can never forget

the efforts of my Brother Muhammad Majid on his kindness and support during critical

time in my life. Words are lacking to express my humble obligation to my wife Saima Abid,

my Sister Rubina Dilwar and my Babhi Samreen Majid for their support in my life.

I feel it a matter of great pride in regarding vigorous tribute to my well-wishers,

sweet and caring nephew Mr. Muhammad Sehal, Zohaib Muhammad, my nice hadia

Shahzadi, my daughter Mahnoor Abid, Minahal Abid and my son Muhammad Fahad on

their innocent prayers and affection which makes my days memorable. May Allah bless

them with health, knowledge in this world and hereafter?

Abid Ali

25

CONTENTS

Ch. No. Title Page No.

Abstract

1 Introduction 1

2 Review of Literature 9

3 Materials & Methods 19

4 Results & Discussions 40

5 Summary 140

References 142

Table of contents

Sr. No. Title Page No.

26

1 Introduction 1

2 Review of literature 9

2.1 Importance of medicinal plants 9

2.2 Uses of medicinal plants in various disorders 9

2.3 Biochemical profiling of Silybum mariamum 11

2.3.1 Therapeutic uses of Silybum mariamum 12

2.3.2 Role of Silybum mariamum in hepatic disorders 12

2.3.3 Use of Silybum mariamum in age related oxidative stress 14

2.3.4 Anti-proliferative role of Silybum mariamum 14

2.3.5 Role of Silybum mariamum on metabolic pathways 15

2.4 Biochemical profiling of Rheum emodi 15

2.4.1 Therapeutic uses of Rheum emodi 16

2.5 Biochemical profiling of Artemesia absinthium 17

2.5.1 Therapeutic uses of Artemesia absinthium 18

3 Materials and Methods 19

3.1 Determination of phytochemical constituents 19

3.1.1 Total phenolic content 19

3.1.2 Total flavonoid content 19

3.2. Antioxidants potential 19

3.2.1 DPPH radical scavenging activity (DPPH assay) 19

3.2.2 Reducing power assay 20

3.2.3 Antioxidant activity by DNA protection assay 20

3.2.3.1 Composition of TAE buffer 21

3.3 Toxicological screening 21

3.3.1 Hemolytic activity 21

3.3.2 Brine shrimp lethality test 22

3.3.2.1 Extraction 22

3.3.2.2 Shrimp hatching process 22

3.3.2.3 Experimental procedure and observation 22

3.3.3 Mutagenicity assay 23

3.3.3.1 Test bacterial strains 23

3.3.3.2 Chemicals 23

27

3.3.3.3 Herbal extracts

23

3.3.3.4 Preparation of reagent mixture

23

3.3.3.5 Mutagenicity assay 23

3.4 Biochemical profiling 24

3.4.1 Liquid chromatography mass spectrometry (LCMS) 24

3.5 Extraction of silymarin from seeds 25

3.5.1 Soxhlet extraction 25

3.5.2 Reflux extraction 25

3.6 Animal model 25

3.6.1 Preliminary trial / evaluation of hepatotoxin 25

3.6.2 Pre-clinical Trial 29

3.6.2.1 Curative potential of medicinal plants 29

3.6.2.2 Blood sampling 29

3.7 Biochemical analysis 29

3.7.1 Serum glutamate pyruvate transaminase (SGPT) 29

3.7.1.1 Principle 30

3.7.1.2 Reagent preparation 30

3.7.2 Serum glutamate oxaloacetate transaminase (SGOT) 30



3.7.3 Serum alkaline phosphatase (ALP) 30



3.7.4 Albumin 31


3.7.4.2 Procedure 31

3.7.4.3 Calculation 31

3.7.5 Total protein 31


3.7.5.2 Procedure 31

3.7.5.3 Calculation 32

3.7.6 Determination of albumin globulin ratio (A/G ratio) 32

28

3.7.7 Antioxidant enzyme assay 32

3.7.7.1 Superoxide dismutase (SOD) 32

3.7.7.1.1 Enzyme assay of superoxide dismutase (SOD) 32

3.7.7.1.2 Required reagents for enzyme assay 32

3.7.7.1.3 Preparation of 0.067 mM phosphate buffer 32

3.7.7.1.4 Preparation of EDTA/NaCN solution 32

3.7.7.1.5 Preparation of riboflavin solution 33

3.7.7.1.6 Preparation of NBT solution 33

3.7.7.1.7 Procedure 33

3.7.7.1.8 Calculation 33

3.7.7.2 Peroxidase (POD) 33

3.7.7.2.1

Preparation of enzyme extract 33

3.7.7.2.2 Enzyme assay of peroxidase 34

3.7.7.2.3 Required reagents for enzyme assay 34

3.7.7.2.4 Preparation of 0.2 M phosphate buffer 34

3.7.7.2.5 Preparation of buffer substrate solution 34



3.7.7.3 Catalase 35

3.7.7.3.1 Isolation of catalase enzyme 35


3.7.7.3.3 Analysis of catalase enzyme activity 35

3.7.7.3.4 Required reagents and chemicals for enzyme assay 35

3.7.7.3.5 Preparation of 60 mM phosphate buffer 36

3.7.7.3.6 Preparation of buffer substrate solution 36



3.8 Histopathology 37

3.8.1 Washing 37

3.8.2 Dehydration 37

3. 8.3 Cleaning 37

3. 8.4 Infiltration 37

29

3.8.5 Embedding 38

3.8.6 Sectioning and mounting 38

3.8.7 Staining 38

4 Results and Discussions 40

4.1. Phytochemical studies 42

4.1.1 Total phenolic and flavonoid content 42

4.2. Antioxidant potential 43

4.2.1 DPPH assay 43

4.2.2 Reducing power 46

4.2.3 DNA protection effect with H2O2 induced oxidative damage on pUC19

DNA

49

4.3 Toxicological profile 52

4.3.1 Hemolytic activity 52

4.3.2 Brine shrimp lethality

55

4.3.3 Mutagenic activity 57

4.4 Metabolomics analysis 61

4.4.1 Biochemical profiling of Silybum marianum 61

4.4.2 Biochemical profiling of Artemesia absinthium 66

4.4.3 Biochemical profiling of Rheum emodi 67

4.5 Quantification of extracted silymarin 72

4.6 Animal model 73

4.6.1 Preliminary evaluation of toxin 73

4.6.1.1 Conclusion preliminary evaluation of toxin 74

4.6.2 Pre-clinical animal trial 76

4.6.2.1 Toxin 76

4.6.2.2 Blood sampling 76

4.6.2.3 Treatment with medicinal plants in combination 76

4.7 Biochemical Analysis 76

4.7.1 Serum glutamate pyruvate transaminase (SGPT) 78

4.7.2 Serum glutamate oxaloacetate transaminase (SGOT) 83

4.7.3 Alkaline phosphatase (ALP) 92

4.7.4 Total protein 93

4.7.5 Albumin 105

30

4.7.6 Albumin globulin (A/G) ratio 112

4.7.7 Antioxidant enzyme 120

4.8 Gross and histopathology 124

5 Summary 140

References 142

List of Tables

Sr. No. TITLE Page No.

3.1 Sample Preparation for DNA protection assay 21

3.2 Set-up of the fluctuation assay 24

3.3 Mode of hepatotoxicity induced 26

3.4 Dosage plan (Combinations of selected medicinal plants) 27-28

4.1 Total phenolic and total flavonoid content of selected medicinal plants in

combinations 44-45

4.2 DPPH and reducing power of selected medicinal plants in combinations 47-48

4.3 Hemolytic activity and brine shrimp lethality of selected medicinal plants

in different combinations 53-54

4.4 Brine shrimp lethality outcomes showing survived shrimps and %

mortality after treatment with extracts 56

31

4.5 Mutagenic activity of methanolic extracts 57

4.6 Statistical analysis of serum glutamate pyruvate transaminase (SGPT) 79

4.7 Statistical analysis of serum glutamate oxaloacetate transaminase (SGOT) 87

4.8 Statistical analysis for alkaline phosphatase (ALP) 94

4.9 Statistical analysis for protein 100

4.10 Statistical analysis for albumin 107

4.11 Statistical analysis for Albumin Globulin (A/G) ratio 113

4.12

Antioxidant enzymes activity offered by different combinations of

selected medicinal plants 121

32

List of Figures

Fig. No. TITLE P. No.

4.1 Plasmid (pUC19) DNA with essential components. 49

4.2

Agarose gel electrophoresis pattern of pUC19 plasmid DNA

treated with 30 mM H2O2 in the presence and absence of different

concentrations of plants extracts. 50

4.3

(A) Blank, (B) Standard mutagen TA98 (K2Cr2O7), (C) Standard

mutagen TA100 (NaN3), (D) Background S. typhimurium TA98

(E) Background S. typhimurium TA100 59

4.4

Anti-mutagenic activity of Milk thistle, Artemesia absinthium and

Rheum emodi against Salmonella typhimurium strain TA98 and TA

100.

60

4.5 Mass spectrum of silychristin 61

4.6 MS2 of 481 at collision induced dissociation (CID) energy (19.00) 62

4.7 MS3 of 481 at CID (20.00) and 463 at CID (20.00) 62



4.10 MS4 of 481 at CID (20), 463 at CID (20) and 419 at CID (30) 63

4.11 Structure of Isosilybin A & Isosilybin B 63

4.12 MS2 of 513 at CID (20.00) showing main peak at 481 64

4.13 MS3 of 513 at CID (20:00) and 463 at (19:00) 64

4.14 MS3 of 513 at CID (20:00) and 453 at (25:00) 64

4.15 Structure of silydianin 65

4.16 MS3 of 513 at CID (20:00) and 481 at CID (20:00) 65

4.17 Proposed fragmentation pattern 66

4.18 MS2 229 at CID 33 peak at 153 showed alfa thoujone 66

4.19 MS peak at 281 showed artemisinin 67

4.20 Structure of absinthin 67

4.21 MS2 at 445 m/z showed the presence of rhein-8-glucoside and physcion at 283

68

4.22 MS showed emodin glycoside at 431 68

4.23 MS peak at 269 showing emodin 68

4.24 MS peak at 253 showing crysophanol 69

4.25 MS peak at 283 showing rhein 69

4.26 Standard curve of standard silymarin (sigma) at various

concentrations 72

4.27 Biochemical markers response of group 1 given normal saline

water 74

4.28 Biochemical markers response of group 2 treated with 30% CCl4 (3

mL/kg body weight twice in a week for two weeks) 75

4.29 Biochemical markers response of group 5 treated with paracetamol

(500 mg / kg body wt. for three consecutive days) 75

4.30 Loading of toxin in syringe 77

4.31 Administration of toxin 77

4.32 Blood sampling 77

4.33 Storing of blood 77

33

4.34 Loading of medicinal plants in syringe 77

4.35 Administration of medicinal plant 77

4.36 Box-whiskers plot for serum glutamate pyruvate transaminase at zero hour

80

4.37 Box-whiskers plot for serum glutamate pyruvate transaminase at 72

hours 80

4.38 Box-whiskers plot for serum glutamate pyruvate transaminase at 168 hours

81

4.39 Box-whiskers plot for serum glutamate pyruvate transaminase at 288 hours

81

4.40 Box-whiskers plot for serum glutamate pyruvate transaminase at 408

hours 84

4.41 Graphically representation of all five times blood sampling analysis of SGPT

84

4.42

Interactive effects of various combinations of selected medicinal

plants on SGPT over time. The levels 100, 200 and 500 mg of

Silybum marianum (MT) and Rheum emodi (RHE) were fixed,

whereas the levels of Artemesia absinthium (AAB) varying, 100

mg (red), 200 mg (Green) and 500 mg (Blue)

85

4.43 Box-whiskers plot for serum glutamate oxaloacetate transaminase at zero hour

88

4.44 Box-whiskers plot for serum glutamate oxaloacetate transaminase at 72

hours 88

4.45 Box-whiskers plot for serum glutamate oxaloacetate transaminase at 168 hours

89

4.46 Box-whiskers plot for serum glutamate oxaloacetate transaminase at 288 hours

89

4.47 Box-whiskers plot for serum glutamate oxaloacetate transaminase at 408

hours 90

4.48 Graphically representation of all five times blood sampling analysis of SGOT

90

4.49


plants on SGOT over time. The levels 100, 200 and 500 mg of

Silybum marianum (MT) and Rheum emodi (RHE) were fixed



91

4.50 Box-whiskers plot for alkaline phosphate (ALP) at zero hour 95

4.51 Box-whiskers plot for alkaline phosphate (ALP) at 72 hours 95




4.55 Graphically representation of all five times blood sampling

analysis of ALP 97

4.56


plants on ALP over time. The levels 100, 200 and 500 mg of




98

4.57 Box-whiskers plot for total protein at zero hour 101

4.58 Box-whiskers plot for total protein at 72 hours 101


34



4.62 Graphically representation of all five times blood sampling analysis of Protein

103

4.63


plants on protein over time. The levels 100, 200 and 500 mg of




104

4.64 Box-whiskers plot for albumin at zero hour 108

4.65 Box-whiskers plot for Albumin at 72 hours 108

4.66 Box-whiskers plot for albumin at 168 hours 109



4.69 Graphically representation of all five times blood sampling analysis of albumin

110

4.70


plants on albumin over time. The levels 100, 200 and 500 mg of




111

4.71 Box-whiskers plot for A/G ratio at zero hour 114

4.72 Box-whiskers plot for A/G ratio at 72 hours 114




4.76 Graphically representation of all five times blood sampling analysis of A

/G ratio 116

4.77


plants on A/G ratio over time. The levels 100, 200 and 500 mg of




117

4.78 Gross pathology of animals treated with A2B2C2 124

4.79 Gross pathology of positive control and absolute control group 124

4.80 Gross pathology of animals treated with A3B2C2 and A3B3C1 124

4.81 Gross pathology of standard (silymarin) and animals treated with

A1B3C3 125



4.84 Photomicrograph of liver of control group showing normal hepatic parenchyma (200 X, H &E staining)

127

4.85 Photomicrograph of liver of group 1 group showing individual cell

necrosis (shown by arrow) and mild degree of congestion in hepatic parenchyma (200 X, H &E staining)

127

4.86 Photomicrograph of liver of group 1 group showing individual cell necrosis (shown by arrow) and biliary hyperplasia (bold arrow) hepatic parenchyma (200 X, H &E staining)

128

4.87 Photomicrograph of liver of group 2 group showing mild degree of vacuolar degeneration (arrow) in cytoplasm of hepatocytes and

128

35

individual cell necrosis (bold arrow) (200 X, H &E staining)

4.88 Photomicrograph of liver of group (G3) group showing normal hepatic parenchyma indicating ameliorative effect of plant extracts (200 X, H &E staining)

129

4.89 Photomicrograph of liver of standard (group 5) showing cell swelling and vacuolar degeneration (arrow) in hepatic parenchyma (200 X, H &E staining)

129

4.90 Photomicrograph of liver of group 6 showing ameliorative effect of plant extracts indicated by normal nuclei of hepatocytes with chromatin material in nucleolus (400 X, H &E staining)

131

4.91 Photomicrograph of liver of group 7 showing mild degree of vacuolar degeneration (arrow) indicating partial ameliorative effect of plant

extracts. (200 X, H &E staining) 131

4.92 Photomicrograph of liver of group 8 showing mild degree of congestion and mild degree of vacuolation (shown by arrow) (200 X, H &E staining)

132

4.93 Photomicrograph of liver of group 8 showing mild degree of congestion and mild degree of vacuolation (shown by arrow) (400 X, H &E staining)

132

4.94 Photomicrograph of liver of group 9 showing mild degree of vacuolar

degeneration (shown by arrow) and cell swelling (200 X, H &E staining) 133

4.95 Photomicrograph of liver of group 9 showing mild degree of vacuolar degeneration (shown by arrow) and cell swelling (400 X, H &E staining)

133

4.96 Photomicrograph of liver of group 10 showing mild degree of vacuolar degeneration (shown by arrow) and cell swelling (400 X, H &E staining)

135

4.97 Photomicrograph of liver of G12 showing normal hepatic parenchyma

indicating good amelioration with plant extracts. (200 X, H &E staining) 135

4.98 Photomicrograph of liver of positive control showing pyknotic changes (Bold arrow) and fibrosis (shown in arrow) indicating chronic changes (200 X, H &E staining)

136

4.99 Photomicrograph of liver of positive control showing pyknotic changes, biliary hyperplasia and fibrosis (shown in arrow) indicating chronic changes (200 X, H &E staining)

136

4.100 Photomicrograph of liver of positive control showing pyknotic (shown in arrow) changes, biliary hyperplasia and fibrosis indicating chronic changes (200 X, H &E staining)

137

List of Abbreviations

SM Silybum marianum

AAB Artemesia absinthium

RHE Rheum emodi

MT Milk thistle

TPC Total phenolic content

TFC Total flavonoid content

36

DPPH 1, 1-Diphenyl-2-picryl-hydrazyl

LCMS Liquid chromatography mass

spectrometry

CCl4 Carbon tetra chloride

SGPT Serum glutamate pyruvate transaminase

SGOT Serum glutamate oxaloacetate

transaminase

ALP Alkaline phosphate

SOD Superoxide dismutase

POD Peroxidase

CAT Catalase

H2O2 Hydrogen peroxide

ROS Reactive oxygen species

CID Collision induced dissociation

NKT Natural killer T

KC Kupffer cells

NF Nuclear factor

HCC Hepatocellular carcinoma

HBV Hepatitis B virus:

HCV Hepatitis C virus

MAP Medicinal and aromatic plants

PLGA NPs Poly (DL-lactide-co-glycolide)

nanoparticles

GAE Gallic acid equivalents

CE Catechin equivalents

NBT Nitro blue tetrazole

AM Aqueous Mixture

NDEA N-nitrosodiethylamine

ABSTRACT

The objective of this study was to find out any alternative natural product to address the hepatic

problems. Though many medicinal plants have been reported for hepatoprotective potential but

still there is a need to explore further their medicinal effect on scientific basis. Hepatoprotective

potential of selected medicinal plants (Silybum marianum, Artemisia absinthium and Rheum

emodi) in different combinations against paracetamol induced liver damage was studied. The

37

phytochemical constituents of selected medicinal plants in combinations were tested by

measuring total phenolic content (276.26 to 356.57 GAE) and total flavonoid content (13.01 to

25.63 CE). The antioxidant activity of selected medicinal plants in combination was evaluated

through DPPH (76.81 to 92.33 % inhibition) and reducing power in term of absorbance (0.964 to

1.652). The antioxidant effect of medicinal plants at cellular level was further explored using

H2O2 induced DNA damage on pUC19 plasmid DNA. It was found that the protective effect of

plants extracts was concentration dependent. The safety of medicinal plants was evaluated

through various toxicological assays including hemolytic, brine shrimp lethality and

mutagenicity. The cytotoxicity findings showed that the plants are safe and may be used as

medicine. Biochemical profiling of selected medicinal plants was performed through liquid

chromatography mass spectrometry (LCMS). The hepatotoxin, paracetamol significantly

increased the levels of SGPT, SGOT and ALP whereas the levels of protein and albumin were

decreased. The combination containing silymarin extracted from S. marianum, aqueous mixture

of both A. absinthium and R. emodi were given to experimental animals with different doses. The

hepatoprotective effect was evaluated by studying various biochemical parameters (SGPT,

SGOT, ALP, Protein and Albumin). The administration of medicinal plants in various

combinations altered the biochemical markers and exhibited significant hepatoprotective activity.

The experimental animals were slaughtered at the end of study. The levels of antioxidant

enzymes, superoxide dismutase, glutathione peroxidase and catalase were decreased in positive

control group (paracetamol treated). The levels of antioxidants enzymes in medicinal plants

treated groups remained in normal limit as in the control group. Histopathological examination of

liver tissues of control and treated animals showed significant difference indicating that the

combinations of selected medicinal plants successfully restored the liver functions. Although

majority of the combinations restored the liver functions but the most suitable combination was

the least quantity i.e. 100 mg of each plant. It is concluded that the medicinal plants in

combination used in this study is an effective and promising formulation against paracetamol

induced liver damage.

38

Chapter No. 1

INTRODUCTION

The treatment of different ailments with medicinal plants as whole or their

derivative/extracts is a common practice worldwide (Shaik et al., 2012). Since beginning

mankind depends on plant based products in order to fulfil their basic humanly needs. The

use of medicinal plants to check their effectiveness on different ailments depends on the

therapeutic properties and their beneficial effects based on the empirical findings of hundreds

and thousands of years ago (Iqbal and Rehman, 2004). There is a small number of plant

species which have been studied scientifically but their complications are not yet known. It is

very important to evaluate the beneficial as well as side effects of the plants used against

different diseases comprehensively (Santos et al., 2008).

Liver is one of the vital organs performing several functions including metabolism,

glycogen storage, plasma protein synthesis and regulation of homeostasis in the body (Opoku

et al., 2007; Maheswari et al., 2008). It also plays a key role in performing other functions

including defense against pathogens, supply of nutrients, production of bile salts and blood

collecting cascade. Liver diseases are major concerns of the health care professionals around

the world and conventional drugs used in the treatment without scientific conformation can

be hazardous (Arhoghro et al., 2009). The liver is highly susceptible to damage from drugs

and other substances because of its role to metabolize substances in close relation with the

gastrointestinal tract (Soni et al., 2011). Gastrointestinal organs directly pass around 75 % of

the total blood to the liver through portal veins which bring drugs and xenobiotics in

concentrated form (Vikramjit and Mrtcalf, 2009). Many studies cited for showing

mechanisms of hepatic injury, still lack exact mechanism which involves direct

hepatotoxicity and adverse immune reactions. Hepatic injury is usually occurred by the bio-

activation of drugs to chemically reactive metabolites that interact with cellular

macromolecules such as nucleic acids, lipids and proteins, leading to DNA damage, lipid

peroxidation, protein dysfunction and oxidative stress (Lynch and Price, 2007).

Owing to damage of hepatocytes and bile duct cells, the quantity of bile acid

increases inside the liver which may promote further liver damage, resulting destruction of

cellular function leading to culminate in cell death and possible liver failure (Blazka et al.,

1995). Liver dysfunction and stress result in the release of signals that stimulate the activation

of other cells. The innate immune system including natural killer (NK) cells, kupffer cells

(KC) and natural killer T (NKT) cells lead to the progression of liver damage by producing

39

pro-inflammatory mediators and secreting chemokine’s. It is established that a variety of

inflammatory cytokines (interferon (IFN)-γ, interleukin (IL)-1β and tumor necrosis factor

(TNF)-α) produced during hepatic injury are involved in promoting tissue damage (Bourdi et

al., 2002; Polyak et al., 2007).

Hepatitis is one of the life threatening diseases that have spoiled a large number of

families. The complications of hepatitis as hepatocellular carcinoma (HCC) have become the

fifth most common cause of death across the world (Yang and Roberts 2010; El-Serag, 2011;

Cornella et al., 2011). Infection with the hepatitis C virus (HCV) and hepatitis B virus are

major health deteriorating source and is a cause of persistent viral infection that leads to the

liver cirrhosis and hepatocellular carcinoma (Shepard et al., 2005; Nazeema and Brindha

2009). The most common cause of developing HCC is linked with risk factors of hepatitis B

and/or C virus (HBV/HCV) (McKillop et al., 2006; El-Serag, 2011; Saeed et al., 2014).

A large percentage (16-20) of Hepatitis C virus (HCV) infected sufferers, even in

Western countries have become patients of cirrhosis with the risk of HCC. The HCV genome

consists of a positive-sense single-stranded RNA approximately 9600 nucleotides long,

which encodes three structural proteins (core, E1, and E2), six non-structural proteins (NS2,

NS3, NS4A, NS4B, NS5A, and NS5B) and the ion channel protein p7. Each of these proteins

have a role in HCV entrance, infection, maturation or replication and are therefore a potential

antiviral target (Bartenschlager et al., 2004; Bartenschlager and Sparacio 2007; Priscilla et

al., 2011).

Herbal products are extensively used to manage the hepatic disorders worldwide

(Chaterrjee, 2000; Jamal et al., 2009). Essential oil extracted from areal parts of A.

absinthium is used for the prevention and treatment of liver diseases (Chinese

Pharmacopoeia, 2005).

Antioxidants play a key role in the human body to develop immunity which helps to

protect against various disorders caused by the reactive oxygen species. Generally ROS

produced by lipid peroxidation that damages the biological membrane and destroy enzymatic

functions (Galhardi et al., 2009; Ayesha et al., 2013). The development and progression of

certain life-threatening diseases and disorders like cancer, atherosclerosis, diabetes,

hyperlipidaemia, neuronal degeneration and hepato-toxicity are due to oxidative stress and

the plants and plant based products being enriched source of antioxidants work well against

such ailments (Alpsoy et al., 2010; Cederbaum, 2010).

Medicinal plants have the capability to produce a wide range of chemicals which are

not only used to carry out some biological functions, but assist in defending against attacks

40

from pest and parasite such as fungi, insects and herbivorous mammals. The major concern is

the safety to improve quality of patients’ life and development of new drugs for different

diseases throughout the world. Synthetic species are being tried on the basis of their chemical

compositions which improve the impact of the safety and efficacy of the herbal products

(Santos et al., 2008).

Phytochemicals from plant sources play a pivotal role in the improvement of human

health (Ahmed et al., 2011). Many of these phytochemicals have long term beneficial effects

on health when consumed by human and can be used effectively to treat human diseases. In

fact, the production of free radicals during metabolism and other activities beyond the

antioxidant ability of a biological system leads to oxidative stress (Astley, 2003) and foods or

medicinal plants rich in natural antioxidants such as phenolic are able to reduce this risk.

Today, the use of synthetic antioxidants in food products is mostly discouraged because of

their adverse effects (Hussain et al., 2008). On the other hand, use of natural antioxidants in

foods is gaining a considerable popularity due to their prospective health benefits and

pharmacological properties (Hamid et al., 2012; Kok et al., 2012; Sobia et al., 2012). While it

is reported that Ayurvedic drugs rarely cause side effects there is little data available to

support this. A possible reason is poor documentation and unawareness by Ayurvedic

physicians to collectively use this information for reports. However adverse effects may

occur due to overdosing of the Ayurvedic drugs (Rastogi, 2007).

Plant secondary metabolites possess a variety of components having pharmacological

actions and therapeutic applications. The phenolic compounds inhibit free radical mediated

processes and also have antioxidant effects (Zhao, 2005). In spite of incredible advancement

made in allopathic medicine, no effective hepatoprotective medicines are available. Plant

based drugs are known to play a vital role in the management of liver diseases (Gupta et al.,

2006; Desai et al., 2010). A variety of traditional remedies or herbal-derived compounds are

investigated for the treatment of hepatic disorders. Results revealed that herbal compounds

are effective because of antioxidant-related properties (Ljubuncic et al., 2005; Raja et al.,

2007; Wu et al., 2007; Wang et al., 2007). Silymarin is widely used for the treatment of liver

diseases worldwide, and its pharmacological effects modulate oxidative-stress-induced liver

injuries (Das and Vasudevan, 2006; Pradhan and Girish, 2006; Brandon-Warner et al., 2012).

Silybum marianum commonly known as milk thistle is widely cultivated due to its

striking medicinal values. It gained importance when it was found that silymarin was the

main active component with hepatoprotective properties (Ramasamy and Agarwal, 2008; El-

http://en.wikipedia.org/wiki/Phytochemicals

41

Kamary et al., 2009). Silymarin, isolated from S. marianum has three main isomeric

flavonolignans namely, silybin, silydianin and silychristin. Silymarin is a membrane

stabilizer that prevents lipo-peroxidation and also acts as free radical scavenger having

antioxidant potential that increases intracellular concentration of glutathione (Shalan et al.,

2005; Fleming, 2005; Wu et al., 2009). Silybin inhibits RNA and protein synthesis in gram

positive bacteria. Silymarin has proved to be a hepatoprotective in many experimental models

of liver damage. It protects the liver in three ways: by enhancing DNA polymerase,

stabilizing cell membranes and scavenging free radicals (Zuber et al., 2002). Clinically

silymarin is used to manage hepatic disorder including chronic inflammatory liver disease

and hepatic cirrhosis. Silybin, isolated from silymarin is used to treat a number of liver

disorders (Gordon et al., 2006; Payer et al., 2010).

Silymarin is well tolerated in subjects with chronic hepatitis C (CHC) and has some

effects on serum HCV RNA. Moreover the silymarin is an ideal choice at this time that is

used for lowering alanine aminotransferase levels and ultimately improves the quality of life

of hepatic patients (Agarwal et al., 2006). Silymarin exerts anti-inflammatory and antiviral

effects in the patients suffering from hepatitis C virus (HCV), thus making it good candidate

for the management of liver disorders (Trappoliere et al., 2009; Wagoner et al., 2010;

Morishima et al., 2010).

Many scientists have demonstrated the anticancer, anti-inflammatory and anti-fibrotic

potential of silymarin as it is able to suppress the proliferation of tumour cells (prostate,

breast, ovary, colon, lung and bladder) (Singh et al., 2004; Brandon-Warner et al., 2012;

Sadava and Kane 2013). As an anti-inflammatory agent both in vivo and in vitro, silibinin

inhibits tumor necrosis factor alpha (TNF-α) production by macrophages and monocytes (Al-

Anati et al., 2009; Bannwart et al., 2010). The cytoprotective, anti-carcinogenic and anti-

inflammatory effects of silymarin is due to inhibition of activation of the kinases and NF

kappa B (Gurley et al., 2006). The bioavailability of enterally administered silymarin is

inadequate because the compound is partially or incompletely soluble in water and only 20-

50 percent is absorbed from the gastrointestinal tract after ingestion. The absorption can be

enhanced significantly if silybin is administered with cross linked polymers (Voinovich et al.,

2009). The flavonolignans in S. marianum work in four different ways to accomplish

beneficial effects in the liver. The first one is anti-inflammatory, antioxidants, scavengers and

regulators of intracellular glutathione content. Secondly, it behaves as membrane stabilizers

and permeability regulators to prevent hepatotoxic chemicals from entering liver cells.

Thirdly, the compounds have the ability to promote RNA synthesis, helping to regenerate the

42

liver. Finally, these compounds can inhibit the transformation of stellate hepatocytes into

myofibroblasts, which is the process that leads to cirrhosis (Fraschini et al., 2002).

In addition to silymarin another plant that has similar therapeutic character is Rheum

emodi (Yen et al., 2000; Semple et al., 2001; Alam et al., 2005; Ibrahim et al., 2008; Radhika

et al., 2010). The root of Rheum emodi is used in curing diarrhea, spleen, inflammation,

dropsy and liver inflammation (Ahmed et al., 2012). Anthraquinone derivatives extracted

from Rheum emodi are used in the management of liver and cancer disorders. Marsupsin and

maesopsin root extracts of Rheum emodi have shown strong antioxidant potential (Krenn et

al., 2003). Methanolic and aqueous extracts of Rheum emodi (root) are reported to possess

antioxidant and anticancer potential (Rajkumar et al., 2010). The anticancer effect of aloe-

emodin has been established in human cancer cell lines, Hep G2 and Hep 3B. Aloe-emodin

inhibits cell proliferation and induces apoptosis in both examined cell lines by different

antiproliferative mechanisms (Kuo et al., 2002). Ethyl acetate extract of Rheum emodi shown

to have immune enhancing activity on cell lines. The effect was dose-dependent as it

increased the release of nitric oxide and cytokine IL-12, TNF-a and decreases in IL-10 in

macrophage cell lines in the presence of extract alone (Kounsar et al., 2011). The

anthraquinone derivatives, such as emodin, aloe-emodin, chrysophanol, rhein and physcion

are reported to possess anti-angiogenic activity and prevent blood vessel formation in zebra

fish embryos (He et al., 2009). Rhizome extract of Rheum emodi exhibited antidiabetic

activity by enhancing the peripheral utilization of glucose, correcting impaired liver and

kidney glycolysis and limiting its gluconeogenic process (Radhika et al., 2010).

Artemesia absinthium commonly known as wormwood is another plant of interest in

the present study which is used in various diseases for its antiseptic, stomachic, carminative,

antispasmodic, febrifuge, cholagogue, anthelmintic and antimicrobial properties (Guarrera,

2005; Kordali et al., 2006; Caner et al., 2008; Tariq et al., 2009; Fiamegos et al., 2011). A.

absinthium exhibited hepatoprotective potential against chemically as well as

immunologically induced acute liver injuries in mice (Amat et al., 2010). Artemesia

absinthium extracts also possess a strong antiradical and antioxidant activity in vitro

(Canadanovic-Brunet et al., 2005). The characteristic bitterness of wormwood is due to the

presence of sesquiterpene lactones such as absinthin, ketopelenolide b, anabsin and

anabsinthin. Flavonoids lignans and polyphenols are also present in extracts of Artemesia

absinthium (Aberham et al., 2010; Ivanescu et al., 2010).

WHO is focusing its attention on the natural assets of the Medicinal and Aromatic

Plants (MAP), as greater than 80% of the world still depends on conventional preparations.

43

Monitoring and categorization of MAP to their natural habitats, in addition to beginning of

"wild" plants into farming should be a strategic priority. As natural resources are abundant

but vary considerable, they should be followed and monitored in normal habitats (Baricevic

et al., 2004; Kumar et al., 2011). Synergistic interactions that occur between compounds in

herbal preparations or Phytomedicines are of great importance. Synergism often explains the

efficacy of a preparation, especially when needed in only small doses. The bioactivity, or

efficacy, of one compound in a herbal mixture often decreases when isolated from the

mixture. This is true both in single plant preparations as well as phytomedicines containing

more than one plant. The use of whole or partially purified extracts containing multiple active

ingredients is essential to the philosophy of herbal medicines (Williamson, 2001).

Hepatotoxicity can be induced in experimental animals by treating with carbon

tetrachloride (CCl4) which is a volatile, odorless and colorless chemical (Tatiya et al., 2012).

Paracetamol or acetaminophen is widely used to reduce pain and fever. It starts its action in

approximately 11-29.5 minutes after taken orally with 1-4 hours half-life (Moller et al.,

2005). Acute overdoses of paracetamol produce toxic effects and cause damage the main

organs such as kidney, brain and liver and in rare cases a normal dose can produce such toxic

effects. One of the main causes of acute liver failure is paracetamol in the Western world

(Khashab et al., 2007; Hawkins et al., 2007; Daly et al., 2008). Drug induced liver injury is

one of the main causative factors that remain a major clinical and regulatory challenge

(Russmann et al., 2009). Paracetamol is firstly converted into non-reactive metabolites in the

liver by glucuranidation and sulphation reactions when took in high doses. These metabolites

are later converted into a reactive metabolite, namely N-acetyl-p-benzo-quinoneimine, known

to be toxic for the liver. The resulting metabolite covalently binds to the oxidized lipids and

sulphydryl groups in the liver tissue and leads to the severe damage of cell membranes

(Kupeli et al., 2006). The main effect of paracetamol intoxication in human and animals is its

acute centro lobular necrosis (Macnaughton, 2003; Hewawasam et al., 2003). Animal models

are helpful to analyze intra hepatic responses resulting from experimental infection of HCV

to test the immunogenicity and efficacy of vaccine candidates (Bukh, 2004; Kremsdorf and

Brezillon 2007).

The literature review supports that if two or more medicinal plants of similar

pharmacological activities are use in combination they might have a potent product owing to

synergistic effect (Li et al., 2014). CGX is a commercially available hepato-therapeutics

consisting of thirteen herbal plants, which is produced from a traditional Chinese formula

http://en.wikipedia.org/wiki/Oral_administration

http://en.wikipedia.org/wiki/Biological_half-life

http://en.wikipedia.org/wiki/Overdose

http://en.wikipedia.org/wiki/Hepatotoxicity

http://en.wikipedia.org/wiki/Acute_liver_failure

http://en.wikipedia.org/wiki/Paracetamol_toxicity

http://en.wikipedia.org/wiki/Western_world

44

meaning “liver cleaning”. This drug has been reported to show hepato-therapeutic properties

in animal models, and is particularly relevant to the treatment of hyper-lipidemia, alcohol

induced liver damage and liver fibrosis (Son et al., 2001, 2003; Shin et al., 2006). CGX has

been recommended for various liver diseases including chronic viral B-type hepatitis, and has

demonstrated a high therapeutic index range in a toxicological study of beagle dogs (Cho et

al., 2001; Choi et al., 2006). “Jigrine CL” another herbal combination of Hamdard

Laboratories, Pakistan which is composed of eight medicinal plants used to manage liver

disorders (Ahmed et al., 2012). An aqueous extract of the three plants namely A. capillaris,

L. japonica and S. marianum in combination successfully restore the liver function against

CCl4 induced liver damaged in rats (Yang et al., 2012). Combination of Menthalongifolia,

Cyperus rotundus and Zingiber officinale are widely used for the treatment of multiple

gastrointestinal diseases, particularly for the treatment of patients with irritable bowel

syndrome (Sahib, 2013). Pharmacological activities of DSH a herbal combination of three

herbs Radix Salviae Miltiorrhizae, Carthamus tinctorius and Fructus Cartaegi was used to

explore its applications in cardiovascular diseases (Li et al., 2014). While some of

biochemical and pharmacological effects of S. marianum, A. absinthium and Rheum emodi

have been reported but there is insufficient data available about their medicinal use in

combination.

Keeping in view the importance of use of medicinal plants in combination, it is

hypothesized that a combination of S. marianum, A. absinthium and Rheum emodi may prove

to be an excellent hepatoprotective product. The present research therefore is focused to

explore the hepatoprotective potential of the selected medicinal plants (S. marianum, A.

absinthium and Rheum emodi) in combination against paracetamol induced liver injury in

rabbits.

Aims and Objective

Keeping in view the above said discussion the following objectives were planned

To explore Potential of selected Medicinal Plants as hepatoprotective

and antioxidant.

To evaluate the synergistic potential of various plants combinations.

45

To explore the toxicity of selected medicinal plants by in vivo and in vitro assays.

Determination of biologically important or active chemical compound in selected

medicinal plants through LC-MS.

Chapter No. 2

REVIEW OF LITERATURE

2.1. Importance of medicinal plants:

Medicinal plants are rich in secondary metabolites and important potential source of drugs to

treat variety of diseases (Wink, 2013; Rasmussen and Ekstrand 2014). These secondary

metabolites include alkoloids, glycosides, coumarins, flavonides, steroids etc. (Zhao, 2005;

Lee et al., 2006; Shibano et al., 2007; Sengul et al., 2011). Medicinal plants are considered to

be more effective and safe alternative source of treatment for liver toxicity (Thirumalai et al.,

2011). These plants provide biologically active molecules that form structures for the

development of tailored derivatives such that they improve activity and hence reduce toxicity.

The medicinal plants are used in two ways: crude formulation of plants to treat various

46

ailments by the local physician in developing countries and use of active ingredients by the

Pharmaceutical companies (Baqar, 2001). The Ayurvedic herbs are commercially used by

hakim in the form of extracts to treat various pathological ailments (Mahmood et al., 2003).

On the other hand the ignorance of common people about the importance of medicinal plants

and which ones are harmful as this causes avoidance of medicinal plants because they use

them as per their need and hence causes eradication of novel medicinal plants (Malik &

Husain, 2007).

2.2. Uses of medicinal plants in various disorders

Herbal products are extensively used in the management of hepatic disorders all over

the world (Desai et al., 2010; Rasool et al., 2014). Silymarin extract is reported in the

biomedical literature to have inflammatory, anti-fibrotic activities, anti-tumor, and

hepatoprotective (Brinda et al., 2012; Colturato et al., 2012). Plant derived compounds are

also used for the treatment of cancer. Silymarin is used to cure liver and gallbladder disorders

including jaundice, hepatitis, cirrhosis (Kren and Walterova 2005; Rainone, 2005). Several

anti-cancer compounds including vinblastine, taxol, camptothecin, vincristine, the

derivatives, topotecan, irinotecan and etoposide derived from epipodophyllo-toxin are in

clinical use all over the world (Butlet, 2004; Ramawat and Merillon, 2008). Anthraquinone

derivatives demonstrate evidence of antimicrobial, antifungal, anti-proliferative, anti-

parkinsons, immune enhancing, antiviral and antioxidant activities (Zargar et al., 2011).

Epidemiological studies suggest that the uses of fruits and vegetables in excess amount that

contain flavonoids and phenolic can reduce the risk of several types of cancer including oral,

breast, lung, larynx, colon, pancreas, and prostate cancer (Ross and Kasum, 2002). Silybum

marianum has cellular modulatory properties, including hepatoprotection and cancer

chemoprevention (Arlene et al., 2013).

Liver is the main organ that performs several functions such as metabolism of

proteins, carbohydrates, lipids, detoxification, storage of glycogen, vitamins A, D and B12,

production of several coagulation factors and excretion (Opoku et al., 2007; Maheswari et al.,

2008; Sharma and Sharma, 2010). Acute viral hepatitis is a common cause of acute hepatitis

and infection with hepatitis viruses that ranges from 80–100% in developing countries and

20–40% in developed countries (Shepard et al., 2005; Meky et al., 2006). Hepatitis is one the

threatening disease that has spoiled a large number of families. One of the complications of

hepatitis is hepatocellular carcinoma (HCC) which became the fifth most common cause of

death across the world (Yang and Roberts, 2010; El-Serag, 2011). The most common cause

of developing HCC is linked with risk factors due to hepatitis B (HBV) and C (HCV) virus

47

(McKillop et al., 2006 and El-Serag, 2011). Infection with the hepatitis C virus (HCV) is a

major global health problem, as persistent viral infection that leads to liver cirrhosis and

hepatocellular carcinoma (Shepard et al., 2005).

Silybum marianum is an annual and biannual plant of the family Asteraceae

(Rambaldi et al., 2005) and this plant grows in warm, dry soil and blooms in July-August.

Each seed is about 5-7 mm long, up to 2-3 mm wide and 1.5 mm thick, with a glossy,

brownish black to greyish, brown husk. The freshly milled fruits have a cocoa-like odor and

an oily and bitter taste (Abenavoli et al., 2010). Rheum emodi commonly known as

revandchini (Prasad and Purohit 2001), is a storehouse of a large number of anthraquinone

derivatives such as physcion, emodin, chrysophanol, rhein, aloe emodin, etc. (Krenn et al.,

2004) are reported for a large number of biological activities including antioxidant (Yen et

al., 2000), anti-microbial (Babu et al., 2003), anti-fungal (Agarwal et al., 2000), larvicidal,

cytotoxic (Yang et al., 2003) and hepatoprotective activities (Akhtar et al., 2009).

The genus Artemisia consists of about 500 species distributed throughout the world,

Artemisia absinthium exhibiting a wide range of biological activities including traditional

medicine (Lachenmeier, 2010). The essential oils of A. absinthium have been reported as

having free radical scavenger (Canadanovic-Brunet et al., 2005), antifungal (Juteau et al.,

2003; Valdes et al., 2008), anti-helmintic (Tariq et al., 2009), hepatoprotective (Gilani and

Janbaz, 1995), antiprotozoal and anti trichinellosis effects (Caner et al., 2008). Thujone-rich

oils have acaricidal (Chiasson et al., 2001) and insecticidal (Kordali et al., 2006). In addition,

organic extracts of this plant species exhibited anti-feedant and toxic effects on Leptinotarsa

decemnlineata (Erturk and Uslu, 2007) and anti-feedant effects on Rhopalosiphum padi

(Halbert et al., 2009). Moreover, water extracts are phytotoxic to Lolium perenne and Bromus

inermis (Corbu and Cachita-Cosma, 2009).

The chemical composition of wormwood oils determines the chemotaxonomy of the

plant (Parejo et al., 2002). The major components of wormwood are myrcene, sabinene (Orav

et al., 2006) thujone, pinene cis chrysanthenol, bornyl acetate (Rezaeinodehi and Khangholi,

2008) and caryophyllene oxide p-cymene 1,8-cineole (Basta et al., 2007 ), and both Z-

epoxyocimene and chrysanthenyl acetate show antifungal activity. Chrysanthenyl acetate,

acts as antiprostaglandins due to their analgesic effect inhibiting the enzyme prostaglandin

synthetase (Badillo et al., 2010).

Multi target therapeutics is a promising paradigm for drug discovery which is

expected to produce greater levels of efficacy with fewer adverse effects and toxicity than

mono therapies. Medical herbs featuring multi-components and multi-targets may serve as

48

valuable resources for network based multi-target drug discovery. An integrated systems

pharmacology platform for drug discovery and combination, with a typical example applied

to herbal medicines in the treatment of cardiovascular diseases was reported in this study.

Firstly, a disease-specific drug-target network was constructed and examined at systems level

to capture the key disease relevant biology for discovery of multi-targeted agents. Secondly,

considering an integration of disease complexity and multi-level connectivity, a

comprehensive data base of literature- reported associations, chemicals and pharmacology for

herbal medicines was designed. Thirdly, a large scale systematic analysis combining

pharmacokinetics, chemo-genomics, pharmacology and systems biology data through

computational methods was performed and validated experimentally, which results in a

superior output of information for systematic drug design strategies for complex diseases (Li

et al., 2014).

2.3. Biochemical profiling of Silybum marianum:

Metabolomic analysis is carried out using liquid chromatography/tandem mass

spectrometry (LCMS) technique which identifies several metabolites, such as amino acids,

nucleotides and lipids (Eckel-Mahan et al., 2012).

Silymarin extracted from fruits and seeds of S. marianum, consists of a mixture of

three structural isomers including the most active component silycristin (20%), silibinin (60-

70%) and silydianin (10%) (Pradhan and Girish, 2006). The main active constituents, namely,

silychristin, silydianin, diastereo-isomers of isosilybin (isosilybin A and B) and diastereo-

isomers of silybin (silybin A and B) are completely separated and quantified by LC/MS (Lee

et al., 2007; Liu and Yue-wei1, 2012). The ultra-performance liquid chromatography (UPLC)

interfaced with the electrospray ionization (ESI) tandem mass spectrometer (MSn) was

developed for the simultaneous determination of silychristin A and B, silydianin, silybin A

and B and isosilybin A and B, from the S. marianum (Wang et al., 2010). Different liquid

chromatographic tandem mass spectrometric technique was used for simultaneous analysis of

silydianin, silychristin, taxifolin, isosilybin A and isosilybin B, silybin A and silybin B in

human plasma (Brinda et al., 2012).

The fragmentation properties of silymarin such as silybin A and silybin B, silychristin

A and silychristin B, isosilybin A and isosilybin B and silydianin was studied by energy

dependent collision induced dissociation (CID) technique. The collision energies dependence

of the fragmentation was studied on the basis of the breakdown curves i.e. the relative

intensities of fragment ions versus collision energy. The fragmentation of isosilybin and

49

silybin were distinct irrespective of minimal difference between their structures (Kuki et al.,

2012). The silymarin was characterized with the aid of several analytical techniques

including; 1H NMR, UV and FTIR. The results demonstrated that the developed alginate

based hydro gel micro particles encapsulating silymarin loaded poly (DL-lactide-co-

glycolide) nanoparticles (PLGA NPs) can be used as biodegradable carriers which confer

sustained oral release of silymarin in addition to enhancing its overall dissolution and oral

bioavailability (Ibrahim et al., 2011).

2.3.1. Therapeutic uses of Silybum marianum

Self-emulsifying pellets to deliver silymarin are the most suitable formulations and

pharmacokinetic data have demonstrated that the extrusion/ spheronisation is a viable

technology to produce self-emulsifying pellets of good quality, capable to improve in vivo

oral bioavailability of main components (Iosio et al., 2011).

2.3.2. Role of Silybum marianum in hepatic disorders (clinical trials)

S. marianum is a blend of at least 7 flavonolignans which has been used for centuries

as adjunct treatment for a variety of liver ailments including cirrhosis, alcoholic liver disease

and hepatitis (Gazak et al., 2007; Brandon-Warner et al., 2012; Rasool et al., 2014).

Hepatocellular carcinoma (HCC) is a global health burden with limited treatment options and

poor prognosis. Silibinin have shown hepatoprotective and anti-tumorigenic effects in vivo

and in vitro by suppressing oxidative stress and proliferation. The results showed that

silibinin exerted marginal hepatoprotective effects in early stages of hepato carcinogenesis

(Brandon-Warner et al., 2012).

The role of silymarin in patients with chronic hepatitis C virus (HCV) infection

relapsed with interferon based therapy. A total number of 154 patients with chronic HCV

infection and serum alanine aminotransferase (ALT) levels of 65 U/L or greater, were non

responder with interferon based therapy. Patients were haphazardly assigned to receive 420

mg silymarin, 700 mg silymarin, or matching placebo administered 3 times per day for 6

months. After 24 weeks of treatment, only 2 patients in each treatment group i.e. 3.8 % for

placebo, 4.0 % for 420 mg silymarin and 3.8 % for 700mg silymarin respectively, met the

primary objective. The primary outcome measure was serum ALT level of 45 U/L or less

(considered within the normal range) or less than 65 U/L, provided this was at least a 50%

decline from baseline values. The mean decline in serum ALT, 4.3 U/L for placebo, 14.4

U/L for 420 mg silymarin, 11.3 U/L for 700 mg silymarin; HCV RNA levels mean change,

0.07 log10 IU/mL for placebo, 0.03 log10 IU/mL for 420mg silymarin, 0.04 log10 IU/mL

50

for 700 mg silymarin. The adverse event profile of silymarin was found to be comparable

with that of placebo (Fried et al., 2012).

In an attempt to generate analogues with improved in vivo properties particularly

reduced metabolic liability a semi synthetic series were prepared in which the hydroxyl

groups of silybin B were alkylated. A total of five methylated analogues of silybin B were

synthesized and the relative potency of all compounds was determined in a 72 h growth-

inhibition assay against a panel of three prostate cancer cell lines (DU-145, PC-3, and

LNCaP), human hepatoma cell line (Huh7.5.1) and compared to natural silybin B.

Compounds were also evaluated for inhibition of both cytochrome P450 2C9 (CYP2C9)

activity in human liver microsomes and hepatitis C virus infection in Huh cell line. The mono

methyl and dimethyl analogues showed enhanced activity in terms of cytotoxicity, CYP2C9

inhibitory potency, and antiviral activity (up to 6-fold increased potency) compared to the

parent compound, silybin B. In a nut shell, these data suggested that methylation of

flavonolignans can increase bioactivity (Arlene et al., 2013).

Silymarin is wide used in treating acute or chronic hepatitis. The inclusion criteria for

patients enrolled in this study were those with had acute clinical hepatitis and serum alanine

aminotransferase (ALT) levels 42.5 times the upper limit of the normal value. The

intervention consisted of three time’s daily ingestion of either a standard recommended dose

of 140 mg of silymarin, or a vitamin placebo for four weeks with an additional four week

follow up. The primary assessment were based on signs and symptoms of acute hepatitis and

liver function tests on days 2, 4, 7, 14, 28 and 56. No side effects were recorded and both

silymarin and placebo were well tolerated. Patients randomized to the silymarin group had

quicker resolution of symptoms related to biliary retention: dark urine, jaundice and scleral

icterus. There was a reduction in indirect bilirubin among those assigned to silymarin but

other variables including direct bilirubin, alanine aminotransferase (ALT) and aspartate

aminotransferase (AST) were not significantly reduced (El-Kamary et al., 2009).

Silymarin is used to prevent the complications of chronic hepatitis C virus infection

and exceptionally in this study silymarin did not improve liver functions. A total number of

177 chronic hepatitis C virus patients were randomly selected to receive either silymarin or

administrated multivitamin supplements only. The recommended dose of silymarin that can

safely be taken for 1 year which improved symptoms and general well-being of patients, but

it produced no effect on hepatitis C virus viremia, serum ALT and ultrasound markers for

hepatic fibrosis (Tanamly et al., 2004).

2.3.3. Use of Silybum mariamum in age related oxidative stress

51

Brain is susceptible to oxidative stress and is associated with age related to brain

dysfunction; the effect of silymarin was evaluated in the brain of aged and young rat. Rats

were administrated with silymarin (200 and 400 mg/kg/day). Both doses were most effective

in the reduction of oxidized proteins in aged brain. These results suggest that silymarin

contributed to the prevention of age related and pathological degenerative processes in the

brain (Galhardi et al., 2009).

2.3.4. Anti-proliferative role of Silybum mariamum

The anti-proliferative effects of the S. marianum were evaluated against different

tumor types. The effect of silibinin on proliferation was studied by performing MTT cell

viability assay. The results are reported as mean values with standard errors and the

experiments were performed in triplicate. The MTT test showed a strong dose dependent

inhibitory effect, 50% inhibition of cell viability (IC50) in Fet- and Geo- lines at 72 h treated

with 75 g/mL. An IC50 dose of 40µg/mL was obtained in HCT116, a poorly-differentiated cell

line, at 72 h. FACS analysis demonstrated statistically significant cell-cycle arrest in all the

cell lines. G2 M phase arrested in Fet- and Geo- cell lines (P < 0.001) and a G1 arrest in

HCT116 (P, 0.005) were noted. Silibinin extensively inhibited proliferation through cell cycle

arrest via inhibition of cyclin CDK promoter activity (Hogan et al., 2007).

Silymarin is used against various liver conditions in both experimental models and

clinical settings. The investigations and mechanistic studies regarding possible molecular

targets of silymarin for cancer prevention were established through a number of studies as

cancer chemopreventive in both in vitro and in vivo models. Silymarin modulates imbalance

between cell survival and apoptosis through interference with the expressions of cell cycle

regulators and proteins involved in apoptosis along with silymarin also showed anti-

inflammatory as well as anti-metastatic activity (Ramasamy and Agarwal, 2008).

2.3.5. Role of Silybum mariamum on metabolic pathways

The effect of silibinin on metabolic pathways is responsible for the maintenance of

glycaemia, mainly glycogenolysis and gluconeogenesis. Silibinin inhibited gluconeogenesis

in the fasted state, inhibited glycogenolysis and glycolysis in the fed state. The mechanisms

by which silibinin exerted these activities were manifold and complex. It inhibited the

pyruvate carrier and activity of glucose 6-phosphatase and reduced the effectiveness of

mitochondrial energy transduction (Parisoto et al., 2011).

2.4. Biochemical profiling of Rheum emodi

Rhubarb (Rheum emodi) is a perennial herb and about 200 compounds with six

different types of skeletons (anthraquinone, anthrone, stilbene, flavonoids, acylglucoside, and

52

pyrone) have so far been isolated from eighteen species of the genus Rheum. These

constituents showed wide ranging pharmacological actions including cathartic, diuretic,

anticancer, hepatoprotective, anti-inflammatory and analgesic effects, as well as toxicological

effects. Chemical fingerprint, LCMS and other analytical techniques have been used for the

quality control of rhubarb (Zheng et al., 2013).

The bioassay guided chemical examination of Rheum emodi based on spectroscopic

and degradative evidence were reported the presence of anthraquinone structures in the

isolation of two new oxanthrone esters, revandchinone-1, revandchinone-2, a new

anthraquinone ether revandchinone-3 and a new oxanthrone ether, revandchinone-4. The

oxanthrone ether was reported to be found for the first time and it was studied for

antibacterial and antifungal activity (Narendra et al., 2005; Min et al., 2007; Malik et al.,

2010; Sharma et al., 2012).

The Rheum emodi was reported to be found as a depot of a large number of

anthraquinone derivatives having enormous biological importance. The researcher studied

interactions between different proteins and natural dye (anthraquinone derivatives based)

isolated from Rheum emodi. The stability parameters were evaluated and there were found a

change in the fluorescence intensity of the dye as a function of temperature due to the

differential interaction of the dye with various conformations of a protein. The effect of the

change in polarity of the solvent on the fluorescence emission spectra of dye was studied and

high quantum yield was observed in alcohol as compared to water. The main compounds

present in it were the anthraquinones glycosides characterized by RP-HPLC (Khan et al.,

2012).

2.4.1. Therapeutics uses of Rheum emodi:

The major bioactive compound present in emodin belongs to anthraquinone class and

its anti-proliferative effect was evaluated against HepG2, MDA-MB231 and NIH/3T3 cells

lines. A large quantity of emodin was isolated from the roots of Rheum emodi. The

derivatives-3 and -12 strongly inhibit the proliferation of HepG2 and MDA-MB231 cancer cell

line with an IC50 of 5.6, 13.03 and 10.44, 5.027, respectively, which is comparable to

standard drug epirubicin (III) available in the market. The compounds 3 and 12 were also

capable of inducing cell cycle arrest and caspase dependent apoptosisin HepG2 cell lines and

exhibit DNA intercalating activity (Narender et al., 2013).

Rheum emodi is traditionally used as diuretic, liver stimulant, purgative/ cathartic,

stomachic, anticholesterolaemic, antitumour, antiseptic and tonic. Various anthraquinone

derivatives including emodin, aloe-emodin, physcion, chrysophanol, rhein, emodin glycoside

53

and chrysophanol glycoside are present as the main constituents. New components such as

sulfemodin 8-O-b-D-glucoside, revandchinone 1, revandchinone 2, revandchinone 3,

revandchinone 4, 6 methyl rhein and 6 methyl aloe emodin are reported from the same

species. Anthraquinone derivatives demonstrated evidence of antimicrobial, antifungal,

antiproliferative, antiparkinson’s, immune enhancing, antiviral and antioxidant activities

(Zargar et al., 2011).

The effect of Rheum emodi as dye and its dyed wool yarns against two bacterial

species (E. coli and Staphylococcus aureus) and two fungal species (Candida albicans and

Candida tropicalis) was investigated. The dyeing was carried out using 5% and 10% o.w.f.

dye concentration in presence and absence of ferrous sulphate, stannous chloride and alum

mordants. The color strength and fastness properties of dyed samples were also assessed.

FTIR spectra of untreated, mordanted and dyed wool yarn were investigated to study the

interaction between fibre, mordant and dye. The structural morphology of wool yarn was

investigated by scanning electron microscopy (SEM). The susceptibility tests for Rheum

emodi were carried out in terms of disc diffusion, growth curve and viability assays against

all the tested micro-organisms. Dyed samples showed very effective antimicrobial properties

showing more than 90% microbial reduction in both bacterial as well as fungal population

(Khan et al., 2012).

2.5. Biochemical profiling of Artemisia absinthium

The principal components in the oils of Artemesia absinthium were myrcene (0.1–

38.9%), 1,8 cineole (0.1–18.0%), sabinene (0.9–30.1%), artemisia ketone (0–14.9%), linalool

and α-thujone (1.1–10.9%), β-thujone (0.1–64.6%), trans-epoxyocimene (0.1–59.7%),

carvone (0–18.5%), trans-verbenol (0–11.7%), curcumene (0–7.0%), (E)-sabinyl acetate (0–

70.5%), neryl butyrate (0.1–13.9%), neryl 2-methylbutanoate (0.1–9.2%), chamazulene 0–

6.6% and neryl 3-methylbutanoate (0.4–7.3%) (Orav et al., 2006).

The composition of the plant extract, carrying volatile and non-volatile oils, was

analyzed by gas chromatography mass spectrometry (GCMS) and HPLC–DAD, respectively.

The anti-feedant and phytotoxic activity of the extracts was tested on insect pests

(Spodoptera littoralis, Myzus persicae and Rhopalosiphum padi) and plants (Lactuca sativa

and Lolium perenne). The supercritical extracts exhibited stronger anti-feedant effects than

the traditional ones (up to 8 times more active) with moderate selective phytotoxic effects on

L. perenne root growth (<50% inhibition) (Martin et al., 2011).

A total of 18 polyphenolic compounds were identified and quantified found in

Artemisia leaves, including hydroxybenzoic acids, hydroxycinnamic acids, flavonols, and

54

catechins. It was noted that total phenolic content of Artemesia annua and Artemesia

Stelleriana leaves were significantly lower than the other four species. Ferulic and caffeic

conjugates acids were the most dominant hydroxycinnamic acid and gallic acid and catechin

were the most dominant hydroxycinnamic acid and catechins respectively (Isabel et al.,

2011).

The chemical composition of the essential oil of Artemisia nanschanica includes 34

compounds (92.26 % of total composition) identified with the major constituents of the oil

were terpenoids (70.86 %), thujone (21.3 %), heptadiene (16.52 %) and linalool (10.94 %).

The content of 1, 8 cineole (9.43 %) and camphor (6.66 %) were more than 6 % (Shang et al.,

2012). The major components of Artemesia absinthium oil were myrcene (8.6-22.7 %), cis-

chrysanthenyl acetate (7.7-17.9 %), a dihydrochamazulene isomer (5.5-11.6 %), germacrene

D (2.4-8.0 %), thujone (0.4-7.3 %), linalool acetate (trace-7.0 %), phellandrene (1.0-5.3 %),

and linalool (5.3-7.0 %), (Farukh et al., 2012).

2.5.1. Therapeutics uses of Artemesia Absinthium:

Qualitative and quantitative phytochemical analysis of the aqueous extract of A.

absinthium was performed on TLC and spectrophotometric assays. The dosage of aqueous

extract (50,100 or 200 mg/kg body weight/day) was administered orally to experimental

mice. The results demonstrated that the pre-treatment with AEAA significantly and dose-

dependently prevented chemically or immunologically induced increase in serum levels of

hepatic enzymes. Histopathology of the liver tissue showed that AEAA decreased the hepato

cellular necrosis and led to reduction of inflammatory cells infiltration. Phyto-chemical

analyses showed the presence of ses-quiterpene lactones, phenolic acids, flavonoids and

tannins in the AEAA (Amat et al., 2010).

Artemisia absinthium is conventionally used as anti-helminthic as whole plants and

plant extracts have demonstrated activity against gastrointestinal nematodes, treated with

1000 mg/kg body weight of A. annua or A. absinthium ethanolic extract or with 300 mg/kg

body weight of Artemesia annua essential oil daily for the five consecutive days. No

significant effects of treatment were seen with artemisinin or any of the extracts of Artemisia

species at the dosages studied (Jill et al., 2011). A. absinthium, aqueous, cold alcoholic and

hot alcoholic extract showed 35%, 55% and 21% inhibition in growth of Plasmodium

falciparum, respectively at 2.00 mg/mL (Saba et al., 2011). Antitussive effect of Echinacea

and Artemisia were evaluated. Four groups of mice were treated with normal saline,

dextromethorphan, Echinacea extract and Artemisia extract respectively before cough

induction. Cough was induced in a 1000 mL special glass chamber with ammonium

55

hydroxide (1 mL of 5% solution) cotton ball. The means of cough was significantly

decreased by dextromethorphan; Echinacea extract and Artemisia extract (Esmailirad et al.,

2012).

Chapter No. 3

MATERIALS AND METHODS

The study was carried out to develop and evaluate a suitable herbal combination to

manage hepatic disorders. In this regard three medicinal plants viz. Milk thistle (Silybum

marianum), Wormwood (Artemisia absinthium) and Rhubarb (Rheum emodi) were selected

and their hepatoprotective effect were studied in different combinations. The methods and

protocols followed in the study are as following.

3.1 Determination of Phytochemical constituents

3.1.1 Total phenolic content

Total phenolic content was determined according to the Folin–Ciocalteu method (Ho et al.,

2010) using gallic acid as the standard. The selected plant parts (10 g of each) were ground

and extracted in 100 mL methanol in orbital shaker keeping for 7 days. After filtration, the

filtrate was reduced using rotary evaporator. The extract (5 mg) was dissolved in 5mL of

methanol/ water (50:50 v/v). The extract solution (500 µL) was mixed with 500µL of 50%

Folin Ciocalteu reagent. The mixture was kept for 5 min followed by the addition of 1.0 mL

of 20% Na2CO3. After 10 min of incubation at room temperature, the mixture was

centrifuged for 8 min (150 g/5400 rpm) and the absorbance of the supernatant was measured

at 730nm. The total phenolic content was expressed as gallic acid equivalents (GAE) in mg/g

of sample.

3.1.2 Total flavonoid content

Total flavonoid content was determined following the procedure described by Barros

et al., 2011. The extract solution (0.125 mg/mL; 0.5 mL) was mixed with distilled water (2

mL) and subsequently with NaNO2 solution (5% of 0.15 mL). After 6 min, AlCl3 solution

(10%, 0.15 mL) was added and allowed to stand for further 6 min; thereafter, NaOH solution

(4%, 2 mL) was added to the mixture. Immediately, distilled water was added to bring the

final volume up to 5 mL. Then the mixture was properly mixed and allowed to stand for 15

min. The intensity of pink colour was measured at 510 nm. (+) Catechin was used to

56

calculate the standard curve (4.5×10-3–2.9×10-1 mg/mL) and the results were expressed as mg

of (+) catechin equivalents (CE) per g DW.

3.2. Antioxidants Potential

3.2.1. DPPH radical scavenging activity (DPPH assay)

The DPPH free radical scavenging activity of the extracts was determined according

to the method reported by Ho et al., 2010. A total amount (10 µL) of the test samples in

methanol, yielding a series of extract concentrations of 5, 10, 50 and 100 µg/mL,

respectively, in each reaction were mixed with 200 µL of 0.1 mM DPPH ethanol solution and

90 µL of 50 mM Tris HCl buffer (pH 7.4). Methanol (10 µL) alone was used as control for

this experiment. After 30 min of incubation at room temperature, the reduction in DPPH free

radicals was measured by reading the absorbance at 517 nm. (+) Catechin, a well-known

antioxidant, was used as the positive control. Three replicates were made for each test

sample. The inhibition ratio was calculated according to the following equation:

% inhibition = [(absorbance of control - absorbance of sample)/ absorbance of control] ×100

3.2.2 Reducing power assay

The reducing power of the extracts was determined according to the procedure

described by Ho et al., 2010 with slight modification by using (+) catechin as the standard. 1

mL of reaction mixture, containing 500 µL of the test sample in 500µL of phosphate buffer

(0.2 M, pH 6.6), was incubated with 500 µL of potassium ferricyanide (1%, w/v) at 50 0C for

20 min. The reaction was terminated by adding trichloro acetic acid (10%, w/v) and then the

mixture was centrifuged at 12,000g for 10 min. The supernatant solution (500 µL) was mixed

with distilled water (500 µL) and 100 µL of ferric chloride (0.1%, w/v) solution, then the

optical density (OD) was measured at 700 nm. The reducing power ability was expressed as

(+) catechin equivalents (CE) in milligrams per gram sample.

3.2.3. Antioxidant activity by DNA protection assay

The antioxidant activity by DNA protection assay was carried out for the plant

extracts following the method described by (Riaz et al., 2012) with some modifications.

For this assay pUC19 DNA 0.5 µg/µL was diluted (0.5 µg /3 µL) using 50 mM sodium

phosphate buffer pH 7.4. The 3µL of diluted pUC19 DNA was treated with 5 µL test

sample. After this, 4 µL of 30% H2O2 was added in the presence and absence of different

concentrations of test samples. The volume was made up to 15 µL with sodium phosphate

buffer (pH 7.4). Different concentrations of absolute methanol extract were used such as

57

1000, 100 and 10 µL/mL in the reaction mixture. The relative difference in migration

between the native and oxidized DNA was examined on 1% agarose by horizontal DNA

gel electrophoresis using a wide mini system (Techview, Singapore). 1gram agarose was

dissolved in 100mL of 1x TAE buffer and placed it in microwave oven for two minutes,

cooled and poured it in casting plate and let it to solidify. After solidification, gel was kept

in the buffer and samples were loaded in the wells one by one (Table 3.1). The gel was

stained with ethidium Bromide. The gels were documented by a Syngene model Gene

Genius unit (Syngene, Cambridge, UK). Protection to DNA was calculated based on the

DNA band corresponding to that of native in the presence and absence of different

concentrations (10µL, 100 µL and 1000 µL) of each plant extract.

Table.3.1. Sample Preparation for DNA protection assay:

Sample No DNA Loading dye H2O2 (30mM) Plant extract

1 3 µL 1 µ 0 0

2 3 µL 3 µL 15 µL 0

3 3 µL 3 µL 0 10 µL (conc. 100 µL)

4 3 µL 5 µL 15 µL 10 µL (conc. 10 µL)

5 3 µL 5 µL 15 µL 10 µL (conc. 100 µL)

6 3 µL 5 µL 15 µL 10 µL (conc. 1000 µL)

3.2.3.1. Composition of TAE buffer

Stock solution in one Liter was prepared by adding the following chemicals

242g of Tris base

57.1g of Glacial acetic acid

100mL of 0.5M EDTA (pH= 8)

The stock solution was diluted to 1X by using formula

C1V1= C2V2

3.3 Toxicological Screening

This part of study was conducted in the Bioassay Section, Medicinal Biochemistry

Lab, Department of Chemistry and Biochemistry University of Agriculture Faisalabad.

3.3.1 Hemolytic activity

The hemolytic of selected medicinal plants extracts were determined following the

method described by Riaz et al., 2012 with some modifications. Three mL of heparinized

58

human blood cells were gently mixed, poured into a sterile 15mL falcon tube and centrifuged

for 5 min at 850 g. The supernatant was poured off and the viscous pellet washed three times

with 5mL of chilled (4 ºC) sterile isotonic phosphate-buffered saline (PBS) solution, adjusted

to pH 7.4, to stabilize the pH mix it almost for half an hour at room temperature (25-30 ºC).

The washed cells was suspended in the 20 mL chilled, saline PBS buffer. Erythrocytes were

counted on haemacytometer (Fisher ultra-plane Neubauer ruling). The blood cell suspension

was maintained on wet ice and diluted with sterile (PBS) the cell count should be 7.068×108

cells/ mL for each assay. Aliquot of 20 µL of plant extract was taken into 2 mL ependoff

tubes. For each assay, Triton X-100 was taken as positive control and phosphate buffer saline

(PBS) as negative control, background (0% lyses). In each 2mL ependoff tube already

contain 20 µL samples and add 180 µL diluted blood cells suspension and mixed it with the

help of pipette tip. Tubes were incubated for 35 min at 37 ºC with agitation (80 rev / min).

Immediately after incubation, the tubes were placed on ice for 5 min then centrifuged for 5

min at 1310g. After centrifugation 100 µL supernatant were taken from the tubes and diluted

with 900 µL chilled PBS. All tubes were maintained on wet ice after dilution. Than 200 µL

into 96 well plates and three replicate was taken in well plate which contain one positive

control (100% of blood lyses) and other negative control (0% of blood lyses). Absorbance at

576 nm was taken at ELISA plate reader (µ Quant). Triton-X 100 (0.1%) were used as

positive control for 100% lyses and PBS buffer as negative control 0% lyses The experiment

was done in triplicate and mean ± S.E. was calculated by using this % hemolysis formula.

% hemolysis = Hb ABS / Hb100 % ABS × 100

3.3.2 Brine Shrimp Lethality test

3.3.2.1. Extraction

The selected plant parts (Seeds of S. marianum, aerial parts of A. absinthium and R.

emodi) of the medicinal plants were ground (10 g) and extracted in methanol (100 mL) at

room temperature for 7 days. The extract was filtered, with Whatman filter paper no. 42 and

the filtrate obtained was concentrated by putting the extract in open air.

3.3.2.2. Shrimp hatching process

Brine Shrimp eggs were hatched in a shallow rectangular dish (22cm×32cm×12cm),

one third of which was filled with saline water. An aluminum divider with several holes was

clamped in the dish to make two unequal compartments. The eggs (50mg) were sprinkled

into the larger compartment that was made darkened and smaller compartment was

59

illuminated. The set was maintained at 30–32 ºC and after 48h the phototropic napulii were

collected with pipette from the lightened side, having been separated by the divider from their

shells (Haq et al., 2012).

3.3.2.3. Experimental procedure and observation

The shrimps were transferred to each sample vial using disposable pipette and saline

water was added to make a total of 5 mL. The napulii could be counted in the stem of the

pipette against a light background. A drop of dry yeast suspension (3mg in 5mL of saline

water) was added as food to each vial. The vials were maintained at room temperature.

Survived shrimps were counted after every 3h up to 24h.

% Death= [(test−control) / 100−control)] ×100

3.3.3. Mutagenicity assay

This test was based on the validated Ames bacterial reverse mutation test but was

performed entirely in liquid culture (fluctuation test).

3.3.3.1. Test bacterial strains

Two mutant strains, S. typhimurium TA98 and S. typhimurium TA100 were

maintained on nutrient agar at 3 ± 1 ºC. The bacteria were inoculated in nutrient broth and

incubated at 37 ºC for 18-24h prior to the test (Razak et al., 2007).

3.3.3.2. Chemicals

The chemicals used for this assay were Davis-Mingioli salt (5.5 times concentrated),

D-glucose (40%, w/v), bromocresol purple (2 mg/mL), D-biotin (0.1 mg/mL), and L-histidine

(0.1 mg/mL). The standard mutagen sodium azide (NaN3, 0.5 µg/100 µL) was used for S.

typhimurium TA100 and the other mutagen Potassium dichromate (K2Cr2O7, 30 µg/100 µL)

was used for S. typhimurium TA98. All chemicals were kept at 3 ± 1 ºC until used.

3.3.3.3. Herbal extracts

Herbal powder (10 g of each plant) was extracted with 75 mL methanol/chloroform

solution. The slurry was stirred with a magnetic stirrer for 24 h at room temperature (22 ± 2

ºC) and then filtered through a Whatman No. 1 filter paper. The filtrate was dried by rotary

vacuum evaporation at 50 ºC. The dried extract was reconstituted in dimethylsulfoxide

(DMSO) to 10 000 μg/mL (stock solution).

3.3.3.4. Preparation of reagent mixture

60

Davis-Mingioli salt (21.62 mL), D-glucose (4.75 mL), bromocresol purple (2.38 mL),

D- biotin (1.19 mL) and L-histidine (0.06 mL) were mixed aseptically in a sterile bottle.

3.3.3.5. Mutagenicity assay

Herbal extract, reagent mixture, sterile distilled water, standard mutagen were mixed

in several bottles at the amount indicated in table 3.2 and were inoculated with an overnight

culture broth of S. typhimurium test strains. The content of each bottle was dispensed into

each well of a 96 well micro titration plate and the plate was incubated at 37 ºC for 4 days.

The results were interpreted; yellow or turbid wells were scored as positive while purple

wells were scored as negative. The extract was considered toxic to the test strain if all wells

in the test plate showed purple coloration. For herbal extract to be mutagenic, the number of

positive well had to be significantly higher than the number of positive well in the

‘background’ plate (spontaneous mutation).

Table.3.2 Set-up of the fluctuation assay

Treatment

Volume added (mL)

Mutagen

standard Extract

Reagent

mixture

Deionized

water

Salmonella

test strain

Blank - - 2.5 17.5 -

Background - - 2.5 17.5 0.005

Standard

mutagen 0.1 - 2.5 17.4 0.005

Test sample - 0.005 2.5 17.5 0.005

3.4. Biochemical Profiling:

Metabolomics analysis was performed using liquid chromatography/tandem mass

spectrometry (LC/MS/MS2) technique (Eckel-Mahan et al., 2012). LCMS analysis was

performed at National Institute of Biotechnology and Genetic Engineering (NIBGE)

Faisalabad Pakistan.

3.4.1. Liquid chromatography mass spectrometry (LCMS)

The selected medicinal plants were analyzed by Liquid Chromatography combined

with Electron Spray Ionization Mass Spectrometry (LC-ESI-MS). The plant extract was

61

filtered through a 0.45 µm syringe filter before analysis. Separation was performed on

Surveyor plus HPLC System equipped with Surveyor auto (Thermo Scientific, San Jose, CA,

USA). The pump equipped with a Luna RP C-18 analytical column (4.6×150 mm, 3.0 µm

particle size) (Phenomenex, USA). Solvent elution consisted of LCMS grade methanol and

acidified water (0.5% formic acid v/v) as the mobile phases A and B, respectively. Solvent

elution consisted of a gradient system run at a flow rate of 0.3 mL/min. The gradient elution

was programmed as follows: from 10% to 30% A in 5 min; from 10% to 50% A in 20 min;

and maintained this till the end of analysis. A 20 min re-equilibration time was used after

each analysis. The column was maintained at 25 °C and the injection volume was 5.0 µL.

The effluent from the HPLC column was directed to the electron spray ionization mass

spectrometer (LTQ XLTM linear ion trap Thermo Scientific River Oaks Parkway, USA). The

mass spectrometer was equipped with an ESI ionization source. Parameters for analysis

were set using negative ion mode with spectra acquired over a mass range from m/z 260 to

800. The optimum values of the ESI-MS parameters were: spray voltage, +4.0 kV; sheath

gas and auxiliary gas were 45 and 5 units/min. respectively; capillary temperature 320 °C;

capillary voltage, -20.0 V and tube lens -66.51 V (Adom and Liu, 2002). The accurate mass

spectra data of the molecular ions was processed through the X-caliber Software (Thermo

Fisher Scientific Inc, Waltham, MA, USA).

3.5 Extraction of Silymarin from seeds

3.5.1. Soxhlet extraction

Silymarin extraction was a two-step process in which powdered seeds were first

defatted. In order to defat finely powdered seeds of Silybum marianum about 20g were first

extracted with n-hexane (300 mL) for 6 hours and then with methanol for 8 hours in a

Soxhlet apparatus. Methanol solution was evaporated under reduced pressure on a rotary

evaporator (50 °C) and extracted silymarin were obtained as soft yellow powder

(Chukeatirote and Saisavoey, 2009; Milk Thistle/officinal Monograph. 2003).

3.5.2. Reflux extraction:

In reflux extraction 50 g of finely powdered seeds of Silybum marianum were defatted

with n hexane (250 mL) in the same flask and heated for 60 minutes. The extract was filtered

and heated again the residue with 250 mL methanol. Methanol solution was evaporated under

reduced pressure on a rotary evaporator at 50 °C (Sultana et. al., 2009).

3.6. Animal Model

3.6.1 Preliminary trial / Evaluation of hepatotoxin

https://us-mg6.mail.yahoo.com/neo/launch?.rand=15ffsh6j3877f#_ENREF_3

62

Objective:

Many chemicals with various concentrations have been reported by the researcher

(Hurkadale et al., 2012; Venkatachalam and Muthukrishnan, 2013; Nurrochmad et al., 2013;

Li et al., 2013) to induce hepatotoxicity. Following chemicals were reevaluated through

animal trial to select the most appropriate hepatotoxin.

1 30% carbon tetrachloride 3 mL/Kg body weight twice in a week for two weeks.

2 100% carbon tetrachloride 0.7 mL/Kg body weight as a single dose.

3 100% carbon tetrachloride 3 mL/Kg body weight as a single dose.

4 Paracetamol 500 mg/kg body weight daily for three consecutive days.

Experimental animals:

Experimental animals were placed in an animal room at National Institute of Food

Science and Technology (NIFST), University of Agriculture Faisalabad. The trial was

initiated ten days after acclimatization at 26 oC (±2) with 12 hours light/dark cycles with food

and water ad libitum. All procedures were conducted in accordance with the guidelines

framed by Institutional Animal Care and Use Committee (IACUC), University of Agriculture

Faisalabad, Pakistan.

Following five groups were made.

Table.3.3

Group No Mode of hepatotoxicity induced

1 Received normal saline served as absolute control

2 Received 30% carbon tetrachloride (CCl4) 3 mL/Kg body weight with liquid

paraffin twice in a week for two weeks.

3 Received 100% carbon tetrachloride 0.7 mL/Kg body weight with liquid

paraffin as single dose.

4 Received 100% carbon tetrachloride 3 mL/Kg body weight with liquid

paraffin as single dose.

5 Received Paracetamol 500mg/kg body weight daily as a single dose for

three consecutive days.

Biochemical analysis:

63

Biochemical parameters such as Serum Glutamate Pyruvate Transaminase (SGPT), Serum

Glutamic Oxaloacetic Transaminase (SGOT) and alkaline phosphate (ALP) in the serum of

rabbits were measured on chemistry analyzer by using commercially available kits.

64

S.

No Treatment Dosage Plan

1 A1B1C1 Extracted silymarin 100mg/kg/b. wt. + AM of Artemisia absinthium 100mg/kg/b. wt. + AM of Rheum emodi 100mg/kg/b.

wt.


wt.


wt.


wt.


wt.


wt.


wt.


wt.


wt.


wt.


wt.


wt.


wt.


wt.


wt.


wt.


wt.


wt.


wt.

65


wt.


wt.


wt.


wt.


wt.

25 A3B3C1 Extracted silymarin 500mg/kg/b. wt. + AM of Artemesia absinthium 500mg/kg/b. wt. + AM of Rheum emodi 100mg/kg/b.

wt.


wt.


wt.

28 Absolute Control No Toxin, No treatment, Only Normal Saline

29 Positive Control Only Toxin for first three consecutive days

30 Siliver 100mg Standard silymarin 100mg



40

3.6.2. Pre-clinical Trial:

3.6.2.1. Curative potential of medicinal plants:

There were 32 different treatment groups (Table 3.3) in preclinical trial including

absolute control, positive control and standard to evaluate the curative potential of medicinal

plants in different combinations. Experimental animals were placed in the animal room at

National Institute of Food Science & Technology, University of Agriculture Faisalabad,

Pakistan. The trial was initiated ten days after acclimatization at 26 oC (±2) with 12 hours

light/dark cycles with food and water ad libitum. All procedures were conducted in

accordance with the guidelines framed by Institutional Animal Care and Use Committee

(IACUC), University of Agriculture Faisalabad, Pakistan.

3.6.2.2. Blood Sampling

The experimental animals were given toxin to damage the liver and then treated with

different combinations of medicinal plants to explore the best combination that restores the

liver function. First blood sample of the experimental animals were drawn and labeled it as

zero hour blood samples and then administration of paracetamol 500 mg per kg body weight

daily for three consecutive days as toxin.

Second blood samplings were taken after 72 hours from all the animals. Medicine plants

combination was started after taking the 2nd blood sample.

Third blood samplings were drawn after 168 hours.

Fourth blood samplings were drawn after 288 hours.

Fifth blood Samplings were drawn after 408 hours.

Blood samples were stored in Jel-clot activator tube manufactured by Guangzhou Improve

Medical Instruments Co.Ltd tubes and placed them in centrifugal machine to separate the

serum. The sera were stored at 2-8 0C and colorimetrical methods were employed to measure

the level of selected biochemical parameter using commercial available kits. All parameters

were assayed through biochemical analyzer by using commercial available kits.

3.7. Biochemical Analysis

Following biochemical markers were evaluated using commercial available kits.

41

3.7.1. Serum glutamate pyruvate transaminase (SGPT)

3.7.1.1. Principle:

Serum alanine aminotransferase (ALT) were determined through kinetic method.

L-alanine + oxoglutrate → Pyruvate + L-glutamate

Pyruvate + NADH + H2 → L-Lactate+NAD+H2O

3.7.1.2. Reagent Preparation:

Reaction mixture was prepared by mixing 9 parts (450µL) of Reagent 1 (R1) and 1 part

(50µL) of Reagent 2 (R2). 50µL serum sample was added in the reaction mixture, mixed and

then read the absorbance at 340 nm after 120 sec. ALT (U/L) were calculated by multiplying

340×1746 (Young, 1975).

3.7.2. Serum glutamate oxaloacetate transaminase (SGOT)

3.7.2.1. Principle:

Serum aspartate aminotransferase (AST) were determined through kinetic method.

Aspartate aminotransferase (AST/GOT) catalyze the following reaction

α-ketoglutrate + L-aspartate ↔ L Glutamate + oxaloacetate

Oxaloacetate + NADH + H+ ↔ L-Malate + NAD+ H2O


Reaction mixture was prepared by mixing 9 parts (450µL) of Reagent 1 (R1) and 1 part

(50µL) of Reagent 2 (R2). 50µL serum sample was added in the reaction mixture, mixed and

then read the absorbance at 340 nm after 120 sec. AST (U/L) were calculated by multiplying

340×1746 (Young, 1975).

42

3.7.3. Serum alkaline phosphatase (ALP)

3.7.3.1 Principle:

Alkaline phosphate catalyzes the hydrolysis of p-nitrophenyl phosphate in p-

nitrophenol and phosphate, in alkaline medium,

p-nitrophenyl phosphate H2O → p-nitrophenol + H3PO4


Reaction mixture was prepared by mixing 4 parts (800µL) of Reagent 1 (R1) and 2

parts (200µL) of Reagent 2 (R2). 10µL serum sample was added in the reaction mixture,

mixed and then read the absorbance at 405 nm after 180 sec. ALP (U/L) were calculated by

multiplying 405×5454 (Young, 1975).

3.7.4. Albumin

3.7.4.1. Principle:

Albumin is bound by the BCG dye to produce an increase in the blue green color in

pH 3.8 acid medium. The color increase is proportional to the concentration of albumin

present in sample. Albumin determination is useful in diagnosis of hepatic and renal

pathologies.

3.7.4.2. Procedure:

1000µL Reagent 2 (R2) was taken and added 10µL distilled water which served as blank.

1000µL Reagent 2 (R2) was taken and added 10µL standard which served as standard.

1000µL Reagent 2 (R2) was taken and added 10µL sample which served as sample. Mixed,

incubated for 5min at room temperature. Read the absorbance of standard tubes.

3.7.4.3. Calculation:

Albumin g/dL = (A) sample/ (A) standard × 4

43

Conversion factor from g/dL × 144.9= µmol (Young, 1975).

3.7.5. Total Protein

3.7.5.1. Principle:

Proteins react with copper ions to produce a blue violet color compound in alkaline medium.

Protein + Cu++ → colored complex

The color intensity is proportional to the concentration of total protein present in the sample.

3.7.5.2. Procedure:

1000µL Reagent 2 (R2) was taken and added 20µLdistilled water which served as blank.

1000µL Reagent 2 (R2) was taken and added 20µL standard which served as standard.

1000µL Reagent 2 (R2) was taken and added 20µL sample which served as sample. Mixed,

incubated for 5min at room temperature. The sample was read and calibrator extinction

(Young et al 1975).


Total Protein g/dL = (A) sample/ (A) calibrator × 6

3.7.6. Determination of albumin globulin (A/G) ratio

Albumin globulin (A/G) ratio were determined as

Globulin = Protein – Albumin

A/G Ratio= Albumin / globulin

3.7.7. Antioxidant enzyme assay

Antioxidant enzymes were evaluated from the liver tissue using following protocols.

3.7.7.1 Enzyme Assay of Superoxide dismutase (SOD):

The activity of superoxide dismutase was determined by measuring its ability to

inhibit the photo reduction of Nitro blue tetrazole (NBT) following the method of

Giannopolitis and Ries (1977).

3.7.7.2. Required reagents for enzyme assay:

44

1. 0.067 mM potassium phosphate buffer, pH 7.8

2. 0.1 M EDTA solution containing 0.3 mMNaCN

3. 0.12 mM Riboflavin

4. 1.5 mM NBT

3.7.7.3. Preparation of 0.067 mM phosphate buffer, pH 7.8:

0.14 g K H2 PO4 was taken in a flask and then 0.74 g K2 H PO4 was added in it and

volume was made up to 80 mL in flask by the addition of distilled water.

3.7.7.4. Preparation of EDTA/NaCN solution:

0.16 g EDTA was taken in a flask and then 0.08 mg NaCN was added in it and

volume was made up to 5.4 mL in flask by the addition of distilled water.

3.7.7.5. Preparation of riboflavin solution:

0.06 mg riboflavin was taken in a flask and its volume was made up to 1.3 mL with

the addition of distilled water and stored in cold dark bottle.

3.7.7.6. Preparation of NBT solution:

3.23 mg NBT was taken in a flask and its volume was made up to 2.64 mL with the

addition of distilled water and stored in cold dark bottle.

3.7.7.7. Procedure:

1 mL buffer was taken in a cuvette as blank and inserted into spectrophotometer to note

the readings of blank, after that spectrophotometer was adjusted at zero at A560 nm. Then 5-6

cuvettes were taken and set them in a light box with an internally mounted light bulb of 30

Watt. Firstly 1 mL of buffer was added to each cuvette, then 0.05 mL enzyme extract and

0.016 mL of riboflavin was added in each cuvette. All the cuvettes were incubated in light

box for 12 minutes. The cuvettes were transferred to the spectrophotometer, where 0.067 mL

of EDTA/NaCN solution and 0.033 mL of NBT were added to the illuminated reaction

mixture. The absorbance was noted after 20 s of reaction. Activity of superoxide dismutase

was determined by measuring the percentage inhibition of NBT. Through the % inhibition of

NBT enzyme activity was calculated then this activity was multiplied with the protein

content (mg) of the extract and specific activity was measured in U/mL/min/mg of protein.

45


% age inhibition = Blank (Abs) - Sample (Abs) × 100

--------------------------------------------

Blank (Abs)

Specific Activity = Enzyme activity × mg of protein

3.7.7.2 Peroxidase (POD):

3.7.7.2.1 Preparation of Enzyme Extract:

To remove the RBCs the dissected organs were rinsed with phosphate buffer of pH

6.5 (0.2 M) and homogenized in cold buffer (1:4 w/v) using a blender. After the

homogenization, organ homogenates were centrifuged for 15 minutes at 10,000 rpm and 4ºC.

After that clear supernatants were stored at 4 ºC for enzyme assay while residue was

discarded.

3.7.7.2.2 Enzyme Assay of Peroxidase

The activity of peroxidase was determined by measuring its ability to reduce the

concentration of H2O2 at A470 nm.

3.7.7.2.3 Required reagents for enzyme assay:

1. 0.2 M phosphate buffer, pH 6.5

2. Guaiacol

3. Hydrogen peroxide

3.7.7.2.4 Preparation of 0.2 M phosphate buffer, pH 6.5:

4 g Na H2 PO4 and 1 g Na2 H PO4 was taken in a flask and dissolved by adding

distilled water. Then volume was raised up to 200 mL and adjusted the pH 6.5.

3.7.7.2.5 Preparation of buffer substrate solution:

Guaiacol (750µL) was added to phosphate buffer (47 mL) and mixed well on vortex

agitator. After agitation H2O2 (0.3 mL) was added to buffer solution. Reaction mixture

contained:

Buffered substrate solution 3 mL

Enzymes extract 0.06 mL

46

Blank: Phosphate buffer used as blank

3.7.7.2.6 Procedure:

A cuvette containing 3 mL of blank solution was inserted into the spectrophotometer

and set it to zero at wavelength of 470 nm. Then a cuvette containing buffered substrate

solution was put into the spectrophotometer and initiation of reaction was occurred by adding

0.06 mL of enzyme extract, the initiation of reaction was occurred. The reaction time is 3

minutes and noted the absorbance at after 3 minutes. Enzyme activity was calculated then

this activity was multiplied with the protein content (mg) of the extract and specific activity

was measured in U/mL/min/mg of protein.

3.7.7.2.7 Calculation:

Activity (Units/ml) = ΔA/3

----------------------------

26.6×60/3000


3.7.7.3. Catalase:

3.7.7.3.1. Isolation of catalase Enzyme:

After 4 weeks trial, the animals were dissected and organs viz. liver was stored for

further analysis to analyze each of the parameters of antioxidant enzyme catalase and three

replicates were made. Catalase (EC 1.11.1.6 pH ≥7.0) is oxidoreductase tetramer enzyme that

contains four equally sized subunits and each subunit contains one ferric heme prosthetic

group. Primarily catalase is found in peroxisomes and it acts as catalyst in the breakdown of

hydrogen peroxide (H2O2) to oxygen (O2) and water (H2O), and gives defense against the

lethal effects of radicals which are produced due to oxygen. In many oxidase catalyzed

reactions, highly reactive oxygen species (ROS) is produced in large number as by products

in electron transport chain. Catalase enzyme is present in all organisms that respire

aerobically.

3.7.7.3.2 Procedure:

47

The organ was weighed.

Added phosphate buffer (pH 7.4) 3 times greater than the weight of organ i.e 1:4.

Homogenized it for 15 minutes with the help of pestle and mortar or homogenizer

with short intermissions.

Passed the homogenized tissues through muslin cloth to remove the biomass.

Filtered the liquid obtained from muslin cloth with the help of Whatman filter paper

no. 1.

Centrifuged the filtrate obtained above step in refrigerator centrifugal machine at

10,000 rpm for 15 minutes.

Separated both, sediments and supernatants for further analysis.

All the enzyme isolation steps were carried out at 4 °C.

The supernatants and sediments were stored at 4 °C until further analysis.

10% glycerol was added

3.7.7.3.3 Analysis of catalase enzyme activity (assay):

The activity of catalase was determined by measuring its ability to decompose H2O2

concentration at 240 nm (Chance and Mehaly, 1977).

3.7.7.3.4. Required reagents and chemicals for enzyme assay:

1. 60 mM phosphate buffer (pH 7.0)

2. 10 mM hydrogen peroxide

3.7.7.3.5. Preparation of 60 mM phosphate buffer (pH 7.0):

0.224 g Na H2PO4 and 0.1632 g Na2HPO4 were taken in a flask and added distilled

water to make volume up to 50mM and pH was adjusted 7.0.

3.7.7.3.6. Preparation of buffer substrate solution:

Buffer substrate solution of 10mM of H2O2 was prepared in 60mM phosphate buffer

by dissolving 0.442 mL H2O2 in 60mM phosphate buffer.

The reaction mixture (2 mL) contains:

Buffered substrate solution 1.95 mL

Enzymes extract 0.05 mL

Blank: 60mM phosphate buffer used as blank.

3.7.7.3.7. Procedure:

48

A cuvette containing 2 mL blank solution was inserted into

spectrophotometer.

Set the reading of spectrophotometer zero at wavelength 240 nm.

Another cuvette containing buffered substrate solution inserted into

spectrophotometer.

Added 0.05 mL enzyme extract into cuvette containing buffered substrate

solution.

Kept the second cuvette for 3 minutes so that reaction was occurred.

Noted the absorbance reading at 240 nm on spectrophotometer after interval

of 1 minute.

3.7.7.3.8. Calculation:

Enzyme activity was calculated then this activity was multiplied with the protein

content (mg) of the extract and specific activity was measured in U/mL/min/mg of protein.

Activity (Units

mL) =

∆A

min×dilution×2 mL

0.04 mM−1cm−1 ×0.05mL

Where,

∆A: Absorbance at 240 nm

Min: Reaction time

2 mL: Amount of enzyme and substrate buffer

0.04 mM-1cm-1: Standard factor

0.05 mL: Enzyme concentration used


3.8. Histopathology:

Reserved organs were processed for Histopathological studies (Bancroft and

Gamble, 2007) as described below:

3.8.1. Washing

After fixation in 10% buffered formalin, 5 mm thick pieces of tissues were taken and

washed under tap water over night to remove all residues of fixative.

3.8.2. Dehydration

Slices of tissues were then dehydrated in ascending grades of ethyl alcohol as follows:

49

Dehydration treatment procedure of histopathology

Dehydrating Agent Time (hours)

Alcohol 70% 8

Alcohol 85% 4

Alcohol 95% 4

Absolute Alcohol-1 2


3. 8.3. Cleaning

This process involved the elimination of dehydrating agent and substitution with some fluid

miscible both with dehydrating agent and the embedding medium as follows:

Clearing treatment procedure of histopathology

Clearing Agent Time (Minutes)

Xylene+ Absolute Alcohol (50+50) 30

Xylene-1 15

Xylene-2 15

3.8.4. Infiltration

Infiltration was carried out in liquid paraffin at 59-60 °C as shown below:

Infiltration treatment procedure of histopathology

Infiltrating Agent Time (hrs.)

Paraffin-1 2

Paraffin-2 2

Paraffin-3 2

3.8.5. Embedding

Centrally placed tissues in the steel mold were poured with the melted wax and then

allowed to solidify at -1 to -5°C in the plastic moults. On solidification of paraffin, the steel

moult was removed from the block.

3.8.6. Sectioning and Mounting

Tissues sectioning was done with the help of microtome as:

50

Thickness of the sections was adjusted to 4-5µm

Block was tightened with the clamp in tissue carrier

Tissue blocks were faced to cutting edge of knife with the help of wheel of

microtome to form the smooth ribbons of sections

These sections were then flattened in the warm water (~50 °C)in water bath

Extremely thin smeared of Meyers egg albumin was made onto glass slide

The slide was mounted by dipping the slide under the section floating in water bath.

Slides were dried in incubator at 45-55°C for 30 minutes to remove fragments of the

paraffin.

3.8.7. Staining

Staining was performed by using H & E stain adopting protocol as:

Detailed procedures of H&E staining protocol for histopathology.

Staining Agent Time (Minutes)

Xylene-1 3

Xylene-2 3



Alcohol 70% 3

Water 4

Hematoxylin 5 – 8

Water 5

Acid Alcohol 1-2 Dips

Water 3

Ammonia Alcohol 3

Water 3

Alcohol 70% 3

Eosin y 1 – 2

Alcohol 70% 3


51


Xylene-1 3

Xylene-1 3

Afterward a drop of DPX was put on the cover slip and it was placed on the stained section

avoiding bubble formation.

52

Chapter No. 4

RESULTS AND DISCUSSIONS

Hepatitis is one of the leading causes of death across the world and economically it

had a devastating effect on a large number of families. The currently available treatment is

not only expensive but also its cure rate is very low, with a lot of side effects. This study

focused, mainly on finding an alternative treatment using natural products to manage hepatic

disorders with little or no side effects. In this regard three medicinal plants viz. Milk thistle

(Silybum marianum), Wormwood (Artemisia absinthium) and Rhubarb (Rheum emodi) were

selected and their hepatoprotective effect were studied in different combinations. The

Silybum marianum is widely used in liver diseases to settle the SGPT levels. Artemisia

absinthium is a popular folk medicine that is used to manage liver disorders. Rheum emodi

has potent anticancer and hepatoprotective properties. All of these three medicinal plants are

mainly prescribed by the alternative medical practitioners. The liver healing potential of all

three selected medicinal plants may be due to the presence of flavonoids which are natural

antioxidants.

The phytochemical constituents of these medicinal plants were evaluated through

total phenolic content (TPC) and total flavonoid content (TFC). Antioxidant profile of the

selected medicinal plants in various combinations was evaluated through DPPH, reducing

power and H2O2 induced oxidative damage on pUC19 DNA. The cytotoxicity of the selected

medicinal plants singly and in different combinations was evaluated using various

toxicological assays including hemolytic, brine shrimp lethality and mutagenic activity.

Biochemical profiling of selected medicinal plants was done through liquid chromatography

mass spectrometry (LCMS). Hepatoprotective potential of the selected medicinal plants in

combinations was evaluated against paracetamol induced liver damage.

Various combinations of medicinal plants were administered to the experimental

animals to check the ameliorative effect on the basis of biochemical analyses, including

SGPT, SGOT, ALP, Protein and Albumin. The hepatoprotective potential of selected

medicinal plants in combination was confirmed by comparative histopathological

53

examination of control and treated animals. The data are presented as mean standard

deviation and depicted in box-whisker plots followed by analysis of variance (ANOVA). In

addition to ANOVA contrast analysis among different treatments to identify the best

combination were also carried out. The results of all the combinations were compared with a

standard drug and control group. Results were analyzed using statistical software R (R Core

Team, 2014).

The results have been presented under the following headings.

4.1. Phytochemical studies

4.1.1 Total phenolic and flavonoid content

4.2. Antioxidant potential

4.2.1 DPPH assay and reducing power

4.2.2 DNA protection effect with H2O2 induced oxidative damage on pUC19 DNA

4.3. Toxicological profile

4.3.1. Hemolytic activity

4.3.2. Brine shrimp lethality

4.3.3. Mutagenic activity

4.4. Metabolomics analysis

4.4.1. Biochemical profiling of Silybum marianum

4.4.2. Biochemical profiling of Artemesia absinthium

4.4.3. Biochemical profiling of Rheum emodi

4.5. Quantification of extracted silymairn

4.6. Animal model

4.6.1. Preliminary evaluation of toxin

4.6.1.1. Conclusion preliminary evaluation of toxin

4.6.2. Pre-clinical animal trial

4.7. Biochemical analysis



4.7.3. Alanine amino transferase (ALP)

4.7.4.. Total protein

4.7.5. Albumin

4.7.6. Albumin Globulin (A/G) ratio

54

4.7.7. Antioxidant enzyme

4.8. Gross and Histopathology

4.1. Phytochemical studies:

The medicinal plants are one of the major sources of novel pharmaceutical

compounds. Phytochemicals aid to maintain excellent health and fighting against diseases.

4.1.1 Total Phenolic and Flavonoid Content

The amount of total phenolic and flavonoid content present in different combinations

(S. marianum, A. absinthium and R. emodi) of the medicinal plants was determined by Folin-

Ciocalteu (FC) method. The results found are expressed in mg/g gallic acid equivalents

(mg/g GAE) and mg/g catechin equivalents (mg/g CE) respectively (Table 4.1). The selected

medicinal plants were used in different combinations containing 100, 200 and 500 mg of

each plant. The total phenolic content ranged from 276.26 to 356.57 (mg/g GAE). The

highest total phenolic content (356.57 mg/g GAE) was recorded in the 27th combination

containing 500 mg of each plant while the lowest phenolic content (276.26 mg/g GAE) was

found in 5th combination containing S. marianum 100 mg, A. absinthium 200 mg and R.

emodi 200 mg. The amount of total flavonoid content was found in the range of 13.01 to

25.63 mg/g CE. The highest quantity of total flavonoid content (25.63 mg/g CE) was

observed in the 27th combination containing 500 mg of each plant, whereas the lowest

quantity of total flavonoid content (13.01 mg/g CE) was observed in the 4th combination

containing S. marianum 100 mg, A. absinthium 200 mg and R. emodi 100 mg.

Phenolic compounds, (viz. flavonoid) are extensively found in food products derived

from plant sources and they possess substantial antioxidant potential. An increase in levels of

flavonoid in the diet could decrease certain human diseases (Nabavi et al., 2008; Jahan et al.,

2012). All phenolics exhibit the antioxidant activity e.g. p-coumaric acid prevents from

oxidative stress and also inhibits the process of lipid peroxidation. This explains why

medicinal plants are used to treat various diseases (Niwa et al., 2001; Tapia et al., 2004). In

vivo and in vitro study of flavonoid content of plants extracts shows different biological

activities (Riaz et al., 2012). Various studies carried out in regard to plants extracts revealed

that there is a relationship between antioxidant potential and presence of phenolic content

55

(Aaby et al., 2004; Silva et al., 2005; Sun and Ho, 2005; Yuan, 2005; Jahan et al., 2012). The

Phenolic content indicates the presence of potent antioxidant agents in plants. Natural

products and their derivatives contribute more than half of all the clinically administered

drugs (Katalinic et al., 2006; Wajdylo et al., 2007).

Flavonoid, natural antioxidant molecules of plants exhibit different therapeutic

applications and pharmacological approaches. Thus phenolic content have contributed in

preventing human from cancer, heart and infectious diseases by scavenging the reactive

oxygen species (Yao et al., 2004; Jiangrong & Jiang, 2007 and Khan et al., 2012). The

amount of total phenolic and total flavonoid content present in the methanolic extracts of S.

marianum was 23.26 mg/GAE/g and 6.96 mg CE/g respectively, (Pereira et al., 2013).

Similarly the amount of total phenolic and total flavonoid content present in the methanolic

extracts of A. absinthium was 131.18 mg GAE/g and 41.21 mg CE/g respectively, (Lee et al.,

2013) and correspondingly their values were 17.21 mg GAE/g and 1.82 mg CE/g

respectively, in Rheum emodi (Singh et al., 2013). The amount of phytochemical

constituents present in the individual plant was lower than that found in our studied

combinations. It is concluded that the quantity of total phenolic and flavonoid content are

greater in combinations and that the higher content of TPC and TFC found in combination (S.

marianum, A. absinthium and R. emodi) may be due to synergism.

4.2. Antioxidant potential

An antioxidant is a substance capable of preventing or slowing down the oxidation of

the other molecules. Generally, an antioxidant can protect against the metal toxicity by

trapping free radicals, hence terminating the chain reaction. Natural antioxidants have the

potential to prevent from many diseases caused by the reactive oxygen species (Poli et al.,

2004). The antioxidant potential in term of DPPH and reducing power are described under

following headings.

4.2.1. DPPH assay

Percent inhibition of DPPH (1,1-Diphenyl-2-picryl-hydrazyl) radical in different

plant combinations (S. marianum, A. absinthium and R. emodi) are presented in Table 4.2.

The percentage inhibition of DPPH among the twenty seven combinations was ranged from

76.81 to 92.33.

56

44

Table.4.1.Total phenolic and flavonoid content of selected medicinal plants in combinations

Sr. No. Treatment Medicinal plants combinations TPC

(mg/g GAE)

TFC

(mg/g CE)

1 A1B1C1 S. marianum 100 mg + Artemisia absinthium 100 mg. + Rheum emodi 100 mg 289.80 ± 2.76 14.90 ± 0.49

2 A1B1C2 S. marianum 100 mg. + Artemisia absinthium 100 mg + Rheum emodi 200 mg 296.17 ± 2.91 16.27 ± 0.56

3 A1B1C3

S. marianum100 mg + Artemisia absinthium 100 mg + Rheum emodi 500 mg 324.60 ± 3.37 15.43 ± 0.61

4 A1B2C1


5 A1B2C2


6 A1B2C3


7 A1B3C1


8 A1B3C2


9 A1B3C3


10 A2B1C1

S. marianum 200 mg + Artemisia absinthium 100 mg + Rheum emodi 100 mg 281.33 ± 2.73 15.28 ± 0.60

11 A2B1C2


12 A2B1C3


13 A2B2C1


14 A2B2C2


15 A2B2C3


All values are an average of triplicate experiment and represented as mean ± SD.

45

Table 4.1 continued

Sr. No. Treatment Medicinal plants combinations TPC TFC

(mg/g GAE/g) (mg/g CE)

16 A2B3C1 S. marianum 200 mg + Artemisia absinthium 500 mg + Rheum emodi 100 mg 295.23 ± 3.29 17.70 ± 0.45









25 A3B3C1 S. marianum 500 mg + Artemesia absinthium 500 mg + Rheum emodi 100 mg 312.57 ± 3.24 23.03 ± 0.70




46

The highest antioxidant activity (92.33 % inhibition) was noted in the 3rd combination

containing S. marianum 100 mg, A. absinthium 100 mg and R. emodi 500 mg whereas lowest

(76.81% inhibition) activity was recorded in another combination containing S. marianum 500

mg, A. absinthium 500 mg and R. emodi 200 mg.

4.2.2. Reducing power assay:

In the present study, the extracts of the selected medicinal plants exhibited effective

reducing capacity at all the concentrations. The results of reducing power are given in term of

absorbance by diluting the concentration of various combinations up to 50 % (Table 4.2).

The natural antioxidants are promising source that prevent from many diseases caused by

the free radicals and reactive oxygen species (Droge, 2002, Lee et al., 2004 and Valko et al., 2007).

The DPPH has an unpaired valence electron atone nitrogen-nitrogen bridge and make a very

stable free radical (Eklund et al., 2005). The antioxidant activity is due to scavenging DPPH free

radical (Alma et al., 2003, Karioti et al., 2004 and Kordali et al., 2005). The potential of antioxidant

activity can be evaluated through the rate of utilization of DPPH free radicals in silybin

containing biological system. If the utilization of DPPH free radical persists and also lasts longer

than 30 minutes then it could be concluded that the plant extract has potential antioxidant activity

(Hadaruga and Hadaruga, 2009). The reducing properties are generally associated with the

presence of reductones which have been shown to exert antioxidant action by breaking the free

radical chain and by donating a hydrogen atom (Gordon et al., 2006). Reductones are also

reported to react with certain precursors of peroxide and prevent peroxide formation (Li et al.,

2007). Our results on the reducing capacity of the extracts suggests that reductone associated and

hydroxide groups of compounds can act as electron donors and can react with free radicals to

convert them to more stable products, and thereby terminate radical chain reactions.

The reported antioxidant potential of S. marianum is due to the presence of silydianin,

silychristin and silybin but unlike quercetin they do not have iron chelating ability in aqueous

solution. Studies showed that the antioxidant potential of A. absinthium was lower than the

results of our studied combinations (Katerere and Eloff, 2005; Craciunescu et al., 2012). The

methanolic and aqueous extracts of R. emodi showed remarkable antioxidant potential in

experimental animals (Rajkumar et al., 2011) but these values were lower than our studied

combinations.

http://www.sciencedirect.com/science/article/pii/S0308814608009710#bib5







47

Table.4.2. DPPH and Reducing power of selected medicinal plants in combinations

S. NO Treatment Medicinal plants combinations DPPH

(% inhibition)

Reducing Power

(Absorbance)

1 A1B1C1 S. marianum100 mg + Artemisia Absinthium 100 mg. + Rheum emodi 100 mg 82.81 ± 1.21 0.964

2 A1B1C2 S. marianum100 mg. + Artemisia Absinthium 100 mg + Rheum emodi 200 mg 89.12 ± 1.37 1.085

3 A1B1C3

S. marianum100 mg + Artemisia Absinthium 100 mg + Rheum emodi 500 mg 92.33 ± 1.15 1.361

4 A1B2C1


5 A1B2C2


6 A1B2C3


7 A1B3C1


8 A1B3C2


9 A1B3C3


10 A2B1C1

S. marianum 200 mg + Artemisia Absinthium 100 mg + Rheum emodi 100 mg 88.43 ± 1.48 1.049

11 A2B1C2


12 A2B1C3


13 A2B2C1


14 A2B2C2


15 A2B2C3



48

Table 4.2.continued

S. No. Treatment Medicinal plants combinations DPPH

(% inhibition) Reducing Power

Absorbance

16 A2B3C1 S. marianum 200 mg + Artemisia Absinthium 500 mg + Rheum emodi 100 mg 90.18 ± 1.96 1.400

17 A2B3C2 S. marianum 200 mg + Artemisia Absinthium 500 mg + Rheum emodi 200 mg 91.39 ± 2.82 1.652

18 A2B3C3


19 A3B1C1


20 A3B1C2


21 A3B1C3


22 A3B2C1


23 A3B2C2


24 A3B2C3


25 A3B3C1

S. marianum 500 mg + Artemesia Absinthium 500 mg + Rheum emodi 100 mg 79.43 ± 0.84 1.328

26 A3B3C2


27 A3B3C3



49

4.2.3 DNA Protection effect with H2O2 induced oxidative damage on pUC19 DNA

The antioxidant potential of studied medicinal plants (viz. Silybum marianum, Artemesia

absinthium and Rheum emodi) was also evaluated by DNA protection assay by using pUC19

plasmid DNA (Fig. 4.1).

Fig.4.1. pUC19 plasmid DNA with essential components

(https://www.neb.com/products/n3041-puc19-vector)

DNA protection effect of different plants methanolic extracts (Silybum marianum, Rheum

emodi and Artemesia absinthium) was determined. Three different concentrations i.e. 10, 100

and 1000 µL/mL of each plant were prepared. DNA, in the presence and absence of plant extract,

in different concentrations is showing the effect of free radicals generated by H2O2 on

supercoiled pUC19 (Fig. 4.2). DNA without any kind of treatment remained intact in its

supercoiled form (Lane 1). The hydroxyl radical of H2O2 damaged the pUC19 DNA and resulted

in a streaking band due to strand cleavage (lane 2). S. marianum at 10 µL/mL concentration

showed complete protective effect (lane 6).

The extract of S. marianum at 100 µL/mL concentration and 1000 µL/mL showed

moderate DNA protection activity (lane 5 and 7). Rheum emodi showed concentration dependent

DNA protection i.e. an increase in concentration has increased its protective effect (lane 9-11).

http://upload.wikimedia.org/wikipedia/commons/8/8d/PUC19.svg

50

Fig.4.2 Agarose gel electrophoresis pattern of pUC19 plasmid DNA treated with 30 mM H2O2 in

the absence and presence of different concentrations of plants extracts.

Lane 1: Normal pUC19 DNA, Lane 2: pUC19 DNA + 30 mM H2O2, Lane 3: DNA Ladder (1 kb, Fermentas), Lane

4: pUC19 DNA + P1 (S. marianum) [100 µL/mL], Lane 5: pUC19 DNA+ 30 mM H2O2+ P1 (S. marianum) [10

µL/mL], Lane 6: pUC19 DNA+ 30 mM H2O2+ P1 (S. marianum) [100 µL/mL], Lane 7: pUC19 DNA+ 30 mM H2O2+

P1 (S. marianum) [1000 µL/mL], Lane 8: pUC19 DNA + P2 (R. emodi) [100 µL/mL], Lane 9: pUC19 DNA+ 30 mM

H2O2 + P2 (R. emodi) [10 µL/mL], Lane 10: pUC19 DNA+ 30 mM H2O2 + P2 (R. emodi) [100 µL/mL], Lane 11:

pUC19 DNA+ 30 mM H2O2 + P2 (R. emodi) [1000 µL/mL], Lane 12: Normal pUC19 DNA, Lane 13: DNA Ladder

(1 kb, Fermentas), Lane 14: pUC19 DNA + 30 mM H2O2, Lane 15: pUC19 DNA + P3 (A. absinthium) [100 µL/mL],

Lane 16: pUC19DNA + 30 mM H2O2 + P3 (A. absinthium) [10 µL/mL], Lane 17: pUC19 DNA+ 30 mM H2O2 + P3

(A. absinthium) [100 µL/mL], Lane 18: pUC19 DNA+ 30 mM H2O2 + P3 (A. absinthium) [1000 µL/mL].

As the concentration of A. absinthium was increased its protective effect gradually

decreased, however it showed complete protective effect at 10 µL/mL concentration. The

protective effect was moderate at 100 µL/mL and poor at 1000 µL/mL concentrations (lane 16-

18). DNA protective potential of the extracts can also be exploited to develop the drugs to

control cancer. Polyphenolic compounds are well established antioxidants mostly present in

plants and vegetables (Pandey and Rizvi, 2009).

51

In this study the pUC19 was used to investigate DNA repair effect of various compounds.

Studies show that when pUC19 is subjected to damage using certain chemicals, the banding

pattern on the gel gives three types of bands which are linear, open circular and supercoiled

which give clear idea of extant of DNA degradation (Drozdz et al., 2011). Results of previous

studies showed a concentration dependent antioxidant ability of selected medicinal plants.

Reactive oxygen species (ROS) could be the reason to damage the cellular genetic material. Free

radical could be the cause of DNA strand disruptions, only if the sugar moiety was damaged and

usually two main types of such changes were observed in DNA at the molecular level which

were altered bases and strand break. Both types of changes, if not repaired, affected the cell

structure and function. The protective effect of medicinal plants on DNA could be explained by

its ability to scavenging ROS due to its property as an antioxidant (Yoshikawa et al., 2006).

The present study has observed DNA damage by free radicals generated by H2O2

conversely to Hosseinimehr and Nemati, (2006) who studied damaged the DNA by e-radiations

and protective effect of hesperidin. But the findings of DNA protection were similar either DNA

is damaged due to free radicals or e-radiation. The protective effect was concentration dependent

in hesperidin as well as in S. marianum, R. emodi and A. absinthium. Hosseinimehr and Nemati,

(2006) also described that hesperidin protected mice against e-radiation induced DNA damage in

bone marrow cells whereas in this study similar results were observed.

The aqueous extract of R. emodi displayed considerably protective activity in comparison

to the methanolic extract which did not show any protective activity. UV photolysis of H2O2 in

control region damaged the entire DNA. It was concluded that UV photolysed H2O2 treatment of

pBR322 obliterated the entire DNA, while 50 μg of the aqueous extract gave partial protection

against DNA damage (Rajkumar et al., 2011). Similar protection of DNA in this study was

observed with H2O2 induced oxidative damage on pUC19 DNA.

This study clearly indicates that extracts of selected medicinal plants are capable of

inhibiting lipid peroxidation. The possible mechanism to do so is by scavenging the free radicals

and preventing hydroxyl radicals from attacking lipids. In addition, DNA protection assay also

supports the hydroxyl radical scavenging activity of the studied plants extracts.

52

4.3. Toxicological Profile:

Toxicological testing is an essential prerequisite for the development of modern

pharmaceutical drugs. The cytotoxicity of selected medicinal plants was evaluated by conducting

hemolytic activity, brine shrimp lethality and mutagenic activity.

4.3.1. Hemolytic Activity:

Hemolytic activity of the methanolic extract of Silybum marianum, Artemesia absinthium

and Rheum emodi as single and also in combination was screened against normal human

erythrocytes using Triton X-100 as positive control. Hemolytic activity of the plant is expressed

in percentage hemolysis and reported as mean ± standard deviation of three replicates. All the

samples exhibited very low hemolytic effect towards human erythrocytes. The percentage

hemolysis of individual plant was 2.76±0.29, 3.34±0.35 and 2.14±0.17 with Silybum marianum,

Artemesia absinthium and Rheum emodi, respectively. The different concentrations of the

medicinal plants in different combinations exhibited different hemolytic activities ranged from

5.14 to 13.30 (Table 4.3). The hemolytic activity was 5.30±0.09 in a combination of medicinal

plants containing A1B1C1 (S. marianum 100 mg, A. absinthium 100 mg and R. emodi 100 mg).

The hemolytic activity was slightly increased (7.17±0.35) in the other combination of selected

medicinal plants containing A1B2C2 (S. marianum 100 mg, A. absinthium 200 mg and R. emodi

200 mg). Highest hemolytic activity (13.30±0.10) was observed in another combination

containing A2B1C2 (S. marianum 200 mg, A. absinthium 100 mg and R. emodi 200 mg). The low

hemolytic activity of the extract suggests the less/no toxicity of the plant on human erythrocytes.

Hemolytic activity could be used as a primary tool for studying the toxicity of any drugs,

since it provides primary information on the interaction between molecules and biological

entities at cellular level. Some other plants also have been studied for the hemolytic activity

towards human erythrocytes. Achyranthes aspera was reported to possess very low hemolytic

activity towards human erythrocytes (Gauthier et al., 2009; Priya et al., 2010). Aqueous extract

of Lantana camara and its various solvent fractions were stated to possess moderate hemolytic

activity towards human erythrocytes (Kalita et al., 2011). Mukherjee and Rajasekaran (2010)

reported the high hemolytic activity of different solvent extracts of Allium stracheyi towards

human red blood cells. Chloroform and aqueous extract of leaves of Acanthus ilicifolius were

reported to possess significant hemolytic activity towards the chick red blood cells

(Thirunavukkarasu et al., 2011).

53

Table No. 4.3. Hemolytic activity and Brine Shrimp lethality of selected medicinal plants in different combinations

S. No Treatment Treatment combinations Brine shrimp

lethality a

Hemolytic

activity b

1 A1B1C1 S. marianum100mg + Artemisia Absinthium 100mg. + Rheum emodi 100mg 18 ± 0.5 5.30 ± 0.09

2 A1B1C2 S. marianum100mg. + Artemisia Absinthium 100mg + Rheum emodi 200mg 33 ± 0.5 5.14 ± 0.06

3 A1B1C3

S. marianum100mg + Artemisia Absinthium 100mg + Rheum emodi 500mg 38 ± 0.3 5.41 ± 0.04

4 A1B2C1


5 A1B2C2

S. marianum100mg + Artemisia Absinthium 200mg + Rheum emodi 200mg 37 ± o.5 7.17 ± 0.35

6 A1B2C3


7 A1B3C1


8 A1B3C2


9 A1B3C3


10 A2B1C1

S. marianum 200mg + Artemisia Absinthium 100mg + Rheum emodi 100mg 36 ± 0.3 9.53 ± 0.20

11 A2B1C2


12 A2B1C3


13 A2B2C1


14 A2B2C2


15 A2B2C3


54

Table No. 4.3. Continued

S. No Treatment Treatment combinations Brine shrimp

Lethality a

Hemolytic

activity b

16 A2B3C1 S. marianum 200mg + Artemisia Absinthium 500mg + Rheum emodi 100mg 54 ± 0. 2 9.89 ± 0.20

17 A2B3C2 S. marianum 200mg + Artemisia Absinthium 500mg + Rheum emodi 200mg 57 ± 0.7 9.37 ± 0.15

18 A2B3C3


19 A3B1C1


20 A3B1C2


21 A3B1C3


22 A3B2C1


23 A3B2C2


24 A3B2C3


25 A3B3C1

S. marianum 500mg + Artemesia Absinthium 500mg + Rheum emodi 100mg 54 ± 0.7 6.57 ± 0.17

26 A3B3C2


27 A3B3C3


Data are presented as means and SD (n=3)

a = % mortality

b = % hemolysis

55

The hemolytic activity ranged from 0.8–19.2 of A. absinthium was slightly higher in

another study against the THP-1 cell line (Yinebeb et al., 2011). Aqueous extract of Aerva lanata

showed very low hemolytic effect with an IC50 value of 24.89 mg/mL (Kumar et al., 2013)

which was higher than our studied combinations. The results of hemolytic activity showed that

the methanolic extracts of Silybum marianum, Artemesia absinthium and Rheum emodi alone and

in combinations are non/less toxic to the human erythrocytes.

4.3.2. Brine shrimp lethality:

The toxicity of selected medicinal plants alone and in combination of different plant

extract was evaluated using brine shrimps test. Every test tube with sample contained 20 larvae

of brine shrimp, including the control group and volume was made up to 5 mL with artificial sea

water. The brine shrimp lethality of individual plant was noted periodically after three hours till

24 hours (Table 4.4). The percentage mortality of selected medicinal plants in various

combinations was determined by counting live larvae after 24 hours (Table 4.3). Lowest

mortality rate was noted (18±0.5) in a combination containing A1B1C1 (S. marianum 100 mg, A.

absinthium 100 mg and R. emodi 100 mg). The mortality rate was increased as the concentration

increases and becomes double (38±0.3) with this combination of selected medicinal plants

containing A1B1C3 (S. marianum 100 mg, A. Absinthium 100 mg and R. emodi 500 mg). The

highest mortality rate (57±0.5) was seen in a combination containing A1B3C3 (S. marianum 100

mg, A. absinthium 500 mg and R. emodi 500 mg). Contrary to the previous trend the mortality

rate at final concentration A3B3C3 (S. marianum 500 mg, A. absinthium 500 mg and R. emodi

500 mg) was 51±0.5 that may be ignored. Mortality rate was proportional to increase of

concentration, which provided linearity in the dose effect relationship of every combination.

The cytotoxicity of some other plants also studied for brine shrimp lethality. Acacia seyal

(12 % mortality) and D. cinerea (17 % mortality) were reported to be safe or non-toxic while A.

indica (34 % mortality), T. indica (36 % mortality) and G. tricocarpa (38 % mortality) were

moderately toxic to A. salina larvae (Nguta et al., 2013). Methanolic extract of flowers of A.

dubia were toxic against newly hatched nauplii (Haq et al., 2012). In another study toxic

potential of S. marianum were evaluated through brine shrimp lethality and found that seeds of S.

marianum has least toxicity at high concentration and safer to be used (Alluri et al., 2005).

56

Table 4.4 Brine shrimp lethality outcomes showing survived shrimps and % mortality after treatment with different extracts

No. of survived shrimps in following intervals

Silybum marianum

Fraction Initial 3 hour 6 hour 9 hour 12 hour 15 hour 18 hour 21 hour 24 hour %

Mortality

Control 20 20±0 20±0 20±0 20±0 20±0 20±0 20±0 20±0 No

activity

10 mg 20 20±0 19.5±0.5 19.5±0.5 19±0 19±0 18.5±0.5 18.5±0.5 18±0 10

25 mg 20 20±0 19±0 19±0 18.5±0.5 18.5±0.5 18±0 17.5±0.5 17.5±0.5 12

50 mg 20 20±0 19.5±0.5 19.5±0.5 19±0 19±0 18±0 17±0 17±0 15

100 mg 20 20±0 19±0 19±0 18±0 18±0 17±0 17±0 16.5±0.5 17

Artemesia absinthium


Mortality

Control 20 20±0 20±0 20±0 20±0 20±0 20±0 20±0 20±0 No

activity

10 mg 20 20±0 19±0.5 18.5±0.5 18±0 17±0 17.5±0.5 17±0 16±0 20

25 mg 20 19±0 19±0 19±0 18±0 18±0 17.5±0.5 16.5±0.5 16±0 20

50 mg 20 19±0 18.5±0.5 18±0 18±0 17±0 16.5±0.5 16±0 15±0 25

100 mg 20 18±0 18±0 17.5±0.5 17±0 16±0 16±0 15±0 14.5±0.5 27

Rheum emodi


Mortality

Control 20 20±0 20±0 20±0 20±0 20±0 20±0 20±0 20±0 No

activity

10 mg 20 20±0 19.5±0.5 19±0 19±0 18.5±0.5 18.5±0.5 18±0 17.5±0.5 12

25 mg 20 19±0 19±0 18.5±0.5 18.5±0.5 18±0 18±0 17.5±0.5 17±0 15

50 mg 20 20±0 19.5±0.5 19±0 19±0 18.5±0.5 18±0 17.5±0.5 17±0 15

100 mg 20 19.5±0.5 19.5±0 19±0 18±0 18±0 17.5±0.5 17±0 16.5±0.5 17

57

The outcomes of brine shrimps lethality revealed that the methanolic extracts of Silybum

marianum, Artemesia absinthium and Rheum emodi alone and in combinations were less toxic to

A. salina larvae.

4.3.3. Mutagenic Activity:

Mutagenic activity was performed by using test strain S. typhimurium TA98 and S.

typhimurium TA98. Both of the standard S. typhimurium TA98 and TA 100 showed significant

increase in positive wells (Fig.4.3. B & C). The numbers of positive wells in background of S.

typhimurium TA98 and S. typhimurium TA100 were 21/96 and 13/96 respectively, (Fig.4.3. D &

E). The plant extracts were non mutagenic and toxic to test strain (Table 4.5) and (Fig.4.4. A-F).

Table 4.5 Mutagenic activity of methanolic extracts

Plant No. of positive wells / total no. of wells Interpretation

S. typhimurium TA98 S. typhimurium TA100

Background 21 / 96 13 / 96

Standard 96 / 96 88 / 96 Mutagenic +

Silybum marianum (MT) 0 / 96 0 / 96 Non-mutagenic -

Artemesia absinthium 1 / 96 1 / 96 Non-mutagenic -

Rheum emodi 0 /96 0 /96 Non-mutagenic -

+, significant increase in the number of positive wells compared to the related control (p <0.05);

–, no significant effect observed.

Toxicological testing is an essential prerequisite for the development of modern pharmaceutical

drugs. All the three selected plant extracts did not show any mutagenic effect against the two

tester bacterial strains. It is known that mutagenic compounds or substances could potentially

damage the germ line leading to fertility complications, mutations in future generations and may

induce cancer (Mortelmans and Zeiger, 2000). Significant higher level of secondary metabolites

such as flavonoids, total phenolics and gallotannins in freshly collected A. oppositifolia extract

compared to the stored one (Amoo et al., 2012). Flavonoids, such as galangin, quercetin and

kaempferol have been shown to be mutagenic in some S. typhimurium strains confirmed by the

Ames test (Boersma et al., 2000; Resende et al., 2012).

58

Acacia karroo, Osyris lanceolata and Pterocarpus angolensis were non-mutagenic

against Salmonella typhimurium strain TA98 while Elephantorrhiza burkei and Ekebergia

capensis showed weak mutagenicity (Mulaudzi et al., 2013). Dose dependent anti-mutagenic

activity of liquid ambar orientalis using TA98 and TA100 strains was observed at 2.5, 0.25 and

0.025 mg/plate concentrations and found strongest anti-mutagenic activity at 2.5 mg/plate

concentration against S. typhimurium TA 98 strain. Only one concentration (0.025 mg/plate) of

the extract did not exhibit any anti-mutagenic effect against S. typhimurium TA 100. The result

showed that extract at concentrations 2.5 mg/plate has a better anti-mutagenic effect (37.7–

85.67%) on the TA98 strain (Sarac and Sen., 2014).

Likewise in another study reported by Aremu et al. (2013) mutagenic activities of ten

long terms stored medicinal plants were evaluated. The freshly collected Acokanthera

oppositifolia plant extract at 5000 μg/ml exhibited mutagenic effects against TA1535 strain and

all other plant extracts did not show any mutagenicity against the three bacterial strains. Loh et

al., 2009 explored the mutagenic and anti-mutagenic activities of aqueous and methanol extracts

of Euphorbia hirta and found that Quercetin (25 g/mL) have strongly mutagenic in S.

typhimurium TA98 in the absence and presence of S-9 metabolic activation. Whereas both the

methanol and aqueous extracts at concentrations up to 100g/mL in the absence and presence of

S-9 metabolic activation were non-mutagen when tested with S. typhimurium TA98 and TA100

strains. The aqueous extracts at 100 g/mL and methanol extracts at 10 and 100 g/mL exhibited

strong anti-mutagenic activity against 2-aminoanthracene, a known mutagen, in the presence of

S-9 metabolic activating enzymes. In another study anti-mutagenicity was found for a number of

extracts from vegetables with higher anti-mutagenic properties of organically vs. conventionally

cultivated vegetables (Ren et al., 2001).

59

B C

D E

Fig.4.3. (A) Blank, (B) Standard mutagen TA98 (K2Cr2O7), (C) Standard mutagen TA100

(NaN3), (D) Background S. typhimurium TA98 (E) Background S. typhimurium TA100

60

A B

C D

E F

Fig. 4.4 Anti-mutagenic activity of Milk thistle, Artemesia absinthium and Rheum emodi

against Salmonella typhimurium strain TA98 and TA 100.

61

4.4. Metabolomics analysis

Metabolomic analysis was carried out using liquid chromatography mass spectrometry (LCMS)

technique.

4.4.1. Biochemical Profiling of Silybum marianum:

LCMS analysis of indigenously cultivated S. marianum was performed to identify

different compounds. The seven flavonolignans compounds present in S. marianum have the

same mol. weight (molecular formula C25H22O10) that’s why the mass chromatogram, ([M−H]−)

at m/z 481 was selected. The HPLC-MS condition was optimized to permit a good separation of

the seven target flavonolignans in silymarin. For MS analysis negative-ion mode of electrospray

ionization (ESI) was selected that provide extensive information via collision induced

dissociation (CID) fragmentations. The molecular ions [M−H]− spectra were obtained by

conducting CID-MS/MS procedure. In CID-MSn experiments product ions at m/z 463, 453

and/or 445, 301, were examined as these key ions peaks differentiated between the seven target

flavonolignans.

Silycristin:

Silychristin A and B were detected at retention times 4.14 and 4.62 min (Fig.4.5). In a

CID MS2 at 481 (m/z) major product ions at m/z 463, 453, 437, 391, 365, 327, 301 and 257

(Fig.4.6) were seen. CID-MS3 at m/z 463 showed three diagnostic fragment ion peaks at m/z

445, 435, 419 along with product ion peaks at 401, 391, 377, 353, 335 and 283 (Fig.4.7). CID-

MS3 at m/z 453 showed 435, 425, 409, 381, 313, 283 and 273 (Fig.4.8). In CID/MS3 at m/z 445

showed four distinctive fragment ions at m/z 430, 427, 417 and 401 (Fig.4.9). In CID/MS4 at m/z

419 showed five characteristic product ions at m/z 401, 389, 377, 335 and 283 (Fig.4.10).

Fig.4.5. Mass spectrum of silychristin

62

OO

O

OH

HO O

OH

O

OH

CH3

OH

Exact Mass: 482.12

Silychristin

Fig.4.6. MS2 of 481 at collision induced dissociation (CID) energy (19.00)

Fig.4.7. MS3 of 481 at CID (20.00) and 463 at CID (20.00)


63


Fig.4.10. MS4 of 481 at CID (20), 463 at CID (20) and 419 at CID (30)

Isosilybin:

The ion peak at m/z 481 showed major product ions at m/z 463, 453, 437, 301, 283 and 257.

O

O

O

OH

HO

O

OH

OCH3

OH

OH

C25H22O10

482.1

Isosilybin A

O

O

O

OH

HO

O

OH

OCH3

OH

OH

C25H22O10

482.1

Isosilybin B

Fig.4.11. Structure of Isosilybin A & Isosilybin B

64

Fig.4.12. MS2 of 513 at CID (20.00) showing main peak at 481

Fig.4.13. MS3 of 513 at CID (20:00) and 463 at (19:00)

Fig.4.14. MS3 of 513 at CID (20:00) and 453 at (25:00)

65

Silydianin:

In another peak at m/z 481 that differ from compound 1 and 2 showed product ions at

m/z 463, 453, 433, 409, 391, 301, 169 and 151 (Fig. 4.12). CID-MS3 on 463 m/z showed

diagnostic fragment ions at m/z 445, 433, 419, 401, 391, 377, 353, and 335 (Fig. 4.13), while

453 m/z showed fragment ions at m/z 435, 425, 409, 391, 381, 327, 313 and 273 (Fig. 4.14).

Fig.4.15. Structure of silydianin

Silybin A & B

In CID/MS2 at 481 m/z produce fragment ions peaks at m/z 463, 453, 435, 395, 301, 257

and 178. CID/MS3 at m/z 463 showed diagnostic fragment ions at m/z 445, 433, 419, 401, 391,

352 and 283. In CID/MS3 at 453 m/z showed fragment ions peaks at m/z 435, 423, 409, 390,

363, 327, 313 and 273. CID-MS4 at 433 m/z showed fragment ions at m/z 417, 415 and 389

(Fig.4.16).

Fig.4.16. MS3 of 513 at CID (20:00) and 481 at CID (20:00)

66

OO

O

OH

O O

OH

O

OH

CH3

OH

Exact Mass: 481.11_H2O

OO

O

OH

O O

OH

O

CH3

OH

Exact Mass: 463.10

OO

O

OH

O O

OHOH

CH3

OH

Exact Mass: 453.12

_CO

_H2O

OO

O

OH

O O

OH

CH3

OH

Exact Mass: 435.11

Fig.4.17. Proposed fragmentation pattern

4.4.2. Biochemical profiling of Aretmesia absinthium

LCMS analysis of Artemesia absinthium showed presence of different compounds such as peak

at 153 represents the alfa thoujone (Fig. 4.18). Peak at 288 represents the Absinthium (Fig. 4.19).

Peak at 281 represents the artemisian (Fig.4.20).

67

CH3

CH3H3C

O

Exact Mass: 154.14(alf a Thoujone)

Fig.4.18 MS2 229 at CID 33 peak at 153 showed Alfa thoujone

O

O

O

H3C

CH3

H

HCH3

H

O O

Exact Mass: 282.15

artemisinin

Fig.4.19 MS peak at 281 showed artemisinin

O

O

O

O

H

H

H

H

HO

H

H

H

H

HO

Exact Mass: 486.30

Absinthin

Fig.4.20 Structure of absinthin

68

4.4.3. Biochemical profiling of Rheum emodi

LCMS analysis of Rheum emodi showed several anthraquinones derivatives. Molecular ion peak

at 445 m/z shows the presence of Rhein-o-glycosides. In CID-MS2 at 445 (m/z) fragment ion peak

at 283 represents physcion (Fig. 4.21). Peak at 431 showed emodin glycoside (Fig. 4.22). Peak at

269 represents emodin having molecular mass 270 (Fig.4.23). Peak at 253 represents

chrysophanol having molecular mass 254 (Fig. 4.24) and peak at 283 showed Rhein (Fig. 4.25).

O

O

O

O OH

O

OHHO

HO

OH

HO

Exact Mass: 446.08

Rein-8-glucoside

OExact Mass: 284.07

Physcion

OH OHO

H3COH

Fig.4.21. MS2 at 445 m/z showed the presence of rhein-8-glucoside and Physcion at 283

O

O

O OH

CH3HO

OHO

HO

OH

HO

Emodin glycosideExact Mass: 432.11

Fig.4.22. MS showed emodin glycoside at 431

69

O

O

OHH3C

OH OH

Exact Mass: 270.05 Emodin

Fig.4.23. MS peak at 269 showing emodin

O

O

Exact Mass: 254.06 Crysophanol

OH OH

CH3

Fig.4.24. MS peak at 253 showing crysophanol

O

O

Exact Mass: 284.03 Rhein

OH OH

OH

O

Fig.4.25. MS peak at 283 showing rhein

70

Xiu-lan et al., 2009 has also reported seven peaks during the analysis of silybum

marianum. The first peak at m/z 303 was labeled as taxifolin while the other six compounds with

the same molecular mass of 482 were labeled as silycristin, silydianin, silybin A and B,

Isosilybin A and B. Flavonoids from Silybum marianum were separated and authenticated using

HPLC with UV combined with ESI/MS detection.

Similar results were reported by Wang et al. (2010) through UPLC coupled with ESI-MS

of the main flavonolignans of Silybum marianum. The main focus of the researcher was the

detailed analysis of fragmentation in CID using MS technique and to discriminate between the

silychristins, silydianin, silybins and isosilybins. The main peak for the seven flavonoligns was at

481. Variation of different compounds was seen on fragmentation pattern. Silychristins A and B

were detected at retention times 3.31 and 3.82 min. In a CIDMS/MS experiment at m/z 481,

major product ions at m/z 463 and 355 along with fragment ions at m/z 451, 433, 419, 337 and

325 were observed. Product ion MS3 at m/z 463 showed three diagnostic fragment ions m/z 445,

435, 419 and 337. In CIDMS4 at m/z 445 showed four fragment ions m/z 430, 427, 417 and 401.

The chief active compound in milk thistle is believed to be silybinin. There was some

evidence that showed silymarin was responsible for pharmacological activities (Skottova et al.,

2000). Different ecological conditions (Temperature, soil texture, rainfall, altitude level etc.) may

affect the silymarine yield. Evidence of genetic differences between ecotypes of milk thistle with

respect to the content of silymarin and its ingredients are also reported (Shokrpour et al., 2007).

Another study performed to explore the simultaneous analysis of the major active components of

silymarin in human plasma found the lower limit of quantification was 2 ng/mL of each analyte.

The ability of quantifying all 7 major bioactive components of milk thistle in any biological

matrix was reported for the first time using sensitive ESI–MS/MS assay to determine taxifolin,

silybin A, silybin B silychristin, silydianin, , isosilybin A, and isosilybin B in human plasma

(Brinda et al., 2012).

Another study reported by James et al., 2006 characterized silymarin contents by LC-ESI

tandem mass spectrometry which provided a unique fragmentation pattern. Peak 1 labeled as

silycrystin and its fragmentation found main peaks at 463, 453, 433, 355, 336, 325, 178 and 124.

Peak 2 labeled as silydianin and its fragmentation pattern represents 463, 453, 409, 301, 179 and

71

151. Peak 3 labeled as silybin A and its fragmentation pattern showed 463, 453, 435, 423, 301,

283, 273, 257,178 and 125. They all have the same molecular formula but differ only in their

fragmentation behavior and retention time.

Phytochemical analyses of the aqueous extract of Artemesia absinthium (AEAA)

revealed the presence of sesquiterpene lactones, flavonoids, phenolic acids and tannins. It

exhibited a protective effect against acute liver injury which may be attributed to its antioxidative

activity and thereby scientifically support its traditional use (Fiamegos et al., 2011). Five known

secondary metabolites, stigmasterol, 24 methylenecicloartanol, sabinyl acetate, sitosterol and

methyl-L-inositol were found in Artemesia sodiroi (Briceno et al., 2011). On the other hand in

methanolic extract of Artemesia sodiroi showed the presence of secondary metabolites including

tannins, alkaloids, sesquiterpene lactones, steroids, and cardiac glycosides (Ordonez, 2006). In

addition to its hepatoprotective potential another pharmacological benefit of Artemesia sodiroi is

its use to treat menstrual discomforts in women that may be associated with the presence of

sitosterol (Burton, 2002). GC/MS analysis of A. absinthium oil revealed the presence of 65

compounds viz. oxygenated monoterpenes (41.20%), oxygenated sesquiterpenes (27.06%),

aromatic hydrocarbons (9.39%), sesquiterpene hydrocarbons (4.99%) and monoterpene

hydrocarbons (8.03%). Major constituents of this oil were camphor (27.40%), davanone

(16.43%), ethyl (E)-cinnamate (5.81%), (E)-nerolidol (4.63%), and chamazulene (4.00%)

(Yinebeb et al., 2011).

72

4.5. Quantification of extracted Silymairn:

Extracted silymarin was quantified using a UV- Vis Spectrophotometer. The percentage

yield was 4.8% and 4.3% with methanol soxhelt and reflux respectively, while silymarin obtained

from reflux is more pure then Soxhlet method. The absorbance was 2.153 and 1.923 with reflux

and Soxhlet respectively. The extracted silymarin was compared with a standard silymarin

purchased from Sigma shown in (Fig.4.27). Extracted silymarin was in dry form and its color was

dark yellow.

Fig.4.26. Standard curve of standard silymarin (Sigma) at various concentrations

y = 0.0311x - 0.2537R² = 0.9977

0

0.5

1

1.5

2

2.5

3

3.5

0 20 40 60 80 100 120

Ab

sorb

an

ce

Concentration (ppm)

73

4.6. Animal Model

4.6.1. Preliminary evaluation of toxin:

A variety of intoxicants are available for use as hepatotoxic agent in experimental

animals. Of these agents, CCl4 at varying doses and paracetamol (500 mg/kg b. wt. daily for

three days) was used in preliminary trial to select appropriate hepatotoxin. The results of a single

dose (500 mg) of paracetamol and various concentration of CCl4 are presented in the Fig. 4.28-

4.30. The levels of SGPT (50.6 ± 5.7 U/L), SGOT (34.16 ± 5.35 U/L) and ALP (179.8 ± 26.5

U/L) in group 1 (absolute control) were lying in the normal range (Fig. 4.27).

In case of group 2 intoxicated with 30% CCl4 (3mL/kg b. wt. twice in a week), the levels

of SGPT with 1st dose of CCl4 was raised up to 128 U/L. With the 2nd dose (after 72 hours) of

CCl4 the levels of SGPT was abruptly raised to around 300 U/L and then sustained (222.6 ± 75

U/L) even up to 12th day. The levels of other biochemical parameters such as SGOT (86.4 ± 16

U/L) and ALP (253.5 ± 29 U/L) were also increased significantly (Fig. 4.28).

In case of group 3 intoxicated with 100 % CCl4 (0.7 mL/kg b. wt.) the levels of SGPT

after 24 hours were abruptly increased to 300 U/L and then up to 600 U/L after 48hours. The

other biochemical markers (SGOT, ALP) also showed significant increase. All animals found

expired on day three and their post-mortem (histopathology of liver tissue) revealed dilation of

sinus, congestion and blood accumulation.

In case of group 4 intoxicated with 100% CCl4 (3 mL/kg b. wt.) the levels of SGPT were

elevated up to 250 U/L after 24 hours. All of the rabbits under trial of this group were expired on

day 2 with this dose and histopathological changes were almost identical with the experimental

rabbits of group 3. None of the dose (0.7 mL/kg b. wt. or 3 mL/kg b. wt.) of 100 % CCl4 with

such high mortality is recommendable in curative studies; however it was observed that these

chemicals could be chosen to assess the preventive studies.

Biochemical analysis of group 5 intoxicated with paracetamol (500 mg/kg b. wt. daily for

three days) have shown gradual increase of all biochemical markers including SGPT, SGOT and

ALP up to 560 U/L, 198 U/L and 482 U/L, respectively on the 12th day (Fig. 4.29). No mortality

was observed in this group and gradual progression of all the biochemical parameters (SGPT,

SGOT and ALP) reflected that paracetamol at this dosage was the most appropriate hepatotoxin.

74

4.6.1.1. Conclusion preliminary evaluation of toxin:

On the basis of biochemical analysis of different groups during preliminary trial it has

been revealed that paracetamol (500 mg/kg body weight daily for three consecutive days) is the

most appropriate chemical to induce hepatotoxicity. Hence, it was decided that paracetamol

would be used as hepatoxin for the pre-clinical trial of this study.

Fig.4.27 Biochemical markers response of group 1 given normal saline water

(absolute control)

0

50

100

150

200

250

1 2 3 4 5 6 7 8 9 10 11 12

Act

ivit

y (

U\L

)

Days

Group 1

SGPT

SGOT

ALP

75

Fig.4.28 Biochemical markers response of group 2 treated with 30% CCl4 (3 mL/kg body

weight twice in a week for two weeks)

Fig.4.29. Biochemical markers response of group 5 treated with paracetamol (500 mg /

kg body wt. for three consecutive days)

0

50

100

150

200

250

300

350

1 2 3 4 5 6 7 8 9 10 11 12

Act

ivit

y (U

\L)

Days

Group 2

SGPT

SGOT

ALP

0

100

200

300

400

500

600

700

1 2 3 4 5 6 7 8 9 10 11 12

Act

ivit

y (

U\L

)

Days

Group 5

SGPT

SGOT

ALP

76

4.6.2. Pre-clinical Animal Trial:

4.6.2.1. Toxin:

Preliminary trials demonstrated that paracetamol damaged the liver gradually but with

less mortality and hence it was used to induce hepatotoxicity in a pre-clinical (animal model)

trial. The experimental animals (Rabbits) were placed in the animal room at National Institute of

Food Science and Technology, University of Agriculture Faisalabad. In order to induce

hepatotoxicity, paracetamol (500 mg/kg body weight) was administrated daily as a single dose

for three consecutive days (Fig.4.30 & 4.31).

4.6.2.2. Blood Sampling:

Blood sampling of the trial rabbits were drawn from the jugular vein. Hairs around the

jugular vein were removed by using the sciser and area around the jugular vein was sterlized

using methylated spirit. Approximately 3 mL blood of each rabbit was taken with the help of

fine needle syringe and stored in Jel-clot activator tube manufactured by Guangzhou Improve

Medical Instruments Co.Ltd (Fig 4.33 & 4.34). The blood samples were processed to separate

the serum which was then used to evaluate different biochemical parameters.

4.6.2.3. Treatment with medicinal plants in combinations:

Treatment with medicinal plants in combination (extracted silymarin, aqueous mixture of

A. absinthium and aqueous mixture of R. emodi) was started following administration of the

toxin to experimental animals in order to evaluate their hepatoprotective potential. The

combination of medicinal plants (Table.3.3) with different dosages were mixed, loaded in a

syringe and administered to the experimental animals (Fig 4.35 & 4.36).

4.7. Biochemical Analysis:

The hepatocellular damage induced by paracetamol and the effect of medicinal plants in

combinations was investigated by measuring the levels of SGPT, SGOT, ALP, protein albumin

in the blood serum. All these biochemical markers are described accordingly.

77

Fig.4.30. Loading of toxin in syringe Fig.4.31. Administration of toxin

Fig.4.32. Blood sampling Fig.4.33. Storing of blood

Fig.4.34. Loading of medicinal plants in syringe Fig.4.35. Administration of medicinal plant

78


The estimation of liver enzymes in a serum is a useful quantitative marker for the

magnitude and type of hepatocellular damage. SGPT is an enzyme which is specific indicator of

liver damage and is advised routinely by medical officers for the determination of liver damage.

A total of five time blood sampling from all the rabbits were drawn with periodic time at 0, 72,

168, 288 and 408 hours. The average values of SGPT of absolute control (40.5 ± 2.5 U/L),

positive control (41 ± 5.5 U/L) and standard (46 ± 10 U/L) at zero hour blood sampling were in

normal range. The overall average values of all groups (48.6 ± 12.74 U/L) and rabbits selected

for treatment with different twenty seven combinations (49 ± 13 U/L) of selected medicinal

plants were also lying in normal range (60 ± 15 U/L) as depicted in Fig.4.36. The non-significant

difference (p=0.878) of SGPT levels at this time prior to any treatment indicates the normal

functioning of the liver of all the rabbits selected for the study (Table 4.6).

The administration of toxin (Paracetamol 500 mg/kg body weight daily for three

consecutive days) was initiated after zero hour blood sampling. The intoxication caused

significant elevation of SGPT in the group of positive control (129 ± 7 U/L), standard (152.5 ±

20.5 U/L) and in all the remaining rabbits to be treated with various combinations of medicinal

plants (116 ± 38 U/L) as compared to absolute control (59 ± 2 U/L) after 72 hours (Fig.4.37).

The significant difference (p=0.051) in SGPT levels of absolute and positive control groups

revealed that the toxin induced deterioration of hepatocytes in the experimental animals (Table

4.6).

The administration of selected medicinal plants in different combinations was started

after 72 hours blood sampling. After 168 hours the relatively lower average values of medicinal

plants treated groups (147 ± 36 U/L) as compared to positive control (234.5 ± 5.5 U/L) and

standard (232 ± 34 U/L) indicate their hepatoprotective potential (Fig.4.38). The treatment with

the combinations of selected medicinal plants prevented the progression of SGPT levels and

statistically the p value of contrast and higher order interaction (S. marianum, A. absinthium and

R. emodi) was also significant (Table 4.6).

79

Table 4.6. Statistical analysis of serum glutamate pyruvate transaminase (SGPT)

Zero Hour 72 Hours 168 Hours 288 Hours 408 Hours

D. f Mean

Sq. Pr.(>F) Mean Sq. Pr.(>F) Mean Sq. Pr.(>F) Mean Sq. Pr.(>F) Mean Sq. Pr.(>F)

Treatment 31 131.292 0.878 1963.579 0.085 3229.533 0.000 2936.554 0.000 3398.652 0.000

Factorial vs Control 1 212.010 0.313 493.414 0.522 6020.658 0.004 8451.052 0.000 22711.270 0.000

Absolut & Positive control vs Standard 1 55.048 0.605 9303.048 0.009 8201.19 0.001 107.440 0.508 11109.000 0.000

Absolute control vs Positive control 1 1.000 0.944 4900.000 0.051 29584 0.000 38612.250 0.000

48841.000 0.000

Factorial 26 138.436 0.832 1690.531 0.172 2029.424 0.001 1591.530 0.000

842.267 0.000

Milk thistle 2 62.389 0.736 2246.741 0.166 3890.848 0.005 4528.594 0.000

1065.131 0.003

Artemisia absinthium 2 271.167 0.276 45.907 0.962 10272.84 0.000 81.052 0.715 47.679 0.729

Rheum emodi 2 13.722 0.934 410.963 0.708 318.707 0.59 765.456 0.056 972.806 0.005

Milk thistle : Artemisia absinthium 4 221.889 0.375 4710.630 0.011 904.558 0.223 1485.980 0.001 462.512 0.032

Milk thistle :Rheum emodi 4 62.861 0.868 1788.102 0.223 741.023 0.313 1333.055 0.002 1175.479 0.000

Artemisia Absinthium :Rheum emodi 4 209.722 0.403 1143.519 0.438 1397.725 0.079 1822.187 0.000

910.147 0.001

Milk thistle:Artemisia Absinthium :Rheum emodi

8 115.861 0.790 997.199 0.570 1453.376 0.039 1508.085 0.000

941.895 0.000

Residuals 27 201.466 1177.052

592.463

238.648

148.759

80

Fig.4.36. Box-whiskers plot for serum glutamate pyruvate transaminase (SGPT) at zero hour

Fig.4.37. Box-whiskers plot for serum glutamate pyruvate transaminase (SGPT) at 72 hours

81



82

The overall decline in the SGPT levels was observed in all the groups treated with

various combinations of medicinal plants when compared with positive control group after 288

hours. Likewise the lower average values of treated groups (113 ± 30 U/L) as compared to

positive control (251.5 ± 12.5 U/L) showed that the combinations (S. marianum, A. absinthium

and R. emodi) of selected medicinal plants initiated restoring of the liver functions. All medicinal

plants treated groups (twenty seven) were comparable with the standard group (silymarin treated)

but the higher average levels of SGPT in standard group indicates the better response of

medicinal plants in combination (Fig.4.39). The p-values of contrast and higher order interaction

(S. marianum, A. absinthium and R. emodi) was also statistically significant (p = 0.000) as shown

in Table 4.6.

The levels of SGPT after 408 hours were decreased further in the medicinal plants treated

groups. The average values of absolute control (54 ± 5 U/L), medicinal plants treated groups (69

± 22 U/L) and standard (75.5 ± 13.5 U/L) were almost in normal range except positive control

(275 ± 15 U/L) group as shown in Fig.4.40. A further decrease in the SGPT levels after 408

hours confirmed the hepatoprotective potential of medicinal plants administered in combination.

The increased levels of SGPT due to paracetamol decreased significantly (p = 0.000) using

various combinations of medicinal plants indicated that some of the plant combinations studied

repaired liver function using SGPT as reference. The graphical representation of all the five

blood samples analysis is shown in Fig.4.41.

Whether the interaction of selected medicinal plants at different doses in twenty seven

different combinations were hepatoprotective in term of SGPT was also studied (Fig.4.42). Three

levels (100, 200 and 500 mg) of each selected medicinal plants (S.marianum (MT), Rheum

emodi (RHE) and A. absinthium (AAB)) in various combinations were used in this study.

In the presence of fixed quantity (100 mg) of both MT and RHE the effect of AAB on

SGPT was gradually improved and at the end of 408 hours all doses 100, 200 and 500 mg of

AAB revealed almost similar results (Fig. 4.42 a). Likewise in another combination when the

quantity of MT and RHE was fixed at 200 mg & 100 mg respectively, faster resolution of the

biochemical marker, under discussion was observed with 100 mg of AAB followed by 500 mg

83

and 200 mg at 168 hours. The level of SGPT was further decreased in this combination with 100

mg of AAB at 288 hours. Conclusively at the end of the experiment (408 hours) AAB at 100 mg

provided a better response as compared to 200 mg and 500 mg (Fig. 4.42 b).

In another combination containing a fixed quantity of MT (500 mg) and RHE (100 mg)

the response of AAB at the end of 408 hours was comparable at all doses of 100, 200 and 500

mg (Fig. 4.42 c). Rapid resolution of SGPT was also observed with 100 mg of AAB in the

presence of fixed quantity of MT (100 mg) and RHE (200 mg) at the end of 408 hours (Fig. 4.42

e). Decrease in SGPT levels with almost similar response at all the three levels of AAB was

noted in the presence of the fixed quantity of MT (500 mg) and RHE (200 mg) at the end of 408

hours (Fig. 4.42 g). The healing effect of AAB with 100 mg was good initially and almost

similar response was observed at all the three levels in another combination containing MT (100

mg), RHE (500 mg) at the end of 408 hours (Fig. 4.42 i). Another combination containing MT

(200 mg), RHE (500 mg) had a similar response to AAB was noted at all three levels at the end

of 408 hours (Fig. 4.42 j).

SGPT analysis of all the five times blood sampling validates that all of the combination

containing 100 mg of any plant displayed almost similar results in all possible twenty seven

combinations. However delayed initial response of AAB at 100 mg was noted in some of the

combinations (Fig. 4.42-e-f-g-h-j-k-l-n) at 168 hours but at the end of 408 hours all these

combinations revealed an almost similar response with regards to resolution of SGPT. The only

exception was a combination containing 200 mg of both MT and RHE in which the levels of

SGPT were not up to mark with 100 mg of AAB at the end of 408 hours (Fig. 4.42 -f). Most of

the combinations reduced the SGPT levels to the normal range. The lowest dose of 100 mg for

each plant proved to be as potent as any one of the other combination in restoring liver functions

against paracetamol induced liver damage. Consequently a dose 100 mg from each plant in

combination is recommended for resolving liver functions.


Owing to a large variation in the reference values of SGOT in rabbits, the least levels of

SGOT observed at zero hour blood sampling was taken as the normal range (47.3 ± 15.25 U/L)

as shown in Fig.4.43.

84


Fig.4.41. Graphically representation of all five times blood sampling analysis of SGPT

85

Fig.4.42. Interactive effects of various combinations of selected medicinal plants on SGPT over time. The levels 100, 200 and 500 mg

of Silybum marianum (MT) and Rheum emodi (RHE) were fixed, whereas the levels of Artemesia absinthium (AAB) varying, 100 mg

(red), 200 mg (Green) and 500 mg (Blue)

86

The average values of the absolute control (46 ± 3 U/L), positive control (35 ± 4 U/L),

standard groups (42.33 ± 8.5 U/L) and in all the remaining rabbits to be treated with various

combinations of medicinal plants (47 ± 15 U/L) were almost similar indicating normal

functioning of rabbits at this time (Fig.4.43). Statistically, p values of all the groups to be studied

including absolute control, positive control, standard and twenty seven groups treated with

various combinations of medicinal plants were non-significant at zero hour analysis (Table 4.7).

The levels of SGOT were significantly increased after 72 hours. The relatively higher

average values of positive control (72 ± 5 U/L), standard (69 ± 12 U/L) and remaining twenty

seven combinations (75 ± 29 U/L) as compared to absolute control (43 ± 8 U/L), mean that toxin

affected the liver functions of rabbits (Fig.4.44). The p values of all the groups were also

statistically significant at this moment (Table 4.7).

The levels of SGOT were further increased after 168 hours. The average values of

positive control (91 ± 6 U/L), standard (93 ± 15 U/L) and medicinal plants treated twenty seven

group (92 ± 40 U/L) were higher than that of the zero hour level as compared to the absolute

control (44 ± 5 U/L) as shown in Fig.4.45. Statistically, significant p values were noted in all the

groups at this time (Table 4.7).

The overall decreasing trend of SGOT levels were observed in the medicinal plants

treated groups and standard group after 288 hours. The average values of absolute control (51 ±

3 U/L), standard (72 ± 19 U/L) and twenty seven medicinal plants treated groups (72 ± 33 U/L)

were close to the normal range, whereas the levels of SGOT in positive control group (123 ± 7

U/L) were significantly higher as compared to absolute control group (Fig.4.46). The least levels

of SGOT in medicinal plants treated groups indicated that treatment with the various

combinations of medicinal plants successfully restored the liver functions at this time and p

values of treated group were statistically significant (Table 4.7).

The levels of SGOT were further decreased after 408 hours. The average values of

absolute control (45 ± 2 U/L), standard (36 ± 11 U/L) and in all remaining twenty seven groups

treated with the combinations of medicinal plants (47 ± 26 U/L) were in normal range except

positive control (143 ± 5 U/L). The decreased SGOT levels exhibited the protective effect of

medicinal plants when compared with positive control (Fig.4.47).

87

Table 4.7 : Statistical analysis of serum glutamate oxaloacetate transaminase (SGOT)


D. f Mean Sq. Pr.(>F) Mean Sq. Pr.(>F) Mean Sq. Pr.(>F) Mean Sq. Pr.(>F) Mean Sq. Pr.(>F)

Treatment 31 138.159 0.991 1323.696 0.000 1738.252 0.192 1136.371 0.184 1394.615 0.002



Absolute control vs Positive control 1 121.000 0.552 784.000 0.080 2162.250 0.199 5112.250 0.018

9702.250 0.000

Factorial 26 144.244 0.983 1487.617 0.000 1886.555 0.146 1099.894 0.214

941.218 0.034

Milk thistle 2 71.722 0.808 849.796 0.041 4340.256 0.045 2189.449 0.084

1255.271 0.082


Rheum emodi 2 140.389 0.661 969.463 0.028 5022.819 0.029 84.546 0.901 387.969 0.439




707.413 0.217


8 111.250 0.946 2278.519 0.000 1255.834 0.454 1932.611 0.043

1097.215 0.042

Residuals 27 333.690 238.276 1246.889 806.037 457.389

88

Fig.4.43 Box-whiskers plot for serum glutamate oxaloacetate transaminase (SGOT) at zero hour

.

Fig.4.44 Box-whiskers plot for serum glutamate oxaloacetate transaminase (SGOT) at 72 hours

89



90


Fig.4.48. Graphically representation of all five times blood sampling analysis of SGOT

91

Fig.4.49. Interactive effects of various combinations of selected medicinal plants on SGOT over time. The levels 100, 200 and 500 mg

of Silybum marianum (MT) and Rheum emodi (RHE) were fixed whereas the levels of Artemesia absinthium (AAB) varying, 100 mg


92

The graphical representation of all the five times blood sampling of SGOT are shown in

Fig.4.48. The p values of all the groups were statistically significant (Table 4.7).

In the presence of fixed quantity (100 mg) of both MT and RHE, the effect of AAB with

all the three levels on SGOT at the end of 408 hours was almost similar and remained in the

normal range (Fig. 4.49 a). In another combination of medicinal plants, when the quantity of MT

and RHE was fixed at 100 mg and 500 mg fast resolution of SGOT levels were observed after 72

hours with 100 mg of AAB. At the end of 408 hours, all doses of AAB provide almost similar

response to bring the SGOT levels in the normal range (Fig. 4.49 c).

All doses of AAB, irrespective of the studied doses of MT and RHE brought the SGOT

levels closer to the values at zero hour, at the end of experiment. Exceptionally, in some

combinations, the increased levels of SGOT with 500 mg and 200 mg of AAB were not resolved

at the end of 408 hours (Fig. 4.49-e-f-m). The combination, comprising 100 mg of each

medicinal plant was not only better one with reference to SGOT, but also found the best healer in

case of SGPT. The overall SGOT effect revealed that medicinal plants in combinations improve

the liver functions significantly and its interaction represents synergism.

4.7.3.1. Alkaline phosphatase (ALP)

Alkaline phosphatase (ALP) is present in a number of tissues including intestine, bone,

liver and placenta. Serum ALP is important in the diagnosis of two main groups of disorders, e.g.

bone disease associated with increased osteoblastic activity and hepatobiliary disease.

Unexpectedly, the levels of ALP at zero hour varied considerably with average values of

absolute control (115 ± 28), positive control (154 ± 58), standard (159 ± 43) and twenty seven

groups to be treated with different combinations of medicinal plants (151 ± 76) were higher than

the reference value (Fig.4.50).

Whatsoever, the value of ALP was at zero hour, the intoxication with paracetamol

further increased its levels at 72 hours. The increased average values of ALP in positive control,

standard and remaining twenty seven groups were 182 ± 64 U/L, 190 ± 59 U/L and 195 ± 65 U/L

respectively, mean that toxin affected the liver functions of rabbits (Fig.4.51).

93

The levels of ALP were sustained after 168 hours and the average values of absolute

control (110 ± 67), positive control (161 ± 39), standard (184 ± 15) and twenty seven

combinations (166 ± 65) were found to be different (Fig.4.52).

The levels of ALP were maintained after 288 hours. The average values of absolute

control (132 ± 61), positive control (161 ± 52), standard (184 ± 31) and twenty seven medicinal

plants treated groups (180 ± 65) were different (Fig.4.53) and this difference was statically non-

significant (Table 4.8).

The elevated levels of ALP were decreased and maintained in almost all combinations

after 408 hours. The average values of absolute control (137 ± 50), positive control (155 ± 42),

standard (176 ± 8) and remaining twenty seven groups (182 ± 57) were close to zero hour range

(Fig.4.54) and statistically p values were non-significant. Although, majority of the combinations

normalized the liver functions but the most suitable combination that brought faster resolution of

biochemical markers (SGPT, SGOT and ALP) was the combination containing 100 mg of each

plant. The graphical representation of all the five times blood sampling is shown in Fig.4.55. The

interaction analysis of medicinal plants at all three levels are presented in Fig.4.56. The p values

of ALP in all possible interaction of selected medicinal plants with periodic time are given in

table 4.8.

4.7.4. Total protein:

Total protein is a measure of the protein content present in blood. Albumin and globulin

are the two main types which are the major contributor of the total protein. Low protein levels

can be a sign of liver abnormality. The average levels of protein in absolute control (5.8 ± 0.2

g/dL), positive control (6.5 ± 0.2 g/dL), standard (6.23 ± 0.13 g/dL) and other twenty seven

groups to be treated with combination of medicinal plants (5.7 ± 0.65 g/dL) were in normal range

at zero hour blood sampling which showed the normal liver functioning (Fig.4.57). All the

values of protein were close to one another and statistically found p values were non-significant

(Table 4.9).

94

Table 4.8: Statistical analysis for alkaline phosphate (ALP)



Treatment 31 8437.646 0.005 5772.408 0.009 3478.850 0.823 3700.811 0.798 3297.658 0.401




324.000 0.745

Factorial 26 9737.513 0.002 6283.719 0.006 3848.022 0.731 4091.199 0.702

3724.722 0.287

Milk thistle 2 3458.389 0.351 2500.796 0.362 900.612 0.833 2553.428 0.608

521.635 0.841


Rheum emodi 2 3206.167 0.378 3857.352 0.214 1470.254 0.743 4621.120 0.412 2361.104 0.464




13826.611 0.006


8 2628.444 0.587 1611.227 0.706 814.242 0.994 1322.603 0.973

661.577 0.984

Residuals 27 3184.621 2373.138 4905.648 5041.241 2991.259

95

Fig.4.50 Box-whiskers plot for alkaline phosphate (ALP) at zero hour

Fig.4.51 Box-whiskers plot for alkaline phosphate (ALP) at 72 hours

96



97


Fig.4.55. Graphically representation of all five times blood sampling analysis of ALP

98

Fig.4.56. Interactive effects of various combinations of selected medicinal plants on ALP over time. The levels 100, 200 and 500 mg of

Silybum marianum (MT) and Rheum emodi (RHE) were fixed whereas the levels of Artemesia absinthium (AAB) varying, 100 mg


99

The protein levels were decreased as a result of intoxication after 72 hours (Fig.4.58).

The average values of protein in positive control (4.85 ± 0.5 g/dL), standard (4.7 ± 0.50 g/dL)

and other twenty seven combinations to be treated with medicinal plants (4.47 ± 0.26 g/dL) were

decreased as compared to absolute control (5.56 ± 0.25 g/dL), which revealed that toxin might

has affected the liver functions.

The levels of protein were retained after 168 hours. The average values of protein in

absolute control (5.55 ± 0.15 g/dL), and twenty seven groups treated with medicinal plants

combinations (4.5 ± 0.38 g/dL) were constant whereas in the positive control (4.2 ± 0.2 g/dL)

and standard (4.3 ± 0.32 g/dL) group protein levels were decreased further (Fig.4.59). The p

values were statistically significant at this time (Table 4.9).

The levels of protein were maintained after 288 hours. The average values of protein in

absolute control (5.5 ± 0.12 g/dL), positive control (3.05 ± 0.15 g/dL), standard (4.2 ± 0.30 g/dL)

and medicinal plants treated groups (4.2 ± 0.36 g/dL) were different. Protein levels were constant

in absolute control and gradual decrease was noted in positive control (Fig.4.60). The statistically

derived p values were significant at this point of time (Table 4.9).

The levels of protein were persistent after 408 hours. The average values of protein in

absolute control (5.55 ± 0.05 g/dL), standard (4.6 ± 0.21 g/dL) and medicinal plants treated

groups (4.55± 0.34 g/dL) were restored whereas protein levels were further reduced in positive

control (3.45 ± 0.15 g/dL). The animals treated with medicinal plants combinations started

restoring the liver functions by preventing further reduction of proteins levels that confirmed the

hepatoprotective effect of medicinal plants (Fig.4.61). The overall five times blood sampling

analysis also showing this trend (Fig. 4.62).

The interactive effect of medicinal plants in combination clearly revealed its

effectiveness when compared with paracetamol treated group. The levels of protein at zero hour

were lying in normal range. The protein levels were decreased as a result of intoxication at 72

hours. The administration of medicinal plants in combinations after 72 hours stabilized the

protein levels and prevented the further deterioration of liver functions. At the end of 408 hours

most of the combinations shows an improving trend in protein levels (Fig. 4.63).

100

Table 4.9: Statistical analysis for protein



Treatment 31 0.485 0.153 0.153 0.000 0.250 0.000 0.302 0.000 0.201 0.000




0.250 0.010

Factorial 26 0.516 0.123 0.129 0.000 0.267 0.000 0.234 0.000

0.215 0.000

Milk thistle 2 0.136 0.668 0.357 0.000 0.006 0.854 0.765 0.000

0.046 0.257


Rheum emodi 2 0.572 0.196 0.054 0.027 0.157 0.026 0.092 0.106 0.602 0.000




0.100 0.031


8 0.703 0.066 0.105 0.000 0.139 0.005 0.348 0.000

0.128 0.003

Residuals 27 0.331 0.013 0.037 0.037 0.032

101

Fig.4.57 Box-whiskers plot for total protein at zero hour

Fig.4.58 Box-whiskers plot for total protein at 72 hours

102

Fig.4.59. Box-whiskers plot for total protein at 168 hours


103


Fig.4.62. Graphically representation of all five times blood sampling analysis of Protein

104

Fig.4.63. Interactive effects of various combinations of selected medicinal plants on protein over time. The levels 100, 200 and 500 mg

of Silybum marianum (MT) and Rheum emodi (RHE) were fixed whereas the levels of Artemesia absinthium (AAB) varying, 100 mg


105

4.7.5. Albumin:

Albumin is an important protein synthesized in liver and low levels are a sign of liver

damage. Serum albumin levels were also measured to see the curative effect of various medicinal

plants combinations. The albumin levels of experimental animals at zero hour were in normal range.

The average values of albumin in absolute control (3.9 ± 0.1 g/dL), positive control (3.95 ± 0.05

g/dL), standard (4.26 ± 0.04 g/dL) and other twenty seven groups to be treated with

combinations of medicinal plants (3.91 ± 0.51 g/dL) were in normal range (Fig.4.64) that showed

the normal functioning of the liver.

The levels of albumin were decreased due to intoxication with paracetamol after 72

hours. The average values of albumin in positive control (3.80 ± 0.05 g/dL) standard (3.53 ± 0.28

g/dL) and in remaining twenty seven groups selected to treat with medicinal plants combination

(3.40 ± 0.21 g/dL) were started decline (Fig.4.65) and statistically p values were significant at

this point of time (Table 4.10).

The levels of albumin were further decreased in positive control (3.05 ± 0.15 g/dL) group

whereas the albumin levels were sustained in absolute control (3.6± 0.2 g/dL), standard (3.56 ±

0.12 g/dL) and medicinal plants treated groups (3.38 ± 0.29 g/dL) after 168 hours (Fig.4.66).

The levels of albumin were further decreased after 288 hours. The average values of

albumin in positive control, standard and twenty seven medicinal plants treated groups were 2.6

± 0.1 g/dL, 3.13 ± 0.1 g/dL and 3.08 ± 0.24 g/dL respectively (Fig.4.67). This difference was

statistically significant and the p values of all the groups were significant (Table 4.10).

The improvement in albumin levels was started after 288 hours (Fig.4.68). This

improvement showed that medicinal plants in combinations restored the liver function and

prevents the further reduction of albumin levels. The average levels of albumin were 3.55 ± 0.05

g/dL, 3.43 ± 0.23 g/dL and 3.43 ± 0.23 g/dL correspondingly in absolute control, standard and

remaining twenty seven medicinal plants treated groups were improved. Albumin levels were

further decreased in positive control (2.55 ± 0.05 g/dL) at the end of 408 hours. The overall five

times blood samples analysis are shown graphical in Fig. 4.69.

106

The interaction analysis clearly shows that the levels of albumin at zero hour were lying

in normal range. The intoxication with paracetamol decreased the albumin levels significantly at

72 hours. The combinations of medicinal plants stabilized the albumin levels after 168 and 288

hours. The levels of albumin were gradually improved at the end of 408 hours (Fig.4.70). All the

selected combinations showed protective effect against paracetamol induced liver damage. The

most suitable combination that brought all the biochemical parameters (SGPT, SGOT, ALP,

Protein and Albumin) at normal level was the least doze i.e. 100 mg of each plant.

107

Table 4.10: Statistical analysis for albumin



Treatment 31 0.385 0.002 0.088 0.000 0.141 0.000 0.080 0.051 0.088 0.001




0.423 0.001

Factorial 26 0.404 0.002 0.079 0.000 0.151 0.000 0.074 0.082

0.079 0.004

Milk thistle 2 1.336 0.000 0.109 0.002 0.084 0.070 0.028 0.524

0.100 0.038


Rheum emodi 2 0.270 0.147 0.095 0.003 0.282 0.001 0.155 0.041 0.039 0.252




0.063 0.082


8 0.371 0.019 0.087 0.000 0.042 0.211 0.062 0.225

0.074 0.024

Residuals 27 0.131 0.013 0.029 0.043 0.027

108

Fig.4.64. Box-whiskers plot for albumin at zero hour

Fig.4.65. Box-whiskers plot for Albumin at 72 hours

109

Fig.4.66 Box-whiskers plot for albumin at 168 hours


110


Fig.4.69. Graphically representation of all five times blood sampling analysis of albumin

111

Fig.4.70. Interactive effects of various combinations of selected medicinal plants on albumin over time. The levels 100, 200 and 500

mg of Silybum marianum (MT) and Rheum emodi (RHE) were fixed whereas the levels of Artemesia absinthium (AAB) varying,

100mg (red), 200 mg (Green) and 500 mg (Blue)

112

4.7.6. Albumin globulin (A/G) ratio:

Albumin and total globulin are the components that make up major portion of the total

protein. The albumin within the body is almost entirely produced by the liver. Albumin is

responsible for variety of biochemical functions and approximately 80% of the colloid-osmotic

pressure between blood and tissue fluids is maintained due to this fraction of protein. A

comparison called the albumin/globulin (A/G) ratio, in which amount of albumin compared with

globulin, is very useful in the evaluation of liver and kidney disease. The A/G ratio can be

decreased in response to a low albumin or to elevated globulins.

The average values of A/G ratio in absolute control (2.55 ± 0.05), positive control (2.55 ±

0.05), standard (2.86 ± 1.61) and twenty seven groups to be treated with medicinal plants

combinations (2.66 ± 1.67) were close to each other (Fig.4.73) and statistically derived p values

was also non-significant at this time (Table 4.11).

The average values of A/G ratio in positive control, standard and twenty seven medicinal

plants treated groups were 3.89 ± 0.44, 3.12 ± 0.95 and 3.5 ± 1.63 respectively, after 72 hours

(Fig. 4.74). This difference was statistically significant (Table 4.11). The levels of A/G ratio in

absolute control (2.63 ± 0.37) remained same after 72 hours as was in zero hour blood sampling.

At the end of 408 hours the average values of A/G ratio in absolute control, positive

control, standard and medicinal plants treated twenty seven groups were 3.91 ± 1.22, 2.52 ± 0.24,

2.95 ± 1.37 and 3.01 ± 1.37 respectively, (Fig.4.77). Graphically representation of all five times

blood sampling analysis of A/G ratio is presented in Fig.4.78. The interaction effect of various

combinations on A/G ratio at different doses of medicinal plants is presented in Fig. 4.79.

Although majority of the combinations showed protective effect against paracetamol induced

liver damage but the most suitable combination that brought all the biochemical parameters at

normal level was the least doze i.e. 100 mg of each plant.

113

Table 4.11: Statistical analysis for albumin globulin (A/G) ratio



Treatment 31 2.722 0.416 3.473 0.008 8.162 0.356 3.882 0.011 2.539 0.004




1.932 0.157

Factorial 26 3.025 0.313 4.020 0.003 4.687 0.852 3.102 0.047

2.937 0.002

Milk thistle 2 0.753 0.743 6.950 0.014 4.893 0.510 4.832 0.066

0.342 0.691


Rheum emodi 2 3.348 0.280 5.589 0.030 5.238 0.487 4.903 0.063 2.569 0.078




1.461 0.203

Milk thistle: Artemisia Absinthium : Rheum emodi

8 2.582 0.439 1.348 0.485 5.458 0.631 3.648 0.052

3.052 0.009

Residuals 27 2.514 1.404 7.079 1.602 0.914

114

Fig.4.71 Box-whiskers plot for A/G ratio at zero hour

Fig.4.72 Box-whiskers plot for A/G ratio at 72 hours

115



116

Fig.4.75. Box-whiskers plot for A/G ratio at 408 hours

Fig.4.76. Graphically representation of all five times blood sampling analysis of A /G ratio

117

Fig.4.77. Interactive effects of various combinations of selected medicinal plants on A/G ratio over time. The levels 100, 200 and 500

mg of Silybum marianum (MT) and Rheum emodi (RHE) were fixed whereas the levels of Artemesia absinthium (AAB) varying, 100


118

In present study hepatoprotective potential of selected medicinal plants in combination

was evaluated against paracetamol induced hepatotoxicity. Intoxication of animals with

paracetamol is one of the most commonly used models to evaluate the magnitude of hepatic

damage (Hurkadale et al., 2012; Venkatachalam and Muthukrishnan, 2013). Paracetamol

induced toxicity is due to the formation of toxic metabolites, N-acetyl–para-benzoquinonimin

(NAPBQ1), when a part of it is metabolized by cytochrome P450 (Mayuren et al., 2010; Ibrahim

et al., 2011). Several mechanisms may be cited to be responsible for either inducing hepatic

injury or worsening the damage process. About 70-80 percent of blood received by liver arrives

directly from gastrointestinal organs and then spleen through portal veins which bring drugs and

xenobiotic in concentrated form (Vikramjit and Mrtcalf, 2009). During hepatic damage cellular

enzymes such as SGPT, SGOT, ALP and other associated enzymes are leaked into serum and

resultantly increase of liver weight and the elevation of their serum concentration (Rajib et al.,

2009). The estimation of liver enzymes in a serum is a useful quantitative marker for the extent

and type of hepatocellular damage. The increased production of serum enzymes in blood stream

was associated with central/ submissive necrosis of liver which caused severe hepatic injury

(Murugesan et al., 2011; Eesha et al., 2011; Madhukiran et al., 2011). Paracetamol significantly

increased SGPT, SGOT, ALP levels and significantly decreased liver GSH-Px activity, when

compared to the control group. FDA committee recommended lowering the daily therapeutic

dose of paracetamol to decrease the high number of cases of liver injury (US-FDA 2009).

SGPT is an enzyme which is specific indicator of liver damage and is routine clinical test

for the determination of liver injury in hepatic patients (Turgut, 2000). In the present study, a

sharp increase in the serum SGPT levels was observed in paracetamol treated rabbits as

compared to absolute control group (Fig.4.40). Previous investigations on acute paracetamol

intoxications revealed that in addition to SGPT levels, serum SGOT and ALP levels are also

increased (Sener et al., 2005; Kupeli et al., 2006; Hurkadale et al., 2012; Venkatachalam and

Muthukrishnan, 2013; Rasool et al., 2014). SGPT is elevated in the serum after acute

hepatocellular necrosiss by N-acetyl–para-benzoquinonimin (NAPBQ1), the toxic reactive

metabolite of paracetamol (Mayuren et al., 2010). Serum SGOT and ALP levels are increased

not only in liver damage but also in case of the damage to various other tissues and organs. Due

to this reason, increased SGPT levels are considered to be specific to liver damage

(Hajimehdipoor et al., 2006). Similarly, in this study, serum SGOT and ALP levels were found

119

to be increased, when compared to the control group (Fig. 4.47 and 4.54). Reinforcing the above

stated mechanisms, biochemical markers and histopathological profile revealed deleterious

effects in paracetamol treated group. Thus, it clearly states that, toxicity is due to either of the

above mechanisms such as depletion of glutathione store or free radical generation or lipid

peroxidation. It is reasonable as agents in most of existing drug combinations have been found to

possess similar therapeutic effects and work in the same or related pathways (Jia et al., 2009; Xu

et al., 2010; Doehmera et al., 2011; Wang et al., 2012). Medicinal plants usually target a group

of proteins and pathways for the specific function (Zhao et al., 2010; Sun et al., 2012), so it was

hypothesized that when two herbs that target the common or related pathway are likely to work

synergistically. The combination of medicinal plants used in this study maintained the normal

architecture with minimal injuries and better protection than standard (silymarin). Among all

combinations the 100 mg of each selected medicinal plant was the most appropriate dose with

least toxicity and better protective effect.

The increased levels of serum SGPT, SGOT, ALP in experimental animals treated with

paracetamol indicate a deterioration of the hepatic functions due to the toxic effects of the drug.

SGOT is present in cytosolic and mitochondrial isoenzymes and is found in the liver, cardiac

muscle, skeletal muscle and kidney. It is less sensitive and specific for the liver (Kanbur et al.,

2009). The Outcomes of our study proved that the combinations of medicinal plants strongly

reduce the increased serum levels of SGPT in rabbits. It has been demonstrated that SGOT

concentration rises as a result of liver damage in paracetamol over dosage in rabbits. The

reduction in levels of SGOT, SGPT by the various combinations of medicinal plants is an

indicator of stabilization of the plasma membrane as well as repair of hepatic tissue damage

caused by paracetamol. The lowering of total protein suggests its inhibiting role on biosynthetic

pathways of various anabolic steps in liver (Piniro and Piniro, 2004) whereas administration of

medicinal plants in combination started restoring the protein level to the normal values.

Alkaline phosphate concentration is related to the functioning of hepatocytes high level

of alkaline phosphatase in the serum related to the increased synthesis of it by cells lining bile

canalliculi usually in response to cholestasis and increased biliary pressure (Nadkarni, 1996).

The levels of ALP increased in the paracetamol treated rabbits, may be due to increased

synthesis in the presence of increasing biliary pressure and it is decreased to baseline levels with

120

the combinations of various medicinal plants. Similarly with D. gangeticum (plant) at 500

mg/mL dose the levels of SGOT, SGPT and ALP was significantly reversed produced by

paracetamol and caused a subsequent recovery towards normalization (Venkatachalam and

Muthukrishnan, 2013).

4.7.7. Antioxidant enzymes:

The effects of medicinal plants in combinations on the level of antioxidant

enzymes in liver homogenate are presented in Table 4.12. The activities of antioxidant enzymes

glutathione peroxidase (POD), superoxide dismutase (SOD) and catalase (CAT) in liver

homogenate were significantly decreased (P < 0.002) in rabbits treated with paracetamol

(positive control group) as compared to the absolute control group. Medicinal plants in

combinations exerted a beneficial effect on antioxidant enzymes since the activities of these

enzymes were found to be significant. The selected medicinal plants in combination successfully

restored the liver function indicating the protection of structural integrity of hepatocytic cell

membrane or regeneration of damaged liver cells. The increase in oxidative stress due to

paracetamol consequently decreases the level of antioxidant enzymes in positive control. The

findings in this group indicated a significant decrease in the activities of antioxidant enzymes as

compared to the absolute control. The results clearly showed that the combination of medicinal

plants significantly restored the liver functions.

The efficacy of any hepatoprotective drug is dependent on its capacity of either

reducing the harmful effect or restoring the normal hepatic physiology affected that has been

distributed by the hepatotoxin (Mujahid et al., 2013). The findings were co-related with the

experiments of Tostmann et al. (2008) that reported significant decrease in the activities of

catalase, superoxide dismutase (SOD) and GPx in anti-tubercular drugs induced rats. The

combination of silymarin and Ginkgo biloba extract (EGb) improved antioxidant activities such

as SOD, GPx and GSH inducing the useful antioxidant abilities in N-nitrosodiethylamine

(NDEA) toxicated experimental animals (El-Mesallamy et al., 2011).

121

Table.4.12.Antioxidant enzymes activity offered by different combinations of selected medicinal plants

S. No Groups SOD

(units/mg protein)

POD (units/mg protein)

CAT (U/min/ mg protein)

1 Absolute control 15.88 ±0.8 4.6±0.3

60±2.17

2 Positive control (Paracetamol) 7.86±0.5 2.1±0.27

32±1.10

3 Extracted silymarin 100 mg + Artemisia Absinthium 100mg + Rheum emodi 500mg 14.76±0.3 4.3±0.5

52±2.1

4 Extracted silymarin 200 mg + Artemisia Absinthium 500mg + Rheum emodi 500mg 13.17±0.9 3.9±0.6

57±2.7

5 Extracted silymarin200mg + Artemisia Absinthium 200mg + Rheum emodi 200mg 14.66±1.1 4.2±0.15 56.1±1.9

6 Standard silymarin 200 mg 11.78±0.74 3.4±0.73 48.1±1.8

7 Extracted silymarin 500 mg + Artemisia Absinthium 100mg + Rheum emodi 100mg 16.18±0.22 4.5±0.14 60.4±2.6




11 Extracted silymarin 500 mg + Artemisia Absinthium 500mg + Rheum emodi 100mg 14.08 ±0.21 4.58±0.13 59.1±1.7



122

Antioxidant enzymes are 1st line of defense for any biological system against free

radicals, peroxides and superoxide anion, which are generated in different reactions within a cell.

Catalase and glutathione peroxidase are the main enzymes which are used to neutralize hydrogen

peroxide, which was induced by the oxidation of GSH to GSSG of superoxide dismutase in

mitochondria and cytoplasm (Park et al., 1994). Antioxidant molecules (GSH, ascorbic acid

(AA), vitamins A, E) as well as antioxidant enzymes such as SOD, glutathione peroxidase,

catalase protect the cells against oxidative stress and free radical damage (Burton et al., 1985).

Destruction of hepatocyte cell membrane and inhibition of hepatic protein caused by the

mushrooms (e. g. the death cup fungus and A. virosa, Amanita phalloides,) contain two toxins: α-

amanatine and phalloidine which resulted in damage and ultimately death. Silymarin provide

protection against these toxins by blocking the toxin’s binding sites and also increase the

regeneration of hepatic cells. Silibinin obtained from milk thistle prevent from the hepatic injury

when used intravenously against toxin (mushrooms) induced liver damage (Desplaces et al.,

1975; Vogel et al., 1984). Silymarin provides hepatoprotection against toxicity induced by

carbon tetrachloride, acetaminophen, phalloidin, halothane, thioacetamid. It also prevents liver

from radiation, iron overload and ischaemic injury (Vizcaya et al., 2009; Akkaya and Yilmaz,

2012).

Silymarin marked as a potential antioxidant against lipid peroxidation and damage caused

by free radicals and reactive oxygen species, so protect the cell against the oxidative damage by

the inhibition of lipid peroxidation and scavenging the free radicals. It is claimed that the

preventive effect of silymarin is due to its enzymatic and non-enzymatic defense system

including superoxide dismutase, catalase and reduced glutathione. Through its antioxidant

system it can protect the brain, liver, heart and many other vital organs from oxidative damage

by the process inhibition of lipid peroxidation and replenish the decreased level of reduced

glutathione level (Das et al., 2008: Akkaya and Yilmaz, 2012; Mujahid et al., 2013:Khan et al.,

2013).

The cell or tissue is stress when there is an increase in the production of reactive oxygen

species (H2O2, O2U and OHU) beyond the capacity of antioxidants in the biological system

(Sies, 1991). The altered level of cell’s own radical scavengers and lipid peroxidation state is

also included in the causes of oxidative stress (Khan et al., 2009).

123

SOD and catalase enzymes give protection to the biological system by scavenging the free

radicals produced by many biological reactions. The function of superoxide dismutase is to

dismutate the superoxide radicals into hydrogen peroxide and oxygen (Fridovich, 1986). Further

detoxification is done through the conversion by catalase and H2O2 that converted into detoxified

form H2O andO2 (Murray et al., 2003). GPX has same function as the catalase so these two

interact mutually against the oxidative stress caused by reactive oxygen species (Bhattacharjee

and Sil, 2006). Due to Diethyl nitrosamine toxicity there was significant decrease in the activities

of antioxidant enzymes due to the enhance usage of these enzymes in deactivating the free

radicals which were produced in response of diethyl nitrosamine metabolism and also due to

increase in lipid peroxidation. Similar reports have shown an elevation in the status of lipid

peroxidation in the liver during diethylnitrosamine treatment (Sanchez-Parez et al., 2005; Khan

et al., 2013) and our results are in accordance with these reports. Recent work is also

representing the same results that there is restoration in the levels of lipid peroxidation after

administration of medicinal plants in combination could be related to its potential ability to

scavenge reactive oxygen species, thus preventing from further damage to membrane lipids. Our

results are in line with previous studies by Ramakrishnan et al. (2006) who have shown that

silymarin exhibits excellent antioxidant property. Therefore this property of silymarin might

have resulted in restoring the activities of the above antioxidant enzymes to normal.

The role of aqueous extracts of Artemisia absinthium (AEAA) was significant as cellular

defense against the deleterious action of ROS such as SOD and GPx. These two enzymes work

mutually as SOD catalyzes the conversion of superoxide free radical to less toxic hydrogen

peroxide and then GPx catalyzes the breakdown of hydrogen peroxide into water, oxygen and

also detoxify lipid peroxides directly. However, administration of AEAA demonstrated that SOD

and GPx were remarkably elevated suggesting that it can restore both enzymes activities in CCl4

damaged liver tissue (Castro and Freeman, 2001).

124

4.8. Gross and Histopathology

The selected animals were slaughtered at the end of study and Gross & Histopathological

examinations of the liver tissues were performed in the department of Pathology, University of

Agriculture, Faisalabad, Pakistan.

Gross Pathology

Fig.4.78. Gross pathology of animals treated with A2B2C2

Fig.4.79. Gross pathology of positive control and absolute control group

125

Fig.4.80. Gross pathology of animals treated with A3B2C2 and A3B3C1

Fig.4.81. Gross pathology of standard (silymarin) and animals treated with A1B3C3


126


Histopathology

Absolute Control

In this group the experimental animals were treated with normal saline, which served as

absolute control. Hepatic parenchyma was normal. Hepatocytes were arranged in fine hepatic

cords. Nuclei of the hepatocytes were normal in appearance having nucleus with prominent

nucleolus and chromatin material (Fig 4.84).

Group 1

Experimental animals included in this group were treated with toxin to induce liver injury

and then treated with medicinal plants combination (extracted silymarin 100 mg/kg/body weight

+ aqueous mixture of Artemesia Absinthium 100 mg/kg/body weight + aqueous mixture of

Rheum emodi 500 mg/kg/body weight). Hepatic parenchyma was quite in normal. Hepatocytes

were arranged in fine hepatic cords, mild degree of congestion was present at few places.

Individual cell necrosis was also present. Sinusoidal spaces were normal, at few places mild

degree of vacuolar degenerations was present but they were hazy in appearance. Partial

ameliorative effect was seen from the histopathology (Fig.4.85 & 4.86).

Group 2

In group 2 the experimental animals were treated with toxin to induce liver injury and

then treated with medicinal plants combination (extracted silymarin 200 mg/kg/body weight +

aqueous mixture of Artemesia Absinthium 500 mg/kg/body weight + aqueous mixture of Rheum

emodi 500 mg/kg/body weight). Mild degree of vacuolar degeneration was present in the

cytoplasm of hepatocytes. At few places individual cell necrosis was also present. It indicates

partial amelioration with this combination of medicinal plants (Fig.4.87).

Group 3

In this group the experimental animals were treated with toxin to induce liver injury and



127

emodi 200mg/kg/body weight). Hepatic parenchyma indicated mild degree of vacuolar

degeneration in the cytoplasm of hepatocytes. Mild degree of congestion was also present. Mild

degree of cellular infiltration was also present in the portal area. It indicated partial ameliorative

effect with this combination (Fig.4.88).

Fig.4.84. Photomicrograph of liver of control group showing normal hepatic parenchyma (200 X, H &E

staining)

128

Fig.4.85. Photomicrograph of liver of group 1 group showing individual cell necrosis (shown by arrow)

and mild degree of congestion in hepatic parenchyma (200 X, H &E staining)

Fig.4.86. Photomicrograph of liver of group 1 group showing individual cell necrosis (shown by arrow) and biliary

hyperplasia (bold arrow) hepatic parenchyma (200 X, H &E staining)

129

Fig.4.87. Photomicrograph of liver of group 2 group showing mild degree of vacuolar degeneration (arrow) in

cytoplasm of hepatocytes and individual cell necrosis (bold arrow) (200 X, H &E staining)

Fig.4.88. Photomicrograph of liver of G3 group showing normal hepatic parenchyma indicating

ameliorative effect of plant extracts (200 X, H &E staining)

130

Fig.4.89. Photomicrograph of liver of standard (group 5) showing cell swelling and vacuolar degeneration

(arrow) in hepatic parenchyma (200 X, H &E staining)

Group 4

Experimental animals included in group 4 were treated with toxin to induce liver injury

and then treated with standard drug (silymarin (siliver, abbot laboratories) 200 mg/kg/body

weight) purchased from local market. In hepatic parenchyma cell swelling was prominent that

was indicated by diminished sinusoidal spaces. Vacuolar degeneration was present throughout

the cytoplasm. Individual cell necrosis was also present; it indicated no ameliorative effect

(Fig.4.89).

Group 5




emodi 100 mg/kg/body weight). Hepatic parenchyma was almost normal. Nuclei of the

hepatocytes were also normal in appearance having nucleus with prominent nucleolus and

chromatin material. It indicated complete ameliorative effect (Fig.4.90).

131

Group 6

Experimental animals included in this group were treated with toxin to induce liver injury

and then treated with medicinal plants combination (extracted silymarin 500 mg/kg/body weight

+ aqueous mixture of Artemesia Absinthium 500 mg/kg/body weight + aqueous mixture of

Rheum emodi 500 mg/kg/body weight). Mild degree of vacuolar degeneration was present. The

sinusoidal spaces were normal indicating moderate ameliorative effect with this combination

(Fig.4.91).

Group 7




emodi 500 mg/kg/body weight). Hepatic parenchyma showed presence of mild degree of

congestion. Sinusoidal spaces were normal. Individual cell necrosis was present indicated partial

ameliorative effect (Fig.4.92 & 4.93).

Fig.4.90. Photomicrograph of liver of group 6 showing ameliorative effect of plant extracts indicated by

normal nuclei of hepatocytes with chromatin material in nucleolus (400 X, H &E staining)

132

Fig.4.91. Photomicrograph of liver of group 7 showing mild degree of vacuolar degeneration (arrow)

indicating partial ameliorative effect of plant extracts. (200 X, H &E staining)

133

Fig.4.92. Photomicrograph of liver of group 8 showing mild degree of congestion and mild degree of

vacuolation (shown by arrow) (200 X, H &E staining)

Fig.4.93. Photomicrograph of liver of group 8 showing mild degree of congestion and mild degree of

vacuolation (shown by arrow) (400 X, H &E staining)

134

Fig.4.94. Photomicrograph of liver of group 9 showing mild degree of vacuolar degeneration (shown by

arrow) and cell swelling (200 X, H &E staining)



135

Group 8

In this group the experimental animals were treated with toxin to induced liver injury and



emodi 200 mg/kg/body weight). Mild degree of vacuolar degeneration was present. Cell swelling

was also present at few places. Sinusoidal spaces were normal at other places less prominent.

Mild degrees of congestion were also present. It indicated partial ameliorative effect with this

combination (Fig.4.94 & 4.95).

Group 9

Experimental animals included in this were treated with toxin to induced liver injury and

then treated with medicinal plants combination (extracted silymarin 500mg/kg/body weight +

aqueous mixture of Artemesia Absinthium 500mg/kg/body weight + aqueous mixture of Rheum

emodi 100mg/kg/body weight). Hepatic parenchyma was normal. Individual cell necrosis was

present. Nuclei of the hepatocytes were normal in appearance having nucleus with prominent

nucleolus and chromatin material. It indicated partial ameliorative effect (Fig.4.96).

Group 10

In group 10 the experimental animals were treated with toxin to induced liver injury and

then treated with medicinal plants combination (extracted silymarin 100mg/kg/body weight +

aqueous mixture of Artemesia Absinthium 100mg/kg/body weight + aqueous mixture of Rheum

emodi 100mg/kg/body weight). Hepatic parenchyma was almost normal. Hepatocytes were

arranged in hepatic cords. Nuclei of the hepatocytes were normal in appearance having nucleus

with prominent nucleolus and chromatin material. It also indicated complete amelioration

(Fig.4.97).

Positive Control

In positive control group the experimental animals were treated with toxin to induced

liver injury. Histopathological examination of this group showed the presence of moderate

degree of vacuolar degeneration in the cytoplasm of the hepatocytes. At few places mild degree

of individual cell necrosis was also present that is indicated by pyknotic nuclei of the

136

hepatocytes. Perivascular coughing, fibrosis and billiary hyperplasia was also present (Fig.4.98,

4.99 & 4.100).



137

Fig.4.97. Photomicrograph of liver of G12 showing normal hepatic parenchyma indicating good

amelioration with plant extracts. (200 X, H &E staining)

138

Fig. 4.98 Photomicrograph of liver of positive control showing pyknotic changes (Bold arrow) and

fibrosis (shown in arrow) indicating chronic changes (200 X, H &E staining)

Fig.4.99. Photomicrograph of liver of positive control showing pyknotic changes, biliary hyperplasia and

fibrosis (shown in arrow) indicating chronic changes (200 X, H &E staining)

139

Fig.4.100. Photomicrograph of liver of positive control showing pyknotic (shown in arrow) changes,

biliary hyperplasia and fibrosis indicating chronic changes (200 X, H &E staining)

In this study, examination of liver function was correlated with the histopathological

changes. Similar changes in liver texture were noted in another study performed to evaluate the

hepatoprotective potential of aqueous extract formula containing Artemisia capillaris, Lonicera

japonica and Silybum marianum (Yang et al., 2012). Liver of the control group was normal in

appearance having smooth surface along with normal hepatic cells without fatty vacuolation and

fibrosis. Among the positive control group the color of the liver was yellowish and tough to the

touch having vacuolated hepatocytes and surrounded by thick fibrotic tissue, causing the

formation of continuous fibrotic septa. The different degrees of fatty vacuolation scattered

throughout the liver tissue that shows CCl4 had successfully induced liver damage. In the

silymarin positive control group, vacuolation was observed but it was less prominent compared

with the CCl4 negative control. In the next group treated with AEF, the vacuolation was greatly

reduced and the proportion of normal cells increased with mild degrees of fibrosis and the

formation of incomplete septa. The main outcome observed in paracetamol intoxication in

humans and animals is acute centrolobular necrosis (Hewawasam et al., 2003; Macnaughton,

140

2003). Previous studies revealed that paracetamol cause mild focal hepatitis in the portal and

lobules areas (Roomi et al., 2008; Ibrahim et al., 2011). Histopathology of damaged liver

showed fatty changes in hepatocytes and fibrosis (Rajib et al., 2009). Histopathology of the liver

tissue showed that aqueous extract of Artemesia absinthium attenuated the hepatocellular

necrosis and led to reduction of inflammatory cells infiltration (Fiamegos et al., 2011). In

another study conducted to explore the hepatoprotective potential of Dodonaea viscosa.

Histopathology of liver showed normal central vein, hepatic cords and sinusoids in the untreated

normal control; pale necrotic areas after CCl4 treatment in the control group (CCl4); protection

by the positive control 200 mg/kg silymarin (CCl4+ silymarin); protection by methanolic extract

of Dodonaea viscosa (CCl4+ MeOH). It was observed that methanolic extract provide better

protection than that of silymarin (Ali et al., 2014).

Kanbur et al., (2009) reported the protective effect of royal jelly (RJ) contrary to

paracetamol induced liver damage in mice and confirmed by histopathology. Among the control

group no significant lesion was seen in the liver cross sections. In another group treated with RJ

Lymphoid cell infiltration in liver parenchyma was observed. Treatment with single dose of

paracetamol showed severely-hyperaemic and large-haemorrhagic areas in the parenchyma.

Alterations ranging from degenerative to necrotic were ascertained in hepatocytes, slight increase

in fibrous tissue and limited lymphoid cell infiltrations were observed in the portal region roll

shaped erythrocyte accumulations were present in the large haemorrhagic areas and some of the

sinusoids, degenerative alterations were observed in the liver parenchyma. These changes were

almost similar in our study positive control group.

Similar results were reported in another study that showed disarrangement and

degeneration of normal hepatic cells with intense centrilobular necrosis among paracetamol

treated groups. Liver sections showed normal cellular architecture with distinct hepatic cells and

sinusoidal space in control group. While rats treated with silymarin, liv-52 and intoxicated with

paracetamol showed comparatively less disarrangement and degeneration of hepatocytes,

indicating marked regeneration activity (Hurkadale et al., 2012).

Conclusion

In conclusion, the hepatoprotective effect of medicinal plants in combinations was

confirmed by the reduction of biochemical parameters of liver impairment caused by

141

paracetamol toxicity. The outcome confirms the therapeutic potential use of medicinal plants in

combination for ameliorating hepatotoxicity. The hepatic parenchyma of treatment groups at the

end of study became as normal as of the control group. Histopathology of liver tissues of control

and treated animals further authenticate the protective effect of medicinal plants in combination.

Besides having the above mentioned hepatoprotective properties, these plants are used in many

herbal formulations for the treatment of different diseases. The plants in this combination could

be further exploited, in order to isolate the various biologically active components responsible

for its activity.

Summary:

Hepatitis is one of the leading causes of death across the world and economically it has

ruined a large number of families. As a rescue to this deplorable situation of hepatic problems

this study evaluated the synergistic hepatoprotective potential of selected medicinal plants i.e.

milk thistle (Silybum marianum), Wormwood (Artemisia absinthium) and Rhubarb (Rheum

emodi). Total phenolic content (TPC) and total flavonoid content (TFC) were evaluated and the

amounts of both of these phytochemical constituents were ranged from 276.26 to 356.57 mg/g

GAE and 13.01 to 25.63 mg/g CE respectively. Likewise antioxidant profile was determined by

performing DPPH, reducing power and H2O2 induced oxidative damage on pUC19 DNA. The

antioxidant activity of selected medicinal plants in term of DPPH was ranged from 76.81 to

92.33 (% inhibition) and reducing power in term of absorbance was 0.964 to 1.652. The finding

142

that H2O2 induced oxidative damage on pUC19 DNA showed concentration dependent

protective effect of plants extracts. The safety of the selected medicinal plants alone and in

different combinations was also evaluated through various toxicological assays including

hemolytic, brine shrimp lethality and mutagenic activity. The percentage hemolysis of individual

plant was 2.76±0.29, 3.34±0.35 and 2.14±0.17 in Silybum marianum, Artemesia absinthium and

Rheum emodi respectively. The combinations of these three medicinal plants with varied doses

exhibited different hemolytic activities in term of percentage hemolysis ranged from 5.14 to

13.30. The brine shrimp lethality in terms of percentage mortality of individual plant was ranged

from 10 to 17, 20 to 27 and 12 to 17 in Silybum marianum, Artemesia absinthium and Rheum

emodi at lowest (10 mg) and highest (100 mg) concentration respectively. The percentage

mortality was ranged from 18±0.5 to 57±0.5 in different combinations of medicinal plants.

Mutagenic activity was performed by using test strain S. typhimurium TA98 and S. typhimurium

TA98. All the three selected plant extracts did not show any mutagenic effect against the two

tester bacterial strains. Furthermore biochemical profiling of selected medicinal plants was

compared by liquid chromatography mass spectrometry (LCMS). The peaks obtained were

dissociated with different collusion energies to confirm the active compounds present in each

plant extract. Silymarin was extracted from the seeds of Silybum marianum using Soxhlet and

reflux apparatus. The extracted silymarin was compared with standard silymarin purchased from

Sigma USA.

A variety of chemicals has been reported that induce hepatotoxicity. Preliminary trials

were conducted to compare paracetamol and CCl4 as hepatotoxicant and of these paracetamol

was selected as the appropriate hepatotoxin. On the basis of the preliminary trial, paracetamol

(500 mg/kg b. wt. daily for three consecutive days) was used as hepatotoxin in pre-clinical trial.

Hepatoprotective potential of selected medicinal plants in different combinations was evaluated

against paracetamol induced liver toxicity. Combinations of medicinal plants were given to the

experimental animals to check the ameliorative effect and confirmed by analysing biochemical

parameters, including SGPT, SGOT, ALP, total protein and albumin. The significant liver

damage was observed when paracetamol treated group and control group compared. The

findings showed that the levels of SGPT, SGOT and ALP were significantly raised while amount

of total protein and albumin were decreased after paracetamol administration. The administration

of medicinal plants in combination gradually restored the disturbed biochemical parameters. The

143

animals were sacrificed at the end of the study for histopathological examination which revealed

significant improvement in medicinal plants treated groups. The activities of antioxidant

enzymes glutathione peroxidase (POD), superoxide dismutase (SOD) and catalase (CAT) in liver

homogenate were significantly decreased in rabbits treated with paracetamol (positive control

group) as compared to the absolute control group. Medicinal plants in combinations exerted a

beneficial effect on antioxidant enzymes. Different combinations effectively restored the liver

functions but the blend containing 100 mg of each plant was the most suitable combination that

has least toxic effect in term of percentage hemolysis (hemolytic activity) and percentage

mortality (brine shrimp lethality) and restored almost all the biochemical markers comparable

with absolute control. The findings of this study clearly showed that paracetamol damaged the

liver significantly, while administration of selected medicinal plants in combinations minimized

the risk of complications such as fibrosis and cirrhosis as observed in positive control group.

These results support the efficacy and safety of the herbal combination (Silybum marianum,

Artemesia absinthium and Rheum emodi) to manage hepatic disorders.

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