CONTROL OF HEPATOTOXICITY USING HERBALDRUGS

Dhiman Deb Nath JoyID# 103 0853 546

A thesis submitted in the partial fulfilment of therequirements for the degree of Bachelor’s inPharmaceutical Sciences

Department of Pharmaceutical Sciences

North South University

Bangladesh

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JANUARY 2015

CONTROL OF HEPATOTOXICITY USING HERBAL DRUGS

Prepared for and Checked by

MOHAMMAD SHOHELLecturerDepartment of Pharmaceutical Sciences

North South University

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Declaration

I hereby declare that this thesis has been prepared by myself andhas not been accepted in any previous application for a degree.The work of which is a record has been performed by myself,unless otherwise stated. All sources of information are properlyacknowledged.

Dhiman Deb Nath Joy

January 2015

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ACKNOWLEDGEMENT

First and foremost, I would take the opportunity to thank myproject supervisor, Mohammad Shohel, Lecuter; Department ofPharmaceutical Sciences, North South University, for hispatience, motivation, enthusiasm, and immense knowledge. Hisguidance helped me in all the time of research. I could not haveimagined having a better advisor and mentor for my undergraduatethesis. I would like to thank you from the bottom of my heart foryour support and understanding over these past four years of myundergraduate study.

I would also like to show my gratitude to Dr. Hasan Mahmud Reza;Professor, Chairman, Department of Pharmaceutical Sciences, NorthSouth University, for his undying support and motivation thatgave me the courage and strength to complete this thesis work.Without his assistance and dedicated involvement in every step

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throughout the process, this paper would have never beenaccomplished.

I would also like to thank Dr. JMA Hannan; Professor, Departmentof Pharmaceutical Sciences for his hard work in teaching us somany of the stuffs about the pharmaceutical world. His motivationand courage had really helped me to accomplish my job with suchbetter results.

I am also indebted to Dr. Rajib Bhattacharjee (AssistantProfessor, North South University), Dr. Md. Nurul Islam(Assistant Professor, North South University) and Dr. PreetiJain (Assistant Professor, North South University) who have givenme their valuable time and energy with their good grace toexpress their rich full experience about the instrumental terms,conditions and working procedures.

I particularly want to acknowledge the tremendously helpful,supportive, creative contributions of our Laboratory Officer Mrs.Junaid Khaleque for her active contribution.

Getting through my thesis work required more than academicsupport, and I have many, many people to thank for listening toand, at times, having to tolerate me over the past four years. Icannot begin to express my gratitude and appreciation for theirfriendship. They have been a wonderful support for me andmentioning a few names would not be enough.

Most importantly, none of this could have happened without myfamily. Their consistent support has made me come this far. Icannot thank them enough for bearing me and being beside methrough highs and lows even when I was unable to be beside themdue to my workload with studies and thesis work. Every time I wasready to quit, they did not let me and I am forever grateful.

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This dissertation stands as a testament to your unconditionallove and encouragement.

AbstractHerbal poly unsaturated drugs are mainly used as blood purifierwhile their components have rich values of substances which canbe used to treat various liver disorders. We have used HamdardIcturn and Icturn for our particular project. In this project wehave worked with female Long Evans rats which have been givenCCl4 to induce hepatotoxicity. For the treatment purposes we haveused the two drugs as named above in the abstract. For inducinghepatotoxicity we have given CCl4 in a 1 ml syringe, out of which40 units of CCl4 along with 40 units of olive oil was taken tomake a volume of 80 units, which has been given the long evansrats weighing on a average of 180gm+. The CCl4 was given at aregular interval of 3 days for a period of 14 days. After thatthe rats were sacrificed and certain vital organs such as liver,heart and kidneys were collected. Furthermore they werehomogenized by using a sonicator and centrifuged to collect theplasma. These plasma are further assayed bu using Bio chemicalAssay, which includes the techniques of MDA, NO and AP-OP. (thecatalyze was not done as the reagents present were quality enoughto provide better results) In an average the control values werecollected and checked which comes in a range of 0.00 to 0.200 asper the standard measurement count. While the drugged one havevalues greater than the control and treatment ones have valuesless than the control whereas treatment + drugged has resultsvery similar to the control data. The histology was procedure was

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carried according to the manual provided and finally the stainsof tissues were collected and pictured under microscope forbetter view.

List of Tables

Table 1: P value comparison of Intial body weight

Table 2: P-value comparison of Final body weight

Table 3: P-value comparison of Intial food intake

Table 4: P-value comparison of final food intake

Table 5: P-value comparison of intial water intake

Table 6: P-value comparison of final water intake

List of Figures

Figure 1: The body weight, Food intake and water intake of therats in the 14 days period.

Figure 2: The organ weight values of heart, kidneys, liver andspleen.

Table of Contents

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Contents Page noDECLERATION

03

ACKNOWLEDGEMENT04

ABSTRACT05

LIST OF TABLES05

LIST OF FIGURES06

CHAPTER ONE 11-15

1. Introduction11

1.1 Drug metabolism system in liver

11

1.1.1 Genetic diversity12

1.1.2 Change in enzyme activity12

1.1.3 Competitive inhibition12

1.2 Adverse drug reaction13

1.3 Patterns of injury13

1.3.1 Zonal Necrosis 14

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1.3.2 Hepatitis 14

1.3.3 Cholestasis 14

1.3.4 Steatosis 14

1.3.5 Granuloma 15

1.3.6 Vascular lesions 15

1.3.7 Neoplasm 15

1.3.8 Composition 15

CHAPTER TWO 17-25

2. Materials and Methods3.

17

2.1 Animals 17

2.2 Materials needed for inducing hepatoxicity 17

2.2.1 Properties 17

2.3 Surgical Instruments 18

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2.4 Phosphate buffer procedure 192.4.1 Phosphate Buffer Materials 192.4.2 Prepare the Phosphate Buffer 192.5 Sodium citrate buffer 21

2.6 Neutral buffer 21

2.7 Histological materials22

2.8 Method23

2.8.1 Animal handling

23

2.8.2 Control Group 23

2.8.3 CCl4 induced group 23

2.8.4 Treatment group 23

2.8.5 Homogenate organs 23

2.8.6 Biochemical Assay 24

2.8.6.1 For NO 24

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2.8.6.2 For MDA 25

2.8.6.3 For APOP 25

2.8.6.4 For catalase 25

CHAPTER THREE 26-42

4. Results26

3.1 Figure of the body weight, food intake andwater intake

26

3.2 Discussion about the graphs27

3.3 Figure of the organ weights27

3.4 Discussion28

3.5 The P-value of Intial body weight29

3.6 The p-value of final body weight30

3.7 the p-value of intial food intake32

3.8 the p-value of final food intake34

3.9 the p-value of intial water intake 36

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3.10 the p-value of final water intake 38

3.11 the P-value comparison of organ (liver) 39

3.12 Icturn plasma APOP value 41

3.13 Icturn plasma MDA value 41

3.14 Icturn plasma NO value 42

3.15 Icturn CCL4 Apop value 42

3.16 Icturn CCL4 MDA value 42

3.17 Icturn CCL4 NO value 42

CHAPTER FOUR 43-45

4.1 Discussion43

4.2 Conclusion44

REFERENCES45

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CHAPTER ONE

INTRODUCTION

1.Introduction

Hepatotoxicity (from hepatic toxicity) means chemical-driven liver damage.

The liver plays a central role in transforming and purifyingchemicals and is susceptible to the toxicity from some agents.Certain medicinal agents, when taken in overdoses and sometimeseven when given within therapeutic ranges, may damage the organ.Other chemical agents, such as those used in laboratories andindustries, natural chemicals (e.g., microcystins) and herbalremedies can also produce hepatotoxicity and chemicals thatcause liver injury are called hepatotoxins.

More than 1100 drugs have been implicated in occuring liverinjury and it is the most common reason for a drug to be bannedfrom the market. Hepatotoxicity and drug-induced liver injuryalso account for a substantial number of compound failures,highlighting the need for drug screening assays, such as stemcell-comes from hepatocyte-like cells, that are capable ofidentifying toxicity early in the drug development process.[2] Chemicals often cause subclinical damage to the liver, whichclears only as abnormal liver enzyme tests. Drug-induced liverinjury is responsible for 10% of all hospital admissions and 50%of all acute liver failures and damages.

1.1Drug metabolism system in liver

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The human body identifies almost all medicinal agents as foreignsubstances (i.e. xenobiotics) and subjects them to variouschemical systems (i.e.metabolism) to make them suitable forelimination. This involves chemical transformations to(a) reduce lipid solubility and (b) to change biologicalactivity. Although almost all tissues in the body have someability to metabolize chemical agents, smooth endoplasmicreticulum in the liver is the principal "metabolic clearinghouse" for both endogenous chemicals (e.g., cholesterol, steroidhormones, fatty acids, proteins,lipids) and exogenous substances(e.g., drugs, alcohol,medicinal agents).[5] The central roleplayed by liver in the clearance and transformation of chemicalsmakes it susceptible to drug-induced liver injury.

Drug metabolism is usually divided into two phases: phase1 and phase 2. Phase 1 reaction is thought to make ready a drugfor phase 2. However many compounds can be metabolized by phase 2directly.Phase 1 reactioninvolves oxidation, reduction, hydrolysis, hydrationand manyother rare chemical reactions. These systems tend to increasewater solubility of the drug and can produce metabolites that aremore chemically pro-active and potentially toxic. Most of phase 2reactions occur in cytosol and involve conjugation withendogenous compounds via transferase enzymes. Chemically activephase 1 products are rendered comperatively inert and suitablefor elimination by this step.

A group of enzymes placed in the endoplasmic reticulum, knownas cytochrome P-450, is the most important family of metabolizingenzymes in the liver area. Cytochrome P-450 is theterminal oxidase component of an electron transport chain. It isa multiple enzyme, but rather consists of a closely relatedfamily of 50 isoforms; six of them metabolize 80% of drugs. Thereis a tremendous diversity of individual P-450 gene products, and

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this heterogeneity permits the liver to perform oxidation on avast array of chemicals (including almost all drugs) in phase 1.Three important features of the P-450 system have roles in drug-induced toxicity.

These are ;-

1.1.1 Genetic diversity:

Each of the P-450 protein is unique and accounts (to some extent)for the different ways in drug metabolism between individuals.Genetic variations (polymorphism) in P-450 metabolism should beconsidered when patients exhibit uncomfortablel sensitivity orresistance to drug effects at normal doses. Such polymorphism isalso accounts for variable drug response among patients ofdiffering ethnic backgrounds.

1.1.2 Change in enzyme activity:

Many substances can effect the P-450 enzyme mechanism. Drugsinteract with the enzyme group in several ways Drugs that modifycytochrome P-450 enzyme are considered to as either inhibitors orinducers. Enzyme inhibitors stop the metabolic activity of one orseveral P-450 enzymes. This effect usually happens immediately.On the other hand, inducers increase P-450 activity by increasingits synthesis. Depending on the inducing chemical agents anddrug's half life, there is usually a delay before enzyme activityincreases.

1.1.3 Competitive inhibition:

Some drugs may share the same P-450 specificity and thuscompetitively stop their bio transformation. This may proceed toaccumulation of drugs metabolized by the enzyme. This type of

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drug interaction may also reduce the rate of generation of toxicsubstrate and chemical substrate.

1.2 Adverse drug reactions

Adverse drug reactions are classified as type A (intrinsic orpharmacological) or type B (idiosyncratic). Type A drug reactionaccounts for 85% of all toxicities.

Drugs   or toxins that have a pharmacological (type A)hepatotoxicity are those that have predictable dose-responsecurves (higher concentrations cause more liver damage) and wellcharacterized mechanisms of toxicity to liver, like directlydamaging liver tissue or blocking a metabolic process. As in thecase of acetaminophen overdose, this type of injury happensshortly after some threshold for toxicity is reached.

Idiosyncratic (type B) injury happens without warning, whenmedicinal agents cause non-predictable hepatotoxicity insusceptible individuals, which is not related to dose and has avariable latency sagment. This type of injury does not have aclear dose-response nor sudden relationship, and most often doesnot have predictive models. Idiosyncratic hepatotoxicity has ledto the removal of some drugs from market even after rigorousclinical testing as part of the FDA approvalsystem; Troglitazone (Rezulin) and trovafloxacin (Trovan) are twoprime examples of idiosyncratic hepatotoxins pulled from medicinemarket.

When used orally, ketoconazole has been associated with hepatictoxicity, including some fatalities.

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1.3 Patterns of injury

Chemicals produce a liver damage. Liver damage is defined as arise in either (a) ALT level more than vast variety of clinicaland pathological hepatic injury. Biochemical markers(e.g. alanine transferase, alkaline phosphatase and bilirubin)are sometimes used to indicate three times of upper limit ofnormal (ULN), (b) ALP level grater than twice ULN, or (c) totalbilirubin level more than twice ULN when associated withincreased ALT or ALP.[18][19] Liver damage is again characterizedinto hepatocellular (predominantly initial Alaninetransferase elevation) andcholestatic (initial alkalinephosphatase rise) types. However they are not mutually exclusiveand mixed types of injuries are often targeted.

Specific histo-pathological patterns of liver injury from drug-induced damage are described below.

1.3.1 Zonal Necrosis

This is the most common type of drug-induced livercell necrosis where the injury is largely constricted to aparticular zone of the liver lobule. It may make simple as a veryhigh level ofALT and severe disturbance of liver function leadingto acute liver failure.

Causes include:Paracetamol, carbon tetrachloride

1.3.2 Hepatitis

In this pattern, hepatocellular necrosis is included withinfiltration of inflammatory cells. There can be three types ofdrug-induced hepatitis. (A) viral hepatitis is the most common,where histological features are same to acute viral hepatitis.(B) in focal or non-specific hepatitis, dispersed foci of cell

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necrosis may accompany lymphocytic infiltration. (C) chronichepatitis is very resemble to autoimmune hepatitis clinically,serologically, and histologically.

Causes:(a) Viral hepatitis: Halothane, isoniazid, phenytoin(b) Focal hepatitis: Aspirin(c) Chronic hepatitis: Methyldopa, diclofenac

1.3.3 Cholestasis

Liver injury leads to damage of bile flow and cases are seen byitching and jaundice. Histology may depict inflammation(cholestatic hepatitis) or it may be bland (withoutany parenchymal inflammation). On rare occasions, it can producefeatures same to primary biliary cirrhosis due to progressivedestruction of tiny bile ducts (Vanishing duct syndrome).

Causes:(a) Bland: Oral contraceptive pills, anabolic steroid, androgens(b) Inflammatory: Allopurinol, co-amoxiclav, carbamazepine(c) Ductal: Chlorpromazine, flucloxacillin

1.3.4 Steatosis

Hepatotoxicity may obvious as triglyceride accumulation, whichleads to either micro-droplet (microvesicular) or macro-droplet(macrovesicular) fatty liver. There is a different types ofsteatosis by which phospholipid accumulation leads to a patternsimilar to the diseases with inherited phospholipid metabolismdefects (e.g., Tay-Sachs disease)

Causes:(a) Microvesicular: Aspirin (Reye'ssyndrome), ketoprofen, tetracycline (especially if expired)(b) Macrovesicular: Acetamenophen, methotrexate(c) Phospholipidosis: Amiodarone, total parenteral nutrition

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1.3.5 Granuloma

Drug-induced hepatic granulomas are usually related withgranulomas in other tissues and patients typically have featuresof systemic vasculitis and hypersensitivity. More than 50 drugshave been involved.

Causes:Allopurinol, phenytoin, isoniazid, quinine, penicillin, quinidine

1.3.6 Vascular lesions

These result from injury to the vascular endothelium.

Causes:Venoocclusive disease: Chemotherapeutic agents, bush teaPeliosis hepatis: Anabolic steroidsHepatic vein thrombosis: Oral contraceptives

1.3.7 Neoplasm

Neoplasms have been represented with prolonged exposure to somemedications or toxins. Hepatocellular carcinoma, angiosarcoma,and liver adenomas are the ones usually noticed.

Causes:Vinyl chloride, combined oral contraceptive pill, anabolicsteroid, arsenic, thorotrast

1.3.8 Composition: Description :

Icturn is an effective herbal preparatiioncontaining Cichorium endivia etc. Cuscuta reflexa,Rheum emodi, Borago officinialis which is effective in liverdisorders, hepatitis, constipation, metritis, ascites,pleurisy, alveolitis and in jaundice.

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Composition:

Each 5ml contains : Cichorium endivia (root)                 400 mg Cichorium endivia (seed)                200 mg Rosa damascene  200 mg Borago officinalis  100 mg Cusctita reflexa  150 mg NympBaea nouchali  100 mg Rheum emodi  125 mg

Indications:

Hepatitis,   jaundice,    ascites,    pleurisy,   alveolitis,    uterine Inflammation & constipation.

Dosage: Adults:2-3 teaspoonful twice daily . Children :  V2-Iteaspoonful twice daily or as directedby the physician

Side effect:

No significant side effect has been observed in properdosage.

Precaution:

Keep out of reach of the children.

Storage :

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Store at cool and dry place protect from light.

Presentation :

Amber glass bottle containing 450 ml & 100 ml syrup.

CHAPTER TWO

MATERIALS AND METHODS

2. Materials and Methods

2.1Animals:

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Long Evans rat weighing 180-200g of female sex , bred in theanimal house were procured and used for the study. Animalswere given food pellets and water and were kept and maintained atstandard lab conditions.

2.2 Materials needed for inducing hepatoxicity:

The chemical we used in our experiment to induce hepatotoxicityis Carbon tetrachloride.

Carbon tetrachloride, also known by many other names (the mostnotable being tetrachloromethane (also recognized by theIUPAC), carbon tet in the cleaning industry, Halon-104 infirefighting and Refrigerant-10 in HVACR), is the compound withthe chemical formula CCl4. It was formerly widely used in fireextinguishers, as a precursor to refrigerants, and as a cleaningagent. It is a colorless liquid with a sweet smell that can bedetected at low levels.

2.2.1Properties: In the carbon tetrachloride molecule,four chlorine atoms which are positioned symmetrically as cornersin a tetrahedral configuration joins to a central carbon atomby single covalent bonds. For this symmetrical geometry, CCl4 istermed as non-polar. Methane gas consist the same structure, whenmaking carbon tetrachloride a halomethane. As a solvent, it ismore soluble to other non-polar compounds, fats, and oils. Iodineis also dissolved in it. It is volatile.

Carbon tetrachloride can be define as one of the mostpotent hepatotoxins (toxic to the liver), and so it is widelyused in scientific research to evaluate hepatoprotective agents.

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CCl4-induced liver damage has been modeled in monolayer culturesof rat primary hepatocytes with a focus on involvement ofcovalent binding of CCl4 metabolites to cell components and/orperoxidative damage as the cause of injury.

I) Hepatocytes was detected with Covalent binding of 14C-labeledmetabolites immediately after exposure to CCl4.

ii) Increase of the reductive metabolism of CCl4 was causedbecause of low oxygen partial pressure.

iii) Lipids and to proteins throughout subcellular fractions wasbounded by[14C]-CCl4. Binding was found to occur preferentiallyto triacylglycerols and phospholipids, with phosphatidylcholinecontaining the highest amount of label.

iv) Compared to other peroxidative substances viz., ADP-Fe3+ andcumol hydroperoxide, respectively the lipid peroxidation potencyof CCl4 revealed subtle differences.

v) CCl4 decreased the rate of triacylglycerol secretion as verylow density lipoproteins.

vi) Prevention of CCl4-induced lipid peroxidation as well ascovalent binding of CCl4 metabolites to cell components is causedby radical scavenger piperonyl butoxide. It also restoreslipoprotein metabolism. The results thus confirm that covalentbinding of the CCl3* radical to cell components initiates theinhibition of lipoprotein secretion and causes steatosis. Hencereaction with oxygen, to form CCl3-OO*, initiates lipidperoxidation. These two processes are independent of each other,and the extent to which either process occurs depends on partialoxygen pressure. The first process may result in adduct formationand, ultimately, cancer initiation, and the second results inloss of calcium homeostasis and, ultimately, apoptosis and celldeath. OLIVE OIL is being used to mix along with the carbontetrachloride to form a mixture of olive oil and carbontetrachloride that is being induced by using 1 ml syringes via

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the oral route to induced hepatotoxicity. Olive oil is used toreduce the potency of the carbon tetrachloride.

2.3 Surgical Instruments:

Scissors

Locking forceps

Forceps

Tweezers

Gross dissection

Blades

Syringes

Test tubes

Sonicator

Centrifugation machine

Phosphate buffer

Sodium citrate buffer

Neutral buffer

2.4 Phosphate buffer procedure:

A phosphate buffer solution is a handy buffer to have around,especially for biological applications. As phosphoric acid hasmultiple dissociation constants, so phosphate buffers near any ofthe three pHs, which are at 2.15, 6.86 and 12.32 is prepared.The buffer is most commonly prepared at pH 7 by using monosodiumphosphate and its conjugate base, disodium phosphate.

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2.4.1Phosphate Buffer Materials

Monosodium phosphate

Disodium phosphate

Water

Phosphoric acid to make the pH more acidic or sodium hydroxide tomake the pH more alkalinePH meter

Glassware

Hot plate with stirring bar

2.4.2 Prepare the Phosphate Buffer

Decide on the concentration of the buffer. Most buffers are usedat a concentration between 0.1 M and 10 M. If you make up aconcentrated buffer solution, you can dilute it as needed.

Decide on the pH for your buffer. This pH should be within one pHunit from the pKa of the acid/conjugate base. So, you can preparea buffer at pH 2 or pH 7, for example, but pH 9 would be pushingit.

Use the Henderson-Hasselbach equation to calculate how much acidand base you need. You can simplify the calculation if you make 1liter of buffer. Select the pKa value that is closest to the pHof your buffer. For example, if you want the pH of your buffer tobe 7, then use the pKa of 6.9:pH = pKa + log ([Base]/[Acid])

Ratio of [Base]/[Acid] = 1.096

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The molarity of the buffer is the sum of the molarities of theacid and conjugate base or the sum of [Acid] + [Base]. For a 1M buffer (selected to make the calculation easy), [Acid] + [Base]= 1[Base] = 1 - [Acid]

Substitute this into the ratio and solve:

[Base] = 0.523 moles/L

Now solve for [Acid]. [Base] = 1 - [Acid] so [Acid] = 0.477moles/L

Prepare the solution by mixing 0.477 moles of monosodiumphosphate and 0.523 moles of disodium phosphate in a little lessthan a liter of water.

Check the pH using a pH meter and adjust the pH as necessaryusing phosphoric acid or sodium hydroxide.

Once you have reached the desired pH, add water to bring thetotal volume of phosphoric acid buffer to 1 L.

If you prepared this buffer as a stock solution, you can diluteit to make up buffers at other concentrations, such as 0.5 M or0.1 M.

2.5 Sodium citrate buffer:

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Sodium citrate, or trisodium citrate, can buffer solutions in thepH range of about 3 to 6.Sodium citrate buffer, or citric acid buffer, is used for RNAisolation because it minimizes base hydrolysis of the RNAstrands. This makes it an important step in the mRNApurification, duringgenomic research or forstudying transcription. Citrate-based buffers can also aidthedetection of antigens in fixed tissue preparations, becausethey break the cross-links formed between the antigens and thefixation media. Instructions below are for making a buffer of pH6.

Mix 2.1 g in just under 1 L distilled water.

Use 1M sodium hydroxide (NaOH) to adjust the pH of the mixture to6.0, while gently stirring the solution using a magnetic stirrer.Make the final volume of the solution up to 1 L with distilledwater using a volumetric.

2.6 Neutral buffer:Neutral buffered formalin, usually simply shortened to NBF, hasbecome the standard fixative for use in a diagnostic setting. Itis more effective than the simple formalin mixtures as thephosphate salts present make it unlikely that erythrocytes will

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be damaged, and the neutral pH inhibits the formation of formalinpigment. The phosphates will adjust the pH to about 7.0 as the"neutral" infers, but there is no need to adjust it to this levelif it is slightly different.

Although the formation of formalin pigment is inhibited it is notstopped altogether, and it may slowly form in very bloodytissues, or in tissues stored for a long time in NBF without itbeing changed.

NBF is useful as a fixative for museum and photography specimensas it permits restoration of natural color to the specimen.

Equipment:500 ml Glass bottleReagents:50 ml 37% Formaldehyde450 ml Distilled Water3.25 gm Sodium Phosphate, dibasic (Na2HPO4)2 gm Sodium Phosphate, monobasic (NaH2PO4)Method:Combine all ingredients and mix well.Store at room temperature.

2.7 HISTOLOGY MATERIALS:

Microscopic slide storage boxes

Embedding cassettes

Microscopic slide storage and folders

Microscopic slide and cover slips

Slide staining system

Embedding base molds

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Cassettes storage drawers

Microtome blades

Dissecting board

Tissues forceps and tweezers

Filter paper

Centrifuge tubes and racks

Funnels

Petri dishes

Micropipettes

Brushes

Lab wipes and gauges

Timer (stopwatch)

Cell counting and measuring tools

Biopsy punches

Applicator sticks

2.8 Methods

2.8.1 Animal handlingThe inbred long Evans female rats were handled with care and fedregularly to maintain their weight.2.8.2 Control Group7 rats were provided with normal food pellets and water for 14days. Weighing of rats, food taken daily and water drank dailywas also done.After 14 days the rats were sacrificed. Their organs such asliver, kidneys, heart, and spleen were collected in test tubesand stored in formalin. Small portion of each organ was cut andstored in eppendorf tube.

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Blood was also collected and later centrifuged to collect theplasma.2.8.3 CCl4 induced group7 rats were provided with normal food pellets and water for 14days. Weighing of rats, food taken daily and water drank dailywas also done. Animals were induced with CCl4 mixed with oliveoil in ratio 1:1 at an interval of 3 days.After 14 days the rats were sacrificed. Their organs such asliver, kidneys, heart, and spleen were collected in test tubesand stored in formalin. Small portion of each organ was cut andstored in eppendorf tube.Blood was also collected and later centrifuged to collect theplasma.

2.8.4 Treatment group7 rats were provided with normal food pellets but instead ofdistilled water Icturn an herbal formulation was given for 14days. Icturn was prepared in 1000ml in which 50ml of Icturn wasmixed with 950ml of water and distributed equally in each bottle.Freshly prepared Icturn was introduced after each 3 days intervalto avoid bacterial contamination. As similar to the disease groupCCl4 was induce at an interval of 3 days. Weighing of rats, foodtaken daily and prepared Icturn drank daily was also done.After 14 days the rats were sacrificed. Their organs such asliver, kidneys, heart, and spleen were collected in test tubesand stored in formalin. Small portion of each organ was cut andstored in eppendorf tube.Blood was also collected and later centrifuged to collect theplasma.

Only Icturn group

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7 rats were provided with normal food pellets but instead ofdistilled water Icturn a herbal formulation was given. Icturn wasprepared in 1000ml in which 50ml of Icturn was mixed with 950mlof water and distributed equally in each bottle. Freshly preparedIcturn was introduced after each 3 days interval to avoidbacterial contamination. Weighing of rats, food taken daily andprepared Icturn drank daily was also done.After 14 days the rats were sacrificed. Their organs such asliver, kidneys, heart, spleen were collected in test tubes andstored in formalin. Small portion of each organ was cut andstored in eppendorf tube.Blood was also collected and later centrifuged to collect theplasma.

2.8.5 Homogenate organsOrgans collected in eppendorf tube are homogenized by ahomogenizer machine present in the laboratory. The organs are cutin small amount such as 1mm before homogenizing them. Then thesolution is centrifuged and the plasma is collected for furtherfor Biochemical assay.

2.8.6 Biochemical Assay

2.8.6.1 for NOReagents required: PBS, sulfonamides, NED, Milli QSulfanamide-0.165g+5ml Milli QNED-0.005g+5ml Milli QProcess:Sample 50 micriolitre+50 microliter PBS.Then add sulfonamide 50microlitre and wait for 5minutes and add 50 microlitre NEDsolution. Again wait for 10 minutes at normal temperature. Thenfinally take absorbance in 500nm.2.8.6.2 for MDA

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Reagents required: PBS (phosphate buffer saline), acetic acid andTBA (thioburbituric acid)TBA-7mg+1ml DMSO+9ml Milli Q

Process:Take 50 micro litre plasma+50 micro litre PBS and 100 micro litreglacial acetic acid. Then incubate for 15minutes at normaltemperature (generally incubating 5-6 at RT, ppt occurrence isknown)Centrifuge at 8000rpm for 15 minutes at 4 degree Celsius if pptoccurs then take supernatant sample in 100 micro litre neweppendorf..If no ppt is formed skip the immediate previous step. Then 100microlitre of TBA solution is added in case of centrifugedsample. But the sample with no ppt formation is introduced with200 micro litre of TBA solution.The eppendorf are then inoculated into boiling water for 10minutes. After 10 minutes it is cooled (pink color may beobserved in diseases group).Then the samples are taken in g6 well plate (200 micro litres).Absorption at 490nm (for only one time i.e. 0 minute)2.8.6.3 for APOPReagents: 3.85gKI+200ml Milli Q and 33.8 microlitres H2O2+50 mlMille QProcess:10 microlitres plasma+90 microlitres PBS (5 microlitre tissue+95microlitre PBS) is added to 50 microlitres acetic acid.Then 50 microlitre KI solutions is added and waited for 2minutes.Absorbance was taken at 405nm.2.8.6.4 For catalaseReagents: 33.8 micolitres H2O2+50ml Milli QProcess:

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10 micro litres plasma+90 micro litres PBS (5 micro litretissue+95 microlitre PBS) is added to 50 micro litres H2O2

solution (5.9 mmol H2O2) and waited for 2 minutes.Absorbance was taken at 405nm in consecutive intervals 0 min, 3mins, and 6 mins. The absorbance should be decreased from 0-6mins which show good result.

Cleaning of g6 well plate:

First rinse it by ethanol well then rinse it by Milli Q H2o threetimes and then dry its surface by tissue paper and dry it.

Cleaning of g6 well plate after use:

Same procedures as above but before that put Ag for sometime andrinse it with acetic acid.

Handling of pipettes:

They should be handled carefully as the final result may vary dueto this only. By (0-10) microlitre measure 5 microlitres liquid.By (10-100) microlitre pipette use it for 40-60 microlitre.

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CHAPTER THREE

RESULTS

Figure 1: The body weight, Food intake and water intake of the rats inthe 14 days period

Here we have 3 prism graphs of the following as noted above,which shows the body weight of the female rats, the food intakeand the water intake for the 14 days period.

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In the first graph we have seen the body weight of the rats to beincreased steadily in a straight in case of control, whileCCl4the graph detoriates from the beginning which shows the weight ofthe rats were not increased but in case of Icturn+control andIcturn+ccl4 we have seen the graph to rise up to certain extentthen detoriates. The graph for icturn+control show more weightgain than the other one.

The 2nd graph shows the food intake of the rats in these 14 days.It shows the rats have a significant change in their diet in alltheir groups, making the graph move up and down quite a number oftimes. So we are not getting a steady graph for this one.

The water intake graph also is similar to food intake one andshows values which has significant changes causing the graph tobe not a steady one.

Figure 2: The organ weight values of heart, kidneys, liver andspleen.

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The liver weight increase in case of CCL4 due to increase ininflammation as it helps to induce the hepatotoxicty. Due to thisreason we have seen high values coming from the CCL4 while incase of control group, the values is quite less as compared toCCL4 as organs of those rats weren’t induce hepatotoxicity. Whilein case of the icturn+control group the values are similar to thecontrol one but lastly in case of the treatment group that isicturn+ccl4 we see the value is quite more than control one whileless than the CCL4 one. This is because of CCL4 inducehepatotoxicity which mainly causes the inflammation while thedrug(icturn) causes the toxicity of the liver to be decreased.While the values of the other organs such as heart, spleen andkidneys were very much similar to the values that we have foundin case of the liver.

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Table 1: Effect of Icturn on body weight, food and water intakeand organ weight of CCL4 treated rats.

Parameters Control Icturn +Control

CCL4 Icturn +CCL4

InitialBodyweight

183.48±0.81

181.42±1.50

183.43±0.74

181.68±1.53

Final Bodyweight

226.92 ±4.52 205.95±6.81

204.63±4.87

212.23±7.66

Foodintake/d

19.02 ±0.34 13.06 ±0.81 19.30 ±0.30 18.29 ±0.46

Waterintake/d

20.10 ±0.37 16.34 ±0.48 19.83 ±0.33 19.60 ±0.36

Liver wetweight

3.21 ±0.14 3.72 ±0.13 3.52 ±0.10 3.32 ±0.15

Kidneyswet weight

0.62 ±0.03 0.31 ±0.02 0.72 ±0.03 0.70 ±0.04

Heart wetweight

0.26 ±0.01 0.13 ±0.00 0.30 ±0.02 0.29 ±0.01

Spleen wetweight

0.30 ±0.02 0.15 ±0.01 0.35 ±0.02 0.33 ±0.02

Table 2: P value comparison of Intial body weight

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Table Analyzed Intial Body Weight

ANOVA summaryF 0.8003P value 0.5083P value summary NsAre differences among meansstatistically significant? (P <0.05) NoR square 0.1072

Bartlett's test

Bartlett's statistic (corrected) 1.044P value 0.7906P value summary nsSignificantly different standarddeviations? (P < 0.05) No

ANOVA table SS DF MS F (DFn, DFd) P value

Treatment (between columns)11.62 3

3.872

F (3, 20) =0.8003

P =0.5083

Residual (within columns)96.77 20

4.839

Total108.4 23

Data summary

Number of treatments (columns) 4Number of values (total) 24Number of families 1Number of comparisons per family 6Alpha 0.05

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Bonferroni's multiplecomparisons test

MeanDiff.

95% CI ofdiff.

Significant?

Summary

Control Group vs. CCL4 0.05-3.667 to3.767 No ns

Control Group vs. Icturn+CCL4 -0.19

-3.907 to3.527 No ns

Control Group vs.Icturn+Control -1.64

-5.357 to2.077 No ns

CCL4 vs. Icturn+ CCL4 -0.24-3.957 to3.477 No ns

CCL4 vs. Icturn+Control -1.69-5.407 to2.027 No ns

Icturn+ CCL4 vs.Icturn+Control -1.45

-5.167 to2.267 No ns

Test detailsMean1

Mean2

MeanDiff.

SE ofdiff.

n1

n2 t DF

Control Group vs. CCL4 183.5 183.4 0.05 1.27 6 60.03937 20

Control Group vs.Icturn+ CCL4 183.5 183.7 -0.19 1.27 6 6

0.1496 20

Control Group vs.Icturn+Control 183.5 185.1 -1.64 1.27 6 6 1.291 20CCL4 vs. Icturn+ CCL4 183.4 183.7 -0.24 1.27 6 6 0.189 20CCL4 vs. Icturn+Control 183.4 185.1 -1.69 1.27 6 6 1.331 20Icturn+ CCL4 vs.Icturn+Control 183.7 185.1 -1.45 1.27 6 6 1.142 20

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Table 3: P-value comparison of Final body weight

Table AnalyzedFianl BodyWeight

ANOVA summaryF 8.382P value 0.0008P value summary ***Are differences among meansstatistically significant? (P <0.05) YesR square 0.557

Bartlett's test

Bartlett's statistic (corrected) 11.15P value 0.011P value summary *Significantly different standarddeviations? (P < 0.05) Yes

ANOVA table SS DF MS F (DFn, DFd) P valueTreatment (betweencolumns) 1820 3

606.8

F (3, 20) =8.382

P =0.0008

Residual (within columns) 1448 2072.39

Total 3268 23

Data summary

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Number of treatments (columns) 4Number of values (total) 24Number of families 1Number of comparisons per family 6Alpha 0.05

Bonferroni's multiplecomparisons test

MeanDiff.

95% CI ofdiff.

Significant?

Summary

Control Group vs. CCL4 22.297.911 to36.67 Yes **

Control Group vs. Icturn+CCL4 17.3

2.921 to31.68 Yes *

Control Group vs.Icturn+Control 19.49

5.111 to33.87 Yes **

CCL4 vs. Icturn+ CCL4 -4.99-19.37 to9.389 No ns

CCL4 vs. Icturn+Control -2.8-17.18 to11.58 No ns

Icturn+ CCL4 vs.Icturn+Control 2.19

-12.19 to16.57 No ns

Test detailsMean1

Mean2

MeanDiff.

SE ofdiff.

n1

n2 t DF

Control Group vs. CCL4 226.9 204.6 22.29 4.912 6 64.538 20

Control Group vs.Icturn+ CCL4 226.9 209.6 17.3 4.912 6 6

3.522 20

Control Group vs.Icturn+Control 226.9 207.4 19.49 4.912 6 6

3.968 20

CCL4 vs. Icturn+ CCL4 204.6 209.6 -4.99 4.912 6 61.016 20

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CCL4 vs. Icturn+Control 204.6 207.4 -2.8 4.912 6 6 0.57 20Icturn+ CCL4 vs.Icturn+Control 209.6 207.4 2.19 4.912 6 6

0.4458 20

We know that the p value shows the significant lavel of differentgroups. The difference between many groups is significant at thelevel of 5%, ie only 5% is due to the chance and 95% is chanceof real difference.

Here we have seen that the P value of initial body weight andfinal body weight. The p value of initial body weight is higherthan .05. It means that the Rat model's weight is very muchrandomized. Initial body weight was taken before treatment. Sothe weight variation from mean value is deviated. Final bodyweight is taking after taking treatment. It shows significant pvalue. we can reject the null hypothesis. The changing weightwith treatment has some relation.

Table 4: P-value comparison of Initial food intake

Table Analyzed Initial Food intake

ANOVA summaryF 154.9

P value < 0.0001

P value summary ****Are differences among means statisticallysignificant? (P < 0.05) YesR square 0.9587

Bartlett's test

Bartlett's statistic (corrected) 39.44P value <0.0001

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P value summary ****Significantly different standard deviations? (P< 0.05)

Yes

ANOVA table SS DF MS F (DFn, DFd) P value

Treatment (betweencolumns) 20942 3 6981

F (3, 20) =154.9

P <0.0001

Residual (withincolumns) 901.2 20

45.06

Total 21843 23

Data summary

Number of treatments(columns) 4

Number of values (total) 24Number of families 1Number of comparisons per

family 6Alpha 0.05

Bonferroni's multiplecomparisons test

Mean Diff.

95% CIof diff.

Significant?

Summary

Control Group vs. CCL4 4.8-6.544

to 16.14 No nsControl Group vs.

Icturn+ CCL40.8

4-10.50

to 12.18 No nsControl Group vs.

Icturn+Control-

66.21-77.55

to -54.87 Yes***

*

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CCL4 vs. Icturn+ CCL4-

3.96-15.30

to 7.384 No ns

CCL4 vs. Icturn+Control-

71.01-82.35

to -59.67 Yes***

*Icturn+ CCL4 vs.

Icturn+Control-

67.05-78.39

to -55.71 Yes***

*

Test detailsMean1

Mean2

MeanDiff.

SE ofdiff.

n1

n2 t DF

Control Group vs. CCL4 16.72 11.92 4.8 3.876 6 61.239 20

Control Group vs.Icturn+ CCL4 16.72 15.88 0.84 3.876 6 6

0.2167 20

Control Group vs.Icturn+Control 16.72 82.93 -66.21 3.876 6 6

17.08 20

CCL4 vs. Icturn+ CCL4 11.92 15.88 -3.96 3.876 6 61.022 20

CCL4 vs.Icturn+Control 11.92 82.93 -71.01 3.876 6 6

18.32 20

Icturn+ CCL4 vs.Icturn+Control 15.88 82.93 -67.05 3.876 6 6 17.3 20

Table 5: P-value comparison of final food intake

Table Analyzed Final Food intakeANOVA summaryF 1399P value < 0.0001P value summary ****Are differences among meansstatistically significant? (P< 0.05) YesR square 0.9953

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Bartlett's test

Bartlett's statistic(corrected) 7.57P value 0.0558P value summary nsSignificantly differentstandard deviations? (P <0.05) No

ANOVA table SS DF MS F (DFn, DFd) P valueTreatment (betweencolumns) 15379 3 5126

F (3, 20) =1399

P <0.0001

Residual (withincolumns) 73.3 20

3.665

Total 15452 23

Data summary

Number of treatments (columns) 4Number of values (total) 24Number of families 1Number of comparisons perfamily 6Alpha 0.05

Bonferroni's multiplecomparisons test

MeanDiff.

95% CI ofdiff.

Significant?

Summary

Control Group vs. CCL4 0.62-2.615 to3.855 No ns

Control Group vs. Icturn+ -1.03 -4.265 to No ns

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CCL4 2.205Control Group vs.Icturn+Control -58.58

-61.82 to-55.34 Yes ****

CCL4 vs. Icturn+ CCL4 -1.65-4.885 to1.585 No ns

CCL4 vs. Icturn+Control -59.2-62.44 to-55.96 Yes ****

Icturn+ CCL4 vs.Icturn+Control -57.55

-60.79 to-54.31 Yes ****

Test detailsMean1

Mean2

MeanDiff.

SE ofdiff.

n1

n2 t DF

Control Group vs. CCL4 19.22 18.6 0.62 1.105 6 60.5609 20

Control Group vs.Icturn+ CCL4 19.22 20.25 -1.03 1.105 6 6

0.9319 20

Control Group vs.Icturn+Control 19.22 77.8 -58.58 1.105 6 6 53 20

CCL4 vs. Icturn+ CCL4 18.6 20.25 -1.65 1.105 6 61.493 20

CCL4 vs.Icturn+Control 18.6 77.8 -59.2 1.105 6 6

53.56 20

Icturn+ CCL4 vs.Icturn+Control 20.25 77.8 -57.55 1.105 6 6

52.07 20

We know that the p value shows the significant lavel of differentgroups. The difference between many groups is significant at thelevel of 5%, ie only 5% is due to the chance and 95% is chanceof real difference.

Here we have seen that the P value of initial food intake andfinal food intake. The p value of initial is lower than .05. Itmeans that is significant. So the variation from mean value is

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not deviated. Final food intake is taking after taking treatment.It shows lower p value. we can reject the null hypothesis. We canconclude that the treatment is significant and has some relation.

Table 6: P-value comparison of intial water intake

Table analysed Initial water intake

ANOVA summaryF 11845P value < 0.0001P value summary ****Are differences among meansstatistically significant? (P <0.05) YesR square 0.9994

Bartlett's test

Bartlett's statistic(corrected) 8.683P value 0.0338P value summary *Significantly differentstandard deviations? (P < 0.05) Yes

ANOVA table SS DF MS F (DFn, DFd) P valueTreatment (betweencolumns) 187199 3 62400

F (3, 20) =11845

P <0.0001

Residual (withincolumns) 105.4 20 5.268Total 187305 23

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Data summary

Number of treatments(columns) 4Number of values (total) 24Number of families 1Number of comparisons perfamily 6Alpha 0.05

Bonferroni's multiplecomparisons test

MeanDiff.

95% CI ofdiff.

Significant?

Summary

Control Group vs. CCL4 -2.06-5.939 to1.819 No ns

Control Group vs. Icturn+CCL4 -1.23

-5.109 to2.649 No ns

Control Group vs.Icturn+Control -205.1

-208.9 to-201.2 Yes ****

CCL4 vs. Icturn+ CCL4 0.83-3.049 to4.709 No ns

CCL4 vs. Icturn+Control -203-206.9 to-199.1 Yes ****

Icturn+ CCL4 vs.Icturn+Control -203.8

-207.7 to-199.9 Yes ****

Test detailsMean1

Mean2

MeanDiff.

SE ofdiff.

n1

n2 t DF

Control Group vs. CCL4 19.57 21.63 -2.06 1.325 6 61.555 20

Control Group vs.Icturn+ CCL4 19.57 20.8 -1.23 1.325 6 6

0.9282 20

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Control Group vs.Icturn+Control 19.57 224.6 -205.1 1.325 6 6

154.7 20

CCL4 vs. Icturn+ CCL4 21.63 20.8 0.83 1.325 6 60.6263 20

CCL4 vs.Icturn+Control 21.63 224.6 -203 1.325 6 6

153.2 20

Icturn+ CCL4 vs.Icturn+Control 20.8 224.6 -203.8 1.325 6 6

153.8 20

Table 7: P-value comparison of final water intake:

Table Analyzed Final waterintake

ANOVA summaryF 24040P value < 0.0001P value summary ****Are differences among meansstatistically significant? (P< 0.05) YesR square 0.9997Bartlett's testBartlett's statistic(corrected) 7.196P value 0.0659P value summary nsSignificantly differentstandard deviations? (P <0.05) No

ANOVA table SS DF MS F (DFn, DFd) P value

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Treatment (betweencolumns)

203869 3

67956

F (3, 20) =24040

P <0.0001

Residual (withincolumns) 56.54 20

2.827

Total203925 23

Data summary

Number of treatments(columns) 4Number of values (total) 24Number of families 1Number of comparisons perfamily 6Alpha 0.05

Bonferroni's multiplecomparisons test

MeanDiff.

95% CI ofdiff.

Significant?

Summary

Control Group vs. CCL4 -1.73-4.571 to1.111 No ns

Control Group vs. Icturn+CCL4 -2.08

-4.921 to0.7613 No ns

Control Group vs.Icturn+Control -214.1

-217.0 to-211.3 Yes ****

CCL4 vs. Icturn+ CCL4 -0.35-3.191 to2.491 No ns

CCL4 vs. Icturn+Control -212.4-215.2 to-209.5 Yes ****

Icturn+ CCL4 vs.Icturn+Control -212

-214.9 to-209.2 Yes ****

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Test detailsMean1

Mean2

MeanDiff.

SE ofdiff.

n1

n2 t DF

Control Group vs. CCL4 17.97 19.7 -1.73 0.9707 6 61.782 20

Control Group vs.Icturn+ CCL4 17.97 20.05 -2.08 0.9707 6 6

2.143 20

Control Group vs.Icturn+Control 17.97 232.1 -214.1 0.9707 6 6

220.6 20

CCL4 vs. Icturn+ CCL4 19.7 20.05 -0.35 0.9707 6 60.3606 20

CCL4 vs.Icturn+Control 19.7 232.1 -212.4 0.9707 6 6

218.8 20

Icturn+ CCL4 vs.Icturn+Control 20.05 232.1 -212 0.9707 6 6

218.4 20

We know that the p value shows the significant lavel of differentgroups. The difference between many groups is significant at thelevel of 5%, ie only 5% is due to the chance and 95% is chanceof real difference.

Here we have seen that the P value of initial water intake andfinal water intake. The p value of initial is lower than .05. Itmeans that is significant. So the variation from mean value isnot deviated. Final water intake is taking after takingtreatment. It shows lower p value. we can reject the nullhypothesis. We can conclude that the treatment is significant andhas some relation.

Table 8: P-value comparison of organ (liver):

Table Analyzed Liver

ANOVA summary

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F 2.139P value 0.1272P value summary nsAre differences among meansstatistically significant? (P< 0.05) NoR square 0.2429

Bartlett's testBartlett's statistic(corrected) 7.257P value 0.0641P value summary NsSignificantly differentstandard deviations? (P <0.05) No

ANOVA table SS DF MS F (DFn, DFd) P valueTreatment (betweencolumns)

1.645 3

0.5484

F (3, 20) =2.139

P =0.1272

Residual (withincolumns)

5.127 20

0.2564

Total6.772 23

Data summaryNumber of treatments(columns) 4Number of values (total) 24

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Bonferroni's multiplecomparisons test

MeanDiff.

95% CI ofdiff.

Significant?

Summary

Control Group vs. CCL4 -0.55-1.406 to0.3056 No ns

Control Group vs. Icturn+CCL4 0.06

-0.7956 to0.9156 No ns

Control Group vs.Icturn+Control 0.09

-0.7656 to0.9456 No ns

CCL4 vs. Icturn+ CCL4 0.61-0.2456 to1.466 No ns

CCL4 vs. Icturn+Control 0.64-0.2156 to1.496 No ns

Icturn+ CCL4 vs.Icturn+Control 0.03

-0.8256 to0.8856 No ns

Test detailsMean1

Mean2

MeanDiff.

SE ofdiff.

n1

n2 t DF

Control Group vs. CCL4 2.97 3.52 -0.55 0.2923 6 61.882 20

Control Group vs.Icturn+ CCL4 2.97 2.91 0.06 0.2923 6 6

0.2053 20

Control Group vs.Icturn+Control 2.97 2.88 0.09 0.2923 6 6

0.3079 20

CCL4 vs. Icturn+ CCL4 3.52 2.91 0.61 0.2923 6 62.087 20

CCL4 vs.Icturn+Control 3.52 2.88 0.64 0.2923 6 6

2.189 20

Icturn+ CCL4 vs.Icturn+Control 2.91 2.88 0.03 0.2923 6 6

0.1026 20

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We know that the p value shows the significant lavel of differentgroups. The difference between many groups is significant at thelevel of 5%, ie only 5% is due to the chance and 95% is chanceof real difference.

Here we have seen that the P value of initial liver damage. The pvalue of initial is higher than .05. It means that is notsignificant. So the variation from mean value is deviated. wecan not reject the null hypothesis. We can conclude that thetreatment is not significant. There is no relation betweentreatment and disease. there might be working other factor.

Table 9: Icturn plasma APOP value:

Icturn CCL4 APOP Plasma

Control

Control +Icturn CCL4

CCL4+Icturn

313.81 316.19 419.37 385.2415.70 21.60 37.06 31.996 6 6 6

Table 10: Icturn plasma MDA value:

Icturn Plasma MDA

Control

Control +Icturn CCL4 CCL4+ Icturn

52.79 60.53 57.97 52.028.64 6.20 4.93 8.466 6 6 6

Table 11: Icturn plasma NO value:

Icturn CCl4 Plasma NOContro Control + CCL4 CCL4+ Icturn

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l Icturn3.02 2.81 7.66 2.890.50 0.34 1.59 0.216 6 6 6

Table 12: Icturn CCL4 Apop value:

Icturn CCL4 APOP Tissue

ControlControl +Icturn CCL4 CCL4+ Icturn

4065.71 3649.84 7911.75 2537.14129.49 655.11 979.78 375.116 6 6 6

Table 13: Icturn CCL4 MDA value:

Icturn Tissue MDA

Control Control + Icturn CCL4 CCL4+ Icturn

119.41 202.49198.51 83.13

7.96 36.97 29.11 9.336 6 6 6

Table 14: Icturn CCL4 NO value:

Icturn CCl4 Tissue NOControl Control + Icturn CCL4 CCL4+ Icturn5.42 5.07 29.19 4.770.40 0.50 11.51 0.536 6 6 6

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CHAPTER FOUR

4.1 Discussion

Liver and its complications is becoming the leading cause ofdeath after cancer and cardiovascular diseases. Many serious sideeffects of medicines and oral drugs necessitate the search fornewer effective and safer class of compounds to overcomehepatotoxic problems. In recent years, herbal products havestarted to gain importance as a source of hepatotoxic medicines.

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It has been estimated that more than 1000 plant species are usedas folk medicine for treating hepatotoxicity though most lackscientific evidence. Our study is directed to evaluate thehepatotoxic property of Icturn in case of females Long Evansrats. Additionally,unpublished, preliminary screening data, ofthis drug, showed highly promising antihepatotoxic activity. Oraltreatment with the different kinds of antioxidants extract showedhepatotoxicity in the female wistar rats.According to establishedstudies, the initiator of oxidants tissue damage is thehepatotoxic states .The cells which are damaged by differentoxidants cannot maintain a constant internal level of glucosewhich ultimately results in altered cellular mechanism and long-term changes in cellular macromolecular content. An increase inthe products of oxidants is another result of hepatotoxic states,which significantly influences the development of hepatotoxicity.Thus, management of hepatotoxic is the most important method ofdiabetes control. Commonly used herbal drugs follow the basicmechanism of reduction of free radical of oxygen. Through ourstudies on Hepatotoxicity, after using several techniques, we aretrying to prove any of the above mentioned mechanism that thisdrug follows.

One of the components of Icturn is Cichorium endivia which isused to block the oxidative stress and cytotoxicity induced bythe t-BHP. Inducing the CCl4 into the rats will cause the rat tobe induced with liver toxicity which can be reduced by thepresence of this component. This component is one of the evidencethat Icturn possess properties which can cause the oxidativestress to be reduced or block it and in turn cause the livertoxicity to reduce to a great level as shown in the results(chapter 3). The p-value has been observed by ANOVA andBonferroni multiple comparision tests which shows the values are<0.05. This is an indication that our drug has worked out. On theother hand we have seen the values in case of biochemical assays

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such as APOP, MDA and NO that in comparison to control the valuesof CCl4 is quite high while in case of using icturn the valuesdecreased quite significantly showing its properties and finallyin case of treatment group it shows greater value than controlgroup while less values than CCl4 group, showing that drug thatwe have used have worked as it was supposed to be. The weight ofthe liver is quite increased in case of CCl4 only as it causesinflammation bt reduced values of liver weighs are observed whilein case of treatment and control groups.

Various in-vivo studies have shown the evidence about Icturn andits components to contain properties that will show graduallyincrease in hepatotoxic conditions as well as improvement inliver.

4.2 Conclusions

Our studies confirm the previous findings showing decrease actionof hepatotoxicity by using Hamdard Icturn. Additionally, we haveelucidated that Icturn(Dinar) has been used to treat otherdisease and also act as a blood purifier. Due to the confirmationof the decrease in the antioxidant level in the body which causesthe hepatotoxicity to be decreased to a significant level.

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Reference:1. Hoffmann M, Marbet UA, Hurni A, Bianchi L, Goldi H. Rezidiveiner medikamentos toxischen Hepatitis. Schweiz Med Forum.2005;5:147–148.

2. Elinav E, Pinsker G, Safadi R, Pappo O, Bromberg M, Anis E,Keinan-Boker L, Broide E, Ackerman Z, Kaluski DN, et al.Association between consumption of Herbalife nutritionalsupplements and acute hepatotoxicity. J Hepatol. 2007;47:514–520.

3. Schoepfer AM, Engel A, Fattinger K, Marbet UA, Criblez D,Reichen J, Zimmermann A, Oneta CM. Herbal does not meaninnocuous: ten cases of severe hepatotoxicity associated withdietary supplements from Herbalife products. J Hepatol.2007;47:521–526.

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4. Jóhannsson M, Ormarsdóttir S, Olafsson S. Hepatotoxicityassociated with the use of Herbalife. Laeknabladid. 2010;96:167–172.

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