Effect of Herbal Coded Drug Hepotin Tablet on ALT and Viral Load in Chronic Hepatitis C

 

Ph.D. Thesis

p 

by 

ASIF IQBAL

 

 

Prof.Dr.Usman Ghani Khan, Research Supervisor 

Prof.Dr.Hk. Abdul Hannan, Co‐ Research Supervisor 

Department of Medicine and Allied Sciences Faculty of Eastern Medicine

HAMDARDUNIVERSITY  

Karachi - 74600 2012

Effect of Herbal Coded Drug Hepotin Tablet on ALT and Viral Load in Chronic Hepatitis C

Thesis submitted for the fulfillment of degree of Doctor of Philosophy

by  

ASIF IQBAL BEMS, M.Phil. (HU)

 

 

Department of Medicine and Allied Sciences 

Faculty of Eastern Medicine HAMDARDUNIVERSITY

Karachi - 74600 2012 

 

    

Dedicated to my mentor and teacher  

Hakim Muhammad Said Shaheed 

Founder Chancellor 

Hamdard University 

and my Parents and Teachers  

 

 

 

 

i

Contents Page No.

Summary ` vii

Acknowledgements x

Chapter I: Diagnosis and treatment of HCV Updates

The Global Burden of Hepatitis C 01

1.1 Epidemiology 05

1.2 Transmission of HCV 08

1.3 Pathogenesis 10

1.4 Structure of Hepatitis C Virus 11

1.5 Genotype of HCV 14

1.6 Diagnostic Tests of HCV (ELISA, RIBA and HCV RNA by PCR) 15

1.7 Flow chart for diagnosis of HCV 18

1.8 Allopathic Treatment 19

1.9 Adverse Reactions to Interferon and Ribavirin 20

1.10 Pegylated Interferon 21

1.11 The Role of Complementary and Alternative Medicine in Chronic Liver Disease 22

1.12 Aim and Objective 24

Chapter II Literature Search 27

2.1 Tamarix gallica 28

2.2 Picrorhiza kurroa 30

2.3 Glycyrrhiza glabra 36

2.4 Phylanthus emblica 40

2.5 Silybum marianum 43 Chapter III Methodology 50

3.1 Aim of the study 51

3.2 Objective 52

3.3 Study Design 52

3.4 Inclusion and Exclusion Criteria 61

3.5 Proforma (Data Sheet) 63

ii

Chapter IV Results and Discussion 69

4.1 Statistical Analysis 70

4.2 Clinical Response 72

4.3 Biochemical Response 87

4.4 Serological Response 92

4.5 Discussion 96

4.6 Conclusion 101

4.7 References 102

iii

CANDIDATE DECLATATION

I, Asif Iqbal, hereby declare that the work reported in this thesis entitled ‘‘Effect of Herbal Coded Drug Hepotin Tablets on ALT and Viral Load in Chronic Hepatitis C’’ has been carried out in the Faculty of Eastern Medicine, Hamdard University Karachi, Pakistan is original and has not been previously submitted for any degree of any university in Pakistan. Asif Iqbal May 2011

iv

Index of Table

Table No. Topic Page No. Table1 Hepatitis C estimated prevalence and number infected by

WHO Region 5

Table 2 Side effects of treatment with Interferon alfa 2b and Ribavirin. 21 Table 3 Work plan 58 Table 4 Survey formulation 59 Table 5 Descriptive characteristics of study sample 60 Table 6 DRUGS * SEX cross tabulation (male and female) 66 Table 7 Age distribution of All Patients, male patients and female

patients 70

Table 8 Comparative data of Hepotin tablets and Interferon alpha 2b and Ribavirin (Anorexia)

76

Table 9 Comparative data of Hepotin tablets and Interferon alpha 2b and Ribavirin (Heart Burn)

78

Table 10 Comparative data between Hepotin tablets and Interferon alpha 2b and Ribavirin (Epigastric Pain)

80

Table 11 Comparative data between Hepotin tablets and Interferon alpha 2b and Ribavirin (Body-Ache)

82

Table 12 Comparative data between Hepotin tablets and Interferon alpha 2b and Ribavirin (Indigestion)

84

Table 13 Comparative data between Hepotin tablets and Interferon alpha 2b and Ribavirin (Burning Micturation)

86

Table 14 Comparative data between Hepotin tablets and Interferon alpha 2b and Ribavirin (Fever)

88

Table 15 Comparative data between Hepotin tablets Interferon alpha 2b and Ribavirin (Burning Palm and Sole)

89

Table 16 Comparative data between Hepotin tablets and Interferon alpha 2b and Ribavirin (ALT)

91

Table 18 Comparative data between Hepotin tablets and Interferon alpha 2b and Ribavirin (BUN)

92

Table 19 Comparative data between Hepotin tablets and Interferon alpha 2b and Ribavirin (Creatinine)

94

Table 20 Comparative data between Hepotin tablets and Interferon alpha 2b and Ribavirin (Serum Interferon alpha 2b and Ribavirin)

96

Table 21 Summary 98

v

Index of Figure

Topic Page no

Figure: 1 Global Epidemiology 6 Figure:2 Structure of HCV Virus 11 Figure:3 The HCV Genome and Expressed Poly protein 12 Figure: 4 Hepatitis C Testing Flow Chart 19 Figure: 5 Tamarix gallica. 30 Figure: 6 Picrorhiza kurroa 33 Figure: 7 Glycyrrhiza glabra 39 Figure: 8 Phylanthus emblica 43 Figure: 9 Silybum marianum 47 Figure:10 Graph 1. Age Distribution 70 Figure:11 Graph 2 Signs and Symptoms 71 Figure:12 Graph 3. Improvement in Anorexia 76 Figure:13 Graph 4. Improvement in Heart Burn 78 Figure:14 Graph 5 .Improvement in Epigastric Pain 79 Figure:15 Graph 6.Improvement in Body Ache 82 Figure:16 Graph 7.Improvement in Indigestion 84 Figure:17 Graph 8.Improvment in Burning Micturation 86 Figure: 18 Graph .9 Improvement in Fever 88 Figure:19 Graph .10Improvement in Burning Palm and Sole 89 Figure: 20 Graph 11. Comparative Data Between Hepotin Tablets and Interferon

alpha 2b Therapy(ALT) 91

Figure: 21 Graph 12. Comparative Data Between Hepotin Tablets and Interferon alpha 2b Therapy (BUN)

92

Figure: 22 Graph 13 Comparative Data Between Hepotin Tablets and Interferon Therapy(Serum Creatinine)

94

Figure: 23 Graph 14. Comparative Data Between Hepotin Tablets and Interferon alpha 2b and Ribavirin

96

vi

Abbreviations

Code Detail ALT Alanine Aminotransferase AST Aspartate Aminotransferase BMI Body Mass Index CHC Chronic Hepatitis C CLD Chronic Liver Disease DGL Deglycyrrhizinated DNA Deoxyribo Nucleic acid DV Dependent Variable ELISA Enzyme Linked Immunosorbent Assays GBD Global Burden of Disease HACEM Hamdard al-Majeed College of Eastern Medicine HBs Ag Hepatitis B Surface Antigen HCC Hepatocellular Carcinoma HCV Hepatitis C virus HIV Human Immunodeficiency Virus ISDR Interferon Sensitivity Determining Region IV Independent Variable LES Lower Esophageal Segment MUSE Metaplasia (M), Ulceration (U), Stricturing (S), and Mucosal Erosion (E) NCR Non Coding Region NS Nonstructural PCR Polymerase Chain Reaction PEG Pegylated PPIs Proton Pump Inhibitors PUO Pyrexia of Unknown Origin RBV Ribavirin RCT Randomized Controlled Trial RFLP Restriction fragment length polymorphism RIBA Recombinant Immunoblot Assays RNA Ribonucleic Acid SGOT Serum Glutamic Oxaloacetic Transaminase SGPT Serum Glutamic Pyruvic Transaminase SNMC Stronger Neominophagen C SVR Sustained Virological Response WHO World Health Organization

vii

Summary

Hepatitis C is emerging as the serious worldwide mailaise and Hepatitis C virus is endemic

throughout the world. It is estimated that HCV affects 170-200 million people worldwide.

Although acute HCV infection resolves spontaneously in some cases, the virus establishes

chronic infection in 55-85% of infected individuals and persists for decades. Left untreated,

approximately 20% of chronically infected individuals will go on to develop cirrhosis and 1-5%

will develop liver failure and hepatocellular carcinoma. Many practitioners have used of

traditional medicine for the treatment of chronic liver disease. In Pakistan medicinal plants are

being used for the treatment of hepatitis, therefore it is intended to find out the evidence based

treatment of hepatitis with the Unani medicine and clinically evaluate the efficacy claims

specifically for Hepatitis C. The human studies have been designed after carefully evaluating the

safety, effectiveness of Unani medicaments components. In addition, this type of study is being

executed due to the fact that many hakims are using different traditional preparation to cure

various forms of viral hepatitis lesions after the failure of allopathic therapy.

Specific Objective Achieved:

• The broad spectrum of pathological investigation (HCV RNA by PCR with Viral load

and biochemical test) for testing the antiviral and hepato-protective profile has been

established for clinical efficacy of herbal medicine and allopathic medicine.

• The study has been conducted on the effect of the coded herbal formulation (Hepotin

tablets) and allopathic Interferon alpha 2b + Ribavirin on the effect on urea, creatinine,

and platelets.

• The negative side effect of both therapy has also been observed and analyzing if any.

viii

Study Design:

Case control randomized clinical trial

Duration:

Three year from 1st June, 2007 to 30th May, 2010.

Setting:

Shifa-ul-Mulk Memorial Hospital for Eastern Medicine Hamdard University,

Muhammad Bin Qasim Avenue Karachi.

Subjects:

Patients of both genders (ages; >25 years and <55 years) with clinical diagnosis of hepatitis C.

Sample sizes for this study included in Hepotin Tab. a total of 80 subjects .In control group 80 in

Interferon therapy for Hepatitis C.

Outcome Measure:

Primary efficacy parameter: clinical response, biochemical response, and serological response.

Results:

Criteria for the assessment of therapeutic evaluation were monitored according to the inclusion

and exclusion parameters. Sign and symptoms recorded were anorexia, heart burn, Epigastric

pain, constipation, body ache, indigestion, burning micturation, fever, burning palm and sole.

Both the drug (test and control) manifestation from clinical perspective was analyzed in each

category of sign and symptoms. The comparative analysis of the test and control drug

accordingly showed that in case of anorexia Hepotin, and Interferon alpha–2b+Ribavirin, the

improvement ratio was 81% and 50% respectively. Similarly in heart burn, epigastric pain,

constipation, body ache, indigestion burning micturation, fever, burning of palm and sole for the

ix

test drugs displayed improvement in test and control drugs from 88% to 90% and from 22% to

57% respectively.

Wilcoxon signed rank tests was used to compare baseline and post treatment values. There was

a significant decrease in serum Alanine Transferase (ALT) from baseline to the end of treatment

(Hepotin tablets 117+34, 54+22 Interferon alpha 2b + Ribavirin, 79+28,67+19; P=0.02); 78% of

subjects had a decrease in serum ALT level. There was no significant change in serum ALT in

Interferon cases. Multivariate logistic regression analysis in a model that included dose, age, sex,

Viral load, elevated ALT. Therefore the Treatment outcome with Hepotin tablets has been

observed as reduced ALT, relive in clinical complaints. The effectiveness as hepatoprotective,

antiviral and anti inflammatory action of Hepotin tablets has been proved. Thus the null

hypothesis was rejected clearly specifying that the clinical efficacy is effective in test drug as

compared to control drug (Interferon alpha 2b+Ribavirin). In addition serological response (HCV

RNA by PCR Viral load) in case of Hepotin was 48% as compared to Interferon alha – 2b and

Ribavirin which was 55% and p value was found to be 0.4.

Conclusion:

The cumulative data of Hepotin tablets revealed clinical, biochemical and serological (HCV RNA by

PCR Viral load) improvements with significant symptomatic control. In addition, there was highly

significant reduction in the mean recovery period. There were no significant adverse events in all

cases has been reported or observed and overall drug compliance was excellent. Therefore, it may be

concluded that Hepotin tablets is effective and safe in the management of hepatitis C.

x

ACKNOWLEDGEMENTS

I remain in gratitude to Almighty Allah for His mercy to execute the clinical research described

in this dissertation and His Prophet Muhammad (Peace be upon him) from whom I take guidance

to lead my life.

I am in deep love and regard to Hakim Mohammed Said Shaheed who was the guiding source

and with whom I learnt Tibb and would always remain indebted for providing me the

opportunity to grow and excel in higher learning and research.

I express my most sincere gratitude to my supervisor Prof. Dr. Usman Ghani Khan, Principal,

HACEM and Head, Basic Clinical Sciences, Faculty of Eastern Medicine, Hamdard University,

Karachi and my co-supervisor Prof.Dr.Hk.Abdul Hannan Dean Faculty of Eastern Medicine for

their invaluable guidance, constant supervision, monitoring, encouragement and instruction to

carry out the research work embodied in this thesis.

I extend my sincere thanks to Mrs. Sadia Rashid, President, Hamdard Foundation Pakistan, Prof. Dr.

Brigadier Naseem A. Khan Vice Chancellor, Hamdard University, Dr. Navaid ul Zafar, Managing

Director, Hamdard Laboratories (Waqf) Pakistan, Prof. Dr. Surgeon. Zakiuddin G. Oonwala Dean

and Prof. Dr.Furqan Ahmad Principal Faculty of Health and Medical Sciences, Hamdard University,

Dr. Tasir Ahmed Mumtaz Assistant Prof. & Consultant Radiologist, Hamdard University Hospital

Taj Medical Complex, Prof.Dr.Rukhsana Abdul Sattar, Dr Qurban Hussain Sheikh and Dr.Maqsood

Jinnah Post graduate Medical Centre & Sindh Medical College and Dr Shahid Karim Aga Khan

University Hospital, Karachi for their cooperation and understanding.

I also wish to express my thanks to Prof.Dr.Rubina Ghani Molecular Biologist at Baqai Medical

University to give me a special favor in laboratory investigations and for her cooperation in my

xi

research work. I like to record my appreciation to the clinical and non-clinical staff of Shifa-ul-

Mulk Memorial Hospital for Eastern Medicine for their assistance.

I am highly grateful to Dr.Irfan Ullha Siddqui Principal Bahria Medical Collegefor his

cooperation and understanding in the statistical analysis and interpretation of results.

At the end, I would like acknowledge the deep love and affection of my late Grandfather

Hk.Feroz-u-Din Ajmali who urged me to pursue education in Tibb and its clinical Practice in

excellence, all my respect for my father Dr.Shaikh Muhammad Iqbal, my Late mother Mrs.Zakia

Iqbal for their help and assistance to complete the higher research and learning in Eastern

Medicine, My special thanks goes to my wife, my children and my beloved uncle Hk.Zia-ur-

Rehman whose love and invaluable encouragement to complete this academic venture.

Asif Iqbal

CHAPTER I

Diagnosis and treatment of HCV Updates

HCV RNA Detection By PCR

 

 

 

 

1

The Global Burden of Hepatitis C

Both industriallized and developing countries are concerned with hepatitis C. Thus, the public

health is related with minimizing of birth and death rate. The domestic as well as all over the

world, the birth and death rate is concerned with HCV infection, based on incidence,

transmission, prevalence, and socioeconomics. To get these purposes an association was

organize to help World health organization (WHO) in estimating global load of HCV .

All over the world almost 170 million people are infectedwith hepatitis C. In fact the liver

cirrhosis,hepatocellular carcinoma and end stage liver disease is due to this.Even the

kidneytransplants are performed due to it in advanced countries. Primarily it is transferred from

parents to children, but transferration from mother to young one is more significant. Partially

because of the genetic diversity of this virus, identification of candidate vaccines is difficult

because of the host immune response different for different varity. Yet, important developments

are to treat chr. hepatitis C. now a days the pegylated interferon alpha 2b and Ribavirin (RBV)

both are the mean cure of hepatitis C virus and it results in longterm elimination of this virus in

about 54% people.Treatment response depends upon the infecting genotype, with 76 to 80% of

those with genotypes 2 and 3 but approximately just 40% with genotype 1 or 4 achieving a

sustained virological response. As the treatment is costly and related with specific adverse

effects, more effective ways for preventing the transferration are required. Especially in the

countries which have limited resources where burden of diseases is on peak.

Even we have made rapid progress in our knowledge about hepetitis C virology and pathogenesis

yet we don’t have much knowledge about its treatment to eradicate this disease from the whole

world. To evolve the better management of effected people and prevention of complexities,

2

careful undrestanding and assessment of hepatitis C with both conventional and traditional

medicine is required.. However it remains a challenge to achieve sustained virological response

in patients of chronic hepatitis C. The rate of response in genotype 1 patients remains less than

50% even by the use of PEG Interferon and Ribavirin (RBV).High doses of drug Ribavirin may

improve the SVR, on the other hand its use is majorly limited by anemia.

In various areas of Pakistan, The burden of HCV and chronic liver disease (CLD) has been

increased. In the present researches it is found that of all patients of cirrhosis, indicates nearly

60 to 70% patients with CLD with anti HCV +ve and 16.6% were anti HCV +ve without

cirrhosis . In Pakistan, almost 50% patients of hepatocellular carcinoma are anti HCV +ve. In the

country the major cause of HCV transmission is blood transfusion as the record of large blood

banks of country tells that almost 25% of them tested blood and blood product donations for

screening of HCV infection are +ve and increasing the transmission of HCV which increases the

global burden. From the most of the studies it has been proved that there is some relationship b/w

use of non sterile needles and transfusion of HCV. Both in the forms of infusion of drips and

injections which are given due to the cultural beliefs there is high practice of parenteral therapy

for treatment. Excessive visits to barbers, Dental practices,ear piercing and use of non-sterile

surgical equipments by unqualified health care workers (quacks) are some important risk factors

for transmission of HCV.In developing countries the mean age of developing CLD is much

lower than that of developed countries.In HCV patient and among those chronically infected,

Cirrhosis may develop in from 5 to 20% of individuals. Currently 13 million patients of hepatitis

C are present in Pakistan. In Pakistan The overall prevalence of the HCV is around 10 to 15

percent ,in lower Punjab and upper Sindh, this prevalence increases to around 40 to 50

percent.250,000 people die per year due to HCV related diseases.

3

The recent survey showed 45.7% cases of chronic liver disease, 43.06% seropositivity in CLD

and cirrhosis combined and 37.7% cases of cirrhosis in Pakistan. The mortality rate due to

chronic liver diseases is high in northern Pakistan. Jaundice and fever has been their initial

presentation. Among 23 (70%) had hepatitis C virus (HCV) positive as the main cause of their

liver disease, 33 patients of CLD, 3 (9%) with undetectable cause of chronic liver disease and 4

(12%) had both hepatitis B and hepatitis C virus infection. At atertiary care hospital in this part

of Pakistan CLD is a major cause of deaths. The main cause of chronic liver diseases is the HCV

infection followed by HBV or combination of these viruses. Portal hypertension,gastrointestinal

bleeding and hepatic failure are some main signs of CLD. The 75% of patients are unable to get

conventional therapy for HCV ie. Interferon alpha 2b + Ribavirin and the 25% patents that get

standard treatment in Pakistan; the sustained virological response (SVR) rate is 60 – 70%. In

Asian countries, such as Thailand, Malaysia and India the prevalence rates are pretty high. This

information related to frequency of HCV in Pakistan the current diagnostic assays, prevalence,

genotypes, treatment outcomes and various therapies. Exact HCV prevalence rate should now be

the further emphasizes, more over development of the guide lines for diagnostic assays based on

local genotype, and search for understanding the correlation between the HCV genotype 3a

genes with cell line genes responsible for HCV pathogenesis. It is necessary to generate

infectious false particle of HCV as a potent vaccine to assess DNA or RNA based vaccine.

4

1.1 Epidemiology

World Health Organization (WHO) has reported that about 170-200 million of the world

population ie. 3% of the total population of the world, are affected with HCV that can lead to

liver cirrhosis and liver cancer. The prevalence of HCV infection in some countries in South-

East Asia, Africa, the Eastern Mediterranean, Western Pacific, North America and Europe is

given in [Table 1].

Table1. Hepatitis C Prevalence and Number,WHORegion2003[17].

WHORegion Total

Population

(Million)

Hepatitis C

prevalence

Rate%

Infected

Population

Africa 602 5.3 31.9

America 785 1.7 13.1

Eastern

Mediterranean

466 4.6 21.3

Europe 858 1.03 8.9

South-East Asia 1500 2.15 32.3

Western Pacific 1600 3.9 62.2

Total 5811 3.1 169.7

The aim of collecting this data is to represent the future hazards threat affect caused by hepatitis

C infection. an important genetic variation is noticed in worldwide populations of HCV, a proof

5

of it is frequent rates ofevolution and rapidmutation.The above mentioned stats from the various

parts of world show a wide range in HCV prevalence[Fig.1]. Higher rates reported in India

(1.5%),Philippines (2.3%) and the Malaysia (2.3%), the Southeast Asian countries. the Northern

European countries, Great Britain, France and Germany showed the lowest prevalence of

hepatitis C. The prevalence of HCV antibodies average less than 1% for the region in blood

donors sa reported by research. The incidence was 1.2% in Japan. In many African nations very

high rates were reported, in Egypt viewing as high as 14.5% in case of hepatitis C. It is

considered that Egypt the use of parenteral antischistosomal therapy to have added in different

regions from 6 to 28 % (average, 22 %) to a prevalence of Ab. against HCV.

-

Figure I: Global Epidemiology

The first edition describing the estimate of global prevalence of HCV was published by WHO in

2003. Data collected from 260 reviewed journal articles revels seroprevalence rates of HCV in

various population . 116 of the most representative studies (one per country) were selected and

according to WHO region countries were grouped after the application of different exclusion

6

criteria. One sixty million persons estimated to be HCV infected upto 1997 worledwide, except

3 countries Zambia, Peru and Botswana where data was available. The data also shows very

high frequency in people such as (blood donors) including the regions such as Burundi,

Bolivia, Rwanda, Egypt, Guinea,Cameroon, and Tanzania. High prevalence of HCV was

reportedin WHO region i.ethe Eastern Mediterranean, Africa, Southeast Asia,and the Western

Pacific. In North America and Europe lower prevalence was found. Twenty present who are get

infection naturally, may get rid of infection in England therefore, was reported as 0.4%. In the

USA, the 1.8 % of the population was +vefor anti-HCV . Viremia is also found in every 3 of 4

seropositive people. In the United States an approximately 2.7 million people possess activeHCV

infection as calculated by available test reports.

7

1.2-Transmission

The contac t to blood, semen and other humors of infected person are responsible for transfusion

of this disease to healthy person, insecure sex with an infected person,close contact and sharing

utensils , drug needles, or razor with an infected person are also its causes. The major cause of

the spreading of this virus is the reused or illegal use of injections and by paramedical staff.The

risk factors strongly concerned with infection include injection and receipt of a transfusion of

blood before 1991, but no any risk factors can be detected in some cases but yet the reasons are

not evident,the razor shaving by hair dresser and transfusion of unsafe blood are major causes

of the propagation of HCV. Usually most of the patients insist the physician to inject them to

overcome their weakness, it is the major mode of transmission that is propagation of hepatitis C.

Pakistan is the country where a large number of people approach barbers for shaving.

Some reports also show that mother to fetal transmission of HCV also occurs but it is infrequent

and mostly related to confection with HIV-1in the mother. Due to some unknown means it was

observed that contact or sexual inter course may lead to the transmission of the hepatitis C virus

but by an inefficient means than in case of HIV. Evenly confection and HIV-1 seems to

increase the risk of both sexual and maternal fetal transmission of hepatitis C virus .

Secretions of infected persons can also be the cause of the transmission of HCV, and

experimentally chimpanzees got infected when being injected with the secretions especially

saliva of infected persons, very inefficient modes of transmission occur through contact with the

secretions of infected persons. The nosocomial transmission, such as from person to person by a

colonoscopy, during any procedure of surgery with dialysis is also documented.

8

Blood transfusion has caused a major risk factor of HCV.”The current risk for the transmission

form blood that is -ve for HCV Ab. is very low ie. 1 in 103,000 transfused in the US”, with the

remaining risk that result from blood transfusion, occur in the interval between the development

of antiHCV calculated to be less than twelve weeks and infection. The risk of transfusion related

to HBV infection would half (1 in 63,000) on the other hand about five times the maximum

risk of HIV-1 infection (only 1 in 493,000).The risk of transfusion associated HCV infection is

decreased by improving the blood screening measures in 1990 and 1992.The risk is now reduced

since new screeningmethods, as direct screening of samples by PCR method. The 3 weeks period

after exposure of HCV should be decreased by it.

The health care workers are not often infected with HCV even with their camparison with whole

population. The transmission rate after aneedle injury involving blood is known to be infected

rangefrom (0-10%) in many studies.

9

1.3 Pathogenesis

HCV is an RNA virus.The family is flaviviridae. Hepatitis G virus,dengue virus, and yellow

fever viruses are most closely related to human viruses.The natural targetsof HCV and possibly

B lymphocytes are hepatocytes.Itsreplication is extremely robust, and morethan ten trillion of

virion particles are calculated to be produced on every day. The replication of HCV occurs

throughan RNA-dependent RNA polymerase that misses a "proofreading function”that results in

rapid evolution of diversed thoughrelated quasi species within infected person.

“In vivo despite thereplication rates is in excess of those observed in HIV-1 and HBV infection,

more efforts to grow HCV in culture had been largely unsuccessful”. The propagationof virus by

inj. of recombinant transcribed HCV RNA in chimpanze have been successful, along with

histological and clinical signs of hepatitis C. The updated genetic expression of the RNA virions

has resulted, from liver cells at high level of multiplication in cell line derived, offering a many

traceable means to study synthesis of protein and HCV RNA.

   

10

1.4 Structure of HCV Virus

An icosahedral protective shell of protein contain core of genetic material (RNA), and then

covered with a lipid envelope. The lipid envelope embedded the two viral envelope

glycoproteins E1 and E2.

In HCV A+ve sense single-stranded RNAgenome is present. This genome has9600 nucleotide

bases length. This single open reading frame is then translated to produce a new single protein

product, which the results in the production of smaller active proteins.

The UTR regions at the 5' and 3' ends of the RNA are very significant for replication and

translation of RNA of virus but they are not translated to protiens. The UTR 5' has a ribosome

binding site.Internal ribosome entry site (IRES) is a very lengthy protein which has about 3000

Figure: 2 Structure of Hepatitis C Virus

Envelope Glycoproteingen

Core

Viral RNA Envelope Approx 60 nm

11

amino acids hat start the translation. Then it is cut down to 10 smaller protiens by viral and

cellular protiens.

Structural proteins made by the hepatitis C virus include E1 and E2; non-structural proteins

include NS2, NS3, NS4, NS4A, NS4B, NS5, NS5A, and NS5B.

Figure: 3HCV RNA Virus

Core and two envelope protiens are present in the structural components. The two parts of

envelope E-2 protein, showed hyper variable regions 1 - 2, and have an high concentration of

mutated patrs that is thaught to be the out come of specific pressure in anti HCV. CD-81binding

site is also present in E2, a tetra spanin shown on liver cells and B lymphocytesthat is known to

 

12

function as a cell receptor or co receptorfor that virus.Virus-specific helicase is also encoded by

HCV, polymerase and protease. Due to the critical functionof these proteins, they indicated the

specific targets for AV. therapy. The both ends of HCV RNA, may represent the promise as

therapeutictargets, therefore they are largely conserved and associated in sensitive steps of viral

replication.

HCV is a single stranded RNA virus of weight 9.5 kb, contain single open reading area and for

un translated parts (UTRs). It encodes the poly protein approximately of 3000 aminoacids, which

is then convert into single proteins through a host signal-peptidase in the specific site and the

HCV proteases innon-structural (NS) site. The st.portion also have the core-protein and 2

envelope proteins (E1-E2). 2 regions in E2 that are called hyper variable regions 1 and 2 (HVR1-

HVR 2), represent the high sequence change, that could be the result of selective pressure by

HCV antibodies. The functions as proteases (in NS-2, NS-3, and NS4 A), helicase (in NS-3),

and RNA dependent, RNA-polymerase (NS-5B)have been assigned by non-structural proteins.

Even though the fine st.of NS-3 and NS-5 is known [32], the fn. and function of proteins are not

defined clearly. Interferon alpha 2b therapy show response by linking with region in NS5A and

called as the INF. sensitivity determining region (I.S.D.R). therefore, it is still not clear the

relevance and importance of this region [34].

13

1.5 Genotypes of HCV

There have been recognized 6 distinct but same (relavent) genotype and multiple sub- types at

the basis of molecular relatedness.Genotypes 1a and 1bare most common in the United States

and Western Europe, followed by genotypes 2 and 3. In Egypt genotype 4, in South-Africa

genotype 5, and Southeast Asia genotype 6 is more common. The geno -type based study is very

much important in term of response to antiviraltherapy[35].

On nucleotide sequence variability hepatitis C virus (HCV) is categorized into different types. In

Karachi it has been reported in one study, five non coding region (NCR) based restriction

fragment length polymorphism (RFLP) genotyping assay was utilized to find out the genotype

distribution in a large series of HCV-infected patients.The anti-HCV by 2nd generation enzyme

immunoassay (EIA) and 48 were confirmed anti-HCV+ve (33- males, 15- females).Over all the

genotype 3 is the most common isolate in HCV-associated chronic liver disease (CLD) in

Pakistan. Genotypes determined for 45/48 anti-HCV+ve study patients; 39/45 (87%) were type

3; four (9%) were type 1; one was type 2; and one was type 5 [36]. A molecular study was

carried out to find out the prevalence of hepatitis C virus genotypes in HCV infected population

of Baluchistan. Out of the 40 HCV sero+ve samples belonging to seven different locations of

Baluchistan collected and the qualitative analysis of these samples using PCR resulted in 28 +ve

samples. Genotyping of 28 samples furnished three different genotypes including 3a, 3b and 1a.

The most prevalent genotype was 3a with rate of 50% followed by genotype 3b and 1a

respectively [37].

14

1.6 Diagnostic Tests of HCV (ELISA, RIBA and HCV RNA by PCR)

Diagnostic tests for HCV infectiondivided into serologicassays for antiHCV and molecular

assesments for viral particles. Recentky new assays based on the molecular detection for the

HCV RNA inducted. These tests can be categorizedas Ql. and Qt., since viral RNA is

unstable,the exact processing of test is critical to minimize the risk of false -ve results. The

diagnostic tests for hepatitis C are given in flow chart I.

A serum ALT level and a PCR should be repeated at 6 months after stopping treatment for

verification, if the patient has a sustained virological response. Patients who are non-responders

to an initial course of interferon management of hepatitis C and Ribavirin therapy need to

determine HCV genotype, quantitative HCV RNA[38].For hepatitis C infection, these include

serological assays for anti-HCV antibody detection and molecular assays that detect and quantify

HCV RNA and determine the HCV genotype. They can be used to test donor’s blood, to

diagnose active infection, guide treatment decisions, assess the virological response to therapy

and establish the prognosis. Genotype 3 is most predominant HCV genotype in Pakistan, with a

prevalence of between 75-90%. PCR assays for HCV RNA is the standard procedure adopted. A

qualitative HCV PCR is repeated only at the end of therapy, in order to document a virological

response. Modern method of the enzyme-linked immune - sorbent assays (ELISA) and the

recombinant immunoblot assays (RIBA) compared with their procedure in terms of sensitivity

and specificity. HCV molecular assays, such as detection of HCV RNA by reverse- transcription

and polymerase chain reaction (PCR), considered in terms of their use in following therapeutic

activity. Quasi-species assesment, geno-typing and serot-yping, used to unravel the genetic

idiosyncrasies of the virus so that more accurate therapeutic strategies. Finally the liver biopsy

expressed in terms of patient prognosis and with it non-invasive markers of histological status,

15

such as hyaluronic-acid and serum pro-collagenase-III peptide assigned their specific time and

place in the treatment of patients with HCV infection. The recent data have shown that the

concentration of HCV antibodies(Ab.)decreases gradually but in the some patients the infection

suddenly resolves. Common origin and expected time of infection assays to Ab. of HCV cleared

after 18-20 years in age of 18 of 43 patientswith sudden clearance of viral infection[39]. The

currently used 2nd and 3rd generation ELISA contain core- protein and non-structural proteins 3

- 4 (the 3rdgn. assay contains non-structural protein-5) and can detect Ab. within four to ten

weeks after exposure to HCV. In risky areas the test loses only 0.5 - 1 % of cases. In addition

false -Ve tests may be found in patients with immune-compromise, like HIV-1 infection, chronic

renal failure, those with HCV-associated cryoglobulinemia. The RIA method used to confirm

+ve-enzyme immuno- assays. It works on the basis antigens similar to those for these EIA. but

in an immunoblot method, so that responses to the specific proteins can be diagnosed. A +ve

assay was explain by the diagnostic method of antibodies against 2 or more Ag. while a non-

detected assay with the determination of Ab. For spacific Ag. . However with the availability of

specific EIA. and new RNA assessment assay, confirm by re-combinant immune-blot (RIA)

assay may become negligible. Technique had a lowest limit of assesment of fewer than hundred

copies of HCV RNA /ml. the gold standard test of viremia and the confirmation of the therapy is

a quantification of PCR assay must be also be used in patients +ve results on EIA in people

acuteinfection is suppose, for the effected persons involved in hepatitis but no cause, while in

those have definite known reasons for false -ve result on Ab. checkup. The early serologic test

of HCV infection is ELISA, there is three sensitivity of consecutive versions increases . The viral

quantification has been appear as relevant to the outcome of antibodies to HCV treatment. The

HCV RNA is usually detectable 2-4 weeks after infection independently the immuneperiod of

16

the patients. The detection of HCV RNA by PCR method proven the past infection. The

quantitative PCR measurement is very useful for determination of efficacy of antiviral therapy.

Quantitative HCV RNA by PCR

A branchedchain RNA assays and two assays involvingreversetranscription PCR systems deliver

reliable, butnot easily comparable, results since no aqurate systemof give the exact viral load has

been established. A single testsystem for each sample for longitudinal observation of the

viralload is the good option.There are varying standard ranges from PCR-based assays which are

more sensitive and the sub-chain RNA assay has a higher range and there is no need for dilution

for the assessment of viral quantity. The outcome of treatmen and therapeutic regimen can be

assess by Viral genotyping. The determination of HCV genotype is important before start of

therapy. There is only clinically relevant distinction among genotype 1,2 and 3, and the various

systems have various results in relation to this distinction. Different methods are present for the

GT. of HCV most of which are based on PCR method. A nonspecific lab. test of HCV-infected

patients measurement by ALT and readily available means of identifying liver disease. It is the

gold standard test for monitoring HCV infection and the outcome of treatment in the periods

between molecular detection[40].

17

Figure:4 Hepatitis C Testing Flow Chart

Positive Risk Factor

OR Clinically Indicated NoYes 

Pos. With

High s/co

Pos. With

Low s/co

NOTE: The s/co Ratio is a calculation done in the laboratory when the HCV antibody testing is performed. Most labs do not routinely report out the s/co Ratio, you must ask for it.

NOTE: An-immuno-compromised state such as Chemotherapy, HIV, or Lupus can cause a false negative anti-HCV result. Repeat testing should be performed in 3-6 months-if-clinically dicated.Some patients can be so immuno-compromised that they never sero-convert and RNA testing may need to be done to demonstrate the patients HCV status.

Negative   Negative

False Positive

Monitor and Repeat Test in 3-6Months if Clinically indicated

Positive Positive 

Positive for HCV at One Time But Has Cleared the Virus Naturally

HCV Antibody

Screen

Monitor Clinically and Repeat Test in 3-6 Months if clinically indicated 

Negative

Monitor Clinically As Needed

Perform RIBA

Report as Positive

Confirm

With RNA 

Actively infected with HCV

18

1.8 Allopathic Treatment

In 1989, the firstf successful treatment documentedfor HCV infection withinterferon alpha 2b

reported, but high ratesof high relapse necessitated treatment, which was not useful. A verity of

interferon have been used, but all have same results. Althoughthe combination therapy with

INF. alpha 2b andRibavirin has improved results, less thanhalf of who with anti HCV may be

expected to have a favorableresponse to the causative agents. The good response of these

therapies can be determine by biochemical response (normalization of ALT-levels), but now the

new method for the detection of viral RNA allows the diagnosis of a virological response(as

defined by a -ve result on a qualitative PCR assayfor HCV RNA). Some studies have also

assessed the histological-response, but in gn. practice there is some need for biopsy in follow-up.

Since results to therapy may not be sustained after treatmentis break, the success of clinical trials

has been evaluatedin terms of the response at the end of therapy (end of treatment outcome) and

6 months followup. Persons with a good sustained virological responsehave a high probability of

having a well virological, serological and histological response[41].

Described the current data on diagnosis, decisions regarding whom to treat, and the

recommended treatments of chronic hepatitis C infection. The acceptable guidelines are ; The

treatment of chronic hepatitis C is in constant flux. the consulting-physicians may deviate from

the strategy and remain within acceptable standards of treatment. Presented here is the current

state of the art for management and treatment of persons with chronic hepatitis C. Moreover,ther

is need the recommendationsshould be revised. There is highly active issue of clinical research in

said topic, and updates appears with increasing high frequency of HCV. [42,43].

19

The possible adverse effects of INF. are listed in table 2.

Table 2: Adverse Effects of Treatment with INF. alpha 2b and Ribavirin.

Fatigue 64% Headache 62%

Depression 31% Irritability 35%

Arthralgias 34% Anorexia 32%

Insomnia 40% Alopecia 36%

Myalgia 56% Injection site reaction 58%

Rigors 48% Nausea 43%

The mostly the optimal treatments cannot be easily searched. Then some other factors, like

gender, age, and viral load in the patient and the results of the liver biopsy, can be used to pilot

therapy The early and sustain virological response to INF. and Rib. therapy should be assessed

after 24th week , since elimination of the virus can occur late with this approach. Persons with a

+ve PCR assay for HCV RNA at week 24 should be considered to have no response to treatment

and therapy should be stopped. Same with HCV genotype 2 or 3 who have a -ve PCR for HCV

RNA can also stop therapy at this time,but an additional 24 weeks of treatment is suggested for

infected persons with different genotypes and -ve PCR . [44].

1.10 Pegylated Interferon

20

The attachment of polyethylene glycol to interferon alpha 2b (pegylatedinterferon alpha 2b)

extends the therapeutic activityand bioavailability of interferon alpha 2b. In contrast to interferon

alpha 2b, the individual dose calculated according to the body weight and pegylated interferon

alpha 2b is given only once a week. Treatment with pegylated interferon alpha 2b results in a

good response than dose conventionalmonotherapy with interferon alpha 2b. Large no. of clinical

trials are under way to evaluating combination of pegylated interferon alpha 2b and Ribavirin

and the results will detection the role of these ingrdients in the treatment with HCV.

Those patients who can’t be treated with Ribavirin can betreated with pegylated interferon alpha

2b. In case ofRibavirin induced anemia the choice of it is therapy for the 20 % of patients who

receive combinationtherapy. The optimal pegylated interferon alpha 2bregimen for different sub-

groups of patients has yet to be diagnosed.49% of patients who relapse after pegylated interferon

alpha 2b have a sustained virological response in combination therapy with INF.& Rib..For

patients who relapse after multi therapy there are not recommended for further treatments. A

prolonged-course of a higher dose of interferon or pegylated interferon alpha 2b alone is another

optionfor patients with contraindications to Ribavirin[45].

< 10 % of patients with no previous response to interferon have a sustained virological response

has no treatment currently available. Even with the use of combination therapythese patients

shouldbe treated in controlled clinical trials [46].

1.11 Herbal Treatment (The Role of Traditional Medicine in Chronic Liver Disease)

The Traditional Medicine are widely utilized for the treatment of hepatitis C. Systematic searches

conducted and cited in the literature citation as follows.

21

Twenty seven randomized clinical trials conducted involving herbal products and supplements.

In 14 of the trials, patients received interferon-α in combination with the Traditional Medicine.

Less than half the trials (11/27) were of good methodological quality. Compared with the control

group, significant improvements in virological and biochemical response seen in trials of

traditional Chinese medicine, and many herbal medicaments including Glycyrrhiza glabra and

oxymatrine [46].

(SNMC) Stronger Neominophagen C active component is glycyrrhizin (extract of licorice) has

been trialed to asses the improvement in liver disorder in Japan. To assess the effectiveness of

interferon (IFN), SNMC combination therapy in patients, who did not respond to IFN therapy

alone, scientist investigate twenty eight patients with histology of CAH- 2B at 12 weeks after

INF. administration. Fifteen patients received INF alone continuously (group A), and thirteen

patients received Interferon with SNMC (group B) for twelve weeks thereafter. Serum ALT level

observed in 33.3% of group A become normal and in 64.3% of group B. Serum HVC RNA was

13.3% in group A and 38.5% in group B has been cleared. In group-A histologically

improvement was not very significant, also in group- B by K. HAI score, but reversal of

histological grade according to Europe classification was noted more frequently in group B. In

this case the INF and SNMC. combination was useful in improving liver function. A case of post

transfusion HCV exacerbated by Interferon therapy is reported. HLA class I antigen was strongly

expressed in the liver tissue after administration of Interferon. [47].

The long-term i.v. glycyrrhizin treatment reportedly reduces the progression of liver disease to

Hepatocellular carcinoma and normalizes the ALT in chronic hepatitis C patients. In another 4

wk. the short-term study feasibility and efficacy on serum ALT of 3-6 times/week i.v.

glycyrrhizin therapy in European patients have been noted. Patients with chronic HCV, non

22

responders or unlikely to respond (genotype 1/cirrhosis) to INF treatment were also included in

this study. Medication was administered i.v. 3-6 times / week for four week; follow-up also

lasted four week. 69/72 treatment courses were completed according to protocol. In the placebo

group no any significant improvement in ALT levels within (n = 13)[48].

No major adverse effects have been observed. Glycyrrhizin treatment induces a significant ALT

decrease in patients with chronic HCV. 6 times per week treatment appears more effective than

three times per week.It appeared feasible to treat European patients with ch. HCV 3 or 6 times /

week with i.v. glycyrrhizin. The mean %age ALT decrease from baseline at the end of treatment

was 26% and 47% for the 3 times / week and 6 times / week treatment group, respectively (both

p< 0.001 vs. placebo). At the end of treatment, 10% (four of 41) and 20% (three of 15) of the

patients reached normal ALT levels for the 3 times / week and 6 times / week treatment group

respectively. At the end of treatment ALT lowering effect disappeared. [49].

Some specific herbal formulations of Ayurvedic medicine and herbal compounds have been

studied for the CLD, including Liv-52, 52-HD03, AO_8 natural formulation, and active alkloids

of E.alba and Ph.amarus. Liv52, comprises Cichorium intybus (wild chicory)Achillea

millefolium (yarrow) Capparis spinosa (capers), , and (Arjuna), Solanum nigrum (black

nightshade), Tamarix gallica, , and Terminalia Arjuna (tararisk). It was thought useful for

treating alcoholic liver disease, but a recent (RCT) Randomized control trial from Europe

showed a detrimental effect on the course of advanced alcohol-induced cirrhosis. 53AO-8 and

HD-03 are herbal formulations that may be useful as an antitoxin and antioxidant, respectively.

Perhaps the most promising agent for liver disease, principally chronic hepatitis. In another

study, Fifty nine patients treated with a extract of Phyllanthus for thirty days became hepatitis B

become -ve, as compared with 4% of placebo end. Screening studies of specific compounds

23

derived from Herbal and Ayurvedic medicines are being performed in an effort to development

of new drug [50].

Treatment of CLD in African traditional system many spices, common medicinal herbs and

dietary vegetables. The Garcinia-kola seeds , Fam. Guttiferae have antiviral and anti-

inflammatory effects. 89% of African rural areas uses traditional herbal medicines[51].

Aim and Objective

A project was made for establishing and evaluating the scientific clinical study of these herbal

formulations to prove their efficacy (Hepatoprotective), anti viral, anti fibrotic and immune

stimulator, increases the serum interferon scientifically. For the people who prefer natural

antiviral over chemical drug treatments, herbs are much plentiful.People rush like bees towards

doctors when a virus strikes. However, opposite to beliefs of the most of the people, most

traditional medicines are not useful against viruses. Yet there are some drugs which decrease the

severity of virus, mostly symptoms are treated and patient is advised to take rest. On the other

hand natural antiviral herbs are very effective against viruses.healing time is reduced and side

effects are practically unheard when antiviral herbs and antiviral essential oils are used, and the

associated symptoms that with the virus are relieved as well because most natural antiviral

treatments have multiple uses, we find Some of the best antiviral herbs include liquorice, green

tea astragalus, mullein, echinacea,schizandra, ,garlic, oregano, and elderberry, ,. The herbal

formulation Hepotin Tablets contains different plant ingredients; the botanical and medical

details are briefed as under. The plants used in the formulation for Hepatitis comprises of

Phylanthus emblica Tamarix gallica, Glycyrrhiza glabra, , Picrorhiza kurroa, and Silybum

marrianum have been utilized for the said purpose. Some of information collected in different

locations is being mentioned as under.

24

Specific objective

To see the clinical, biochemical, serological, and sustained virological response of herbal

formulation Hepotin in patients of chronic hepatitis C.

1. To compare the effect of Herbal verses Allopathic therapies on Platelets.

2. To relates the basic theme to compare the variable efficacy between two frequent groups and

evaluate the adverse effect of both drugs on ALT.

3. To compare the effect (Child plug score) and clinical complain of herbal drug and Interferon

therapy.

Background

The medicinal plants are increasing tremendously in the developing countries of the world. By

keeping this, we have tried to enlist the Indian medicinal plants which are highly useful in the

treatment of viral infections and associated conditions. Viral infections can be one of the biggest

nightmares for medical doctors. And the use of antiviral herbs have some specific benefits over

the use of drugs. Many herbal products are thought to be advantageousfor the liver and hepatitis

C patients who are not benefitted with conventional drug therapy, cannot bear its co-effects or

simply want to support their body's fight against the disease may use such products.

25

Chapter II

Ethno botanical/Medical Review of Test Drug

26

2.1 Picrorhiza kurroa Royle ex Benth

Ethno-Medical Information on Picrorhiza kurroa

Picrorhiza kurroa has been medicinally utilized as antibilious, cholagogue, digestive, hepato-

protective, also reported as antispasmodic and antioxidant [54].It is traditionally used for

hepatitis,and acute viral, infections[55], indigestion and constipation [56]. It acts as antioxidant

[57,58] and increases bile production in the liver [59]. It has glycosides like kutkoside and picroside-

I androsin and apocynin, who have the ability to inhibit antiallergic powers [60] and can be used

to treat asthma and joint inflammation [61]. Kutkoside and picroside-I have an immunostimulating

quality effect in hamsters that prevent infections [62]. PK is a good protector against several

potent liver toxins as compared to milk thistle [63,64]. It is a chemical induced liver cancer cell

inhibitor[65]. A series of cases were reportedly treated successfully by a combination of PK with a

variety of minerals [66,67].

Figure 6:Picrorhiza kurroa

27

Picrorhiza kurroaadministered in decoction with liquorices is regarded as a favorite remedy in

bilious disorder accompanied by fever and is raisins and Melia azadirachtaLinn. (Neem) bark

combined with aromatics given to alleviate dyspepsia and dysentery, loss of appetite, to improve

the peristaltic movement of the intestines and also improve liver function, often prescribed to get

rid of worms from alimentary canal. Powdered root in warm water sweet syrup is recommended

doses acts as gentle aperients. In dropsy and chronic persistent bilious fevers, the powdered root

alone or powdered root with neem bark proves effective anti periodic. In liver affection for

relieving symptoms of constipation. Use of single drug is considered clinically active for

improving total bilirubin levels to treat effectively infective hepatitis with jaundice and as

hydrocholeretic agent, also effective for sick liver cells.It is also recommended in hepatocelluler

carcinoma (HCC)[68].

In an animal study, the galactosamine-induced liver injury in rats, Poke at a dose of 200 mg/kg),

Picrorhiza kurroa (Pk) showed a significant reduction (p<0.05) in liver lipid content, SGOT and

SGPT (ALT).In a randomized, double-blind placebo controlled trial in patients diagnosed to

have acute viral hepatitis (HBsAg -ve), a root powder 375 mg of Picrorhiza kurroa (Pk)was

given three times a day for 2 weeks (n = 15) or was given a matching placebo (n = 18). The

Difference was significant between Pk and placebo groups the values of SGOT, SGPT and total

billuribin.. The total serum bilirubin was drop in required time to average value of 2.5 mg% in

placebo was 75.9 days versus 27.44 days in Pk group. The efficacy of Pk has shown a biological

plausibility by present study as supported by clinical trial for viral hepatitis, in animal study the

model of hepatoprotection and for standardizing extracts the approach based on picroside content

[69].

28

Arogya-wardhani with its major ingredient Pk (50%) was significantly shown to reduce

transaminases and serum bilirubintransaminases`in patients with viral hepatitis by some of the

current authors in the late seventies, in double-blind trial. At the same period, adouble-blind

placebo-controlled trial had showed Pk to be as effective as Arogya-wardhani. In 1981,the

Annual Conference of the Association of Physicians of India had presented the findings. In India

and some abroad more work was also carried out at various centres [70].

In adult male albino rats the hepatoprotective activity of picroliv, it was determined the irridoid

glycoside mixture from Picrorhiza kurroa. As evidence by many biochemical and

histopathological observations the pretreatment with picroliv had prevented the hepatotoxic

effects of galactosamine and paracetamol. With daily oral doses of 6 and 12 mg/kg maximum

hepatoprotective effect was noted for 7 or 8 days. Picroliv’s antihepatotoxic action of (active

constituents of pk) was seems likely due to an change in the biotransformation of the toxic

substances that result in less formation of reactive metabolites [71]. In another double-blind (RCT)

of thirty three people suffering from acute viral hepatitis, use of the Picrorhiza kurroa herbat a

dose of 375 mg three times daily was significantly increase the recovery time as compared to

placebo group[72]. P. kurroa (PK.) has also useful in the management of bronchial asthma, some

other formulation of PK. to protect the liver against toxins, potent antioxidant activity,

hepatoprotective properties,modulates liver enzyme levels, anti-allergy and anti-inflammatory

action action [73]. In India antiviral activity studied and dried rhizomeorallywith dose of 2.0

gm/day was used on adult humans on both sexes. Dried rhizome used in India has antiviral

activity, interferon alpha 2b and Ribavirin in vitro study on both groups with dose of 2.0 gm/day

[74].

29

Dosage

Between 400 to 1,500 mg of powder is recommended dose and twice per day 1-2 ml of fluid

extract.

Side Effects

Loose stools and colic have been reported when un-prepared picrorhiza rhizomes are used as

medicine.No other adverse effects have been reported with picrorhiza but during pregnancy and

breast feeding it is prohibited. However extracts in alcohol have shown much less tendency to

cause such effects.

Active Constituents

In 1993 the TLC method reported by Dwivedi uses densitometric evaluation of picroside-I and

kutkoside after visualization of spots with ceric sulphate spraying reagent (1% solution in

2 M sulphuric acid), that reduces the sensitivity and reproducibility of the method. At various

stages the derivatisation of the plate imparts variations like the amount and concentration of

spraying reagent, drying after derivatisation, immersion or spraying time, heating (time and

temperature) and waiting time before detection. Therefore, an exact aquracy and reproducible

TLC method in the present investigation an attempt has been made to develop without

derivatisation for the analysis of picroside-I and kutkoside in Picrhiza extract.

It has analyzed by the proposed method and results reported in Table-4, the samples of extraction

efficiency study. The results suggested that more than 95% of the picroside-I and kutkoside were

30

extracted in the first cycle of extraction only. There was no interference observed from the

glassware and filter paper used during the sample preparation.

Development of optimum mobile phase:

The mobile phase for TLC was optimized by testing different solvent compositions of varying

polarity. The selected mobile phase showed good resolution with Rf values 0.42 ± 0.03 and 0.61

± 0.03 for kutkoside and picroside-I respectively (Fig.2).\The best results obtained using ethyl

acetate: methanol: glacial acetic acid: formic acid (25:5:1:1, /v) as a mobile phase composition.

The scanning wavelength selected was 265nm, which is the absorption maximum for picroside-I

and kutkoside. The selected mobile phase showed good resolution with Rf values 0.42 ± 0.03 and

0.61 ± 0.03 for kutkoside and picroside-I respectively (Fig.2).

Analysis of kutkoside and picroside-I in Kutki extract

The content of picroside-I and kutkoside determined by the proposed method is reported in table

3

Table3: Comparison of Results Obtained by TLC and HPLC Method (n+3)

TLC HPLC

Name Content (%) %RSD Content (%) %RSD

Kutkoside 2.18 .93 2.26 0.79

Picroside-1 1.90 1.0 2.43 0.43

31

The proposed TLC method is simple, selective, accurate and rapid. The method avoids the use of

any spraying reagent, which improves the accuracy and sensitivity of the method. Method

validation proves that the method is precise and reproducible.The assay can be performed

without any special pretreatment of the sample to be analyzed. A large number of extract

samples can be analyzed at the same time without compromising accuracy. For the routine

analysis of commercial Kutki extracts the method can be used as a quality control tool [75].

32

2.2 Tamarix gallica Roxb.ex Roth

Tamarix (Jhao)

Introduction:

Twigs,Bark and galls are used in medicines generally as astringent, the manna use as detergent

and aperients. The bark is astringent, tonicand bitter. The twigs and leaves are regarded as

vulnerary,hepatoprotective (resolvent of hepatic and splenic inflammations), diuretic and

carminative.Ashes of this tree when it grow near/beside river banks, contains rich quantities of

sulphate of soda).

Figure 5:The general morphology of Tamarix.

Ethno-Medical Information on Tamarix gallica

Locally all the parts of plant act as sedative, desiccative and resolvent . It acts as aperients and

resolves the inflammations slowly. Tamarix wood is beneficial against splenic inflammations.

Therapeutic response of infectious hepatitis is found better when a preparation of Tamarix is

33

used in liver disorders, even jaundice.It also prescribed by traditional and modern system

practitioners simultaneously.

The use of Tamarix gallica (TG)presently for prevention and control of chronic liver diseases

(CLD) is in the focus of attention for both the doctor and the patients; the reasons for such shift

toward the use of traditional herbals include the expensive cost of conventional drugs, adverse

drug reactions, and their inefficacy [52]. Tamarix gallica, commonly known as Jhavuka in Hindi,

has been effectively used in traditional medicines as a diuretic, alaxative and in the treatment of

diarrhea and dysentery. This study was conducted to investigating the hepatoprotective role of

50% ethanolic extract of TG leaves against acetaminophen induced hepatocellular damage in

albino mice [53].

At Tehran Hepatic Center the efficacy of herbal medicine T.gallica with herbal extracts of C.

spinosa, Terminalia arjuna, Achillea millefolium) Cichorium intybus, and Solanum nigrum,on

liver cirrhosis outcomes compared with the placebo for six months in 36 cirrhotic patients. The

outcome measures included childpugh score (ascities Portal hypertention, ALT, AST, total

billirubin (T.Bil.), albumin (Alb.), prothrombin time (P.Time), platelet count and white blood

cells counts (TLC). The indices were recorded in all patients before and after 6 months of drug

and placebo treatment. The results demonstrate that the patients treated with TG. compound for 6

months had significantly better child-pugh score, Tamarix gallica can be attributed to the, anti-

inflammatory, immune-modulating, diuretic and anti-oxidative, properties of the component

herbs [54].

34

2.3 Glycyrrhiza glabraLinn.

Licorice (Common name Assal-ul-soos) contains the glycoside, glycyrrhizin which has a

structure and activity similar to the adrenal steroids. Licorice is too acts like the hormone,

ACTH, causing potassium depletion,sodium retention, and water retention [76].Indeed,

Hippocrates, Theophrastus, Pliney the Elder and Galen had cited the extract of the root of

Glycyrrhiza glabra for healing of ulcers and wounds and quenching thirst [77,78].The medicinal

use of Glycyrrhiza glabra L. root has been continued about 2000 years even during the time of

Hammurabi [77].A biological active constituent glycyrrhizic acid and its aglycone glycyrrhetinic

acid are found in the root extract[79]. It shows anti-bacterial, anti-inflammatory, antiviral, and

anti-arrhythmic activity[80]. It may cause hypertension, hypokalemia, myoglobinuria,lethargy,

hypertensive encephalopathy, mineralocorticoid effects, inhibition of the renninangiotensin

system, andparaparesis quadriplegia (body paralysis) [81].

Figure: 7Glycyrrhiza glabra

In whole Licorice the deglycyrrhizinated (DGL) form is most often used to avoid the

hypertensive side effects of the glycyrrhetinic acid. Licorice and DGL have a mild laxative effect

and can protect the intestinal lining by increasing the production of mucus, ulcers and thus

alleviating heart burn. In treatment for chronic hepatitis glycyrrhizin had been used for more than

35

20 years in Japan as Hepatoprotective. Recently, these short-term effects have been amplified by

a well-conducted retrospective study suggesting that long-term usage of glycyrrhizin prevents

development of hepatocellular carcinoma in CLD. In RCT the glycyrrhizin induced a significant

reduction of s.aminotransferase (ALT) and an improvement in liver histology (Biopsy) compared

to placebo group.Stronger Neominophagen C (SNMC) is a Japanese formulation that contains

0.2% glycyrrhizin, 0.1% cytokine, and 2% glycerin. SNMC is primarily acts as an anti-

inflammatory or cytoprotective drug. SNMC may prevent the development of hepatocellular

carcinoma (HCC) in patients with chronic hepatitis C. SNMC does not reduce mortality among

patients with cirrhosis or CLD. It improves mortality in patients with sub acute liver failure and

improves liver functions (LFT)in patients with sub acute hepatic failure, chronic active hepatitis

and cirrhosis with activity. [82,83].

In licoricethe quantity of the glycyrrhizin and its aglicone, glycyrrhetinic acid was determined by

HPLC [84], gas chromatography [85], TLC densitometry [86],and capillary electrophoresis [87].

The 18β-glycyrrhetinic acid is clearly observed at 10.3 >1 minutes in the chromatogram of

standard in HPLC analysis,which matches with licorice extract.The HPLC analysis was

completed using a C-18 reversed phase column (VP-ODS, (250×4.6 mm, 5 mm) and LC-10AD

pump with a 20 μl sample loop.Qualitatively (Qt) the TLC fingerprint had shown the Rf. of 18β-

glycyrrhetinic acid in both standard and extract samples was the same equal (Rf =0.12). In figure

3there is shown the HPLC chromatograms of standard and extract. The retention time of 18β-

glycyrrhetinic acid was 10.3 >1 min which is desired and economical. Quantitatively HPLC

analysis, the calibration function was determined by linear regression over the range 0.0078-1

mg/ml.The regression equation was Y=311.01X + 1.6184, where X = concentration of standard

samples (mg/ml), and the correlation factor = 0.9995 (figure8). The recoveries are 99.60 % -

36

102.81% with relative standard deviations between 0.01 %and 1.58%, which are in acceptable

ranges.The amount of 18β-glycyrrhetinic acid was determined in licorice extract and according

to the analysis it is 0.022% per 100mg. The outcome of studying the accuracy and precision are

shown in table 1 and 2. Also, the relative standard deviation(SD) of repeatability test is

acceptable (2.96%).

Figure 8:The TLC chromatogram of the licorice extract and 18β-glycyrrhetinic acid

standard.

The method is a accurate¸simple rapid, safe and economical for determination of 18 β-

glycyrrhetinic acid. In this method 2 safe solvents(e.g. Dioxin and THF) [88,89] used as mobile

stage and it is preferred over the methods using toxic or mix solvents system [90].

37

Quantitation of Glabridin

High performance liquid chromatography a reverse phase is developed using C-18 column with

acetonitril with water containing 02 % Ac-OH (70:30) as eluent for the quantitation of glabridin

in Glycyrrhiza glabra. Glabridin is detected by UV absorption at 280 nm after separation by the

chromatographic system. Good linearity was obtained in the working range of the concentration

(0.01–0.1 mg mL), with correlation coefficients 0.999. Limit of detection and limit of

quantitation were 0.0195 and 0.065 mg ml) [91].

In HPLC study, we developed rapid analytical method for quantification of glabridin in

Glycyrrhiza using reverse phase HPLC [93, 94].

38

2.4 Phylanthus emblica Linn

Ethno-Medical information on Phylanthus emblica

Traditionally the Phylanthus emblica (common name Amla) has been used to treat jaundice,

dysentery and diabetes. It has been used topically as a treatment for skin ulcers, and itchiness [95].

Current studies has focused on Phylanthus emblica potential for the treatment of hepatitis B.

Indeed, studies also suggest that it may suppress the growth and replication of the virus Modern

research with Phylanthus emblica focuses on its potential for fighting viruses, specifically the

hepatitis B virus, presented at the 3rd International Congress on Phytomedicine in Munich. [96].

Figure9: Phylanthus emblica

Medical assistance should always be sought in hepatitis infections.It also helping the body fight

hepatitis B, Phylanthus emblica(PE) also support the overall health of the liver. Phylanthus

emblica can decreases the amount of hepatitis B virus (Viral load) found in the blood stream, it

39

has not been reported to remove the entire virus and should not be considered a cure. Some other

studies have looked at the potential use of Phylanthus emblica in protecting against liver injuries,

as well as cancerous tumors of the liver [97].Attempts have been made to isolate Phylanthus

emblica possess antiviral activity. Traditional Herbal preparations that stronger the immune

system help the body fight off invading viruses that could other wise cause infection and are of

particular interest to the scientific community [98]. Promise for arabinoxylan, a compound derived

from Phylanthus emblica shown by current research [99]. Water extract of Phyllanthus amarus

studied in India used on human adult’s at the dose of 600.0mg. Activity studies on hepatitis B

virus M-RNA transcription and replication proved that it slow down its replication.In some vivo

study of water extract of dried roots studied in USA as a DNA polymerase inhibition and study

on hepatocellular carcinoma of hepatitis B patient conducted in Philadelphiaare also reported

[100].

HPLC analysis

“The EtOAc (Ethyl Acetate)extract of Emblica showed strong scavenging activity on NO (IC-50

= 39.27±1.27 μg/ml). The Nitric oxide sc. activity directed fractionation and isolation experiment

confirmed the presence of NO sc.active ingridients in the Et.OAc extract of Emblica. 5 alkloids

isolated from the active EtOAc extractwere Gallic acid, Furosin , Corilagin, Methyl gallate, and

Geraniin by column chromatography on Sephadex LH-20. By spectroscopic evidence their

structures were confirmed and by matching the data with the literature standard values. HPLC.

analysis of the alkloids was carried out with gradient mobile step and showed best separation of

tested compounds (Fig.2). Detection of purified alkloids was accomplished by matching

FAB.mass and NMR. spectral data. Gallic acid (1): amorphous powder; FAB. MS, m/z 171 [M +

H] +, and 193 [M + Na] +; 1H NMR (270 MHz, CD3COCD3) δ 7.18; 13C NMR (68 MHz,

40

CD3COCD3) δ 111, 123, 138, 146 and 169. Outcome for the NO radical scavenging activity and

the quantitiy of confirmd alkloids by HPLC from Emblica.The data are reported by proven

values literature values [101].Methyl Gallate is white amorphous powder [102].

Figure 10:Structural formula of compounds isolated from Emblica.

Corilagin (3) White amorphous powder; [103, 104].

Furosin (4) Yellow crystalline powder; [105,106].

The present paper showed the detection of alkloids were present in Emblica in clear quantities

and may be the radical scavenging principles of medicinal value [107]. NO or reactive nitrogen

species, formed during their reaction with O2 or with super-oxides, such as NO-2, N2O-4, N3O-

4, and NO-3 − are highly reactive. Testing of NO by the compounds was depend on

concentration. It was observed that all the compounds have significant NO scavenging activity.

These compounds (alkloids)are responsible to altering the functional and structural functions of

many components of cell. Incubation period of solutions of sodium nitro-prusside in (PBS) at 25

˚C for 2 hr. resulting in linear time dependent nitrite production, which was condenced by the

proven alkloids. The efficacy was in the order; Geraniin > Corilagin > Furosin > Gallic acid >

Methyl gallate. Reference antioxidant, Curcumin present only less activity among the Proven

alkloids. [108]The traditional herbal products have the properties to counteract the effect of NO

41

formation and in turn, may be of special interest in preventing the ill effects of excessive NO

generation in the human body. NO is also implicated as anti-inflammatory, anti cancer in

pathological stats[109]. Moreover, the sc. activity may also help to block the chain of reactions

initiated by excess generation of NO that are detrimental to the human health.

Antioxidative features and free radical binding activity of phenolic compounds found mainly due

to the occurrence of hydroxyl groups. Many reports from previous data have enumerate that the

phenolic ingriedients have many therapeutics effects, specipif for anti-oxidant activity.

Therefore, quantitatively analysis of the phenolics in Emblica and compared their (NO) radical

sc. function in vitro. Therefore it explain the frequent use of this medicinal plant in medicinal

preparations prescribed in a variety of pathological conditions.

Toxicity

There was no side effects have been reported even after prolonged use of this drug [110].

2.5 Silybum marianum L.Gaertn.

Milk thistle (scientific name Silybum marianum, common name Oont Kutara) is a plant from the

Asteraceae family. Milk thistle has been used in Europe as a treatment for liver disease (CLD)

and jaundice since the 16th century. In the USA, Silymarin is the most popular CAM product

taken by people with liver disease. Silymarin, the active extract of milk thistle, is believed to be

responsible for the herb's medicinal qualities. [111].

Silybum marianum has been used for centuries as an herbal medicine for the liver disorders

“excellent for carrying off bile”[112].It contains silymarin, which consists of the silybin,

silychristine, silydianin and flavanolignans. The concentration is high in the fruit portion of the

42

plant, leaves and seeds[113]. The seeds also contain trimethylglycine, essential fatty acids and

betaine, which make it hepatoprotective and anti-inflammatory[114].

Fig 11: Silybum marianum

The laboratory studies have suggested that the milk thistle benefit the liver bypromoting and

protecting the growth of liver cells, fighting oxidation, and inhibiting inflammation [72,115].

The HALT_C study define the above found that the silymarin useful for hepatitis C was

associated with fewer symptoms of CLD and pts got better quality of life, but there was no

change in virus ctivity. The researchers emphasize on retrospective study, not a RCT. Milk

thistle is well tolerate and has shown little side effects in RCT on patients with ch.HCV.

Milk thistle mainly used as an CAM to cure CLD because of its iron chelating, antioxidant,and

purported anti-inflammatory properties. Most of studies, clinical trials and in vivo studies prove

the effectiveness and anti fibrotic and anti-inflammatory, Hepatoprotective action of Silybum

marianum

37 patients with Ch.HCV and fibrosis stage II/ III and IV were randomized to 1 of 3 doses of

I.d.B- 1016 for tewelve weeks. S.ferritin, S.iron, total iron- binding- capacity and transferrin-

43

iron saturation were measured before and after four weeks . Wilcoxon signed rank tests were

used for evaluation of results. No significant decrease in s.ferritin from before and after of

treatment (mean, 244- vs. 215- mug/L, median, 178- vs. 148 mug/L, P=0.005) 78 percent of

subjects had a decrease in serum ferritin level. No response in s.iron or transferrin-iron. Meta-

analysis(MA.) in a model that included, age, gender, dose, HFE- genotype, H/O long term

alcohol use, and high baseline ferritin levels explain the stage III to IV fibrosis was

independently associated with decreased post-treatment serum ferritin level.

The treatment with silymarene associated with reduced body iron stores, especially among

patients with (CLD) or late fibrosis stage. [73-74, 116-117].

HPLC TEST (High Performance Liquid Chromatography)

HPLC is the abbreviation of the High Performance Liquid Chromatography. It is used to separate

the chemical constituents of the compound [118].Light scattering detection may analyzed on

288nm ultraviolet detection. Its analysis of non-chromophoric and chromophoric and compounds

alike as its response is independent of the spectral properties. With it analyse response more

convenient and accurately reflects the relative abundance of sample constituents. The ELSD_LT

is not a spectroscopic detector, but instead makes a light scattering measurement of analysed

ingredients (particles) after dried through evaporation. As per INA. method 70mg powdered

silybum. extract was placed in 100 ml experimental graded flask. Approx.

Seventy ml of methanol was added and the mixture was associated for thirty min. shaking. The

mixture was diluted to 100ml with methylalcohol and then filtered by syringe (0.45m;Nylon) and

placed into an auto sampler vial for injection.

Analytical Conditions (as per INA. Method)

Mobile Phase: A- 1.00% Formic Acid in 80/20 H2O / Methanol

44

B- 1.00% Formic Acid in 20/80 H2O / Methanol*

Gradient: (Time, %B) (0-15) (5-15) (20-45) (40-45) (41-15) (55-15)

Column: Shimadzu Premier C18, 5m, 150 x 4.6mm**

Injection Volume: 10L

Flow Rate: 1mL/min.

Column Temp: 40˚C

Detector Settings: Gain = 7; Temp = 80˚C; Press = 250/kPa

Formic acid is a volatile modifier and provides the necessary pH adjustment for selectivity.INA-

115.000 for phosphoric acid for pH. Settlement. The Shimadzu Premier C18, 5m, 150 x 4.6mm

column offers similar phase attributes and comparable selectivity.

Phosphoric acid giving no better results in ELS (Evaporative Light Scattering) detection as it is a

mineral origin and non-volatile. **INA 115.000 calls for either an YMC-Pack ODS-A, 5m, 150

x 4.6mm or a Phenomenex-Luna C18, 5m, 150 x 4.6mm, both hydro-phobically end-capped,

higher carbon load” .

45

Results

Figure 12:UV and ELSD Chromatogram Comparison ofSilybum marianum.

The ELSD chromatogram shows two peaks (X1 and X2) that are not found in UV at 288nm,

similar to Silybinin A and B, prove ELSD as a complementary detector for HPLC [119].

Isolation of Chemical Constituents

4 diastereo-isomeric flavor-nolignans, of silybin A and B [120] and iso_silybin A [121] and B [122]

from the seeds of milk thistle achieved for the first time using a preparative reversed phase

HPLC. In addition, 3 flavonolignans,that are silychristin [123] iso-silychristin [124] and silydianin

[125] and a flavonoid, taxifolin [126] have been isolated. Structures including absolute stereo-

chemistries of silybins and iso-silybins were confirmed using CD spectroscopy[127].

Milk Thistle by UV and ELSD-LT

UV 288nm 

ELSD-LT

Minutes

46

Mechanisms of Action

It exhibits several anti inflammatory effects including inhibition of leuko triene (LT) and prosta

glandin synthesis (PG) [128], Kupf. cell inhibition [129], mast cell(Monocytse) stabilization [130],

and inhibition of neutrophil migration[131]. In addition, silymarin has been shown to increase

hepatocyte protein synthesis. It is anti oxidant through inhibition of lipidperoxidation [132]. It

decreases the conversion of hepatic stellate cells into myofibroblasts, slowing or even reversing

fibrosis [136] to have immune-modulatory effects on the diseased liver [137]. It increases the liver

detoxification by inhibition of enhanced glucuronidation [133,134] and protection of glutathione

depletion [135].

Studies conducted in Austria have showed silymarin resulted in a normalization of serum liver

enzyme (ALT) and total bilirubin levels in patients with alcoholic liver disease (ALD), in

addition to improved histology of hepatocytes[141]. In patients with cirrhosis, long-term at least

3.4 year administration of silymarin at 420 mg / day resulted in a significant improvement in

survival as compared to the placebo end [142].

Silymarin is’t water soluble, making decoction effective therefore it is usually administered

orally in as encapsulated form because absorption of silymarin from the gastrointestinal

tract(GIT) is only moderate (23-47%), it is best administered as a standardized extract of 70-80

percent silymarin. Studies have shown silymarin to be effective in the treatment of both acute

and chronic HCV. Studies using various animal tumor models has shown that silymarin

possesses chemo- preventive effects against chemo-carcinogenesis as well as photo-

carcinogenesis[143]. In animals and humans, peak plasma levels are reached in 4-6 hours after an

oral dose. Silymarin is excreted primarily through the bile but some clearance is also achieved

through Renal path. The clearance half-life of silymarin is 6-8 hours [138,139].

47

In acute phase of viral hepatitis, silymarin shortened the treatment duration and lowered serum

bilirubin, AST and ALT. In patients with chronic hepatitis and CLD, 420 mg silymarin / day for

six months also yielded improved serum liver enzymes(ALT) [140].

Dosage/Toxicity

Silymarin is mild allergic and laxative effect on high dosage but non-toxic drug in therapeutic

dose. [144,145].

   

48

CHAPTER III

METHODOLOGY

49

METHODOLOGY

Hepatitis C is a worldwide disease, particularly common in tropical countries and places where

public living and sanitation are poor. Viral infection is caused by hepatitis C virus that usually

spread through blood transfusion, sexual contact and surgical instruments.

3.1Aim of the study

To study the effect of herbal treatment on human sufferer and treat the patient by effective and

cost effective herbal medicinal preparation. To study the effect of Interferon therapy and evaluate

side effect of both drugs.

3.2 Material and Method

The study was carried out on the patients of age 20 to 55 year for 3 year. The trial was conducted

on 160 patients irrespective of socioeconomic status at outpatient department of different centre.

The study was carried out from June 2007 to May 2010. The trials were executed in Taj Medical

complex (HamdardUniversityHospital), Matab Hamdard and Shifaul Mulk Memorial hospital for

Eastern Medicine. The participants who were sustaining the inclusion criteria were selected for

this research project. A patient questionnaire, which also served as a data sheet was documented

by the principal investigator with the subject at entry. The major ethical dilemma in such or trial

was to decide about using best known treatment. It was of phase 3 trials and referred as

Randomized controlled trial (RCT), in which there was a clear control group (Treated with

Interferon alpha 2b follow up and all other conditions were kept similar for these groups.

3.2 Objective

It relates to the use of Herbal drug coded as Hepotin Tablets, and Allopathic drug Interferon

alpha 2b, Ribavirin with the dosage and the time duration of the treatment. The basic theme to

50

compare the variable efficacy between two frequent groups and evaluate the adverse effect of

both drugs.

3.3Study Design

Randomized Control Trial

3.3.0 Case Series

Describe the group of patients with similar diagnosis that is hepatitis C.

3.3.1 Alternate Hypothesis: H1

The herbal formulation for the treatment of hepatitis C has better clinical, biochemical and

serological response than Interferon alpha 2b and Ribavirin.

3.3.2 Alternate Hypothesis: H2

The Interferon alpha 2b and Ribavirin for the treatment of hepatitis C has better clinical,

biochemical and serological response than herbal formulation.

3.3.3 Null hypothesis: H0

The herbal coded formulation Hepotin Tablets is of the same value as Interferon alpha 2b and

Ribavirin and there is no difference between these two medicines and is evenly effective for the

treatment of hepatitis C.

51

3.3.4 Point of error or level of significance: (α)

It stands for probability and expressed as the level of significance in the study. The smaller the p

value more significant the result and it means less is the chance of making error. The error is

deduced to occur if null hypothesis is being rejected but when the null is accepted then the type

of wrong decision is type 1 or error. The p value is the function of two factors.

• The magnitude of difference between two medications.

• The size of sample

• (α) = 0.05

• The statement p=0.05 means that there is less than 95% risk that Hepotin Tablets is miser

present active sample of the null hypothesis is true which would be reasonable evidence for

concluding that the null hypothesis is false.

3.3.5 Variable

To evaluate the hypothesis, statistical analysis was made by applying independent variable (IV)

to dependent variable (DV) and confounding variables.

The variable here are independent/ predictor variable: Hepotin Tablets and Interferon alpha 2b

and Ribavirin dependent / outcome variable symptoms of hepatitis C or level of improved after

the treatment.

CAUSEEFFECT

MEDIATING VARIABLE DEPENDENT VARIABLE

↑---------TREATMENT -------------↑

52

3.3.6 INDEPENDENT VARIABLE

Mediating variable: Hepatitis C mediating variable are those that we expect to affect some

variable and to be improved by other variables.

3.3.7 Bias

Bias is referred to as any systematic error that results in an incorrect estimation of the association

between the controlled and experimental group.

3.3.8 Blinding

To alleviate the possibility of experimental bias blinding used and in this experimental study

single blind study is used in which two investigators were involved in the collection of data.

The principal investigator performed assessment including identification (diagnosis) of hepatitis

C and reduction in symptoms of hepatitis C. An independent observer to verify if full improved

had occurred in addition clinical inspection of side effects. This observation is noted at the

maximum level of test and reliability and ultra was subjected to reliability assessment of the

observer.

53

3.3.8 WORK PLAN

Table:4 Work Plan

ACTIVITY OUT LINE DISCRIPTION

No. of drugs for

comparison

2 Test drugs Hepotin Tablets and

Interferon alpha 2band

Ribavirin

No. of patients 160 both sex

No. of test Blood Urea, Creatinine,

CBC, ESR, ALT, HCV

RNA by PCR Viral Load

As in Performa

Time period 6 months 6 month follow up

Total duration 3.5 years 1st June 2007 to 30thMay

2010

3.3.9 Normal and Numerical Data

The variable is divided into different categories like total number of patients, distribution of

individual signs and symptoms and levels of the improved of all tests and control and

experimental medicines. Here the patient’s sex and sign and symptoms are mediating variable

54

while the number of patients, age, temperature and blood pressure are regarded as numerical

variable.

3.3.10 Frequency Distribution

To make the data more manageable for evaluation, the description has been presented in tabular

form. It gives the frequency with which a particular volume appears in the data which can be

statistically calculated. A visual display of numerical values ranging from the lowest to the

highest showing the numbers of times (frequency) each values occurs has also been mentioned.

3.2.12 Confidence Interval

A confidence interval level identifies a range of values that include the true population value of a

particular characteristic at a specified probability level (usually 95%).

55

Table 5: Survey Formulation

Diseases Test Drug Formulation

(Hepotin Tablets) Control Drugs

Hepatitis C

Tamarix gallica, 500 mg

Picrorhiza kurroa, 500 mg

Silybum marianum, 250mg

Glycyrrhiza glabra, 250mg

Phylanthus emblica,250mg

Interferon alpha 2b (Uniferon).

Interferon alpha 2b 3MIU injections three

times a week for 6-12 months. Ribavirin

(Ribazole).

For patients of hepatitis C according to patient

body weight.

• Less than 55kg (Patient body weight)

Ribavirin 400mg bid.

• Between 55 and 75kg Ribavirin 500mg bid.

• Greater than 75kg Ribavirin 600mg bid.

Table 6: Descriptive Characteristics of Study Sample

56

3.3.14 Description Characteristic of Study Sample (160)

Variables Hepatitis C

Total

Total 160

Test Drug 80

Control Drug 80

Gender Male 33

Female 47

Age distribution for Test

Drug

< 25 8

26 – 43 48

> 40 24

Age distribution for

Control Drug

< 25 8

26 – 43 52

> 40 20

Dosage Test Drug 2 tablets twice a day 250 ml of water twice

a day (for 6 months).

Control Ribavirin (Ribazole)

For patients of hepatitis C according to

patient body weight.

• Less than 55kg (Patient body weight)

Ribavirin 400mg bid.

• Between 55 and 75kg Ribavirin 500mg

bid.

• Greater than 75kg Ribavirin 600mg bid.

3.3.15 Correlation Coefficient

57

Correlation Coefficient(CC) is a measure of the degree of relationship between two variables. A

correlation coefficient lies setmeea +1.0 (indicating a perfect+ve relationship) though (indicating

no relationship between two variables to – 1.0 (a perfect -ve relationship).

3.3.16 Inferential Statistics

Inferential statistics use logic and mathematical processes in order to test hypothesis relating to a

specific population based on data gathered from a sample of the population of interest.Statistics

that allow the P.Investigator to make inferences about whether relationships observed in a

sample are likely to occur in the maximum population from which required types of sample was

drawn.

3.3.17 Population

A well-defined group of patients with symptoms of liver disorder, biochemically and

serologically diagnosedHCV patients registered who fulfilled the inclusion criteria and willing to

receive the treatment.

3.3.18 Sample Selection

The selection of a subgroup (shows level of improved persons) from the population patients to

represent the entire population. It was a simple random sampling which gives an equal chance of

being selected in the sample random procedures employed to select a sample using a sampling

frame.

3.3.19Funding

All materials were supplied free of charge by the investigator. HamdardUniversity component

and funds provided by the research grant of the HamdardUniversity.

58

Dr.Rubina Ghani Karachi Lab provided laboratory investigation on very nominal charges.

Alamgir Welfare Trust provided the patients. All the institutions are gracefully acknowledged.

3.3.20 Description of Methodology

The study is a quasi experimental, randomized clinical trial. The study was carried out from

1stJune 2007 to 30thMay 2010. The trials were executed in Matab Hamdard,

ShifaulMulkMemorialHospital for Eastern Medicine and Taj medical complex. The Patients who

were sustaining the inclusion criteria were selected for this research project. A patient

questionnaire, which also served as a data sheet was documented by the principal investigator

with the subject at entry. This recorded information containing all information about the sex and

age of the subject, medical history, clinically, biochemically and serologically carried out

investigations.

3.3.21Statistical Analysis

All the data were collected at ShifaulMulkMemorialHospital for Eastern Medicine, Hamdard

University, Karachi. To find out results data so obtained were entered into different software like

SPSS14 (Pearson chi square, Bayesian analysis, Kaplan Meir test, Wilcoxon Renksum test and

Mall Whitney-U test) for Windows Vista version and Microsoft Office XP2007 and EPI info.

3.3.22 Clinical Diagnosis of Hepatitis C

Hepatitis C diagnosed by complete history and biochemical and hematological and serological

investigation with the criteria as shown in proforma.

59

3.3.24 Treatment

In this research study the patients selectively enrolled are those who were carefully diagnosed to

have chronic active hepatitis C as per rule through laboratory investigations. Clinical findings

usually consist of mild to moderate anemia, moderate leukocytosis with a shift to the left and

slightly abnormal liver tests. Serological testingmost commonly with liver profile and are +ve in

100% of patients such as Hepatitis C. The patients were divided into control and test groups.

Controlled group received treatment interferon alpha 2b and Ribavirin while the test group

Hepotin Tablets.

3.3.25 Criteria for Assessment of Therapeutic Evaluation

Completely Improved

Where there is a complete relief from all the clinically (signs/symptoms), biochemically and

serological within a period of 6 months with hepatitis C.

No Improvement

No noticeable response of the drugs is observed clinically, biochemically and serologically from

the above sufferings within a period of 12 weeks with hepatitis C.

3.4 Inclusion Criteria

• The cases suffering from only chronic hepatitis C were selected on the following lines.

• Patients between age group of 20 to 55 years.

• Patients having no obvious pathological findings on routine examination.

• Patients living in Karachi, Pakistan.

• All socio-economical classes including lower, middle and upper.

60

3.8.4 Exclusion Criteria

The cases suffering from Chronic Medical illness were excluded on the following lines. Patients

with concurrent physical illness for example uncontrolled hypertension and diabetes or other

complication of chronic hepatitis C like esophageal varises, ascities, bleeding disorders etc.

• Patients having hyper pyrexia (103o F or more).

• Patient having liver abscess. Patients with hepatic or renal impairment and cardiac disorder.

•

• 3.5 Tab

Assessm

Name Age Gender ID(National) Farter Name Address Date and Visi

CAss

Anorexia

Heart Burn

Epigastric P

Body ach

Indigestion

Burning Mi

Fever

Burning Pal

Other

Biochemi

ALT

Urea

Creatinine

Hb%

Platelets

SeroRe

HCBy PCR

Standard Dru

Test Drug

Placebo

Response of D

ble 7 :PRO

ment the effi

it No.

linical sessment

Pain

cturation

lm and Sole

ical Assessment

ological esponse

CV RNA

R Viral Load

ug

Drug

OFORMA

icacy of Herb

Baseline

Base

Side Effect If

Side Effect If

Side Effect If

Improved

bal coded druCh.Hep

2nd week

e Line

any

any

any

61

ug Hepotin tpatitis C

4th weak 8th

wee

After Treatm

Not Improved

tablets on AL

ID: Date Start of

Date End of T

Cell.

ek

16th we

ment 6 mo

Breakth

LT and Viral

f Therapy

Therapy

eek 20th week

onth Follow up

hrough

l load in

24th week

62

Table 8. DRUGS * SEX Cross Tabulation (Male and Female)

SEX TOTAL

Female Male

DRUGS

Count 47 33 80

Hepotin Tablets % within DRUGS 58% 42% 100.0%

Count 47 33 80

Interferon alpha 2b

and Ribavirin

% within DRUGS

58% 42% 100.0%

Total Count 94 66 160

% within DRUGS 58% 42% 100.0%

The data managed by calculation taking formulae for Range = Largest observation of patient’s

age – Smallest observation of patient’s age, according to the formula, largest observation of

patients age is 55 while smallest observation of patients' age is 20, therefore, the range calculated

was 35. To determine the class interval of patient’s age distribution the formula used as Class

Interval (C-I) = 1 + 3.3 Log (n) “n= total number of patients” after putting the values, class

interval was approximately 6 and to find out the class size the formula applied is C-S = Range /

Class Interval and the class size was approximately 6 as shown in calculation among 160 patients

of hepatitis C.

63

Calculations of Class Size and Class Interval for Distributions

Total number of Patients =160

Largest Observation of Patients' age

= 55

Smallest Observation of Patients' age

= 20

Range = 35

To Find Class Interval

= 1 + 3.3 Log (n)

Class Interval

= 1 + 3.3 Log

16

0

Class Interval

= 6

Class Size

= Range

C – I

Class Size

= 35

6

Class Size

= 6

64

A comparative study was conducted for herbal coded drug Hepotin Tablets with Interferon

alpha 2b and Ribavirin

The present study was undertaken to evaluate the therapeutic efficacy of these medicinal

preparations for hepatitis C.The therapeutic evaluation of these medicines were conducted on

160 patients clinically, biochemically and serologically diagnosed cases of hepatitis C at

ShifaulMulkMemorialHospital, for Eastern Medicine, Matab Hamdard. The patients were

registered from the general OPD and hospitalized to the clinical research ward of the hospital.

All the patients selected for the study were thoroughly observed and clinical history was

recorded in the prescribed Performa case sheet (Annexure I) this proforma designed on

Microsoft Access XP database program. The therapeutic evaluation of the drug was made on the

basis of improved subjective signs and symptoms, clinical observations and pathological

investigation at periodic interval during the course of treatment. In general the cases established

the supportive treatment in the form of complete rest and nutritious diet free from spices.

CLINICAL ANALYSIS

The hospitalized cases were given restricted diet and investigations were carried out before

treatment, during treatment and after complete therapy.

The age distribution of patients was classified in different class intervals ranging from 20 years

to 55 years. The age distribution of 160 patients recorded having 6 class intervals accordingly

viz. 20 – 25, 26 – 31, 32 – 37, 38 – 43, 44 – 49, and 50 - 55 as shown in Table 9 and Graph 1.

Between 20-25 years of age only 13 patients were enrolled. In the class interval between “26 –

31” only patients were registered, males were 9 and females 15. 30 Patients were registered

between age of “32 – 37” having 10 males and 16 females. Among the class interval from 38 –

43, 31 patients were recorded out of which 15 patients were male and 16 were female. Between

44 – 49 c

were fem

female. T

– 55 pat

female pa

also fema

Table 9.

Class – I

20 ---25

26 ---31

32 --- 37

38 --- 43

44 --- 49

50 --- 55

class interva

male. 34 pati

This specifie

ients were 2

atients the in

ale in the cla

Age distrib

Interval

Freq

uenc

y of

Pat

ient

s

als the total n

ients enrolle

ed that a maj

24 and 38 c

ncidence is m

ass interval o

bution of All

All Patients

13

24

30

31

28

34

160

Fig

Graph 1. Age Dis

All

numbers of p

d in the last

or occurrenc

correspondin

more among

of 50 - 55.

l Patients, M

s Frequ

3

9

10

15

13

16

66

ure 13:Gra

65

0

5

10

15

20

25

30

35

20 ---25

26 ---31

32 ---37

38 --43

1324

30 31

3

9

10

1

1015

16

Class Intervals

stribution

l Patients Frequency of

patients were

t class interv

ce of disease

ngly, while

g female in th

Male Patien

uency of Ma

ph 1. Age D

-- 44 ---49

50 ---55

28

34

15 1316

16 15

22

f Male Frequency of Fe

e 28, out of

val from 50

e in the class

considering

he class inte

ts and Fem

ale Freq

10

15

16

16

15

22

94

Distribution

emale

which 13 w

– 55 having

s intervals o

the ratio be

rval of 26 –

ale Patients

quency of Fe

were males an

g 16 male an

f 26 – 31 an

etween male

31 years ag

s

emale

nd 15

nd 22

nd 50

e and

e and

Signs an

The para

pain, bod

Figure14

The data

49 patien

patients

patients

patients w

The thera

and symp

the cours

nd Symptom

ameters taken

dy ache, indi

4, Graph 2.

a showed in

nts were reg

were registe

were record

were register

apeutic evalu

ptoms, clinic

se of treatme

Freq

uenc

yof

Patie

nts

ms

n in signs an

igestion, bur

Fig.13 regar

gistered with

ered with bo

ded with bur

red with bur

uation of the

cal observat

ent.

Figure 14

Freq

uenc

y of

Pat

ient

s

nd symptoms

rning mictur

rding signs

h heart burn,

ody-ache, ag

rning mictur

rning palm a

e drug was m

tions and pat

4: Graph 2

0

10

20

30

40

50

60

70 57

66

s for hepatiti

ration, fever

and symptom

, 45 patients

gain 53 pati

ration, 13 p

nd sole.

made on the

thological in

. Signs and

49 45

63

53

24

is C were an

and burning

ms, 57 patie

s were recor

ients were e

patients were

basis of imp

nvestigations

Symptoms

13

47

norexia, hear

g palm and s

ents presente

rded with ep

enrolled with

e showed w

proved in the

s at periodic

rt burn, epiga

sole as prese

ed with anor

pigastric pai

h indigestion

with fever an

e subjective

c intervals d

astric

ent in

rexia,

n, 63

n, 24

nd 47

signs

during

67

RESULT AND DISCUSSION

Hepatitis C is quite prevalent in both developing and developed countries. Therefore, the global

mortality and morbidity related to chronic hepatitis C poses a serious threat to public health

around the globe. In Pakistan, hepatitis C is taking its toll and that patients in Pakistan those

cannot afford are usually treated with traditional medicine and those who can afford are being

offered the conventional treatment. The drug of choice in conventional are anti HCV therapy

(Interferon alpha 2b + Ribavirin) and of the 25% that do receive such treatment, the sustained

virological response (SVR) rate is 60 - 70%. However the traditional regime to treat hepatitis C

is many and varied. But those who were treated with conventional therapy exhibited 43.06%

seropositivity in chronic liver diseases and cirrhosis combined, with 45.7% cases of chronic liver

disease and 37.7% cases in cirrhosis. The mortality pattern pertaining to chronic liver disease

(CLD) in Pakistanis determined to be around 120 million which is rather quite alarming and in

addition those that are infected with HCV and are at risk of developing liver cirrhosis and liver

cancer could add to the misery. Enough statistics have been cited in the literature to demonstrate

the enormous threat posed by hepatitis C. Hepatitis C shows rapidly genetic variation, evidence

of its frequent rates of mutation and rapid evolution. The disease transmit through contact or

secreation of infected person's (blood, semen or other body fluids, sexual contact), sharing drug

needles, living with someone having the disease and sharing tooth-brush or razor of infected-

person of HCV. Therefore it appears that majority of the population in Pakistan are treated with

traditional medicine but no concrete scientific clinical trial or evidence based study has been

reported to prove the efficacy and effectiveness of the same. Hence one of the objectives of

present study was to correlate the effect on viral load with the herbal coded treatment and also to

substantiate the claims of earlier preliminary study carried out in our laboratory (M.Phil. Thesis

68

Asif Iqbal 2005) for the treatment of hepatitis C. The end purpose of the present investigation is

also to compare the herbal medicine with allopathic medicine so to assess its ultimate effects for

its curative function.

Controlled studies include a group given a comparison treatment of placebo or another

therapeutic agent (authentic conventional medicine) or both if the comparison agent is an

established therapeutic agent recognized as effective and safe by standard parameters then the

study does not need a placebo arm. If however the comparison is not established then the

benchmark is insufficient and placebo arm is required. As such the constituents of dosage form

design of coded drug Hepotin Tablets as reported on the formulation comes under the broad

category of therapeutic agent therefore this study has been compared with standard therapy such

as Interferon alpha 2b and Ribavirin. The study conducted is described as follows.

4.1Statistical Analysis (Clinical Comparative Study)

A comparative study of Hepotin Tablets with Interferon alpha 2b and Ribavirin was conducted in

different multicentre setups. Multicentre trial was the only practical mean of accruing sufficient

subjects to satisfy. The trial objective within reasonable time frame. These clinical studies

enlisted 160 patients which were selected by manually randomization method. Basically it is the

drug development phase III on Hepotin Tablets, is to estimate the efficacy, large population of

patient (180) and clinical trial compared with standard treatment. Trial follow up is up short

duration. Type of clinical trial design is based on parallel group and intervention compare to

therapeutic agents singly blind study trials where in only the investigator was aware of such

treatment in subjective patient was receiving and participants were not aware of the treatment

assignment. The parallel group design randomized control trial with effective randomization

afforded, the best method eliminating the bias. This study trial was supervised by the ethical

69

committee of the HamdardUniversity. Safety of the subject in clinical trial based on hepatitis C

was derived from the earlier studies on hepatitis C. Scope of the trial was on the population that

was residing in the Karachi only. Multiple primary variables in the sign and symptoms were

quite sufficient to cover the range effect of the therapy.

Applying the designed format collected the data. Out of 160 patients treated for hepatitis C, 80

patients were treated with herbal coded drug Hepotin Tabletsand 80 patients treated with

Interferon alpha 2b and Ribavirin. The parameters taken in signs and symptoms for hepatitis C

were anorexia, heart burn, epigastric pain, body-ache, indigestion, burning micturation, fever and

burning palm and sole.

70

4.2 Clinical Response

Anorexia

Anorexia complain has been recorded on the herbal test drug Hepotin Tablets and control drug

Interferon alpha 2b and Ribavirin and the effects observed on the patients are shown in table 8

and graph 10. Patients presenting with complaint of anorexia exhibited improvement of this

condition in 75% of patients and 25% of patient did not improve with Hepotin Tablets. The

effects of Interferon alpha2b and Ribavirin in respect to anorexia only 9% of the patient

indicated complete improvement as compared 91% patients showed no improvement and in

addition 4% showed reverse effect. The overall effects of Hepotin Tablets were better than

Interferon alpha 2b and Ribavirin. This response of Hepotin Tablets is due to Tamarix gallica

and Glycyrrhiza glabra which is documented and proven drug for gastritis, these medicinal

substances are carminative, anti-inflammatory and hepatoprotective action. It is widely used by

practitioner of traditional medicine.Traditionally milk thistle fruits another content of Hepotin

Tablets have been used for disorders of the liver, spleen and gall bladder such as jaundice and

gall bladder colic. Milk thistle has also been used for indigestion; it is stated to possess

hepatoprotective, antioxidant and choleretic properties. Current interest has been focused on the

hepatoprotective activity of milk thistle and its use in the prophylaxis and treatment of liver

damage and disease [146-148].

For anorexia, after applying the test of significance chi-square significant difference between

these two drugs was found and chi-square test was deduced (Yates correct) 5.06 and p-value was

0.01 as shown in Table 10[149].

Table 1

(Anorexi

Num

ber

of P

atie

nts

Level of

Hepotin T

Interferon

and Riba

10,Compara

ia)

0%

20%

40%

60%

80%

100%

Improved

Tablets

n alpha 2b

avirin

H

ative data o

Figure 1

%

%

%

%

%

%

75%

Graph 3

Complet

Improve

75%

9%

Hepotin Tablets

of Hepotin

15: Graph 3

25%

Levels of

3: Improveme

In

e

d

Not

25%

91%

71

Tablets an

. Improvem

9%

Improvement

ent in Anore

nterferon alpha 2

Improved

%

%

nd Interfero

ment in Anor

91%

t

exia

2b and Ribvirin

p valu

0.01

on alpha2b

rexia

ue

b and Riba

avirin

72

Heart Burn

Heartburn is acommon complain in which there is burning sensation in the chest that can extend

to the neck, throat and face; it is worsened by bending or lying down , some times along with

burning eructation, it is the primary symptom of gastro esophageal reflux, which is the

movement of stomach acid into the esophagus. On rare occasions, it is due to gastritis, most

common complaint in chronic hepatitis C[150].Presented with the complaint of heart burn showed

that Hepotin Tablets was effective in 61% of the patient and not effective in 39%.Interferon

alpha 2b and Ribavirin was found completely effective in 5% of the patients and not improved in

95% and 3% patients recorded with reverse effect. By observing overall response rate of these

drugs Hepotin Tablets was more effective than Interferon alpha2b and Ribavirin it shows that

Interferon alpha 2b and Ribavirin is not effective as compare to herbal coded drug Hepotin

Tablets. After applying the test of significance chi-Square test there was found highly significant

difference between these two drugs when Chi-Square Test (Yates correct) 17.72 and p-value was

found to be 0.00, which is shown in Table 11, Graph 15. The German tradition meidicine was

found to be equivalent to cisapride and also significantly superior to metoclopramide at

decreasing the symptoms of the functional dyspepsia over a 4 week period.A 2004 meta-analysis

from 3 double-blind placebo controlled studies (PCT) found the multiple herbal extract Ibero-

gast to be significantly more effective than placebo (p value = 0.001) in functional dyspepsia

through the targeting of multiple gastric pathologies.

Table 11

(Heart B

Level o

Hepotin

Interfero

2band R

F

Num

ber

of P

atie

nts

1. Compara

Burn)

f Improved

n Tablets

on alpha

Ribavirin

Figure: 16Gr

ative data o

d Complet

7

raph4. Imp

0%

20%

40%

60%

80%

100%

7

Grap

Hepotin

of Hepotin

e Improved

71%

5%

rovement in

71%

29%

Leve

ph 3: Improv

n Tablets 

73

Tablets an

d Not Imp

2

9

nHeart Bur

els of Improve

vement in He

Interfero

nd Interfero

proved

29%

95%

n

5%

95%

ement

art Burn

on alpha 2b an

on alpha 2b

p value

0.00

nd Ribavirin 

b and Ribaavirin

74

Epigastric Pain

Result of the efficacy of the drugs Hepotin Tablets and Interferon alpha 2b and Ribavirin in

patients presenting with complaint of epigastric pain was recorded as follows. Epigastric pain

observed as completely improved in 84% of the patient and not improved 16% were noticeable.

Interferon alpha 2b and Ribavirin were effective in 29% of the patient and no effect in 71%

where 20% of patient exhibited the reverse effect. Overall comparative result of the data showed

that herbal coded formulation drug was Hepotin Tablets more efficacious and compliance rate is

much better than Interferon alpha 2band Ribavirin. Such type of studies showed the significant

response of herbs in functional dyspepsia, and abdominal pain. In another randomized control

trail the content of Hepotin show better response than famotidine[80, 81]. After applying the test of

significance Chi-Square there was highly significant difference between these two drugs Chi-

Square Test (Yates correct) 8.01 and p-value was found to be 0.00 as shown in Table 12,

Graph 5.

Table: 1

Ribaviri

Level o

Hepotin

Interfero

and Rib

Num

ber

of P

atie

nts

12. Compa

in (Epigastr

f Improved

n Tablets

on alpha 2b

bavirin

H

rative data

ric pain)

d Complet

84%

29%

Figure 17:

0%

20%

40%

60%

80%

100%

8

Graph 5. Im

Hepotin Table

a between

e Improved

Graph 5 Im

84%

16%

Level

mprovement

Complet

No Impr

ets  

75

Hepotin Ta

d No Impr

16%

71%

mprovement

29%

ls of Improvem

in Epigastric

te Improvement

ovement

Interfe

ablets and

roved

t in Epigastr

%

71%

ment

c Pain

ron alpha 2b 

Interferon

p value

0.00

ric pain

and Ribavirin

n alpha 2b

n 

and

76

Body-ache

The comparative data of the two drugs Hepotin Tablets and Interferon alpha 2b and Ribavirin on

patients presenting with complaint of body-ache represents that 89% of the patient treated by the

drug Hepotin Tablets showed complete Improved and 11% of the patients showed no response.

Interferon alpha 2b and Ribavirin were completely effective in 25% of the patients while no

effect was noticed in 75% of the patients where as 75% showed reverse effect. By comparing the

success rate of these drugs it was analyzed that Hepotin Tablets was better than Interferon Alpha

2b and Ribavirin in patients showing complain of body-ache. Various well-controlled clinical

trials that have independently confirmed the clinical efficacy and safety of the constituents that

have been used in Hepotin. Differentclinical studies that have shown the different effects of the

single plant extracts on molecular mechanisms that are discussed as the underlying manifestation

of symptoms. [151]After applying the test of significance Chi-Square there was found highly

significant difference between these two drugs Chi-Square Test (Yates correct) 13.33 and p-

value was found to be 0.00 as shown in Table 13, Graph 6.

Table 13

Ribaviri

Level o

Hepotin

Interfero

and Rib

Num

ber

of P

atie

nts

3. Compa

in (Body-ach

f Improved

n Tablets

on alpha 2b

bavirin

0%

20%

40%

60%

80%

100%

arative data

he)

d Complet

89%

25%

Figure: 1

%

%

%

%

%

%

89%

Graph

Hepot

a between

e Improved

8. Graph 6.

11%

Levels of

6. Improvem

Complet

No Impro

in Tablets

77

Hepotin T

d No Impr

11%

75%

. Improvem

25%

f Improvement

ment in Body

e Improvement

ovement

Interferon

Tablets and

roved

ment in Body

75%

y-Ache

alpha 2b and

Interferon

p value

0.00

y-ache

d Ribavirin

n alpha 2b

and

78

Indigestion Indigestion is a disease that occurs when stomach acids containing pepsin abnormally reflux

back to the esophagus, which causes irritation. If left untreated, it will take a very long time

before the esophagus completely heals. Worse, it will develop into ulcer and some form of

cancer. It is more common in patient with chronic hepatitis C.

Patients presented with the complaint of Indigestion showed that Hepotin Tablets was

completely effective in 81% and 19% of patients showed no Improved. Interferon alpha 2b and

Ribavirin were found effective in 38% of the patients and no Improved was found in 62% of the

patient and Reverse effect showed in 20% of the patient. By observing at overall response rate of

these drugs Hepotin Tablets was more effective than Interferon alpha 2b and Ribavirin, same

results also found in various randomized studies in Philadelphia[152]. After applying the test of

significance Chi-Square was found highly significant difference between these two drugs Chi-

Square Test (Yates correct) 8.01 and p-value was found to be 0.00 as shown in Table 14, Graph

7.

Num

ber

of P

atie

nts

Table 14

Ribaviri

Level of

Hepotin T

Interferon

and Riba

1

4. Comparat

in (Indigesti

Improved

Tablets

n alpha 2b

avirin

0%

20%

40%

60%

80%

100%

75

Graph 7

tive data be

ion)

Complet

75%

5%

Figure 19

%

25%

Levels o

7: Improvem

etween Hepo

e Improved

9: Graph 7.I

79

5%

of Improveme

ment in Indig

otin Tablets

d No Impr

25%

95%

Improveme

95%

ent

etion

s and Interf

roved

ent in Indige

feron alpha

P value

0.00

estion

2b and

80

Burning Micturation

By comparing the data collected regarding the effect of the herbal coded drug Hepotin Tablets

and Interferon alpha 2b and Ribavirin on the patients with complain of burning micturation. It

was observed that Hepotin Tablets was effective in 79% of patients and 21% of the patients was

in failure rate whereas in case of Interferon alpha 2b and Ribavirin 30% of patients were

recorded completely improved and 70% of the patient displayed in failure rate and 50% showed

the reverse effect. By comparing the success rate of Hepotin Tablets and Interferon alpha 2band

Ribavirin it was observed that Hepotin Tablets was much more effective than Interferon alpha 2b

and Ribavirin in patients with complain of burning micturation. In some other pilot studies it has

been proved the efficacy of the drug in the complain of burning micturation and urine detail

report of patients was also recorded before and after treatment and revealed either the complete

elimination of complain or improvement in the ph in most of the patients

[153]. After applying the test of significance Chi-Square was found highly significant difference

between these two drugs Chi-Square Test (Yates correct) 14.10 and p-value was found to be 0.00

as shown in Table 15, Graph 8

Table 1

Ribaviri

Level of

Hepotin T

Interferon

and Riba

1

2345678

910

Num

ber

of P

atie

nts

5. Compar

in (Burning

Improved

Tablets

n alpha 2b

avirin

Fig

0%10%

20%30%40%50%60%70%80%

90%00%

79

rative data

Micturatio

Complete

79%

30%

gure 20: Gra

9%

21%

Graph 8:

Hepotin Tab

between H

on)

Improved

aph 8. Impr

Levels of Im

: Improveme

blets 

81

Hepotin Ta

No Impro

21%

70%

rovement in

30%

7

mprovement

ent in Burnin

Interferon alp

ablets and

oved

n Burning M

70%

ng Micturatio

pha 2b and R

Interferon

p value

0.00

Micturation

on

ibavirin 

n alpha 2b

and

82

Fever

Result of the efficacy of the both groups drugs Hepotin Tablets and Interferon alpha 2b and

Ribavirin in patients with complaint of fever was recorded as under: Hepotin Tablets was

observed completely improved in 87% of the patient and no Improve in 13%. Interferon alpha 2b

and Ribavirin was effective in 13% of the patients and no effect in the patients were recorded in

87% of the patient wear as reverse effect appeared in 85% of the patients. By observing overall

respond rate of these drugs Hepotin Tablets was found more effective than Interferon alpha 2b

and Ribavirin. Picrorhiza kurroa is a well-known herb in the traditional system of medicine and

has been traditionally used to treat disorders of the liver and upper respiratory tract, reduce

fevers. In most of studies it has been monitored that the efficacy of these plant effective even in

pyrexia of unknown origin (PUO)[154]. After applying the test of significance Chi-Square test

there was fond highly significant difference between these two drugs Chi-Square Test (Yates

correct) 18.36 and p-value was found to be 0.00 as shown in Table 16, Graph 9.

Table 16

Ribaviri

Level o

Hepotin

Interfero

and Rib

Burning

By com

Ribavirin

observed

of patien

Num

ber

of P

atie

nts

6. Comparat

in (Fever)

f Improved

n Tablets

on alpha 2b

bavirin

g Palm and S

mparing the

n regarding t

d that in case

nts not respo

0%

20%

40%

60%

80%

100%

tive data be

d Complet

Improve

87%

13%

Figure

Sole

data of the

their effects

e of Hepotin

onded to the

%

%

%

%

%

%

87%

13

Graph 9:

Hepotin T

etween Hepo

ely

d

e: 21Graph

herbal coded

on the patie

Tablets 71%

treatment. I

%

Levels of I

: Improveme

Tablets 

83

otin Tablets

No Improv

13%

87%

9. Improve

d drug Hepo

ents with co

% of patients

In case Inter

13%

87%

mprovement

ent in Fever

Interferon 

s and Interf

ved P va

0.00

ment in Fev

otin Tablets

omplain of b

s recorded co

rferon alpha

alpha 2b and

feron alpha

alue

ver

and Interfer

burning palm

ompletely im

a 2b and Rib

d Ribavirin 

2b and

ron alpha 2b

m and sole, it

mproved and

bavirin 5% o

b and

t was

d 29%

of the

patients w

as revers

was foun

Chi-Squa

Square T

10.

Table17.

(Burning

Level o

Hepotin

Interfero

Ribaviri

 

Num

ber

of P

atie

nts

were observ

se effect app

nd better tha

are test ther

Test (Yates c

. Comparat

g Palm and

f Improved

n Tablets

on alpha 2b

in

Figu

ved as compl

peared in 5%

an Interferon

re was found

correct) 4.3 a

tive data b

Sole)

d

and

ure: 22 Grap

0%

20%

40%

60%

80%

100%

7

Graph

Hepotin

letely improv

%. By compa

n alpha 2b an

d highly sig

and p-value

between Hep

Improved

71%

5%

ph10.Impro

71%

29%

Levels

h 10: Improv

n Tablets 

84

ved, 95% pa

aring the suc

nd Ribavirin

gnificant dif

was found t

potin Table

d Not

29%

95%

ovement in B

5%

s of Improvem

vement in Bur

Interfero

atients were

ccess rate of

n. After app

fference betw

to be 0.01 as

ets Interfer

t Improved

%

%

Burning Pa

%

95%

ment

rning Palm a

on alpha 2b an

observed no

f both drugs

lying the tes

ween these

s shown in T

ron alpha2b

p value

0.01

alm and Sole

and Sole

nd Ribavirin 

ot effected w

s Hepotin Ta

st of signific

two drugs

Table 17, G

b and Riba

e

where

ablets

cance

Chi-

Graph

avirin

85

4.3 Biochemical Response

Comparative Study Biochemically

A comparative study of Hepotin Tablets with Interferon alpha 2b and Ribavirin was conducted in

different setups. This Biochemical study enlisted 160 patients at random applying the designed

format collected from the data. The number of female and male was 27 and 33 respectively.

Out of 160 patients treated for hepatitis C 80 patients were treated from herbal coded drug

Hepotin Tablets and 80 patients treated with Interferon alpha 2b and Ribavirin.

By the comparing the data regarding the effects of herbal coded drug Hepotin Tablets and

Interferon alpha 2b and Ribavirin observed on the patients. It was revealed that in case of

Hepotin Tablets 79% of patients recorded completely improved and 21% of patients observed no

improvement of the their complaint. In case of Interferon alpha 2b and Ribavirin 72% of the

patient observed complete improved, 28% patients observed the failure rate. By comparing it

was found Hepotin Tablets better than Interferon alpha 2b and Ribavirin. It has been proved the

same results biochemical improvement in patients contracted with hepatitis C in numerous

studies in India, Japan and China that have been already mentioned in the literature search and

discussion [155,156]. After applying the test of significance Chi-Square test there was found

significant difference between these two drugs with Chi-Square test p-value was found to be 0.02

as shown in Table 18, Graph 11.

Table 18

Ribaviri

Level o

Hepotin

Interfero

and Rib

Figur

Blood Ur

Out of 16

Ribavirin

treatment

Num

ber

of P

atie

nts

8. Comparat

in (ALT)

f Improved

n Tablets

on alpha 2b

bavirin

re 23,Graph

rea Nitroge

60 patients t

n no patient

t it has obser

0%10%20%30%40%50%60%70%80%

Graph 11

 

tive data be

d Comp

79%

72%

h 11. Compa

en

treated for h

have renal im

rved 16 pati

79%

. Comparat2b

         Hepotin

etween Hepo

plete Improv

arative data

Ribavirin

hepatitis C 8

mpairment b

ent showed m

%

21%

Levels o

tive data betand Ribavir

n Tablets         

86

otin Tablets

ved No I

21%

28%

a between H

therapy on

0 patients w

before treatm

mild increas

72

of Improvement

tween Heporin therapy

    Interfe

s and Interf

mproved

Hepotin and

(ALT)

were treated

ment accordi

sed BUN.

2%

28%

otin and Inte(ALT)

ron alpha 2b 

feron alpha

p valu

0.5

Interferon

with Interfe

ing to exclus

erferon alph

and Ribavirin

2b and

ue

alpha 2b an

ron alpha 2b

sion criteria,

ha

n 

nd

b and

after

87

By the comparing of the data of the herbal coded drug Hepotin Tablets and Interferon alpha 2b

and Ribavirin about their effects observed on the patients revealed that incase of Hepotin

Tablets 0% of patients recorded mild increase in blood urea nitrogen and 100% of patients

observed with no affect of the their BUN. In case of Interferon alpha 2b and Ribavirin 2% of the

patient observed mild increase in BUN, 98% patients observed that no significant changes found

in BUN [157]. By comparing the success rate of both drugs Hepotin Tablets and Interferon alpha

2b and Ribavirin. After applying the test of significance Chi-Square test there was found

significant difference between these two drugs with Chi-Square p-value was found to be 0.2 as

shown in Table 20, Graph 12.

Table 19. Comparative data between Hepotin Tablets and Interferon alpha 2b and

Ribavirin (BUN)

Level of Improved Affected Not affected p value

Hepotin Tablets 0% 100%

0.4 Interferon alpha 2b and

Ribavirin 1% 99%

24

Serum C

Out of 16

Ribavirin

treatment

the herba

observed

before an

observed

of Hepot

been prov

of signifi

with Chi-

Table 2

Ribaviri

Num

ber

of P

atie

nts

4: Graph 12

Creatinine

60 patients t

n no patient

t it has obse

al coded drug

d on the patie

nd after the

d increase cr

tin Tablets a

ved that the

ficance Chi-S

-Square test

0. Compar

in (Creatinin

0%

20%

40%

60%

80%

100%

Graph 12.

2.Comparat

treated for h

have renal im

rved 1% pat

g Hepotin T

ents revealed

treatment. I

eatinine, 99%

nd was foun

Hepotin dru

Square test t

p-value was

rative data

ne)

. Comparatalpha 2b a

H

tive data bet

Ribavirin

hepatitis C 80

mpairment b

tient showed

Tablets and

d that in cas

In case of I

% patients o

nd better than

ug is safe in

there was fo

s found to be

between H

100%

Levels of

tive data betand Ribavir

Hepotin Table

88

tween Hepo

ntherapy on

0 patients w

before treatm

d mild increa

Interferon a

se of Hepotin

nterferon alp

observed the

n Interferon

aspect of ren

ound signific

e 0.4 as show

Hepotin Ta

f Improvement

tween Heporin therapy

ts   Int

otin and Inte

n BUN

were treated

ment accordi

ased creatinin

alpha 2b and

n Tablets n

pha 2b and

e normal. By

alpha 2b an

nal toxicity

cant differen

wn in Table

ablets and

99%

otin and Inte(BUN)

erferon alpha

erferon alph

with Interfe

ing to exclus

ne. By comp

d Ribavirin a

o change in

Ribavirin 1

y comparing

nd Ribavirin

[158]. After

nce between

21, Graph

Interferon

erferon

a 2b and RibaFi

ha 2b and

eron alpha 2b

sion criteria,

paring the da

about their ef

serum creat

1% of the pa

g the success

. Therefore i

applying th

n these two d

13.

n alpha 2b

avirin igure

b and

after

ata of

ffects

tinine

atient

s rate

it has

he test

drugs

and

Level o

Hepotin

Interfero

Ribaviri

Figure 2

123456789

10

Num

ber

of P

atie

nts

f Improved

n Tablets

on alpha 2b

in

25: Graph 1

0%10%20%30%40%50%60%70%80%90%00%

Graph 13. Interfe

d

and

13 comparat

0%

10

Comparativeron alpha 2

Hepotin

Affected

Before trea

0%

01%

tive data be

Cr

00%

Levels of Imp

ve data betw2b and Riba

Creatinin

n Tablets 

89

atment

No

Af

10

99

etween Hepo

reatinine)

1%

provement

ween Hepotavirin therapne)

Interfero

ot Affected

fter Treatm

00%

9%

otin and Int

99%

tin Tablets apy (Serum

on alpha 2b an

ment

p

0

terferon 2bt

and

nd Ribavirin 

p value

0.4

therapy (Se

erum

90

4.4 Serological Response (Serum HCV RNA by PCR Viral Load)

A comparative study of Hepotin Tablets with Interferon alpha 2band Ribavirin was conducted in

different setups. This serological study enlisted 160 patients at random, applying the designed

format collected from data. The number of female and male was 27 and 33 respectively. 80

patients were treated from herbal coded drug Hepotin Tablets and 80 patients treated with a

medicine Interferon alpha 2b and Ribavirin.

By the comparing of the data of the herbal coded drug Hepotin Tablets and Interferon alpha 2b

and Ribavirin about their effects observed on the patients it was revealed that incase of Hepotin

Tablet 48% of patients recorded completely improved and 52% of patients observed no

improved of their complaint. In case of Interferon alpha 2b and Ribavirin 55% of the patient

observed complete improved, 45% patients observed the failure rate. By comparing the success

rate of both drugs Hepotin Tablets was found not better than Interferon alpha 2b and Ribavirin

in early virological response but the sustained response is better than Interferon therapy therefore

it needs more time and studies to evaluate the accurate picture. The active components of

Hepotin Tablets also have been proved highly efficacious in thousand of studies[85]. There is well

defined and established their antiviral and immune stimulant activity [159, 160]. After applying the

test of significance Chi-Square test there was founds no difference between these two drugs with

Chi-Square Test p-value was found to be 0.2 such as shown in Table 22.

Table 21

Ribaviri

Level o

Hepoti

Interfer

Ribavir

Figure:

Num

ber

of P

atie

nts

1. Comparat

in (SerumH

of Improved

n Tablets

ron alpha 2b

rin

26. Graph 1

0%

10%

20%

30%

40%

50%

60%

Graph 14

tive data be

CV RNA by

d Co

48

band

55

14. Compar

%

%

%

%

%

%

%

48%

. ComparatInterfero

Hep

etween Hepo

y PCR Vira

omplete Imp

%

%

rative data b

and

%52%

Levels o

tive data beton alpha 2b

potin Tablets 

91

otin Tablets

al Load)

proved

between He

d Ribavirin

55

of Improvement

tween Hapoand Ribavir

  Inter

s and Interf

No Impr

52%

45%

epotin Table

5%

45%

otin Tablet arin

rferon alpha 2

feron alpha

roved

ets and Inte

and

Complete Im

No Improvem

2b and Ribavi

2band

p value

0.24

rferon alph

mprovement

ment

rin 

ha 2b

92

Table 22: Summary

Hepotin Tablets Interferon alpha 2b and Ribavirin

Age-yr 40.5+12 40.5+12

Weight-kg 83.4+15.8 83.4+15.8

Sex-no. N=80

M=33 F=47

N=80

M=33 F=47

Clinical Study

Hepotin Tablets Interferon alpha 2b and Ribavirin

Before

treatment

After treatment

Before treatment

After treatment

P value

(A)* (NA)**

(A)* (NA)**

Anorexia (%) 57 13 75% 74 66 9% .005

Heart burn 49 11 72% 44 10 05% .005

Epigastric pain 45 13 86% 34 06 05% .02

Body- ache 63 17 71% 76 19 05% .01

Indigestion 53 05 75% 67 09 05% .00

Burning micturation 24 04 86% 43 06 05% .00

Fever 13 03 87% 22 20 05% .02

Burning palm and sole

49 11 71% 72 68 05% .01

93

Table 24: Biochemical Study

Hepotin Tablets

Before treatment After treatment

Interferon alpha 2b and Ribavirin

Before treatment After treatment

P value

(A)* (NA)**

%age (A)* (NA)**

%age

ALT 80(A)*

117 + 34

14(NA)**

54+ 22

82% 72

79+28

52(NA)**

67+19 40%

.06%

BUN 00 00 100% 01 01 100%

.05%

Creatinine 00 00 100% 00 02 -50% .5%

Hb% 09

11+3

02

12+2

100% 07

10+2

12

-68% .1%

Platelets 187000+23000 200000+21000 195000+20000 119000+18000 -50% .1%

Table 25: Serological Study

Hepotin Tablets Interferon alpha 2b and Ribavirin

Before treatment

(A)*

After treatment

(NA)**

Imp.

***

Before treatment

(A)*

After treatment

(NA)**

Imp

***

P valu

e

HCV RNA by PCR

(Viral load)

80 38

48%

80 42

55% 0.4 3730000+290000

IU/ml

65000+19000

IU/ml

3980000+

238000

23000 +11000

IU/ml

(A)*=Affected cases (NA) **=Not affected cases Imp***=Improved

94

4.5Discussion The World Health Organization has previously estimated that approximately 170 million people

throughout the world are infected with hepatitis C[161]. The detail description on the status of

hepatitis C in Pakistan and the world has already been focused and it appears that hepatitis C is

quite prevalent in Pakistan. The epidemiology with particular reference to the clinical feature in

hepatitis C and its complications has also been under lined. A review of literature both from bio

activity and ethno medical point of view on the various ingredients of herbal medicine are

designated as Hepotin Tablets with Interferon alpha2b and Ribavirin are also documented [162].

Experimental design include clinical diagnosis, laboratory investigation are given as illustration.

Materials and Methods uses in this study are given in quite detail. The patients treated with test

and control drugs and data so generated through statistical methods and computer programming

are presented here with in chronological order of events. The data was collected in the years

from 1stJune 2007 – 30thMay 2010 which completed the clinical trial protocol and there were

total 160 patients, the frequency of male patients were 33 (percentage of male 42%) while, 27

were of female patients (percentage of female 58%) were enrolled into the study as shown in

Table 2. The overall, 160 patients were subjected to coded Hepotin Tablets and Interferon alpha

2b and Ribavirin study trials. These 160 patients have been selected after the adjustment made

according to exclusion and inclusion decisive factor.

The test drug Hepotin Tablets comprise of Phyllanthus amarus, Picrorhiza kurroa, Glycyrrhiza

glabra, Silybummarianum and Tamarix gallica was compared with Interferon alpha 2b and

Ribavirin with the dosage and time duration for the efficacy and adverse effects. Criteria for the

assessment of therapeutic evaluation were monitored according to the inclusion and exclusion

parameters. Sign and symptoms recorded were anorexia, heart burn, epigastric pain, constipation,

body ache, indigestion, burning micturation, fever, burning palm and sole. Both the drug

95

manifestation from clinical perspective was analyzed in each category of signs and symptoms.

The comparative analysis of the test and control drug accordingly showed that in case of

anorexia Hepotin Tablets and Interferon alpha 2b and Ribavirin, the improvement ratio was 81%

and 50% respectively. Similarly in heart burn, epigastric pain, constipation, body ache,

indigestion burning micturation, fever, burning of palm and sole for the test drugs displayed

improvement between 88% to 90% (P=<0.02) and as control drug between 22% to 57%(P=>

0.2) respectively. The levels of significance in case of Hepotin Tablets were compared to

Interferon alpha 2b and Ribavirin. Thus null hypothesis was rejected clearly specifying that the

clinical efficacy is effective in test drug as compared to control drug. Furthermore biochemical

response analysis such as ALT complete improvement was observed in 95% patients and p

values calculated 0.02. There was a significant decrease in serum Alanine Transferase (ALT)

from baseline to end of treatment (Hepotin Tablets BT* 117+34. AT* 54+22 Interferon alpha 2b

+ Ribavirin, BT*79+28, AT* 67+19L; P=0.02); 78% of subjects had a decrease in serum ALT

level. There was no significant change in serum ALT in Interferon cases.

In addition serological response (RNA) in case of Hepotin Tablets was 48% as compared to

Interferon alpha 2b and Ribavirin which was 55% and p value was found to be 0.4. Overall the

efficacy of Hepotin Tablets versus the Interferon alpha 2b and Ribavirin were assessed in the

overall effects to bring about curative effects for the treatment of hepatitis C. The antiviral

effectiveness of Hepotin Tablets or serological response (HCV RNA by PCR Viral load) in case

of Hepotin Tablets BT* 780000+290000 AT*65000+19000Iu/ml, IU/ml was 48% as compared

to Interferon alpha 2b and Ribavirin which BT*3980000+238000, AT*23000 +11000 IU/ml was

55% and p value was found to be (P= 0.4) The cumulative data revealed clinical, biochemical

and Serological (HCV RNA by PCR Viral Load) improvements with significant symptomatic

96

control. In addition, there was highly significant reduction in the mean recovery period. There

were no reported or observed significant adverse events in all cases of Hepotin Tablets and the

overall drug compliance was excellent. Therefore it may be concluded that Hepotin Tablets are

effective and safe in the management of hepatitis C.

The modern medicine consultans have been using the traditional medicine that has change the

traditional context [163].In the most different Hospitals (Multicentre) RCT, the peg.INF with

Ribavirin of high bio-avalibility has sustained virological response (the loss of the HCV-RNA,

after completion of treatment at least 6 months) in 56% of patients after completion of treatment.

A sustained virological response that is asumed to be a highly significant end point. The

naturopathic approach would be to strengthen and healing of the liverfor managing hepatitis C.

In Chinese system of medicine in context of the Yang and Yin practitioners use herbs for

treatment . In future clinical trials will be aimed to analyzing other kind of treatment must have

these pharmacological laments, it is nothing more than pharmacologic studies of a drug but the

drug derived from a plant. Interferon alpha 2b, in particular cases depression, fatigue, and

irritability that may lead to dose limitation. Also Americans in the context of prayer and

ceremony of Ayurvedic practitioners in the context of the 4 - Vedas. Thus, in Traditional herbal

system of medicine simply prescribing pills is common. Very good and high rate of compliance

are observed that may be associated to the symptoms based response. In such studies, the end

point of beneficial response to CAM would be measures of side effects, symptoms and total dose

of conventional therapy tolerated. It is studied that some CAM therapies showed the biological

effects that should include anti-oxidant, immune modulator anti- fibrotic activity that may help to

amelio rate the disease. [164].

97

The therapeutic benefit believed to be various herbal products and mixtures. There has been

found that various herbal products considered helpful in treating liver diseases and as mention of

potential interest of product derived from diverse geographic plants that are used in local

practices and ext. of S.marianum use throughout the Europe, North America and Asia.Since 16th

centurythe Silymarin has been used in Europe and it continues for the treatment of liver

disease[165].It has been evaluated from experimental data that the silymarin acts as free-radical

scavenger and an antioxidant and it prevents glutathionine depletion and also free-radical

formation. The patients that are treated with silymarin and the vitamin placebo (77% vs 67% at

twoyears and 58% vs 39% at four years; P _ .036), silymarin had no measured significant side

effects. At the end the results of this trial was a major impactin Europe to widespread use of

silymarin for all forms of liver disease [166].

Glycyrrhizin that is an aqueous extract of Glycyrrhiza glabra, that is endogenous to European

and western Asia.It has been found that licorice root (licorice radix), in traditional medicine for

the treatment of cough, liver inflammation, bronchitis and gastritis [167]. It has also antioxidant

activity that inducing glutathionine-smc-transferase and catalase activity and by decreasing the

rate of formation of polymorph nuclear cell oxidative product. Glycyrrhizin treatment controls

the ALT elevations and suppresses fibrosis in animals, possibly by blocking the activation and

action of the nuclear factor B in result to injury signals [168]. Japanese study has illustrated that

S.N.M.C in hepatitis C, the development of cirrhosis after about 15 years in 21% of patients after

treatment that is compared with 37% of untreated controls and whereas hepatocellular carcinoma

arose in 25% of controls and only 12% of treated persons, this trial was not proven that

glycyrrhizin is a main ingridient of many herbal medications including TJ_9, and herbal

formulation 861[169]. In a study of 37 hepatitis C patients, Gomisin A had reduced the HCV RNA

98

levels in 21% of the patients.51 accordingly; TJ108 is now being evaluated in Japan as a

complement to the Western treatments [170].

There are some herbal formulations and also their extracts trial for the CLD that are including

Liv52, AO8 and 52 HD03 herbal formulations and the extracts of P. amarus and E. alba.Liv52

are used in India as an well known hepato-protective agent, it is a tradiotional medicine that has

comprises of C. spinosa (capers), Terminalia arjuna (arjuna),Solanum nigrum,Cichorium intybus

(wild chicory),Achillea millefolium and also Tamarix gallica (tararisk). 53AO_8 and HD_03 are

the herbal medicine that may be useful an anti-oxidant and the anti-toxin respectively [171]. The

seeds of Garcinia kola (Fam. Guttiferae) have been shown the antiinflammatory antiviral

properties [172]. All these above mention studies indicate the more or less results as has been

described in Hepotin Tablets but the detail study on active principle of the ingredients of Hepotin

Tablets and broad spectrum multicentre randomized clinical trials are still needed to confirm its

antiviral, anti-inflammatory and anti fibrotic activity. The new therapies is generally need to

randomized controlled trials (RCTs) that evaluate the new treatment with standard therapy.

99

4.6 Conclusion

The cumulative data revealed clinical, biochemical and serological (HCV RNA by PCR viral

load) improvements with significant symptomatic control. In addition there was highly

significant reduction in the mean recovery period. There were no significant adverse events in all

cases as has been reported or observed and overall drug compliance was excellent. Therefore, it

may be concluded that Hepotin Tablets is effective and safe in the management of hepatitis C.

100

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