FIELD OF THE INVENTION
The present invention relates to a novel herbal composition, a process for the preparation of the composition, a method of treating acute viral Hepatitis and therapeutic effects of Hepatitis B virus infection and its use as a hepatoprotective agent. The present novel herbal composition is derived essentially from four plants namely (1) Phyllanthns amarus (2) Eclipta alba (3) Andrographis paniculata (4) Picrorhiza kurroa. In addition, the present invention provides a novel antiviral and hepatoprotective composition comprising essentially extracts from plants namely (1) Phyllanthus amarus (2) Eclipta alba (3) Andrographis paniculata (4) Picrorhiza kurroa and (5) Rheum emodi.
The present novel composition is the safe and effective herbal pharmaceutical preparation for the treatment of acute and chronic Hepatitis, Hepatitis B virus (Hepadna viridae) infection, and also as a hepatoprotective agent as well as an agent for improving the cell line functions.
BACKGROUND OF THE INVENTION
The Ayurvedic system of Indian medicine provides many formulations for treating many disorders / diseases in human beings and animals. A few of the plants have already been investigated for the beneficial medicinal properties.
Hepatitis B virus (HBV): Man is the natural source and reservoir of Hepatitis B virus infection. Hepatitis B virus is an Hepadna virus with a size of 42 run. Morphologically, the virus is complex, double shelled with surface and core antigen comprising the outer and inner with surface and core antigen stranded
DNA with (+) and (-) strands with 3200 bp and overlapping gene sequences. There are four open reading frame on (-) strands. S = HBsAg. C = HBcAg,P=DNA-P,X-Unknown. HBV needs reverse transcriptase for its replication. The virus is difficult to grow and is non cytopathic and is inactivated at boiling temperatures. The source of infection is HBV infected blood or transmission by sexual route from infected mother to newborn (perinatal). All age groups and both sexes are susceptible. Endemicity exists and high risk groups include blood recepients, hamophilics, dialysis patients, intravenous drug users, heterosexuals, health care workers and children born to HBV infected mothers. The incubation period of acute HBV hepatitis is 4-18 weeks. The first marker appearing in blood is HBsAg followed by a high risk in transaminase levels. HBV infection may lead to both asymptomatic and severe form of liver diseases. Around 5-10% of acute HBV infected population become chronic carriers. Fulminant hepatic failure occurs in 1-3% of acute cases and 10% of acute infection go to subacute hepatic failure leading to cirrhosis. HBV carrier stage is common if exposure occurs in earlychildhood. Most of the infections are acquired in early child hood, usually as mother to child transmission and results in a persistent HBV carrier stage. HBV carriers have 200 times more risk of liver cancer.
Man is the natural host and reservoir of the infection. So far, no effective drug therapy against HBV has been available. Hepatitis B virus is endemic in some population and hyperendemic in many parts of world. The only way of gradually eradicating the infection is by (1) active universal immunisation in childhood and/or (2) development of treatment modalities for persistently infected persons.. Therapeutic studies conducted over the past 15-20 years had only marginal success once the infection is established. Several compounds, such as intreferon, adenine arabinoside, acyclovir, ganciclovir, zidovudine and immunomodulatory regime
administered alone or in combination have been evaluated. Most of these agents have transient effects on viremia, with rebound occurrence on withdrawal of drug. Significant toxicity have been observed with long term therapy. Though, HBV was discovered to be the causative agent of Hepatitis B almost 30 years ago. however, a detail understanding of the biology of this clinically important virus has only developed in the last ten years. Among the problems faced by the early researchers were limited host range, and lack of tissue culture system in which to propagate the virus. The advent of molecular cloning techniques and the discovery of HBV like viruses in certain animals lead to rapid advances in late -1970s. Soon after the structure and sequence of HBV-DNA was determined, HBV like viruses in animals other than man were discovered. HBV and similar DNA viruses which differ from known class of animal DNA viruses were collectively grouped under Hepadna viridae(Gust. et al. 1986). Among the features which define the family are unique viron ultrastructure, polypeptide and antigenic composition, common genome size, similar structure and replicating mechanism in hepatocyte and extrahepatic tissue. The first animal hepadna virus was isolated from Woodchuck by Summers and coworkers in 1978. This followed an earlier observation by-Robert Snyder that a high proportion of Woodchucks which died at Philadelphia Zoo had liver tumours. Viral particles isolated from woodchuck sera exhibited a similar size and morphology to those described for HBV. The woodchuck virus particles were shown to be serologically related to HBV particles, and also contain an endogenous polymerase activity and a genome of similar size and structure to HBV. A similar high incidence of liver tumour in domestic ducks in China lead to identify a second animal Hepadna virus - Duck Hepatitis B Virus. Attempts to transmit the duck hepatitis B virus (DHBV) revealed unexpected results that approximately 10% of domestic ducks in the United States were congenitally infected with \irus(summers 1987). At about the same time, a third hepadna virus was identified in beechy ground squirrel (GSHV) in California(Mono« et al.
1980). Hepadna virus have also been reported in tree sqwrre\s(Mehrotra et al. 1990, Feitelson et al. 1990). Duck hepatitis B virus (DHBV) infection is accompanied by the presence of large number of viral specific particles in the blood The basic structure of DNA genome of Hepadna viridae is conserved in DHBV(Muo« et al. 1980). The average transmission occurs through egg from a carrier mother. In avians the host immune system develops 3 to 5 days post hatch and therefore 24 hours old ducklings are ideal candidates for transmission studies. If the DHBV infection occurs just after egg hatching the animal become persistently viremic. It is therefore, possible using this experimental model of HBV to evaluate any compound/preparation for its effects on carrier state. DHBV has been used as a model for the evaluation of antiviral chemotherapies against human hepatitis B virus infection(Zuckerman 1987) and also the natural plant products(Mzw.s/M et al. 1993, 1994). Several antiviral agents have been evaluated in chronically infected ducks(Hirota et al. 1987). DHBV infection has been associated with a broad spectrum of liver pathology e.g. chronic hepatitis, cirrhosis and hepatocellular carcinoma(Mehrotra et al. 1987, Duflot et al. 1995).
SUMMARY OF INVENTION
The invention provides a novel herbal composition which is useful in the treatment of hepatitis, the said composition consists essentially of extracts from plants namely Phyllanthus amarus, Eclipta alba, Andrographis paniculata and Picrorhiza kurroa.
HEPATOPROTECTION
Liver is constantly exposed to abuse by several hepatotoxic agents. These hepatotoxic agents have been used to study the hepatoprotective effects of herbs in the animal models. A few of the most important animal models are
i) Galactosamine acute Hepatotoxicity model, ii) Thioacetamide acute hepatotoxicity model, iii) Alfatoxin B 1 acute hepatotoxicity model and iv) Rifampicin & Isoniazid acute hepatotoxicity model.
The invention also provides a novel hepatoprotective and anti viral composition comprising extracts of Phyllanthus amarus, Eclipta alba, Andrographis paniculata, Picrorhiza kurroa and Rheum emodi used for the treatment of acute or chronic viral hepatitis (Hepatitis B virus infection).
The invention further provides a novel hepatoprotective and anti Hepatitis B virus composition optionally includes one or more extracts of plants selected from Fumaria ojficinalis, Tinospora cordifolia, Terminalia chebula, Cichorium intybus Tephrosea purpurea and Boerhaavia diffusa.
Furthermore, the invention relates to a novel herbal composition which is useful in the treatment of acute viral hepatitis infection, the composition consists essentially of aqueous extractable components of Phyllanthus amarus, Eclipta alba, Andrographis paniculata, Picrorhiza kurroa and /or Rheum emodi.
The invention also provides a novel hepatoprotective and anti viral composition comprising extracts of Phyllanthus amarus, Eclipta alba, Andrographis paniculata, Picrorhiza kurroa and Rheum emodi. As used herein the expression
water extractable components of above plants includes extracts prepared from whole plants or parts thereof.
The invention also relates to a method of treating patients suffering from hepatitis B virus infection, which comprises administering to the patients the present novel herbal pharmaceutical preparation.
Further, the invention relates to a pharmaceutical preparation which produces effects on animal model of Hepatitis B virus persistent carrier state: Duck hepatitis B virus (Hepadna viridae).
The invention also relates a pharmaceutical preparation which provides protection from the acute hepatotoxic effects of hepatotoxines such as paracetamol, carbontetrachloride, oral contraceptives, Galactosamine, Thioacetamide, Aflatoxin B 1, Rifampicin and Isoniazid to the liver.
Further, the invention provides a novel composition useful in the clinical treatment of acute viral hepatitis, which comprise of fractions of Phyllantus amarus. Eclipta alba, Andrographis paniculata, Picrorhiza kurroa and/or Rheum emodi with optional extracts of plants such as Fumaria officinalis, Tinospora cordifolia, Terminalia chebula, Cichorium intybus, Tephrosea purpurea, and Boerhaavia diffusa, said fractions contain aqueous extractable components of the above plants or parts thereof.
Furthermore, the invention also relates to a novel herbal composition having an effect on the complete clearance/intermittent effects on the viremia of HBV animal model i.e. duck hepatitis B virus (DHBV). It does not produce any toxicity to
systemic parenchymatous organs of the body e.g. Heart, Kidney, lung, pancreas and Gastrointestinal tract.
The invention also provides a novel herbal composition which is useful in the hepatoprotection in the acute hepatotoxicity produced due to well known hepatotoxic agents e.g. Galactosamine, Thioacetamide. Aflatoxin Bl, Rifampicin and Isoniazid as demonstrated in experimental studies.
Moreover, the invention also relates to a process for the preparation of a novel herbal composition by mixing in any known manner the extracts of Phyllamhus amarus, Eclipta alba, Andrographis paniculata, Picrorhiza kurroa and/or Rheum emodi. In addition, the process includes adding optional ingredients such as extracts of plants namely Fumaria officinalis, Tinospora cordifolia, Terminalia chebula, cichorium intybus Tephrosea purpurea and Boerhaavia diffusa.
DETAILED DESCRIPTION OF THE INVENTION
The invention describes a novel herbal composition and a process for the preparation of aqueous extract of plants and its usefulness at least in (i) treatment of acute viral hepatitis, (ii) therapeutic effects on Hepatitis B virus infection (Hepadna viridae) and (iii) as a hepatoprotective agent against wide range of chemicals/drugs used in animals such as human beings.
Pharmaceutical preparation of plant extracts
The plant extracts which are mixed together to obtain the present novel composition can be obtained by any known manner. All batches of plants used in the preparation the present novel composition were botanically authenticated. The
solvent used in the extraction of the plants may be any suitable solvent such as ethanol, methanol, chloroform and water.
Preferably, the plants extract can be obtained by any known manner. Most preferably, the whole plants and parts thereof were powdered and extracted with water. The extract was evaporated under reduced pressure below 50°C leaving a residue. For human administration, the residue is mixed with pharmaceutically acceptable neutral excepients and converted into suitable oral dosage form. As regards the animal studies, the residue was mixed with water, kept overnight at 37°C with stirring and extract was centrifuged at 10,000 rpm. The solution thus obtained was vacuum dried and dry weight of extract of each plant was determined This dry extract was dissolved in a fixed volume of normal saline to make a stock solution and stored at 4°C. The stock solution of each plant extract was used to prepare the present novel composition.
Alternatively, the method of preparing the novel compositions comprising blending and extracting in any known ratio of all the plants or plant materials together by any known manner.
The hepatoprotective herbs used for the formulation of the
present composition contains the extracts, preferably aqueous extracts of the
following:
Common Name Botanical Name Range of Extract in mg
per dose
Bhumyama Laki PhyUanthus amarus 25 - 250
Bringrqj Echpta alba 25- 250
Kalmegh . Andrographis paniculata 25-250
Kutki Picrorhiza kurroa 25- 250
What is unexpected in the present invention is that earlier, the above four plant extracts were combined in the range of 10 to 20 mg individually, the hepatoprotective property of this combination is identified only when the amounts of these ingredients are enhanced to the range of 25 to 250 mg per dose. In other words, the applicants for the first time noticed the hitherto unknown hepatoprotective property of the composition comprising extracts of plants namely Phyllanthiis amarus, Eclipta alba, Andrographis paniculata and Picrorhiza kurroa when the amounts of these plant extracts are enhanced to the range of 25 to 250 mg per dose.
The present invention also provides a hepatoprotective and anti hepatitis B virus
composition comprising the following plant extracts:
Common Name Botanical Name Range of Extract in mg
per dose
]Bhumyama Laki Phyllanthus amarus 25 - 250
Bringraj Eclipta alba 25- 250
Kalmegh Andrographis paniculata 25- 250
Kutki Picrorhiza kurroa 25- 250
Rheum emodi Rheum emodi Wall 25-250
The above novel compositions also comprising optional ingredients such as extracts, preferably aqueous extracts of the following plants:
Common Name Botanical Name Range of Extract in mg
per dose
Pitpapra Fumaria officinalis 5-50
Gilo Tinospora cordifolia 5-50
Haritaki Terminalia chebula 5-50
Kasni Cichoriumiinlybus 10-50
Sarphunka Tephrosea purpurrea 10-50
Punarnava , Boerhaavia diffusa 10-50
The present composition is a synergistic composition exhibiting surprising
properties such as enhanced hepatoprotective and anti hepatitis B viral properties.
The present invention is illustrated with reference to the following examples and
such examples should not be construed at any extent to limit the scope of the
invention.
Example 1: describes the usefulness of the present composition (with Rheum
emodi) in the treatment of acute viral hepatitis.
Clinical Trials of the present composition on patients of Acute Viral Hepatitis
The present composition was tried for clinical efficacy on the patients of classical
acute viral hepatitis (95 patients) and results are summarised in table 1, 2. The
mean values of biochemical investigations are tabulated below in table 1.
Table 1: Summary of Biochemical Investigations from
Clinical Trials Data on the present composition in the patients of Classical Acute Viral Hepatitis
(Table Removed)
Table 2: Effect of the present composition on Physical signs and symptoms of acute classical Hepatitis (No. of patients= 95)
(Table Removed)
There was definite significant improvement in subjective symptoms as well as objective parameters by of the present composition. Significant improvement was reported in anorexia, abdominal discomfort, loss of appetite, and nausea within 2-3 weeks while complete recovery from jaundice and hepatomegaly was also observed within 4 weeks. SGOT, SGPT and serum bilirubin levels showed significant reduction after 4 weeks of treatment. It was also observed that out of 60 patients who were positive for HBV, 30 patients became negative after 12 to 18 weeks of therapy with this formulation.
The present composition thus promotes the rate of recovery in subjective symptoms and objective parameters in patients of acute classical viral hepatitis and it reduces the period of clinical convalescence.

Example II
This example describes the effects of the present composition (with Rheum emodi) on Hepatitis B virus (HBV) infection using duck hepatitis B virus (DHB V) animal model.
Material and Methods:
1. Production of DHBV viremic and non viremic animals for
experimentation
The Indian strain of DHBV has been characterised by cloning and sequencing and a persistent viremicVnon-viremic colony of the animals were maintained for experimentation. The eggs from both the groups (DHBV viremic/DHBV non viremic) were obtained marked on shell and hatched for 30 days in the laboratory to obtain ducklings. The ducklings were raised on the food and water adlib and when attaining the adulthood at age of 12 weeks were incorporated in the experiment described below.
2. Preparation of the present composition for administration to DHBV
viremic/non-viremic ducks
The preparation of the present composition for experimentation was similar as described above.
3. Experimental Design:
A total of 7 three month old viremic ducks were given of the present preparation (100 mg/kg body weight) twice daily orally for a period of twelve weeks. Post treatment follow up of the animals was carried out upto a maximum period of six weeks after stopping the therapy.
4. Blood samples were obtained from the wing vein from all the ducks and
stored at -70°C after serum separation, the samples were collected prior to the start
of the treatment, at weekly interval during the present treatment and after cessation
of the treatment. All the samples were subjected to determination for the presence
of DHB V-DNA by Dot Blot hybridization. The animals were sacrificed at the end
of their follow up period and an autopsy was performed.
5. Controls: Ten ducks viremic and ten non-DHBV viremic ducks were used as
controls for of the present composition experiment. During the experimentation
they were given normal phosphate buffered saline (placebo) in a similar protocol
as with the experimental group and sacrificed at the end of the study as in 4.
6 Studies on serum: DHBV-DNA in serum was analysed by dot blot hybridization.
7. Histopathological Studies: Liver, kidney, pancreas, heart and lungs were collected at autopsy from each animal for histopathological examination. The tissue samples were fixed in buffered formalin, paraffin embedded in a routine standard laboratory techniques and cut at 4 ji and stained with hematoxylin and eosin for examination.
8. Result: These have been described in Table 3 below as (a) effect of the present composition on the persistent DHBV viremia and (b) DHBV status in control group.
(a) Present composition: the effect on the serum DHBV DNA is shown in
table 3. Twelve weeks therapy with preparation of the present composition
produced DBV-DNA clearance in 40% animals. There was intermittent periods of
DHBV-DNA clearance in 20% animals while in 40% animals there were no effects.
(b) Controls: All the DHBV viremic and non viremic animals maintained their
serum DHBV status during the study.
Table 3: Summary of Effects of preparation of the present composition (with Rheum emodi) on the serum Hepatitis B virus using duck hepatitis B virus carrier animal model
Duration of therapy = 12 weeks (Table Removed)

(c) Toxicity Study: Animals receiving the present preparation did not reveal any histopathological abnormality in liver, kidney, lung, pancreas and gastrointestinal tract at the end of study. Similarly in the control group there were no histopathological alteration in the above parenchymatous organs. Example 111
This example describes the hepatoprotective effects of the present preparation comprising Phyllanthus amarus, Eclipta alba, Andrographis paniculata and Picrorhiza kurroa using acute hepatotoxicity experimental models. The following animal models were used: i) Galactosamine acute Hepatotoxicity model, ii) Thioacetamide acute hepatotoxicity model, iii) Alfatoxin B 1 acute hepatotoxicity model, and
iv) Rifampicin & Isoniazid acute hepatotoxicity model; and the details of these models are given here below:
GALACTOSAMINE (GLN) ACUTE HEPATOTOXICITY MODEL
First reported by Keppler et al. in 1966 as a cause of hepatocellular injury, GLN has been found to cause acute hepatocyte necrosis. The hepatic injury caused by GLN is an extremely interesting model and the lesion resembles that of acute viral hepatitis in humans. A single dose of GLN leads to acidophilic degeneration and necrosis of Hepatocytes in 4 to 6 hours. By 24 hours, there are multiple focal areas of necrosis often accompanied by inflammatory response of neutrophils and plasma cells. At this time the non-necrotic areas show prominent acidophilic degeneration, free acidophilic bodies, and ballooning. Enlargement and increase in number of Kupffer cells is apparent in 6 hrs and prominent by 24 hours. By 48 hours the lesion is maximal with marked dissociation of Liver cell plates and
extensive areas of necrosis. Portal areas show oedema, inflammatory infiltration and bile ductular proliferation. The serum biochemical manifestation of the acute liver injury induced by GLN are characterised by an elevated serum transaminase (ALT) and alkaline phosphatase levels with or without a mild rise in serum bilirubin.
THIOACETAMIDE ACUTE HEPATOTOXICITY MODEL
Thioacetamide (Thio.) is a white crystalline organic compound and a single dose (200 mg/kg) leads to centrizonal hepatocyte necrosis by 12 hours which by 24 to 30 hours is maximum and diffuse involving the complete liver lobule along with mononuclear inflammatory response.
AFLATOXIN BI ACUTE HEPATOTOXICITY MODEL
Aflatoxin are a group of Furano Coumarins and at least 13 have been identified. Six of these (AFB, AFG, AFB2, AFG2, AFB20, AFGa2) are found in cultures of A. flaviis. AFB I is the most potent hepatotoxic and hepatocarcinogenic member of aflatoxins. Ducks are the most susceptible animal model system to study the effects of acute hepatotoxicity due to AFB 1. A single dose leads to hepatotcyte necrosis, fatty change, sinusoidal infiltration by mononuclear cells and proliferation of bile ductules in the portal areas.
RIFAMPICIN & ISONIAZID ACUTE HEPATOTOXICITY MODEL:
Rifampicin (RTF) is the member of rifamycin group of antibiotics with a wide spectrum activity and has achieved 'first line' status in the treatment of tuberculosis. In general it is an agent of low toxicity, but can have pathological
effects in liver. Isoniazid (INH) is also antituberculosis agent, and used in combination with RTF, INH alone can also result in liver injury, but KEF-INH combination appears to lead a higher incidence of hepatic injury than either drug alone. This is based on experimental studies that RIF & INH together are more hepatotoxic than either drug alone.
Hepatoprotective studies of the present composition were done on the duck Hepatitis B virus (DHBV) carrier ducks. The following preliminary studies were done prior to detail study for Hepatoprotective effects of the present composition in acute hepatotoxicity animal models.
ESTABLISHMENT OF OPTIMAL DOSES FOR ACUTE HEPATOTOXICITY MODELS
The aim of these experiments was to establish the optimal doses of each hepatotoxicant which was required to produce a damage to the liver which was compatible with the survival of the animal to study the hepatoprotective effects of the present composition. Before the experimentation the baseline levels of serum alkaline phosphatase and ALT. were determined by collecting blood samples from eight healthy DHBV viremic ducks. The serum was separated and the estimation was done using commercial kits (Bayer Diagnostics) as per manufacturer's instructions. The mean levels of the two enzymes were as under:
Mean level Range
Serum Alkaline Phosphatase
(IU/L) 61 52-79
ALT-(IU/L) 34.6 30-38
Preliminary Acute hepatotoxicity studies in both viremic and non viremic DHBV animals were carried out using Galactosamine (GLN), Thioacetamide (Thio), Aflatoxin B (AFB 1), Rifamycin (RIF) + Isoniazid (INH). Blood samples were collected before and at the time of sacrificing animals for estimation of serum alkaline phosphatase and transaminase (ALT) using the commercial kits (Bayer Diagnostics), Baroda (India) utilising Technicon RA50 autoanalyser. At the end of each experiment, animals were sacrificed and liver was obtained for the histopathological study. The results established that the following optimal single doses were required to produce the desired acute hepatotoxicity in animals
i) Galactosamine = 500 mg/kg body weight
ii) Thioacetamide = 500 mg/kg body weight
iii) Aflatoxin B 1 = 0.1 mg/kg body weight
iv) Rifamycin & isoniazid combination = (250 mg RDF + 150 mg DMH/kg body
weight)
In the preliminary experiments used to standardise the above doses it was revealed that the acute hepatotoxicity produced by GLN, Thio, AFB 1, REF+INH was more marked in DHBV viremic as compared to non viremic animals. Hence, only DHBV viremic animals were utilised for demonstration of hepatoprotection.
Priming of the animals with the preparation was done in Galactosamine, Thioacetamide and Aflatoxin B 1 models since these agents produce hepatotoxicity which is acute in nature and occurs within 48 hrs after administration of the respective toxic agent. In Rifampicin and isoniazid toxicity model, the preparation was administered simultaneously to the animals.

Hepatoprotective studies with each of the above model using the known preparation has been described and illustrated as below:
GALACTOSAMINE ACUTE HEPATOTOXICITY MODEL Experimental Design
1. The present preparation (without Rheum emodi) treated and
galactosamine challenged animals
Six Duck hepatitis B virus (DHBV) persistently carrier adult ducks were primed with the present preparation by administering 100 mg/kg of body weight preparation orally by intragastric tube twice daily for a period of six days. On sixth day each animal received 500 mg/kg of body weight galactosamine (Sigma Chemical, St. Louis, USA) as Intraperitoneal (EP) single injection. The animals were continued with the present preparation administration for 6, 7 and 8 day and were subsequently followed by sacrifice and an autopsy performed.
2. Control Group: Saline treated and galactosamine challenged animals.
Six Duck hepatitis B virus (DHBV) persistent carrier ducks were given 1 ml of normal saline orally by intragastric tube twice a day for six days. A single dose of galactosamine 600 mg/kg of body weight was given intraperitoneal (IP) injection on the sixth day. The animal received saline treatment for 6, 7 and 8 day and were followed by sacrifice and autopsy performed.
Result :
(A) Liver histopathology
The composition of the present invention protected and Galactosamine challenged animals: The hepatocytes were well preserved and arranged in well defined cell plates. The overall architecture of the liver parenchyma was normal.
Saline and galactosamine treated animals (Control):
A marked lobular disarray of the hepatocytes was noted. There was degeneration, ballooning of the hepatocytes, along with necrosis. Foci of bile ductular proliferation were also present.
(B) Serum ALT & Alkaline Phosphatase levels in controls and the present
composition protected animals: have been shown in the Table No. 4.
Table 4: Galactosamine acute hepatotoxicity model, mean values of serum alkaline phosphatase and ALT in controls and the present composition
(without Rheum emodi) protected animals
(Table Removed)
THIOACETAMIDE EXPERIMENTAL MODEL:
The present composition (without Rheum emodi) protected and thioacetamide challenged group:
Six Duck hepatitis B virus (DHBV) persistent carrier ducks were primed with the present preparation (without Rheum emodi) orally by intragastric tube (100 mg/kg of body weight) twice a day for a period of six days, followed by single dose of thioacetamide (500 mg/kg of body weight intraperitoneal injection) on the sixth day. The animals were given the present composition twice a day for 7, 8 and 9 days subsequently sacrificed and an autopsy was performed. Control: Saline treated and thioacetamide challenge animals:
Six Duck hepatitis B virus (DHBV) carrier ducks were given normal saline (I ml) for a period of six days, followed by a single dose of thioacetamide (500 mg/kg of body weight intraperitoneal injection). The animals received saline treatment on 7,8 and 9 days subsequently sacrificed and an autopsy performed.
Results
(A) Liver histopathology
The present preparation protected and thioacetamide challenged animals
The liver parenchyma was restored to normal except for the presence of fat vacuoles in the hepatocyte in some focal areas, the portal tracts were preserved
Saline treated and thioacetamide challenged animal (Control):
There was a diffuse and marked hepatocyte changes in the liver parenchyma which were characterised by necrosis, cytoplasmic degeneration and ballooning of cells. Some of the hepatocytes also showed steatosis (fatty change).
(B) Serum ALT and Alkaline Phosphatase levels in controls and the present preparation protected animals have been shown in Table No. 5.
Table 5: Thioacetamide acute hepatotoxicity model, mean values of serum alkaline phosphatase and ALT in control and the present preparation protected animals
(Table Removed)
The values represent the mean of the six animals.
AFLATOXIN BI ACUTE HEPATOTOXICITY
Experimental design:
The present preparation (without Rheum emodi) protected and Aflatoxin Bl (AFBL) treated animals:
Six Duck hepatitis B virus (DHBV) persistent carrier ducks were primed with the present preparation (100 mg/Kg of body weight) twice a day orally by intragastric tube for three days. On the third day, each animals received Aflatoxin B 1 (Sigma Chemicals, St. Louis, USA). 0.1 mg/Kg of body weight as intraperitoneal injection. The present composition (100 mg/ Kg of body weight) twice a day was continued for another 6 days and the animals were sacrificed and an autopsy was performed.
Saline treated and Aflatoxin (AFBL) challenged animals (control):
Six Duck hepatitis B virus (DHBV) carrier ducks received phosphate buffered saline orally by intragastric tube for 3 days followed by Aflatoxin Bl (0.1 mg/Kg of body weight as intraperitoneal injection). The phosphate buffered saline treatment was continued for another 6 days and animals were sacrificed and autopsy performed.
RESULTS:
(A) Liver histopathology
The present composition (without Rheum emodi) protected and Aflatoxin (AFB1) challenged animals:
The lobular architecture of the liver was preserved and the hepatocytes were arranged in well defined cell plates, focal proliferation as seen in the control group was absent in the portal area.
Saline treated and Aflatoxin (AFB1) challenged animal (control):
There were a diffuse hepatocyte changes characterised by cytoplasmic degeneration, necrosis, and ballooning of the cells. In the portal tracts proliferation of bile ductules accompanied by infiltration by mononuclear inflammatory cells was present.
(B) Serum ALT & Alkaline phosphatase levels have been shown below in table No. 6.
Table 6: Aflatoxin Bl acute hepatotoxicity model, mean values of serum alkaline phosphatase and ALT in control and protected animals.
(Table Removed)
The values represent mean of six animals.
RIFAMPICIN & ISONIAZID ACUTE HEPATOTOXICITY
Experimental design:
The present preparation (without Rheum emodi) protected and Rifampicin (RIF) and isoniazid (INH) treated group:
Six Duck hepatitis B virus (DHBV) persistent carrier ducks were given the present composition orally by intragastric tube (100 mg/Kg of body weight twice a day) along with a combination of RTF 250 mg + INH 150 mg per Kg body weight (Lupin Laboratories India) daily orally for a period of seven days simultaneously, and were sacrificed at the end of study and an autopsy was performed.
Controls: Saline treated and Rifampicin and Isoniazid challenged:
Six Duck hepatitis B virus (DHBV) persistent carrier ducks were given 1 ml. of phosphatase buffered saline twice a day and RIF 250 mg + INH 150 mg per Kg of body weight daily orally for a period of seven days and were sacrificed at the end of the study and an autopsy was performed.
RESULTS:
A) Liver Histopathology:
The present preparation (without Rheum emodi) protected and Rifampicin and Isoniazid challenged group:
The lobular architecture of the liver was restored to normal. The hepatocytes were arranged in well defined cell plates. Only a few hepatocytes showed fat vacuoles in their cytoplasm.
Saline treated and Rifampicin and Isoniazid challenged group (Control):
There was diffuse marked macro vesicular type steatosis (fatty change) involving the cell cytoplasm.
(B) Serum ALT & Alkaline phosphatase levels have been shown below in table No. 7
Table 7: Rifampicin and Isonized acute hepatotoxicity mean values of serum alkaline phosphatase and ALT in control and protected animals.
(Table Removed)
The values represent mean of six animals.
Hepatoprotection provided by the present preparation in acute hepatotoxicity experimental model.
The four acute Hepatotoxicity experiment models described above eg. Galactosamine, thioacetamide, Aflatoxin Bl, Rifampicin and Isoniazid demonstrate that the present preparation provided hepatoprotection as illustrated by the liver histopathological examination and serum biochemical parameters of the liver enzymes.
Example IV
Hepatoprotective effects of known composition (Cl) and the present composition with Rheum emodi (C2) and without Rheum emodi (C3)
A total of 80 DHBV-viremic animals were used in the study. For each acute hepatotoxicity models, the animals were primed by giving either composition Cl, C2 or C3 followed by acute hepatotoxic dose of toxicant and subsequent administration of composition Cl or composition C2 or composition C3 twice daily. Blood samples were collected from each animal prior to the introduction in the study, during priming with either of these preparations and at subsequent intervals and at the sacrifice. Serum samples were separated and subjected to estimation for serum bilirubin, alkaline phosphatase, and transaminases. At the autopsy, the liver, spleen, kidney, lung and heart were removed in toto and subjected to histopathological examination.
Results:
The following tables 8, 9 and 10 summarise the results of hepatoprotective effects of the above-identified composition based on the liver function tests and liver histopathology. The hepatoprotection is graded as absent (-) or present (+) with each preparation.
Table 8: Showing the hepatoprotective effects of known composition (Cl) (Table Removed)
Table 9: Showing hepatoprotective effects of present composition with Rheum emodi (C2) (Table Removed)

Table 10 - Showing hepatoprotective effects of present composition without Rheum emodi(C3) (Table Removed)

The above experiments clearly indicate that the present composition comprising essentially (1) Phyllanthus amarus (2) Eclipta alba (3) Andrographis paniculata and (4) Picrorhiza kurroa in an amount ranging from 25 to 250 mg per dose has excellent hepatoprotective property when compared with known composition. In addition, the novel composition of the present invention comprising essentially the plant extract derived from (1) Phyllanthus amanis (2) Eclipta alba (3) Andrographis paniculata (4) Picrorhiza kurroa and (5) Rheum emodi showed excellent hepatoprotective property along with anti hepatitis B viral property.

We Claim;
1. A synergistic polyherbal composition useful for treating acute Hepatitis E virus^
Hepatitis B infection as a hepatoprotective agent, said composition comprising
extract of plant Rheum emodi Wall in the range of 25-50(mg), Phyllanthus amarus
Linn in the range of 25-50(mg)., Eclipta alba Hassk in the range of 25-250 (mg).,
Andrographis paniculate Nees in the range of 25-250 (mg)., and Picrorhiza
kurroa Royle ex Benth in the range of 25-250 (mg)., and optionally Fumaria
offcinalis in the range of 5-50 mg, Tinospora cordifolia Miers in the range of 5-50
(mg)., Terminalia chebul Retz is in the range of (5-50 mg)., Cichorium intybus
Linn, in the range of 10-50 (mg), and Boerhaavia diffusa Linn in the range of 10-
50 (mg); wherein the whole plant or parts thereof are powdered and extracted with
water under reduced pressure and temperature below 50°C and if desired, the
residue thus obtained is mixed with pharmaceutically acceptable neutral agents
and converted into suitable pharmaceutically acceptable dosage form.
2. A composition as claimed in claim 1, wherein the amount of Fumaria officinalis
Linn., Tinospora cordifolia Miers., Terminalia chebulia Retz., Cichorium intybus
Linn., Tephrosea purpurea Linn, and Boerhaavia diffma ranges from 5 to 50 mg.
3. A composition as claimed in claim 1, wherein the composition comprises of:
Ingredients Qty.
Revand chini (Rheum emodi Wall) 170 mg
Bliringraj (Eclipta alba Hassk 300 mg
Bhumyamalaki (Phyllanthus amarus Linn.) 300 mg
Sarpanunkha (Tephroseapurpurea Linn,) 180 mg
Kasni (Cichorium intybus Linn.) 180 mg
Punarnava (Boerhaavia diffusa Linn.) 100 mg
Gilo (Tinospora cordifolia Miers) 72 mg
Haritaki (Terminalia chebula Retz.) 72 mg
Kalmegh (Andrographis peniculate Nees.) 60 mg
Kutki (Picrorrhiza kurroa Royle ex Benth. i) 60 mg
Pitpapra (Fumaria officinalis Linn.) 30 mg
4. A composition as claimed in claim 1, wherein the composition is a powder, tablet,
syrup or granules,
5. A process for preparing a composition as claimed in claim 1, comprising the steps
of:

a) preparing an extract of Rheum emodi Wall., Phyllanthus amarus Linn., Eclipta
alba Hassk., Andrographis paniculate Nees., and Picrorhiza kurroa Royle ex
Benth;
b) optionally preparing an extract of Fumaria offcinalits, Tinospora cordifolia Miers.,
Terminalia chebul Retz., Cichorium intybus Linn., and Boerhaavia diffusa Linn,
and;
c) mixing the extracts of step (a) and step (b) to obtain a composition.
6. A process as claimed in claim 5, wherein the extract is a solvent extract.
7. A process as claimed in claim 5, wherein the solvent for extraction is selected from
ethanol, methanol, chloroform hexane, ethyl acetate, methyl chloride, carbon
tetrachloride, toluene, acetone , water and mixtures thereof.
8. A process as claimed in claim 5, wherein the residue is mixed with water, kept
overnight at 37°C with stirring and centrifuged, preferably at 10,000 rpm, the
solution thus obtained was vacuum dried and dissolved in a fixed volume of
normal saline to make a stock solution of the plants and stored at 4!C and if
desired, such extract is mixed with suitable pharmaceutically acceptable additives
to prepare the composition.
9. A pharmaceutical composition and process for preparing the same substantially as
herein described with reference to the forgoing examples.