Title of Invention	"A PROCESS FOR THE PREPARATION OF A SYNERGISTIC NOVEL HERBAL PHARMA-CEUTICAL COMPOSITION USEFUL IN THE TREATMENT OF HEPATITIS `B` VIRAL INFECTION IN MAMMALS"
Abstract	A process for the preparation of a synergistic novel herbal pharmaceutical composition useful in the treatment of hepatitis B viral infection in mammals, said process comprising the steps of: a) preparing a solvent extract from the plant parts of Rheum emodi in a method such as herein described; b) evaporating the extract under reduced pressure below 50° C to obtain a residue; c) mixing the residue obtained with water; d) keeping the aqueous extract overnight at 37° C with stirring and repeated centrifugation; e) drying the solution obtained under vacuum and dissolving it in normal saline for preparation of a stock solution , and optionally mixing the solution with pharmaceutically acceptable excepients or additives to obtain a synergistic herbal pharmaceutically composition, and if desired, converting the same into a suitable oral dosage form such as tablets, capsules or syrups by any known method.

Title of Invention

"A PROCESS FOR THE PREPARATION OF A SYNERGISTIC NOVEL HERBAL PHARMA-CEUTICAL COMPOSITION USEFUL IN THE TREATMENT OF HEPATITIS `B` VIRAL INFECTION IN MAMMALS"

Abstract

A process for the preparation of a synergistic novel herbal pharmaceutical composition useful in the treatment of hepatitis B viral infection in mammals, said process comprising the steps of: a) preparing a solvent extract from the plant parts of Rheum emodi in a method such as herein described; b) evaporating the extract under reduced pressure below 50° C to obtain a residue; c) mixing the residue obtained with water; d) keeping the aqueous extract overnight at 37° C with stirring and repeated centrifugation; e) drying the solution obtained under vacuum and dissolving it in normal saline for preparation of a stock solution , and optionally mixing the solution with pharmaceutically acceptable excepients or additives to obtain a synergistic herbal pharmaceutically composition, and if desired, converting the same into a suitable oral dosage form such as tablets, capsules or syrups by any known method.

Full Text	FILED OF THE INVENTION The present invention relates to a process for the preparation of a synergistic novel herbal pharmaceutical composition derived from a plant namely Rheum emodi, which is useful for the treatment of Hepatitis B virus infection, a process for the preparation of an extract from Rheum emodi and a composition comprising extract of Rheum emodi. BACKGROUND OF THE INVENTION Rheum emodi is a plant grown in sub-Himalayan regions of India and its neighbouring countries ('Chopra RN, Nayar SL and Chopra IC. 1059/ The characteristics of this plant are given below: Rheum emodi Botanical Name : Rheum emodi wall Family Polygonaceae Hindi Name Revand chini English Name Indian Rhubarb Habitat and Distribution : Sub-alpine and alpine Himalayas upto 12,000ft.above sea level Botanical Description : A stout herb with woody large roots, Radicle leaves, long petioles, very large orbicular or broadly ovate flowers in panicles. Fruits ovoid oblong. Himalayan species of Rhubarb are found at altitudes of 4,000 to 12,000 feet and in States of Kashmir and Sikkim in India as well as in Bhutan. The traditional preparations consist of the dried rhizome, or underground stem of the plant, either whole or cut pieces of suitable length. The roots are dug up, cut transversely into short pieces which are dried in the sun or by artificial means. Constituents - Rhubarb root contains a large proportion of Chrysophanic acid, sometimes called Chrysophan, an allied substance Emodin, a glucoside rhapanliein, a tannin named Rheo tannic acid several resins, an albuminoid principle, mucilage, extractives, tannic and gallic acids, sugar, starch, pectin, lignan, calcium oxalate and various inorganic salts. The well known grittiness of the root is due to crystals of calcium oxalate. Leaves contain oxalic acid. Action & Uses - Rhubarb is largely employed in modem medicine as a purgative and is a good laxative. It never produced sickness in any one. Rhubarb is stomachic, bitter, tonic and cathartic so that its secondary effect is confined to bowels movements.. Hepatitis B virus (HBV): Man is the natural source and reservoir of Hepatitis B virus infection (Sherlock S and Dooley J. Disease of the liver and biliary system Page 269-280, 1993 Blackwell Scientific Publications Oxford, England./ Hepatitis B virus is an Hepadna virus with a size of 42 nra. Morphologically, the virus is complex, double shelled with surface and core antigen comprising the outer surface and inner core antigen. 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 of human beings are susceptible to this infection. Endemicity exists and high risk groups include blood recepients, hamophiliacs, dialysis patients, intiavenous 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 rise 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 carrier. 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 early childhood. Most of the infections are acquired in early childhood, usually as mother to child transmission and results in a persistent HBV carrier stage. HBV carriers have 200 times more risk of liver cancer. The status of serological markers of HBV infection in man and their clinical significance is summarised as follows: Acute infection : HBsAg, anti HBc IgM Chronic Infection : HBsAg persisting more than 6 months Virus replication HBeAg HBV-DNA Carrier stage : HBsAg Protection : Anti HBs Infection on Wane : Anti HBe 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 (I) active universal immunisation in childhood and/or (2) development of treatment modalities for persistently infected persons. Intenuption of perinatal transmission is critical in prevention of chronic infection. For this purpose, either vaccine, or post exposure prophylaxis in form of drug will be very important. However, therapeutic studies conducted over the past 15-20 years had only marginal success once the infection is established. Several compounds, such as antiviral agents, biological response modifiers and immune stimulants regime administered alone or in combination have been evaluated (Hoofnagle JH. 1991, ). Most of these agents have transient effects on viremia, with rebound occurrence on withdrawal of drug. Significant toxicity has been observed with long term therapy. Though, HBV was discovered to be the causative agent of Hepatitis B almost 30 years ago, a detailed 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 to propagate the virus. The advent of molecular cloning techniques and the discovely 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 wese collectively grouped under Hepadna viridae(Gust ID, Burrell CJ, Coulpis AG et al. 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 vims was isolated from Woodchuck. 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 woodihuck sera exhibited a similar size and moiphology 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 4 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 vims(DHBV) revealed unexpected results that approximately 10% of domestic ducks in the United Slates were congenially in¬fected with virus(summers 1987). At fbout the same time, a third hepadna virus was identified in beechy ground squirrel (GSHV) in California(Morion etal. 1980). Hepadna virus have also been reported in tree squirrels(Mehrotra etal. 1990, Feitelson et al. 1990). Duck hepatitis B vims (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 {Mason el al. 1980). The average transmission occurs through egg from a carrier mother. The experimental DHBV transmission studies have established that within Hepadna viridae, the age at which infection occurs determines whether the resulting viremia will be persistent or transient. Earlier the age of embryo in ducks greater is viremia(Lamhert el al. 1991). This situation is similar to that of HBV infection in man in that infection before or at birth and infancy results in high carrier rate. 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 becomes persistently viiemic. 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 vims infection and also the natural plant products(Munshi et al. 1993(a) (h), 1994). Several antiviral gents have been evaluated in chronically infected ducks. DHBV infection has ieen associated with a broad spectrum of liver pathology e.g. chronic hepatitis, cirrhosis and hepatocellular carcinoma (Duflot A, Mchrotra R, Yu SZ el al. 1995). Hepatitis B virus infection and Natural plant products Many plant products have been in use in the treatment of liver diseases since time immortal. In Indian system of traditional medicine (Ayurvedia), natural plant products have been used in the treatment of jaundice and liver disorders. The use of various plants have been described in ancient Indian Ayurvedic literature. In South India, the plants of genus Phyllanthus and Eclipta alba are commonly used as a traditional treatment for clinical jaundice including that of viral hepatitis and are commercially available (Thyagarajan, 1986; Thyagarajan & Jayaram, 1992). Phyllanthus species are also used in China, the Phillippines, Cuba, Nigeria, Guam, Bast and West Africa, the Carribean, Central America and South America. The above two plants have been recently evaluated for their action on Hepatitis B virus. The first screening strategy used was to test the ability of plant extract to coat viral HBsAg and thereby inhibit the reaction with antibody to HBsAg (anti HBs) (Unander & Blumberg, 1991; Blumberg et al, 1990). The rationale was that such an inhibition might have effect on pathogenesis of Hepatitis B virus in vivo in man (Mehrolra et ai, 1990, 1991). This observation was also supplemented by the ability of plant extiact to inhibit DNA polymerase (DNAp) of Hepatitis B virus in vitro. The crude extracts of Eclipta alba when mixed with HBsAg brings about its interaction, thereby suggesting the presence of anti Hbs like activity (Thyagarajan et ai, 1982). The extracts of Phyllanthus niruri now known as Phyllanthus a mams inhibit human HBV-DNAb as well as HBsAg and anti HBs interaction. Also, the closely related animal hepadna viruses, e.g. Wookchuck hepatitis virus, and duck hepatitis B virus are also affected by P. amarus (Blumberg et ai, 1990). In a series of studies on long term carriers of woodchucks infected with woodchuck hepatitis B virus it was observed that the P. amarus extract generally eliminates or decrease the level of vims (Venkaleshwaran et ai, 1987; Venkateswaran and Blumberg -CS Patent No. 4673575, 1987). However, subsequent studies using another animal model of Human Hepatitis B virus; Duck hepatitis B virus failed to conlnni the findings reported earlier [Nitu et al, 1990; Munshi et al, J993(a),(b)J. Clinical studies in humans using P. amarus, on Hepatitis B virus has also been carried out. In 1988, it was reported from Madras that dried milled P. amarus was successful in clearing Hepatitis B surface antigen from 59% carriers of Hepatitis B virus (Thyagarajan et al, 1988). However, this observation was followed by another report wherein the success rate of treatment with P. amarus was reduced to 20% (Thyagarajan et al, 1990). In subsequent clinical studies on Hepatitis B virus carriers with P. amarus, results were not reproducible by other workers in different other countries (Brook, 1988; LeeLarasamee et al, 1990; Mei-Xia et al, 1991; Milne et al, 1993). Traditional plant medicines have also been tested for suppression of viral antigen secretion by Alexander cell line (PLC7PRF/5), obtained from human hepatocellular carcinoma which contain several integrated copies of HBV genome and produces small amount of HBsAg apparently in the form of 22 nm particles. Goto et al (1996) using the above cell line as a model for persistently HBV infected liver tested forty three (43) extracts of herbal medicines for suppression of secretion of HBsAg in vitro. Of the various extracts found to have appreciable effects, Rheum Palmatum strongly suppressed the secretion of HBsAg. The Alexander cell line model has also been used to evaluate the inhibition Suppression of HBsAg by P. Amarus (Jayaram & Thyagarajan 1996). The ultimate aim of therapy with HBV infection is to render patients less infectious which helps in recovery, thereby the development of chronic sequelae (Cirrhosis, hepatocellular carcinoma) can also be prevented. A positive therapy should aim towards reduction in HBV replication. This activity can be assessed by demonstrating a decrease in serum HBV-DNA, clearance of HBeAg and HBsAg. So far, there is no effective and economical treatment for Hepatitis B virus infection and its associated pathological complications. Therefore, after much research, the applicants have detected for the first time that the extract of Rheum emodi can cure Hepatitis B virus infection, a hitherto unknown property of the herb Rheum emodi. OBJECTS OF THE INVENTION The main object of this invention relates to a pharmaceutical composition comprising a plant namely Rheum emodi in any form for treating hepatitis B virus infection in animal or human being. Another object of the invention is for providing a pharmaceutical composition comprising extracts of Rheum emodi for the tieatment of acute and chronic hepatitis B viral infection in animal or human being. Yet another object of the invention relates to a pharmaceutical composition useful for the tieatment of hepatitis B vims infection, which comprises of fraction of Rheum emodi, said fraction contains extractable components of Rheum emodi, said fraction having anti HBsAg, anti HBeAg like activity and also produces an effect on HBV-DNA. Further object of the invention relates to a pharmaceutical preparation for treatment of hepatitis B virus infection which comprises of aqueous extract of Rheum emodi, said fraction having effect on clearance/intermittent effects on viremia of HBV in human beings and animal model such as Duck hepatitis B virus. Another object of the invention provides a pharmaceutical preparation comprising, of a fraction of Rheum emodi, said fraction prevents the development of the hepatits B virus (DHBVO carrier state in animals, for example non-infected ducklings. Still another object of the invention relates to a process of preparing pharmaceutical aqueous extract of Rheum emodi which does not produce any toxicity to systemic parenchymatous organs of the body e.g. Heart, kidney, lung, pancreas and gastrointestinal tract. A further object of the invention relates to a process for the preparation of a composition comprising extract, preferably aqueous extract of Rheum emodi for the treatment of hepatitis B virus infection. To achieve the above objects, the present invention provides a process for the preparation of a synergistic novel herbal pharmaceutical composition useful in the treatment of hepatitis B viral infection in mammals, said process comprising the steps of preparing a solvent extract from the plant parts of Rheum emodi in a method such as herein described; evaporating the extract under reduced pressure below 50°C to obtain a residue; mixing the residue obtained with water; keeping the aqueous extract overnight at 37°C with stirring and repeated centrifugation; drying the solution obtained under vacuum and dissolving it in normal saline for preparation of a stock solution, and optionally mixing the solution with pharmaceutically acceptable excepients or additives to obtain a synergistic herbal pharmaceutical composition, and if desired, converting the same into a suitable oral dosage form such as tablets, capsules or syrups by any known method. DETAILED DESCRIPTION OF THE INVENTION The above objects are achieved by providing a pharmaceutical herbal formulation including extract of Rheum emodi. Preferably, the pharmaceutical preparation is an aqueous extract of Rheum emodi. The solvent used in the extract of Rheum emodi can be any suitable solvent including water. The suitable solvent may be selected from ethanol, methanol, chloroform, water, hexane, ethyl acetate, carbon, tetrachloride, toluene, acetone etc. and the mixtures thereof, in the extraction process, the whole plant and parts thereof powered and extracted with water or with any other suitable solvent. The extract was evaporated under reduced pressure below 50°C leaving a residue. For human administration, the residue was 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. Preferably, the stock solution of the plant extract was used to prepare the present novel composition by mixing with any suitable additives. The extract obtained from Rheum emodi is mixed with pharmaceutically acceptable reagents to obtain a composition. The composition thus obtained is a synergistic composition having unexpected properties. The ratios of the aqueous extract with the pharmaceutically acceptable reagents will vary depend on the requirements and other conditions. A process for the preparation of a synergistic pharmaceutical composition is comprising an extract of Rheum emodi and pharmaceutically acceptable reagents. The novel composition containing Rheum emodi is converted into forms such as tablet, capsule or solution and the average dosage per day to human being can vary from 250 to 900 ml and such dosage can be administered 2 to 5 times in equal or unequal divided portions. BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS In the drawings : Fig.l: represents TLC Chromatogram photograph of Rheum emodi deducted under UV at 254nm. Fig.2 : represents TLC Cliromatogram photograph of Rheum emodi deducted under UV at 366nm. Fig. 3 : represents. HPLC Chromatogram photograph of Rheum emodi deducted under UV at 254nm. The plant Rheum emodi was botanically authenticated and the applicants have taken pains to analytically standardize the extract of Rheum emodi, which is locally called Revand Chini in India. The finger print profile of Rheum emodi is given below : Finger print profile of the REVAND CHINI (Rheum emodi) Rhubarb roots of Revand chini (Rheum emodi) are used here. It contains Chrysophanic acid, an allied substance EMODIN", a glueoside Rhapantiein, a Tannin named Rheotannic acid. A method of finger print by Thin Layer Chromatography (TLC) and high performace liquid chromatography (HPLC) method were developed. THIN LAYER CHROMATOGRAPHY (TLC) Using a silica gel F254 precoated plates (Merck silica gel 60 F254 are suitable) and a mixture of 50 volumes of ethyl acetate, 7 volumes of Formic acid, 3 volumes of Glacial acetic acid, 3 volumes of ethyl methyl ketone, 10 volumes of water as the mobile phase. Apply separately to the plate 10 µ1 of the following solutions. For solution (1) of extract, separately weigh about 1 gm of extract in a beaker, add 2.5 ml of ethyl acetate, shake for about 30 minutes and filter. After removal of the plate, allow it to dry in air and examine under ultravoilet light at 254 nm and 366 µm. OBSERVATIONS: In UV 254 nm (TLC chroinalograin photograph No, I accompanying the specification) orange, yellow and some other zones are visible. The broad zone in the upper Rf (at about 0.56) represents mixture of glycosides of emodin and chrysophanic acid. In 366nm (TLC chromatogram photograph No.2 accompanying the specification) some aglycones migrate with the solvent front to an Rf at about 0.80 and some other zones also appear in the lower Rf range of 0.36-0.42. HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC) Using the following solution (1) Weigh about 1 gm of sample in a beaker, add 20 ml of mobile phase, warm, shake and filter. The chromatographic procedure are carried out using (a) A stainless steel column ODS (C18) (300 mm X 4.6 mm), (b) a mixture of 50 volumes of 0.1% of V/V of oilhophosphoric acid and 50 volumes of methanol as mobile phase with a flow rate 1 ml per minute and (c) detection wavelength of 254 nm. OBSERVATIONS: There (HPLC chromatogram, Figure No.3) are about 10 components which are separated out, these are in different concentrations. These components are correlated to the components of reference extract. These components are most probably aglycones and one of them in chrysophanic acid. Hepatitis B virus (HBV) infection Human Hepatitis B virus (HBV) infection is a public health problem. HBV is associated with both acute and cliionic liver diseases including the development of cirrhosis and hepatocellular carcinoma. There are about 300 million healthy carriers of HBV worldwide about half have ongoing viral replicatiein and are at work for the development of progressive liver disease. There is no effective drug which is effective against HBV infection except interferon in modem medicine. Treatment with interferan alpha can lead to viral replicatien and romessian of active liver disease in nearly 407 of selected patients. However, individuals who are immunosuppersed, and have high level of viral rfeplicatien or low levels of serum amino transferases are unlikely to benefit from this therapy. Further, administration of interferan is cimbersome, costly and limited by side effects. No therapy is available for the interruption of persistent HBV carrier state in man HBV is a DNA vims belonging to Hepadna viridae family, which has a number of animal prototypes, one of which is Duck Hepatitis B virus (DHBV) present in special breed of ducks: Pekin ducks. The DHBV shows more than 75% homology with HBV and has been successfully used as an experimental model to screen various agents and plant products which might have an effect against HBV. and thereby useful and beneficial for man. Screening of pharmaceutical preparation of Rheum emodi extract in DHBV model The Pharmaceutical preparation in the form of extract of Rheum emodi can be obtained by any known method and all parts or the whole of the plant Rheum emodi can be used to obtain extracts thereof The Pharmaceutical preparation from the Indian plant Rheum emodi has been obtained and screened in DHBV model to (I) test the effects on clearance of persistent DHBV viremia. (2) prevention of the development and establishment of DHBV carrier state in non viremic ducklings, and to (3) demonstrate in vitro effects on HBV. Prolonged administration of aqueous(preferably aqueous extract) extract of Rheum emodi shows an encouraging response on the persistent DHBV viremia by producing DHBV clearance in 30% animals receiving therapy as well as leads to intemiittent periods of non viremia in DHBV carrier ducks during treatment. Further, a prolonged administration of extract(preferably aqueous extiact) of Rheum emodi does not produce any toxic effects on the parenchymatous organs of the animals receiving treatment. The in vitro treatment of extract(preferably aqueous extract) of Rheum emodi with the infective inoculum of DHBV and its subsequent intraperitoneal administration to 72 hrs old DHBV negative ducklings inhibits the development of persistent DHBV carrier state in animals as compared to their control. The extract(preferably aqueous extract) of Rheum emodi also has anti HBs and anti HBe like activity as demonstrated by in vitro effects on HBV by producing the inhibition of HBV surface antigen (HBsAg), Hepatitis B virus e' antigen (HBeAg) and also has an effect on HBV-DNA. The prefened extiact here is the aqueous extiact. The invention also covers a process of pharmaceutical preparation of extract of Rheum emodi and effects of extract of Rheum emodi on Hepatitis B Virus (HBV) infection. Here again the extract is an aqueous extiact since it is cheap and easy to prepare. Further, the invention provides a method of heating Hepatitis B virus infection by administering a pharmaceutical herbal preparation comprising Rheum emodi. The treatment includes administering the extract of Rheum emodi for accute and chronic Hepatitis B viral infection in animals or human beings. The present method of treatment includes administering a pharmaceutical composition comprising fraction of Rheum emodi, said fraction contains exhactable components of Rheum emodi, said fraction having anti HBsAg, anti HBeAg like activity and also produces an effect on HBV-DNA. Furthermore, the present invention comprises administering to a patient a pharmaceutical preparation for the cliarance/intermittent effects on viremia* of HBV in said patient. The patient can be a human being or animal model such as Duck hepatitis B virus. The treatment includes administering to a patient a pharmaceutical preparation comprising of a fraction of Rheum enwdi tliereby preventing the development of the hepatitis B virus (DHBV) carrier State in the patient which may be a non-infected duckling. The preparation of Rheum emodi can toe in any form such as tablet, capsule or solution. The average dosage per day to a human being can vary from 250 to 900 ml and such dosage can be administered 2 to 5 times in equally or unequally divided portions. The treatment may vary from 3 to 6 months and for certain acute cases the period of treatment may be extended. The present invention is illustrated with reference to the following examples andj these examples should never be conitrued to limit the scope of the present invention. In other words, the following examples are merely to illustrate the present invention and such examples do not in any manner limit the scope of the invention. Example No. 1 describes the (i) process of pharmaceutical preparation and effects of aqueous extiact of Rheum emodi on the persistent DHBV viremia in adult animals, and (ii) the prevention of the development of DHBV viremia in DHBV non-infected ducklings. Example No. 2 describes the in vitro effects of aqueous extract of Rheum emodi on Hepatitis B Vims (HBV). Example 3 describes the controlled clinical trial of Rheum emodi in patients of acute viral hepatitis (AVH) associated with Hepatitis B virus infection. Example 4 describes that Rheum emodi does not possess any antihepatotoxic properties as demonstrated in experimental studies. Example 4 also describes that aqueous extracts of Rheum emodi does not produce any damage to hepatocytes at cellular level. EXAMPLE 1 This example is illustrated by experiment 1 and experiment II as described below. Experiment No. I: Pharmaceutical preparation and elucidation of the effects of aqueous extract of Rheum emodi on the DHBV carrier state. MATERIAL AND METHODS Pharmaceutical Preparation of aqueous extract of Rheum emodi An aqueous extract of Rheum emodi can be prepared by any known manner such as mixing the plant material obtained from any part of the plant with water. Preferably, all the batches of Rheum emodi used in the study were collected from Sikkim and botanically authenticated. The roots were dried, powdered and extracted with water. The extract was evaporated under reduced pressure below 50 degree Celsius leaving a solid residue. The solid residue was mixed with water, kept overnight at 37°C with stirring and supernatant was extracted by repeated centrifugation at 10,000 rpm. The solution thus obtained was vacuum dried and the dry weight of extract was determined. This dry extract was dissolved in a fixed volume of normal saline to make a stock solution and stored at 4C. The stock solution of the extract was diluted with phosphate buffered saline (pH 7.4) to obtain the desired strength required for experimentations. The piefened strength/quantum required for treatment can be 50 mg to 200 mg per kg / body weight and it can be administered orally daily for 12 to 20 weeks. The sequence of treatment can be daily in continuous manner for the stipulated period of treatment. Production of DHBV viremic and nonviremic animals for experimentation The Indian strain of DHBV has been; characterised by cloning and sequencing (Munshi et al. 1994) and a persistent viremic/non-viremic colony of the animals were maintained. The eggs from boil the groups (DHBV viremic/DHBV non viremic) were obtained, marked on shell and hatched for 30 days to obtain ducklings. The ducklings were raised on the food and water ad lib and when attaining the adulthood at age of 12 weeks were incorporated in the experiment described below. Experimental Design: A total of 7 three month old viremic ducks were given aqueous extract of Rheum emodi (100 µg/kg body weight) as prepared in 2, twice daily orally for a period of twelve (11) weeks. Post treatment follow-up of the animals was carried out upto a maximum period of six (6) weeks after stopping the therapy. 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 intervals, during the aqueous extract of Rheum emodi treatment and after cessation of the treatment. All the samples were subjected to determination for the presence of DHBV-DNA by Dot Blot hybridization. The animals were sacrificed at the end of their follow-up period and an autopsy was performed. Controls: Ten ducks viremic and ten non-DHBV viremic ducks were used as controls for aqueous extract of Rheum emodi experiment. During the experimentation they were given normal phosphate buffer saline (placebo) in a similar protocol as with the experimental group and sacrificed with the treated animals at the end of the study. Studies on serum: Detection of DHBV DNA by Dot Blot hybridization DHBV-DNA in serum was analyzed by molecular hybridization as described before (Dot Blot hybridization method). Histopathological Studies: Liver, kidney, pancreas, heart and lungs were collected at autopsy from each animal for histopathological examination to rule out any toxic effects of the aqueous extriet of Rheum emodi treatment. The tissue samples were fixed in buffered formalin paraffin embedded using routine standard laboratory techniques and cut at 4 p. and stained with hematoxylin and eosin for examination. RESULTS These have been described below as (i) effect of aqueous extract of Rheum emodi on the persistent DHBV viremia and (fe) DHBV in control group. (a) The effect on the serum DHBV DNA during aqueous extract of Rheum emodi therapy and the subsequent follow up after stoppage of therapy. The results have been summarized in the table 1. Twelve weeks therapy with aqueous extract of Rheum emodi produced DHBV-DMA clearance in 40% animals. There were intermittent periods of DHBV-DNA clearance in 20% animals during therapy, while in 40% there was no effect. (b) Controls: All the DHBV virenasic and non viremic animals maintained their serum DHBV status during the study. (c) Toxicity study: Animals receiving aqueous extract of Rheum emodi 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 was no histopathological alteration in the above parenchymatous organs. Table 1 : Effects of therapy by aqueous extract of Rheum emodi on persistant DHBV infection (Table Removed) Experiment 11: Prevention of establishment of DHBV viremia by aqueous extract of Rheum emodi in non infected ducklings. Material and Methods The pharmaceutical preparation of the aqueous extracts of Rheum emodi for experiment were similar to those described in experiment I. In vitro treatment of DHBV with aqueous extract of Rheum emodi: 200 pL of DHBV positive serum (as detected by Dot Blot hybridization) obtained from a persistently DHBV viremic duck was mixed with 200 µ1 of aqueous extract of Rheum emodi (0.5 to 6 mg/ml preferably 4 mg/ ml) and this mixture was incubated in a moist water bath at 37°C for two hours followed by centrifugation at 2000 rpm and the supernatant was used as innoculum for ducklings (50 µ1/animal) as described below. Animals: 20 ducklings were obtained by hatching the eggs obtained from DHBV negative female ducks. The ducklings were bled immediately after hatching and serum were tested for DHBV. All the 20 ducklings were found to be negative for DHBV-DNA and were divided into following two groups: Group A: 10 ducklings in this group were innoculated at 72 hours, with 50 pL of aqueous extract of Rheum emodi treated DHBV-DNA positive serum intraperitoneally. Group B : 10 ducklings in this group were innoculated at 72 hours, with 50 p.L of DHBV-DNA positive and PBS treated serum intraperitoneally. Bleeding: All the animals from Group A were bled at 7 days intervals post-inoculation, upto period of 28 days and at the end of the experiments they were sacrificed and liver, heart, kidney, lung, intestine were subjected for histopathological study. Results: Table 2 summarises the findings in the establishment of DHBV carrier state after in vitro treatment of DHBV-DNA positive serum sample and its control. The percentage protection of DHBV-DNA negative ducklings inoculated with DHBV-DNA positive serum in vitro treated with aqueous extract of Rheum emodi from viremia development at the end of 28 days was only 20% as compared to the control (100%). (In other words, 80% of ducklings among the Rheum emodi treated group were protected against the development of subsequent Viremia) Table 2: Shows the effects of aqueous extract of Rheum emodi on prevention of the establishment of DIIBV vircmia after in vitro treatment of the iuuoculum (Table Removed) The above findings of the experiment 11 demonstrate that aqueous extract of Rheum emodi when in vitro heated with DHBV-DNA positive serum samples, the applicants presume that a sort of binding occurs with the virus thereby making latter non infectious. This observation was further extended to evaluate the effect of the duration of aqueous extract of Rheum emodi in vitro treatment in hours with DHBV-DNA positive serum and the subsequent development of DHBV viremia in ducklings. The DHBV-DNA positive serum samples were treated with Rheum emodi extract for 1, 3 and 6 hours at 37°C in a moist water bath and subsequently utilised as innoculum in DHBV-DNA negaitve duckling obtained from DHBV negative mothers as described above and followed for a period of 28 days. It was observed that the duration of aqueous extract of Rheum emodi in vitro treatment with DHBV-DNA was independent of its effects produced on DHBV infectivity and the results were similar to the two hours invitro treatment as described above. EXAMPLE 2 In vitro effects of aqueous extract of Rheum emodi on Hepatitis B virus: The following three sets of experimints demonstrate the presence of in vitro effects of aqueous extract of Rheum emodi extract on Hepatitis B virus surface antigen (HBsAg), Hepatitis B virus e'antigen (HBeAg) and Hepatitis B virus DNA (HBV-DNA). a) In vitro Effects of aqueous extract of Rheum emodi on HBsAg: The preparation of the extract of Rheum emodi for the use in the experiment were similar as described in Example 1 of Experiment 1. Clinical Material: The HBsAg positive serum samples were obtained from healthy HBV carriers (20-45 yrs age group) patients of acute viral hepatitis (AVH), and chronic liver diseases (Cirrhosis of liver). Each serum sample was serially two folds diluted in phosphate buffered saline and titrated for the presence of HBsAg to determine HBsAg title. All the sera were negative for rheumatoid factor. HBsAg Assay Method: A double sandwich solid phase enzyme immune assay (ELISA) kit using monoclonal antibody (Wellcome Diagnostics, UtK.) were used to test HBsAg. The tests were performed as per manufacturer's instructions and results were read on ELISA reader (Organon, Holland). Treatment of Serum Samples with aqueous extract of Rheum emodi : A 0.5-6 mg/ml, preferably 4 mg/ml concentration of Rheum emodi extract was used in the study. Serum samples were mixed withiequal volume of Rheum emodi extract and incubated for 12 hours at 37°C in a moist chamber. This mixture was centrifuged at 2000 ipm for 10 minutes and subsequently subjected to HBsAg screening by ELISA as described above. With each set of scrum, controls comprising of untreated samples (saline treated) were simlarly incubated for 12 hours and tested for HBsAg. The mean fall in serum HBsAg after aqueous extract of Rheum emodi treatment was calculated. Results: The results of individual samples are shown in Table 3. The mean HBsAg titre of serum fell after aqueous extract of Rheum emodi treatment. The range of HBsAg reduction after in vitro treatment varied from 5 to 12 folds (mean 6.75). Table 3: Shows the folds reduction in HBsAg titre after in vitro treatment of serum samples with aqueous extract of Rheum emodi in the serum samples obtained from various HBV associated conditions. (Table Removed) b) In vitro effects of aqueous extract of Rheum emodi on HBeAg: The preparation of the extiact of Rheum emodi for experiment was similar as described in example 1 of experiment 1. Clinical Material: HBeAg positive sera were obtained from eight healthy HBV carriers patients, of Acute HBV viral hepatitis and HBV associated Liver Cirrhosis. These serum samples were serially two fold diluted in phosphate buffered saline and tested for the presence of HBeAg to determine its titre. All sera were negative for Rheumatoid factor. HBeAg Assay Method: A double sandwich solid phase enzyme immune assay (ELISA) kit supplied by Wellcome Diagnostics, U.K. was used to determine HBeAg. The tests were performed as per manufacturer's instructions and results were read on ELISA reader (Organon, Holland). Teatment of serum samples with aqueous extract of Rheum emodi : A 0.5 to 6 mg/ml, preferably 4 mg/ml concentration of aqueous extract of Rheum emodi was used in the study. Serum samples serially diluted in phosphate buffered saline were mixed with equal volumes of aqueous extract of Rheum emodi and incubated for 12 hours at 37°C in a moist water bath. This mixture was centrifuged at 2000 rpm for 10 minutes and subsequently subjected to HBeAg screening by ELISA as described above. With each set of samples, control comprised of untieated samples (saline treated) and similarly incubated and tested for HBeAg. The mean fall in serum HBeAg after aqueous extract of Rheum emodi treatment was calculated. Results : The results of the individual serum samples is shown in Table 4. The mean HBeAg titre of serum samples was 1/452.88 which fell to 1/22.80 after aqueous extract of Rheum emodi treatment. The range of HBeAg reduction after in vitro treatment varied from 4 to 7 folds (mean 5.25). Table 4: Shows fold reduction in HBeAg titre after in vitro treatment of serum samples with Rheum emodi in samples obtained from various HBV associated conditions (Table Removed) c) In vitro effects of aqueous extract of Rheum emodi on HBV-pNA: The preparation of the aqueous extract of Rheum emodi for experiment were similar to those described, in example 1 experiment I. Clinical material: Four semm sanpipies from patients of acute viral hepatitis B positive for HBV-DNA (mean concentration 90 pg/ml) were used. HBV-DNA determination: A radiological molecular hybridization assay for the detection and quantitation of Hepatitis B viral DNA in serum assay (Abbott Genostics Hepatitis B Viral DNA, Abbot's diagnostics, North Chicago, USA) was used as per manufacturer's instruction to determine the HBV-DNA in serum samples both before and after in vitro treatment with aqueous extract of Rheum emodi. The results were expressed as mean percent inhibition of HBV-DNA. Treatment of serum samples with aqueous extract of Rheum emodi: HBV-DNA positive serum samples (4) were mixed with 0.5 to 6 mg/ml, preferably 4 lng/ml aqueous extract of Rheum emodi and incubated for 12 hrs at 37°C in a moist chamber. Control comprised of saline treated samples and treated similarly. At the end of incubation HBV-DNA was determined in both aqueous extract of Rheum emodi treated and untreated samples (controls). The results were expressed as percent inhibition of HBV-DNA concentration. Results: The mean HBV DNA of serum samples was reduced from 90 pg/ml to 28.55 pg/ml after in vitro aqueous cxtiact of Rheum emodi teatment representing 31.72% reduction. EXAMPLE 3 CONTROLLED CLINICAL fRIAL OF RHEUM EMODI AQUEOUS EXTRACT IN PATIENTS OF ACUTE VIRAL HEPATITIS (AVH) ASSOCIATED WITH HEPATITIS B VIRUS (HBV) Material and Methods Five patients (3 male, 2 female) of a mean age 31.50 (range 18-45 years) presenting within one week of the onset of their clinical symptoms suggestive of classical acute viral hepatitis (AVH) were included in the study. The diagnosis of AVH was established by the clinical symptoms and abnormal biochemical features (Sherlock & Dooley, 1993). The diagnosis of acute HBV infection was done on the basis of presence of HBsAg along with anti HBc IgM in the patient. The other possible causes which might have lead to a similar clinical presentation e.g. surgical jaundice, drug induced cholestasis, heipes, cytomegalovirus infection (as demonstrated by the absence of the tespective IgM antibodies) were absent in the study patients. In all five patients the liver function tests (Serum bilirubin, ALT) and HBsAg and anti Hbc IgM (Elisa) were carried out to establish the diagnosis before the start of their therapy with Rheum emodi, and subsequently at eveiy four week intervals duiing therapy upto a period of twenty (20) weeks. Prior consent of all the patients were obtained ant they were explained about the treatment protocol and follow up procedure. The preparation of the Rheum emodi aqueous extract for clinical use in the above patients were similar as described in example 1 of experiment 1. Each patient was given a dose of 250 mg four times a day packed in gelatin capsules which correspond to a total Rheum emodi dose of 1000 mg per day for each patient. However, with this dose all the five patient just after one day, complained of loose motions. Therefore, from the second day onward the dose of Rheum emodi aqueous extract was reduced to 150 mg four times a day corresponding to a total dose of 600 mg per day for each patient. This dose was well tolerated by all the five patients without any gastrointesinal disturbances and therefore was continued by all the five patients upto a period ©f 20 weeks. Results In all the five AVH patients associated with HBV infection a clinical as well as biochemical (Table 5, 6) improvement was noted starting from the second week onwards during Rheum emodi aqpeous extract therapy. A complete clinical recovery occurred in all five patients at the end of 4 weeks of therapy. The biochemical improvement as demo\|strated by the nonnalisation of ALT levels and fall in serum bilimbin was noted $t the beginning of the second week. Both the biochemical parameters returned to normal levels between 8 to 12 weeks of therapy. In three out of five patients, HBsAg disappeared after 4 weeks, while in the remaining two after eight weeks of therapy (Table 7). Table 5 Effects of the Rheum emodi aqueous extract (150 mg four times a day x 20 weeks) on physical signs and symptoms of patients of acute classical viral hepatitis associated with Hepatis B virus infection (Number of patients = 5) Rheum emodi aqueous extractTreatment (weeks) (Table Removed) Table 6: Mean levels of serum filtrubin, ALT in patients of acute viral hepatitis associated with Hepatitis virus infection receiving Rheum emodi (150 mg x 4 #mes/day x 20 weeks) (Table Removed) Normal values; Serum Bilirubin = 0.31-1.0 mg/dl, ALT - 10-40 IU/L. Table 7: Status of Hepatitis B virus in patients of Acute viral hepatitis associated with Hepatitis B infection receiving Rheum emodi (150 mg x 4 times/#ay x 20 weeks) (Table Removed) Acute viral hepatitis associated with Hepatitis B virus was diagnosed on the basis of presence of HBsAg + anti Hbc IgM. All patients were negative for Hepatitis A, Hepatitis D and Hepatitis C virus infection. EXAMPLE 4 This example illustrates that the Rheum emodi does not possess anti hepatotoxic property as demonstrated by the failure to protect the liver in acute experimental hepatotoxic models. This observation is illustrated by using known acute hepatotoxic experimental models e.g. galactosamine, and Paracetamol. The pharmaceutical preparation of the aqueous extracts of Rheum emodi for experiments in example 4 were simtar to those described in experiment 1. Material and Methods Animals: Adult male albino rats of % body weight 125 + 15 g were used in the study. During the course of experiment 4 animals were kept in one cage and fed adlib with standard pellet diet and allowel free access to water. Histopathological analysis: At the end of each experiment the animals were sacrificed, their liver was removed in tola and subjected to histopathological examination. Experiment 1 Acute Paracetamol Hepatotoxicity and Rheum emodi aqueous extract Paracetamol tablets (Calpol, 500 mg; Wellcome) were purchased and a dose of 2 gm/kg body weight was prepared ill normal saline. Rats were administered single dose of paracetamol orally by intragastric tube. Four rats each were kept in the following four groups as follows: Group (a): Rheum emodi aqueous extract primed and Paracetamol challenged group: A twice a day dose schedule (100 mg/kg body weight) was used for the Rheum emodi aqueous extract treatment to paracetamol toxicated rats. In this schedule, rats were administered Rheum emodi aqueous extract, 48 hours prior to start of experiment and upto 7 days after receiving the single dose of Paracetamol toxication. Animals were sacrificed 7 days after the toxicant and Rheum emodi aqueous extract treatment. Group (b) : Saline treated and Paracetamol challenged group: Four rats were given saline (500 µ1) for 2 days followed by single dose of Paracetamol. Saline treatment was continued upto seven days followed by sacrifice. Group (c) : Saline treated group: four rats were given saline (500 µl) twice a day and sacrificed after seven days. Group (d) : Rheum emodi aqueous extract treated group: Four rats were given Rheum emodi aqueous extract 100 rag/kg body weight twice a day for seven days and sacrificed. RESULTS Group (a) and Group (b) Animals: The histopathological features were similar in all the animals in these two groups. The significant histopathological alterations were presence of areas characterised by centrizonal hepatocyte necrosis of vaiying degree, hepatocyte degeneration with cellular inflammation (Polymorphs, mononuclear cells) focal haemorrhages, acidophilic degeneration and presence of ballooned hepatocytes. The above findings establish that Rheum emodi aqueous extract does not provide any hepatoprotection activity in against Paracetamol hepatotoxicity. Group (c) (Saline Treated) and Rheum emodi aqueous extract treated group (d): The histopathological features in all the animals in both the two groups were similar. The lobular architecture of liver was preserved and there was no evidence of any pathology. These findings suggest that Rheum emodi aqueous extract administration in rats does not lead to any damage to hepatocytes. Experiment 2 Galactosamine Hepatotoxicity and Rheum emodi aqueous extract Galactosamine (Sigma Chemical Company, St. Louis, USA) was used as 800 mg/kg body weight intraperitonealy as single dose. The experiment comprised of following four groups: (a) Rheum emodi aqueous extract primed and galactosamine challenged group: Four rats were given Rheum emodi twice a day (100 mg/kg body weight) for seven (7) days, followed by a single dose of galactosamine, alongwith one day additional Rheum emodi aqueous extract therapy Animals were sacrificed twenty four (24) hours after galactosamine administration. (b) Saline treated and galactosamine challanged group: Four rats were given saline (500 µl) twice a day for seven days followed by "a single dose of galactosamine and sacrificed after twenty four (24) hours. (c) Saline treated group: Four rats were given saline (500 µ1) twice a day treatment for seven days and sacrificed (d) Rheum emodi aqueous extract treated group: Four rats were given Rheum emodi aqueous extract 100 mg/kg twice a day for seven days and sacrificed. Results Group (a) and (b) Animals: The histopathological featuers in the liver of all the animals with or without Rheum emodi aqueous extract treatment and galactosamine challenge were similar. The significant features observed were, hepatocyte balloon degeneration, centiizonal hepatocyte necrosis, foamy hepatocyte vacuolation, acidophilic degeneration, mononuclear cell infiltration and occasional fatty change and portal inflammation. These findings establish that the Rheum emodi aqueous extract does not provide any hepatoprotection in galactosamine hepatotoxicity. Saline treated Group (c) and Rheum emodi treated group (d): The histopathological features of animals receiving either Rheum emodi or saline treatment were similar. The lobular architecture of liver was preserved and there was no evidence of any pathology. These findings suggest that Rheum emodi administration in rats does not lead to any damage to hepatocytes at cellular level. Conclusion of hepatoprotective studies with Rheum emodi aqueous extract Both the acute hepatotoxicity experiments described above demonstrate the following two features (Table 8). (a) Rheum emodi aqueous extract does not act as antihepatotoxic agent (c) Rheum emodi aqueous extract per se does not produce any liver toxicity. Table 8: Summary of Example 4 (Table Removed) [Method of treating hepatitis B virus The treatment relates to two kinds of hepatitis B virus infections namely acute (recently acquired) infection and chronic carriers (infected more than 6 months). In case of acute infection a daily dose of 0.5 to 1.5 mg of the present aqueous composition can be orally administered. Preferably, 300 mg to 1.5 mg of the composition can be given orally in 2 to 4 divided dosages for a period of 4 to 9 months. On the other hand, regarding chronic infection, the dosage indicated above can be given for a longer duration ranging from 6 months to 2 years. We claim: 2. A process for the preparation of a synergistic novel herbal pharmaceutical composition useful in the treatment of hepatitis B viral infection in mammals, said process comprising the steps of: a) preparing a solvent extract froln the plant parts of Rheum emodi in a method such as herein described; b) evaporating the extract under reduced pressure below 50°C to obtain a residue; c) mixing the residue obtained with water; d) keeping the aqueous extract overnight at 37°C with stirring and repeated centrifugation; e) drying the solution obtained under vacuum and dissolving it in normal saline for preparation of a stock solution, and optionally mixing the solution with pharmaceutical^ acceptable ixcepients or additives to obtain a synergistic herbal pharmaceutical composition, and if desired, converting the same into a suitable oral dosage form sucfc as tablets, capsules or syrups by any known method. 2. A process as claimed in claim 1 wherein the plant parts are selected from nodes, stem or roots of Rheum emodi. 4 A process as claimed in claim I wherein the plant parts are dried and powdered before preparation of the solvent extract. 5 A process as claimed in claim 1 wherein the solvent is selected from water ethanol, methanol, chloroform, hexane, ethyl acetates, carbon tetrachloride, toluene, acetone and mixtures thereof. 6 A process as claimed in claim 1 wherein the residue is mixed with pharmaceutically acceptable additives in pharmaceutically acceptable amounts to obtain herbal composition suitable for oral administration. 7. A process as claimed in claim 1 wherein the extract is centrifuged at 10,000 rpm. 8. A process as claimed in any one of the preceding claims wherein the novel composition containing Rheum emodi is converted into forms such as tablet, capsule or solution and the average dosage per day to human being can vary from 250 to 900 ml and such dosage can be administered 2 to 5 times in equal or

Full Text

FILED OF THE INVENTION
The present invention relates to a process for the preparation of a synergistic novel herbal pharmaceutical composition derived from a plant namely Rheum emodi, which is useful for the treatment of Hepatitis B virus infection, a process for the preparation of an extract from Rheum emodi and a composition comprising extract of Rheum emodi.
BACKGROUND OF THE INVENTION
Rheum emodi is a plant grown in sub-Himalayan regions of India and its neighbouring countries ('Chopra RN, Nayar SL and Chopra IC. 1059/ The characteristics of this plant are given below:
Rheum emodi
Botanical Name : Rheum emodi wall
Family Polygonaceae
Hindi Name Revand chini
English Name Indian Rhubarb
Habitat and Distribution : Sub-alpine and alpine Himalayas upto
12,000ft.above sea level
Botanical Description : A stout herb with woody large roots,
Radicle leaves, long petioles, very large
orbicular or broadly ovate
flowers in panicles. Fruits ovoid oblong.
Himalayan species of Rhubarb are found at altitudes of 4,000 to 12,000 feet and in States of Kashmir and Sikkim in India as well as in Bhutan. The traditional preparations consist of the dried rhizome, or underground stem of the plant, either

whole or cut pieces of suitable length. The roots are dug up, cut transversely into short pieces which are dried in the sun or by artificial means.
Constituents - Rhubarb root contains a large proportion of Chrysophanic acid, sometimes called Chrysophan, an allied substance Emodin, a glucoside rhapanliein, a tannin named Rheo tannic acid several resins, an albuminoid principle, mucilage, extractives, tannic and gallic acids, sugar, starch, pectin, lignan, calcium oxalate and various inorganic salts. The well known grittiness of the root is due to crystals of calcium oxalate. Leaves contain oxalic acid.
Action & Uses - Rhubarb is largely employed in modem medicine as a purgative and is a good laxative. It never produced sickness in any one. Rhubarb is stomachic, bitter, tonic and cathartic so that its secondary effect is confined to bowels movements..
Hepatitis B virus (HBV): Man is the natural source and reservoir of Hepatitis B virus infection (Sherlock S and Dooley J. Disease of the liver and biliary system Page 269-280, 1993 Blackwell Scientific Publications Oxford, England./ Hepatitis B virus is an Hepadna virus with a size of 42 nra. Morphologically, the virus is complex, double shelled with surface and core antigen comprising the outer surface and inner core antigen. 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 of human beings are susceptible to this infection. Endemicity exists and high risk groups include blood recepients, hamophiliacs, dialysis patients, intiavenous 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 rise 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 carrier. 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 early childhood. Most of the infections are acquired in early childhood, usually as mother to child transmission and results in a persistent HBV carrier stage. HBV carriers have 200 times more risk of liver cancer.
The status of serological markers of HBV infection in man and their clinical significance is summarised as follows:
Acute infection : HBsAg, anti HBc IgM
Chronic Infection : HBsAg persisting more than 6 months
Virus replication HBeAg
HBV-DNA
Carrier stage : HBsAg
Protection : Anti HBs
Infection on Wane : Anti HBe
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 (I) active universal immunisation in childhood and/or (2) development of treatment modalities for persistently infected persons. Intenuption of perinatal transmission is critical in

prevention of chronic infection. For this purpose, either vaccine, or post exposure prophylaxis in form of drug will be very important. However, therapeutic studies conducted over the past 15-20 years had only marginal success once the infection is established. Several compounds, such as antiviral agents, biological response modifiers and immune stimulants regime administered alone or in combination have been evaluated (Hoofnagle JH. 1991, ). Most of these agents have transient effects on viremia, with rebound occurrence on withdrawal of drug. Significant toxicity has been observed with long term therapy. Though, HBV was discovered to be the causative agent of Hepatitis B almost 30 years ago, a detailed 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 to propagate the virus. The advent of molecular cloning techniques and the discovely 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 wese collectively grouped under Hepadna viridae(Gust ID, Burrell CJ, Coulpis AG et al. 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 vims was isolated from Woodchuck. 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 woodihuck sera exhibited a similar size and moiphology 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
4
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 vims(DHBV) revealed unexpected results that approximately 10% of domestic ducks in the United Slates were congenially in¬fected with virus(summers 1987). At fbout the same time, a third hepadna virus was identified in beechy ground squirrel (GSHV) in California(Morion etal. 1980). Hepadna virus have also been reported in tree squirrels(Mehrotra etal. 1990, Feitelson et al. 1990). Duck hepatitis B vims (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 {Mason el al. 1980). The average transmission occurs through egg from a carrier mother. The experimental DHBV transmission studies have established that within Hepadna viridae, the age at which infection occurs determines whether the resulting viremia will be persistent or transient. Earlier the age of embryo in ducks greater is viremia(Lamhert el al. 1991). This situation is similar to that of HBV infection in man in that infection before or at birth and infancy results in high carrier rate. 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 becomes persistently viiemic. 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 vims infection and also the natural plant products(Munshi et al. 1993(a) (h), 1994). Several antiviral gents have been evaluated in chronically infected ducks. DHBV infection has ieen associated with a broad spectrum of liver pathology e.g. chronic hepatitis, cirrhosis and hepatocellular carcinoma (Duflot A, Mchrotra R, Yu SZ el al. 1995).

Hepatitis B virus infection and Natural plant products
Many plant products have been in use in the treatment of liver diseases since time immortal. In Indian system of traditional medicine (Ayurvedia), natural plant products have been used in the treatment of jaundice and liver disorders. The use of various plants have been described in ancient Indian Ayurvedic literature. In South India, the plants of genus Phyllanthus and Eclipta alba are commonly used as a traditional treatment for clinical jaundice including that of viral hepatitis and are commercially available (Thyagarajan, 1986; Thyagarajan & Jayaram, 1992). Phyllanthus species are also used in China, the Phillippines, Cuba, Nigeria, Guam, Bast and West Africa, the Carribean, Central America and South America. The above two plants have been recently evaluated for their action on Hepatitis B virus. The first screening strategy used was to test the ability of plant extract to coat viral HBsAg and thereby inhibit the reaction with antibody to HBsAg (anti HBs) (Unander & Blumberg, 1991; Blumberg et al, 1990). The rationale was that such an inhibition might have effect on pathogenesis of Hepatitis B virus in vivo in man (Mehrolra et ai, 1990, 1991). This observation was also supplemented by the ability of plant extiact to inhibit DNA polymerase (DNAp) of Hepatitis B virus in vitro. The crude extracts of Eclipta alba when mixed with HBsAg brings about its interaction, thereby suggesting the presence of anti Hbs like activity (Thyagarajan et ai, 1982). The extracts of Phyllanthus niruri now known as Phyllanthus a mams inhibit human HBV-DNAb as well as HBsAg and anti HBs interaction. Also, the closely related animal hepadna viruses, e.g. Wookchuck hepatitis virus, and duck hepatitis B virus are also affected by P. amarus (Blumberg et ai, 1990). In a series of studies on long term carriers of woodchucks infected with woodchuck hepatitis B virus it was observed that the P. amarus extract generally eliminates or decrease the level of vims (Venkaleshwaran et ai, 1987; Venkateswaran and Blumberg -CS Patent No. 4673575, 1987). However, subsequent studies using another animal model of Human Hepatitis B virus; Duck hepatitis B virus failed to conlnni the

findings reported earlier [Nitu et al, 1990; Munshi et al, J993(a),(b)J. Clinical studies in humans using P. amarus, on Hepatitis B virus has also been carried out. In 1988, it was reported from Madras that dried milled P. amarus was successful in clearing Hepatitis B surface antigen from 59% carriers of Hepatitis B virus (Thyagarajan et al, 1988). However, this observation was followed by another report wherein the success rate of treatment with P. amarus was reduced to 20% (Thyagarajan et al, 1990). In subsequent clinical studies on Hepatitis B virus carriers with P. amarus, results were not reproducible by other workers in different other countries (Brook, 1988; LeeLarasamee et al, 1990; Mei-Xia et al, 1991; Milne et al, 1993). Traditional plant medicines have also been tested for suppression of viral antigen secretion by Alexander cell line (PLC7PRF/5), obtained from human hepatocellular carcinoma which contain several integrated copies of HBV genome and produces small amount of HBsAg apparently in the form of 22 nm particles. Goto et al (1996) using the above cell line as a model for persistently HBV infected liver tested forty three (43) extracts of herbal medicines for suppression of secretion of HBsAg in vitro. Of the various extracts found to have appreciable effects, Rheum Palmatum strongly suppressed the secretion of HBsAg. The Alexander cell line model has also been used to evaluate the inhibition Suppression of HBsAg by P. Amarus (Jayaram & Thyagarajan 1996).
The ultimate aim of therapy with HBV infection is to render patients less infectious which helps in recovery, thereby the development of chronic sequelae (Cirrhosis, hepatocellular carcinoma) can also be prevented. A positive therapy should aim towards reduction in HBV replication. This activity can be assessed by demonstrating a decrease in serum HBV-DNA, clearance of HBeAg and HBsAg. So far, there is no effective and economical treatment for Hepatitis B virus infection and its associated pathological complications.

Therefore, after much research, the applicants have detected for the first time that the extract of Rheum emodi can cure Hepatitis B virus infection, a hitherto unknown property of the herb Rheum emodi.
OBJECTS OF THE INVENTION
The main object of this invention relates to a pharmaceutical composition comprising a plant namely Rheum emodi in any form for treating hepatitis B virus infection in animal or human being.
Another object of the invention is for providing a pharmaceutical composition comprising extracts of Rheum emodi for the tieatment of acute and chronic hepatitis B viral infection in animal or human being.
Yet another object of the invention relates to a pharmaceutical composition useful for the tieatment of hepatitis B vims infection, which comprises of fraction of Rheum emodi, said fraction contains extractable components of Rheum emodi, said fraction having anti HBsAg, anti HBeAg like activity and also produces an effect on HBV-DNA.
Further object of the invention relates to a pharmaceutical preparation for treatment of hepatitis B virus infection which comprises of aqueous extract of Rheum emodi, said fraction having effect on clearance/intermittent effects on viremia of HBV in human beings and animal model such as Duck hepatitis B virus.
Another object of the invention provides a pharmaceutical preparation comprising, of a fraction of Rheum emodi, said fraction prevents the development of the

hepatits B virus (DHBVO carrier state in animals, for example non-infected ducklings. Still another object of the invention relates to a process of preparing pharmaceutical aqueous extract of Rheum emodi which does not produce any toxicity to systemic parenchymatous organs of the body e.g. Heart, kidney, lung, pancreas and gastrointestinal tract.
A further object of the invention relates to a process for the preparation of a composition comprising extract, preferably aqueous extract of Rheum emodi for the treatment of hepatitis B virus infection.
To achieve the above objects, the present invention provides a process for the preparation of a synergistic novel herbal pharmaceutical composition useful in the treatment of hepatitis B viral infection in mammals, said process comprising the steps of preparing a solvent extract from the plant parts of Rheum emodi in a method such as herein described; evaporating the extract under reduced pressure below 50°C to obtain a residue; mixing the residue obtained with water; keeping the aqueous extract overnight at 37°C with stirring and repeated centrifugation; drying the solution obtained under vacuum and dissolving it in normal saline for preparation of a stock solution, and optionally mixing the solution with pharmaceutically acceptable excepients or additives to obtain a synergistic herbal pharmaceutical composition, and if desired, converting the same into a suitable oral dosage form such as tablets, capsules or syrups by any known method. DETAILED DESCRIPTION OF THE INVENTION
The above objects are achieved by providing a pharmaceutical herbal formulation including extract of Rheum emodi. Preferably, the pharmaceutical preparation is an aqueous extract of Rheum emodi. The solvent used in the extract of Rheum emodi can be any suitable solvent including water. The suitable solvent may be

selected from ethanol, methanol, chloroform, water, hexane, ethyl acetate, carbon, tetrachloride, toluene, acetone etc. and the mixtures thereof, in the extraction process, the whole plant and parts thereof powered and extracted with water or with any other suitable solvent. The extract was evaporated under reduced pressure below 50°C leaving a residue. For human administration, the residue was 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. Preferably, the stock solution of the plant extract was used to prepare the present novel composition by mixing with any suitable additives.
The extract obtained from Rheum emodi is mixed with pharmaceutically acceptable reagents to obtain a composition. The composition thus obtained is a synergistic composition having unexpected properties. The ratios of the aqueous extract with the pharmaceutically acceptable reagents will vary depend on the requirements and other conditions. A process for the preparation of a synergistic pharmaceutical composition is comprising an extract of Rheum emodi and pharmaceutically acceptable reagents. The novel composition containing Rheum emodi is converted into forms such as tablet, capsule or solution and the average dosage per day to human being can vary from 250 to 900 ml and such dosage can be administered 2 to 5 times in equal or unequal divided portions.

BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS
In the drawings :
Fig.l: represents TLC Chromatogram photograph of Rheum emodi deducted under UV at 254nm.
Fig.2 : represents TLC Cliromatogram photograph of Rheum emodi deducted under UV at 366nm.
Fig. 3 : represents. HPLC Chromatogram photograph of Rheum emodi deducted under UV at 254nm.
The plant Rheum emodi was botanically authenticated and the applicants have taken pains to analytically standardize the extract of Rheum emodi, which is locally called Revand Chini in India. The finger print profile of Rheum emodi is given below :
Finger print profile of the REVAND CHINI (Rheum emodi)
Rhubarb roots of Revand chini (Rheum emodi) are used here. It contains Chrysophanic acid, an allied substance EMODIN", a glueoside Rhapantiein, a Tannin named Rheotannic acid. A method of finger print by Thin Layer Chromatography (TLC) and high performace liquid chromatography (HPLC) method were developed.
THIN LAYER CHROMATOGRAPHY (TLC)
Using a silica gel F254 precoated plates (Merck silica gel 60 F254 are suitable) and a mixture of 50 volumes of ethyl acetate, 7 volumes of Formic acid, 3 volumes of Glacial acetic acid, 3 volumes of ethyl methyl ketone, 10 volumes of water as the mobile phase. Apply separately to the plate 10 µ1 of the following solutions. For solution (1) of extract, separately weigh about 1 gm of extract in a beaker, add 2.5 ml of ethyl acetate, shake for about 30 minutes and filter. After removal of the plate, allow it to dry in air and examine under ultravoilet light at 254 nm and 366 µm.
OBSERVATIONS: In UV 254 nm (TLC chroinalograin photograph No, I accompanying the specification) orange, yellow and some other zones are visible. The broad zone in the upper Rf (at about 0.56) represents mixture of glycosides of emodin and chrysophanic acid. In 366nm (TLC chromatogram photograph No.2 accompanying the specification) some aglycones migrate with the solvent front to an Rf at about 0.80 and some other zones also appear in the lower Rf range of 0.36-0.42.
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC)
Using the following solution (1) Weigh about 1 gm of sample in a beaker, add 20
ml of mobile phase, warm, shake and filter. The chromatographic procedure are
carried out using (a) A stainless steel column ODS (C18) (300 mm X 4.6 mm), (b)
a mixture of 50 volumes of 0.1% of V/V of oilhophosphoric acid and 50 volumes
of methanol as mobile phase with a flow rate 1 ml per minute and (c) detection
wavelength of 254 nm.
OBSERVATIONS: There (HPLC chromatogram, Figure No.3) are about 10 components which are separated out, these are in different concentrations. These components are correlated to the components of reference extract. These components are most probably aglycones and one of them in chrysophanic acid.
Hepatitis B virus (HBV) infection
Human Hepatitis B virus (HBV) infection is a public health problem. HBV is associated with both acute and cliionic liver diseases including the development of cirrhosis and hepatocellular carcinoma. There are about 300 million healthy carriers of HBV worldwide about half have ongoing viral replicatiein and are at work for the development of progressive liver disease. There is no effective drug which is effective against HBV infection except interferon in modem medicine. Treatment with interferan alpha can lead to viral replicatien and romessian of active liver disease in nearly 407 of selected patients. However, individuals who are immunosuppersed, and have high level of viral rfeplicatien or low levels of serum amino transferases are unlikely to benefit from this therapy. Further, administration of interferan is cimbersome, costly and limited by side effects. No therapy is available for the interruption of persistent HBV carrier state in man HBV is a DNA vims belonging to Hepadna viridae family, which has a number of animal prototypes, one of which is Duck Hepatitis B virus (DHBV) present in special breed of ducks: Pekin ducks. The DHBV shows more than 75% homology with HBV and has been successfully used as an experimental model to screen various agents and plant products which might have an effect against HBV. and thereby useful and beneficial for man.
Screening of pharmaceutical preparation of Rheum emodi extract in DHBV model
The Pharmaceutical preparation in the form of extract of Rheum emodi can be obtained by any known method and all parts or the whole of the plant Rheum emodi can be used to obtain extracts thereof
The Pharmaceutical preparation from the Indian plant Rheum emodi has been obtained and screened in DHBV model to (I) test the effects on clearance of persistent DHBV viremia. (2) prevention of the development and establishment of DHBV carrier state in non viremic ducklings, and to (3) demonstrate in vitro effects on HBV. Prolonged administration of aqueous(preferably aqueous extract) extract of Rheum emodi shows an encouraging response on the persistent DHBV viremia by producing DHBV clearance in 30% animals receiving therapy as well as leads to intemiittent periods of non viremia in DHBV carrier ducks during treatment. Further, a prolonged administration of extract(preferably aqueous extiact) of Rheum emodi does not produce any toxic effects on the parenchymatous organs of the animals receiving treatment. The in vitro treatment of extract(preferably aqueous extract) of Rheum emodi with the infective inoculum of DHBV and its subsequent intraperitoneal administration to 72 hrs old DHBV negative ducklings inhibits the development of persistent DHBV carrier state in animals as compared to their control.
The extract(preferably aqueous extract) of Rheum emodi also has anti HBs and anti HBe like activity as demonstrated by in vitro effects on HBV by producing the inhibition of HBV surface antigen (HBsAg), Hepatitis B virus e' antigen (HBeAg) and also has an effect on HBV-DNA. The prefened extiact here is the aqueous extiact.
The invention also covers a process of pharmaceutical preparation of extract of Rheum emodi and effects of extract of Rheum emodi on Hepatitis B Virus (HBV) infection. Here again the extract is an aqueous extiact since it is cheap and easy to prepare.
Further, the invention provides a method of heating Hepatitis B virus infection by administering a pharmaceutical herbal preparation comprising Rheum emodi. The treatment includes administering the extract of Rheum emodi for accute and chronic Hepatitis B viral infection in animals or human beings.
The present method of treatment includes administering a pharmaceutical composition comprising fraction of Rheum emodi, said fraction contains exhactable components of Rheum emodi, said fraction having anti HBsAg, anti HBeAg like activity and also produces an effect on HBV-DNA.
Furthermore, the present invention comprises administering to a patient a pharmaceutical preparation for the cliarance/intermittent effects on viremia* of HBV in said patient. The patient can be a human being or animal model such as Duck hepatitis B virus.
The treatment includes administering to a patient a pharmaceutical preparation comprising of a fraction of Rheum enwdi tliereby preventing the development of the hepatitis B virus (DHBV) carrier State in the patient which may be a non-infected duckling.
The preparation of Rheum emodi can toe in any form such as tablet, capsule or solution. The average dosage per day to a human being can vary from 250 to 900 ml and such dosage can be administered 2 to 5 times in equally or unequally divided portions. The treatment may vary from 3 to 6 months and for certain acute cases the period of treatment may be extended.
The present invention is illustrated with reference to the following examples andj these examples should never be conitrued to limit the scope of the present
invention. In other words, the following examples are merely to illustrate the present invention and such examples do not in any manner limit the scope of the invention.
Example No. 1 describes the (i) process of pharmaceutical preparation and effects of aqueous extiact of Rheum emodi on the persistent DHBV viremia in adult animals, and (ii) the prevention of the development of DHBV viremia in DHBV non-infected ducklings.
Example No. 2 describes the in vitro effects of aqueous extract of Rheum emodi on Hepatitis B Vims (HBV).
Example 3 describes the controlled clinical trial of Rheum emodi in patients of acute viral hepatitis (AVH) associated with Hepatitis B virus infection.
Example 4 describes that Rheum emodi does not possess any antihepatotoxic properties as demonstrated in experimental studies. Example 4 also describes that aqueous extracts of Rheum emodi does not produce any damage to hepatocytes at cellular level.
EXAMPLE 1
This example is illustrated by experiment 1 and experiment II as described below.
Experiment No. I: Pharmaceutical preparation and elucidation of the effects of aqueous extract of Rheum emodi on the DHBV carrier state.

MATERIAL AND METHODS
Pharmaceutical Preparation of aqueous extract of Rheum emodi
An aqueous extract of Rheum emodi can be prepared by any known manner such as mixing the plant material obtained from any part of the plant with water.
Preferably, all the batches of Rheum emodi used in the study were collected from Sikkim and botanically authenticated. The roots were dried, powdered and extracted with water. The extract was evaporated under reduced pressure below 50 degree Celsius leaving a solid residue. The solid residue was mixed with water, kept overnight at 37°C with stirring and supernatant was extracted by repeated centrifugation at 10,000 rpm. The solution thus obtained was vacuum dried and the dry weight of extract was determined. This dry extract was dissolved in a fixed volume of normal saline to make a stock solution and stored at 4C. The stock solution of the extract was diluted with phosphate buffered saline (pH 7.4) to obtain the desired strength required for experimentations.
The piefened strength/quantum required for treatment can be 50 mg to 200 mg per kg / body weight and it can be administered orally daily for 12 to 20 weeks. The sequence of treatment can be daily in continuous manner for the stipulated period of treatment.
Production of DHBV viremic and nonviremic animals for experimentation The Indian strain of DHBV has been; characterised by cloning and sequencing (Munshi et al. 1994) and a persistent viremic/non-viremic colony of the animals were maintained. The eggs from boil the groups (DHBV viremic/DHBV non viremic) were obtained, marked on shell and hatched for 30 days to obtain ducklings. The ducklings were raised on the food and water ad lib and when

attaining the adulthood at age of 12 weeks were incorporated in the experiment described below.
Experimental Design: A total of 7 three month old viremic ducks were given aqueous extract of Rheum emodi (100 µg/kg body weight) as prepared in 2, twice daily orally for a period of twelve (11) weeks. Post treatment follow-up of the animals was carried out upto a maximum period of six (6) weeks after stopping the therapy.
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 intervals, during the aqueous extract of Rheum emodi treatment and after cessation of the treatment. All the samples were subjected to determination for the presence of DHBV-DNA by Dot Blot hybridization. The animals were sacrificed at the end of their follow-up period and an autopsy was performed.
Controls: Ten ducks viremic and ten non-DHBV viremic ducks were used as controls for aqueous extract of Rheum emodi experiment. During the experimentation they were given normal phosphate buffer saline (placebo) in a similar protocol as with the experimental group and sacrificed with the treated animals at the end of the study.
Studies on serum: Detection of DHBV DNA by Dot Blot hybridization DHBV-DNA in serum was analyzed by molecular hybridization as described before (Dot Blot hybridization method).
Histopathological Studies: Liver, kidney, pancreas, heart and lungs were

collected at autopsy from each animal for histopathological examination to rule out

any toxic effects of the aqueous extriet of Rheum emodi treatment. The tissue samples were fixed in buffered formalin paraffin embedded using routine standard laboratory techniques and cut at 4 p. and stained with hematoxylin and eosin for examination.
RESULTS
These have been described below as (i) effect of aqueous extract of Rheum emodi on the persistent DHBV viremia and (fe) DHBV in control group.
(a) The effect on the serum DHBV DNA during aqueous extract of Rheum emodi therapy and the subsequent follow up after stoppage of therapy. The results have been summarized in the table 1. Twelve weeks therapy with aqueous extract of Rheum emodi produced DHBV-DMA clearance in 40% animals. There were intermittent periods of DHBV-DNA clearance in 20% animals during therapy, while in 40% there was no effect.
(b) Controls: All the DHBV virenasic and non viremic animals maintained their serum DHBV status during the study.
(c) Toxicity study: Animals receiving aqueous extract of Rheum emodi 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 was
no histopathological alteration in the above parenchymatous organs.

Table 1 : Effects of therapy by aqueous extract of Rheum emodi on persistant DHBV infection
(Table Removed)
Experiment 11: Prevention of establishment of DHBV viremia by aqueous extract of Rheum emodi in non infected ducklings.
Material and Methods
The pharmaceutical preparation of the aqueous extracts of Rheum emodi for experiment were similar to those described in experiment I.
In vitro treatment of DHBV with aqueous extract of Rheum emodi: 200 pL of DHBV positive serum (as detected by Dot Blot hybridization) obtained from a

persistently DHBV viremic duck was mixed with 200 µ1 of aqueous extract of Rheum emodi (0.5 to 6 mg/ml preferably 4 mg/ ml) and this mixture was incubated in a moist water bath at 37°C for two hours followed by centrifugation at 2000 rpm and the supernatant was used as innoculum for ducklings (50 µ1/animal) as described below.

Animals: 20 ducklings were obtained by hatching the eggs obtained from DHBV negative female ducks. The ducklings were bled immediately after hatching and serum were tested for DHBV. All the 20 ducklings were found to be negative for DHBV-DNA and were divided into following two groups:
Group A: 10 ducklings in this group were innoculated at 72 hours, with 50 pL of aqueous extract of Rheum emodi treated DHBV-DNA positive serum intraperitoneally.
Group B : 10 ducklings in this group were innoculated at 72 hours, with 50 p.L of DHBV-DNA positive and PBS treated serum intraperitoneally.
Bleeding: All the animals from Group A were bled at 7 days intervals post-inoculation, upto period of 28 days and at the end of the experiments they were sacrificed and liver, heart, kidney, lung, intestine were subjected for histopathological study.
Results: Table 2 summarises the findings in the establishment of DHBV carrier state after in vitro treatment of DHBV-DNA positive serum sample and its control. The percentage protection of DHBV-DNA negative ducklings inoculated with DHBV-DNA positive serum in vitro treated with aqueous extract of Rheum emodi from viremia development at the end of 28 days was only 20% as compared to the control (100%). (In other words, 80% of ducklings among the Rheum emodi treated group were protected against the development of subsequent Viremia)

Table 2: Shows the effects of aqueous extract of Rheum emodi on prevention of the establishment of DIIBV vircmia after in vitro treatment of the iuuoculum
(Table Removed)

The above findings of the experiment 11 demonstrate that aqueous extract of Rheum emodi when in vitro heated with DHBV-DNA positive serum samples, the applicants presume that a sort of binding occurs with the virus thereby making latter non infectious. This observation was further extended to evaluate the effect of the duration of aqueous extract of Rheum emodi in vitro treatment in hours with DHBV-DNA positive serum and the subsequent development of DHBV viremia in ducklings. The DHBV-DNA positive serum samples were treated with Rheum emodi extract for 1, 3 and 6 hours at 37°C in a moist water bath and subsequently utilised as innoculum in DHBV-DNA negaitve duckling obtained from DHBV negative mothers as described above and followed for a period of 28 days. It was observed that the duration of aqueous extract of Rheum emodi in vitro treatment with DHBV-DNA was independent of its effects produced on DHBV infectivity and the results were similar to the two hours invitro treatment as described above.

EXAMPLE 2
In vitro effects of aqueous extract of Rheum emodi on Hepatitis B virus:
The following three sets of experimints demonstrate the presence of in vitro effects of aqueous extract of Rheum emodi extract on Hepatitis B virus surface antigen (HBsAg), Hepatitis B virus e'antigen (HBeAg) and Hepatitis B virus DNA (HBV-DNA).
a) In vitro Effects of aqueous extract of Rheum emodi on HBsAg:
The preparation of the extract of Rheum emodi for the use in the experiment were similar as described in Example 1 of Experiment 1.
Clinical Material: The HBsAg positive serum samples were obtained from healthy HBV carriers (20-45 yrs age group) patients of acute viral hepatitis (AVH), and chronic liver diseases (Cirrhosis of liver). Each serum sample was serially two folds diluted in phosphate buffered saline and titrated for the presence of HBsAg to determine HBsAg title. All the sera were negative for rheumatoid factor.
HBsAg Assay Method: A double sandwich solid phase enzyme immune assay (ELISA) kit using monoclonal antibody (Wellcome Diagnostics, UtK.) were used to test HBsAg.
The tests were performed as per manufacturer's instructions and results were read on ELISA reader (Organon, Holland).

Treatment of Serum Samples with aqueous extract of Rheum emodi : A 0.5-6 mg/ml, preferably 4 mg/ml concentration of Rheum emodi extract was used in the study. Serum samples were mixed withiequal volume of Rheum emodi extract and incubated for 12 hours at 37°C in a moist chamber. This mixture was centrifuged at 2000 ipm for 10 minutes and subsequently subjected to HBsAg screening by ELISA as described above. With each set of scrum, controls comprising of untreated samples (saline treated) were simlarly incubated for 12 hours and tested for HBsAg. The mean fall in serum HBsAg after aqueous extract of Rheum emodi treatment was calculated.
Results: The results of individual samples are shown in Table 3. The mean HBsAg titre of serum fell after aqueous extract of Rheum emodi treatment. The range of HBsAg reduction after in vitro treatment varied from 5 to 12 folds (mean
6.75).
Table 3: Shows the folds reduction in HBsAg titre after in vitro treatment of serum samples with aqueous extract of Rheum emodi in the serum samples obtained from various HBV associated conditions.

(Table Removed)
b) In vitro effects of aqueous extract of Rheum emodi on HBeAg:
The preparation of the extiact of Rheum emodi for experiment was similar as described in example 1 of experiment 1.

Clinical Material: HBeAg positive sera were obtained from eight healthy HBV carriers patients, of Acute HBV viral hepatitis and HBV associated Liver Cirrhosis. These serum samples were serially two fold diluted in phosphate buffered saline and tested for the presence of HBeAg to determine its titre. All sera were negative for Rheumatoid factor.
HBeAg Assay Method: A double sandwich solid phase enzyme immune assay (ELISA) kit supplied by Wellcome Diagnostics, U.K. was used to determine HBeAg. The tests were performed as per manufacturer's instructions and results were read on ELISA reader (Organon, Holland).
Teatment of serum samples with aqueous extract of Rheum emodi : A 0.5 to 6 mg/ml, preferably 4 mg/ml concentration of aqueous extract of Rheum emodi was used in the study. Serum samples serially diluted in phosphate buffered saline were mixed with equal volumes of aqueous extract of Rheum emodi and incubated for 12 hours at 37°C in a moist water bath. This mixture was centrifuged at 2000 rpm for 10 minutes and subsequently subjected to HBeAg screening by ELISA as described above. With each set of samples, control comprised of untieated samples (saline treated) and similarly incubated and tested for HBeAg. The mean fall in serum HBeAg after aqueous extract of Rheum emodi treatment was calculated.
Results : The results of the individual serum samples is shown in Table 4. The mean HBeAg titre of serum samples was 1/452.88 which fell to 1/22.80 after aqueous extract of Rheum emodi treatment. The range of HBeAg reduction after in vitro treatment varied from 4 to 7 folds (mean 5.25).

Table 4: Shows fold reduction in HBeAg titre after in vitro treatment of serum samples with Rheum emodi in samples obtained from various HBV associated conditions
(Table Removed)

c) In vitro effects of aqueous extract of Rheum emodi on HBV-pNA:
The preparation of the aqueous extract of Rheum emodi for experiment were
similar to those described, in example 1 experiment I.
Clinical material: Four semm sanpipies from patients of acute viral hepatitis B
positive for HBV-DNA (mean concentration 90 pg/ml) were used.
HBV-DNA determination: A radiological molecular hybridization assay for the detection and quantitation of Hepatitis B viral DNA in serum assay (Abbott Genostics Hepatitis B Viral DNA, Abbot's diagnostics, North Chicago, USA) was used as per manufacturer's instruction to determine the HBV-DNA in serum samples both before and after in vitro treatment with aqueous extract of Rheum emodi. The results were expressed as mean percent inhibition of HBV-DNA. Treatment of serum samples with aqueous extract of Rheum emodi: HBV-DNA positive serum samples (4) were mixed with 0.5 to 6 mg/ml, preferably 4 lng/ml aqueous extract of Rheum emodi and incubated for 12 hrs at 37°C in a moist chamber. Control comprised of saline treated samples and treated similarly. At the end of incubation HBV-DNA was determined in both aqueous extract of Rheum emodi treated and untreated samples (controls). The results were expressed as percent inhibition of HBV-DNA concentration.
Results: The mean HBV DNA of serum samples was reduced from 90 pg/ml to 28.55 pg/ml after in vitro aqueous cxtiact of Rheum emodi teatment representing 31.72% reduction.
EXAMPLE 3 CONTROLLED CLINICAL fRIAL OF RHEUM EMODI AQUEOUS EXTRACT IN PATIENTS OF ACUTE VIRAL HEPATITIS (AVH) ASSOCIATED WITH HEPATITIS B VIRUS (HBV)
Material and Methods
Five patients (3 male, 2 female) of a mean age 31.50 (range 18-45 years) presenting within one week of the onset of their clinical symptoms suggestive of classical acute viral hepatitis (AVH) were included in the study. The diagnosis of AVH was established by the clinical symptoms and abnormal biochemical features

(Sherlock & Dooley, 1993). The diagnosis of acute HBV infection was done on the basis of presence of HBsAg along with anti HBc IgM in the patient. The other possible causes which might have lead to a similar clinical presentation e.g. surgical jaundice, drug induced cholestasis, heipes, cytomegalovirus infection (as demonstrated by the absence of the tespective IgM antibodies) were absent in the study patients. In all five patients the liver function tests (Serum bilirubin, ALT) and HBsAg and anti Hbc IgM (Elisa) were carried out to establish the diagnosis before the start of their therapy with Rheum emodi, and subsequently at eveiy four week intervals duiing therapy upto a period of twenty (20) weeks. Prior consent of all the patients were obtained ant they were explained about the treatment protocol and follow up procedure.
The preparation of the Rheum emodi aqueous extract for clinical use in the above patients were similar as described in example 1 of experiment 1. Each patient was given a dose of 250 mg four times a day packed in gelatin capsules which correspond to a total Rheum emodi dose of 1000 mg per day for each patient. However, with this dose all the five patient just after one day, complained of loose motions. Therefore, from the second day onward the dose of Rheum emodi aqueous extract was reduced to 150 mg four times a day corresponding to a total dose of 600 mg per day for each patient. This dose was well tolerated by all the five patients without any gastrointesinal disturbances and therefore was continued by all the five patients upto a period ©f 20 weeks.
Results
In all the five AVH patients associated with HBV infection a clinical as well as biochemical (Table 5, 6) improvement was noted starting from the second week onwards during Rheum emodi aqpeous extract therapy. A complete clinical recovery occurred in all five patients at the end of 4 weeks of therapy. The

biochemical improvement as demo|strated by the nonnalisation of ALT levels and fall in serum bilimbin was noted $t the beginning of the second week. Both the biochemical parameters returned to normal levels between 8 to 12 weeks of therapy. In three out of five patients, HBsAg disappeared after 4 weeks, while in the remaining two after eight weeks of therapy (Table 7).
Table 5
Effects of the Rheum emodi aqueous extract (150 mg four times a day x 20
weeks) on physical signs and symptoms of patients of acute classical viral
hepatitis associated with Hepatis B virus infection (Number of patients = 5)
Rheum emodi aqueous extractTreatment (weeks)
(Table Removed)

Table 6: Mean levels of serum filtrubin, ALT in patients of acute viral hepatitis associated with Hepatitis virus infection receiving Rheum emodi
(150 mg x 4 #mes/day x 20 weeks)
(Table Removed)

Normal values; Serum Bilirubin = 0.31-1.0 mg/dl, ALT - 10-40 IU/L.
Table 7: Status of Hepatitis B virus in patients of Acute viral hepatitis associated with Hepatitis B infection receiving Rheum emodi (150 mg x 4
times/#ay x 20 weeks)
(Table Removed)

Acute viral hepatitis associated with Hepatitis B virus was diagnosed on the basis of presence of HBsAg + anti Hbc IgM. All patients were negative for Hepatitis A, Hepatitis D and Hepatitis C virus infection.

EXAMPLE 4
This example illustrates that the Rheum emodi does not possess anti hepatotoxic property as demonstrated by the failure to protect the liver in acute experimental hepatotoxic models. This observation is illustrated by using known acute hepatotoxic experimental models e.g. galactosamine, and Paracetamol.
The pharmaceutical preparation of the aqueous extracts of Rheum emodi for experiments in example 4 were simtar to those described in experiment 1.
Material and Methods
Animals: Adult male albino rats of % body weight 125 + 15 g were used in the study. During the course of experiment 4 animals were kept in one cage and fed adlib with standard pellet diet and allowel free access to water.
Histopathological analysis: At the end of each experiment the animals were sacrificed, their liver was removed in tola and subjected to histopathological examination.
Experiment 1
Acute Paracetamol Hepatotoxicity and Rheum emodi aqueous extract
Paracetamol tablets (Calpol, 500 mg; Wellcome) were purchased and a dose of 2 gm/kg body weight was prepared ill normal saline. Rats were administered single dose of paracetamol orally by intragastric tube. Four rats each were kept in the following four groups as follows:

Group (a): Rheum emodi aqueous extract primed and Paracetamol challenged group: A twice a day dose schedule (100 mg/kg body weight) was used for the Rheum emodi aqueous extract treatment to paracetamol toxicated rats. In this schedule, rats were administered Rheum emodi aqueous extract, 48 hours prior to start of experiment and upto 7 days after receiving the single dose of Paracetamol toxication. Animals were sacrificed 7 days after the toxicant and Rheum emodi aqueous extract treatment.
Group (b) : Saline treated and Paracetamol challenged group: Four rats were given saline (500 µ1) for 2 days followed by single dose of Paracetamol. Saline treatment was continued upto seven days followed by sacrifice.
Group (c) : Saline treated group: four rats were given saline (500 µl) twice a day and sacrificed after seven days.
Group (d) : Rheum emodi aqueous extract treated group: Four rats were given Rheum emodi aqueous extract 100 rag/kg body weight twice a day for seven days and sacrificed.
RESULTS
Group (a) and Group (b) Animals: The histopathological features were similar in all the animals in these two groups.
The significant histopathological alterations were presence of areas characterised by centrizonal hepatocyte necrosis of vaiying degree, hepatocyte degeneration with cellular inflammation (Polymorphs, mononuclear cells) focal haemorrhages, acidophilic degeneration and presence of ballooned hepatocytes.

The above findings establish that Rheum emodi aqueous extract does not provide any hepatoprotection activity in against Paracetamol hepatotoxicity.
Group (c) (Saline Treated) and Rheum emodi aqueous extract treated group
(d): The histopathological features in all the animals in both the two groups were similar. The lobular architecture of liver was preserved and there was no evidence of any pathology. These findings suggest that Rheum emodi aqueous extract administration in rats does not lead to any damage to hepatocytes.
Experiment 2
Galactosamine Hepatotoxicity and Rheum emodi aqueous extract
Galactosamine (Sigma Chemical Company, St. Louis, USA) was used as 800 mg/kg body weight intraperitonealy as single dose. The experiment comprised of following four groups:
(a) Rheum emodi aqueous extract primed and galactosamine challenged group:
Four rats were given Rheum emodi twice a day (100 mg/kg body weight) for seven (7) days, followed by a single dose of galactosamine, alongwith one day additional Rheum emodi aqueous extract therapy Animals were sacrificed twenty four (24) hours after galactosamine administration.
(b) Saline treated and galactosamine challanged group: Four rats were given saline (500 µl) twice a day for seven days followed by "a single dose of galactosamine and sacrificed after twenty four (24) hours.
(c) Saline treated group: Four rats were given saline (500 µ1) twice a day treatment for seven days and sacrificed

(d) Rheum emodi aqueous extract treated group: Four rats were given Rheum emodi aqueous extract 100 mg/kg twice a day for seven days and sacrificed.
Results
Group (a) and (b) Animals: The histopathological featuers in the liver of all the animals with or without Rheum emodi aqueous extract treatment and galactosamine challenge were similar.
The significant features observed were, hepatocyte balloon degeneration, centiizonal hepatocyte necrosis, foamy hepatocyte vacuolation, acidophilic degeneration, mononuclear cell infiltration and occasional fatty change and portal inflammation. These findings establish that the Rheum emodi aqueous extract does not provide any hepatoprotection in galactosamine hepatotoxicity.
Saline treated Group (c) and Rheum emodi treated group (d): The
histopathological features of animals receiving either Rheum emodi or saline treatment were similar. The lobular architecture of liver was preserved and there was no evidence of any pathology.
These findings suggest that Rheum emodi administration in rats does not lead to any damage to hepatocytes at cellular level.
Conclusion of hepatoprotective studies with Rheum emodi aqueous extract
Both the acute hepatotoxicity experiments described above demonstrate the following two features (Table 8).
(a) Rheum emodi aqueous extract does not act as antihepatotoxic
agent

(c) Rheum emodi aqueous extract per se does not produce any liver toxicity. Table 8: Summary of Example 4
(Table Removed)

[Method of treating hepatitis B virus
The treatment relates to two kinds of hepatitis B virus infections namely acute (recently acquired) infection and chronic carriers (infected more than 6 months). In case of acute infection a daily dose of 0.5 to 1.5 mg of the present aqueous composition can be orally administered. Preferably, 300 mg to 1.5 mg of the composition can be given orally in 2 to 4 divided dosages for a period of 4 to 9 months. On the other hand, regarding chronic infection, the dosage indicated above can be given for a longer duration ranging from 6 months to 2 years.

We claim:
2. A process for the preparation of a synergistic novel herbal pharmaceutical composition useful in the treatment of hepatitis B viral infection in mammals, said process comprising the steps of:
a) preparing a solvent extract froln the plant parts of Rheum emodi in a method such as herein described;
b) evaporating the extract under reduced pressure below 50°C to obtain a residue;
c) mixing the residue obtained with water;
d) keeping the aqueous extract overnight at 37°C with stirring and repeated centrifugation;
e) drying the solution obtained under vacuum and dissolving it in normal saline for preparation of a stock solution, and optionally mixing the solution with pharmaceutical^ acceptable ixcepients or additives to obtain a synergistic herbal pharmaceutical composition, and if desired, converting the same into a suitable oral dosage form sucfc as tablets, capsules or syrups by any known method.
2. A process as claimed in claim 1 wherein the plant parts are selected from nodes,
stem or roots of Rheum emodi.

4 A process as claimed in claim I wherein the plant parts are dried and powdered before preparation of the solvent extract.
5 A process as claimed in claim 1 wherein the solvent is selected from water ethanol, methanol, chloroform, hexane, ethyl acetates, carbon tetrachloride, toluene, acetone and mixtures thereof.
6 A process as claimed in claim 1 wherein the residue is mixed with pharmaceutically acceptable additives in pharmaceutically acceptable amounts to obtain herbal composition suitable for oral administration.

7. A process as claimed in claim 1 wherein the extract is centrifuged at 10,000 rpm.
8. A process as claimed in any one of the preceding claims wherein the novel composition containing Rheum emodi is converted into forms such as tablet, capsule or solution and the average dosage per day to human being can vary from 250 to 900 ml and such dosage can be administered 2 to 5 times in equal or

Documents:

797-del-1996-abstract.pdf

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797-del-1996-complete specification (granted).pdf

797-del-1996-correspondence-others.pdf

797-del-1996-correspondence-po.pdf

797-del-1996-description (complete).pdf

797-del-1996-description (provisional).pdf

797-del-1996-drawings.pdf

797-del-1996-form-1.pdf

797-del-1996-form-2.pdf

797-del-1996-form-26.pdf

797-del-1996-form-4.pdf

797-del-1996-form-5.pdf

797-del-1996-form-6.pdf

797-del-1996-form-9.pdf

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Patent Number

189717

Indian Patent Application Number

797/DEL/1996

PG Journal Number

16/2003

Publication Date

19-Apr-2003

Grant Date

27-Jan-2004

Date of Filing

15-Apr-1996

Name of Patentee

DABUR RESEARCH FOUNDATION

Applicant Address

22, SITE IV, SAHIBABAD, GHAZIABAD 201 010, INDIA, WITH ITS REGISTERED OFFICE AT 8/3, ASAF ALI ROAD, NWE DELHI-110002, INDIA.

Inventors:

#	Inventor's Name	Inventor's Address
1	RAJ MEHROTRA	9, SHAHMINA ROAD, LUCKNOW-226 003, INDIA.

PCT International Classification Number

A61K 31/00

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA