| Title of Invention | A PHARAMACEUTICAL FORMULATION FOR THE TREATMENT OF HEPATITIS & VIRAL INFECTIONS AND A PROCESS FOR ITS PREPARATION |
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| Abstract | The invention disclosed in this application relates to a Chemo - biologically Standardised pharmaceutical formulation which comprises (i) an extract of the plant Phy//anthus amarus obtained by using a polar solvent alone other than water (ii) an extract of the plant Phy//anthus amarus obtained by using a mixture of a polar solvent and water and (iii) an extract of the plant Phy//anthus amarus obtained by using water alone. The invention also provides a process for its preparation. The composition is useful for the treatment of Hepatitis B [both acute and chronic], Hepatitis C [both acute and chronic] and other related viral infections of the liver. |
| Full Text | This invention relates to a pharmaceutical formulation useful for the treatment of Hepatitis viral infections and a process for its preparation. This invention particularly relates to a pharmaceutical formulation useful for the treatment of acute and chronic Hepatitis B & C virus infections prepared from the Indian biotype medicinal plant Helianthus amours. This invention also relates to a process for the preparation of the pharmaceutical formulation useful for the treatment of acute and chronic Hepatitis B and Hepatitis C and other viral infections of the liver from the medicinal plant Phyllanthus amarus. It is needless to stress the need for a successful drug that would keep the liver functioning at its optimum or the one that would be selectively active against the currently known etiological agents of acute viral diseases of the liver. This is important because the disease of the liver throw the entire human body out of gear. The exciting alphabet of viral Hepatitis includes a wide range of totally unrelated often highly unusual pathogenic human viruses like Hepatitis A virus (HAV), Hepatitis B virus (HBV), Hepatitis C virus (HCV), Hepatitis D virus (HDV), Hepatitis E virus (HEV) etc. Of the viruses it has been dearly established that HBV, HCV and HDV are the ones that are associated with the development of chronic persistent/active hepatitis, cirrhosis of the liver and even hepatocellular carcinoma besides being associated with culminant hepatitis and sub acute hepatic failure. The natural disease course in HBV is being summarized to understand the need for the effective management and treatment of Hepatitis B in a country. For normal adults with low viral production and an early immune response, the disease course is self limiting and usually asymptomatic (60-80% of all HBV infections). Individuals who replicate the virus in larger quantities, with a relatively late immune response have a self-limiting symptomatic acute hepatitis. Irrespective of whether initially symptomatic or asymptomatic, the infection becomes chronic in 5-10% of the individuals, 20-30% of them developing clinical sequelae such as chronic hepatitis, cirrhosis or hepatica within years or decades. In neonates, however, the immune defense is still lacking (induction of tolerance), so that infected individuals do not develop acute hepatitis, but more of them become chronic carriers (80-90%). Such carriers also progress frequently to chronic clinical sequelae faster. Between these Uvo extremes are immunocompromised individuals, such as intravenous drug users, haemodialysis patients or transplant recipients, who are more likely to become chnjnic carriers than are healthy adults (10-60%). (WHO Tech. Report. Series 1987;754:18) Based on substantial body of data, HBV has been proved as a major pathogen producing chronic liver diseases. It has also been proved that there are over 400 mllliCKi healthy earners of HBV all over the worid and one tenth of these carriers (40 millions) t)eing in India alone. These carriers besides acsng as htrnian reservoirs of HBV infection also act as primary soun::e of spread of HBV Infection to the community and was also shown to have 200 times increased risk of develc^ng chronic liver diseases and/or hepatocellular carcinoma. With the above documented international HBV scenario, the HBV epidemiology in India is to be considered as alarming since there are definite data on pre«ilence pattern of HBV in asymptomatic population (4%) high risk groups (13%), significant inwslvement of HBV in Indian acute and sub-acute liver feilure cases (42 & 45%). 70% of the chronic hepatitis cases, 40-80% of ctrrtrasis cases and over 60% of primary liver cancer cases. Although effective vaccines have been devdoped against HBV and successfully adopted, the need for effective treatment of acute and chronic Hepatitis B has become universal public health emergency since vaccines as on date are neit^ier capable of inducing Immunity in a carrier nor able to diminate HBV carrier status. Research conducted from the mid 70s have delineated several agents to have treatinent potential in chronic HBV infections which has be^ illustrated In tiie Table 1 given below. Table 1 Agents that have been studied in the treatment of HBV Infection (Lau etal.. Gut. Suppl. 1991;S47-S62) Immunosuppressive Corticosta^ds Anti-virals Interferons Alf^a interferon Beta interferon Gamma interferon Immunostimu lators BCG vaccination Levamisole Interleukin-2 Interferon-gamma Thymosin Tumor necrosis factor Tumour necroas fector Adenine arabino^de (Ara-A) Acyclovir, deoxyacydovir Zidovudine Suramin Ribavirin Phosphonoformate Quinacrine (+) -cyanidanol-3 Lamuvidine Phyflanthus amarus However, except ttie interferons, Lamuvidine and the latest entry PhyllanUius amarus, the others seem to be ftr from successful. The limited success rate, prohibitive cost, profound side effects and the non-accessibility of interfenans and lamuvidine in developing and underdeveloped countries have necessitated further search for newer antihepatitis B agents. ACUTE AND CHRONIC HEPAimS-C: 1. Acute HCV Infection Persons with acute HCV infection typically are either asymptomatic or have a mild clinical illness ; 60%-70% have no discernible symptoms ; 20%-30% might have jaundice ; and 10%-20% might have nonspecific symptoms (e.g. anorexia, malaise or abdominal pain). Qinical illness in patients with acute hepatitis C who seek medical care is similar to tJiat of other types of viral hepatitis, and serologic testing is necessary to determine the etiology of hepatitis in an individual patient. In a 20% of these patients, onset of symptoms might precede anti-HCV saoconversion. Average time period from exposure to symptom onset is 6-7 weeks, w/hereas average time period from e)qx>sure to seroconversion is 8-9 weeks. Anti-HCV can be detected in 80% of patients within 15 weeks after exposure, in > 90% witiiin 5 months after exposure, and in > 97% by 6 months after exposure. Rarely, seroconversion might be delayed until 9 months after ej^sure. The course of acute hepatitis C Is variable, although elevations in semm ALT levels often in a fluduating pattern, are its most characteristic feature. Normalization of ALT levels might ocojr and suggests fliti recovery, but this is frequentiy fbilowed by ALT elevations tfiat indicate progression to chronic disease. Fulminant hepatic feilure following acute hepatitis C Is rare. However, in developing country especially India, HCV in FHF was reported significantiy. 2. Chronic HCV Infection After acute Infection, l5%-25% of persons appear to resolve their infection without sequelae as defined by sustained absence of HCV RNA in serum and normalization of ALT le\«ls. Chronic HCV infection devel{^ in most persons (75%-85%), with persistent or fluctuating ALT elevations indicating active liver disease devdoping in 60%-70% of chronically infected persons. In the remaining 30%-40% of chronically Infected persons, ALT levels are normal. No clinical or epidemic^ogic features among patients with acute infection have been found to be predictive of either persstoit infection or chronic liver disease. Moreover, various ALT patterns have been otiserved in these patients during follow-up, and patients might have jXDlonged periods ( ^ 12 months) of normal ALT activity even though they have histologically confimied dironic b^Mtitis. Thus a single ALT detemiination cannot be used to exclude ongoing hepatic injury, and long term follow-up of patients witii HCV infection is required to determine their clinical outcome or prognosis. The course of chronic liver disease is usually insidious, progressing at a slow rate without symptoms or physical signs in the majority of patients during the first two or more decades after inl^ction. Frequentiy, chrcwiic hepatitis C is not recognised until asymptomatic persons are identified as HCV positive during blood donor screening or elevated ALT levels are detected during routine physical examinations. Most studies have reported that cirrhosis develops in i0%-20% of persons with chronic hepatitis C over a period of 20-30 years, and HCC in i%-5%, with striking geographic variations in rates of this disease. This difference is more discemable also in the SEA region countries. Although factors predicting severity of liver disease have not been well defined, recent data indicate that increased alcohol intake, being aged >40 years at infection, and being male are associated with more severe liver disease. In particular, among persons with alcoholic liver disease and HCV infection, liver disease progresses more rapidly ; among those with cirrhosis, a higher risk for development of HCC exists. Furthermore, even intake of moderate amounts (>10 g/day) of alcohol in patients with chronic hepatitis C might enhance disease progression. More severe liver injury observed in persons with alcoholic liver disease and HCV infection possibly is attributable to alcohol induced enhancement of viral replication or increased susceptibility of cells to viral injury. The cur^nt treatment guidelines of HCV infection are presented below : . Current efifective therapy for HCV infection is IFN based, with or without other therapeutic agents such as ribavirin. Ribavirin monotherapy is not recwnmended. . For the interim, patients with acute hepatitis should receive IFN-a, 3-6 million units (or 9-15 pg) thrice weekly for atleast 6 months until more effective regimens emerge. . The standard treatment for previously untreated (naive) patients with chronic hepatitis C is IFN-a, 3-6 million units (or 9-15 pg) thrice weekly fw 12 months. However, recent data indicate that a regimen of IFN-a and ribavirin for 6 months or IFN-a monotherapy using different schedules and/or higher doses may significantiy improve sustained response rates and become preferred options for treatment in the future. . Adverse side-effects to IFN and ribavirin are tolerable, but a fatal outcome (suicide, liver failure, sepsis) has been observed primarily in patients with cirrriosis. Less severe side-effects occur in less than 10% of the treated patients and include flu-like symptoms, fetigue, thinning of hair, myalgia, bone marrow suppression requiring dose reduction, neuropsychiatric effects, such as depression and autoimmune disease (thyroid). All patients must be carefully monitored by the prescribing doctor for side-effects by using appropriate biochemical, haematotogical and immunological tests. Appropriate medical records should be maintained. Newer Search of Antiviral Agents Against Hepatitis B and Hepatitis C One of these searches for the last two decades has been in the development of a promising antiviral agent against hepatitis B and hepatitis C and otiier viral infections of liver from the plant, Phyllanthus amaais. Phyllanthus niruri Linn, as it has been indexed in majority of published ethnobotanlcal reviews, until recently, belongs to the family Euphorbiaceae. Phytlanthus is one of the largest genera of the family Euphorbiaceae containing about 700 species. It has been shown that about 24 species of Phyllanthus are active against clinical Hepatitis (Jaundice) as indicated in TaWe 2, out of which 8 have been used in India. Table 2 Ust of Phvllanthus soedes used in dinical jaundice (Unanderetal., 3 Etfinopharmacol 1991;34:97-133) 1. Phyilantttus niruri 2. P. amarus 3. P. fi-atsmus 4. P. mimiajs 5. P. debilis 6. P. urinaria 7. P. can^ini&isis 8. P. abnormis 9. P. airy-shavii 10. P. tenelius 11. P. gasstmemi 12. P. gunni 13. P. similus 14. P. tiiymodes 15. P. hirtdlus 16. P. sdpulatus 17. P. nirurddes 18. P. rheedi 19. P. acutifdius 20. P.hutdiinosdianus 21. P. cantoniensis 22. P. vityatus 23. P. corcovadensis 24. P. palanessis TAXONOMY OF PHYLLANTHUS AMARUS: This plant has recently been delineated as a mixture of three distinct species namely Phyilantiiusamarus, Phyilanthus frat&nusand Phyflanttiusdebilis. It was later idenlafied that the drumtropical weed P. amanjs is the predominant species in South India, particularly in Tamilnadu. P. amarus are erect annual herbs, 10-60 cm tall ; main stem simple or branched, terrete smooth or scabridulous in younger parts. Cataphylls, stipules 1.5-1.9 mm long, ddtoid acuminate blade 1-1.5 mm long, subulate acuminate. Deciduous branchlets 1.5-14 cms long, subserete, smooth or a few lower nodes sometimes scabridulous witii 13-30 distichous leaves. Leaves 3-11 x 1.5 x 6 mm elliptic oblong obovate, oblong, or even obovate, obtuse, or minutely apiculate at apex, obtuse or slightiy Inequilateral at base, petioles 0.3-0.5 mm long , stipules 0.8-1.1 mm long triangular accuminate. Flowers in axillary, unisexual and bisexual cymles on deciduous branches. Prodmal 2-3 axils with unisexual cymules, each consisting of 1 male and 1 female or 2(-3) males and fenale or 1 male and 2 female flower or combination thereof ; male flowers pedicals at anthesis ca 1 mm long. Calyx lobe 5, subequal each c3 0.7 x 0.3 mm elliptic or oblong elliptic and abruf^y acute at apex hyaline with unbranched mid ribs. Disc segments 5, roundish stames 3 (rarely 2): filaments connate into a column 0.2-0.3 mm high autheros sessile a top dehiscing longitudinally. Female flowers; pedicles 0.8-1 mm long, obtusely 4 gonous, dialated above, ca 1.5 mm in fruits, calyx 5 lobes, subequal. Ovate-oblong, acute at apex, midsepaline band green. Disc flat, deeply 5 lobed. Lobes sometimes toothed at apex. Styles 3, free, more or less spreading, shallowly bifid at apex ; arms divergent (Mitra & Jain, Bull Bot Surv Ind 1985;27:167-176). HISTORICAL USE OF P. NIRURI IN JAUNDICE Even though clinical uses of P. nimri and other species viz. P. amarus dted for over a century in the Ayurvedha and Siddha literatures, scientific evaluatory studies have been attempted only during the last 50 years for its efRcacy in the treatment of jaundia;/viral hepatitis. A logical approach towards Identification of the active principles of P. amarus is to fractionate the plant extracts and identify biologically active compounds and to chemically characterise them. STUDIES ON P.NIRURI/P. AMARUS AGAINST HBV: The first ever designed invitro antiviral shjdy on Phyllanthus niruri against any hepatitis virus with HBV as model was reported by Thyagarajan in 1979 fhxn Madras CThyagarajan, Ph.D. Thesis, University of Madras 1979), India. Subsequentiy, Thyagarajan et al (1982) have shewn the whole plant extract of P. ninjri thnxjgh several solvents bn^ught about binding of Hepatitis B surface antigen (HBsAg) (Thygarajan et al., Ind J Med Res 1982; 76(Suppl.): 124-130. This plant from Tamilnadu, India was later identified taxonomically by Unander as P. amarus. Venkateswaran et al (1987) (Proc Nati Acad Sci USA 1989;14:195-201) and Blumberg et al (1989) (Cancer detection and prevention 1987;84:274-278). fn^m United States using the P. amarus plants provided by Thyagarajan have shown that the plants collected from Madras, India whose aqueous exbacts bound the surface antigen of HBV invitro, have inhibited tiie viral DNA polymerase (DNAp) of HBV and Woodchuck hepatitis virus (WHV) invitro. When administered intraperltoneally to WHV infected woodchucks, acutely infected animals lost the viral surface antigen; the surfece antigen titre dropped in some chronically infected animals; the liver cancer rate in treated chronically infected animals was lower than the untreated controls. Based on these findings they had secured an Australian patent numbered AV-A-56530/86 for a composition of matter useftjl in the treatment of Hepatitis B vinjs infection consisting essentially of the methanol extractable components of P. niruri L. Yanagi et al., (Meeting on Hepatitis viruses, Sept 25-28, 1989, Cold Spring Haror Laboratory, NY, 1989,77) ft^m Japan have reported that aqueous extracts of high dilutions of P. amarus collected from South India inhibited HBV DNAp, DNApI, T4-DNAp, the Klenow fragment and reverse transcriptase of avian myeloblastosis virus. Shead et al (1990) (1990 International symposium on viral Hepatitis and liver diseases, April 4-8, 1990, Houston, TX, USA; A602) from Australia have shown tfie aqueous extracts to inhibit the endogenous DNAp of DHBV at high dilutions. Niu etal (1990) fbrm Australia in collatwration vi/ith Thyagarajan from India using P. amams collected from Madras, Tamilnadu, on treatment of 4-5 week old ducks congenitally infected with Duck hepatitis B virus (DHBV) with suitable controls after a period of 10 weeks treatment showed transient reduction of wral DNA in serum but no effect on the level of virus DNA or suface antigen in the liver (J Med Virol 1990;32:212-218). Jayaram et al (1996) (Ind J Pathol Microbiol 1996;39(3);211-215) reported invitro inhibition of HBsAg secr^on by PLCyPRF/5 (Alexander) cell line for 48 Mrs when the cellline was treated with 1 mg/ml concentration of P. amarus as a single dose. Lee et al (1996) (European J Qin Invest 1996; 26:1069-76) from USA in collaboration with TTiyagarajan have shown that P. amarus down-regulates Hepatitis B vims m RNA transcription and replication using transgenic mice and transgenic celllines. The continuation of this collaboration by Ott et al (1997) has shown the cellular and molecular mechanism of HBV suppression by P. amarus to be by interrupting interactions between HBV enhancer I and cellular transcription factors (European J Clin Invest 1997; 27-.908-915). The biosafety studies on P. amarus dates back to 1971 when Mokkhasmit et al from Thailand using P. niruri have reported it to be non toxic to mice at 10 gms/kg body weight (Bull of Dept of Medical Science NAPRALERT Chicago, IL, 1971;12:36-65). Rao (1985) from Andhra Pradesh, India reported 20% aqueous extract of P. ninjri leaves to be effective to be as an oral pretreatment of 0.2 ml/lOO mg body weight against CCU induced hepatotoxicity in rats (Probe 1985;115-119). Syamasundar et al (1985) from Uttar Pradesh, India showed the hexane extracted compounds Phytlanthin and Hypo phyllanthin reduced Ca4 or galatosamine induced cytotoxicity to cultijred rat hepatocytes (J Ethnopharmacol 1985;14:41-44). Jayaram et al (1987) from Madras, India using the aqueous extract of dried whole plant showed no chronic toxicity in mice at 0.2 mg daily dose per animal for 90 days as revealed by physiological, biochemical and histopathological parameters. There was also no cybDtonic or cytotoxic changes when tested in a tissue culture model using vero cell line (Biomedicine 1987;7:9-16). Venkateswaran et al (1987) fi"om USA demonstrated its invivo safety using woodchucks as animal models, while Niu et al (1990) from Australia have shown P. amanjs to be non toxic in Pekin ducks chronically infected with duck Hepatitis B virus. 3ayaram and Thyagarajan (1994) studying ttie effect of P. amarus on p-galactosamine induced hepatotoxicity on isolated rat hepatocytes have shown that a) P. amarus by itself did.not bring about any hepatotoxidty on rat hepatocytes. b) At 1 mg/ml concentration the aqueous extract were shown to protect isdated rat hepatocytes significantly from p-galatosamine induced hepatotoxidty thus ptiving the anti hepatrtoxic potentials of P. amanjs (Ind J Med Microbiol 1994;12(4):247-250). In all the traditional medidne systems, there has bewi several formulatory medicines for the treatment of jaundice in general without taking into consideration their virat etiology. Even though P. niruri was one of the constituents of such medicines, these wens always been multiherbaf ^Keparatkms oontaining anywhere upto 12 medicinaf hertas and most of the treatment evaluations were based on clinical Improvement onfy. On the other hand, there is no documented blal report on their use In chronic liver disease patients. It was in this context, Thyagarajan and his collaboratore, after proving the Invitro and invivo efficacy and safety of P. amams, conducted 2 open dinical trials In acute viral hepatitis cases and seven cElnical trials (2 of th&r\ being double blind trials and the others Phase I/II open trials) In chronic carriers of Hepatitjs B vims (HBV) Jayanthi et al (1988)CJ Gasfroenterol and Hepatxrf 1988; 3:533-534) In a control dinical trial in acute viral hepatitis (AVH) using P. nimri on one arm, and other hertjal medicines in other groups have shown a signiflcantiy greater decrease In transaminases after two weeks treatment with P. ninjri in both HBsAg positive and negative groups. In a virologically characterised AVH dinical trial, Geetiia et at (1992) (J Gen Medidne 1992;4(2): 53-58) have ^lown that a) P. amarus treatmeit has brought about significantiy fester biochemical nonnalcy in botii hepatitis A and B ; b) there was a higher rate of HBsAg dearance in P. amarus treated AVH-B cases than ottrer treatment modalities and c) there was no observable side effects due to P. amarus treatment. The dinical trials were conducted by Thyagarajan et al between 1988 and 1997. While the first dial 1988 (Lancet 1988; 2:764-766) reported 59% HBsAg clearance in the P. amarus treated group, as against 4% in tiie placebo gnsup, the second open trial (1990) (Lancet 1990;2: 949-950) showed 20% HBsAg clearance and 63.6% loss of Infectivity Indicated by HBeAg sero-conversion. Paralldy, Investigators from other countries like l-eelarasamee et al (1990) (Lancet 1990;l:1600-160i) from Thailand, Wang Me Xia et al from China (1991) (Hepatology RIR 1991;21(5):22-24) have reported tfie non produdbility of treatment efficacy by the local variety of P. amanjs grown in tiieir respective countries. CONCEPT & HYPOTHESIS It was the feeling that the non-reproducibliity of treatment efficacy of tiie extract of the plant Phyllanthus amams was due to the absence in the extract of all the under mentioned antiviral and biological properties which are essentially required for the efficient treatment of Hepatitis B (both acute and chronic) Hepatitis C and other rdated viral infections of the liver. (i) HBsAg binding property fecilitating the inactivation of the virus in circulation ultimately leading to viral dearance. (li) HBV-DNA pdymerase enzyme inhibiting potential, thus acting as anti-wral preventing the multiplication of HBV. (iii) Reverse Transcriptase enzyme inhibition also required for the initiation of HBV replication, (iv) Inhibition of HBsAg secretion from HBV transinfected liver cells thus possessing activity against virus infected chrcffiic live- disease conditions, (v) Hepatoprotective and antih^jabatoxic properties against the liver cell toxidty Ixought abOit by all hepatitis viruses (A,BAD &. E) and other hepatotoxic agents, (vi) Immunomodulating property to potentiate the immune system of HBV infected patients towards virus clearance and protective antibody (and HBs) responses. In the light of the above mentioned studies coupled with the above mentioned observations, it was necessary to explain tiie reasons for non-reproducibiiity of the dinical efficacy of P. amanjs on one hand and to conduct further dinlcal trials indepCTdently in different places using the P. amarus preparatim of Thyagarajan. Accordingly clinical trials were condurted on a total of 153 dironic HBV carriers ( 3 in Chennai (Madras), 1 at Vellore and 1 at Glasgow, UK). The results are presented in Table 3. Table 3-. SutTimarv of seven clinical trials conducted bv Thvaoaraan and his collaborators on HMnrian HBV carriers using P. amanjs grown in TamilnadM- For co^vffl^^e^ce it is termed as "Universiy,^ preparation." Clinical Authors/year Dosage/ Dura Number treated HbsAg clearance HBeAg sero- Trial TO. Mgms/tds tion % conversion % Test Placebo Test PlacetJO Test Placebo 1. Published Thyagarajan et al (1988) 200 Im 40 38 59 4 ND ND 2. Madras Thyagarajan et al (1990) Madras 250 3m 20 Nil 20 63.6 3. Unoublished Benjamin Samuel et al (1991), Vellore 250 2m 10 12 20 8.3 37.5 0 4. Thyagarajan etal (1992), Madras 250 6m 72 Nit 25 54.0 5. Thyagarajan etal (1993), Madras 500 3m 8 8 25 0 71.4 16.0 6. Eric W^ker et al (1993-95) Glasgow 500 4-6 m 26 Nil 11.6 18.9 45.4 7. Thyagarajan et al (1995-97), Madras 500 6 m 37 Nil 60.0 Total 213 58 25.6 3.4 55.3 1.7 ND - Not done In summary, these trials have shown a mean HBsAg clearance rate of 25.6 % and mean HBeAg seroconversion rate of 55.3%. It was finally decided to recommend a schedule of 500 mg dosage of P. amarus preparation in capsules given orally for ttiree times daily for six months. PHYLLANTHUS AMARUS TREATMENT IN ACUTE AND CHRONIC HEPATXnS C Available lilsrature in the public domain did not reveal any report on the use of Phyllanthus amarus in the treatment of acute and chronic hepatitis C virus infection which is another major liver pathogen leading to significant morbidity and mortality. Hence the formulation of Phyllanthus amams envisaged by the present invention v^tas utilised to conduct two clinical trials to treat acute and chronic tiepatitis C. The first clinical trial was conducted on two cases by Dr.Eric Walker and his co-workers at Glasgow, UK during the period 1996-99 using the earlier "University preparation" of Phyllanthus amarus in the ftwm of capsules provided by Thyagarajan et al from Chennai, India. The preliminary results obtained by Eric Walker et al (unpublished) are presented in Table 4. Since there are no experimental animal/tissue culture models available for testing specific antjvirai properties against Hepatitis C virus (HCV), dirert studies on human volunteers infected with HCV were conducted. Ethical clearance and Infwmed consents from the study participants were obtained based on the high safety profile of the preparation of P.amaru5 prepared by the applicants as described above. Table.4. Case Studies on the efficacy of the formulation of Phyllanthus f amarus treatment on chronic hepatitis C infections conducted at Glasgow,UK (Eric Walker et al 1996-99, unpublished) * 1. Patient having the Date of Birth 07/02/49 Summary: The major finding has been the marked improvement in symptoms when the patient was treated with the preparation of Phyllanthus amarus whk^ symptoms relapsed on 2 occasbns when the treatment was withdrawn. The liver enzymes deteriorated when the preparation of Phyllantiius amams was witiidrawn and improved when it was started again. The patient has always been PCR positive. Appearance is of someone who is getfing a "liver protective effect but witiiout elimination of virus. Source was blood transfusion. 29/01/96- Liver biopsy- some inflammation and hepatic fibrosis consistent witti chronic hepatitis C infec^on. Itch, joint pains, lethargy were the main symptoms. Not keen on interieron- Treatment with tiie preparation of Phyilantiius amams was started 23/0496 - The weight of the patient increased by 2kgs. Markedly improved energy and symptoms. Little change in liver enzymes (AZT 42,ALT 50). PCR positive 15/05/96 treatment with the preparation of thy composition containing Phyllanthus amarus was stopped 17/06/96- Itch, joint pains and lettiargy returned {AST 46, ALT 50) 11/07/96 - Treatment with the composition containing Phyllanthus amarus was restarted 10/09/96- Symptoms were much improved (AST46, ALT 71) 01/07/97 - Stilf hepatitis C PCR positive(genotype 3). Repeat liver biopsy shows little change (inflammation and fibrosis but no active cinliotic changes) 05/06/98 - weight of the patient was found to be steady and the patient felt well (AST 42. ALT 59) 08/08/98 - Treatment with the preparation of Phyllanthus amarus was stopped 31/12/98 - The patient was to be depressed and "run down"{AST 62, ALT 75) 21/01//99 - Treatment with the preparation of Phyllanthus restarted following which a marked improvement in the patient"s feeling of well being was observed. 29/12/99 - The patient felt well (AST 50, ALT 69), remained PCR positive 2. Patient having a Date of Birth 20/06/54 Source of HCV infection was intravenous dmg use This patient was one of the first to clear HBsAg and e antigens after treatment with the preparation of Phyllanthus amanjs Back in 1990- he remains negative (1999) He was not tested him for hepatitis C until 1999( April) when he was found to have antibody but was PCR positive He remained well Based on the encouraging clinical efficacy of Phyllanthus amarTjs treatment observed by Eric Walker et al on Scottish cases of chronic Hepatitis C and to confirm the improved efficacy of the new pharmaceutical formulation proposed in the present application, Thyagarajan et al at Chennai, India conducted tile second clinical trial and confirmed the efficacy of the new formulation for the treatment of acute and chronic Hepatitis B & C and other related infections of liver. THE INVENTION: We observed that if all the above said six essential properties are made availaWe in a single fomiulation, the resulting formulation will have uniform and stable antiviral and biological potentials which will be beneficial for the treatment of acute and chronic hepatitis B & C and other viral diseases of the lever. Based on the above findings the R & D work for the developm«it of a new formulation of P. amanjs extract was initiated which formulation will be active significantly against both acute Hepatjtis B and against chronic HBV carriers, Accordingly R&D was directed towands developing such a formulation from the plant Phyllanthus amanjs containing all the above said essential characteristics which have not only efficient dinical and biological efficacy against acute and chronic Hepatitis B; acute and chronic Hepatitis C and Cither viral infections of the liver but also have uniform and stable antiwral and bioactive properties. Objectives of the invention Accordingly, the main objective of the present invention , therefore , is to prowde a pharmaceutical formulation having uniform and statue anitviral and biological efficacy which is useful for the treatment of acute and chronic Hepatitis B, Hepatitis C, chronic HIV carriers and other related viral infections of the liver from the plant, Phyllanthus amanjs Anoth©" c^jective of the present invention is to provide a pharmaceutical fomiulation useful fiar the treatment of Hepatitis B, Hepatitis C, chnanic HIV carriers and other related viral infections of the liver from the plant Phyllanthus amarus which brings about binding of Hepatitis B surface antigei (HBsAg) of the Hepatitis B virus, thus fecititating the inactivation of the virus in circulation ultimately leading to viral clearance. Yet another objective of the present invention is to provide a pharmaceutical farmulation useful for the treatment of Hepatitis B, Hepatitis C, chronic HIV carriers and other related viral infections of tiie liver from the plant P. amarus which inhibit the HBV-DNA polymerase enzyme required for the replication for the virus, thus acting as antiviral preventing the multiplication of the virus itself. still anotfier objective of ttie present invention is to provide a pharmacsutical fomnulation useful fbr tile hutment of Hepatitis B, Hepatitis C, chronic HIV carriers and other related viral infections of Oie liver from the plant P. amarus which inhibits the Reverse Transcriptase enzyme which is also required for the initiation of HBV replication and is the chief enzyme required for the replication of the AIDS vinjs, Human Immunodeficiency virus (HIV). Another objective of the present invention is to provide a process fbr the preparation of a pharmaceutical formication useful fbr the treatment of Hepatitis B, Hepatitis C, chronic HIV earners and other related viral infections of tiie tiver from the plant P. amarus which is hepatc^Jrotective and also possess antihepabDxic properties against the liver ceil toxicity brought about by all Hepatitis viruses (A,B,C,D & E) and other hepatotoxic agents including chemicals and aflatoxins. Yet another objective of the present invention is to pnavide a pharmaceutical formulation useful fbr tiie treatment of acute and chrcffiic Hepatitis B; acute and chronic Hepatitis C chronic HIV carriers and otiier related viral infiactions of the liver from tJie plant P. amarus which is anti-inflammatory and also possesses the property of normalising the transaminase enzymes level indicating antihepatotoxic and liver cell regenerating potentials of the formulation. Stilt another objective of the present invention is to provide a pharmaceutical formulation useful fbr the treatment of acute and chnonic Hepatitis B; acute and chronic Hepatitis C, chronic HIV carriers and other rdated viral infections of the \\y&r fhDm plant P.amarus which inhibited the Hepatitis C vims replication as revealed by conversion from HCV-RNA positivity to HCV-RNA negatitvity in patients treated by the fomiulation. Another objective of the present invention is to provide a pharmaceutical formulation useful for tiie treatment of acute and chronic Hepatitis B; acute and chnanic Hepatitis C chronic HIV carriers and ether related viral infections of the liver from plant P.amarus which is immunomodulatory as revealed by increased T-cell proliferation index and stimulation of protective antibody (anti-HBs) in P.amarus treated chronic HBV carriers who cleared HbsAg as shown in Table 5 Table 5. Anti- HBS Pattern in Pre and Post-Treatment samples of HBsAg cleared and refractoryHBV carriers (Thygarajan et al ; "Proceedings of Indo-UK Symposium on Updates in Viral Hepatitis":1990,63-77) indicative of Immunomodulatory potentials of Phyllanthus amarus. Groups Anti"HBs 1 ntres(IU/l) Pre-treatment Post-treatment HbsAg cleared (15) 15.2 ± 23.6 78.6 ± 43.5 HbsAg persistent (10) 6.8 ± 7,2 10.3 ± 11.4 Still another objective of the present invention is to provide a pharmaceutical formulation useful for the treatment of acute and chronic Hepatitis B, acute and chronic Hepatitis C and other related viral infections of the liver from the plant P. amarus in which the antiviral and biological activities are uniform Yet another objective of the present invention is to provide a process for the preparation of a pharmaceutical formulation useful for the treatment of acute and chronic Hepatitis B acute and chronic Hepatitis C and other related viral infections of the liver from the plant P. amarus with antihepatotoxic, liver cell regenerating and immunomodulating potentials. The invention is based on our surprising findings that when the parts of the plant Phyllanthus amarus are extraded separately with a polar solvent alone, other than water, mixture of polar solvent and water and water alone and when the extracts so obtained are mixed together , the resultant formulation is found to have all the under mentioned properties which are required for the efficient treatment of Hepatitis B (both acute and chronic). Hepatitis C (both acute and chronic) and other related viral infections of the liver, (i) HBsAg binding property facilitating the inactivation of the virus in circulation ultimately leading to viral clearance, (ii) HBV-DNA polymerase enzyme inhibiting potential, thus acting as anti-viral preventing the multiplication of HBV. (iii) Reverse Transcriptase enzyme inhibition also required for the initiation of HBV replication. (iv) Inhibition of HBsAg secretion from HBV transinfected liver cells thus possessing activity against virus infected chronic liver disease conditions, (v) Hepatoprotective, antiinflammatory, antihepatotoxic and liver ceil regenerative properties against the liver cell toxicity brought about by all hepatitis viruses (A,B,C,D &. E) and other hepatotoxic agents, (vi) Immunomodulating property to potentiate the immune system of HBV infected patients towards virus clearance and protective antibody (anti HBs) responses, (vii) HCV-replication inhibition as shown by converting HCV-RNA positivity to HCV- RNA negativity thus possessing activity against HCV infected chronic liver disease conditions. It is observed that the individual extracts of the plant Phyllanthus amarus namely the extract obtained by using a poiar solvent alone , extract obtained by using a polar solvent and water and the extract obtained by using water alone , themselves , does not possess a\\ the above said essential characteristics . But the combination of the above mentioned extracts imparts to the resulting formulation all the above mentioned essential properties which are required for the efficient treatment of Hepatitis B (both acute and chronic), Hepatitis C (both acute and chronic) and other related viral infections of the liver. These properties are acquired by the formulation due to the biological synergism of the different components contained in the individual extracts when combined to form the formulation. The pharmaceutical formulation of the present invention is not, therefore , a mere admixture of the individual components resulting in the aggregation of the properties of the individual components but is a novel pharmaceutical formulation having biological synergism of all the required efficacious antiviral and biological properties of the components employed. Accordingly, the present invention provides a pharmaceutical formulation useful for the treatment of Hepatitis B (both acute and chronic) Hepatitis C (both acute and chronic) and other related viral infections of the liver which comprises (i) an extract of the plant Phyllanthus amarus obtained by using a polar solvent alone other than water (ii) an extract of the plant Phyllanthus amarus obtained by using a mixture of a polar solvent and water and (iii) an extract of the plant Phyllanthus amarus obtained by using water atone According to another feature the present invention provides a pharmaceutical formulation useful for the treatment of Hepatitis B (both acute and chronic). Hepatitis C (both acute and chronic) and other related viral infections of the liver which comprises (i) 10 to 40% w/w of an extract of the plant Phyllanthus amarus obtained by using a polar solvent alone, other than water, (ii) 10 to 40% w/w of an extract of the plant Phyllanthus amarus obtained by using a mixture of a polar solvent and water wherein the ratio of the solvent to water ranges from 20 to 80 and 80 to 20% w/w respectively and ((("() 10 to 40% w/w of an extract of the plant Phyllanthus amarus obtained by using water alone In another preferred embodiment of the invention, there is provided a pharmaceutical formulation useful for the treatment of Hepatitis B (both acute and chronic). Hepatitis C (both acute and chronic) and other related viral infections of the liver which comprises (i) 20 to 30% w/w of an extract of the plant Phyllanthus amarus obtained by using a polar solvent alone, other than water, (ii) 20 to 40% w/w of an extract of the plant Phyllanthus amarus obtained by using a mixture of a polar solvent and water wherein the ratio of the solvent to water ranges from 80 to 20% and 20 to 80% w/w respectively (iii) 20 to 40-% w/w of an extract of the plant Phyllanthus amarus obtained by using a polar solvent and water wherein the solvent to water ratio ranges from 20 to 80 % and 80 to 20 % w/w respectively and (iv) 20 to 30% w/w of an extract of the plant Phyllanthus amarus obtained by using water alone In still another preferred embodiment of the present invention there is provided a pharmaceutical formulation useful for the treatment of Hepatitis B (both acute and chronic). Hepatitis C (both acute and chronic) and other reiated viral infections of the liver which comprises one part each of an extract of the plant Phyllanthus amarus obtained by using a polar solvent a\one , an extract of the p\ant Phyllanthus amarus obtained by using a mixture of a polar solvent and water and an extract of ttie plant Phyllanthus amarus using water alone In yet another preferred embodiment of the present invention there is provided a pharmaceutical formulation useful for the treatment of Hepatitis B (both acute and chronic). Hepatitis C (both acute and chronic) and other related viral infections of the liver which comprises one part each of (i) 10 to 40% w/w of an extract of the plant Phyllanthus amarus obtained by using a po\ar solvent alone, other than water, (ii) 10 to 40% w/w of an extract of the plant Phyllanthus amarus obtained by using a mixture of a polar solvent and water wherein the ratio of the solvent to water ranges from 20 to 80 and 80 to 20% w/w respectively and (iii) 10 to 40% w/w of an extract of the plant Phyllanthus amarus obtained by using water alone In another preferred embodiment of the present invention there is provided a pharmaceutical formulation useful for the treatment of Hepatitis B (both acute and chronic). Hepatitis C (both acute and chronic) and other related viral infections of the liver which comprises one part each of (i) 20 to 30 %w/w of an extract of the plant Phyllanthus amarus obtained by using a polar solvent alone, other than water, (ii) 20 to 40 %w/w of an extract of the plant Phyllanthus amarus obtained by using a mixture of a polar solvent and water wherein the ratio of the solvent to water ranges from 80 to 20% and 20 to 80 % w/w respectively (iii) 20 to 40 % w/w of an extract of the plant Phyllanthus amarus using a mixture of a polar solvent and water wherein the ratio of the solvent to water ranges from 20 to 80 % w/w and 80 to 20 % w/w respectively and (iv) 20 to 30 % w/w of an extract obtained using water alone In still another preferred embodiment of the present invention there is provided a pharmaceutical formulation useful for the treatment of Hepatitis B (both acute and chronic). Hepatitis C (both acute and chronic) and other related viral infections of the liver which comprises two parts (i) of an extract of the plant Phyllanthus amarus obtained by using a polar solvent alone an extract of the plant Phyllanthus amarus obtained by using a polar solvent other than water and one part each of (ii)of an extract of the plant Phyllanthus amarus obtained by using a mixture of a polar solvent and water and (iii) an extract of the plant Phyllanthus amarus using water aione In yet another preferred embodiment of the present invention there is provided a pharmaceutical formulation useful for the treatment of Hepatitis B (both acute and chronic), Hepatitis C (both acute and chronic) and other viral infections of the liver which comprises two parts (i) of 10 to 40 %w/w of an extract of the plant Phyllanthus amarus obtained by using a polar solvent, other than water and one part each (ii) of 10 to 40 % w/w of an extract of the plant Phyllanthus amarus obtained by using a mixture of a polar solvent and water wherein the ratio of the solvent to water ranges from 20 to 80% and 80 to 20 % w/w respectively (Iii) of 10 to 40% w/w of an extract of the plant Phyllanthus amarus obtained by using water alone In yet another preferred embodiment of the present invention there is provided a pharmaceutical formulation useful for the treatment of Hepatitis B (both acute and chronic). Hepatitis C (both acute and chronic) and other viral infections of the liver which comprises two parts (i) of 20 to 30 % W/W of an extract of the plant Phyllanthus amarus obtained by using a polar solvent alone, other than water and one part each (Ii) of 20 to 40 % w/w of an extract of the plant Phyllanthus amarus obtained by using a mixture of a polar solvent and water wherein the ratio of the solvent to water ranges from 80 to 20% and 20 to 80 % w/w respectively (iii) of 20 to 40 % w/w of an extract of the plant Phyllanthus amarus obtained by using a mixture of polar solvent and water wherein the ratio of the solvent to water ranges from 20 to 80% w/w and 80 to 20% w/w respectively and . (iv) of 20 to 30 % w/w of an extract of the plant Phyllanthus amarus obtained by using water alone. In yet another preferred embodiment of the invention there is provided a process for the preparation of pharmaceutical formulation useful for the treatment of Hepatitis B (acute and chronic), Hepatitis C (both acute and chronic) and other related viral infections of the liver which comprises mixing (i) an extract of the plant Phyllanthus amarus obtained by using a polar solvent alone (ii) ar) extract of the plant Phyllanthus amarus obtained by using a mixture of a polar solvent and water and (iii) an extract of the plant Phyllanthus amarus obtained by using water alone According to another feature the present invention provides a process for preparing a pharmaceutical formulation useful for the treatment of Hepatitis B (both acute and chronic). Hepatitis C (both acute and chronic) and other related viral infections of the liver which comprises mixing (i) 10 to 40% w/w of an extract of the plant Phyllanthus amarus obtained by using a polar solvent alone, other than water (ii) 10 to 40% w/w of an extract of the plant Phyllanthus amarus obtained by using a mixture of a polar solvent and water wherein the ratio of the solvent to water ranges from 20 to 80 and 80 to 20% w/w respectively and (iii) 10 to 40% w/w of an extract of the plant Phyllanthus amarus obtained by using water alone In another preferred embodiment of the invention, there is provided a process for the preparation of a pharmaceutical formulation useful for the treatment of Hepatitis B (both acute and chronic), Hepatitis C (both acute and chronic) and other related viral infections of the live which comprises mixing (1) 20 to 30% w/w of an extract of the plant Phyllanthus amarus obtained by using a polar solvent alone, other than water (ii) 20 to 40% w/w of an extract of the plant Phyllanthus amarus obtained by using a mixture of a polar solvent and water wherein the ratio of the solvent to water ranges from 80 to 20% and 20 to 80% w/w respectively (iii) 20 to 40-% w/w of an extract of the plant Phyllanthus amarus obtained by using a polar solvent and water wherein the solvent to water ratio ranges from 20 to 80 % and 80 to 20 % w/w respectively and (iv) 20 to 30% w/w of an extract of the plant Phyllanthus amarus obtained by using water alone In still another preferred embodiment of the present invention there is provided a process for the preparation of pharmaceutical formulation useful for the treatment of Hepatitis B (both acute and chronic). Hepatitis C (both acute and chronic) and other related viral infections of the liver comprises mixing one part each of (i) an extract of the plant Phyllanthus amarus obtained by using a polar solvent alone, other than water (ii) an extract of the plant Phyllanthus amarus obtained by using a mixture of a polar solvent and water and (iii) an extract of the plant Phyllanthus amarus using water alone In yet another preferred embodiment of the present invention there is provided a process for the preparation of pharmaceutical formulation useful for the treatment of Hepatitis B (both acute and chronic), Hepatitis C (both acute and chronic) and other related viral infections of the liver which comprises mixing one part each of (i) 10 to 40% w/w of an extract of the plant Phyllanthus amarus obtained by using a polar solvent alone, other than water (ii) 10 to 40% w/w of an extract of the plant Phyllanthus amarus obtained by using a mixture of a polar solvent and water wherein the ratio of the solvent to water ranges from 20 to 80 and 80 to 20% w/w respectively and (iii) 10 to 40% w/w of an extract of the plant Phyllanthus amarus obtained by using water alone In another preferred embodiment of the present invention there is provided a process for the preparation of a pharmaceutical formulation useful for the treatment of Hepatitis B (both acute and chronic), Hepatitis C (both acute and chronic) and other related viral infections of the liver which comprises mixing one part each of (i) 20 to 30 %w/w of an extract of the plant Phyllanthus amarus obtained by using a polar solvent alone, other than water (ii) 20 to 40 %w/w of an extract of the plant Phyllanthus amarus obtained by using a mixture of a polar solvent and water wherein the ratio of the solvent to water ranges from 80 to 20% and 20 to 80 % w/w respectively (iii) 20 to 40 % w/w of an extract of the plant Phyllanthus amarus using a mixture of a polar solvent and water wherein the ratio of the solvent to water ranges from 20 to 80 % w/w and 80 to 20 % w/w respectively and (iv) 20 to 30 % w/w of an extract obtained using water alone In still another preferred embodiment of the present invention there is provided a process for the preparation of a pharmaceutical formulation useful for the treatment of Hepatitis B (both acute and chronic), Hepatitis C (both acute and chronic) and other related viral infections of the liver which comprises mixing two parts (i) of an extract of the plant Phyllanthus amarus obtained by using a polar solvent alone, other than water an extract of the plant Phyllanthus amarus obtained by using a polar solvent and one part each of (ii) , of an extract of the plant Phyllanthus amarus obtained by using a mixture of a polar solvent and water and (iii) an extract of the plant Phyllanthus amarus using water alone In yet another preferred embodiment of the present invention there is provided a process for the preparation of a pharmaceutical formulation useful for the treatment of Hepatitis B (both acute and chronic), Hepatitis C (both acute and chronic) and other viral infections of the liver which comprises mixing two parts (i) of 10 to 40 %w/w of an extract of the plant Phyllanthus amarus obtained by using a polar solvent, other than water and one part each (ii) of 10 to 40 % w/w of an extract of the plant Phyllanthus amarus obtained by using a mixture of a polar solvent and water wherein the ratio of the solvent to water ranges from 20 to 80% arxi 80 to 20 % w/w respectively (Iii) of 10 to 40% w/w of an extract of the plant Phyllanthus amarus obtained by using water alone In yet another preferred embodiment of the present invention there is provided a process for the preparation pharmaceutical formulation useful for the treatment of Hepatitis B (both acute and chronic), Hepatitis C (both acute and chronic) and other viral infections of the liver which comprises mixing two parts (1) of 20 to 30 % W/W of an extract of the plant Phyllanthus amarus obtained by using a polar solvent alone, other than water and one part each (ii) of 20 to 40 % w/w/ of an extract of the plant Phyllanthus amanjs obtained by using a mixture of a polar solvent and water wherein the ratio of the solvent to water ranges fn^m 80 to 20% and 20 to 80 % w/w respectively (ill) of 20 to 40 % w/w of an extract of the plant Phyllanthus amarus obtained by using a mixture of polar solvent and water wherein the ratio of the solvent to water ranges from 20 to 80% w/w and 80 to 20% w/w respectively and . (iv) of 20 to 30 % w/w of an extract of the plant Phyllanthus amarus obtained by using water alone. Extraction of the components of the formulation: Parts sudi as leaves, stems, seeds and roots of the taxonomically identified collections of the plant Phyllanthus amanjs were dried and kept in an oven heated to a temperature in the range of 50 to 80 degree C for a period in the range of 3 to 5 hrs a day. for a period of 3 to 6 successive days. The dried parts of the plants are then powdered. The powder thus obtained is used for the extraction of different components of the preparation of the formulation of the present invention. Before subjecting the powder for extraction procedures, each batch of powder is subjected for sterility testing to rule out any bacterial or fungal contamination as per standard methods. One portion of tfie powder is extf3cted with polar solvent. TTie polar solvent employed may be methanol, ettianot, hexane, butanol and the like. The extraction may be carried out at a temperature in the range of 37 to 60 degree C for a period ranging from 2 hours to 18 hours preferably at a temperature in the range of 37 to 60 degree C. This extract may be used as the component CO of the pharmaceutical formulation of the present invention. Another portion of the powder is extracted with a mixture of polar solvent ancj water. The polar solvent employed may be methanol, ethanol, Hexane and the like. The ratio of the solvent and water used fo^ extraction may range from 20 to 80 and 80 to 20% w/w respectively , The ratio may preferably range frcim 30 to 50% and 50 to 30% w/w respectively. The extracBon may be carried out at a temperature in the range of 4 to 40 degree C for 2 to 18 hours more preferably at a temperature in the range of 37 to 60 degree C for 2 to 4 hours. This extract may be used as the component (li) of the pharmaceutical formulation of the present invention. Yet another portion of the powder is extracted with water alone. The extraction may be carried out at a temperature in the range of 37 to 60 degree C fbr 2 to 18 hours. This extract may be used as the component (iii) of the pharmaceutical formulation of the invention The extracts so detained may now be mi)«d together to certain the phamiaceutical fonnufation of the present invention. The mixing may be effected in a vertex mixer or heating mantle and stirring it tiioroughly till a homogenous fomiulation is obtained. The mixing may be effected at a temperature in the range of 37 to 60 degree C for 15 to 30 minutes. The details of the invention are given in the Examples provided bdow which are given to illustrate the invention only and therefore should not be consbued to limit the scope of the present invention. Example 1 40 gms of the powder obtained as explained above Is extracted with 200 ml of ethanol at a tfimperature of 35 to 37 degree C for 2 hrs and 56 degree C for another 2 hrs under shaking. Yield of the extract is 90ml (EXTRACT-I) 40 gms of ttie powder curtained as explained abo^« is extracted with a mixtijre of 160 ml of ethanol and 40 ml water total volume being 200ml. The extraction was effected at a temperature of 35 to 37 degree C for 2 hrs and 56-60 degree C for another 2 hrs under shaking. Yield of the extract 65 ml {EXTRACT-II) 40 gms of the powder prepared as exi^ained above is extracted with 200mt water at temperature of 35 to 37 degree C fbr 2 hrs and 56-60 degree C for another 2 hrs under shaking. Yield of the extract 50 ml (DCTRACT-III) Extracts I, II, and III were mixed in 1:1:1 ratio (i.e., 50 ml each) in a conical flask in an environmental shaker kept at 37°C for 15-30 min. TTie resultant formulation was dried in a dessicator/"Vertis" vaccum drier until ttie solvents get fully evaporated. The yield of the powdery formulation was 28 gms (20%). Example 2 40 gms of the powder obtained as explained above is extracted with 200 ml of ethanol at a temperature of 35 to 37 degree C for 2 hrs and 56-60 degree C for another 2 hrs under shaking. Yield of the extract 90 ml (DfTRACT-I) 40 gms of the powder obtained as explained above is extracted with a mixture of 160 ml of ethanol and 40 ml water total volume being 200ml. The extraction was effected at a tonperature of 35 to 37 degree C for 2 hrs and 56-60 degree C for another 2 hrs under shaking. Yield of the extract 65 ml (EXTRACT-II) 40 gms of the powder prepared as explained above is extracted with 200ml water at temperature of 35 to 37 degree C for 2 hrs and 56-60 degree C for another 2 hrs under shaking. Yield of the extract 50 ml (E)CrRACr-III) The extracts I, II &IIIin 2:1:1 ratio (i.e., 80 ml of extract!: 40ml of extract 11 and 4aml of extract III) were mixed in a conical flask in an environmental shako- kept at 37""C for 15-30 min. The resultant formulation was dried in a desacator/"Vertis" vaccum drier until the solvents get fully evaporated. The final yield when dried was 36 gms (25%). Example 3 30 gms of the powder obtained as explained above is extracted wiUi 200ml of methanol at a temperature of 35 to 37 degree C for 2 hrs and 56-60 degree C for another 2 hrs under shaking. Yield of the exh^ct 90 ml (EXTRACT-I) 30gms of the powder obtained as explained above are extracted with a mixture of 60 ml of methanol and 140 ml water. The extraction was effected at a temperature of 35 to 37 degree C for 2 hrs and 56-60 degree C for another 2 hrs under shaking. Yield of the extract 70nnl (EXTRACT-II) 30 gms of the powder prepared as explained atxjve is extracted with 200ml water at temperature of 35 to 37 degree C for 2 hrs and 56-60 degree C for another 2 hrs under shaking. Yield of the extract 50 ml (E)aRACr-III) The Brtracts I,II and III were mixed in 1:1:1 ratio (i.e., 50 ml each) in a conical flask in an environmental shaker kept at 37*0 for 15-30 min. TTie resultant formulation was dried in a dessicator/"Vertis" vaccum drier until the sc^vents get fully evaporated, "me yield of the powdery formulation was 25 gms (15%). Example 4 30 gms of the powder obtained as ejqitained above is exd^cted with 200ml of methanol at a temperature of 35 to 37 degree C for 2 hrs and 56-60 degree C for another 2 hrs under shaking. Yield of the ©(tract 90ml (BCTRACT-I) 30gms of the powder otstained as explained above are extracted with a mixture of 60 ml of methanol and 140 ml water. The extraction was effiected at a temperabjre of 35 to 37 degree C for 2 hrs and 56-60 degree C for another 2 hrs under shaking. Yield of the extract 70ml CEXTTRACT-II) 30 gms of the powder prepared as explained above is extracted with 200ml water at temperature of 35 to 37 degree C for 2 hrs and Sfr-M degree C for another 2 hrs under shaking. Yield of the ©ctract 50 ml (EXTRACT-III) The extracts I, II and HI in 2:1:1 ratio (i.e., 80 ml of exb3ct I: 40ml of II and 40ml of III) were mixed in a conical flask in an environmental shaker kept at 37""C for 15-30 min. The resultant formulation was dried in a dessicator/"Vertis" vaccum drier until the solvents get fully evaporated. The final yield was 32 gms (22%). ExamF^e5 40 gms of the powder obtained as explained above is exti^cted with 200ml of methanol at a temperature of 56 to 60 degree C for 4 hrs under ^laking. Yi^d of &ie extract 90ml (EXTRACT-I) 35 gms of the pcwder obtained as explained above is extracted with a mixture of iOOml of methanol and 100 ml water, total volume being 200ml. The extinction was effected at a temperature of 35 to 37 degree C for 2 hrs and 56-60 degree C for another 2 hrs under shaking. Yield of the extract 70ml (DCmACT-II) 30gms of the powder prepared as explained above is extracted with 200ml water at temperature of 56 to 60 degree C for 4 hrs under shaking. Yield of the extract 60ml (EXTRACT-III) Extracts I, II and III were mixed in 1:1:1 ratio (I.e., 50 mi each) in a conical ftask in an environmental shaker kept at 37°C for 15-30 min. This fonnulation was dried in a dessicator/"Vertis" vaccum drier until the solvents get ftjHy evaporated. The yield of the powdery formulation so obtained was 25 gms (15%). Example 6 40 gms of the powder obtained as explained above is extracted with 200ml of methanol at a temperature of 56 to 60 degree C for 4 hrs under shaking. Yi^d of the extract 90ml (EXTRACr-I) 35 gms of the powder obtained as explained above is extracted with a mixture of 100 ml of methanol and 100 ml water, total volume being 200ml. The extraction was eifected at a temperature of 35 to 37 degree C for 2 hrs and 56-60 degree C for another 2 hrs under shaking. Yield of the extract 70ml (EXTWVCT-II) 30gms of the powder prepared as explained above is extracted with 200ml water at temperature of 56 to 60 degree C for 4 hrs under shaking. Yield of the extract 60ml (EXTRACr-III) The Extracts I, II, III were mixed in 2-.l-.lratio(i.e., 80 ml of extract!: 40ml of extract n and 40ml of extract III) were mixed in a conical flask in an environmental shaker kept at S?^ for 15-30 min. This formulation was dried in a dessicator/"Vertis" vaccum drier until the solvents get fully evaporated.TTie final yield when dried was 40 gms (25%). Example 7 40 gms of the powder obtained as explained above is extracted witii 200ml of methanol at a temperature of 35 to 37 degree C for 2 hrs and 56-60 degree C for another 2 hrs under shaking. Yield of the extract 90ml (E)CTRACT-I) 40 gms of the powder prepared as explained above is extracted with 200ml water at a temperature of 35 to 37 degree C for 2 hrs and 56-60 degree C for another 2 hrs under shaking. Yield of the extract 60ml (E)Cri^CT-n) 35 gms of the powder obtained as explained above is © The Extracts I, II, III and IV were mixed in 1:1:1:1 ratio (i.e., 50 ml each) in a conical fiask in an envinjnmental shaker kept at 37°C for 15-30 min. The resultant fonnulation was dried in a dessicator/"Vertis" vaccum drier until the solvents get fully evaporated. The yield of the powdery formulations so obtained was 40 gms (25%). Example 8 40 gms of the powder obtained as explained above is exfracted with 200ml of methanol at a temperature of 35 to 37 degree C for 2 hrs and 56-60 degree C for another 2 hrs under taking. Yield of the extract 90ml (DCTRACT-I) 40 gms of the powder prepared as explained above is actracted with 200ml water at a temperature of 35 to 37 degree C for 2 hrs and 56-60 degree C for another 2 hrs under shaking. Yield of the exbact 60ml (eORACT-II) 35 gms of the powder obtained as explained above is eftracted with a mixture of 100 ml of methanol and 100 ml water, total volume being 200 ml. The extraction was effected at a temperature of 56 to 60 degree C for 4 hrs under shaWng. Yield of the extract 70ml (EXTRACT-ni) 30 gms of the powder obtained as explained above is extracted with a mixture of 60 ml of methanol and 140 ml water total volume being 200ml. TTie extraction was effected at a temperature of 56 to 60 degree C for 4 hrs shaking. Yield of the extract 65 ml (EXTRACT-IV) The extracts I, H, III & IV were mixed in 2:1:1:1 ratio (I.e., 80 ml of extract I: 40m( of extract II, 40ml of extract III and 40ml of extract IV) were mixed in a conical flask in an environmental shaker kept at 37»C for 15-30 min. The resultant formulation was dried in a dessicator/"Vertis" vaccum drier until the solvents get fully evaporated. The final yield when dried was 48 gms (30%). The examples given below are provided to compare the properties of the extract of the plant Phyllantiius amarus obtained using polar solvent alone and water alone with the properBes of the pharmaceutical fbrmulation of the present Invention. Example 9 40 gms of the powder obtained as explained above is ex&acted with 200 ml of methanol at a temperature of 35 to 37 degree C for 2 hrs and 56-60 degree C for another 2 hrs under shaking. Yield of the extract 90ml This extract as a formulation was dried in a dessicator/"Vertis" vacuum drier until the solvents get fully evaporated. The yield of the powdery ftarmulations so obtained was 15 gms (12.5%). Example 10 40 gms of the powder prepared as explained above is ©dracted with 200ml water at a temperature of 35 to 37 degree C for 2 hrs and 56-60 degree C for another 2 hrs under shaking. Yield of the extract 50 ml This extract was dried in a dessicator/"Vertis" vacuum drier until the water get fully evaporated. The yidd of the powder so otrtained was 8 gms (10%). BIOLOGICAL EFFICACY OF THE PHARMACEUTICAL FORMULATION WITH PARTICULAR REFERENCE TO THE FORMULATION OBTAINED BY THE PROCESS DESCRIBED IN EXAMPLES 1 to 10 The powdery formulations obtained in the Examples 1 to 10 were used for the biological studies to confirm the presence of the above mentioned essential properties . For these studies Phosphate buffered saline (PBS) as vehicle in w/v concentration was employed . The solutions of the formulations prepared as mentioned above were used for the following assay methods for confirming the presence of the essential properties mentioned above A. HBsAg binding property of the pharmaceutical formulation of the invention Prindole: The test is an en2yme immunoassay based on the sandwich principle. HBsAg in the plasma is neutralised or bound by pre-incubation with anti-HBs like substance, and hence no longer reacts with the antibody coated in the wells. The presence of HBsAg binding activity in the formulation of the present inventiwi is demonstrated by reduction of colour or a negative ELISA result The presents of unbound HBsAg in a test sample is demonstrated by an increase in the colour or a positive ELISA result. Procedure: Pre-incubatlon with HBsAg plasma: Equal volume of pre-titrated HBsAg positive plasma and 5mg/ml concenb^tion of the formulation of the present invention was mixed and inojbated at 37""C for 5 days. The mixture was assayed daily for the presence of bound/unbound HBsAg using Hepanostika or any other commercial EQSA kit Control tubes containing solvent (PBS) and plasma (solvent control) and P. amams and plasma (positive control) w&e set up in each batch. ELISA was performed as per the instructions of the manufacturer given in the kit. Calculation: 1. Cut-off value: the cutHDff value was calculated as mean negative conbx)l+0.025 2. Values equal to or greater than the cut-^Dff should be taken as positive The results of the assays of the pharmaceutical formulation of the present invention prepared by the process described in the Examples 1 to 8 are given in Table 5 B. HBV - DNA polymerase inhibiting properties of the pharmaceutical formulation of the invention Principle: Replication of hepadna viruses involves a viral DMA polymerase, which is a potential target for chemotherapy against HBV. In the presence of HBV-DNA polymerase, compiemoitary bases are added to the template (HBV-DNA), the addition of which are quantltated with the help of tritiated thymidine triphosphate. Reduction in the count of 50% or more in tiie test is noted as inhibitory activity. r Virus preparation: Pre-titrated HBsAg and HBeAg positive serum was centrifuged at 35,000 rpm for 3 hrs using SW 41 rotor. The pellet was washed in PBS and again centrifuged at 35,000 rpm. The peilet got in this was dissolved in PBS and stored at - 20°C. Procedure: The procedure foilowed was as described by l-O^ren et al (1989). Prior to the assay, the virus preparation was pre-treated with 1/8 volume of 2% mercapto ethanol and 10% NP-40 for 15-30 min at room temperature. Aliquotes of 25MI were then incubated at 37°C for 3 hrs together with 25pl reaction mixture containing Tris-HO (pH 8.0) lOOmM, Mga2 20mM, KQ 200mM, df^TPs lOmM each and "H dTTP lOmM and 25tJl of DNase and RNase free water and the fiMmulation of the present invention which is to be studied. After incubation, txi SOpI of reaction mixture lOpI of 0.2M EDTA was added and spotted onto a Whabman DE81 filter paper discs and processed for a radioactivity measurement. The results of the assa\ra of the phanmaceufical formulation of the present invention prepared by the process described in the Examples 1 to 8 are given in Table 4 C. Reverse Transcriptase InhiUting property of the pharmaceutical fbrmulation of the invention This property was evaluated by Electrophoretic and Isotopic RT-lnhibltion assays. . ELECTROPHOREnC RT-INHIBITION ASSAY The elediophoretic RTHnhibition was conducted to screen and identify the anti-retrowral potentials of formulations of the present invention prepared by the process described in Examples 1 to 8. Moloney murine leukaemia virus reverse transcriptase (MMLV RT) was used for cDNA synthesis. Princiole: Reverse transcriptase (RNA-dependent DNA polymerase) is an enzyme in retroviruses and piays an important role in their multiplication by transaibing the viral RNA into cDNA which is required for the proviral synthesis. This test is performed to find the ability of an extract to inhibit Ri. me presence or this property is determined by the fiDmiatJon or non-formation of cDNA. Procedure: The electrophorebc RT -inhibition assay was performed in a reaction mixture that contained the following in a final volume of 25ijl. Tris HCt (pH 8.3) 50 mM, MgOi 6mM, KCi 40mM, P(rA)(dT)i2.i8 0.5 pg, MMLV RT 5 Units. To this 2 pi of the fonnulation of the present invention prepared by the process described in tiie Bamples 1 to 10 was added immediately before incubation. A positive control and solvent control were set up using Azidothymidine (1 pg/ml) and the solvent used for reconsdtution of extract, respectively. All the tubes were inoibated at 37""C for one hour, and the reaction was stopped using O.l M EDTA. 10 pi of each assay mix was loaded onto a 1% gel and run for 30 minutes at 60 V. The gel was stained in Ethidium bromide (0.5 pg/ml) and viewed in UV transilluminator. The presence of absence of the cDNA band indicates the nonnnhibiton of inhibition of RT respectively. Each sample was tested three times for the reproducibility of its activity if present The procedure followed Is a modification of cDNA synthesis kit procured from Amersham International Uri. Burminghamshire, UK. (a) Isotopic Enzyme Assays Standard Isotopic RT-InhibiOon Assay principle: as described in the eledrophoretic RT-inhibition assay. The inhibitory activity is identified by using an isotope, triOated thymidine triphosphate. A 50% or more reduction in the radioisotope uptake between the control and the test is taken as a positive inhibitory activity. Procedure: The procedure followed was as described by Ono et al. (1989). The assay was perfomned in a reaction mixture that contained the following in a final volume of 50 pi. Tris HO 50 mM, P(rAXdT^i2-i810 pl/ml, BSA 10 pg/ml, ^H-dTTP 0.5 mM, DTT 10 mM, MgCI; 3mM, MMLV RT 1 unit. In the test, a known concentration of the formulation of the present invention prepared by the pnxess described in tiie Examples 1 to 8 was added \o the reaction mixture and incubated. Similariy a positive control (0.1 pg/ml AZT), a negative control (distilled water) and a solvent control (solvent used in exti-act) were set up. Each set of test and conljols were run in triplicate. After 30 minutes the reaction was stopped by adding 10 pi of ice cold EDTA (0.2M) and immereing the mixture in ice immediately. Processing fiDr radioactivity measurement: After termination of the reaction, the DNA was precipitated using 10 pi of cold 5% TCA and 0.1 M sodium pyrophosphate. 50 [i\ of the reaction mixture was then filtered through Whatman DE81 filter paper. The filter paper was later washed thrice in 3 ml of 5% TCA and three times in absolute alcohol. The filters were then air dried, and radioactivity measured using a toluene-based scintillation coct^il. A reduction of 50 % of more In the radioactive count of the test from the negative control is taken as present of RT inhibition activity. Tlie results are presented In Table 5. D. Anti hepatotoxic potentials of the pharmaceutical formulation of the present invention And hepatotoxic potentials of fDrmulation of the present invention prepared by the process described in the Examples 1 to 8 was assessed after challenging isolated rat hepatocytes witin a known hepatotoxic compound p- galactosoamine. Isolation of rat hepatocytes An adult rat of known weight preferably more than 125 gm was anaesthetized with ether and a midline incision were made. The liver was perfused ttirough the portal vein with 30-50 m! erf cold sodium citrate (0.027M) in calcium free locke"s solution. During the perfLislon, the liver gets blanched and fully distended. Perfusion is generally completed within 5 minutes of anaesthetizing the animal. The perfused liver Is excised and washed well with the perfusion fluid and pressed In folds of sterile filter paper, weighed and cut into several small pieces and with a pair of scissors. A known wet weight of the liver (usually between 2-6 gm) was transfer^ with 5 volume of cold 0.25M sucrose Into a sterile glass homogenizer and finely grounded. The cell suspension was filtered once, without application of pressure through 200-mesh brass gauze to remove strands of connective tissue and clumps of cells. The suspension contained some cell debris and blood cells at this stage, which were removed by centrifugation at a low speed C100-200g) for 2 minutes. After removal of the supernatant, the cell sediment was resuspended in a known volume of the minimum essential medium for further studies. Cell count 10Q)il of the cells are teten and to this 300^1 of 4% trypan blue Is added and this is mixed and then viewed in the haemocytometer within 5 minutes of staining. Ihe cells, which have not taken the dye, are live cells, which are counted. The cells were inoculated into the culture medium which Is omposed of Eagle"s MEM supplemented with 10% heat inactivated calf serum penicillin (lOOIU/ml), str^Jtomydn ( lOOIU/ml), 10-6M Dexamethosome and 10-8 units Insulin. InooJla of 5x 10* cells /O.lml/cm" were seeded into plastic dishes and preinoJbated in a humidified incubator at 37°c under 5% COz in air for 24 hours and medium is replaced. Study design Four sets of isolated rat hepatocyte cultures were put up. Set I acted as control, Set II was treated with Img/ml concentration of the fiDrmulation of the present invention prepared by the process described in the Examples l to 8. Set III with 0.5mM concentration of p-gatactosamine, a known hepatoxic agent and set IV was treated with p-galactosamine and then protected with the formulation of the present inventiOTi. Culture supematants of all the sets were assayed for glutamic pyruvate transaminase levels as per standard procedures. Results 1. The study revealed that the formulation of tiie present invention by itself did not bring about any hepatotoxicity on rat hepatocytes, 2. p-^aladosamine was proved as a profound hepatotoxic chemical. 3. The fbmiulation of the present invention at Img/ml concentration was shown to protect isolated rat hepatocytes significantly ftom p-galacbasamine induced hepatotoxicity. 4. Thus the study has proved that the formulation of the present invention has significant an« hepatotoxic potentials (p The results of the assay by the different fomiulations of the present inventlwi prepared by the process described in the Examples 1 to 8 are given in Table 5 E. InvJtro inhibition of HBsAg secretion by the pharmaceutical fomiulatjon of the present invention Alexander cell line (345) was kindly provided by Dr. Tim Hanison, Academic School of Medicine, Royal Free Hospital, l_ondon which is a continuos cell line of human hepatic cellular carcinoma cells (PLC/PRF/5). The cell line was cultured from a cancer patient who was also an HBsAg earner. These cells grown in \^tro secrete only HBsAg witiiout any infectious virus. Cultivation of Alexander cell line Alexander ceil line was grown as per the procedure adopted for the cultivation of Vero cell line described in section 4.6.2.2. 10% foetal calf senjm (Sigma Chemical Company, USA) was used instead of 5% inactivated goat serum. Study design Six sets of Alexander cell line were grown in LetghtiDn tubes. On day 1 of the experiment, the culture medium was decanted, and fresh medium was added. Img/ml concenti^on of formulation of the present invention prepared by the pnDcess described in the Examples 1 to 8 was added to each tube. The culture medium (supernatant) was assayed daily at varying doubling dilutions starting from neat t 1/128 dilution tia check for the inhibition of secretion of HBsAg by the cell line. Distilled water was added to the control tubes. HBsAg detection from the supernatant was done using Hepanostika HBsAg kits as per ttie procedure described eariier. Results. Inhibition of HBsAg secretion was observed for 48 hours when the cell line was treated with Img/ml concentration of the formulation as a single dose. However HBsAg was detected from the culture medium at lower dilutions after 72 hojrs. The details of ttie results of the formulations of the present invention prepared by the process described in the examples 1 to 10 are given in Table 6. F. STUDY OF XMMUNOMODUUTORY POTENTIALS OF THE PHARMACEUHCAL FORMULATION OF THE PRESENT INVENTION (a) The isolation of lymphocytes was done by ficoll-paque method using 0 group Rh+ve human blood. After centrifugation of the blood sample layered onto the Ficoll-paque at 400g for 20min at 18-20""C in a refrigerated centrifuge, lymphocytes are s^jerated and suspended gentiy in 6-8 ml of balanced salt solution. It was centrifuged at lOOg for 10 min at IS-ao^C. After removing the supernatant, the lymphocytes are suspended in RPMI medium. (b) Lymphocyte vtabHity test: A cell suspension containing 5X 10-6 cell$/ml was prepared in RPMI medium. 0.5ml of 0.4% trypan blue soiution was transferred to a test tube. To this C.3ml of RPMI medium and 0.2ml of cell suspension were added and mixed thoroughly. The mixture was allowed to stand for 5 minutes. The ajspension was viewed through a haemocytometer and looked for waWe cells. The vlatHe cells do ncA take up the dye. % N^ability was calculated by the fonnula = total viaUe cells (unstained) X100 total cells {stained & unstained) (c) T-cell proliferation assay: To test by in-vitro method the T-cell proliferation Inhibition/accelaation potentials of fomiulation of the present invention prepared by the process described in the Examples 1 to 8, which may be indicative of immunomodulatory potentials. Requirements for the assay: 1. Peripheral Wood lymphocytes (PBL) 2. Phytohaemaggluljnin (PHA) 3. MTT (3,4,5-dlmethyl thyol-2-yl-di phenyl tetrayolium bromide) 4. Acid propanol (0.40M HO in isopropanol) 5. RPMI 1640 medium 6. F^Bl calf serum 7. Antibiotics Vancomycin -25Mg/ml Gentamycin-20pg/ml Procedure of the assay; 1. Peripheral blood lymphocytes (PBL) were obtained by the Ficoll-hypaque method 2. Under sterile condition SOpi of PBL suspenaon (5x106 cells/ml), SOul of sample dilutions and 50pl of PHA (33Mg/ml) were added in the 96-well flat bottomed nnicrotitre plate 3. Incubate the plates at 37°C and 5% CO2 for 48 hrs 4. After incubation cell growth was quantified by adding 25pl of MTT to each well 5. Incubate the plates at ST"C for 4 hrs 6. SOpI of acid propanol was added and the content of each well was mixed thoroughly 7. Plates were read on automatic ELISA reader at 550nm. ContnDls & tests in duplicate: 1. PBL+ PHA ^ 100% activity 2. PBL+ RPMI ^ 0% activity 3. PBL+ Picroliv ^ known Poative control 4. PBL+ formulations ^ Test samples of the present invention RESULTS: The extracts were assessed for the lymphocyte proliferation activity by a) Microscopic: Multiwell plates were observed under inverted phase contrast microscope for any observable indudjon of proliferation. A minimum of 10 fields was observed. b) l^nr calorimetnc assay: The multiwell plates were incubated with l^fTT for 4 hrs. Then the plates were centrifuged at 100 rpm for 10 min. The supernatant was aspirated, SOpI of acid propanol was added and the content of each well was mixed thoroughly. The plates were read in an automated ELISA reader at 550nm. The proliferation of the lymphocytes was assessed by comparing with known immunomodulatory control (Picroliv) The details of the results are given in Table 6. Table 6i ANTIVIRAL AND BIOLOGICAL EFFICACY OF THE FORMULATIONS OBTAINED BY THE PROCESS DESCRIBED IN EXAMPLES 1T010 EXAMPLES ANTIVIRAL & BIOLOGICAL PROPERTIES (MIC /ml concentration) HBsAg HBV-DNAp RT- Inhibition of Ant- Inimuoomod binding inhgiition inhibition HBsAg secretion hqjatotoxicity Illation Example-l 2.5 mgm 400 ng 200 ng 1 nigm 1 mgm 400 iig Example-2 1.25 mgm 100 tig 100 ng 0.5 mgm t mgm 100 |ig Example-3 2.5 mgm 400 Mg 200 Jig 1 mgm 1 mgm 400 fig Exainple-4 L25mgm 200 MS lOOng 1 mgm 1 mgm 200 ng Example-5 5.0 mgm 400 fjg 400 ^g 2 o^m 1 mgm 400 (ig Example-d 2.5 mgm 200 Mg 100 ng 1 mgm 1 mgm 200 ng Example-7 2.5 mgm 400 Mg 200 (ig 2 mgm 1 mgm 400 ng Example-8 1.25 mgm 200 MQ 100 ng 1 mgm 1 mgm 200 fig Exainple-9 2.5 mgm 400 pg 400 iig 2 mgm - - Examptc"lO 5.0 mgm - - - 1 mgm 400 fig Table 7 summarises the case studies on the efficacy of the new formulation of the present invention prepared by the process described in Example 1 for the treatment of acute and chronic Hepatitis C infections conducted at Chennai, India by Thyagarajan et al (1996-99) using the formulation Table 7. Case studies on the efficacy of the new formulation of P. amanjs for the treatment of acute and chronic Hepatitis C infections conducted at Chennai, India using the formulation drained by the process described in Example 1 CThyagarajan et al, 1996-99, unpublished) !si. No Pt. Identity Age/ Sex Diagnosis &. History HCV Pre-ireUincnt parameters P.amarus treatment period (months) HCV Prc-trealmcnl parameters Ab RNA SGOT lU SGPT lU SAP lU S. biltnibin Mgm% Ab RNA SGOT SGPT lU SAP RJ S, bilirubin mgm% 1 Mrs.M 45/F PTHB&C After cardiac suTRery + + 210 ln\ 78 5-6 2m + 25 34.2 44 1.2 2 Mrs. U.B 42/F PTH-CCPH after Caesarian + ND 200 146 80 0.6 9m + ND 69 72 22 0.6 3 Mr. V,R 52/ M PTH-C CLDCaidiac siffBfirv + ND 237 290 34 14.3 3m + ND 45 62 12 2.8 4 Mrs. L.M 38/F PTH-C AVH + + 562 360 34 0.9 3m + - 62 48 18 0.8 5 Mrs. V.O 44/F PTH-B&C AVH + + 111 173 44 0.9 4m + - 55 42 20 0.8 6 Mrs. J. A 6W PTH-C AVH + + 162 213 123 0.9 3m + + 112 146 84 0.9 7 Mrs. R.K 38/F CAH Sxirgeiy + Traiisfiisio!i+ + + 404 581 88 1.5 6m + + 340 491 52 0.5 8 Mr, R.I.R 32/ M PTH-C + + 52 95 201 0.6 3m + + 42 38 62 0.5 9 Mr.G.N 37/ M Cinlio^s (B&C) Transfiision + Surgery+ Jaundice^ + ND 60 100 444 13 6m + ND 56 72 120 5.2 10 Mr.B.M 24/ M Recurrent VH + - 69 108 ND 3,1 9m + - 35 24 - 1,0 11 Mrs. M.K 29/ M PTH-AVH-C Surgery + + + 250 90 ND 5,7 5m + + 46 "42~ ND 1,8 1 12 Mr. SMA W i5t M CLD + + 76 47 " 127 1.9 6in + + 72 44 86 1.9 ! n Mr.R 25/ PTH-AVH-C &)ood trans+ + ND 79 m - 2.4 3m + ND 42 35 - 0.9 Mr. K.P.J 63/ M PTH-AVH-C Surger>+ Biood uans+ + ND 207 233 280 1.6 3ni -^ ND 48 42 51 0.9 1 ]-"" Mr.S.V 29/ M CRF: Dialysis + + 185 509 376 0.7 3m + + 61 85 78 0.7 16 Mr. MC 55/ M CAH + + 27 125 139 1.2 6m + + 20 54 115 0.9 1 17 Dr. S.K 29/ M Asymptomatic C ? occupational + + 52 66 72 0.6 6m + + 38 55 42 0.56 1 IS 1 Mrs.V, R 50/F PTH-AVH-C Blood (rans+ + ND 482 809 62 18.6 3m + ND 83 128 ~40 1 4.0 Inference: Total cases studied :18; Normalisation of enzymes :14/18 (77.8%); HCV-RNA negativity: 4/12 (33.3%) (By Qualitative PCR only) ; Physical well being and clinical improvement 17/18 (94.4%); Observable side effects : nil [Dosage: SOOmgms oral capsules of P.amanjs-university preparation x thrice dally x peroids mentioned against each case] In summary, the above findings confirm that when parts of medicinal plant, PhyUanthus amarus are extracted separately with a polar solvent alone, polar solvent and vi/ater in specific ratios and when such extracts are mixed together, the resultant formulation has all the essential antiviral and biological properties while the individual polar or aqueous extracts alone does not possess one or more of these properties. It is our finding that if all the above said essential properties are made available in a single formulation, the resulting formulation will have uniform and stable antiviral and biological properties G. BIOLOGICAL AND CHEMICAL STANDARDISATION OF THE PHARHACEiniCAL FORMULATION OF THE PRESENT INVENTION As the antiviral actiwty of P.amarus was found to differ between collections made from different areas, the fDnnulation preparation to be used in the clinical trial was standardized by evaluating ther antiviral properties and matching them using the HPLC pattern upon fractionation, initially and later upon the preparation of the phamiaceutical formulations of the present invention prepared by the process mentioned in Examples 1 to 8. Collection of plant material: PhyUanthus amams was collected Irom different places within Chennai dty and within Tamilnadu and Bangalore. They were Salem, Mathuranthagam, Coimbatore, Tinjchy, Tiruttani, Bangalore from outside Madras; Annanagar, Thinjvanmiyur and TTruvatriyur from in and around Madras. Ail these plant materials were identilied based on the taxonomical system of nomenclature. Preparation of the formulation for HPLC: The formulations of the present invention prepared by the process explained in Examples 1 to 8 fnam various collections as described eariler under the head "embodiments". The formulations so obtained were filtered and made upto 50ml. 10ml of each extract was used as such for chemical standardisation study. The rest was dried and used for antiviral and biological testing by a battery of tests as described in A to F. Chemical fingerprinting was perfonned using a High performance liquid chromatogram for each of the extract and formulation. HPLC analysis; Requirements*. 1. HPLC grade methanol 2. HPLC grade water ^ Rreverse phase ., Dr^nnn^K^ kit (4.6 mmid x 25 cm, partiae Equipment required". HPU:- A Shlmadzu chromatographic system comprising 1. LC-6A liquid pump 2. SCL-6B system controller 3. Model 7125 injector 4. SPD-6A UV detector Chromatographic system: Column Mobile phase Flow rate Detection Injection volume Shimpack Pr^pODS (K) kit (4.6 mmid x 25 cm, particle dla S-ym Reverse phase column) Methanol: water gradient 2ml/min UV 225 nm lOpI Ptpc^urg: The formulations of the present invention prepared for the study was filtered through 0.2p membrane filter discs. The filtrate was used for the study. After the column was washed, to achieve a basrfine, the gradient parameters were set and the run was started by injecting iOpI of the fomiulation. The chrexnatogram was plotted. Similarly chnDmatographic patterns were plotted for all the formulations. The chromatograms got were compared and analysed for tiieir maft^ing antiviral and biological pnaperties as described in A to F. The formulations prepared as per the process explained in Examples I to 8 were found to have all the above mentioned properties optimally as shown in Table 4 and to possess the HPLC pattern depicted in Fig 1 to 10 of the drawings accompanying this specification which ^presents the HPLC pattern of the formulation prefared by the process described in Example 1 to 10. The pharmaceutical formulation of the present invention can uc u-,.iinistered to a person who requires such administration by the nomal means such as tablets , capsules, oral suspensions and the like. The dosage of administration of the formutatjon may range from 250 mg to 500 mg thrice daily for a duration of 1 to 6 months time depending upon the clinical conditions of the person who requires the administration of the formulation Advantages of the invention The pharmaceuticai formuladon of the present invention can find applications fix the treatment of acute and chranic Hepatitis B; acute and chronic Hepatitis C and other related viral infections of ttie liver because 1. The formulation has all the essential antiviral and biological prc^rties against Hepatitis B virus; Hepatitis C vinjs and other related viral infections of the liver. 2. The formulation is non-toxic at tissue and cellular level; with proven safety for human use by experimental studies and dinical titals. 3. The fbmiulation possess r^produdWe dinkal efficacy tn the treatment of acute and chronic Hepatitis B; acute and chrt)nic Hepatitis C and other related viral infections of the liver. We Claim 1. A pharmaceutical formulation useful for the treatment of Hepatitis B (both acute and chronic), Hepatitis C (both acute and chronic) and other related viral infections of the liver with antihepatotoxic and liver cell regenerating potentials and immunomodulating properties which comprises (iv) an extract of the plant Phyllanthus amarus obtained by using a polar solvent alone, other than water (v) an extract of the plant Phyllanthus amarus obtained by using a mixture of a polar solvent and water and (ill) an extract of the plant Phyllanthus amarus obtained by using water alone 2. A pharmaceutical formulation as claimed in claim which comprises (i) 10 to 40% w/w of an extract of the plant Phyllanthus amarus obtained by using a polar solvent alone other than water (ii) 10 to 40% w/w of an extract of the pliant Phyllanthus amarus obtained by using a mixture of a polar solvent and water wherein the ratio of the solvent to water ranges from 20 to 80 and 80 to 20% w/w respectively and (iii) 10 to 40% w/w of an extract of the plant Phyllanthus amarus obtained by using water alone 3. A pharmaceutical formulation as claimed in claims 1 & 2 which comprises (i) 20 to 30% w/w of an extract of the plant Phyllanthus amarus obtained by using a polar solvent alone, other than water (ii) 20 to 40% w/w of an extract of the plant Phyllanthus amarus obtained by using a mixture of a polar solvent and water wherein the ratio of the solvent to water ranges from 80 to 20% and 20 to 80% w/w respectively (iii) 20 to 40-% w/w of an extract of the plant Phyllanthus amarus obtained by using a polar solvent and water wherein the solvent to water ratio ranges from 20 to 80 % and 80 to 20 % w/w respectively and (iv) 20 to 30% w/w of an extract of the plant Phyllanthus amarus obtained by using water alone 4. A pharmaceutical formulation as claimed in Claims 1 to 3 which comprises one part each of (i) an extract of the plant Phyllanthus amarus obtained by using a polar solvent alone, other than water (ii) an extract of the plant Phyllanthus amarus obtained by using a mixture of a polar solvent and water and (iii) an extract of the plant Phyllanthus amarus using water alone 5. A pharmaceutical formulation as claimed in claims 1 to 4 which comprises one part each of (i) 10 to 40% w/w of an extract of the plant Phyllanthus amarus obtained by using a polar solvent alone, other than water (ii) 10 to 40% w/w of an extract of the plant Phyllanthus amarus obtained by using a mixture of a polar solvent and water wherein the ratio of the solvent to water ranges from 20 to 80 and 80 to 20% w/w respectively and (ill) 10 to 40% w/w of an extract of the plant Phyllanthus amarus obtained by using water alone 6. A pharmaceutical formulation as claimed in claims 1 to 5 which comprises one part each of (i) 20 to 30 %w/w of an extract of the plant Phyllanthus amarus obtained by using a polar solvent alone, other than water (ii) 20 to 40 %w/w of an extract of the plant Phyllanthus amarus obtained by using a mixture of a polar solvent and water wherein the ratio of the solvent to water ranges from 80 to 20% and 20 to 80 % w/w respectively (iii) 20 to 40 % w/w of an extract of the plant Phyllanthus amarus using a mixture of a polar solvent and water wherein the ratio of the solvent to water ranges from . 20 to 80 % w/w and 80 to 20 % w/w respectively and (iv) 20 to 30 % w/w of an extract obtained using water alone 7.A pharmaceutical formulation as claims in claims 1 to 6 which comprises two parts (i) of an extract of the plant Phyllanthus amarus obtained by using a polar solvent alone, other than water and one part each of (ii) of an extract of the plant Phyllanthus amarus obtained by using a mixture of a polar solvent and water and (iii) an extract of the plant Phyllanthus amarus using water alone 8. A pharmaceutical formulation as claimed in claims 1 to 7 which comprises two parts (i) of 10 to 40 %w/w of an extract of the plant Phyllanthus amarus obtained by using a polar solvent, other than water and one part each (ii) of 10 to 40 % w/w of an extract of the plant Phyllanthus amarus obtained by using a mixture of a polar solvent and water wherein the ratio of the solvent to water ranges from 20 to 80% and 80 to 20 % w/w respectively (iii) of 10 to 40% w/w of an extract of the plant Phyllanthus amarus obtained by using water alone 9. A pharmaceutical formulation as claimed in claims 1 to 8 which comprises two parts (I) of 20 to 30 % W/W of an extract of the plant Phyllanthus amarus obtained by using a polar solvent alone, other than water and one part each (ii) of 20 to 40 % w/w of an extract of the plant Phyllanthus amarus obtained by using a mixture of a polar solvent and water wherein the ratio of the solvent to water ranges from 80 to 20% and 20 to 80 % w/w respectively (iii) of 20 to 40 % w/w of an extract of the plant Phyllanthus amarus obtained by using a mixture of polar solvent and water wherein the ratio of the solvent to water ranges from 20 to 80% w/w and 80 to 20% w/w respectively and (iv) of 20 to 30 % w/w of an extract of the plant Phyllanthus amarus obtained by using water alone. 10. A process for the preparation of pharmaceutical formulation useful for the treatment of Hepatitis B (acute and chronic), Hepatitis C (both acute and chronic) and other related viral infections of the liver with antihepatotoxic and liver cell regenerating potentials and immunomodulating properties which comprises mixing (i) an extract of the plant Phyllanthus amarus obtained by using a polar solvent alone, other than water (ii) an extract of the plant Phyllanthus amarus obtained by using a mixture of a polar solvent and water and (iii) an extract of the plant Phyllanthus amarus obtained by using water alone 11. A process as claimed in claim 10 which comprises mixing (i) 10 to 40% w/w of an extract of the plant Phyllanthus amarus obtained by using a polar solvent alone, other than water (ii) 10 to 40% w/w of an extract of the plant Phyllanthus amarus obtained by using a mixture of a polar solvent and water wherein the ratio of the solvent to water ranges from 20 to 80 and 80 to 20% w/w respectively and (iii) 10 to 40% w/w of an extract of the plant Phyllanthus amarus obtained by using water alone 12. A process as claimed in claims 10 & 11 which comprises mixing (i) 20 to 30% w/w of an extract of the plant Phyllanthus amarus obtained by using a polar solvent alone, other than water (ii) 20 to 40% w/w of an extract of the plant Phyllanthus amarus obtained by using a mixture of a polar solvent and water wherein the ratio of the solvent to water ranges from 80 to 20% and 20 to 80% w/w respectively (iii) 20 to 40-% w/w of an extract of the plant Phyllanthus amarus obtained by using a polar solvent and water wherein the solvent to water ratio ranges from 20 to 80 % and 80 to 20 % w/w respectively and (iv) 20 to 30% w/w of an extract of the plant Phyllanthus amarus obtained by using water alone 13. A process as claimed in claim 10 to 12 which comprises mixing one part each of (i) an extract of the plant Phyllanthus amarus obtained by using a polar solvent alone, other than water (ii) an extract of the plant Phyllanthus amarus obtained by using a mixture of a polar solvent and water and (iii) an extract of the plant Phyllanthus amarus using water alone 14. A process as claimed in claims 10 to 13 which comprises mixing one part each of (i) 10 to 40% w/w of an extract of the plant Phyllanthus amarus obtained by using a polar solvent alone, other than water (ii) 10 to 40% w/w of an extract of the plant Phyllanthus amarus obtained by using a mixture of a polar solvent and water wherein the ratio of the solvent to water ranges from 20 to 80 and 80 to 20% w/w respectively and (iii) 10 to 40% w/w of an extract of the plant Phyllanthus amarus obtained by using water alone 15. A process as claimed in claims 10 to 14 which comprises mixing one part each of (i) 20 to 30 %w/w ofan extract of the plant Phyllanthus amarus obtained by using a polar solvent alone, other than water (ii) 20 to 40 %w/w of an extract of the plant Phyllanthus amarus obtained by using a mixture of a polar solvent and water wherein the ratio of the solvent to water ranges from 80 to 20% and 20 to 80 % w/w respectively (iii) 20 to 40 % w/w of an extract of the piant Phyllanthus amarus using a mixture of a polar solvent and water wherein the ratio of the solvent to water ranges from 20 to 80 % w/w and 80 to 20 % w/w respectively and (iv) 20 to 30 % w/w ofan extract obtained using water alone 16. A process as claimed in claims 10 to 15 which comprises mixing two parts (i) of an extract of the plant Phyllanthus amarus obtained by using a polar solvent alone, other than water and one part each of (ii) of an extract of the plant Phyllanthus amarus obtained by using a mixture of a polar solvent and water and (iii) an extract of the plant Phyllanthus amarus using water alone 17. A process as claimed in claims 10 to 16 which comprises mixing two parts (i) of 10 to 40 %w/w of an extract of the plant Phyllanthus amarus obtained by using a polar solvent, other than water and one part each (ri) of 10 to 40 % w/w of an extract of the plant Phyllanthus amarus obtained by using a mixture of a polar solvent and water wherein the ratio of the solvent to water ranges from 20 to 80% and 80 to 20 % w/w respectively (iii) of 10 to 40% w/w of an extract of the plant Phyllanthus amarus obtained by using water alone 18.A process as claimed in claims 10 to 17 which comprises mixing two parts (i) of 20 to 30 % W/W of an extract of the plant Phyllanthus amarus obtained by using a polar solvent alone, other than water and one part each (ii) of 20 to 40 % w/w of an extract of the plant Phyllanthus amarus obtained by using a mixture of a polar solvent and water wherein the ratio of the solvent to water ranges from 80 to 20% and 20 to 80 % w/w respectively (iii) of 20 to 40 % w/w of an extract of the plant Phyllanthus amarus obtained by using a mixture of polar solvent and water wherein the ratio of the solvent to water ranges from 20 to 80% w/w and 80 to 20% w/w respectively and (iv) of 20 to 30 % w/w of an extract of the plant Phyllanthus amarus obtained by using water alone. 19. A process as claimed in claims 10 to 18 wherein the extracts are prepared from the powder obtained by drying the parts such as leaves, stems, seeds and roots of the taxonomically identified collections of the plant Phyllanthus amarus , keeping it in an oven heated to a temperature in the range of 50 to 80 degree C for a period in the range of 3 to 5 hrs a day for a period of 3 to 6 successive days and powdering 20. A process as claimed in claim 19 wherein before subjecting the powder for extraction procedures, each batch of powder is subjected for known sterilising treatment , if required for removing any bacterial or fungal contamination 21. A process as claimed in claims 10 to 20 wherein the extraction is carried out at a temperature in the range of 37 to 60 degree C for a period ranging from 2 hours to 18 hours 22, A process as claimed in claims 10 to 21 wherein the polar solvent such as methanol, ethanol, hexane and butanol is used for the extraction . 23. A pharmaceutical formulation useful for the treatment of Hepatitis B {both acute and chronic); Hepatitis C (both acute and chronic) and other related viral infections of the liver with antihepatotoxic and liver cell regenerating potentials and immunomodulating properties substantially as herein described with reference to the Examples 1 to 8. 24. A process for the preparation of a pharmaceutical formulation useful for the treatment of Hepatitis B (both acute and chronic); Hepatitis C (both acute and chronic) and other related viral infections of the liver with antihepatotoxic and liver cell regenerating potentials and immunomodulating properties substantially as herein described with reference to the Examples 1 to 8. |
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0405-mas-1999 claims-duplicate.pdf
0405-mas-1999 correspondence-others.pdf
0405-mas-1999 correspondence-po.pdf
0405-mas-1999 description (complete)-duplicate.pdf
0405-mas-1999 description (complete).pdf
| Patent Number | 202193 | |||||||||
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| Indian Patent Application Number | 405/MAS/1999 | |||||||||
| PG Journal Number | 05/2007 | |||||||||
| Publication Date | 02-Feb-2007 | |||||||||
| Grant Date | 03-Oct-2006 | |||||||||
| Date of Filing | 12-Apr-1999 | |||||||||
| Name of Patentee | M/S. UNIVERSITY OF MADRAS | |||||||||
| Applicant Address | CHEPAUK CHENNAI 600 005 | |||||||||
Inventors:
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| PCT International Classification Number | A61K 35/78 | |||||||||
| PCT International Application Number | N/A | |||||||||
| PCT International Filing date | ||||||||||
PCT Conventions:
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