This invention relates to a simple process for. preparing reproducibly an active fraction from Phvllanthus amarus with an assured in vitro HBsAg binding activity which can be used in the treatment of Hepatitis viruses, especially Hepatitis B.
There have been several clinical trials on chronic carriers of Hepatitis B using Phvllanthus amarus from different sources. However there has been wide variation in the results obtained because the active principle(s) have not been identified and the material has not been standardised in the terms of the active principle, although one trial in New Zealand has used material standardised in terms of geranial, which however, was not claimed to be the active principle (A.Milne et.al., New Zealand Medical J. 107(980) 243, 1994)
Phyllanthus amarus has been used in Indian Systems of medicine especially in Ayurveda and Siddha as powdered whole plant, paste of fresh plant, and decoctions to treat jaundice of varied a etiology.
Blumberg et al [U.S.Patent, 4, 673, 575 (1987)] used the methanol extract of Phyllanthus amarus after extraction with hexane and benzene to obtain material which was claimed useful for the treatment of Hepatitis B infection and shown in woodchucks to reduce HBsAg titres. This extract showed both HBsAg binding and HBV DNA polymerase inhibitory activity. However the active constituents have not been identified by Blumberg et al.
Mehrotra et al (Indian J. Med. Res. [A] 93. 71-73 March 1991) have fractionated cold ethanol extract of whole plant of Phyllanthus amarus' followed by solvent fractionation and shown that the butanol fraction showed maximum in vitroHBsAg binding, HBeAg binding and maximum inhibitory activity against HBV DNA. The butanol extract was further fractionated; however no active constituents were identified.
In both the above cases, neither has the chemical class of the active principle nor the identity of the active principle been identified.
The process proposed herein is different from the known processes involving Phyllanthus amarus; besides this process also identifies the active principles.
2

In the present invention it has been possible to obtain a polar fraction consisting mostly of tannins and flavonoids which has an enhanced HBsAg binding activity as compared to starting extracts. Thus extraction of powdered aerial parts of Phvllanthus amarus using a 1:1 mixture of methanol and water gave an extract showing HBsAg binding at a concentration of 2.5 mg/ml. Solvent fractionation using different solvents like hexane, chloroform, ethylacetate and bitingly leads to concentration of the activity in the ethylacetate fraction which had a HPTLC pattern shown in Fig.1. Further purification of the ethylacetate fraction by chromatography twice on Spandex LH-20 gave fractions which showed an increase in activity in certain of the fractions. No HBsAg binding activity is found in the n-butanol fraction.
It has also been possible to obtain enhancement of activity by directly loading the methanol: water (1:1) extract, after removal of methanol, on Sephadex LH-20 and chromatography using water followed by increasing amounts of methanol. The active fractions get eluted with 80-100% methanol showing an enhancement of activity. Further enrichment by rechromatoQraphy of one of the fractions over Sephadex LH-20 gave a fraction containing 85% of the tannin geranial the HPLC curve of which is shown in Fig. 2. This compound has been earlier identified from the same plant but has not been identified as a major active HBsAg binding principle. All fractions showing activity contain geraniin. Fractions showing enhanced activity contain increased amounts of geraniin. Thus, geraniin appears to be one of compounds present in major amount which contributes to the activity. However there are probably other inhibitory principles which synergetically contribute to the activity. Thus fraction of composition shown by HPTLC profile in Fig. 4 obtained by rechromatography of another fraction also shows enhanced HBsAg binding and consists in major amounts of corilagin, frozen and ellagic acid. Identification of peal’s was done by comparison with isolated compounds whose structure was established by spectroscopic comparison with literature data.

1) Reproducible preparation of an enriched active fraction
2) Identification of the major components of the active fraction
3) Identification of geraniin as a major HBsAg binding principle
4) Simple process for the preparation of the active fraction
5) Identification of the relative amounts of the various compounds present and thus standardizing the extract composition.
Thus we Identify and conclude that presence of geraniin in major amounts in the extracts contribute to the enhanced activity.
It is noteworthy that:
(a) according to this invention, the process for the preparation of an enriched fraction from Phyllanthus amarus for use in the treatment of hepatitis comprises the steps of carrying out at least one extraction of the plant material with a solvent selected from (i) methanol (ii) methanol-water mixture containing at least 30% of methanol; removing the methanol from the extract of the plant material; and fractionating the residual aqueous portion of the said extract by purification procedures.
(b) The plant material and solvent in the process as set out against item (a) above are preferably in the proportion 1:5 to 1:10 weight/volume.
(c) Whenever the solvent used is methanol, in the
process as set out against item (a) or item (b)

above the extract is mixed with preferably 10 to 20 times by weight of water, before purification.
(d) In the process as set out against any one of the above items above the methanol-water mixture preferably contains 50% of methanol.
(e) In the process as set out against any one of the above mentioned items, the purification procedure comprises the steps of carrying out three to five extractions of the residual aqueous portion of the extract of plant material with ethyl acetate after carrying out
three to five preliminary extractions of the said residually portion of the extract of plant material with hexane and chloroform.
(f) The residual aqueous portion of the extract
of plant material and ethyl acetate, in the
process as set out against item (e) above, are
preferably in the proportion 1:0.5 to 1:3 by
volume.
(g) The residual aqueous portion of the extract of
plant material and hexane, in the process as set
out against item (e)above, are preferably in the
proportion 1:0.5 to 1:3 by volume.
(h) The residual aqueous portion of the extract of

plant material and chloroform, in the process as set out against items(e)above, are preferably in the proportion 1:0.5 to 1:3 by volume. (1) The purification procedure according to the process set out against any one of the items (e) to (h) above, comprises the steps of chromatography of the ethyl acetate fraction over Sephadex LH-20 or other polymeric adsorbents such as Sephadex-G25, MCI Gel CHP-20P or DIAION HP-20.
(j) On the other hand, the purification procedure according to the process set out against any one of the items (a) to (d) above, comprises the steps of separating the residual aqueous portion by chromatography over Sephadex LH-20 or other polymeric adsorbents such as Sephadex G-25, MCI Gel CHP-20P or DIAION HP-20.
(k) In the process as set out against item (j) above, one of the fractions obtained after chromatography is further fractionated by chromatography over Sephadex LH-20 using methanol to give a fraction containing substantially greenling identified as the major active principle. (1) In the process as set out against item (j)

above, the other fraction obtained is further purified by chromatography over Sephadex LH-20 using methanol to give an active fraction comprising major amounts of corilagin, furosin and ellagic acid.
Thus in a typical experiment powdered aerial parts of Phyllanthus amarus were extracted with methanol water containing from 30 to 100% of methanol. After keeping for 24 hours with the solvent mixture, the methanol was removed under reduced pressure and the aqueous phase extracted successively with hexane, chloroform and ethylacetate. HBsAg binding activity is concentrated in the ethylacetate fraction. Further fractionation by twice chromatography on LH-20 leads to a further enrichment of activity and increased percentage of geraniin in the active fractions. A suitable procedure for obtaining active fractions is illustrated in Fig. 3.
The process is further illustrated by the following examples which are provided by way of illustration and therefore not to be construed to limit the scope of invention.
Example-1
This example describes the preparation of the active extract of Phyllanthus amarus and method of fractionation to get enriched fractions containing geraniin which were tested to determine the HBsAg binding activity.

Preparation of extract
Aerial parts of Phvllanthus amarus were dried and powdered. 250g of the powdered material was extracted with 2x1750 ml of methanol: water (1:1) at ambient temperature for 2x24 hours. The solution which was brown in colour was filtered and divided into two equal lots (Lot-1 & Lot-2).
Fractionation
The extract (Lot-1) was concentrated under reduced pressure to remove methanol and the residual aqueous portion was extracted sequentially with hexane (3x150 ml), chloroform (3x150 ml), ethylacetate (3x150 ml) and n-butanol (4x100 ml). The remaining aqueous portion was evaporated to dryness. The ethylacetate fraction was found to have significant HBsAg binding activity. The ethylacetate fraction was separated by chromatography over Sephadex LH-20. HBsAg binding activity was found in two fractions each weighing 110 mg and 260 mg.
HPTLC Studies
The active ethylacetate fraction was subjected to HPTLC studies .using Silica gel GF254 as adsorbent and ethylacetate:formic acid acetic acid: water (100:11:11:27) mixture as the developing solvent. A typical HPTLC profile of the extract is shown in Fig. 1.

Example-2
Phvllanthus amarus was extracted as under ExampIe-1.
The extract (Lot-2) was concentrated- under reduced pressure to remove methanol and loaded directly on Sephadex LH-20. Elution was done using water followed by increasing amounts of methanol in water. Two fractions with significantly enhanced HBsAg binding activity each weighing 386 mg and 600 mg were obtained. Further purification of the latter fraction over Sephadex LH-20 using methanol resulted in a fraction with enhanced activity containing ~ 85% of geraniin identified on the basis of comparison of spectroscopic data (1H-NMR, 13C-NMR, [α] with literature information whereas purification of the former fraction gave a fraction with a HPTLC profile shown in Fig.4, HPTLC analysis of which was carried out as described earlier in Example-1. This fraction consists of various tannins including in major amounts corilagin, furosin and ellagic acid which were identified by spectroscopic comparison with literature data.
HPLC Studies
The active fraction is essentially composed of material having an elution time less than 8 minutes by HPLC on a ODS reverse phase column using a solvent system of 15:85 (acetonitrile:0.01M potassium dehydrogenate phosphate) pH=3. HPLC curve of the fraction is shown in Fig. 2.

We claim:
A Process for the preparation of an enriched fraction from Phyllanthus amarus for use in the treatment of hepatitis comprising the steps of carrying out at least one extraction of the plant material with a solvent selected from (I) methanol, (ii) methanol-water mixture containing at least 30% of methanol, removing the methanol from the extract of the plant material; and fractionating the residual aqueous portion of the said extract by purification procedures. —; ',x f
A process as claimed in Claim 1 wherein the plant material and solvent are in the proportion of 1:5 to 1:10 weight/volume.
A process as claimed in Claim 1 or Claim 2 wherein the extract is mixed with 10 to 20 times by weight of water, before purification, whenever the solvent used is methanol.
A process as claimed in any one of the preceding Claims wherein the methanol-water mixture contains 50% of methanol.
A process as claimed in any one of the preceding Claims wherein the purification procedure comprises the steps of carrying out three to five extractions of the residual aqueous portion of the extract of plant material with ethyl acetate after carrying out three to five preliminary extractions of the said residual aqueous portion of the extract of plant material with hexane and chloroform.
A process as claimed in Claim 5 wherein the residual aqueous portion of the extract of plant material and ethyl acetate are in the proportion 1:0.5 to 1:3 by volume.
A process as claimed in Claim 5 or Claim 6 wherein the residual aqueous portion of the extract of plant material and hexane are in the proportion 1:0.5 to 1:3. by \^c>luir)G. .
A process as claimed in any one of the Claims 5 to 7 wherein the residual aqueous portion of the extract of plant material and chloroform are in the proportion 1:0.5 to 1:3. by Wofurv)^.

A process as claimed in any one of the Claims 5 to 8 wherein the purification procedure comprises the steps of chromatography of the ethyl acetate fraction over Sephadex LH-20 or other polymeric adsorbents as Sephadex-G25, MCI Gel CHP-20P or DIAION HP-20.
A process as claimed in any one of the Claims 1 to 4 wherein the purification procedure comprises the steps of separating the residual, aqueous portion by chromatography over Sephadex LH-20 or otherf polymeric as sorbents such as Sephadex G-25, MCI Gel CHR -20P or DIAION HP-20.
A process as claimed in Claim 10 wherein one of the fractions obtained after chromatography is further fractionated by chromatography over Sephadex LH-20 using methanol to give a fraction containing substantially; geraniin identified as the major active principle.
A process as claimed in Claim 10 wherein the other fraction obtained is
further purified by chromatography over Sephadex LH-20 using methanol
to give an active fraction comprising major amounts of corilagin, furosin
and ellagic acid. ^
A process for the preparation of an enriched fraction from Phyllanthus amarus for use in the treatment of hepatitis substantially as herein described and illustrated.
Enriched fraction prepared from Phyllanthus amarus for use in the treatment of hepatitis whenever prepared by a process as claimed in any of the preceding Claims.