This invention relates to a simple process for the preparation of active fractions from Phvllanthus amarus with 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 employing the aerial parts or whole plant However the wide variation in results obtained points to the necessity of identifying compounds contributing to activity and to have a basis for structure activity relationship.
In Indian systems, medicine especially in Ayurveda and Siddha Phvllanthus amarus is used as powdered whole plant, paste of fresh plant and decoctions to treat jaundice of varied etiology. Also used are roots or leaves of the plant to treat jaundice.
Thus in order to correlate activity with different parts of the plant - stem, leaves, fruits and roots of Phvllanthus amarus were extracted with 50% methanol in water and the in-vitro HBsAg activity tested. The stem and the root showed HBsAg binding, the roots being more active than the stem, 2.5 mg/ml as against 5 mg/ml. This result correlates well with the results obtained by Unander et.al (D.W.Unander, P.S.Venkateswaran, I.Millman, H.H.Bryan and B.S.BIumberg, In J.Janick and J.E. Simon [eds] Advances in New Crops, Timber Press, Portland, Oregan 1990 pp 518-521) who found that roots showed significantly mqre_.DNA polymerase.inhibitory activity than shoots. However, the active fractions and the chemical nature of the compounds present have not been identified.
(n the present invention, the root was extracted with various solvents like hexane, chloroform, methanol, methanol:water{1:1) and water in order to optimise the activity and methanol - water 1:1 gave good results, with HPTLC profile as in Fig.1. The methanol - water extract was then fractionated by further solvent extraction using hexane, chloroform, ethylacetate and butanol, the residual aqueous portion being evaporated to dryness under reduced pressure. The hexane, butanol and aqueous fractions which showed HBsAg binding had HPTLC finger prints as shown by Fig.2, 3 & 4, thus the composition of the active fractions are defined.

he main advantages of the present invention are :
1) Identification of the root as the part showing maximum HBsAg binding
2) Process for preparation of the active fraction from the root
3) Identification of the fractions contributing to the activity
4) Defining Composition of the active fractions by means of HPTLC
chromatograms thus defining the composition which could lead to consistent composition and hence to consistent results.
Thus in a typical experiment powdered root of Phvllanthus amarus was extracted with methanol:water mixture containing 50% methanol v/v. After keeping for 24 hours with the solvent mixture, the procedure is repeated and the methanol is removed from the combined extracts under reduced pressure. The residual aqueous phase is centhfuged and the centrifugate is extracted successively with hexane, chloroform and ethylacetate and butanol and the solvents removed under reduced pressure. The hexane, butanol and aqueous fractions show HBsAg binding. A suitable flow-chart for obtaining active fractions is illustrated in Fig.5.
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 an active extract of Phvllanthus amarus and the method of fractionation to get fractions active in-vitro against HBsAg.

Preparation of extract
Roots of Phvllanthus amarus were dried and powdered. 500g of the powdered material was extracted with 2x3500 ml of methanol:water (1:1) at ambient temperature for 2x24 hours. Removal of solvent gave an active extract with HPTLC profile shown in Fig.1. Instead of evaporation the combined extracts are filtered and further worked-up as given below:
Fractionation
The filtered extract was concentrated under reduced pressure to remove methanol and the residual aqueous portion was centrifuged. The centrifugate was extracted sequentially with hexane (3x400 ml), chloroform (3x400 ml), ethylacetate (4x500 ml) and n-butanol (3x500 ml) and the solvents removed under reduced pressure. The remaining aqueous portion was evaporated to dryness under reduced pressure. The hexane, n-butanol and the residual aqueous fractions showed HBsAg binding activity.
HPTLC Studies
The active hexane fraction was subjected to HPTLC studies using silica gel GF254 as adsorbent and hexane:ethylacetate (2:1) mixture as the developing solvent. A typical HPTLC profile of the extract is shown in Fig. 2.
The active n-butanol and residual aqueous fraction were subjected to HPTLC studies using silica gel GF 254 as adsorbent and ethylacetate:formic acid:acetic acid:water (100:11:11:27) mixture as the developing solvent. A typical HPTLC profile of the n-butanol and residual aqueous fractions are shown in Fig.3 and Fig.4 respectively.

We Claims
1. A process for the preparation of active fractions from the plant Phyllanthua Amarus for the treatment of hepatitis comprising the steps of carrying out at least one extraction of the powdered root material of the
said plant with a solvent comprising a methanol-water
mixture containing' at least 40-60 X methanol;
removing the methanol from the resulting extract; an
fractionatingNthe residual aqueous portion of the saidextract by; purification procedures^ ,■'-..,
2. ft process as claimed in Claim 1 wherein the said
root material and, solvent are in the proportion ts5 to
1:10 weight/volume.
3v A process as claimed in Claim 1 or Claim 2 wherein the purification procedure comprises the steps of carrying out three to five extractions of the residual aqueous portions of the root material successively with hexane, chloroform, ethylacetate and butanol.
4. f\ process as claimed in Claim 3 wherein the residual aqueous portion of the extract of the ssid root

material and bexane are in the proportion Isi to ls3
/*

34'A process as claimed in Claim 3 wherein the residual
aqueous portion of the extract of the said root
material and chloroform are in the proportion ltl to
1:3 v/v.
A process as claimed in Claim 3 wherein the residual
aqueous portion of the extract of the said root
material and ethyl acetate are in the proportion 1:1 to
1:3 v/v. s~i ,
7. f\ process as claimed in Claim 3 wherein the residual
aqueous portion of the extract of the said root
materi al and butanol are in the proportion 1:1 to

-■$■.—
8. p process as claimed in any one of the Claims 3 to 7
wherein the residual aqueous phase after successive
extractions is evaporated to dryness under reduced
pressure.
9. ft process for the preparation of /active fractions
I
F

from the plant phyllanthus Amarus for the treatment of hepatitis substantially as herein described and as i1lustrated.
10. Active fractions prepared from the plant .
PhylIan thus Amarus for the treatment of hepatitis
/^' whenever prepared by a process as claimed in anyone of
the preceding Claims
Dated this the 21ot day of May 1999