FORM-2
THE PATENTS ACT, 1970
(39 of 1970)
& THE PATENTS RULES, 2003
COMPLETE SPECIFICATION
(See section 10; rule 13)
A PROCESS FOR THE PREPARATION OF WITHANIA SOMNIFERA FRACTION
SERUM INSTITUTE OF INDIA LTD.,
an Indian Company
of Off Soli Poonawala Road, 212/2 Hadapsar, Pune 411 028,
Maharashtra, India;
THE FOLLOWING SPECIFICATION PARTICULARLY DESCRIBES THE INVENTION AND THE MANNER IN WHICH IT IS TO BE PERFORMED

FIELD OF THE INVENTION
The present invention relates to a process for preparing an enriched plant fraction containing a mixture of Withaferin A and Withanolide A, obtained from the roots of plant Withania Somnifera.
Particularly, the present invention relates to a process for preparing a withanolide rich fraction from polar extracts.
BACKGROUND OF THE INVENTION
Withania Somnifera is commonly known as Ashwagandha, Indian ginseng, Winter cherry and Ajagandha. It is mainly used in herbal formulations of the Ayurvedic or Indian system of medicine for treating memory loss, tumors, inflammation, arthritis, asthma and hypertension.The main constituents of ashwagandha are alkaloids and steroidal lactones. Among the various alkaloids, withanine is the main constituent. The other alkaloids are somniferine, somnine, somniferinine, withananine, pseudo-withanine, tropine, pseudo-tropine, 3-a-gloyloxytropane, choline, cuscohygrine, isopelletierine, anaferine and anahydrine. Two acyl steryl glucoside viz. sitoindoside VII and sitoindoside VIII have been isolated from root. The leaves contain steroidal lactones, which are commonly called withanolides. The withanolides have C28 steroidal nucleus with C9 side chain, having six membered lactone ring. Apart from leaves withanolides are also present in roots and berries.
Existing Knowledge
Following patents/applications disclose processes for the preparation of Withania Somnifera extract.
US 6713092 discloses a process of making an Withania Somnifera extract composition which comprises (a) providing root stock and leaves of a Withania Somnifera plant which is about 1-2 years old, (b) extracting said root stock and leaves

with an aqueous-alcoholic solvent in the presence of a exogenous saccharide, c) concentrating the extract under vacuum, (d) treating the residue with an apolar organic solvent to remove free withanolide A aglycones therefrom, (e) vacuum the insoluble residue of such treatment below about 60.degree. C. to provide a dry solid, and (f) pulverizing the solid under controlled temperature and humidity conditions, to obtain the desired powder product.
US Patent No. 6153198 discloses a process of making the extract of Withania Somnifera which comprises (a) providing root stock of a Withania Somnifera plant which is about 1-2 years old, (b) extracting said root stock with an aqueous-alcoholic solvent, (c) concentrating the extract under vacuum, (d) treating the residue with an apolar organic solvent to remove free withanolide A aglycones therefrom, (e) vacuum drying the insoluble residue, of such treatment below about 60° C to provide a dry solid, and (f) pulverizing the solid under controlled temperature and humidity conditions, to obtain the powder product. The aqueous-alcoholic solvent used is water-methanol or water-ethanol and the organic solvent used is chloroform or ethyl acetate.
US 20040033273 discloses a method for obtaining a composition having immune stimulating activity or anti-tumor activity from Withania Somnifera comprising: (a) contacting Withania Somnifera plant or plant part with a first medium polar solvent to produce a particulate suspension; (b) clarifying the particulate suspension to produce a clarified First solution and a first residue; (c) evaporating the solvent from the first clarified solution to produce a fraction, denoted fraction A; (d) resuspending the first residue in a second polar solvent thereby producing a second solution and a second residue; (e) clarifying the second solution to produce a second clarified solution; (f) evaporating the second polar solvent from the second clarified solution to produce a fraction, denoted fraction B; (g) resuspending the second residue in a third solvent

more polar than the second polar solvent thereby producing a third solution and a third residue; (h) clarifying the third solution to produce a third clarified solution; (i) evaporating the third solvent from the third clarified solution to produce a fraction, denoted fraction C; (j) combining fractions A, B and C to produce an extract; (k) resuspending the extract in a solution to produce a fourth alkaline solution; and (1) fractionating the fourth solution with a non polar solvent and removing the solvent to produce a composition having immune stimulating activity or anti-tumor activity. The first residue is resuspended in a solvent having about 50% ethanol or about 40 to 60% isopropyl alcohol. The first medium polar solvent comprises acetone, tetrahydrofuran or ethylacetate. The second solvent comprises a mixture of water and isopropyl alcohol (IPA).
US7108870 discloses a process for isolation of withaferin-A from plant materials, said process comprising the steps of: (i) extracting the plant materials in an aqueous alcohol extraction solvent, (ii) defatting the extract, as obtained in step (i), with partitioning with n-hexane followed by chromatographic separation to obtain a withanolide preparation, (iii) portioning out withanolide aglycones from the withanolide preparation, as obtained in step (ii), into chloroform followed by evaporation of said chloroform to obtain a chloroform extract, and (iv) dissolving the chloroform extract as obtained in step (iii) in methanol followed by chromatographic separation to obtain withaferin-A..
The extraction is performed using a 60:40 methanol:water extraction solvent.
The prior art discusses crude Withania Somnifera extract preparation methods which are not sufficient to produce a biologically active product.
Accordingly, it is desirable to develop a process for the preparation of Withania
Somnifera fraction which is enriched with withanolides. I

Further, it is desirable to develop a simple, economic and high yielding process for the preparation of Withania Somnifera fraction.
OBJECTS OF THE INVENTION
It is an object of the present invention is to provide a novel process for the preparation of Withania Somnifera fraction enriched with withanolides.
It is another object of the present invention to provide a process for preparing an enriched plant fraction containing a mixture of Withaferin A and Withanolide A, obtained from the roots of plant Withania Somnifera.
It is still another object of the present invention to provide a Withania Somnifera fraction which contains a specific ratio of Withanolide A to Withaferin A in combination with other components.
It is yet another object of the present invention to provide a simple process for the preparation of Withania Somnifera fraction .
It is a further object of the present invention to provide a process for the preparation
of Withania Somnifera fraction which is high yielding.

It is still further object of the present invention to provide a process for the preparation of Withania Somnifera fraction which is economic.
SUMMARY OF THE INVENTION
In accordance with the present invention there is provided a process for preparing a Withania Somnifera fraction enriched in Withanolides; said process comprising the following steps:

a) obtaining the coarse root material from Withania Somnifera plant and refluxing with hot water to obtain a slurry;
b) filtering and vacuum concentrating the slurry to obtain concentrated aqueous extract;
c) liquid -liquid successive partitioning of concentrated aqueous extract with at least one non-polar organic solvent & at least one polar organic solvent to obtain an organic fraction of the extract; and
d) concentrating and co-distilling the an organic fraction under vacuum to
remove the traces of polar organic solvent followed by drying the fraction at a
temperature not more than 70°C to obtain a Withania Somnifera fraction
enriched in Withanolides.
Typically, the non polar organic solvent is selected from the group consisting of n-hexane, toluene and benzene.
Typically, the polar organic solvent is selected from the group consisting of butanol, dichloromethane, dichloroethane and chloroform.
In accordance with another aspect of the present invention there is also provided a Withania Somnifera fraction enriched with Withanolide.
. . .1
In accordance with the present invention the Withania Somnifera fraction comprises
a) Withanolide A of about 0.5-1%; b) Withaferin A of about 0.1-0.6%; c) Withanolide
B of about 0.01-0.1%; d) Withanoside IV of about 0.8-1.2%; e) Withanoside V of
about 0.5-0.8%; and f) 12-deoxy withastramonolide of about 0.8-1.2%.

Typically, the ratio of Withaferin A and Withanolide A is in the range of about 1:2 to 1:5.
BRIEF DESCRIPTION OF THE DRAWINGS
The invention will now be described with reference to the accompanying drawing in which:
Fig.l illustrates a process for the preparation of a Withania Somnifera fraction enriched with withanolide in accordance with the present invention.
Fig. 2 illustrates identification of marker compounds on the basis of retention time and UV spectral matching with standard markers.
Fig. 3 illustrates identification of marker compounds on the basis of LC-MS analysis.
Fig. 4 illustrates HPLC-DAD chromatograms of various fractions obtained from successive fractionations of Withania Somnifera extract (WSE) and reference standards.
Fig. 5 illustrates chemo-profiling of Withania Somnifera fraction in accordance with the present invention.
Fig. 6 illustrates chemo-profiling of the extract disclosed in Indian Patent application No.l253/MUM/2003.
Fig. 7 illustrates chemo-profiling of the fraction disclosed in US20040033273.
DETAILED DESCRIPTION OF THE INVENTION
In accordance with the present invention there is provided a process for preparing a Withania Somnifera fraction enriched in Withanolides; said process comprising the following steps:

First step is obtaining the coarse root material from Withania Somnifera plant and refluxing with hot water to obtain a slurry. The obtained slurry is then filtered and vacuum concentrated to obtain concentrated aqueous extract.
The next step is liquid -liquid successive partitioning of concentrated aqueous extract with at least one non-polar organic solvent and at least one polar organic solvent to obtain an organic fraction of the extract. The obtained the organic fraction is then concentrated and co-distilled under vacuum to remove the traces of polar organic solvent followed by drying the fraction at a temperature not more than 70°C to obtain a Withania Somnifera fraction enriched in Withanolides.
Typically, the non polar organic solvent is selected from the group consisting of n-hexane, toluene and benzene.
Typically, the polar organic solvent is selected from the group consisting of butanol, dichloromethane, dichloroethane and chloroform.
In accordance with another aspect of the present invention there is also provided a Withania Somnifera fraction enriched with Withanolide.
In accordance with the present invention the Withania Somnifera fraction comprises a) Withanolide A of about 0.5-1%; b) Withaferin A of about 0.1-0.6%; c) Withanolide B of about 0.01-0.1%; d) Withanoside IV of about 0.8-1.2%; e) Withanoside V of about 0.5-0.8%; and f) 12-deoxy withastramonolide of about 0.8-1.2%.
Typically, the ratio of Withaferin A and Withanolide A is in the range of about 1:2 to
1:5.
Following examples illustrate the invention, but are not intended to limit the scope of the present invention.

EXAMPLES
Example 1
Chemical characterization of Withania Somnifera fraction of the present invention (SIIL-F-4)
LC-MS fingerprint of Withania Somnifera fraction was developed towards identification of constituents.
a) Sample Preparation for LC-MS analysis:
Sample was prepared by sonication-extraction of weighed amount of extract (0. 4 gm) with 10 ml of methanol at a room temperature for 10 minutes. Marker compounds (1 mg/ml) in methanol were also sonicated for 10 minutes at room temperature for maximum dissolution. The resulting Withania Somnifera extract (WSE) suspension was filtered through a 0.22 u membrane filter (Pall Corporation, Mumbai, India) prior to analysis. The volume of injection was optimized at 10 ul.
b) Identification and quantification of Marker compounds in the Withania
Somnifera fraction of the present invention
Marker compounds were identified and quantified in the Withania Somnifera fraction by using HPLC. (Table 2)

Table 2

Column , RP C-l 8 Luna phenomenex (250 x 4.6 mm)
Column oven i 25oC
temperature
Mobile phase 1.36 g of anhydrous Potassium dihydrogen orthophosphate (KH2P04) was dissolved in 900 ml of HPLC grade water (obtained from Millipore, Milli-Q Water purification system) and the pH was adjusted to 2.58 using 10.0% orthophosphoric acid solution. The volume was adjusted to 1000 ml with water and was then filtered through 0.45 u membrane and degassed in a sonicator for 3 minutes.

B-Acetonitrile
Flow rate 1.5 ml
Injection volume 25.0ul
Gradient Time A cone B.Con
0.01 95 5
18.00 55 45
25.00 20 80
28.00 20 80
35.00 45 45
40.00 95 5
45.00 95 5
45.00 Stop
Detector ; SPD- M 10 Avp Photo diode array detector
Wave length ; 227 nm
Standard preparation Withanoside-IV - 5.83 mg :Withanoside V-4.9 mg ; Withaferin A-2.72 mg ; 12 deoxy withastramonolide-2.82mg ; Withanolide-A 2.45 mg & Withanolide-B- 2.35 mg were dissolved in 25 ml methanol & then volume was adjusted to 50 ml.
Sample preparation -20002.8 mg of extract was transferred to a 100ml volumetric flask and dissolved in methanol followed by sonication for 10 to 15 minutes. Further the sample was boiled using water bath for 15 to 20 minutes and cooled. Then the volume was adjusted to 100ml with methanol and filtered through 0.45 u membrane filter paper.
Separations were carried out using reversed column BDS Hypersil C-l8 column (particle size, 5 um; dimensions, 250 X 4.6 mm, Thermo electron corporation)

maintained at 50 °C. The mobile phase consisted of water (A) and mixture of methanol and reagent alcohol (B) in the ratio of 1:1. Reagent alcohol is denatured ethanol and consists of ethanol, methanol and 2- propanol in the ratio of 90.6: 4.5: 4.9.
Analysis was done using gradient elution as 65A/35B to 55A/45B during run time of 25 min. Flow rate was kept at lml/min and each run was followed by wash with 100 % of acetonitrile (B) for 5 min and equilibration period of 10 min. The results are shown in table 3.
Table 3

Analysis Specification Results
Identity HPLC-PDA detection
Total withanolide by HPLC NLT 2 % 4%
(As calculated on the basis of w/w content of i
Withanolide A, withaferin A, Withanolide B,
withanoside IV, Withanoside V and 12-deoxy i
withastramonolide)
Content of withanolides %(w/w)-
Withanolide A 0.74
Withaferin A 0.17
Withanolide B 0.07
withanoside IV 1.06
Withanoside V 0.54
12-deoxy withastramonolide 0.86

c) LC-MS/MS details
LC-MS analysis was performed in the positive atmospheric pressure electrospray ionisation (ESI) API-2000 (Applied Biosciences/MDS SCIEX, USA) equipped with triple quadrupole detection mode. (The optimized MS conditions: ionization source voltage 5500 V and source temperature 450°C). Scanning range was kept from m/z 50-1200 (amu). The data acquisition was done using Analyst 1.4.2 software (MDS SCIEX).
LC-MS fingerprint shows the presence of 10 peaks (Table 4)
Table 4

Peak no tR MS/MS data (m/z) Identification if any
1 4.60 940; 919; 897; 834; 715.3; 475.4; 453.3; 307.2; 180.1 Not available in literature
2 5.52 832.3; 778.4; 721.5; 549; 473.3; 344; 252.2; 177.3 Not available in literature
3 8.09 816.3; 675.2; 670.4; 473.2; 437; 187.2 Not available in literature
4 9.97 950.5; 592.3; 549.3; 511.3; 506.2; 489.3; 439.3; 185.4 Not available in literature
5 12.98 872.5; 821.4; 816.4; 799.2; 637.2; 457.3; 439.2; 258.1; 149.3 Not available in literature
6 13.73 856.4; 800.4; 783.4; 621.3; 473.3; 459.3; 441.3; 391.3; 247.1; 130.3 Not available in literature
7 14.55 963.4; 941.5; 493.2; 488.2; 471.3; 341.4; 299.1; 281.0 Marker I (external standard)
8 16.03 488.3; 471.1; 459.2; 435.4; 399.3; 315.2; 299.4; 263.1 Marker III (literature report)
9 17.75 958.3; 488.4; 471.3; 453.2; 417.3; 289.3; 262.9 Marker II (external standard)
10 19.71 954.1; 791.3; 659.4; 643.5; 459.1; 441.2; 351.2; 155.1 Not available in literature
tR: retention time

d) LC-MS profile of Withania Somnifera fraction of the present invention (SIIL-
F-4)
i) Marker compounds were identified on the basis of retention time and UV spectral matching with standard markers as shown in Figure 2 wherein, A= HPLC chromatogram of standard marker compounds; B= HPLC chromatogram of Withania Somnifera fraction of the present invention (SIIL-F-4); C= Spectral overlays of compound 1; D= Spectral overlays of compound II.
ii) Marker compounds were confirmed on the basis of LC-MS analysis as shown in Figure 3 wherein, A= Total ion chromatogram of standard compound; B= Total ion chromatogram of SIIL-F-4; C= Mass spectra of compound IA and D= Mass spectra of compound II
e) Content Analysis:
Detection of Withaferin A and withanolide A contents in various fractions obtained
from successive fractionation of Withania Somnifera extract.
The fractions, marker, extract were analyzed for Withaferin A & Withanolide A using HPLC-DAD analysis and are reported here as mg/lOOmg of test material. HPLC-DAD chromatograms of various fractions obtained from successive fractionations of WSE and reference standards are shown in Figure 4, wherein A= HPLC chromatograms of n-butanolic fraction; reference standards such as withanolide A (B) and withaferin A(C) ; D= methanolic fraction; and E= aqueous fraction
f) Marker content analysis of Withaferin A and Withanolide A in various
fractions of Withania somnifera(WS) extract is provided in table 5.

Table 5

Sr Name of fraction Withaferin A Withanolide A
no. (mg/100 mg ± SD) (mg/100 mg ± SD)
1 WS- n-butanol ( Present invention) 0.853 ± 0.005 1.75 ±0.009
2 WS Methanol P ND ND
3 WS aqueous P ND ND
4 WSE (patent
application
1253/MUM/2003) 0.021325 ±0.005 0.04375 ± 0.009
*ND = Not detectable
g) Marker content
Marker content in Withania Somnifera fraction of the present invention was
calculated by HPLC and is shown in table 6. '
Table 6

Batch Withaferin A (mg/100 mg) Withanolide A
(mg/100 mg)
FRACTION (Lab scale) 0.385 ±0.003 0.742 ± 0.007
FRACTION (Scaled up) 0.853 ± 0.005 1.750 ±0.009
Example 2
Comparative Chemo-profiling of Withania Somnifera fraction of the present invention and extract used in Indian patent application No,1253/MUM/2003 is shown in Figure 5 and 6.
The content of Withaferin A and Withanolide A in the extract used in Indian patent application No.l253/MUM/2003 and Withania Somnifera fraction of the present invention is provided in table 7.

Table 7

Batch Withaferin A Withanolide A
(mg/100 mg) (mg/100 mg)
TM-l(1253/MUM/2003) 0.04585 ± 0.0005 0.04785 ±0.0001
SIIL-F-4(Withania Somnifera 0.853 ± 0.005 1.750 • 0.009
fraction of the present invention)
(Scaled up)
Results suggest that the fractionation has resulted in 20 and 40 fold higher enrichment in Withaferin A and Withanolide A respectively.
Example 3
Comparative Chemo-profiling of Withania Somnifera fraction of the present invention (SIIL-F-4) and fraction disclosed in US20040033273 is shown in Figure 5 and 7. The content of Withaferin A and Withanolide A in the fraction used in US20040033273 and Withania Somnifera Fraction of the present invention is provided in table 8.
Table 8

Batch Withaferin A Withanolide A
■ (mg/100 mg) (mg/100 mg)
US20040033273(Withasol) Below detection limit 0.063 ± 0.0006
Withania Somnifera fraction of the 0.853 ± 0.005 1.750 ±0.009
present invention (SIIL-F-4) (Scaled
up)
Result indicates that Withania Somnifera fraction fingerprint is more enriched in Withaferin A and Withanolide A.

TECHNICAL ADVANCEMENT
In accordance with the present invention there is provided a process for obtaining a Withania Somnifera fraction enriched with Withanolide-A and Withaferin-A from a crude aqueous Withania Somnifera root extract. In particular the Withania Somnifera fraction obtained by the present process is enriched with 20 to 40 times more withanolides as compared to the crude extracts of prior art disclosures.
While considerable emphasis has been placed herein on the specific features of the preferred embodiment, it will be appreciated that many additional features can be added and that many changes can be made in the preferred embodiment without departing from the principles of the invention. These and other changes in the preferred embodiment of the invention will be apparent to those skilled in the art from the disclosure herein, whereby it is to be distinctly understood that the foregoing descriptive matter is to be interpreted merely as illustrative of the invention and not as a limitation.

We claim;
1. A process for preparing a Withania Somnifera fraction enriched in
Withanolides ; said process comprising the following steps:
a) obtaining the coarse root material from Withania Somnifera plant and refluxing with hot water to obtain a slurry;
b) filtering and vacuum concentrating the slurry to obtain concentrated aqueous extract;
c) liquid -liquid successive partitioning of concentrated aqueous extract with at least one non-polar organic solvent and at least one polar organic solvent to obtain an organic fraction of the extract; and
d) concentrating and co-distilling an organic fraction under vacuum to remove the traces of polar organic solvent followed by drying the fraction at a temperature not more than 70°C to obtain a Withania Somnifera fraction enriched in Withanolides.

2. The process as claimed in claim 1, wherein the non polar organic solvent is selected from the group consisting of n-hexane, toluene and benzene.
3. The process as claimed in claim 1, wherein the polar organic solvent is selected from the group consisting of butanol, dichloromethane, dichloroethane and chloroform.
4. A Withania Somnifera fraction enriched with Withanolide; said fraction comprises a) Withanolide A of about 0.5-1%; b) Withaferin A of about 0.1-0.6%; c) Withanolide B of about 0.01-0.1%; d) Withanoside IV of about 0.8-

1.2%; e) Withanoside V of about 0.5-0.8%; and f) 12-deoxy withastramonolide of about 0.8-1.2%.
5. A Withania Somnifera fraction enriched with Withanolide as claimed in claim 4, wherein the ratio of Withaferin A and Withanolide A is in the range of about 1:2 to 1:5.