FORM 2
THE PATENT ACT 1970
(39 of 1970)
&
The Patents Rules, 2003
COMPLETE SPECIFICATION
(See section 10 and rule 13)
1. TITLE OF THE INVENTION:
"A Process For Identification And Isolation Of Hecogenin And Tigogenin Glycosides From Safed Musli Root"
2. APPLICANT
(a) NAME: NANDAN BIOMATRIX LIMITED
(b)NATIONALITY: Indian Company incorporated under the Indian Companies ACT, 1956
(c) ADDRESS: C-002/C-003, Ground Floor, Gokul Plaza, Thakur Complex, Kandivli (East), Mumbai - 400 101, Maharashtra, India

3. PREAMBLE TO THE DESCRIPTION The following specification particularly describes the invention and the manner in which it is to be formed:

This application is Divisional Application of Indian Patent Application No. 1255/MUM/2006 titled "Novel Process for Isolation and Identification of Hecogenin and Tigogenin Glycosides From Safed Musli Root"
Related Applications: The contents of the specification of the following related patent . applications may be treated as incorporated herein by reference. 1262/MUM/2003 titled "Processes for extraction of Safed musli and characterization of the extracts thereof." 603/MUM/2004 titled "Safed musli compositions and process for preparation thereof
TECHNICAL FIELD OF THE INVENTION:
The present invention relates to the processes for extraction, isolation, identification and standardization of steroidal saponins and sapogenin from Safed musli (Chlorophytum borivilianum Santapau & Fernanes) roots. The present invention specifically relates to improved methods for identification and isolation of hecogenin and tigogenin glycosides. The present invention further relates to the use of hecogenin and tigogenin glycosides as nutraceuticals.
BACKGROUND AND PRIOR ART:
The medicinal properties of Safed Musli root:
Search for effective aphrodisiac drug is dates back to centuries. Aphrodisiacs are classified as agents that increase libido, potency, and/or sexual pleasure. Various substances of animal and plant origin have been used as aphrodisiacs in different cultures. Pharmacological basis of some has been worked out, that has been resulted in the understanding of their mechanisms of action. For increasing libido, ambrein, a major Chemical constituent of Ambra grisea, is used in Arab countries. This tricyclic triterpene alcohol increases the concentration of several anterior pituitary hormones and serum testosterone. Bufo toad skin and glands containing bufotenine is called as 'love stone' and it is popular in West Indies and in the Chinese medication chan su. For increasing potency, Panax ginseng is often used in traditional Chinese medicine, it works as an antioxidant by enhancing nitric oxide synthesis in the endothelium of

many organs. Furthermore, Cantharidin, a chemical compound derived from blister beetles (especially from"Spanish fly"), has been used as a sexual stimulant for increasing sexual pleasure. Its mode of action is believed to be by inhibition of the phosphodiesterase and protein phosphatase activity and stimulation of beta-receptors, inducing vascular congestion and inflammation.
Sildenafil citrate (Viagra®) is a synthetic compound that has been effective for erectile dysfunction. Safed Musli is a traditional medicinal plant in India, which is used as an aphrodisiac drug since long time.
Safed Musli has been described in the ancient Indian Traditional Medicine literature such as Bhavaprakash Nigantu and Rasendra Sarangrah, Raja Ballabh Nigantu as "Vajikaran" or aphrodisiac.
References: i) Triveni, A., Rasendrasarasangrah: Vajikarandhikar. Nutan Press, Rajkot, India. Pages: 617-643; ii) Kirtikar, KR. And Basu, BD., Indian Medicinal Plants. 2nd Edition. India. Lalith Mohan Basu, Allahabad, 1956, Pages: 235-246; iii) Sharma, SK Chunekar, KC. and Paudal, K. Plants of Sharangdhar Samhita. RAV publications, Director, Rastriya Ayurveda Vidyapeeth, New Delhi: Pages: 221-222.
The Indian system of medicine, Ayurveda conceptualizes a category of drug activity known as "Rasayana" the word Rasayana is composed of two words "Rasa" meaning elixir and "Ayana" meaning house. The word therefore signifies property of the plant that helps to rejuvenate the system, i.e., adaptogenic activity. The adaptogenic property of Safed Musli root is well proved
Reference: Handa, SS. Rasayana Drugs (in: Supplement to Cultivation and
utilization
of Medicinal Plants. Vol. I, Jammu Tawi Regional Research laboratory, 1996, Pages
509-510).
The root of Safed Musli is employed as "Rasayana" (an adoptogen)
References: i) Puri, HS. Rasayana- Ayurvedic Herbs for longevity and Rejuvenation.

Taylor and Francies, London. 2003, Pages: 212-224.: ii) Mishra, SN. Bhaisaya Ramavali. I Edition. Chaukambha Surbharti Prakashan, 2005 Pages: 1008-1133).
Efficacy of Safed Musli root on the androgenic and sexual behavour of male rats was
studied by Thakur and Dixit.
Reference: i) (Thakur, M. and Dixit, VK. Indian Drugs 2006; Pages 300-306.
Standardization and determination of antioxidant activity of Safed Musli is well
documented
Reference: Govindarajan R, Sreevidya N, Vijayakumar M, Thakur M Dixit
VK.,Malhotra S., Naturall Product Science 2005, Vol. 11- Pages: 165-169
The immunomodulatory activity of Safed Musli root is investigated and reported by Takur and others. Reference: Thakur, M., Bhargava, S., and Dixit, VK., eCAM, 2006. Pages: 1-5.
Use of Safed Musli root for improving the overall resistance of body against common
infections and pathogens has been described
Reference: Kothari, S.K., Safed Musli Revisited. Journal of Medicinal & Plant
Science,
2004; Vol. 26, Pages: 60-63).
Distribution of Safed Musli in India:
The genus Chlorophytum Ker-Gawl belongs to the botanical family Liliaceace. There are about 200 species reported to be distributed mainly in tropical and sub-tropical countries. It is documented that about 15 species are occurring in Peninsular India. About 7 species of Chlorophytum species are recorded in India and named as Safed Musli and they are Chlorophytum borivilianum, C. armdinaceum, C. tuberosum, C. laxum, C. malabericum, C. attenuatum, and C. breviscapum. Of these, Chlorophytum borivilianum, C. arundinaceum C. laxum and C. tuberosum are extensively cultivated in the states of Andhra Pradesh, Tamil Nadu, Madhya Pradesh, Karnataka, Maharashtra and Chattisgarh.
Chlorophytum borivilianum (Safed Musli) is an herb with sub erect lanceolate leaves. It flowers in the August and early September and the flowers are white in colour. The leaves are dried in the month of December / January and it remains dormant during

the rest of the year (early winter till break of monsoon). The propagation through seeds is very rare. The propagation by roots is feasible and it is the regular cultivation practice in India.
Safed musli grows as a wild plant in Bastar forest (M.P.), Dangs forest (Gujarat), Mount Abu, Mahi, Aravalli hills (Rajasthan) and in the forests of Western and Eastern Ghats of India.
In India about eight plant species are reported as Safed musli; and they are Chlorophytum borivilianum, Chlorophytum arundinaceum, Chlorophytum tuberocum, Chlorophytum malabericum, Chlorophytum attennatum, Chlorophytum breviscapum, Asparagus adscendens, A. gonoclados. Of these Chlorophytum borivillianum, Chlorophytum arundinaceam and Chlorophytum tuberosum are collected by our tribes from the forest and using them as aphrodisiac drug.
Chlorophytum borivilianum is the only species which is brought under commercial cultivations and is widely cultivated in different parts of India like Andhra Pradesh, Karnataka, Tamil Nadu, Rajasthan, Madhya Pradesh, Maharashtra, and Gujarat. It is a potent herb whose root tubers are useful for the aphrodisiac and health promoting properties. It contains alkaloids, phytosterols, steroidal saponins, polysaccharides, carbohydrates, proteins, minerals, vitamins. Safed Musli root contains saponins (4-7%), stigmasterol (1.9-3.5%), sugars (arabinose, galactose, glucose, rhamnose, xylose, and carbohydrates) and reducing sugar and non-reducing sugars.
Saponins are sapogenin glycosides and they are one of the major bioactive
compounds present naturally in a number of plants. Each saponin consists of a
sapogenin (the aglycon moiety of the molecule), and a sugar. There are two major
groups of sapogenins reported and they are (1) triterpenoids and (2) steroids. Saponins
have considerable potential use as pharmaceutical and as nutraceutical agents.
Saponins, from a variety of sources, have been shown to have hypocholesterolemic,
anti-coagulant, anticarcinogenic, hepatoprotective, hypoglycemic,
immunomodulatory, neuroprotective, anti-inflammatory and antioxidant activity.
Hecogenin and tigogenin are the steroidal sapogenins which are the important precursors in the synthesis of steroid hormones, sexual hormones (progesterone) and protein anabolic hormones. Hecogenin and tigogenin steroidal sapogenins are the

natural steroidal metabolites having pharmacological activities similar to phytosterols. They are reported in few plants species like Agave Americana, Agave intermixitxta, Agave sisalana, Cissus sicyoides, Furcarea macrophylla.
The health benefits of hecogenin glycoside reported are, they possess aphrodisiac activity, they strengthen the immune system, useful as anti-arthritis and antidiabetic agent, increase physical stamina, also possess cholesterol lowering property, anti-tumour and anti-inflammatory properties. Hecogenin glycosides and its derivatives ■ isolated from Tribulus terrestris possess antimicrobial activity on Candida albicans, and other Candida species and Cryptocococcus neoformans. Japanese researchers reported such sapogenins in Chlorophytum cosmosum. . But no such steroidal sapogenins are reported from Safed Musli (Chlorophytum borivilianum).
Mimaki, Y. et al., have reported the isolation of steroidal saponins from the roots of Chlorophytum cosmosum and studied their activity on tumor promoter - induced phospholipids metabolism in HeLa cell lines (Reference: Phytochemistry Vol.41(5): pp.1405-1410.)
US Patent No. 3981867 discloses a process for obtaining sapogenin particularly hecogenin from plant material such as agave sisalana leaves.
US2006062863 discloses compositions for anti-obesity, health-restorative and health-promotional benefits comprising extracts of Chlorophytum species in the form of a tablet, syrup, elixir or capsule, or any other pharmaceutically acceptable form. The patent discloses extraction isolation, characterization and quantification of the extracted compounds, of the distinct compounds which constitutes spirosta-steroidal saponins comprising diosgenin, tigogenin, neotigogenin and sarsasapogenin as the major genin components and mono-, di- and oligosaccharides, comprising glucose, rhamnose, arabinose, galactose and xylose as glycosidic components from the fresh tuber-roots of a cultivated variety.
US Patent No. 7,160,866 discloses a process for isolation of a novel saponin, tigogenin penta glycoside isolated from the aerial parts of Chlorophytum nimonii, and process there of as well as its use in anti-hyperglycemic and hypolipidemic activities.

The present invention discloses an isolation process of hecogenin and tigogenin sapogenins from Safed Musli roots {Chlorophylum borivilianum) and also evaluates their nutraceutical uses.
OBJECT OF THE INVENTION:
The main object of the present invention is to identify, isolate and standardize steroidal saponins especially hecogenin and tigogenin glycosides in Safed musli roots (Chlorophylum borivilianum).
Another object of the present invention is to provide an efficient process for improving the quantities of sapogenins, specifically hecogenin and tigogenin from Safed musli (Chlorophytum borivilianum).
Yet another object of the invention to blend Safed musli (Chlorophytum borivilianum) extracts containing hecogenin and tigogenin with aphrodisiac and anti-obesity formulations for nutraceutical uses.
SUMMARY OF THE INVENTION:
The present invention discloses processes for extraction isolation and characterization of sapogenin, mainly hecogenin and tigogenin from Safed Musli {Chlorophytum borivilianum) roots. The present invention discloses improved and efficient processes for extraction of sapogenins particularly hecogenin and tigogenin in improved quantities from Safed Musli {Chlorophytum borivilianum) roots. The hecogenin and tigogenin glycosides of the present invention can be blended with aphrodisiac and anti-obesity formulations for nutraceutical uses. The processes for extraction involve extraction of wet Safed musli roots with water and acid digestion; and enzymatic digestion method by using a mixture of enzymes.
Extraction of Safed Musli roots with water and acid includes acid digestion with hydrochloric acid at pH in the range of 0.5 to 2.0 followed by extraction with the combination of solvents containing a less polar solvent and high polar solvent at a temperature range of about 60-70°C and drying the top solvent layer by evaporation

under reduced pressure. The invention further discloses purification of crude hecogenin and tigogenin by preparative TLC, MPLC and column chromatography and identification, of the said glycosides by TLC, melting point, specific rotation, UV and IR spectra.
Enzymatic digestion method includes treatment with mixture of starch hydrolyzing
enzymes cellulases, hemicellulases and other enzymes like protease, pectinase and
lipase or their complexes followed by extraction with the combination solvent
containing a less polar solvent and high polar solvent at a temperature of about 60-
70°C and drying the top solvent layer by evaporation under reduced pressure. The invention further discloses purification of crude hecogenin and tigogenin by preparative TLC, MPLC and column chromatography and identification of the said glycosides by TLC, melting point, specific rotation, and UV, IR, MASS, H1 and C13 spectra.
DESCRIPTION OF DRAWINGS:
Fig 1 shows the purity of Hecogenin by HPLC chromatogram.
Fig 2 shows the purity of Tigogenin by HPLC chromatogram.
Fig 3 shows UV-Spectral analysis of Hecogenin.
Fig 4 Shows UV-spectral analysis of Tigogenin
Fig 5 shows IR spectrum of Hecogenin.
Fig 6 shows IR Spectrum of Tigogenin
Fig 7 shows Mass spectrum of Hecogenin.
Fig 8 shows Mass spectrum of Tigogenin
Fig 9 shows H1 NMR spectrum of Hecogenin.
Fig 10 shows H1 NMR spectrum of Tigogenin
Fig 11 shows C1 3 NMR spectrum of Hecogenin
Fig 12 shows C13 NMR spectrum of Tigogenin.

DETAILED DESCRIPTION OF THE INVENTION:
The invention will now be described in detail in connection with certain preferred and optional embodiments, so that various aspects thereof may be more fully understood and appreciated.
Accordingly, the present invention describes the processes for extraction, isolation and identification of the sapogenin, mainly hecogenin and tigogenin from Safed Musli (Chlorophytum borivilimum) roots. The other seven Safed Musli species i.e., Chlorophytum arundinaceum, Chlorophytum tuberocum, Chlorophytum malabericum, Chlorophytum attenuatum, Chlorophytum breviscapum, Asparagus adscendens and A gonoclados are also included in this investigation.
The present invention describes improved processes for extraction, isolation, identification and standardization of hecogenin and tigogenin glycosides and nutraceutical uses of hecogenin and tigogenin glycosides when blended with aphrodisiac and anti-obesity formulations. Further, the comparative study between the sapogenin enriched Safed Musli product and normal Safed Musli product is carried out.
The present invention describes different processes of isolation and identification of
the said glycosides from safed musli roots:
I. Extraction of wet Safed Musli roots with water and acid digestion. It is a
different technique from the convention hot extraction method.
II Enzymatic digestion method by using a mixture of enzymes preferably starch
hydrolyzing enzymes, especially cellulases, hemicellulases and also other enzymes like protease, pectinase and lipase or their complexes.
According to a preferred embodiment, a process for isolation of sapogenin glycosides, mainly hecogenin and tigogenin from wet Safed Musli (Chlorophytum borivilicmum) roots using water extraction and acid hydrolysis method comprising the steps of:
a) preparing the paste of small broken roots of Safed Musli using multi mill;
b) adding 20 volumes of water followed by stirring for 1 hour;
c) adding 2% charcoal followed by heating up to 60-70°C for 2 hour under
constant stirring;

d) filtering the water extract through filter press or nutch filter;
e) acidifying the water extract by adjusting the pH in the range of 0.5 to 2.0.using Hydrochloric acid;
f) heating the reaction mixture up to 150°C to 250°C at a pressure from 25 to 125 p.s.i / g. for 2 to 4 hours to produce a hydrolysate of crude sapogenins containing hecogenin and tigogenin;
g) separating the water insoluble mass by filtering through 5 micron filter;
h) drying & milling the water insoluble mass into a coarse powder;
i) charging the powder into a cone shaped stainless steel reactor fitted with agitator;
j) repeatedly treating the powder with a combination solvent containing a less polar solvent and a high polar solvent at a temperature of 60-70°C under constant stirring for 1 hour;
k) cooling down the reaction mass to room temperature with out stirring;
1) separating the top solvent layer and evaporating the solvent under reduced pressure to obtain dry powder contains the crude sapogenins including hecogenin and tigogenin.
According to another preferred embodiment, a process for isolation of sapogenin glycosides, mainly hecogenin and tigogenin from wet Safed Musli (Chlorophytum borivilianum) roots using enzymatic digestion method comprising the steps of:
a) preparing the paste of small broken roots of Safed Musli using multi mill;
b) adding 20 volumes of water followed by stirring for 1 hour;

c) adding 2% charcoal followed by heating up to 60-70°C for 2 hour under constant stirring;
d) filtering the water extract through filter press or nutch filter followed by collecting the water extract in a suitable stainless steel fermentor; .
e) adding sufficient quantity (10 to 15 g per liter) of enzyme complex
containing the proportionate mixture of cellulase, hemicellulase, protease
and lipase;

f) agitating the reaction mass for 30 minutes for every 4 hour interval at room
temperature up to 24 hour;
g) allowing the reaction mass to stand for 24 hour at room temperature;
h) separating water insoluble mass by filtering through 5 micron filter;
i) drying & milling the water insoluble mass into a coarse powder;
j) charging the powder into a cone shaped stainless steel reactor fitted with agitator;
k) repeatedly treating the powder with a combination solvent containing a less polar solvent and a high polar solvent at a temperature of 60-70°C under constant stirring for 1 hour;
1) cooling down the reaction mass to room temperature with out stirring;
m) separating the top solvent layer; and
n) evaporating the solvent under reduced pressure to obtain dry powder contains the crude sapogenins including hecogenin and tigogenin.
The non-polar solvent used in the above two processes is selected from hexane, heptane or petroleum ether. The polar solvent used in the above two processes is selected from methanol, ethanol, butanol or isopropyl alcohol. The molar ratio of the said non- polar to polar solvent used in the above processes is 9:1.
In yet another preferred embodiment, the invention provides methods of identification of sapogenins particularly, hecogenin and tigogenin:
In, Thin Layer Chromatography (TLC) 5 prominent spots are observed and the hecogenin and tigogenin spots are more prominent than the other three spots. Hecogenin and tigogenin spots are identified with the help of authentic samples. Hecogenin tigogenin are further identified by performing the following tests comparing with authentic sample; melting point, specific rotation; UV and IR spectra.

Purification of hecogenin and tigogenin is achieved by preparative TLC, MPLC and column chromatography. The purity of the isolated compounds, hecogenin and tigogenin is carried out by HPLC.
HPLC of Hecogenin: (Fig 1)
Mobile phase: methanol (HPLC grade); Column: C18; Wave length: 234nm; Flow
rate: 0.7ml/min; Run time: 15 min; Retention time: 6.30 min.
The percentage of the purity of the compound (Hecogenin) is: 99.02.
HPLC of Tigogenin (Fig 2)
Mobile phase: methanol (HPLC grade); Column: C18; Polarity: + (RI Detector);
Flow
rate:l ml/min; Run time: 15 min; Retention time: 7.9 min
The percentage of the purity of the compound (Tigogenin) is: 98.99.
Further, the spectral analyses of the compounds are done and the same is briefly described below.
Spectral analysis of Hecogenin:
UV SPECTROSCOPY (fig.3)
UV-Spectroscopy analysis shows that the molecule contains unsaturation which is confirmed. Strong absorption at 254 nm indicating the π- Π* transition.
INFRARED SPECTROSCOPY (Fig.5)
IR spectrum shows the peak at 3398 cm-1, which indicates O-H group (alcohol
group).
Similarly, the peak at 2958 - 2858 cm-1 indicates the molecule containes alkane (C-H
stretching) and the peak at 1701 cm-1 confirmed the presence of Carbonyl group
(0=C<).
MASS SPECTROSCOPY (Fig.7)
Based on LC-MS spectral analysis the molecular formula is confirmed as C27H42O4
and
the molecular weight is 430.

H'NMR (Fig.9)
The peak at 5 7.21 indicates proton attached to carbonyl group. The pentate at 5 4.35 indicates proton attached to adjacent oxygen. The multiplate at 8 3.5 indicates H1 attached to (O-C-C) oxygen (p) group.
C13NMR:(Fig.ll)
Singlet at 213.5 indicates carbonyl group C (Carbon).
Peak at 109 indicates C bonded with 2 oxygen molecules.
Peak at 79.1 indicates C bonded with Oxygen.
Peak at 66 indicates attachment of carbonyl group carbon with other carbons.
Peak at 55 is indicates C attached with carbonyl group carbon.
Based on the above spectral data the molecular structure is confirmed as

HECOGENIN
Spectral analysis of Tigogenin
UV SPECTROSCOPY (Fig.4)
Based in UV spectral analysis the molecule does not contains unsaturation (it shows n-π* transition).
INFRARED SPECTROSCOPY(Fig.6)
Based on IR spectrum the peak at 3520 cm-1 indicate O-H group (alcohol group). The peak at 2968 - 2873 cm-1 indicates the molecule containes alkane (C-H stretching) group.

MASS SPECTROSCOPY(Fig.8)
Mass spectrum shows the molecular ion peak at 416 indicates M+l peak. The molecular formula is confirmed as C27H44O3. Molecular weight: 416
H1 NMR(Fig.lO)
Based on proton NMR Spectroscopy The peak at 5 4.3 singlet indicates carbonyl
group. The pentate at 5 4.35 indicates proton attached to adjacent oxygen.
The multiplate at 5 0.9-1.8 indicates 1H1 attached to symmetric environment SP3
hybridized protons.
The triplet at 5 3.5 indicates H1 attached to oxygen molecule
C13NMR(Fig.l2)
At 213.9 no signal is appeared, which clearly indicates that the molecule carbonyl group carbon is not present. Based on the above spectral data the structure of the molecule is determined as

TIGOGENIN
The following examples, which include preferred embodiments, will serve to illustrate the practice of this invention, it being understood that the particulars shown are by way of example and for purpose of illustrative discussion of preferred embodiments of the invention.

Example 1:
Water extraction and acid hydrolysis method (Commercial scale process): Safed Musli roots (100 kg) were broken into small pieces followed by made into a paste with the help of a multi mill. 20 volume of water was added and stirred for 1 hour. 2% ( 20 kg) charcoal was added to above and heated up to 60-70°C for 2 hour under constant stirring. The water extract was filtered through the filter press or nutch filter. The pH of the water extract was adjusted to a range of 0.5 to 2.0with Hydrochloric acid. The reaction mixture was heated up to 150°C to 250°C at a pressure from 25 to 125 p.s.i / g. for 2 to 4 hours to produce a hydrolysate of crude sapogenins containing hecogenin and tigogenin. The water insoluble mass was separated by filtering through 5 micron filter. The insoluble mass was dried and milled it to a coarse powder. The powder was charged to a cone shaped stainless steel reactor fitted with agitator and treated the same with combination solvent containing a less polar solvent such as hexane, petroleum ether and heptane and a high polar solvent such as methanol, ethanol, butanol, isopropyl alcohol in the ratio of preferably 90:10 at a temperature of 60-70°C under constant stirring for I hour. Finally, the stirring was stopped and cooled the reaction mass to room temperature. The top solvent layer was separated and evaporated to obtain dry powder containing crude sapogenins including hecogenin and tigogenin. Yield: 5.5 kg (5.5%)
Example 2:
Enzymatic digestion method (Commercial scale process):
Safed Musli roots (100 kg) were broken into small pieces followed by made into a paste with the help of a multi mill. 20 volume of water was added and stirred for 1 hour. 2% (20 kg) charcoal was added to above and heated up to 60-70°C for 2 hour under constant stirring. The water extract was filtered through the filter press or nutch filter. The water extract taken in a suitable stainless steel fermentor and added sufficient quantity (10 to 15 g per liter) of enzyme complex containing proportionate mixture of cellulase, hemicellulase, protease and lipase. The reaction mass was agitated for 30 minutes for every 4 hour interval at room temperature up to 24 hour. The reaction mass was allowed to stand for 24 hour at room temperature. The water insoluble mass was separated by filtering through 5 micron filter and dried the same and milled it in to a coarse powder. The powder was charged into a cone shaped

stainless steel reactor fitted with agitator and charged with a combination solvent containing a less polar solvent such as hexane, petroleum ether, heptane and a high polar solvent such as methanol, ethanol, butanol, isopropyl alcohol in the ratio of preferably 9:1 at a temperature of 60-70°C under constant stirring for 1 hour.. Finally, the stirring was stopped and cooled the reaction mass to room temperature. The top solvent layer was separated and evaporated to obtain dry powder containing crude sapogenins including hecogenin and tigogenin. Yield: 6.5 kg (6.5%)
These improved processes are efficient and produces higher quantities of sapogenins, especially hecogenin and tigogenin in the final products of Safed Musli extracts. The conventional method includes hot extraction of dried root.
It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative examples and that the present invention may be embodied in other specific forms without departing from the essential attributes thereof, and it is therefore desired that the present embodiments and examples be considered in all respects as illustrative and not restrictive, reference being made to the appended claims, rather than to the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.

We Claim,
1. A process for isolation of Hecogenin and Tigogenin from Safed Musli {Chlorophytum borivilianum) roots using enzymatic digestion method comprising the steps of:
a) preparing the paste of small broken wet roots of Safed Musli using multi mill;
b) adding 20 volumes of water followed by stirring for 1 hour;
c) adding 2% charcoal followed by heating up to 60-70°C for 2 hour under constant stirring;
d) filtering the water extract through filter press or nutch filter followed by collecting the water extract in a suitable stainless steel fermentor;
e) adding sufficient quantity (10 to 15 g per liter) of enzyme complex containing the proportionate mixture of cellulase, hemicellulase, protease and lipase;
f) agitating the reaction mass for 30 minutes for every 4 hour interval at room temperature up to 24 hour;
g) allowing the reaction mass to stand for 24 hour at room temperature;
h) separating water insoluble mass by filtering through 5 micron filter;
i) drying & milling the water insoluble mass into a coarse powder;
j) charging the powder into a cone shaped stainless steel reactor fitted with agitator;
k) repeatedly treating the powder with a combination solvent containing a less polar solvent and a high polar solvent at a temperature of 60-70°C under constant stirring for 1 hour;
1) cooling down the reaction mass to room temperature with out stirring;
m) separating the top solvent layer; and
n) evaporating the solvent under reduced pressure to obtain dry powder containing hecogenin and tigogenin,

2. The processes as claimed in claim 1, wherein, said non-polar solvent is selected from hexane, heptane or petroleum ether.
3. The processes as claimed in claims 1, wherein said polar solvent is selected from methanol, ethanol, butanol or isopropyl alcohol.
4. The processes as claimed in claims 1, wherein the molar ratio of the said non-polar to polar solvent is 9:1.
5. The process for isolation of hecogenin and tigogenin from Safed Musli {Chlorophytum borivilianum) roots and characterization of said hecogenin through HPLC, UV Spectroscopy, Infrared Spectroscopy, Mass Spectroscopy, H1 NMR, C13 NMR as in Figures 1, 3, 5, 7, 9, 11 respectively and characterization of said tigogenin through HPLC, UV Spectroscopy, Infrared Spectroscopy, Mass Spectroscopy, Hl NMR, C13 NM.R as in Figures 2, 4, 6, 8, 10, 12 respectively.