Title of Invention

A PROCESS FOR ISOLATION OF HECOGENIN AND TIGOGENIN FROM SAFED MUSLI ROOTS

Abstract The present invention disclosed novel processes for extraction, isolation, identification and standardization of sapogenin glycoside, mainly hecogenin and tigogenin from Safed musli roots (Chlorophytum borivillianum).
Full Text FORM 2
THE PATENT ACT 1970
(39 of 1970)
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The Patents Rules, 2003
PROVISIONAL SPECIFICATION
(See section 10 and rulel3)
1. TITLE OF THE INVENTION:
"Novel 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

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 novel processes for extraction, isolation, identification and standardization of steroidal saponins and sapogenin from Safed musli roots {Chlorophytum borivillianum). 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:
Safed musli belongs to Liliaceae family and grows as a wild plant in Bastar forest (M.P.), Dangs forest (Gujarat), Mount Abu, Mahi, Aravalli hills (Rajasthan) of India. In India about eight species of Safed musli are reported; Chlorophytum borivillianum, Chlorophytum arundinaceum, Chlorophytum tuberocum, Chlorophytum malabericum, Chlorophytum attenuatum, Chlorophytum breviscapum, Asparagus filicinus, A. gonoclados. Of these Chlorophytum borivillianum, Chlorophytum arundinaceam and Chlorophytum tuberosum are collected by our tribes from the forest.
Chlorophytum borivillianum is the only species which is under commercial cultivations and is widely cultivated in different parts of India like Andhra Pradesh, Rajasthan, Madhya Pradesh, Maharashtra, and Gujarat. It is a potent herb whose root tubers is useful for its aphrodisiac and health promoting properties. It contains alkaloids, steroid, saponin, 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), reducing sugar and non-reducing sugars.
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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 triterpenoids and steroids. Saponins have considerable potential 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, improves arthritis and antidiabetic properties, 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. But these steroidal sapogenins are not reported from Safed Musli {Chlorophytum borivilianum Sant. & F ). Japanese researchers reported such sapogenins in Chlorophytum cosmosum
US13935P discloses a new and distinct cultivar of Chlorophytum plant, botanically known as Chlorophytum comosum. The said Chlorophytum is a naturally-occurring whole plant mutation of Chlorophytum comosum 'Variegatum'.
US Patent No. 3981867 discloses a process for obtaining sapogenin particularly hecogenin from plant material such as agave sisalana leaves.
US Patent No.2006062863 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
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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.
The present invention discloses novel isolation process of hecogenin and tigogenin sapogenins from Safed Musli roots (Chlorophytum 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 (Chlorophytum 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).
It is 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 and isolation of sapogenin, mainly hecogenin and tigogenin from Safed Musli {Chlorophytum borivilianum) roots. The present invention discloses improved and efficient processes for extraction of improving the quantities of sapogenins particularly hecogenin and tigogenin 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
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nutraceutical uses. The processes for extraction involves 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 solvent 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 and IR spectra.
Detailed description of the invention:
The present invention describes process for identifying sapogenin, mainly hecogenin and tigogenin from Safed Musli {Chlorophytum borivilianum) roots. The other seven Safed Musli species i.e., Chlorophytum arundinaceum, Chlorophytum tuberocum, Chlorophytum malabericum, Chlorophytum attenuatum, Chlorophytum breviscapum, Asparagus filicinus and A. gonoclados are also included in this investigation.
The present invention describes improved processes for identification, isolation 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.
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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. 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.
Water extraction and acid hydrolysis method includes the steps as follows:
1. Safed Musli roots are broken into small pieces.
2. They are made into a paste with the help of a multi mill.
3. Add 20 volume of water and stir for 1 hour.
4. Add 2% charcoal with this and heat it up to 60-70°C for 2 hour under constant stirring.
5. Filter the water extract through filter press or nutch filter.
6. Acidify the water extract with Hydrochloric acid to a pH in the range of 0.5 to 2.0.
7. Heat 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.
8. Separate water insoluble mass by filtering through 5 micron filter.
9. Dry the water insoluble mass and mill it to a coarse powder.

10. Charge the powder to a cone shaped stainless steel reactor fitted with agitator.
11. Charge a 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.
12. Heat the reactor up to 60-70°C under constant stirring for 1 hour.
13. Stop stirring and cool down the reaction mass to room temperature.
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14. Separate the top solvent layer. (Solvent extract I)
15. Repeat the step No. 9 to 12. (Solvent extract II)
16. Combine both the solvent extracts I & II
17. Evaporate the solvent under reduced pressure.
18. The resultant dry powder contains the crude sapogenins including hecogenin and tigogenin.
Enzymatic digestion method includes the steps as follows:
1. Safed Musli roots are broken in to small pieces.
2. Make it in to a paste with the help of a multi-mill
3. Add 20 volume of water and stir for 1 hour.
4. Add 2% charcoal in to the reaction mixture and heat it up to 60-70°C for 2 hour under constant stirring.
5. Filter the water extract through filter press or nutch filter.
6. Collect the water extract in a suitable stainless steel fermentor.
7. Add sufficient quantity (10 to 15 g per liter) of enzyme complex containing proportionate mixture of cellulase, hemicellulase, protease and lipase.
8. Agitate the reaction mass for 30 minutes for every 4 hour interval at room temperature up to 24 hour.

8. Allow the reaction mass to stand for 24 hour at room temperature.
9. Separate water insoluble mass by filtering through 5 micron filter.

10. Dry the water insoluble mass and mill it in to a coarse powder
11. Charge the powder in to a cone shaped stainless steel reactor fitted with agitator.
12. Charge 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.
13. Heat the reactor up to 60-70°C under constant stirring for 1 hour.
14. Stop stirring and cool down the reaction mass to room temperature.
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15. Separate the top solvent layer (solvent extract I).
16. Repeat the step No. 9 to 12 one more time (Solvent extract II).
17. Combine both the solvent extracts I & II.
18. Evaporate the solvent under reduced pressure.
19. The resultant dry powder contains the crude sapogenins including hecogenin and tigogenin.
Identification of sapogenins particularly, hecogenin and tigogenin includes:
Thin Layer Chromatography (TLC) wherein 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.

Dr. Gopakumar G. Nair Agent for the Applicant
Dated this 7th day of August 2006

Documents:

1255-MUM-2006-ABSTRACT(5-10-2010).pdf

1255-mum-2006-abstract(7-8-2007).pdf

1255-MUM-2006-ABSTRACT(AMENDED)-(5-10-2010).pdf

1255-MUM-2006-ABSTRACT(GRANTED)-(23-9-2011).pdf

1255-MUM-2006-CANCELLED PAGES(11-7-2011).pdf

1255-mum-2006-claims(7-8-2007).pdf

1255-MUM-2006-CLAIMS(AMENDED)-(11-7-2011).pdf

1255-MUM-2006-CLAIMS(AMENDED)-(5-10-2010).pdf

1255-MUM-2006-CLAIMS(AMENDED)-(8-3-2011).pdf

1255-MUM-2006-CLAIMS(GRANTED)-(23-9-2011).pdf

1255-MUM-2006-CLAIMS(MARKED COPY)-(11-7-2011).pdf

1255-MUM-2006-CLAIMS(MARKED COPY)-(8-3-2011).pdf

1255-MUM-2006-CORRESPONDENCE(21-7-2008).pdf

1255-MUM-2006-CORRESPONDENCE(25-8-2006).pdf

1255-MUM-2006-CORRESPONDENCE(5-9-2011).pdf

1255-mum-2006-correspondence(7-8-2007).pdf

1255-MUM-2006-CORRESPONDENCE(IPO)-(23-9-2011).pdf

1255-mum-2006-correspondence-received.pdf

1255-mum-2006-description (provisional).pdf

1255-mum-2006-description(complete)-(7-8-2007).pdf

1255-MUM-2006-DESCRIPTION(GRANTED)-(23-9-2011).pdf

1255-mum-2006-drawing(7-8-2007).pdf

1255-MUM-2006-DRAWING(GRANTED)-(23-9-2011).pdf

1255-MUM-2006-FORM 1(5-10-2010).pdf

1255-MUM-2006-FORM 1(8-3-2011).pdf

1255-MUM-2006-FORM 1(8-8-2006).pdf

1255-MUM-2006-FORM 18(21-7-2008).pdf

1255-mum-2006-form 2(7-8-2007).pdf

1255-MUM-2006-FORM 2(GRANTED)-(23-9-2011).pdf

1255-MUM-2006-FORM 2(TITLE PAGE)-(5-10-2010).pdf

1255-mum-2006-form 2(title page)-(7-8-2007).pdf

1255-MUM-2006-FORM 2(TITLE PAGE)-(COMPLETE)-(7-8-2007).pdf

1255-MUM-2006-FORM 2(TITLE PAGE)-(GRANTED)-(23-9-2011).pdf

1255-MUM-2006-FORM 2(TITLE PAGE)-(PROVISIONAL)-(8-8-2006).pdf

1255-mum-2006-form 26(4-9-2006).pdf

1255-mum-2006-form 5(7-8-2007).pdf

1255-mum-2006-form-1.pdf

1255-mum-2006-form-2.doc

1255-mum-2006-form-2.pdf

1255-mum-2006-form-3.pdf

1255-MUM-2006-MARKED COPY(5-10-2010).pdf

1255-MUM-2006-OTHER DOCUMENT(11-7-2011).pdf

1255-MUM-2006-OTHER DOCUMENT(25-8-2011).pdf

1255-MUM-2006-REPLY TO EXAMINATION REPORT(5-10-2010).pdf

1255-MUM-2006-REPLY TO EXAMINATION REPORT(8-3-2011).pdf

1255-MUM-2006-REPLY TO HEARING(11-7-2011).pdf

1255-MUM-2006-REPLY TO HEARING(25-8-2011).pdf

1255-MUM-2006-SPECIFICATION(AMENDED)-(5-10-2010).pdf


Patent Number 249024
Indian Patent Application Number 1255/MUM/2006
PG Journal Number 39/2011
Publication Date 30-Sep-2011
Grant Date 23-Sep-2011
Date of Filing 08-Aug-2006
Name of Patentee NANDAN BIOMATRIX LIMITED
Applicant Address C-002/C-003, GROUND FLOOR, GOKUL PLAZA, THAKUR COMPLEX, KANDIVALI (EAST), MUMBAI-400 101
Inventors:
# Inventor's Name Inventor's Address
1 BHAVANASI, JAYAKUMAR NO.8-2-615/A&B, FLAT NO.204 ROAD NO.11, MEENAKSHI ROYAL COURT APTS., BANJARA HILLS, HYDERABAD-500 034.
2 SINNATHURAI, GOPINATHAN FLAT NO.408/A, SAI DHAMAM GARDEN APARTMENT, TARA NAGAR, LINGAMPALLY, HYDERABAD-500 050.
PCT International Classification Number A61K31/01
PCT International Application Number N/A
PCT International Filing date
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 NA