Title of Invention	"A PROCESS FOR THE ISOLATION OF FRACTION CONTAINING SIXTEEN CONSTITUENTS MAINLY CONTAINING FLAVANOIDS & GLYCOSIDES POSSESSING ADAPTOGENIC ACTIVITY FROM PLANT CENTELLA ASIATICA"
Abstract	Isolation of fraction possessing adaptogonic activity from plant centella asiatica comprises : i) Crushing and homogenising of fresh whole plant of C.asiatica. ii) Extracting the said homogenised plant using an aqueous alcohol. iii) Concentrating the said aqueous alcoholic extract to minimum volume by conventional method and partitioning with different organic solvents of increasing polarity and drying the polar fraction completely, iv) Subjecting the said polar fraction to conventional chromatographic method employing silica gel of mesh size 230-400.

Title of Invention

"A PROCESS FOR THE ISOLATION OF FRACTION CONTAINING SIXTEEN CONSTITUENTS MAINLY CONTAINING FLAVANOIDS & GLYCOSIDES POSSESSING ADAPTOGENIC ACTIVITY FROM PLANT CENTELLA ASIATICA"

Abstract

Isolation of fraction possessing adaptogonic activity from plant centella asiatica comprises : i) Crushing and homogenising of fresh whole plant of C.asiatica. ii) Extracting the said homogenised plant using an aqueous alcohol. iii) Concentrating the said aqueous alcoholic extract to minimum volume by conventional method and partitioning with different organic solvents of increasing polarity and drying the polar fraction completely, iv) Subjecting the said polar fraction to conventional chromatographic method employing silica gel of mesh size 230-400.

Full Text	The present invention relates to a process for the isolation of fraction containing sixteen constituents mainly containing flavanoids & glycosides possessing adaptogenic activity from plant Centella Asiatica. The fraction so obtained has been designated by us as 'Centelloglycotonide. Centelloglycotonide is comprised of complex mixture of sixteen components with ten constituents comprising mainly of flavonoids and their corresponding glycosides showing presence in the range of 0.8 to 25.4% as monitored by preparative high pressure liquid chromatography (HPLC). Centella asiatica (Linn) urban (Syn. Hydrocotyle asiatica Linn Sans. Mandukaparni; Hindi, Brahma-manduki or Brahmi; N.O. Apiaceae) is cosmopolitan in its natural distribution. It is listed drug in the Ayurvedic, [ (1992) the wealth of India-Raw Materials, vo!3 (Revised) 428; Publication and Information Directorate (CSIR), New Delhi ], Chinese, [Tang, W.; Eisenbrand, G. (1992): Chinese Drugs of plant origin, Chemistry, Pharmacology and use in Traditional and Modern.Medicine, 273]and several pharmacopoeias [ (1993) The Martindale Extra Pharmacopoeia, JUL.U Saiv-ion, 756.3] of the world. C.asiatica a prostrate, faintly aromatic, stolcnif erous, perennial herb upto 2. m long is widely distributed in India. It is commonly found as weed in crop fields and occurs wild in marshy banks of rivers, streams, ponds and other waste places throughout India upto an atitude of 600 m. In the traditional system of medicine the plant is given internally as digestible, laxative, cooling, tonic, alterative, antipyretic and also for improving appetite, voice, memory, anaemia, diseases of blood, bronchitis and asthma. [Kiritikar, K.R. and Basu, B.D. (1988) Indian Medicinal plants vol. II (3rd Ed.) 1194 (International book Dept; Dehradaun]. Externally cold poultice of fresh plant is applied in rheumatism, elephantiasis and hydrocele [(1992) The wealth of India-Raw materials, vo!3 (Revised) 429 (Publication and Information Directorate, CSIR, New Delhi)] Though the plant has numerous other uses mentioned in the literature but the most important of all, is that in the classical Ayurvedic system of medicine it is used as 'RASAYANA' (Rejuvenator) to improve the texture of the human skin, for enhancing learing and memory and increasing the life span [ Singh, R.H., Shukla, S.P., and Misra, B.K., (1981) J. Res. Ayurv. and Sidda 2(1), 1, kakar, K.K., (1990) Probe 29(3), 176]. Antianxiety and antistress activities of the drug have also been reported [Upadhyay, S.C., Khosa, R.L., Sharma, N.D., Kumar, M and Chansauria, J.P.N. (1991) Ind. Drug 28 (8) 388] . Previous studies on this plant have revealed the presence of several triterpenoid saponins and sapoge-nins derived from ursanoic acid have been isolated and characterised [ Polonsky, J. (1953) Bull. Soc. Chem. Fr. 173; Polonsky, J. Sach, E. Laderer, E. (1959) Bull. Soc. Fr. 880 and Luo, SQ and Chin, H.F. (1980) Chin Trad. Herb. Drugs 11 ] Brahmic acid isolated from Indian plant was found structurally indentical to madecassic acid obtained from madagascar variety [Pinhas, H (1969) Bull. Soc. Chem. Fr. 173]. In addition to triterpenoid constituents, some mono and ses-quiterpenoids [ Asakawa, Y; Matsuda, R. and Takamoto, T. (1982) Phytochemistry 21, 2590 ] and some polyacety-lenic compounds [ Bohlmann F. and Zdero, C (1975) Chem. Ber. 108(2), 511 ] were reported. Recently three flavo-noid glycosides were obtained from leaves of the plant growing in Madagascar and have been identified as 3-glucosylquercetin, 3-glucosylkaempferol and 7-glucosyl-kaempferol Prum, N. ; Illel, B. ; Raynaud, J. (1983) Pharmazie 38, 423. Keeping in view the high reputation enjoyed by C. asiatica in traditional system of medicine in India we have carried out research work on this plant with the main objective to provide a process for the preparation of a fraction containing constituents responsible for adaptogenic activity. Aqueous alcoholic extract of C.asiatica (fresh whole plant) on submitting to preliminary pharmacological screening showed significant antistress activity. The active extract was subjected to successive partitioning between water and different organic solvents with increasing polarity resulting into a number of fractions on detailed pharmacological evaluation, maximum activity was located in the polar fraction (butanol fraction). The fraction was subjected to medium pressure liquid chromatography (MPLC) using silica gel (230-400 mesh) and eluting the fraction with mixture of organic solvent in order of increasing polarity to yield a fraction which has been code marked as 'F' showing most potent antistress activity. The bioactive fraction (F) so obtained was designated by us as 'Centelloglycotonide1. Pharmacological testing of Centelloglycotonide elicited dose-dependant activity against physical and chemical stress in experimental animals following standard procedures. The effect was on swimming performance, anti-fatigue, survival time against swimming, hypoxia, protection against dose induced liver damage and antiinflammatory activities. Doses from 12.5 to 100 mg/kg given orally for two weeks to different groups of animals showed increase in survival swimming time upto 51%, hypoxia upto 53% with 100% animals showing antifatigue effect. There was no mortality among the drug treated animals in three hour continuous swimming. It also exhibited hepatoprotective and antiinflammatory activities. Its effects when 'tested were comparable to some presently available drugs in the market like 'Plus 30', Dabur's 'Ashwagand-ha' and Doosan's "Ginseng tea1. Preparative reversed phase HPLC of the fraction (F) designated as centello-glycotonide was carried out on U Bonda pack C-18 column using UV (light scattering)detector. Three solvent systems (I) MeOH-H20 (15:85), (II) (52:48) and (III) pure menthanol were used in sequence. As a result sixteen peaks were observed of which ten constituents were in the range of 0.8 to 25.4%. Which comprising of carbohydrates, phenols and flavonoids. It failed to respond to Lieberman-Burchard reaction. Accordingly the present invention provides a process for the isolation of fraction containing sixteen constituents mainly containing flavanoids & glycosides possessing adaptogonic activity from plant centella asiatica comprises : i) Crushing and homogenising of fresh whole plant of C.asiatica. ii) Extracting the said homogenised plant using an aqueous alcohol. iii) Concentrating the said aqueous alcoholic extract to minimum volume by conventional method and partitioning with different organic solvents of increasing polarity and drying the polar fraction completely, iv) Subjecting the said poler fraction to conventional chromatographic method employing silica gel of mesh size 230-400 and eluting the fraction with required solvent mixture to yield the said fraction having adaptogenic activity. In the prefered embodiment of the present invention the choice of the drug as in the step (i), the solvent used in step (ii) for preparing the alcohol extract may be methanol, ethanol, propanol, butanol, or their mixtures. In the step (iii) polar solvents of appropriate polarity is selected. In the chromatograph-ic method in step (iv) for the separation of Centello-glycotonide solvent can be selected from chlorinated solvents, ethylacetate, acetone, methanol and their mixtures in order of increasing polarity. The invention is described in detailed by .the set of examples given below which should not however be construed to limit the scope of present invention. Homogenity and reproducibility of the activity fraction is monitored by reversed phase HPLC. Example I The homogenised fresh whole plant (2 kg) was well stirred for 10-15 minutes in a percolator with 6 litres of methanol and allowed to stand the contents for 18 hours and extract was drawn. Two more extractions were taken by adding each time 5 litres of methanol for a period of twenty four hours. The combined 3 extracts were concentrated. The resultant concentrate (300-350 ml) was partitioned with hexane, chloroform, ethylac-tate & butanol in order of increasing polarity. All these fractions were tested for their antistress activity. The polar fraction (butanol extract) yielded 12g dry mass which showed dose dependant antistress activity. The part of the active fraction (10 g) was subjected to medium pressure liquid chromatography (MPLC) over silica gel 250 g (230-400 -mesh) and each fraction collected was of 250 ml. The column was eluted successively with (a) Chloroform 750 ml (b) Chloroform-methanol 2 litres (9:1) (c) Chloroform-methanol 2 li tres to (8:2) (d) Chloroform-methanol 2.25 litres (7:3) (e) Chloroform-methanol 500 ml (6:4) (f) Chloroform-methanol 2.5 litres (4:6) and Chloroform-methanol 500 ml (2:8) (g) pure MeOH 500 ml and (h) finally with methanol-water 250 ml (85:15). The eluants of fraction (f) were pooled and concentrated to yield centelloglycotonide (0.95) Preparative HPLC of fraction (f) revealed it to be a complex mixture of unidentified sixteen constituents with ten showing presence in range of 0.8 to 25.4%. The detailed HPLC (waters-991) data given as cloumn used U Bonda pack C-18 column'; solvent systems-(1) MeOH-H2O (15:85) (2) (52:48) and (3) pure methanol with UV light scattering detector concentration 4 mg/ml injection; total peaks 16 numbers with ten coded peaks percentage given in the parenthesis. YgS,[3.75], Vgl2, [11.45]; Vg 6 and I"?, [8.30]; Vgl3, [5.50]; Vgl4, [4.35], Vg 15, [3.95]; Vg 8, [25.40]; Vg 16, [3.95], Vg 10, [0.8] & Vg 9, [6.80]. Cenlloglycotonide showed qualitatively the presence of carbohydrates, phenols and flavonoids. pH of its aqueous solution was recorded as 4.5. It failed to respond to Lieberaman-Burchard reaction showing.absence of a steroid or a triterpenoid. Example 2 The homogenised fresh whole plant (2 kg) was charged into percolator provided with stirrer and extracted with menthanol (3x5 litres) for a period of 18-42 hours. The combined percolates was concentrated and the resultant dark green Viscous concentrate (350-370 ml) was partitioned with hexane, chloroform, ethy-lacetate and butanol in that order. A maximum activity was located in the polar butanol fraction (12 g) when tested biologically. A portion of polar fraction (10 g) was subjected to column chromatography using silica gel (100-200 mesh) and eluting, the column successively with (a) Chloroform 750 ml (b) Chloroform-methanol 2 litres (9:1) (c) Chloroform-methanol 2 litres (8:2) (d) Chloroform-methanol 2.5 litres (6; 4) (e) Chloro-form.-MeOH 1 litre (4:6) (f) Chlorof orm-methanol 3 litres (2:8) (g) pure methanol 1 litre and finally (h) methanol-water 1 litre (85:15) in that order. Each eluate of 250 ml was collected. The eluant from fraction (f) was poled and concentrated to yield brownish yellow material (0.89 g) is designated as Centellogly-cotonide possessing antistress activity and identical chemical behaviour as cited in example I. Example 3 Fresh drug (2 kg) was extracted with methanol (3x5 litres) in a homogeniser as in examples 1 and 2 and combined percolate was concentrated and the resultant dark brown concentrate (400 ml) was extracted with (4x320 ml) a mixture of ethylacetate and methanol (85:15). The combined extract distilled and dried to yield centelloglycotonide (0.81 g) with similar antis- tress activity and chemical behaviour as observed in the compound isolated in examples 1 & 2 as monitored by HPLC. We claim 1. A process for the isolation of fraction containing sixteen constituents mainly containing flavanoids & glycosides possessing adaptogonic activity., from plant centella asiatica comprises : i) Crushing and homogenising of fresh whole plant of C.asiatica. ii) Extracting the said homogenised plant using an aqueous alcohol. iii) Concentrating the said aqueous alcoholic extract to minimum volume by conventional method and partitioning with different organic solvents of increasing polarity and drying the polar fraction completely, iv) Subjecting the said polar fraction to conventional chromatographic method employing silica gel of mesh size 230-400 and eluting the fraction with required solvent mixture to yield the said fraction having adaptogenic activity. 2) A process as claimed in claim (1) where the alco hol used for the extraction in step (ii) is selected from methanol, ethanol or their mixtures. 3) A process as claimed in claims 1-2 wherein the polar organic used in step (iii) is selected -From hexane, chloroform, ethylacetate, propanol, butanol. 4) A process as claimed in 1 - 3 wherein the isolation is effected by using medium pressure liquid chromatog- raphy/column chromatographic technique/solvent parti tioning. 5) A process for the isolation of fraction containing sixteen constituents mainly containing flavanoids & glycosides possessing adaptogonic activity from plant centella asiatica substantially as herein described with references to examples.

Full Text

The present invention relates to a process for the isolation of fraction containing sixteen constituents mainly containing flavanoids & glycosides possessing adaptogenic activity from plant Centella Asiatica. The fraction so obtained has been designated by us as 'Centelloglycotonide.
Centelloglycotonide is comprised of complex mixture of sixteen components with ten constituents comprising mainly of flavonoids and their corresponding glycosides showing presence in the range of 0.8 to 25.4% as monitored by preparative high pressure liquid chromatography (HPLC).
Centella asiatica (Linn) urban (Syn. Hydrocotyle asiatica Linn Sans. Mandukaparni; Hindi, Brahma-manduki or Brahmi; N.O. Apiaceae) is cosmopolitan in its natural distribution. It is listed drug in the Ayurvedic, [ (1992) the wealth of India-Raw Materials, vo!3 (Revised) 428; Publication and Information Directorate
(CSIR), New Delhi ], Chinese, [Tang, W.; Eisenbrand, G.
(1992): Chinese Drugs of plant origin, Chemistry, Pharmacology and use in Traditional and Modern.Medicine, 273]and several pharmacopoeias [ (1993) The Martindale Extra Pharmacopoeia, JUL.U Saiv-ion, 756.3] of the world. C.asiatica a prostrate, faintly aromatic, stolcnif erous, perennial herb upto 2. m long is widely distributed in India. It is commonly found as weed in crop fields and occurs wild in marshy banks of rivers, streams, ponds and other waste places throughout India

upto an atitude of 600 m. In the traditional system of medicine the plant is given internally as digestible, laxative, cooling, tonic, alterative, antipyretic and also for improving appetite, voice, memory, anaemia, diseases of blood, bronchitis and asthma. [Kiritikar, K.R. and Basu, B.D. (1988) Indian Medicinal plants vol. II (3rd Ed.) 1194 (International book Dept; Dehradaun]. Externally cold poultice of fresh plant is applied in rheumatism, elephantiasis and hydrocele [(1992) The wealth of India-Raw materials, vo!3 (Revised) 429 (Publication and Information Directorate, CSIR, New Delhi)]
Though the plant has numerous other uses mentioned in the literature but the most important of all, is that in the classical Ayurvedic system of medicine it is used as 'RASAYANA' (Rejuvenator) to improve the texture of the human skin, for enhancing learing and memory and increasing the life span [ Singh, R.H., Shukla, S.P., and Misra, B.K., (1981) J. Res. Ayurv. and Sidda 2(1), 1, kakar, K.K., (1990) Probe 29(3), 176]. Antianxiety and antistress activities of the drug have also been reported [Upadhyay, S.C., Khosa, R.L., Sharma, N.D., Kumar, M and Chansauria, J.P.N. (1991) Ind. Drug 28 (8) 388] .
Previous studies on this plant have revealed the presence of several triterpenoid saponins and sapoge-nins derived from ursanoic acid have been isolated and

characterised [ Polonsky, J. (1953) Bull. Soc. Chem. Fr. 173; Polonsky, J. Sach, E. Laderer, E. (1959) Bull. Soc. Fr. 880 and Luo, SQ and Chin, H.F. (1980) Chin Trad. Herb. Drugs 11 ] Brahmic acid isolated from Indian plant was found structurally indentical to madecassic acid obtained from madagascar variety [Pinhas, H (1969) Bull. Soc. Chem. Fr. 173]. In addition to triterpenoid constituents, some mono and ses-quiterpenoids [ Asakawa, Y; Matsuda, R. and Takamoto, T. (1982) Phytochemistry 21, 2590 ] and some polyacety-lenic compounds [ Bohlmann F. and Zdero, C (1975) Chem. Ber. 108(2), 511 ] were reported. Recently three flavo-noid glycosides were obtained from leaves of the plant growing in Madagascar and have been identified as 3-glucosylquercetin, 3-glucosylkaempferol and 7-glucosyl-kaempferol Prum, N. ; Illel, B. ; Raynaud, J. (1983) Pharmazie 38, 423.
Keeping in view the high reputation enjoyed by C. asiatica in traditional system of medicine in India we have carried out research work on this plant with the main objective to provide a process for the preparation of a fraction containing constituents responsible for adaptogenic activity.
Aqueous alcoholic extract of C.asiatica (fresh whole plant) on submitting to preliminary pharmacological screening showed significant antistress activity. The active extract was subjected to successive partitioning between water and different organic solvents

with increasing polarity resulting into a number of fractions on detailed pharmacological evaluation, maximum activity was located in the polar fraction (butanol fraction). The fraction was subjected to medium pressure liquid chromatography (MPLC) using silica gel (230-400 mesh) and eluting the fraction with mixture of organic solvent in order of increasing polarity to yield a fraction which has been code marked as 'F' showing most potent antistress activity. The bioactive fraction (F) so obtained was designated by us as 'Centelloglycotonide1. Pharmacological testing of Centelloglycotonide elicited dose-dependant activity against physical and chemical stress in experimental animals following standard procedures. The effect was on swimming performance, anti-fatigue, survival time against swimming, hypoxia, protection against dose induced liver damage and antiinflammatory activities. Doses from 12.5 to 100 mg/kg given orally for two weeks to different groups of animals showed increase in survival swimming time upto 51%, hypoxia upto 53% with 100% animals showing antifatigue effect. There was no mortality among the drug treated animals in three hour continuous swimming. It also exhibited hepatoprotective and antiinflammatory activities. Its effects when 'tested were comparable to some presently available drugs in the market like 'Plus 30', Dabur's 'Ashwagand-ha' and Doosan's "Ginseng tea1. Preparative reversed

phase HPLC of the fraction (F) designated as centello-glycotonide was carried out on U Bonda pack C-18 column using UV (light scattering)detector. Three solvent systems (I) MeOH-H20 (15:85), (II) (52:48) and (III) pure menthanol were used in sequence. As a result sixteen peaks were observed of which ten constituents were in the range of 0.8 to 25.4%. Which comprising of carbohydrates, phenols and flavonoids. It failed to respond to Lieberman-Burchard reaction.
Accordingly the present invention provides a process for the isolation of fraction containing sixteen constituents mainly containing flavanoids & glycosides possessing adaptogonic activity from plant centella asiatica comprises :
i) Crushing and homogenising of fresh whole plant of C.asiatica.
ii) Extracting the said homogenised plant using an aqueous alcohol.
iii) Concentrating the said aqueous alcoholic extract to minimum volume by conventional method and partitioning with different organic solvents of increasing polarity and drying the polar fraction completely, iv) Subjecting the said poler fraction to conventional chromatographic method employing silica gel of mesh size 230-400 and eluting the fraction with required solvent mixture to yield the said fraction having adaptogenic activity.

In the prefered embodiment of the present invention the choice of the drug as in the step (i), the solvent used in step (ii) for preparing the alcohol extract may be methanol, ethanol, propanol, butanol, or their mixtures. In the step (iii) polar solvents of appropriate polarity is selected. In the chromatograph-ic method in step (iv) for the separation of Centello-glycotonide solvent can be selected from chlorinated solvents, ethylacetate, acetone, methanol and their mixtures in order of increasing polarity.
The invention is described in detailed by .the set of examples given below which should not however be construed to limit the scope of present invention. Homogenity and reproducibility of the activity fraction is monitored by reversed phase HPLC. Example I
The homogenised fresh whole plant (2 kg) was well stirred for 10-15 minutes in a percolator with 6 litres of methanol and allowed to stand the contents for 18 hours and extract was drawn. Two more extractions were taken by adding each time 5 litres of methanol for a period of twenty four hours. The combined 3 extracts were concentrated. The resultant concentrate (300-350 ml) was partitioned with hexane, chloroform, ethylac-tate & butanol in order of increasing polarity. All these fractions were tested for their antistress activity. The polar fraction (butanol extract) yielded 12g

dry mass which showed dose dependant antistress activity. The part of the active fraction (10 g) was subjected to medium pressure liquid chromatography (MPLC) over silica gel 250 g (230-400 -mesh) and each fraction collected was of 250 ml.
The column was eluted successively with (a) Chloroform 750 ml (b) Chloroform-methanol 2 litres (9:1) (c) Chloroform-methanol 2 li tres to (8:2) (d) Chloroform-methanol 2.25 litres (7:3) (e) Chloroform-methanol 500 ml (6:4) (f) Chloroform-methanol 2.5 litres (4:6) and Chloroform-methanol 500 ml (2:8) (g) pure MeOH 500 ml and (h) finally with methanol-water 250 ml (85:15). The eluants of fraction (f) were pooled and concentrated to yield centelloglycotonide (0.95) Preparative HPLC of fraction (f) revealed it to be a complex mixture of unidentified sixteen constituents with ten showing presence in range of 0.8 to 25.4%. The detailed HPLC (waters-991) data given as cloumn used U Bonda pack C-18 column'; solvent systems-(1) MeOH-H2O (15:85) (2) (52:48) and (3) pure methanol with UV light scattering detector concentration 4 mg/ml injection; total peaks 16 numbers with ten coded peaks percentage given in the parenthesis. YgS,[3.75], Vgl2, [11.45]; Vg 6 and I"?, [8.30]; Vgl3, [5.50]; Vgl4, [4.35], Vg 15, [3.95]; Vg 8, [25.40]; Vg 16, [3.95], Vg 10, [0.8] & Vg 9, [6.80]. Cenlloglycotonide showed qualitatively the presence of carbohydrates, phenols and flavonoids. pH of its aqueous solution was recorded as 4.5. It failed to respond

to Lieberaman-Burchard reaction showing.absence of a steroid or a triterpenoid. Example 2
The homogenised fresh whole plant (2 kg) was charged into percolator provided with stirrer and extracted with menthanol (3x5 litres) for a period of 18-42 hours. The combined percolates was concentrated and the resultant dark green Viscous concentrate (350-370 ml) was partitioned with hexane, chloroform, ethy-lacetate and butanol in that order. A maximum activity was located in the polar butanol fraction (12 g) when tested biologically. A portion of polar fraction (10 g) was subjected to column chromatography using silica gel (100-200 mesh) and eluting, the column successively with (a) Chloroform 750 ml (b) Chloroform-methanol 2 litres (9:1) (c) Chloroform-methanol 2 litres (8:2) (d) Chloroform-methanol 2.5 litres (6; 4) (e) Chloro-form.-MeOH 1 litre (4:6) (f) Chlorof orm-methanol 3 litres (2:8) (g) pure methanol 1 litre and finally (h) methanol-water 1 litre (85:15) in that order. Each eluate of 250 ml was collected. The eluant from fraction (f) was poled and concentrated to yield brownish yellow material (0.89 g) is designated as Centellogly-cotonide possessing antistress activity and identical chemical behaviour as cited in example I. Example 3
Fresh drug (2 kg) was extracted with methanol (3x5

litres) in a homogeniser as in examples 1 and 2 and
combined percolate was concentrated and the resultant
dark brown concentrate (400 ml) was extracted with
(4x320 ml) a mixture of ethylacetate and methanol
(85:15). The combined extract distilled and dried to
yield centelloglycotonide (0.81 g) with similar antis-
tress activity and chemical behaviour as observed in
the compound isolated in examples 1 & 2 as monitored by
HPLC.

We claim
1. A process for the isolation of fraction containing sixteen constituents mainly containing flavanoids & glycosides possessing adaptogonic activity., from plant centella asiatica comprises :
i) Crushing and homogenising of fresh whole plant of C.asiatica.
ii) Extracting the said homogenised plant using an aqueous alcohol.
iii) Concentrating the said aqueous alcoholic extract to minimum volume by conventional method and partitioning with different organic solvents of increasing polarity and drying the polar fraction completely, iv) Subjecting the said polar fraction to conventional chromatographic method employing silica gel of mesh size 230-400 and eluting the fraction with required solvent mixture to yield the said fraction having adaptogenic activity.
2) A process as claimed in claim (1) where the alco
hol used for the extraction in step (ii) is selected
from methanol, ethanol or their mixtures.
3) A process as claimed in claims 1-2 wherein the polar
organic used in step (iii) is selected -From
hexane, chloroform, ethylacetate, propanol, butanol.
4) A process as claimed in 1 - 3 wherein the isolation
is effected by using medium pressure liquid chromatog-
raphy/column chromatographic technique/solvent parti
tioning.

5) A process for the isolation of fraction containing sixteen constituents mainly containing flavanoids & glycosides possessing adaptogonic activity from plant centella asiatica substantially as herein described with references to examples.

Documents:

510-del-1996-abstract.pdf

510-del-1996-claims.pdf

510-del-1996-correspondence-others.pdf

510-del-1996-correspondence-po.pdf

510-del-1996-description (complete).pdf

510-del-1996-form-1.pdf

510-del-1996-form-2.pdf

510-del-1996-form-4.pdf

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Patent Number

188975

Indian Patent Application Number

510/DEL/1996

PG Journal Number

48/2002

Publication Date

30-Nov-2002

Grant Date

19-Sep-2003

Date of Filing

11-Mar-1996

Name of Patentee

COUNCIL OF SCIENTIFIC & INDUSTRIAL RESEARCH

Applicant Address

RAFI MARG,NEW DELHI-110001,INDIA.

Inventors:

#	Inventor's Name	Inventor's Address
1	KEWAL KRISHEN ANAND	REGIONAL RESEARCH LABORATORY,JAMMU ,INDIA.
2	BAL KRISHAN GUPTA	REGIONAL RESEARCH LABORATORY,JAMMU ,INDIA.
3	BUPINDER SINGH JAGGI	REGIONAL RESEARCH LABORATORY,JAMMU ,INDIA.
4	ASHOK SHARMA	REGIONAL RESEARCH LABORATORY,JAMMU ,INDIA.
5	GOPAL KRISHAN GUPTA	REGIONAL RESEARCH LABORATORY,JAMMU ,INDIA.
6	KANYA LAL DHAR	REGIONAL RESEARCH LABORATORY,JAMMU ,INDIA.

PCT International Classification Number

B01D 11/00

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA