Title of Invention	"A NOVEL OLIGOSPIROSTANOSIDE"
Abstract	The present invention relates to a novel oligospirostanoside of the structure 1, 3-0-[α-L-rhamnopyranosyl-(1->2)-α-L-rhamnopyranosyl-(1->4)-0-ß-D-glucopyranosyl]-25(S) -5beta -spirostan - 3beta - ol isolated from Asparagus racemous and to a pharmaceutical composition containing novel oligospirostanoside.

Full Text

This invention relates to a novel oligospirostanoside. It particularly relates to a novel chemical entity Immunoside, white amorphous powder, mo 275°CA.ialphal -90,2fC Q.,5% p,yridine}, molecular composition C45H74O16derived from FABMS. MS (M+Na)+ m/z 893, elemental analytical data and I3C-NMR, CPD and DEPT spectral data and structurally constructed as 3-0-[alpha-L-rhamnopyranosyl-(l—>2)-alpha-L~ rhamnopyranosyl-0—>4)-0-beta-D-glucopyranosyl]-25(S)- 5-beta-spirostan-3-beta-ol on the basis of IR, 'H and 13C-NMR Data (CPD, DEPT, HOMOCOR, HETCOR and COLOC), isolated from Asparagus racemosus and biologically evaluated as potent immunomodulatory agent. The present invention also relates to a process for isolation of novel oligospirostanoside from Asparagus racemosus. The present invention also relates .to a pharmaceutical composition containing the novel oligospirostanoside and to a method for immunomodulation using said oligospirostanoside. The Ayurvedic crude drug, Shatavari of commerce comprises of decorticated roots of Asparagus racemosus willd [Kanitkar, U.K., Dange, P.S. and Pendse, G.S. J. Res. Indian Med. 3 (1969) 123; Medicinal Plants of India vol. 1, ed. by Satyavati, G.V., Raina, M.K. and Sharma, M. Indian Council of Medical Research, New Delhi (1976) 101]. Phytochemical investigations on the plant Asparagus racemosus have resulted in isolation and characterization of steroidal glycosides [Ravikumar, P.R., Soman, R., Chetty, G.L. Pandey, R.C. and Sukhdev, Indian J. Chem. 26 B (1987) 1012', Kar, Deepak Kumar and Sen Sumitra, Cell Chromosome Res. 1 (1984) 10], a novel cage type pyrrolizidine alkaloid, asparaginine [Sekine, T., Fukasawa, N., Kashiwagi, Y., Ruangrungsi, N. and Murakoshi, I. Chem. Pharm. Bull. 42 (1994) 1360] and a 9,10-dihydrophenanthrene derivative [Sekine, T., Fukasawa, N., Murakoshi, I. and Ruangrungsi, TV. Phytochemistry, 44 (1997) 763]. Immunoside responded positively to the Liebermann-Burchard Reaction [Liebermann, G. (1885)5er. Deut. Chem. Ges. 18, 1804; Burchard, H. (1890) Chem. Zentbl. 1, 25], negatively to the Ehrlich test [Kiyosawa, S and Hutoh, M. (1968) Chem. Pharm. Bull. 16, 1162; Tschesche, R; Siedel, L., Sharma, S.C. and Wulff, G. (1972) Chemische Berichte, 105,3397] and positively to Molisch's test indicating it to be spirostanol glycoside. The main object of the present invention is to provide novel oligospirostanoside. Another object of the present invention is to provide and characterize a novel sarsasapogenin glycosides Immunoside, an oligospirostanoside, isolated from the aqueous extract of Asparagus racemosus, present in the range of 0.0023-0.0045% w/w in the dried plant material. Yet another object of the present invention is to provide a process for isolation of novel oligospirostanoside from Asparagus racemosus. Accordingly, the present invention provides a novel oligospirostanoside 3-0-[ α -L-rhamnopyranosyHl—>2)- α -L-rhamnopyranosyl-(l—>4)-0- ß -D-glucopranosyl]-25(S)~ 5-beta spirostan-3ß -ol. of formula 1 of the drawing accompanying the specification. In an emobodiment of present invention, pharmaceutical composition comprising an effective amount of novel oligospirostanoside ,3-0-[ α-L-rhamriopyranosyl-(l—>2)-α -L-rhamnopyranosyl-(l—>4}-0- ß -D-glucopranosyl]-25(S)-;5-beta-spirostan-3-beta-ol contained in a pharmaceutically acceptable carrier. hi another emobodiment of present invention, composition wherein the amount of ,3-0-[α-L-rhamnopyranosyl-(l—>2)- α -L-rhamnopyranosyl-(l—>4)-0- ß -D-glucopranosyl]-25(S)- 5-beta spirostan-3ß -ol is in the range of 0.006 to 0.0125 mg per kg of body weight of subject to be treated. In yet another emobodiment of present invention, method for immunomodulation in a immune suppressed animal comprising administering a pharmaceutically effective amount of ,3-0-[ α-L-rhamnopyranosyl-(l—>2)- α -L-rharnnopyranosyl-(I—>4)-0- ß -D-glucopranosyl]-25(S)- 5-beta-spirostan-3-bcia-ol In yet another emobodiment of present invention, a method wherein the amount of ,3-0-[ α -Ixhamnopyranosyl-(l—>2> α -L-rhamnopyranosyl-(l—>4)-0- ß -D-glucopranosyl]-25(S)-5-beta-spirostan-3-beta-ol administered to the said animal comprises 0.006 to 0.0125 mg per kg of body weight oFfhe animal In an emobodiment of present invention , a method wherein the animal is human The present invention also provides a process for isolation of immunoside, 3-0-[α-L-rhamnopyranosyl-(l—> 2)- α- L-rhamnopyranosyl - (1—>4)-0-ß-D- glucopyranosyl]-25(S)-5~beta spirostan-3ß -ol; which involves various steps such as: a). Extraction of dried and powdered roots of Asparagus racemosus may be carried out with a polar solvents like water, methanol, ethanol or their mixtures with or without prior extraction with EtOAc. b). Clarified extracts (Filtering or centrifugation) may be subjected to desolventation in a spray dryer, hot air oven or a rotavapour at 50±5 C . c). Dry residue may be dissolved in water and subjected to partitioning with CHCl3 EtOAc and n-BuOH sequentially or n-BuOH saturated with water alone, d). n-BuOH extract may be desolventated by distillation under reduced pressure to get a dry residue, e). Dry residue may be resolved into pure constituents by adsorption, gel permeation chromatography using isotropic or graded elution or reverse phase purification on Lichroprep RP-8. f). TLC homogeneous fractions (Rf 0.53, EtOAc : MeOH : H2O :: 75:13.5:10) maybe pooled and distilled under reduced pressure, g). Crystallisation of the residue may be carried out from methanol or ethanol repeatedly to get pure immunoside. In another embodiment of the invention, physical constants and spectral data ('H-NMR, 13C-NMR, MS and IR spectral data) which may be used for characterization of the novel isolate. In yet another embodiment, corroboration of the assigned structure may be done by permethylation and hydrolysis to get aglycone and partially methylated sugars to confirm linkage site and sequence of sugar units. In yet another embodiment estimation of the compound in the dried plant material may be carried out by HPTLC densitometer scanning (0. 0023-0.0045%). In another embodiment, the compound may be used for authentication of immunomodulatory formulation from Asparagus racemosus. In yet another embodiment, the novel oligospirostanoside may be evaluated for immunomodulatory activity (Table 1). The following examples for the process of extraction are given byway of illustrations and therefore should not be construed to limit the scope of present invention: Example 1: Dried underground part of plant material Asparagus racemosus (1 Kg) was ground to a coarse powder. Coarse powder was extracted with deionised water at 98°C for 2 hrs. Extraction process was repeated thrice using total water (7+4+4 Litre, three extractions) in 1:15 ratio w/v with respect to the plant material. All the three extracts were pooled. The pooled aqueous extracts were centrifuged, clear supernatant was evaporated to dryness on a wiped film evaporator at 50+/- 5 deg. C residue 480 g (extractive value 48%). Aqueous extract residue was dissolved in deionised water (4 Litre) and the resulting solution was extracted with CHC13, EtOAc and n-BuOH (6x1 Litre each) successively. CHC13 and EtOAc extracts were 0.2 and 0.3 gm respectively whereas n-BuOH extract residue (40 gm) was rich in quantity and chemical constituents. n-BuOH extract was subjected to adsorption chromatography. 31.0 g of n-BuOH extract dissolved in minimum quantity of MeOH, was adsorbed on SiO2 gel, 100-200 mesh (100 gm.) Solvent was completely removed to get free flowing material. A glass column of 1* " dia. was packed with 300 gm SiO2 gel, 100-200 mesh in CHCl3 . The adsorbed extract was charged in the column. The column was eluted with solvents by gradually increasing the %age of MeOH in CHCl3. hi all 105 fractions of 100 ml each were collected and pooled on the bases of TLC patterns using EtOAc: MeOH:H2O : : 75:13.5:10 as developing solvent. Spots were visualised by spraying with 1% cericammonium sulphate followed by heating at 110 C for 20 minutes. Fractions 23-29 showed same TLC pattern. These fractions were pooled, dried and subjected to rechromatography using 100-200 mesh SiO2 gel column (1:20 ratio) and eluted with CHCl3 : MeOH mixtures of increasing polarity. In all 60 fractions of 200 ml each were collected. Fractions 37-44 were pooled on the bases of TLC and again subjected to chromatography. 30 fractions of 100 ml each were collected. Fractions 23-28 were concentrated under reduced pressure. Residue was repeatedly crystallised from MeOH, a colourless amorphous powder soluble in CHCl3 : MeOH mixture was obtained. Compound Rf 0.53, (solvent system EtOAc:MeOH:H2O:: 75:13.5:10) was named as immunoside. Example 2 Air dried roots (1 Kg) of A. racemosus Willd. were ground and extracted with 75% aqueous methanol three times (75% MeOH, 3x5 Litre) for 12 hrs. each. The combined extracts were concentrated to dryness under reduced pressure. The residue (407 gm) was dissolved in water (4 litre) and extracted successively with EtOAc and n-BuOH (6x1 Litre each) to yield the correspondending fractions (0.3 gm, 38 gm ). The n-BuOH ext. was chromatographed on a column of silica gel (60-120 mesh) eluted with a gradient of MeOH in CHC13. The CHC13 : MeOH (5:1) eluate was rechromatographed on a silica gel (100-200 mesh) column using CHCl3-MeOH: H2O (6:1:0.1) as solvent. Fractions homogeneous on TLC were pooled, dried and charged on a sephadex LH-20 column, eluted with MeOH to produce two fractions of 500 ml each. Second fraction containing mainly the target compound was subjected to further purification over Lichroprep RP-8 CC eluted with MeOH: H2O (3:2) to afford a fraction, which on repeated crystallisation from EtOH yielded a colourless amorphous powder soluble in CHCl3-MeOH mixture.Compound Rf 0.53 (solvent system EtOAc:MeOH:H2O:: 75:13.5:10) was named as immunoside. Example 3 A. racemosus root powder (4 Kg) was extracted with EtOAc (12Lx2) in a Soxhlet for 36 hr. each. The marc was next extracted with 80% aq. ethanol (10 L x 2) for 24 hr. each. The aq. alcoholic extract was distilled under reduced pressure to dryness. The residue (700 g) was dissolved in water (IL) and extracted with n-BuOH saturated with water (250 ml x 8). The combined BuOH extractions were desolvented to get saponin mixture (46 gm.) The mixture was subjected to column chromatography over neutral A12O3 using n-BuOH saturated with water as the packing solvent and eluent to get six major fractions (500 ml each). Fractions were monitored on TLC using CHC13 : MeOH : H2O : : 65:35:10; (lower phase) as developing solvent. Spots were visualised by spraying the plate with 1% cericammonium sulphate followed by heating at 110° C for 20 minutes. Residues from fractions, 3 and 4 were pooled and chromatographed on ODS silica gel eluting with MeOH: H2O (3:2) to give two fractions of 350 ml. each. Residue from 2nd fraction was subjected to flash CC on SiO2 gel (230-400 mesh) eluting with CHC13 : MeOH: H2O (30:10:1) to give the desired compound with traces of impurities. Final purification was achieved by repeated crystallisations from MeOH to get colourless amorphous powder oluble in CHCl3-MeOH mixture. Compound Rf 0.53 (TLC developing system: EtOAc:MeOH: H2O::75: 13.5:10) was named as immunoside. Immunoside, white amorphous powder, mp 275 deg. C,[α]d21 -90.2 [C 0.5% Pyridine], MS: FABMS, [M+Na]+ m/z 893. Molecular composition derived to be C45H74O16 from MS and elemental analytical data [calcd for C45H74O16 : C, 62.06;H, 8.50; Found : C. 61.98; 8.47] and 13C-NMR, CPD and DEPT. The IR spectrum of the saponin indicated the existence of hydroxyl groups (3400-3350 cm-1) and the characteristic absorption bands of (25S)-spiroketal at 919 and 896 cm-1 with the absorption at 919 cm-1 being of greater intensity than at 896cm-1 [Wu G, Jiang S, Jiang F, Zhu D, Wu H and Giang C. (1996) Phytochemistry 42, 1677; Li XC, Wang D Z and Yang C R, (1990). Phytochemistry 29, 3899]. 25(S)-Spirostane skeleton of Immunoside was also suggested by the occurrence of a resonance at 6 109.85 (C-22) in 13C-NMR spectrum. In addition, *H and 13C-NMR (200.13 MHz) and 50.32 MHz), C5D5N) spectra displayed three anomeric proton signals at 4.87 (d, J=7.7 Hz, 1H), 5.73 (brs, 1H) and 6.40 (brs, 1H) corresponding to three anomeric C-atoms at 5101.60, 102.23 and 103.01 respectively indicating that Immunoside contained one glucosyl and two rhamnosyl units in the oligosaccharide function. The anomeric configuration of the glucosyl unit was indicated to be p based on J, 2 (7.7Hz). The anomeric configuration of two rhamnosyl units was assigned on a based on there C-5 chemical shifts at 8 69.52 and 70.57 respectively [Soe, S., Tomita, Y., Toti, K. and Yoshimura, Y., J.Am. Chem. Soe. 100 (1978) 3331]. A comparison of 13C-Chemical shifts of the sugar units with those reported for methylglycopyranosides, revealed glycosylation shifts by 5 +6.00 for C-2 and +6.79 for C-4 of glucose unit, thus indicating the presence of one 2,4-disubstituted glucose unit.These data indicated that 2-rhamnose units are linked to glucose moiety at position 2- and 4-. Further proof to the site of interlinkage amongst sugar units and to the sapogenin was provided by hydrolysis of permethylated Immunoside [Hakomori, S., J. Biochem. 55 (1964) 205]. Acid catalyzed hydrolysis of permethylated rmmunoside yielded sapogenin, 3,5,6-tri-O-methyl-D-glucose and 2,3,4-tri-O-methyl-L-rhamnose. This established linkage of two a-L-rhamnose units to glucosyl moiety as 1—>2 and 1—>4. The sapogenin was identified as sarsasapogenin by direct comparison on TLC, mmp, co-ir with authentic sample. These data made us to construct Immunoside as 3-0-[ a -L-rhamnopyranosyl-(l—>2)- α -L-rhamnopyranosyl-(l—>4)-0-ß-D-glucopyranosyl]-25(S)-,' 5-beta-spirostan-3-beta-ol, a new chemical entity. Biological activity: Oral administration of Immunoside potentiated antibody synthesis and enhanced cell mediated immune response in immunecompromised experimental animals by 55.55-69.44% and 77.77-102.22% respectively in dose levels of 0.0062-0.0125 mg per Kg (Table 1). In the drawing accompanying the specifications figure I represents the novel chemical compound, Immunoside, having potent immunomodulatory activity. . The draft spectroscopy data. (IR; !H, 13C-NMR, MS) alongwith physical constants (mp, [α] D21 ) have been provided for characterization of a novel molecule,3-0-[ α -L- rhamnopyranosyl-(l—>2)- α -L-rhamnopyranosy]-(l—>4)-0- ß-D-glucopranosyl]-25(S)- 5-beta spirostan-3ß-ol. The assigned structure has been corroborated by chemical degradation data i.e., permethylation and hydrolysis. The immunomodulatory activity, both in humoral and CMI models, of the compound has been evaluated in the product. Table-1 EFFECT OF IMMUNOSIDE ON HUMORAL & CELL-MEDIA TED IMMUNE RESPONSES IN IMMUNECOMPROMISED MICE (Table Removed) Lev: Levamisole DTH: Delayed type hypersensitivity Number of observations = 12 *P Treatment Schedule 0 day = Sensitization with 0.2ml of 5x 109 SRBC/ml Lp. 0-4 day = Drug treatment 4th day = Challenged with 20 ul of SxlO9 SRBC/ml into right hind foot pad for DTH reaction only. 5th day = Measurement of foot thickness /Haemagglutinatioil antibody litre We claim: 1. A novel oligospirostanoside of formula 1, 3-0-[α-L-rhamnopyranosyl- (1->2)-a-L-rhamnopyranosyl-(l->4)-0-ß-D-glucopyranosyl]-25(S) -5 beta- spirostan-3ß-ol of formula 1 of the drawing accompanying the specification. 2. A novel oligospirostanoside substantially as herein described with reference to the examples and figures accompanying this specification.

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