EP1523315A1 - 3-o-[alfa-l-rhamnopyranosyl-(1-2)-alfa-l-rhamnopyranosyl-(1-4)-3beta-d-glucopyranosyl]-25(s)-spirostan-3beta-ol, process for its isolation, and its use as immunomodulator - Google Patents

3-o-[alfa-l-rhamnopyranosyl-(1-2)-alfa-l-rhamnopyranosyl-(1-4)-3beta-d-glucopyranosyl]-25(s)-spirostan-3beta-ol, process for its isolation, and its use as immunomodulator

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Publication number
EP1523315A1
EP1523315A1 EP02708616A EP02708616A EP1523315A1 EP 1523315 A1 EP1523315 A1 EP 1523315A1 EP 02708616 A EP02708616 A EP 02708616A EP 02708616 A EP02708616 A EP 02708616A EP 1523315 A1 EP1523315 A1 EP 1523315A1
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EP
European Patent Office
Prior art keywords
rhamnopyranosyl
spirostan
glucopyranosyl
oligospirostanoside
novel
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP02708616A
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German (de)
French (fr)
Inventor
Sukhdev Swami Handa
Om Parkash Suri
Vishwa Nath Gupta
Krishan Avtar Suri
Naresh Kumar Satti
Vikram Bhardwaj
Kasturi Lal Bedi
Anamika Khajuria
Anpurna Kaul
Krishnakant Zandu Pharm. Works Ltd. PARIKH
Prabhakar Zandu Pharm. Works Ltd. KULHE
Ulhas Zandu Pharmaceuticals Works Ltd. SALUNKHE
Raman Zandu Pharm. Works Ltd. KRISHNAMURTHY
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Zandu Pharmaceutical Works Ltd
Council of Scientific and Industrial Research CSIR
Department of Science and Technology of Ministry of Science and Technology India
Original Assignee
Zandu Pharmaceutical Works Ltd
Council of Scientific and Industrial Research CSIR
Department of Science and Technology of Ministry of Science and Technology India
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Application filed by Zandu Pharmaceutical Works Ltd, Council of Scientific and Industrial Research CSIR, Department of Science and Technology of Ministry of Science and Technology India filed Critical Zandu Pharmaceutical Works Ltd
Publication of EP1523315A1 publication Critical patent/EP1523315A1/en
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J71/00—Steroids in which the cyclopenta(a)hydrophenanthrene skeleton is condensed with a heterocyclic ring
    • C07J71/0005—Oxygen-containing hetero ring
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators

Definitions

  • the present invention relates to a novel oligospirostanoside.
  • the present invention particularly relates to a novel chemical entity oligospirostanoside (hereinafter referred to as Immunoside) and structurally constructed as 3-0-[ ⁇ -L-rhamnopyranosyl-(l->2)- -L- rhamnopyranosyl-(l->4)-0- ⁇ -D-glucopyranosyl]-25(S)-spirostan-3 ⁇ -ol (on the basis of IR, 1H and 13 C-NMR Data (CPD, DEPT, HOMOCOR, HETCOR and COLOC), isolated from Asparagus racemosus and biologically evaluated as a potent immunomodulatory agent.
  • Immunoside novel chemical entity oligospirostanoside
  • the present invention also relates to a process of isolation of a novel oligospirostanoside from Asparagus racemosus.
  • the present invention also relates to a process of isolation of a 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 present invention also relates to the use of a novel oligospirostanoside 3-0-[ ⁇ -L- rharr opvranosyl-(l- 2)- ⁇ -L-rhamnopyranosyl-(l- ⁇ -4)-0- ⁇ -D-glucopyranosyl]-25(S)- spirostan-3 ⁇ -ol in immunomodulation in animals.
  • Background of the invention is a novel oligospirostanoside 3-0-[ ⁇ -L- rharr opvranosyl-(l- 2)- ⁇ -L-rhamnopyranosyl-(l- ⁇ -4)-0- ⁇ -D-glucopyranosyl]-25(S)- spirostan-3 ⁇ -ol in immunomodulation in animals.
  • the Ayurvedic crude drug, Shatavari comprises decorticated roots of Asparagus racemosus wild [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 of 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.
  • the main object of the present invention is to provide a novel oligospirostanoside. Yet another object of the present invention is to provide a process for isolation of a novel oligospirostanoside from Asparagus racemosus.
  • Another object of the invention is to provide and characterize a novel sarsasapogenin glycoside Immunoside, an oligospirostanoside isolated from aqueous extract of Asparagus racemosus, present in the range of 0.0023-0.0045% w/w in the dried plant material. Summary of the invention
  • the present invention provides a novel oligospirostanoside, 3-0-[ ⁇ -L- rhamnopyrano syl-( 1 -»2)- ⁇ -L-rhamnopyranosyl-( 1 ->4)-0- ⁇ -D-glucopyranosyl] -25 (S) spirostan-3 ⁇ -ol.
  • the novel oligospirostanoside of the invention is of the formula 1 below :
  • the present invention also relates to a a process for isolation of immunoside, 3-0-[ ⁇ -L- rhamnopyranosyl-(l- ⁇ 2)- ⁇ -L-rhamnopyranosyl - (l-»4)-0- ⁇ -D- glucopyranosyl]-25(S) - spirostan-3 ⁇ -ol which comprises:
  • step (b) clarifying the extract obtained above in step (a)
  • step (c) subjecting the clarified extract obtained from step (b) to desolventation to obtain a dry residue
  • the oligospirostanoside of the invention is of the formula 1 below:
  • the clarified extract is obtained by filtering or centrifugation.
  • the clarified extract is subjected to desolventation in a spray dryer, hot air oven or a rotavapour at 50+5°C.
  • the dry residue of step (e) is resolved into pure constituents in step (f) by adsorption, gel permeation chromatography using isotropic or graded elution, reverse phase purification on Lichroprep RP-8 or pooling and distillation of
  • the dry residue obtained at the end of step (e) is repeatedly crystallised using methanol or ethanol to get pure immunoside.
  • the present invention also relates to a pharmaceutical composition
  • a pharmaceutical composition comprising an effective amount of the novel oligospirostanoside 3-0-[ ⁇ -L-rhamnopyranosyl-(l- ⁇ -2)- ⁇ -L- rhamnopyranosyl - (1— »4)-0- ⁇ -D- glucopyranosyl]-25(S) - spirostan-3 ⁇ -ol contained in a pharmaceutically acceptable carrier.
  • the amount of 3-0-[ ⁇ -L-rharnnopyranosyl-(l-»2)- ⁇ -L-rhamnopyranosyl - (l- 4)-0- ⁇ -D- glucopyranosyl]-25(S) - spirostan-3 ⁇ -ol is in the range of 0.006 to 0.0125 mg per kg of body weight of subject to be treated.
  • the present invention also provides a method for immunomodulation in a immune suppressed animal comprising administering a pharmaceutically effective amount of 3-0-[ -L- rhamnopyranosyl-(l- 2)- ⁇ -L-rhamnopyranosyl - (1— >4)-0- ⁇ -D- glucopyranosyl]-25(S) - spirostan-3 ⁇ -ol.
  • the amount of 3-0-[ ⁇ -L-rhamnopyranosyl-(l- 2)- ⁇ -L-rhamnopyranosyl - (l-»4)-0- ⁇ -D- glucopyranosyl]-25(S) - spirostan-3 ⁇ -ol administered to the said animal comprises 0.006 to 0.0125 mg per kilogram of body weight of the animal.
  • the invention also relates to use of 3-0-[ ⁇ -L-rharrmopyranosyl-(l— »2)- ⁇ -L- rhamnopyranosyl - (l-»4)-0- ⁇ -D- glucopyranosyl]-25(S) - spirostan-3 ⁇ -ol for the preparation of a pharmaceutical composition for immunomodulation in animals.
  • 3-0-[ ⁇ -L-rharrmopyranosyl-(l— »2)- ⁇ -L- rhamnopyranosyl - (l-»4)-0- ⁇ -D- glucopyranosyl]-25(S) - spirostan-3 ⁇ -ol for the preparation of a pharmaceutical composition for immunomodulation in animals.
  • the compound of the invention can be used for authentication of immunomodulatory formulation from Asparagus racemosus.
  • the novel oligospirostanoside was evaluated for immunomodulatory activity and the results are given in Table 1.
  • the compound of the invention is obtained as a white amorphous powder, mp 275°C, [ ⁇ ] D 21 -90.2[C 0.5% pyridine], molecular composition C 45 H 7 O ⁇ 6 derived from FABMS, MS (M+Na) + m/z 893, elemental analytical data and 13 C-NMR, CPD and DEPT spectral data.
  • Aqueous extract residue was dissolved in deionised water (4 Litre) and the resulting solution was extracted with CHC1 3 , EtOAc and «-BuOH (6x1 Litre each) successively.
  • CHCI3 and EtOAc extracts were 0.2 and 0.3 gm respectively whereas «-BuOH extract residue (40 gm) was rich in quantity and chemical constituents.
  • ⁇ -BuOH extract was subjected to adsorption chromatography. 31.0 g of n-BuOH extract dissolved in minimum quantity of MeOH, was adsorbed on SiO 2 gel, 100-200 mesh (100 gm) Solvent was completely removed to get free flowing material.
  • a glass column of ⁇ dia. was packed with 300 gm SiO 2 gel, 100-200 mesh in CHCI3.
  • the adsorbed extract was charged in the column.
  • the column was eluted with solvents by gradually increasing the %age of MeOH in CHC1 3 .
  • 105 fractions of 100 ml each were collected and pooled on the bases of TLC patterns using EtOAc: MeOH:H 2 O : : 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 SiO 2 gel column (1:20 ratio) and eluted with CHC1 3 : MeOH mixtures of increasing polarity.
  • Air-dried roots (1 Kg) of A. racemosus illd. 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 w-BuOH (6 x 1 Litre each) to yield the corresponding fractions (0.3 gm, 38 gm).
  • the ra-BuOH extract was chromatographed on a column of silica gel (60-120 mesh) eluted with a gradient of MeOH in CHC1 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 w-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 Al 2 O 3 using rc-BuOH saturated with water as the packing solvent and eluent to get six major fractions (500 ml each).

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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Engineering & Computer Science (AREA)
  • Medicinal Chemistry (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Pharmacology & Pharmacy (AREA)
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  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

The present invention provides a novel oligospirostanoside structurally constructed as 3-0[α-L-rhamnopyranosyl-(1→2)-α-L-rhamnopyranosyl-(1→4)-0-β-D-glucopyranosyl]-25(S)-spirostan-3β-ol isolated from Asparagus racemosus and biologically evaluated as a potent immunomodulatory agent and to a process of isolation of a 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 present invention also relates to the use of a novel oligospirostanoside 3-0-[α-L-rhamnopyranosyl-(1→2)-α-L-rhamnopyranosyl-(1→4)-0-β-D-glucopyranosyl]-25(S)-spirostan-3β-ol in immunomodulation in animals.

Description

- 0- ' ALFA-L-RHAMNOPYRANOSYL- ( 1-2 ) -ALFA-L-RHAMNOPYRANOSYL- ( 1-4 ) -3BETA-D- GLUCOPYRANOSY1 ! -25 ( S ) -SPIROST AN-3BETA-OL , PROCESS FOR ITS ISOLATION, AND ITS USE AS IMMUNOMODULATOR
Field of the invention The present invention relates to a novel oligospirostanoside. The present invention particularly relates to a novel chemical entity oligospirostanoside (hereinafter referred to as Immunoside) and structurally constructed as 3-0-[α-L-rhamnopyranosyl-(l->2)- -L- rhamnopyranosyl-(l->4)-0-β-D-glucopyranosyl]-25(S)-spirostan-3β-ol (on the basis of IR, 1H and 13C-NMR Data (CPD, DEPT, HOMOCOR, HETCOR and COLOC), isolated from Asparagus racemosus and biologically evaluated as a potent immunomodulatory agent.
The present invention also relates to a process of isolation of a novel oligospirostanoside from Asparagus racemosus. The present invention also relates to a process of isolation of a 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 present invention also relates to the use of a novel oligospirostanoside 3-0-[α-L- rharr opvranosyl-(l- 2)-α-L-rhamnopyranosyl-(l-→-4)-0-β-D-glucopyranosyl]-25(S)- spirostan-3β-ol in immunomodulation in animals. Background of the invention
The Ayurvedic crude drug, Shatavari comprises decorticated roots of Asparagus racemosus wild [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 of 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. 7 (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, N. Phytochemistry, 44 (1997) 763]. Objects of the invention
The main object of the present invention is to provide a novel oligospirostanoside. Yet another object of the present invention is to provide a process for isolation of a novel oligospirostanoside from Asparagus racemosus.
Another object of the invention is to provide and characterize a novel sarsasapogenin glycoside Immunoside, an oligospirostanoside isolated from aqueous extract of Asparagus racemosus, present in the range of 0.0023-0.0045% w/w in the dried plant material. Summary of the invention
Accordingly, the present invention provides a novel oligospirostanoside, 3-0-[α-L- rhamnopyrano syl-( 1 -»2)-α-L-rhamnopyranosyl-( 1 ->4)-0- β-D-glucopyranosyl] -25 (S) spirostan-3β-ol.
In one embodiment of the invention, the novel oligospirostanoside of the invention is of the formula 1 below :
FORMULA 1
The present invention also relates to a a process for isolation of immunoside, 3-0-[α-L- rhamnopyranosyl-(l-^2)-α-L-rhamnopyranosyl - (l-»4)-0-β-D- glucopyranosyl]-25(S) - spirostan-3β-ol which comprises:
(a) extracting dried and powdered roots of Asparagus racemosus with a polar solvent selected from the group consisting of water, methanol, ethanol and any mixture thereof with or without prior extraction with EtOAc.
(b) clarifying the extract obtained above in step (a) (c) subjecting the clarified extract obtained from step (b) to desolventation to obtain a dry residue
(d) dissolving said dry residue in water and subjected the solution to partitioning with CHC13, EtOAc and n-BuOH sequentially or «-BuOH saturated with water alone to obtain an alcoholic extract,
(e) subjecting the said alcoholic extract to desolventation by distillation under reduced pressure to get a dry residue,
(f) resolving the dry residue to obtain pure constituents and separating the constituents to obtain 3-0-[α-L-rhamnopyranosyl-(l- 2)-α-L-rhamnopyranosyl - (1— 4)-0-β-D- glucopyranosyl]-25(S) - spirostan-3β-ol.
In another embodiment of the invention, the oligospirostanoside of the invention is of the formula 1 below:
FORMULA 1 In one embodiment of the invention, the clarified extract is obtained by filtering or centrifugation.
In another embodiment of the invention, the clarified extract is subjected to desolventation in a spray dryer, hot air oven or a rotavapour at 50+5°C. In a further embodiment of the invention, the dry residue of step (e) is resolved into pure constituents in step (f) by adsorption, gel permeation chromatography using isotropic or graded elution, reverse phase purification on Lichroprep RP-8 or pooling and distillation of
TLC homogeneous fractions (Rf 0.53, EtOAc : MeOH : H2O : : 75:13.5:10) under reduced pressure.
In a further embodiment of the invention, the dry residue obtained at the end of step (e) is repeatedly crystallised using methanol or ethanol to get pure immunoside.
The present invention also relates to a pharmaceutical composition comprising an effective amount of the novel oligospirostanoside 3-0-[α-L-rhamnopyranosyl-(l-→-2)-α-L- rhamnopyranosyl - (1— »4)-0-β-D- glucopyranosyl]-25(S) - spirostan-3β-ol contained in a pharmaceutically acceptable carrier. /
In one embodiment of the invention, the amount of 3-0-[α-L-rharnnopyranosyl-(l-»2)- α-L-rhamnopyranosyl - (l- 4)-0-β-D- glucopyranosyl]-25(S) - spirostan-3β-ol is in the range of 0.006 to 0.0125 mg per kg of body weight of subject to be treated. The present invention also provides a method for immunomodulation in a immune suppressed animal comprising administering a pharmaceutically effective amount of 3-0-[ -L- rhamnopyranosyl-(l- 2)-α-L-rhamnopyranosyl - (1— >4)-0-β-D- glucopyranosyl]-25(S) - spirostan-3β-ol.
In one embodiment of the invention, the amount of 3-0-[α-L-rhamnopyranosyl-(l- 2)- α-L-rhamnopyranosyl - (l-»4)-0-β-D- glucopyranosyl]-25(S) - spirostan-3β-ol administered to the said animal comprises 0.006 to 0.0125 mg per kilogram of body weight of the animal.
The invention also relates to use of 3-0-[α-L-rharrmopyranosyl-(l— »2)-α-L- rhamnopyranosyl - (l-»4)-0-β-D- glucopyranosyl]-25(S) - spirostan-3β-ol for the preparation of a pharmaceutical composition for immunomodulation in animals. Detailed description of the invention
The process for isolation of a novel oligospirostanoside of formula 1 which comprises extraction of dried and powdered roots with a polar solvent with or without prior extraction with EtOAc, subjecting aqueous sol. of the desolvented extract to partitioning with CHC13, EtOAc, n-BuOH and resolution of n-BuOH extract residue to adsorption/gel permeation chromatography using isotropic or graded elution, reverse phase purification on Lichroprep RP-8 and repeated crystallizations from methanol or ethanol. Physical constants and spectral data (1H-NMR, 13C-NMR, MS and I R spectral data) are used for characterization of novel isolate. Corroboration of assigned structure is done by permethylation and hydrolysis to get aglycone and partially methylated sugars to confirm linkage site and sequence of sugar units.
Estimation of the compound in the dried plant material is carried out by HPTLC densitometer scanning (0. 0023-0.0045%). The compound of the invention can be used for authentication of immunomodulatory formulation from Asparagus racemosus. The novel oligospirostanoside was evaluated for immunomodulatory activity and the results are given in Table 1. The compound of the invention is obtained as a white amorphous powder, mp 275°C, [α]D 21-90.2[C 0.5% pyridine], molecular composition C45H7 Oι6 derived from FABMS, MS (M+Na)+ m/z 893, elemental analytical data and 13C-NMR, CPD and DEPT spectral data. Immunoside responded positively to the Liebermann-Burchard Reaction [Liebermann, G. (1885) Ber. 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 following examples for the process of extraction are given by way 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°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 «-BuOH (6x1 Litre each) successively. CHCI3 and EtOAc extracts were 0.2 and 0.3 gm respectively whereas «-BuOH extract residue (40 gm) was rich in quantity and chemical constituents. κ-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 \ dia. was packed with 300 gm SiO2 gel, 100-200 mesh in CHCI3. The adsorbed extract was charged in the column. The column was eluted with solvents by gradually increasing the %age of MeOH in CHC13. In 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 CHC13 : 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 CHC13 : 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 illd. 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 w-BuOH (6 x 1 Litre each) to yield the corresponding fractions (0.3 gm, 38 gm). The ra-BuOH extract 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 CHCk-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 w-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 Al2O3 using rc-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 CHCI3 : 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 soluble 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°C, [α]21 -90.2 [C 0.5% Pyridine], MS:
DFABMS, [M+Na]+ m/z 893. Molecular composition derived to be C45H7 Oι6 from MS and elemental analytical data [calcd for C45H74Oι6 : 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 δ 109.85 (C-22) in 13C-NMR spectrum. In addition, 1H 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, IH), 5.73 (brs, IH) and 6.40 (brs, IH) corresponding to three anomeric C-atoms at δlθl.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 β based on J1)2 (7.7Hz). The anomeric configuration of two rhamnosyl units was assigned on α based on there C-5 chemical shifts at δ 69.52 and 70.57 respectively [Soe, S., Tomita, Y., Toti, K. and Yoshimura, Y., J.Am. Chem. Soc. 100 (1978) 3331]. A comparison of 13C- Chemical shifts of the sugar units with those reported for methylglycopyranosides revealed glycosylation shifts by δ +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 Immunoside yielded sapogenin, 3,5,6-tri-0-methyl-D-glucose and 2,3,4-tri-0- methyl-L-rhamnose. This established linkage of two α-L-rhamnose units to glucosyl moiety as l- 2 and l-»4. The sapogenin was identified as sarsasapogenin by direct comparison on TLC, mmp, co-ir with authentic sample. These data confirm the presence of construct Immunoside as 3-0-[α-L-rhanmopvranosyl-(l→2)-α-L-rhamnopyranosyl-(l- 4)-0-β-D-glucopyranosyI]- 25(S)-spirostan-3β-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 Kg'1 (Table 1). The draft spectroscopy data (IR; !H, 13C-NMR, MS) along with physical constants (mp, [α]o21) result in the characterization of the novel molecule as 3-0-[α-L-rhamnopyranosyl-(l-^-2)-α-L- rhamnopyranosyl-(1^4)-0-β-D-glucopranosyl]-25(S)-spirostan-3β-ol. The assigned structure was 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 and cell mediated immune response in immunecompromised mice
LEV: Levamisole; DTH: Delayed type hypersensitivity
Number of observations: 12
*P<0.05; **P<0.01; ***P<0.001
Treatment schedule
0 day: sensitization with 0.2 ml of 5 x 109 SRBC/ml i.p. 0 - 4 day: drug treatment
4th day: challenged with 20μl of 5 x 109 SRBC/ml into right hind footpad for DTH reaction only
5th day: measurement of foot thickness/Haemagglutination antibody titre

Claims

We Claim:
1. Novel oligospirostanoside 3-0-[α-L-rhamnopyranosyl-(1^2)-α-L-rhamnopyranosyl- (l->4)-0-β-D-glucopyranosyl]-25(S) -spirostan-3β-ol
2. Novel oligospirostanoside as claimed in claim 1 of the formula 1 below:
FORMULA 1.
3. Process for the isolation of novel oligospirostanoside 3-0-[α-L-rlιarnnopyranosyl-(l-»2)- α-L-rhamnopyranosyl-(l→4)-0-β-D-glucopvranosyl]-25(S) -spirostan-3β-ol which comprises:
(a) extracting dried and powdered roots of Asparagus racemosus with a polar solvent selected from the group consisting of water, methanol, ethanol and any mixture thereof with or without prior extraction with EtOAc. (b) clarifying the extract obtained above in step (a)
(c) subjecting the clarified extract obtained from step (b) to desolventation to obtain a dry residue
(d) dissolving said dry residue in water and subjected the solution to partitioning with CHC13, EtOAc and n-BuOΕL sequentially or «-BuOH saturated with water alone to obtain an alcoholic extract,
(e) subjecting the said alcoholic extract to desolventation by distillation under reduced pressure to get a dry residue, (f) resolving the dry residue to obtain pure constituents and separating the constituents to obtain 3-0-[α-L-rhamnopyranosyl-(l- 2)-α-L-rhamnopyranosyl - (l-»4)-0-β-D- glucopyranosyl]-25(S) - spirostan-3β-ol.
4. Process as claimed in claim 3 wherien the oligospirostanoside obtained is of the formula 1 below:
FORMULA 1
5. Process as claimed in claim 3 wherein clarified extract is obtained by filtration or centrifugation.
6. Process as claimed in claim 3 wherein the clarified extract is subjected to desolventation in a spray dryer, hot air oven or a rotavapour at 50+5°C.
7. Process as claimed in claim 3 wherein the dry residue of step (e) is resolved into pure constituents in step (f) by adsorption, gel permeation chromatography using isotropic or graded elution, reverse phase purification on Lichroprep RP-8 or pooling and distillation of TLC homogeneous fractions (Rf 0.53, EtOAc : MeOH : H2O : : 75:13.5:10) under reduced pressure.
8. Process as claimed in claim 3 wherein the dry residue obtained at the end of step (e) is repeatedly crystallised using methanol or ethanol to get pure immunoside.
9. Pharmaceutical composition comprising an effective amount of novel oligospirostanoside 3-0-[α-L-rhamnopyranosyl-(l->-2)-α-L-rhamnopyranosyl - (l- 4)-0-β-D- glucopyranosyl]-25(S) - spirostan-3β-ol contained in a pharmaceutically acceptable carrier.
10. Composition as claimed in claim 10 wherein the amount of 3-0-[α-L-rhamnopyranosyl- (l-»2)-α-L-rhamnopyranosyl - (l-> )-0-β-D- glucopyranosyl]-25(S) - spirostan-3β-ol is in the range of 0.006 to 0.0125 mg per kg of body weight of subject to be treated.
11. Method for immunomodulation in a immune. suppressed animal comprising administering a pharmaceutically effective amount of 3-0-[α-L-rhamnopyranosyl-(l-»2)-α-L- rhamnopyranosyl - (l-»4)-0-β-D- glucopyranosyl]-25(S) - spirostan-3β-ol.
12. Method as claimed in claim 11 wherein the amount of 3-0-[α-L-rhamnopyranosyl-(l— »2)- α-L-rhamnopyranosyl - (l->4)-0-β-D- glucopyranosyl]-25(S) - spirostan-3β-ol administered to the said animal comprises 0.006 to 0.0125 mg per kilogram of body weight of the animal.
13.Use of 3-0-[α-L-rharnnopvranosyl-(l->2)-α-L-rhamnopyranosyl - (l-»4)-0-β-D- glucopyranosyl]-25(S) - spirostan-3 β-ol for immunomodulation in an animal.
14.Use as claimed in claim 13 wherein the animal is a human.
EP02708616A 2002-03-21 2002-03-21 3-o-[alfa-l-rhamnopyranosyl-(1-2)-alfa-l-rhamnopyranosyl-(1-4)-3beta-d-glucopyranosyl]-25(s)-spirostan-3beta-ol, process for its isolation, and its use as immunomodulator Withdrawn EP1523315A1 (en)

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CN1315866C (en) * 2004-05-09 2007-05-16 上海正祥天然药物科技有限公司 Radix asparagi steroid saponin extract and its preparing process and application
WO2009007774A1 (en) * 2006-07-07 2009-01-15 Avestha Gengraine Technologies Pvt. Ltd. Asparagus racemosa extracts for treating osteoporosis and the extraction process thereof
CN117244054B (en) * 2023-10-16 2024-06-07 国药中生生物技术研究院有限公司 Adjuvant use of compounds having spirostane-O-gal structure

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US6649745B2 (en) * 2002-03-20 2003-11-18 Council Of Scientific And Industrial Research Oligospirostanoside, pharmaceutical composition containing novel oligospirostanoside and method for immunomodulation using said oligospirostanoside

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Publication number Priority date Publication date Assignee Title
US6649745B2 (en) * 2002-03-20 2003-11-18 Council Of Scientific And Industrial Research Oligospirostanoside, pharmaceutical composition containing novel oligospirostanoside and method for immunomodulation using said oligospirostanoside

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Chemical Abstracts (online), database accession no. 2003:692769. *
DHULEY, J. N.: "Effect of some Indian herbs on Macrophage Functions in Ochratoxin A Treated Mice", J. ETHNOPHARMACOLOGY, vol. 58, 1997, pages 15 - 20, XP002196030 *
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