Title of Invention

"AN IMPROVED PROCESS FOR THE ISOLATION OF A BIOCTIVE FRACTION FROM VITEX NEGUNDO"

Abstract

An improved process for the Isolation of bioactive fraction from vitex negundo containing irideid glycosides of formula 1 & 2 as shown in accompanying drawing from vitex negundo which comprises .defalting the plant parts with non polar solvent, extracting the plant part or defatted plant parts by polar solvents, removing the solvent by evaporation, washing the residue obtained from plant parts with non-polar solvents such as herein described to get the said bioactive fraction at least with 15% yield.

Full Text

This invention relates to an improved process for the isolation of a bioactive fraction from Vitex negundo containing iridoid glycosides of formula 1 & 2 as shown in accompanying drawing from plant Vitex negundo. Particularly this relates to a process for the isolation of a bio-active fraction containing two iridoid glycosides, agnuside and negundoside having health restorative effects including marked hepatoprotective, immunorestorative ( one that restores the depressed immune system) and mild antistress activities, from Vitex negundo Linn. Vitex negundo Linn, (family: Verbenaceae) is one of the common plants used in the indigenous system of medicine in India. Almost all its parts are used as medicine but leaves and roots find maximum use. Various medicinal properties are ascribed to these two parts. The leaves are aromatic, tonic and vermifuge [Chopra, R.N. Nayar, S.L. and Chopra, I.C., Glossary of Indian Medicinal Plants, CSIR, New Delhi, 1956,p. 256; Wealth of India : Raw Materials, CSIR, New Delhi, 1976, Vol.X.p.522; Kirtikar, K.R. and Basu, B.D, Indian Medicinal Plants, M/s Bishen Singh Mahendra Pal Singh, New Connaught Place, Dehra Dun, Ed.2, 1980 Vol.Ill, 1937]. Though V.negundo has been recommended for a long time as a valuable medicinal plant, nothing was known about its constituents except about a trace of an alkaloid in the leaves before 1936. From the leaves Ghosh and Krishna isolated a number of compounds viz. glucononitol, p-hydroxybenzoic acid, 5-hydroxyi-sophthalic acid and 3, 4 - dihydroxybenzoic acid along with two glucosides and an amorphous alkaloid [Ghosh, T.P. and Krishna, S.J.Indian Chem.Soc. 1936, 13, 634]. Masilungan ported the presence of the terpenes, A-pinene, camphene, citral and Q>- caryophyllene in the essential oil of the leaves [Masilungan, V.A., Philip. J. Sci. 1955, 84, 275]. A large number of flavonoids have been isolated from the leaves and twigs. These are casticin, orientin, isoorientin, luteolin, luteolin-7-0-glucoside, corymbosin, gardenins. A and B, 3-0-desmethylartemetin, 5-0-desmethylnobiletin, 3',4',5,5',6,7, 8-heptamethoxy-flavone, 3',5-dihydroxy-4',7,8- trimethoxyflavanone and 3', 5-dihydroxy -4',6,7-trimethoxyflavanone[Sirait,LM., Rimpler, H. and Haensal, R., Experientla 1962, 18, 72; Haensal, Ret al. Phytochemistry 1965, 4,19; Banerji A. et al. Phytochemlstry 1969, 8,511; Ferdous, A.J.et al. Bangladesh Acad.Sci. 1984, 8,23; Dayrit, F.M..ef al. Philipp.J.Sci. 1987, 116, 403; Banerji, J. et al. Indian J.Chem .1988; 27B, 597; Achari, B.ef al. Phytochemistry. 1984, 23, 703. Stem-bark afforded five new flavone glycosides along with luteolin and acerosin. The new flavone glycosides are 6ß-glucopyranosyl -7-hydroxy-3',4',5, 8-tetramethoxyflavone -5-O-A-L-rhamnopyranoside, 3',7- dihydroxy-4',6,8-trimethoxyflavone-5-0-(6"-0-acetyl-ß-D-glucopyranoside), 3',4',6,7- pentamethoxyflavone 5'-0- (4"-0-ß-D-glucopyranosyl-A-rhamnopyranoside, 4,5, 7-tri-hydroxy-flavone-8-(2"-caffeoyl-ß-glucopyranoside) and 3',5, 5',7-tetrahydroxy- 4-methoxyflavone -3'-0-(4"-0--ß-D- galactopy- ranosyl) galactopyranoside [Rao, V.K.et al. Indian J. Pharm. 1977,39, 41; Subramamian, P.M. and Misra, G.S. Indian J.Chem. 1978, 16B, 615; Subramaniam, P.M. and Misra, G.S. J. Nat. Prod. 1979, 42, 540]. A diterpenoid, 5 ß-hydro-8,11,13-abieta-trien-6 A - ol and three triterpenoids, 2A, 3A- dihydroxyoleana-5 12- dien-28-oic acid, 2ß, A- diacetoxyoleana-5, 12-dien-28-oic acid, 2A,3ß-diacetoxy-18-ydroxyoleana- 5,12-dien-28-oic acid have been isolated from the seeds. These compounds exhibited antiinflammatory activity [Chawla, A.S., Sharma, A.K., Handa, S.S. and Dhar, K.L. Indian J. Chem. 1991, 30B, 773 and J.Nat. Prod. 1992,55,163]. Leaves, however, did not yield any triterpenoids but from the roots acetyloleanolic acid was isolated [Vishnol, S.P.,Shoeb, A., Kapil,R.S. and Popli, S P Phytochemistry 1983, 22, 597] Five iridoid glucosides have been isolated from the leaves of V negundo These are aucubin, agnuside of the formula 1 shown in the drawing accompanying the specification, nishindaside, negundoside of the formula 2 and 6'-p-hydroxybenzoylmussaenosidic acid In view of the strong hepatoprotective and immunorestorative activities exhibited by the iridoid glucosides of the roots of Picrorhiza kurrooa viz kutkin, picroside and kutkoside [Ansari, RA et al Indian J Med Research, 1988, 87, 401, Sharma, ML Ph D Thesis entitled "Evaluation of Immunomodulatory activity of some medicinal plants in mammals", 1991, submitted at the University of Jammu] it was thought desirable to evaluate the iridoid glycosides of V negundo for hepatoprotective and immunorestorative activities All these glycosides showed marked hepatoprotective and immunorestorative activities Of these five glycosides the major two are agnuside of the formula 1 and negundoside of the formula 2 An application for a patent on a process for the isolation of an immunostimulatory agent from the leaves of plant Vitex negundo has been submitted from Regional Research Laboratory, Jammu ( Jogeshwar Lai Sun et al 777-DEL/92) However the isolation of this immunostimulating agent which is obtained in about 3% yield involves extraction, fractionation of the extract followed by chromatographic purification/separation This immunostimulating gent contains agnuside, negundoside and some unidentified substances, the first two iridoid glycosides making about 70-80% of it In the course of our work on medicinal plants aimed at developing hepatoprotective agents we reinvestigated V negundo and it was possible to isolate a bioactive composition( fraction) in about 15 per cent yield ( as compared to the immunostimulatory agent obtained in 3% yield 70-80% of which are the two iridoid glycosides, agnuside and negundoside) The bioactive composition isolated in 15% yield in the course of the present investigation had an immunorestorative activity comparable with the product isolated by J.L.Suri et al. (Patent application No.777-DEL/92). This bioactive composition was also found to contain the two iridoid glycosides, agnuside and negundoside in the range of 3-7% each, the variation in their percentage content being due to variation in habitat and season of collection of the plant materials.The bioactive fraction showed marked hepatoprotective and immunorestorative activities and a mild antistress activity. A comparison with known hepatoprotective agent silymarin on multidose treatment (p.o) revealed that it is almost as effective as silymarin, reducing the elevated levels of serum bilirubin,glutamic-pyruvic transaminase (GPT) and glutamic oxaloacetic transaminase (GOT) induced by CCI , paracetamol and galactosamine by 67.50%, 75.6% and 53.5% respectively the corresponding values with silymarin being 70%,73% and 59% respectively. It was found to protect the drug metabolizing enzymes on single dose treatment as observed in hexobarbitone induced sleeping time by 72% as compared to 81% with silymarin and zoxazolamine induced paralysis time by 73.9% as compared to 73.4% with silymarin. In addition to hepatoprotective activity the active composition showed marked immunostimulatory activity stimulating both the cell mediated delayed type hypersensitivity (DTH) and humoral response (antibody titre) and immunorestorative activity in animals treated with cyclophosphamide, a known immunsuppressive chemotherapeutic agent. The increase in humoral response was found to range from 1.4 to 11.2 fold at dose of 12.5 to 100 mg/kg (p.o). It also showed a mild antistress activity increasing the hypoxia time and swimming survival time by 26.4% and 29.6% respectively following a two-week treatment on a dose of 100 mg/kg/day (p.o). It is extremely safe having a therapeutic index of more than 50. The main object of the present invention is to provide an improved process for the isolation of bioactive fraction from Vitex negundo. Another object of the present invention is to provide a process for the isolation of a bioactive fraction, containing two iridoid glycosides, agnuside of the formula 1 and negundoside of the formula 2 in the range of 3 to 7% each, from the whole plant or the aerial parts or leaves of Vitex negundo, having marked hepatoprotectivejmmunorestorative and mild antistress activities. Accordingly the present invention provides an improved process for the isolation of bioactive fraction from Vitex negundo containing iridoid glycosides of formula 1 & 2 as shown in accompanyiydrawing from Vitex negundo which comprises defatting the plant parts with non-polar solvent, extracting the plant part or defatted plant parts by polar solvents, removing the solvent by evaporation, washing the residue obtained from plant parts with non-polar solvents such as herein described to get the said bioactive fraction at least with 15% yield. In an embodiment of the present invention non polar solvents may be used for defatting and washing the residue such as hexane, petroleum ether pentane and mixture thereof. In another embodiment of the present invention polar solvents used for extraction may be such as methanol, ethanol, butanol and mixture thereof. The defatting of the plant material may be effected by washing the whole plant or the aerial parts or leaves of V.negundo with petroleum ether/n-hexane in a percolator at room temperature. The defatted plant material is then extracted with ethanol or methanol by percolation. The treatment is repeated till the extraction is complete.Thereafter the solvent may be removed by evaporation under reduced pressure. The active fraction (composition) prepared by the process of the present invention has the following characteristics. i) it is a dark brown crisp powder; ii) it is hygroscopic; iii) about 80% of the active composition is soluble in water; when shaken with water it gives a brown solution and the insoluble mass slowly settles at the bottom; iv) on TLC (silica gel) it gives a minimum of three spots of Rf values 0.68, 0.32 and 0.18 [solvent system: chloroform-methanol (85:15)] and Rf values 0.88, 0.55 and 0.37 [ solvent system: chloroform-methanol (70:30)] the upper two spots of which viz. Rf values 0.68 (0.88) and 0.32 (0.55) correspond to negundoside and agnuside respectively; v) On HPLC analysis the total content of negundoside and agnuside is found to be not less than 10% and the percentage ranges of these compounds are Negundoside (3-7%) Agunside (3-7%) HPLC column - C - Bondapak Solvent system - methanol - water ( 40:60) with uv detection at 254 nm,flow rate 1 ml/ minute. The process of the present invention is illustrated by the following examples which are, however, not to be construed to limit the scope of the present invention. All the samples of the bio-active composition on analysis were found to conform to the characteristics described above. However, in case any of the samples of the bioactive composition falls short of the required amounts of agnuside and negundoside these iridoid glycosides should be externally added to the sample to make up for their deficiencies. Example 1 The finely ground leaves of Vitex negundo ( 20 mesh powder, 2.25 kg) were charged in a percolator, treated with methanol (11.25 litres) and left overnight. The percolate ( 6.75 litres ) was drained and the marc was extracted thrice by cold percolation each time with 6.75 litres of methanol. The combined percolates (27 litres) were evaporated to dryness in a thin film evaporator keeping the temperature of the bath below 35°C. The reside thus obtained was suspended in 2 litres of 25% aqueous methanol and extracted thrice with n-hexane ( 3x1 litre) in a separatory funnel. The lower aqueous layer was evaporated to dryness, first in a thin film evaporator (to remove organic solvents) at a temperature below 35°C and finally in a freeze dryer; yield 348 g.The product on HPLC analysis was found to contain 6.8 % of agnuside and 4.3% of negundoside. Example 2 Vitex negundo ( whole plant, 2.25 kg) was powdered and the material charged in a percolator. It was first extracted with 11.25 litres of petroleum ether and then twice with 6.75 litres of the same solvent to remove fatty and inactive nonpolar components. Petroleum ether was removed from the marc under reduced pressure. The marc was then extracted with 11.25 litres of ethanol in a percolator at room temperature and then thrice with ( 6.75 litres each time) the same solvent. Evaporation of ethanol in a rotary film evaporator yielded 364 g of the dark brown active composition. The product on HPLC analysis was found to contain 6.3% of agunside and 4.5% of negundoside. Example 3 V.negundo (aerial parts (leaves & stems), 2.25 kg) was powdered and the material charged in a percolator. It was thrice washed with n-hexane (11.25, 6.77 and 6.75 litres). The solvent from the marc was removed under reduced pressure at a temperature below 35°C. It was then extracted with 11.25 litres of methanol in a percolator and then thrice with the same solvent using 6.75 litres each time. Evaporation of methanol at 30-35°C under reduced pressure yielded the bioactive product as dark brown crisp powder; yield 363 g. The product on HPLC analysis was found to contain 7.0% of agnuside and 4.5% of negundoside. Example 4 The finely ground leaves of V.negundo (2.25 kg) were leached thrice in a percolator with petroleum ether ( 60-80°) (11.25 litres for first leaching and 6 75 litres each for two subsequent leachings). Petroleum ether was removed from the marc on a water bath at 30-35°C under reduced pressure. The marc was then charged in a percolator and extracted four times with methanol at room temperature using 11.25 litres of the solvent for the first extraction and 6.75 litres each for the three subsequent extractions. The combined percolates were distilled on a water bath at 30-35°C under reduced pressure to remove methanol. The last traces of methanol were removed under high vacuum to yield the bioactive composition as a dark-brown solid;yield 360 g.The product on HPLC analysis was found to contain 6.7% of agnuside and 4.1 % of negundoside. Example 5 The finely ground aerial parts (leaves & stems) of V.negundo (2.25 kg) were charged in percolator and extracted with 11.25 litres of ethanol at room temperature. The marc was then extracted thrice with 6.75 litres each of ethanol. The combined percolates ( 27 litres) were evaporated to dryness on a water-bath at 30-35°C under reduced pressure. To the residue 25% aqueous ethanol (2 litres) as added and the mixture was washed thrice with n-hexane (3x750 ml ) to remove undesirable constituents. The lower aqueous layer containing the bio - active material was first ] distilled at 30-35°C under reduced pressure to remove organic solvents and then evaporated to dryness in a freeze-dryer to yield 350 g of the bio-active composition The product on HPLC analysis was found to contain 6 5% of agnuside and 5 1 % of negundoside Example 6 Finely ground aerial parts (leaves & stems)of V negundo (2 25 kg) were extracted in a percolator such as column pre-packed with said finely ground aerial parts of vitex with 11 25 litres of methanol at room temperature The marc was then extracted thrice with 6 75 litres each of methanol The combined percolates were distilled on a water-bath at 30-35°C under reduced pressure to remove methanol The residue was taken up in 2 litres of 25% aqueous ethanol and extracted thrice with 750 ml each of n-hexane to remove fatty and non-polar constituents The aqueous suspension was then evaporated to dryness first under reduced pressure to remove organic solvents and finally in a freeze-dryer to yield 355 g of the bioactive composition The product on HPLC analysis was found to contain 7 0% of agnuside and 6 1 % of negundoside We Claim: 1. An improved process for the isolation of bioactive fraction from Vitex negundo containing iridoid glycosides of formula 1 & 2 as shown in accompanying drawing from Vitex negundo which comprises defatting the plant parts with non-polar solvent, extracting the plant part or defatted plant parts by polar solvents, removing the solvent by evaporation, washing the residue obtained from plant parts with non-polar solvents such as herein described to get the said bioactive fraction at least with 15% yield. 2. An improved process as claimed in claim 1 wherein any part of the plant may used for extraction. 3. An improved process as claimed in claims 1 to 2 wherein the non-polar solvents for defatting and washing the residue are such as hexane, pentane, petroleum ether and mixture thereof. 4. An improved process as claimed in claims 1 to 3 wherein the polar solvents used for extraction are such as methanol, ethanol, butanol and mixture thereof. 5. A improved process as claimed in claims 1 to 4 wherein the extract containing the bioactive constituents is evaporated by conventional methods such as using a thin film evaporator or by evaporation under reduced pressure. 6. An improved process for the isolation of bioactive fraction from Vitex negundo substantially as herein described with reference to the examples.

Documents:

116-del-1998-abstract.pdf

116-del-1998-claims.pdf

116-del-1998-correspondence-others.pdf

116-del-1998-correspondence-po.pdf

116-del-1998-description (complete).pdf

116-del-1998-drawings.pdf

116-del-1998-form-1.pdf

116-del-1998-form-2.pdf

abstract.jpg