Title of Invention

"AN IMPROVED PROCESS FOR ENRICHMENT OF BABACHI DRUG FROM THE SEEDS OF BABACHI (PSORALEA CORYLIFOLIA L L.)

Abstract 1. An improved process for ennchment of babchi drug from the seeds of babchi (Psoralea Corylifolia L.) which comprises: a) soaking babchi seeds in acidic (5%HCL) Methanol (MeOH) for 12 hr. with occasional stirring in order to hydrolyze the glycosidic active principle such as herein described, b) extracting the active principle and reducing to 20-30% (WAA/) and treating with chloroform (CHCL3) for discarding impurities, c) and further treating with a mixture of CHCL3 and petroleum ether (30:70 (VA/) ratio followed by hot extraction with same mixture yielding 50% enriched babchi drug in the range of 1.5-2.5% (WAA/), d) and further enrichment of babchi drug upto 70-80% in the range of 1-1.5% (WAA/) is done by treating the above mixture with hot petroleum ether at 60-80% temperature,
Full Text Details of invention;
This invention relates to the improved process for the enrichment of babchi drug from the seeds of Psoralea corylifolia L which has proven potential use sn therapeutic as well as in stored grain protection against insect infestation The term babcht drug refers to an enriched fraction containing psoralen/ isopsoraien alone or in combination. The object of the present invention is to provide an improved process of enrichment of psoralen/isopsoralen mixture as babchi drug
Complete Specifications:
The following specification (particularly) describes the nature of invention and the manner in which it is to be performed
This invention relates to the improved process for tne enrichment of babcht drug from the seeds of Psoralea corylifolia L which has proven potential use In therapeutic as well as in stored grain protection against insect infestation The term babchi drug refers to an ennched fraction containing psoralen/ isopsoraien alone or in combination. The object of the present invention is to provide an improved process of enrichment of psoralen/isopsoralen mixture as babchi drug
BACKGROUND OF THE INVENTION:
The seeds of Psoralea corylifolia are weii known for their therapeutic use in vanous kinds of ailments (Krishnamurthr 1969) it has been recognised in agnculture sector for its uses as pulse protectant (Chander and Ahmad, 1982) against insect infestation, as antifeedent for the larvae of pest of castor [Ricinus communis) (Mohanty et al., 1988), and as growth regulator in siik worms {Bombyx man) (Murugan et a!., 1998). The plant extract of Psoralea corylifolia L could be a cheap and abundant source of insect hormones and can be used to increase silk yield in commercial silk worm reahng (Gowda et ai. 1997)
The major groups of compounds identified in the seeds and other parts of the Psoralea corylifolia L plant include furanocoumanns, coumestans, flavones/ chalcones, terpenes, saponins/ sterols hydrocarbons and fats (Siddiqui and Ansari, 1995). Out of these, the furanocoumanns including psoralen and its angular isomer, isopsoraien are of immense importance in the medical science as well as tn agriculture.
Various modern instrumental techniques of estimation and isolation of these compounds have been evolved (Lu et al 1999; Spencer et al 1987) but a very high
isolation cost associated with these techniques has always been a limiting factor in the common use of these techniques. It requires a handy, easy and cheap solution to their problems. A pure constituent is not commonly required. The gravimetric method of isolation of these compounds is a key to success not only of these users but also the small scale entrepreneurs.
The available processes of isolation of psoralen and isopsoralen mixture are based on the retention or elution of the desired compound from a medium (silica gel, aluminium oxide etc.) against a polar or non-polar eluent, thus narrowing down the selection of the compound by discarding other unwanted compounds. There is an array of solvents, which when used in altered sequence or in the form of mixture change their polarity and thereby their solubilizing efficiency towards a compound. How quickly and effectively a desired compound is isolated depends upon selection of various parameters and factors like solvent, mixtures of various solvents in varying proportions, heating, non heating or cooling, sequence of extraction or elution with solvent or solvent mixture etc.
The present process of isolation of psoralen and isopsoralen mixture is a set of sequential processes like soaking in mild acidic medium, extraction with a mixture of organic solvents in fixed proportion, say 60:40 or 70:30 or 75:25, followed by hot extraction with the same mixture and further another organic solvent at a higher temperature, say 75 to 85°C or 80°C and followed by removal of psoralen/isopsoralen free oily/waxy part of the extract in chilled extraction.
DESCRIPTION OF THE PRIOR ART
Isolation methods of psoralen and isopsoralen mixture from the seeds of Psoralea corylifolia have been developed and patented in the past.
In one method (Indian patent 59265 Sept. 17, 1958) dried whole seeds are soaked in petroleum ether (62°C-82°C) over night. The solvent is then drained off. The
process is repeated twice. The seeds, free from solvent, are coarsely ground and extracted in soxhiet apparatus with petroleum ether for 20 hr. The extract is condensed to 500 ml and kept in cold for 7-10 days. Practically all psoralen and isopsoralen separated out, filtered and washed with a little petroleum ether to remove adhering oil and coloured impurities..
In another method (Indian patent 59266 Nov. 26, 1958) improved by the same inventor the seeds are extracted with petroleum ether for 12 hr. in soxhiet apparatus. The seeds are then coarsely pulverised and powder is soaked in water for 7-10 days and then allowed to dry. The dry powder is extracted with petroleum ether for 6 hr. The extract is concentrated to a small volume and kept cold and psoralen and isopsoralen mixture crystallizes out and is separated by filtration and washed with a little ether to remove adhering coloured material.
In another method ( Indian J. Appl. Chem. 1959, 22, 82-5) psoralen and isopsoralen were isolated from both water soluble glycosidic and ether soluble non-glycosidic fraction. The seeds are defatted with petroleum ether and then dried ground seeds are percolated with ethyl alcohol. The concentrated ethyl alcohol extract was further extracted with solvent ether (Et2O) to separate non-glycosidic fraction. The glycosidic fraction was separated by hydrolysing with ale. HCI. After extraction of the hydrolysis mixture with ether, washing with NaOH and drying over Na2SO4, the ether was evaporated and a white crystalline material isolated. The psoralen was recrystallized with methyl alcohol. From the mother liquor isopsoralen was isolated by crystallization. Remaining quantity of psoralen and isopsoralen could be obtained by chromatography on acetic acid (HoAc) deactivated alumina by eluting with petroleum ether-benzene mixture.
In another method (Indian patent 84828, 14 Sept, 1974) psoralen-isopsoralen mixture was isolated. Seeds washed with hot petroleum ether and the residue was steam distilled. The residue was crystallized from rectified spirit to give psoralen, the distillate was cooled to give isopsoralen.
In another method (Brit. 1,212,134(cl. C07d, A61k), 11 Nov. 1970) seeds were crushed sieved washed with water. The powder extracted with toluene, benzene or chloroform, the extract washed repeatedly with dilute aqueous NaOH, the solvent distilled off, kerosene or mineral spirit added to precipitate psoralen and mother liquor chilled to separate isopsoralen.
In another method (Hascher.Antoine U.S. 355323605 Jan 1971) seeds were purified with water, extracted with toluene or benzene, filtered, filtrate washed with caustic soda, and the oil removed by decanting, the solvent is washed with water filtered and distilled under reduced vacuum. Psoralen and its isomer isopsoralen remained in the flask. Kerosene added to separate psoralen, filtered and isopsoralen crystallized from mother liquor.
The present invention is distinct from all above investigations, where pre-treatments are pre-requisites. In the present invention, the seeds are directly acid hydrolysed reducing various steps as contained in the above methods. The enrichment of drug up to 70-80% with a yield of 1-1.5% (w/w) has been obtained in very few simple steps without involving any sophisticated lab equipments. The procedure is simple, quick and high yielding.
METHOD OF ENRICHMENT OF BABCHI DRUG
1 Soak the powdered seed (50 gram) in acidic (5% HCI v/v) methanol (MeOH) (150 ml) for 12 hr. with occasional shaking.
2 Separate the MeOH extract and concentrate (remove MeOH completely).
3 Extract the above MeOH soluble residue with chloroform (CHCI3) (50ml x 5 lots =250 ml) or till TLC test is positive for complete extraction of babchi drug.
4 Extract the above CHCI3 soluble residue with a mixture of CHCl3+petroleum ether (boiling range 60-80°C (30 :70 ratio) (200ml) followed by hot extraction with the same mixture(300 ml or till TLC test is positive) to obtain 50% enriched babchi drug.
5 Extract the above enriched babchi drug mixture with pure (600 ml or till TLC test Is positive) hot petroleum ether to obtain 70-80% enriched babchi drug.
6 Add some petroleum ether to the above enriched babchi drug and chill the concentrated material in refrigerator over night and discard the chilled petroleum ether soluble part.
7 The residue, called babchi drug (1-1.5% of the total seed weight), contains ordinarily about 70-80% w/w psoralen/isopsoralen which were further isolated by the known method (Khastagir et al. 1959). The UV and IR spectra of the isolated babchi drug along with standard psoralen are enclosed.
Further isolation of pure constituents such as psoralen, isopsoralen from the babchi drug is a known art. Further objects and advantages of this invention will be more apparent from the ensuing examples:
Example-1.
1. Soak the powdered seeds (50 gram) in acidic (5% HCI v/v) methanol (MeOH) (150 ml) for 12 hr. with occasional shaking.
2. Separate the MeOH extract and concentrate (remove MeOH completely).
3. Extract the above MeOH soluble residue with chloroform (CHCI3) (50ml x 5 lots =250 ml) or till TLC test is positive for complete extraction of babchi drug.
4. Extract the above CHCI3 soluble residue with a mixture of CHCl3+petroleum ether (boiling range 60-80°C (30:70 ratio) (200ml) followed by hot extraction with the same mixture (300 ml or till TLC test is positive) to obtain 50% enriched babchi drug.
5. Extract the above mixture soluble concentrated residue with pure (600 ml or till TLC test is positive) hot petroleum ether to obtain 70-80% enriched babchi drug.
6. Add some petroleum ether and chill the concentrated material obtained above in refrigerator over night and discard the chilled petroleum ether soluble part.
7. The babchi drug obtained above (1-1.5% of the total seed weight) contains
ordinarily about 70-80% w/w psoralen/isopsoralen which may be further isolated
by the known methods.
Example-2.
1. Soak the powdered seeds (100 gram) in acidic (2% HCI v/v) ethanol (350 ml) for 24 hr. with occasional shaking.
2. Separate the ethanol extract and concentrate (remove ethanol completely).
3. Extract the above ethanol soluble residue with benzene (50ml x 8 lots =400 ml) or till TLC test is positive for complete extraction of babchi drug.
4. Extract the benzene soluble residue obtained above with CHCl3+petroleum ether (boiling range 60-80°C (40:60 ratio) mixture (400ml) followed by hot extraction
(500 ml or till TLC test is positive) with 20:80 mixture of the same solvents and concentrate it.
5. Add some petroleum ether and chill the concentrated material obtained above in refrigerator over night and discard the chilled petroleum ether soluble part
6. The residue obtained above (1-2% of the total seed weight) contains 40-50% (w/w) psoralen/isopsoralen which may be further isolated by the known methods
Exannple-3.
1. Soak the powdered seeds (500 gram) in acidic (6% HCI v/v) methanol (MeOH) (1.2 L) for 12 hr. with occasional shaking.
2. Separate the MeOH extract and concentrate (remove MeOH completely).
3. Extract the above methanol soluble residue with ethyl acetate (100ml x 6 lots =600 ml) or till TLC test is positive.
4. Extract the ethyl acetate soluble residue obtained above with CHCL3+hexane (50+50 v/v) with 500ml mixture or till TLC test is positive and concentrate it.
5. Extract the CHCI3+hexane (50:50 v/v) mixture soluble residue with 1000 ml mixture of CHCl3+hexane (10:90 v/v) or till TLC test is positive and concentrate it.
6. Chill the concentrated material obtained above in refrigerator over night and discard the chilled petroleum ether soluble part
7. The residue obtained above (about 2% of the total seed weight) contains about 45-55% w/w psoralen/isopsoralen, which may be further isolated by the known methods.
SUMMARY OF THE INVENTION
At the outset of the description which follows, it is to be understood that the ensuing description refers to a particular form of invention. Such particular form in only an exemplary embodiment without intending to imply any restriction to the scope of the invention. Any suitable modification or improvement occurring to a man skilled in the art is also with in the scope of the invention.
The present invention is related to the isolation of psoralen and isopsoralen mixture from the seeds of Psoralea corylifolia by direct acid hydrolysis of the seeds for the minimum possible time and using optimum hydrolysing agent. Various steps as described in the past methods have been reduced, thus saving the time and cost of isolation.
In the present invention is comprised of two key steps viz., extraction cum acid treatment followed by enrichment of the babchi drug from the total extractives thus obtained. Thus, in the present invention, dried ground babchi seeds are acid hydrolysed in an organic solvent comprising of one of the following; methanol, ethanol, acetonitrile, benzene, chloroform, acetone or the like, which practically extract all the psoralen and isopsoralen from the seeds to the extent of 30% yield on dry weight basis. The duration and temperature of hydrolysis and the proportion of the hydrolysing agents were optimised.
The total extract obtained above (about 30% yield on dry weight basis) is then subjected to the next step of enrichment to obtain the babchi drug. Thus, the moisture free total extract was extracted with a solvent or a mixture of solvents having dielectric constant of 0-33 (at 25°C) range under a temperature range between 25° C to 80° C. The solvent can be chosen from among dichloromethane, chloroform, toluene, ethyl acetate, acetone, light ligroin, n-hexane, n-pentane, benzene etc. Thus, 50-80% enriched desired babchi drug can be obtained in 1-2.5% yield on the dry weight basis of the seeds.
REFERENCES
1. Krishnamurthi, A. 1969. Wealth of India, Vol. VIII, pp -295-298
2. Chander, H. and Ahmed, S.M. 1982. Extractives of medicinal plants as pulse protectants against Callosobruchus chinensis I. infestation. Journal of Food Science and Tech. 19 (2): 50-52.
3. Mohanty, K.K., Chakraborty, D.P. and Roy, S. 1988. Antifeedant activity of oil fraction of seed of some leguminous plants against Diacrisia oblique. Indian J. Agric. Sci. 58 (7): 579-580.
4. Murugan, K., Jeyabalan, D., Kumar, N.S., Nathan, S.S., Shivaprakasam, N. and Pandey, A. (Eds.) Soccol, C.R. and Josh, V.K. 1998. Growth promoting effects of plant products on silk worm- a biotechnological aproach. J. Sci. & Indus. Res. 57: 10-11.
5. Gowda,R. R., Gopalan, M., Jayaraj, S. and Natarajan, N. 1997. Field performance of plant extracts on mulberry silk worm (Bombyx mori L.) Entomon. 22 (3-4): 235-238.
6. Siddiqui, A.A. and Ansari, S.H. 1995. Phyto-chemica! and pharmacological investigation on Psoralea corylifolia. Hamdard Medicus. 38 (3): 109-115.
7. Lu, Feng., Liu.LiLi., Li, Ling., Wu, Yutian., Lu, F., Liu, L.L., Li, L. and Wu, Y.T. 1999. Determination of psoralen and isopsoralen in Psoralea corylifolia L. by supercritical fluid chromatography. Acta Pharmaceutica Sinica. 34(4): 301-303.
8. Spencer, G.F., Tjarks, L.W. and Powell, R.G. 1987. Analysis of linear and angular furanocoumarins by dual column High Performance Liquid Chromatography. J. Agric. Food. Chem. 35 (5): 803
9. Khastagir, H.N., Duttagupta, P.C. and Sengupta, P. 1959. Psoralen and Isopsoralen from Psoralea Corylifolia, Indian J. Applied Chemistry 22, 82-85





I/We Claim
1. An improved process for enrichment of babchi drug from the seeds of
babchi (Psoralea Corylifolia L.) which comprises:
a) soaking babchi seeds in acidic (5%HCL) Methanol (MeOH) for 12 hr. with occasional stirring in order to hydrolyze the glycosidic active principle such as herein described,
b) extracting the active principle and reducing to 20-30% (WAA/) and treating with chloroform (CHCL3) for discarding impurities,
c) and further treating with a mixture of CHCL3 and petroleum ether (30:70 (V/V) ratio followed by hot extraction with same mixture yielding 50% enriched babchi drug in the range of 1.5-2.5% (WAA/),
d) and further enrichment of babchi drug upto 70-80% in the range of 1-1.5% (W/W) is done by treating the above mixture with hot petroleum ether at 60-80%temperature,
2. The process as claimed in Claim 1 wherein active principle is
psoralen/isopsoralen.

Documents:

965-del-2003-abstract.pdf

965-del-2003-claims.pdf

965-del-2003-correspondence-others.pdf

965-del-2003-correspondence-po.pdf

965-del-2003-description (complete).pdf

965-del-2003-form-1.pdf

965-del-2003-form-19.pdf

965-del-2003-form-2.pdf

965-del-2003-form-3.pdf

965-del-2003-form-4.pdf


Patent Number 244028
Indian Patent Application Number 965/DEL/2003
PG Journal Number 47/2010
Publication Date 19-Nov-2010
Grant Date 15-Nov-2010
Date of Filing 05-Aug-2003
Name of Patentee INDIAN COUNCIL FOR AGRICULTURAL RESEARCH
Applicant Address KRISHI BHAWAN, DR. RAJENDRA PRASAD ROAD, NEW DELHI-110 001, INDIA
Inventors:
# Inventor's Name Inventor's Address
1 DR. MOHAMAD ASIM KIDWAI (PRINCIPAL SCIENTIST (RETIRED)), 5/2 INDIAN ZAKIR NAGAR, JAMIA NAGAR, NEW DELHI-110025, INDIA
2 DR. CHITRANGAD SINGH RAGHAV TECHNICAL OFFICER, NBPGR, PUSA INDIA CAMPUS, NEW DELHI-110012
3 MR. BRIJ MOHAN SINGH PRINCIPAL SCIENTIST (RETIRED), A/18F, INDIAN DDA FLATS, MIG, MAYAPURI,NEW DELHI-110064, INDIA
4 DR. PREM LAL GAUTAM FORMER DIRECTOR NBPGR, PUSA CAMPUS, NEW DELHI-110012, INDIA
PCT International Classification Number A61K 35/78
PCT International Application Number N/A
PCT International Filing date
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 NA