The present invention relates to a process for the isolation of heraclenin from Aegle marmelos Correct. The present invention also relates to a process for the isolation of a low molecular weight linear furanocoumarin from the mature/immature mesocarp of fresh/dry Aegle marmelos Correa fruits. The compound belongs to the linear furanocoumarin group of compounds. The compound (9-(3,3-dimethyloxiran-2-yl)methoxy)-7H-furo[3,2-g]chromen-7-one is a furano derivative of benzo-alpha-pyranocoumarin and is found in the A.marmelos in the free state.
BACKGROUND AND OBJECTS OF THE INVENTION:
Aegle marmelos Correa is a moderately sized, slender, aromatic tree, 6.0-7.5 m in height and 90-120 cm in girth, native to the Indian sub continent. The plant grows wild throughout the deciduous forests of India and in the sub-Himalayan forests, Central and South India. Aegle belongs to the family (Rutaceae). Fruits of A. marmelos Correa are 5.0-7.5 cm in diameter, globose, oblong pyriform, rind is gray or yellow; pulp sweet, thick yellow, orange to brown in color. Seeds are numerous.and arranged in the cells surrounded by slimy transparent mucilage. The unripe or half-ripe fruit is regarded as astringent, digestive and stomachic. The fruit is reputed to be a valuable Ayurvedic medicine for chronic diarrhea and dysentery and said to act as a tonic for heart and brain. Clinical trials of unripe fruits showed anti-viral activity against Ranikhet disease virus, hypoglycaemic activity and significant results against parasites. The pulp, diluted with water and added with requisite amount of sugar and tamarind, forms a delicious cooling drink. The Bael fruit is one of the most nutritious edible fruits, rich in carotenoids, riboflavin and pectin, used for preparation of a number of products like candy, squash, toffee, slab, pulp powder and nectar (The Wealth of India vol.5, 1990-94 and Compendium of Indian Medicinal Plants vol. 1-5, (1962-1998). Furanocoumarins (also called furocoumarins) are a class of widely occurring phenolic compounds, especially abundant in citrus fruits. Furanocoumarins have several interesting biological activities, such as analgesic, antiinflammatory, antibacterial, antiviral, anticoagulant in addition to their well-known photosensitizing effect.
Heraclenin (C16H14O5), a linear furanocoumarin, was isolated from the ethyl acetate extract of the roots of Prangos pabuhria has been reported for use in optoelectronics and as fluorophores in biological imaging applications(Javid et ah, Heraclenin: A Potential Optoelectronic Device Material from Prangos pabularia, Journal of Electronic Materials, 2013, 42(8),2489-2503). Heraclenin, a furocoumarin with an epoxide group in its side chain, has induced structural chromosome aberrations and sister-chromatid exchanges (SCEs) in human lymphocytes in vitro (Gudrun Abel and Oskar Schimmer; Chromosome-damaging effects of heraclenin in human lymphocytes in vitro; Mutation Research/Genetic Toxicology; 1986, 169(1-2); 51-54).

Heraclenin has inhibited T cell-receptor-mediated proliferation in human primary T cells in a concentration-dependent manner. Heraclenin also induced apoptosis (Appendino, Giovanni., et al., 2004. Coumarins from Opopanax chironium. New dihydrofurano coumarins and differential induction of apoptosis by imperatorin and heraclenin. Journal of natural products. 67(4): 532-6). Treatment of arg- or strd mutant cells of Chlamydomonas reinhardii with heraclenin, plus UV-A resulted in a decrease in survival and a UV-A dose-dependent increase in induced Arg+ or Strs revertants. The involvement of the epoxide moiety of heraclenin in mutagenicity was reported (Schimmer 0, Abel G; Mutagenicity of a furocoumarin epoxide, heraclenin, in Chlamydomonas reinhardii; Mutat Res. 1986 Jan-Feb;169(l-2):47-50). Heraclenin has also been identified in Heracleum candicansWall. ex DC. (Apiaceae) which is widely used in Indian system of medicines as an aphrodisiac, nerve tonic and also in the treatment of skin diseases with reports of being rich in furocoumarins which are extensively used in pharmaceutical industry for their photosensitizing activity on human skin (Rawat et al, Separation and Identification of Furocoumarin in Fruits of Heracleum candicans DC. by HPTLC; Journal of Chemistry, 2013, doi.org/10.1155/2013/915762). It has also been isolated from Halocnemum strobilaceum (Miftakhova et al., Coumarins from the aerial part of Halocnemum strobilaceum Fitoterapia. 2001 Mar;72(3):319-21).
Despite the plant Aegle marmelos Correa having widely used in India for medicinal purposes as well as for making cooling drink, not many patents exist related to Aegle marmelos Correa. A few patents, which exist, relate to herbal catalytic compositions using Aegle marmelos Correa for pollution control and energy saving of fuel used for automobile, and anti- diabetic compositions (IPR Bulletin, Vol. 7, No. 3-4, March - April 2001).The Bael fruit is relatively free from pests and diseases except for the fungi causing deterioration in storage. It is used in several indigenous systems of medicine in India, China, Burma and Sri Lanka. The natives use several parts of the plant. A kind of thick sherbet is prepared, which in India, is much esteemed as a laxative, tonic and digestive effects A decoction of the unripe fruit, with fennel and ginger, is prescribed in cases of hemorrhoids. The mucilage around the seed serve as an adhesive expands greatly in water, used as an additive, acts as a versatile suspending agent, a surfactant and an emulsifying agent. It is used as a varnish for pictures and adds brilliancy to water-colour paints. Seed oil has pesticidal property and can be used as ear drops. A yellow dye is prepared from the rind when the fruit is ripe. For medicinal use, the young fruits, while still tender, are commonly sliced horizontally and sun dried and sold in local markets. The thick rind of the unripe fruit is astringent and used in India for dysentery, diarrhea etc. Since the fruit is astringent, it is used as one of the ingredient in herbal hair oils.
Objectives of the invention
The main objective of the present invention is to provide a novel cost-effective, inexpensive, efficient and commercially feasible process for the isolation of heraclenin from fresh/dry

mesoearp of mature/immature Aegle marmelos Correa. Another objective of the present invention is to select a cost-effective solvent for selective extraction of heraclenin.
BRIEF STATEMENT OF THE INVENTION:
The present invention accordingly provides a novel and cost-effective process for the extraction and isolation of heraclenin from Aegle marmelos Correa. The invention more particularly provides a process, which gives a cheaper and higher yield of potent drug candidate, from the natural source. The present invention provides a process for the isolation of heraclenin from mature/immature fruits of Aegle marmelos Correa, the process comprising: (a) extracting fresh/dried powdered pulp of fruits with a solvent directly; (b) concentrating the extracted solvent extract to 10 to 20% of its original volume (c) drying the extract over anhydrous sodium sulphate (e) cooling down to 0 to 5 C to crystallize the compound.

In another embodiment of the invention the fresh/dried powdered pulp of fruits are extracted directly with a non polar hydrocarbon solvent, halogenated solvent or with monohydric alcohol at ambient temperature for 24 to 72 hrs with a pulp: solvent ratio in the range of 1 :3 to 1 :6. In another embodiment of the invention the fresh/dried powdered pulp of fruits are extracted with a non-polar hydrocarbon solvent, halogenated solvent or monohydric alcohol in a Soxhlett apparatus for 5 to 10 hrs with a pulp to solvent ratio of 1:4.
In another embodiment of the invention the solvent used for direct extraction is selected from the group consisting of petroleum ether, n-hexane, dichloromethane, chloroform, carbon tetrachloride, ethylene dichloride and methanol.
In another embodiment of the invention the heraclenin remaining in mother liquor after crystallization is subjected to column chromatography over silica gel (230- 400 mesh) in the ratio of 1 :5 to 1 :7 for complete isolation of pure heraclenin.
In another embodiment of the invention the yield of heraclenin, isolated from fresh mature fruits is in the range of 0.62% to 1.25% (dry weight basis) by direct process of 2 days cold percolation with petroleum ether (pulp -.solvent 1:3).