CLIAMS:We claim:
1. Pongamol in high yield of at least 2% isolated from Karanja oil.

2. Pongamol as claimed in claim 1 in high yield of 2.16 to 2.25% isolated from Karanja oil.

3. Pongamol as claimed in claim 1 or 2 isolated from Karanja oil containing 0.2 to 0.9% pongamol.

4. Karanja oil extract having high pongamol content of at least 5%.

5. Karanja oil extract as claimed in claim 4 having pongamol content of 5.12 to 14.77%.

6. Karanja oil extract as claimed in claim 4 or 5 isolated from Karanja oil having pongamol content of 0.2 to 0.9% .

7. A process for the preparation of Karanja oil extract having high pongamol content, comprising extracting Karanja oil with ethanol in the w/v ratio 1:1 at 50 to 60 °C, removing the ethanol from the ethanol extract, treating the ethanol extract with concentrated organic acid in the weight ratio 5 to 1000 and at 50 to 120°C.

8. The process as claimed in claim 7 for the isolation of Karanja oil extract having high pongamol content of at least 5%.

9. The process as claimed in claim 7 for the isolation of Karanja oil extract having high pongamol content of 5.12 to 14.77%.

10. The process as claimed in claim 7, wherein the Karanja oil contains 0.2 to 0.9% pongamol.

11. A process for the isolation of pongamol in high yield from Karanja oil, comprising extracting the oil with ethanol in the w/v ratio 1:1 at 50 to 60 °C, removing the ethanol from the ethanol extract, treating the ethanol extract with a concentrated organic acid in the weight ratio 5 to 1000 and at 50 to 120°C and recovering the pongamol from the acid treated extract.

12. The process as claimed in claim 11, for the isolation of pongamol in high yield of at least 2% by weight.

13. The process as claimed in claim 11 for the isolation of pongamol in high yield of 2.16 to 2.25% by weight.

14. The process as claimed in claim 11, wherein the Karanja oil contains 0.2 to 0.9% pongamol.

15. The process as claimed in claim 7 or 11, wherein the ethanol contains a moisture content upto 50% by weight.

16. The process as claimed in claim 7 or 11, wherein the ethanol contains a moisture content of 5% by weight.

17. The process as claimed in claim 7 or 11, wherein the ethanol is removed from the ethanol extract by vacuum distillation of the ethanol extract.

18. The process as claimed in claim 7 or 11, wherein the ethanol extract and organic acid are treated in the weight ratio 100 - 200%.

19. The process as claimed in claim 7 or 11, wherein the ethanol extract and organic acid are treated in the weight ratio 200%.

20. The process as claimed in claim 7 or 11, wherein the ethanol extract and organic acid are treated at 60 - 100°C.

21. The process as claimed in claim 7 or 11, wherein the ethanol extract and organic acid are treated at 75 – 85°C.

22. The process as claimed in claim 7 or 11, wherein the organic acid is selected from concentrated acetic acid, ascorbic acid, formic acid and lactic acid.

23. The process as claimed in claim 11, wherein pongamol is recovered from the acid treated extract by removing the acid from the acid treated extract, subjecting the acid treated extract to column chromatography, removing the eluent solvent from the acid treated extract, ageing the acid treated extract with hexane at 15 to 20°C to precipitate out the pongamol crystals, filtering out the pongamol crystals, washing the crystals with hexane and drying the crystals to remove the hexane.

24. The process as claimed in claim 23, wherein the acid is removed from the acid treated extract by vacuum distillation of the extract.

25. The process as claimed in claim 23, wherein the column chromatography is carried out with solid absorbent, silica and hexane in combination with or without ethyl acetate as the eluent solvent.

26. The process as claimed in claim 25, wherein ethyl acetate is used upto 5% by volume of the hexane.

27. The process as claimed in claim 23, wherein the pongamol crystals are dried at 80 - 90°C.
,TagSPECI:FORM 2
THE PATENTS ACT, 1970
(39 of 1970)
As amended by the Patents (Amendment) Act, 2005
&
The Patents Rules, 2003
As amended by the Patents (Amendment) Rules, 2014
COMPLETE SPECIFICATION
(See section 10 and rule 13)

TITLE OF THE INVENTION

Pongamol in high yield from Karanja oil and process for the isolation thereof

APPLICANTS

Kancor Ingredients Ltd, an Indian Company, Rajan House, Appasabheb Marathe Marg, Prabhadevi, Mumbai 400 025

PREAMBLE TO THE DESCRIPTION
The following specification describes the nature of the invention and the manner in which it is to be performed.

FIELD OF THE INVENTION
This invention relates to pongamol in high yield from Karanja oil and process for the isolation thereof.

This invention also relates to Karanja oil extract having high pongamol content and process for the preparation thereof.

BACKGROUND OF THE INVENTION
Karanja oil is extracted from the Millettia pinnata seeds (Pongamia glabra seeds) by expeller or cold pressing or solvent extraction and is yellowish-orange to brown in colour. Besides the crystalline component named pongamol, Karanja oil also contains another crystalline compound named karanjin. Pongamol, commercially known as pongamia extract, has the chemical name 1-(4-methoxy-5-benzofuranyl)-3-phenyl 1,3 propanedione and the structural formula shown in the following Fig 1:

Fig 1

Karanja oil is styptic, antimicrobial and anthelmintic and is reported to be effective for treating diseases like leprosy, piles, ulcers, chronic fever, liver pain, rheumatism arthritis, scabies and whooping cough. (Warrier PK, Nambiar VPK, Ramankutty C.Indian Medicinal Plants. Vol.IV. Orient Longman Ltd., Madras 1995: 339p; Prasad G, Reshmi MV. A manual of medicinal trees. Agrobios India 132 p.a Propagation Methods. Foundation for Revitalization for Local Health Tradition, India 2003. CSIR. The Wealth of India: raw materials. Vol. 1-10. Council of Scientific and Industrial Research (CSIR), New Delhi, India 1948-98).

Karanja oil is highly recommended for inclusion in moisturizers and hair care products where emolliency is desired. In skin care, Karanja oil is used to treat eczema, psoriasis, skin ulcers and dandruff and to promote wound healing. WO 2014016349 A1 describes usage of Karanja oil for a photo protective composition. Madhavarao Buddepu et al reported that Karanja oil extract can be used to formulate highly effective sunscreen preparations as it enhances UV absorbing properties of sunscreens and helps in broadening the UV protection ability of the sunscreens. (Madhavarao Buddepu. Determination of In -Vitro Sunscreen Activity of Millettia pinnata (L.) Essential Oil. Drug Invention Today 01/2011; Vol 3:197-199). EP 0431755, DE 10140538 , WO 2009125182, EP 0521651 and US 5152983 reported pongamol as an ingredient for topical application to human skin and hair to provide enhanced protection from ultra violet light.

Recent research has revealed that pongamol is capable of inducing increase in glucose uptake in L6 myotubes, driven by a PI-3-K/AKT dependent mechanism. (Molecular and Cellular Endocrinology, 2011, 339(1-2), 90-104). It also reported to have antihyperglycemic activity (J. Ethnopharmacol. 2008, 118, 435-439). It has been used as an anticonvulsant CNS depressant (Ind. J. Pharma. Sci. 1994, 56, 163-167) and as potent intestinal alpha-glucosidase inhibitor (Bioorg Med Chem. 2009, 17(14), 5170-5175).

Different methods are reported for isolation of pongamol from Karanja oil. N.V.Bringi teaches a process for isolation of pongamol by molecular distillation of Karanja oil using high vacuum (J. Oil Tech. Ass. 1976, 8(3), 81). In another process Karanja oil is stored under refrigeration to remove precipitated karanjin and then it is extracted with alcohol to isolate pongamol. Alcohol extract of Karanja oil is stored and the precipitated material is taken in ether and crystallized to get pongamol (Ind J Pharm 1941, 3, 3 and Proc. of Ind. Acad. Sci 1942, 15A, 437).

R N Khana and T R Seshadri isolated pongamol from the alcohol extract using column chromatography with alumina as stationary phase and light petroleum as eluant. Pongamol was isolated from the light petroleum by counter current distribution between aqueous acetic acid and light petroleum. (Tetrahedron 1963, 19, 223). In another improvement the chromatographic step in the isolation of pongamol from the alcohol extract was eliminated and instead the isolation of pongamol from the alcohol extract was carried out by repeated partitioning of karanjin removed alcohol extract of Karanja oil between light petroleum and aqueous acetic acid. (R Aneja, R N Khana and T R Seshadri (J. Chem. Soc., 1963, p. 163)).

In another process for extraction of pongamol from Karanja oil, dry hydrogen chloride is passed through the alcohol extract of Karanja oil, and the precipitate separated is fractionally crystallized (J. Ind. Chem. Soc, Industrial and News Edition, 1954, 17, 39). GB2237805 describes extraction of pongamol from Karanja oil with organic acid followed by crystallization from solvents. Sangli variety (a region in Maharashtra State, India) Karanja oil (2000 g) was extracted with aqueous acetic acid (800 ml diluted with 200 ml water) at ambient temperature ( 25 - 30°C). Extract was aged at ambient temperature (25 - 30°C) for 3 days to obtain pongamol in 0.9% yield. The oil after isolation of pongamol is reported to be used for making soap. GB2237805 teaches extraction of Karanja oil with aqueous acid (v/v ratio 80 : 20) in the w/v ratio 20:1 at ambient temperature followed by ageing at ambient temperature. Pongamol yield reported is 0.9%,

Parmar et al reported 0.85% pongamol content in Karanja oil while V.K. Gore et al reported 0.9% pongamol content in Karanja oil. (Parmar BS, Shrawat KL and Mukerjee SK, Pongamia glabra: constituents and uses, J Sci Ind Res, 1976, 35, 608-611; V.K. Gore, P. Satyamoorthy. Determination of Pongamol and Karanjin in Karanja Oil By Reverse Phase HPLC. Analytical Letters.Vol. 33, Iss. 2, 2000). It is reported that different Karanja oil samples by HPLC analysis gave pongamol in the range of 0.85 to 0.9%. (Analytical Letters, 2000, 33(2), 337-346). However, chemical analysis of commercially available Sangli variety Karanja oil in our own laboratory showed a varying pongamol content of ~0.2-0.9%.

Extensive research and development activities are being carried out in the field of natural products, especially for use in pharmaceutical and cosmetic and dermatogical industries because of their being comparatively safe and cheap and naturally available. Therefore, there is need for natural products like Karanja oil having increased pongamol content, pongamol in high yield from Karanja oil and Karanja oil extract having high pongamol content and processes for the preparation thereof for various uses, especially pharmaceutical and cosmetic and dermatological applications.

DESCRIPTION OF THE INVENTION
According to the invention there is provided pongamol in high yield of at least 2%, preferably 2.16 to 2.25% from Karanja oil, which may contain 0.2 to 0.9% pongamol.

According to the invention there is also provided Karanja oil extract having high pongamol content of at least 5%, preferably 5.12 to 14.77% from Karanja oil, which may contain 0.2 to 0.9% pongamol.

According to the invention there is also provided a process for the preparation of Karanja oil extract having high pongamol content, comprising extracting Karanja oil with ethanol in the w/v ratio 1:1 at 50 to 60 °C, removing the ethanol from the ethanol extract, treating the ethanol extract with concentrated organic acid in the weight ratio 5 to 1000 and at 50 to 120°C.

According to the invention there is also provided a process for the isolation of pongamol in high yield from Karanja oil, comprising extracting the oil with ethanol in the w/v ratio 1 : 1 at 50 to 60 °C, removing the ethanol from the ethanol extract, treating the ethanol extract with a concentrated organic acid in the weight ratio 5 to 1000 and at 50 to 120°C and recovering the pongamol from the acid treated extract.

The ethanol used for extraction may contain upto 50% by weight moisture, preferably 5% by weight moisture. Preferably the ethanol is removed from the ethanol extract by vacuum distillation of the ethanol extract. The ethanol also can be removed from the ethanol extract by other known procedures. The ethanol extract and organic acid are treated preferably in the weight ratio 100 - 200%, still preferably 200% and preferably at 60 - 100°C and still preferably at 75 – 85°C. Preferably, the organic acid is selected from concentrated acetic acid, ascorbic acid, formic acid and lactic acid. However, other concentrated organic acids also can be used for the acid treatment.

In one variation of the process of the invention, pongamol is recovered from the acid treated extract by removing the acid from the acid treated extract, subjecting the acid treated extract to column chromatography, removing the eluent solvent from the acid treated extract, ageing the acid treated extract with hexane at 15 to 20°C to precipitate out the pongamol crystals, filtering out the pongamol crystals, washing the crystals with hexane and drying the crystals to remove the hexane. Preferably, the acid is removed from the acid treated extract by vacuum distillation of the extract. However, other known procedures also can be used for the acid removal. Preferably, the column chromatography is carried out with solid absorbent, silica and hexane in combination with or without ethyl acetate as the eluent solvent. Ethyl acetate is preferably used upto 5% by volume of the hexane. Preferably the pongamol crystals are dried at 80 - 90°C.

It is understood that Karanja oil can be extracted with any other oil immiscible organic solvent such as methanol or propanol. Ethanol is only a preference. The scope of the invention should be construed and understood accordingly.

Pongamol in high yield and high purity and Karanja oil extract having high pongamol content as obtained according to the invention can be used inter alia in pharmaceutical industry for therapeutic purposes. In cosmetic and dermatological industry, it can be used as a UVA absorber. However, the pongamol in high yield and high purity and Karanja oil extract having high pongamol content also can be used for other purposes. Activity increases with amount of the pongamol content so to reduce the amount of pongamol required and to give cost benefits. The process of the invention is simple to carry out and is very cost effective.

The following experimental examples are illustrative of the invention but not limitative of the scope thereof:

Example 1
Sangli variety Karanja oil (200 g) having 0.6% pongamol content was extracted with ethanol (200 ml) at 50-60°C by stirring for about 1 hr. Oil with ethanol layer was cooled to 25 – 35°C to separate the ethanol layer from the oil layer. Oil layer was extracted with ethanol four more times in the same manner as mentioned above. Total volume of ethanol used was 1000 ml, 95% ethanol with 5% moisture content. Ethanol layers were washed with water and the washings were combined and vacuum distilled to remove ethanol and get ethanol extract (33 g).

Ethanol extract as obtained above was treated with different proportions of glacial acetic acid at 75 - 85°C for about 1 hr and the results were as shown in the following Table 1:

Table 1
ethanol extract % glacial acetic acid % pongamol %
0.51 g 5% ( 0.026g)
5.12
0.50 g 14% ( 0.07g)
6.53
0.51 g 100% ( 0.51g) 14.47

0.51 g 150% (0.77 g) 14.58

0.50 g 200% ( 1 g) 14.75

0.50 g 1000% (5 g) 14.77

Table 1 shows that the pongamol content of the oil increased substantially with acid treatment of the oil in the weight percentages 50 to 1000 of the acid thereby indicating conversion of the various other unknown compounds in the oil into pongamol under the selective process parameters and conditions of the invention. Table 1 also shows that conversion of the various other unknown compounds in the oil into pongamol reaches saturation with acid percentage of 1000.

Example 2
Ethanol extract of Karanja oil (7g) obtained by ethanol extraction as stated in Example 1 was treated with 200% acetic acid ( 14 g) as stated in Example 1. Acid treated extract was vacuum distilled to remove acetic acid and to obtain acid treated extract ( 7.16 g) with 14.64% pongamol content. Extract was loaded over silica gel (140 g; mesh size 100-200,1:20 ratio) column with l/d 40 and eluted with hexane (200ml) to remove non-polar fractions and then with hexane (900 ml) containing ethylacetate 5% to get pongamol in fractions. Pongamol fractions were concentrated separately under vacuum distillation to yield oily pongamol (2.57 g) which on ageing with 1:1 hexane at 15-20°C for 7 - 10 hrs, pongamol crystals were precipitated out. Pongamol crystals were filtered out and washed with hexane (1 ml) twice followed by oven drying at 80-90°C to yield 0.9753g (2.25%) pongamol with 98% purity.

Example 3
Sangli variety Karanja oil (100 g) having 0.6 % pongamol content was extracted with ethanol at 50 - 60°C by stirring for about 1hr. Oil and ethanol was cooled to 25 - 35°C to separate the ethanol layer from the oil layer. Oil layer was extracted with ethanol four more times in the same manner as mentioned above. Total volume of 95% ethanol used was 500 ml, 95% ethanol with 5% moisture content. Ethanol layers were washed with water and the washings combined and vacuum distilled to remove ethanol.

Ethanol extract 18.174 g with 4.06 % pongamol content as obtained was treated with 200 % glacial acetic acid (36.348 g) at 75 - 85°C for about 1 hr and acid was removed from the extract by vacuum distillation to give extract (18.24g) with 12.3 % pongamol.

Extract was loaded over silica gel (360 g, mesh size 100-200, 1:20 ratio), column with l/d 40 and eluted with hexane (500 ml) to remove non-polar fractions and then with hexane (2500ml) containing ethyl acetate 5% to get pongamol in fractions. Pongamol fractions were concentrated separately under vacuum distillation to yield oily pongamol (6.7 g) which on ageing with 1:1 hexane at 15-20°C for 7 - 10 hrs, precipitated out as crystals pongamol. Pongamol crystals were filtered and washed with hexane (5 ml) twice followed by oven drying at 80-90°C to yield 2.16 g (2.16%) of pongamol with 98% purity.

Example 4
Sangli variety of Karanja oil (100 g) having pongamol content of 0.6% was extracted with 80% aqueous acetic acid in the weight ratio 1:0.5 at 25 to 30°C according to the teaching of GB 2237805. The combined miscella was aged for 3 days at 25 to 30°C. Pongamol crystals precipitated were filtered out and washed with hexane ( 3 x 3.5 ml) and dried in vacuum (5 mm) at ambient temperature to obtain 0.60% of pongamol with 98% purity.

It is quite clear from Examples 1 to 3 that the pongamol content increased susbtantially when treated according to the process of the invention as compared to the conventional process of Example 4.

We claim:
1. Pongamol in high yield of at least 2% isolated from Karanja oil.

2. Pongamol as claimed in claim 1 in high yield of 2.16 to 2.25% isolated from Karanja oil.

3. Pongamol as claimed in claim 1 or 2 isolated from Karanja oil containing 0.2 to 0.9% pongamol.

4. Karanja oil extract having high pongamol content of at least 5%.

5. Karanja oil extract as claimed in claim 4 having pongamol content of 5.12 to 14.77%.

6. Karanja oil extract as claimed in claim 4 or 5 isolated from Karanja oil having pongamol content of 0.2 to 0.9% .

7. A process for the preparation of Karanja oil extract having high pongamol content, comprising extracting Karanja oil with ethanol in the w/v ratio 1:1 at 50 to 60 °C, removing the ethanol from the ethanol extract, treating the ethanol extract with concentrated organic acid in the weight ratio 5 to 1000 and at 50 to 120°C.

8. The process as claimed in claim 7 for the isolation of Karanja oil extract having high pongamol content of at least 5%.

9. The process as claimed in claim 7 for the isolation of Karanja oil extract having high pongamol content of 5.12 to 14.77%.

10. The process as claimed in claim 7, wherein the Karanja oil contains 0.2 to 0.9% pongamol.

11. A process for the isolation of pongamol in high yield from Karanja oil, comprising extracting the oil with ethanol in the w/v ratio 1:1 at 50 to 60 °C, removing the ethanol from the ethanol extract, treating the ethanol extract with a concentrated organic acid in the weight ratio 5 to 1000 and at 50 to 120°C and recovering the pongamol from the acid treated extract.

12. The process as claimed in claim 11, for the isolation of pongamol in high yield of at least 2% by weight.

13. The process as claimed in claim 11 for the isolation of pongamol in high yield of 2.16 to 2.25% by weight.

14. The process as claimed in claim 11, wherein the Karanja oil contains 0.2 to 0.9% pongamol.

15. The process as claimed in claim 7 or 11, wherein the ethanol contains a moisture content upto 50% by weight.

16. The process as claimed in claim 7 or 11, wherein the ethanol contains a moisture content of 5% by weight.

17. The process as claimed in claim 7 or 11, wherein the ethanol is removed from the ethanol extract by vacuum distillation of the ethanol extract.

18. The process as claimed in claim 7 or 11, wherein the ethanol extract and organic acid are treated in the weight ratio 100 - 200%.

19. The process as claimed in claim 7 or 11, wherein the ethanol extract and organic acid are treated in the weight ratio 200%.

20. The process as claimed in claim 7 or 11, wherein the ethanol extract and organic acid are treated at 60 - 100°C.

21. The process as claimed in claim 7 or 11, wherein the ethanol extract and organic acid are treated at 75 – 85°C.

22. The process as claimed in claim 7 or 11, wherein the organic acid is selected from concentrated acetic acid, ascorbic acid, formic acid and lactic acid.

23. The process as claimed in claim 11, wherein pongamol is recovered from the acid treated extract by removing the acid from the acid treated extract, subjecting the acid treated extract to column chromatography, removing the eluent solvent from the acid treated extract, ageing the acid treated extract with hexane at 15 to 20°C to precipitate out the pongamol crystals, filtering out the pongamol crystals, washing the crystals with hexane and drying the crystals to remove the hexane.

24. The process as claimed in claim 23, wherein the acid is removed from the acid treated extract by vacuum distillation of the extract.

25. The process as claimed in claim 23, wherein the column chromatography is carried out with solid absorbent, silica and hexane in combination with or without ethyl acetate as the eluent solvent.

26. The process as claimed in claim 25, wherein ethyl acetate is used upto 5% by volume of the hexane.

27. The process as claimed in claim 23, wherein the pongamol crystals are dried at 80 - 90°C.
Dated this 29th day of September 2014

(Jose M A)
of Khaitan & Co
Agent for the Applicants
Reg No IN/PA-44