Title of Invention

"AN ENZYMATIC PROCESS FOR THE PREPARATION OF CURCUMIN GLYCOSIDE"

Abstract

The present invention relates to an enzymatic method for the preparation of curcumin glycoside. The present invention particularly relates to the preparation of curcumin glycoside by using amyloglucosidase in non-polar solvents In the present invention involves a method whereby large scale conversions into α and ß curcumin glucosides are possible in a single experimental set up with higher yields. Readily available commercial enzymes like amyloglucosidase from Rhizopus mold and other glycosidases have been used.

Full Text

The present invention relates to an enzymatic method for the preparation of curcumin glycoside. The present invention particularly relates to the preparation of curcumin glycoside by using amyloglucosidase in non-polar solvents. Curcumin (diferuloylmethane) is a yellow pigment of turmeric (dried rhizome of Curcuma longa, Family - Zingiberaceae). It has been used primarily as a food colorant and also as a pharmacologically active principle of turmeric in folk medicine. Reference may be made to D.C.F. Gomes, L.V. Alegrio, L.L. Leon and M.E.F. de Lima. Arzneim.-Forsch. 52 (2002), pp. 695-698, wherein the potent pharmacological activities of curcumin are reported to be anti-oxidative, anti-inflammatory and anti-leishmanial. Reference may be made to A.A. Nanji, K. Jokelainen, G.L. Tipoe. A. Rahemtulla, P. Thomas and A.J. Dannenberg. Am. J. Physiol. Gastrointest. Liver Physiol. 284 (2003). pp. G321-G327, wherein the ability to reduce alcohol-induced liver disease is reported. Reference may be made to H.H. Tonnesen and J. Karlsen. Z Lebensm.-Unters.-Forsch. 180, 1985, pp. 132-134 , wherein the solubilities of curcumin at different pH are given. At acidic and neutral pH, curcumin is insoluble in water. Although it is soluble in alkali, it undergoes rapid hydrolytic degradation at pH values above neutral. Curcumin is also highly lipophilic. This low water solubility limits further pharmacological exploitation and practical application of curcumin. Selective synthesis of these compounds using chemical reagents do not lead to products arising out of reactions at specific positions of the sugar molecules employed. Few references are available for the synthesis of phenolic glycosides. Reference may be made to M. Hergenhahn (2002), German patent application, DE 2337, wherein curcumin-4,4'-0-glucoside and curcumin-4-O-monoglucoside were chemically synthesized by condensing curcumin with α-D-acetyl-bromoglucose followed by deacetylation. But the yields were only 3% and 8% respectively. Reference may be made to Yasuhisa Kaminaga, Akito Nagatsu, Takumi Akiyama, Naoki Sugimoto, Takeshi Yamazaki, Tamio Maitani and Hajime Mizukami, FEBS Letters, 555(2), 311-316, 2003, wherein Catharanthus roseus cell suspension cultures converted exogenously supplied curcumin to a series of corresponding glucosides. The glucoside yield was 2.5 umol/g fresh weight of the cells at an optimal culture condition. Reference may be made to Takashi Kometani, Hidenori Tanimoto. Takahisa Nishimura, Isao Kanbara and Shigetaka Okada, Bioscience Biotechnology Biochemistry. 57(12), 2192-2193,1993, wherein capsaicin was converted into capsaicin-P-D-glucoside by cell suspension cultures of Coffea arabica. An yield of 15.8mg (10.3%) capsaicin glucoside was obtained from Coffea arabica cells from 153 mg of capsaicin for a 48h culture. Reference may be made to Shinji Inomata, Mineyuki Yokoyama. Susumu Seto and Mitsuo Yanagi, Applied Microbiology and Biotechnology, 36. 315-319.1991. wherein plant cell suspensions of Catharanthus roseus effectively converted exogenously supplied hydroquinone into arbutin (hydroquinone-ß-D-glucopyronoside). The yield obtained was 9.2 g/1 ( 3.5 % with respect to weight of the cell) in a period of 4 days. Reference may be made to Takashi Kometani, Hidenori Tanimoto, Takahisa Nishimura and Shigetaka Okada, Bioscience Biotechnology Biochemistry, 57(8), 1290-1293,1993, wherein vanillin was converted into the corresponding glucoside in cell suspension cultures Coffea arabica. The conversion yield of 7 mg (4.6 %) was obtained by addition 152 mg of vanillin from a 24 h culture. Reference may be made to Hajime Mizukami, Toshimitsu Terao, Hiroshi Miura and Hiromu Ohashi, Phytochemistry, 22(3) 679-680,1983, wherein isosalicin was identified as a product of glucosylation of salicyl alcohol in Lithospermum erythrorhizon suspension cultures and in Datura innoxia. Gardenia jasminoides could produce salicin from salicyl alcohol and 30% of salicyl alcohol was converted to their glucosides in 4 days by addition of 150µmol of salicyl alcohol to the media. Reference may be made to Mamoru Tabata, Fumiaki Ikeda, Noboru Hiraoka and Masao Konoshima, Phytochemistry, 15,1225-1229,1976, wherein three isomers of dihydroxy benzene (hydroquinone, resorcinol and catechol) were readily converted into their corresponding mono-ß-glucosides by Datura innoxia suspension cultures. The feeding experiments showed that the cultured cells possess a remarkably high capacity for glucosylation of hydroquinone to arbutin. A conversion of 70% hydroquinone to arbutin was accomplished in 6 days period. Major drawbacks of the above mentioned enzymatic methods are : 1. The methods which involve plant cell culture systems have employed lower amounts of substrates. 2. The conversion yields are less and in most cases only mono ß - glucosides are formed. 3. Larger periods of incubation were required. Novelty of the present invention is that it involves a method whereby large scale conversions into a and ß curcumin glucosides are possible in a single experimental set up with higher yields. The main object of the present invention is to provide an enzymatic method for the preparation of curcumin glycosides which obviates the above mentioned drawbacks. Yet another object of the present invention is to employ readily available commercial enzymes like amyloglucosidase from Rhizopus mold and other glycosidases. Still another object of the present invention is to use low boiling solvents in the boiling point range 50°C to 75°C. Yet another object of the present invention is to carry out the reaction at a large scale in a single experimental set up. Still another object of the present invention is to obtain glycosides in greater yields. Yet another object of the present invention is that unprotected mono and disaccharides can be employed. Still another object of the present invention is to obtain glycosides wherein both α and ß anomers can be glycosylated. Yet another object of the present invention is that the glycoside yields obtained (2.9 - 47.5%) by this procedure is much higher. Accordingly, the present invention provides an enzymatic process for the preparation of curcumin glycoside which comprises: (a) digesting a mixture of carbohydrates and curcumin with the enzyme glucosidase, under reflux conditions in a non-polar solvents, for a period of 2-5 days at pH ranging from 4.0 - 8.0 and at a temperature in the range of 50°C - 75°C in order to obtain reaction product having curcumin glycoside ; (b) distilling the product obtained in step (a) in order to remove the solvent; (c) holding the reaction mixture in a water bath at a temperature in the range of 80-100 deg C for a period of 5-10 minutes in order to inactivate the enzyme glucosidase; (d) adding water to the product obtained in step (c ) followed by evaporation of the water to obtain a dry mass having curcumin glycoside and un-reacted glucose ;. (e) removing un-reacted glucose by repeatedly extracting curcumin glycoside with dichloromethane by a known method to obtain the desired product. In another embodiment of the present invention, the alcohols employed may be curcumin and its homologues. In yet another embodiment of the present invention, the carbohydrate employed may be a monosaccharide such as D-glucose, D-fructose, D-galactose, D-mannose, D-arabinose, ribose and deoxyribose and disaccharides like lactose, maltose and trehalose. In still another embodiment of the present invention, the enzyme used may be amyloglucosidase from Rhizopus mold , almond ß-glucosidase and other glycosidases. In yet another embodiment of the present invention, the glycosylation may be conducted under reflux conditions with stirring in the temperature range 50°C - 75 °C in an experimental set up involving a two necked round or flat bottomed flask fitted with a cold water circulated condenser. In still another embodiment of the present invention, the solvent may be the one with boiling points in the range 50°C - 75°C like n-pentane, di-isopropyl ether, ethyl methyl ketone, chloroform, hexane and petroleum ether (40°C - 60°C). In yet another embodiment of the present invention, the period of reaction employed may be 2-5 days. In still another embodiment of the present invention, the buffer employed may be in the pH range 4.0 - 8.0 and the volume of the buffer added may be in the range 0.1 ml - 1 ml and the concentration of the buffer may be in the range 5mM - 100mM. In yet another embodiment of the present invention both a and (3 - glycosides of curcumin are formed. In still another embodiment of the present invention the curcumin glucoside produced is soluble in water and its solubility in water is 0.066%. In yet another embodiment of the present invention the total color of the aqueous solution of curcumin glucoside produced was found to be 2.7. In still another embodiment of the present invention, a 2.68 mM solution of the curcumin glucoside exhibited an antioxidant activity of 83.9% by the DPPH method which compared to an antioxidant activity of 78.6% for free curcumin at 5.3 mM. In yet another embodiment of the present invention, the curcumin glucoside produced exhibited antibacterial activity against Micrococcus luteus and Bacillus subtilis. Amyloglucosidase from Rhizopus mold was purchased from M/S Sigma Chemical. Co. Mo, USA. Curcumin (0.0001 - 0.001 mol) and a mono or disaccharide (0.0005 - 0.002 mol) were taken in a 100 ml two necked round or flat bottomed flask containing 30 - 70 ml of a solvent with a boiling point in the range 50°C -75°C in presence of 10 - 80% (by weight of mono or disaccharides) amyloglucosidase and 0.1-1.0 ml of lOmM buffer pH 4.0 - 9.0 and refluxed with stirring on a magnetic stirrer with a hot plate for a period of 2-5 days. The solvent was distilled off and the reaction mixture held in a water bath for 5 - 10 min to denature the enzyme. About 10- 15 ml of water was added and the whole reaction mixture was evaporated to dryness. The dry mass was repeatedly extracted with dichloromethane to remove unreacted curcumin. The dried residue consisting of curcumin glycoside and unreacted glucose was subjected to HPLC analysis to determine the extent of conversion. The products were monitored by HPLC on a aminopropyl column using acetonitrile : water (80:20 v/v) and Refractive Index detector. The retention times are : glucose -5.24 min, curcumin-3.0 min and curcumin glucoside - 7.0min . Curcumin glucoside was characterized by recording one-dimensional *H and 13C NMR and two-dimensional Heteronuclear Multiple Quantum Coherence Transfer spectrum on a Bruker DRX-400 and 500 NMR instruments operating at 40° C. The samples were dissolved in DMSO-d6 and the signals were referenced to DSS. The NMR analysis showed the formation of both α and ß curcumin glucosides. The position of glucosylation was inferred to be C8 and C8' . Glucosylation occured at the phenolic OH position. Curcumin: 1H NMR (ppm) : (DMSO-d6) 3.84( 6H, s, 2-OCH3), 6.05 (1H, s, H-l), 6.76 (2 H, d, J=15.8Hz, H-3,3'), 7.57(2 H, d, J=15.8, H-4,4'), 7.32 (2 H, s, H-6,6'), 6.82 (2 H, d, J=8.2Hz, H-9,9'), 7.15 (2 H, dd, H-10, 10'). 13C NMR (ppm) : 56.7 (OCH3), 101.8 (CI), 184.3 (C2,C2'), 122.2 (C3.C3'). 141.7 (C4,C4'), 127.1 (C5,C5'), 112.5 (C6,C6'), 149.1 (C7,C7'), 150.4 (C8,C8'), 116.8 (C9,C9'), 124.1 (C10, C10') Curcumin glucoside: 1H NMR : 3.85( 6H, s, 2-OCH3), 6.06 (1H, s, H-l), 6.71 (2 H, d, J=15.8Hz, H-3,3'), 7.51(2 H, d, J=15.8, H-4,4'), 7.25 (2 H, s, H-6,6'), 6.81 (2 H, d, J=8.2Hz, H-9,9'), 7.11 (2 H, dd, H-10,10'), 4.66 (H-la), 3.15 (H-2a), 2.90 (H-2P), 3.73 (H-3a), 3.75 (H-4a), 3.53 (H-6a). 13C NMR : 56.1 (OCH3), 101.2 (CI), 183.5 (C2,C2'), 121.5 (C3,C3'), 141.0 (C4,C4'), 126.8 (C5,C5'), 111.6 (C6,C6'), 148.4 (C7,C7'), 149.6 (C8,C8'), 116.1 (C9,C9'), 123.4 (C10, C10'), 99.0 (Cla), 72.2 (C2a), 75.0 (C2p), 73.6(C3a), 70.6 (C4a), 61.3 (C6a) and two cross at *H 2.06 and 13C 31.0, lH 5.63 and 13C 54.9. The solubility of curcumin glucoside in water at 25°C was found to be 0.066%. The total color of the aqueous solution of curcumin glucoside produced was found to be 2.7. A 2.68 mM solution of the curcumin glucoside exhibited an antioxidant activity of 83.9% by the DPPH method which compared to an antioxidant activity of 78.6% for free curcumin at 5.3 mM. The curcumin glucoside produced exhibited antibacterial activity against Micrococcus luteus and Bacillus subtilis. An inhibition zone of 15 mm for the curcumin glucoside and 16 mm for curcumin was observed when a 75 ul of 5.3 mM of the glucoside and curcumin were applied to wells of size 10 mm on agar plates. The following examples are given by way of illustration of the present invention and therefore not be construed to limit the scope of the present invention. EXAMPLE 1 A mixture of 0.001 mol of curcumin and 0.001 mol of D-glucose was taken in a 25ml conical flask along with 5 ml of carbon tetrachloride solvent in presence of 50% (w/w D-glucose) amyloglucosidase and 0.4 ml of lOmM acetate buffer pH 5.0 and incubated in temperature control shaking incubator for a period of 72h at 150 rpm. The reaction mixture held in a water bath for 5 - 10 min to denature the enzyme. About 10-15 ml of water was added and the whole reaction mixture was evaporated to dryness. The dry mass was repeatedly extracted with dichloromethane to remove unreacted curcumin. The dried residue consisting of curcumin glycoside and unreacted glucose was subjected to HPLC analysis to determine the extent of conversion. Conversion yield was found to be 10.1 %(101.1µ mol). EXAMPLE 2 A mixture of 0.0005 mol of curcumin and 0.001 mol of D-glucose was taken in a 100 ml two necked round or flat bottomed flask along with 40-60 ml of di-isopropyl ether in presence of 50% (w/w D-glucose) amyloglucosidase and 0.4 ml of lOmM acetate buffer pH 4.0 and refluxed with stirring on a magnetic stirrer with a hot plate for a period of 72h. The solvent was distilled off and the reaction mixture held in a water bath for 5 - 10 min to denature the enzyme. About 10- 15 ml of water was added and the whole reaction mixture was evaporated to dryness. The dry mass was repeatedly extracted with dichloromethane to remove unreacted curcumin. The dried residue consisting of curcumin glycoside and unreacted glucose was subjected to HPLC analysis to determine the extent of conversion. Conversion yield was found to be 28.7 % ( 287.3 u mol). EXAMPLE 3 A mixture of 0.0005 mol of curcumin and 0.001 mol of D-glucose was taken in a 100 ml two necked round or flat bottomed flask along with 40-60 ml of di-isopropyl ether in presence of 50% (w/w D-glucose) amyloglucosidase and 0.4 ml of lOmM acetate buffer pH 5.0 and refluxed with stirring on a magnetic stirrer with a hot plate for a period of 72h. The solvent was distilled off and the reaction mixture held in a water bath for 5 - 10 min to denature the enzyme. About 10- 15 ml of water was added and the whole reaction mixture was evaporated to dryness. The dry mass was repeatedly extracted with dichloromethane to remove unreacted curcumin. The dried residue consisting of curcumin glycoside and unreacted glucose was subjected to HPLC analysis to determine the extent of conversion. Conversion yield was found to be 17.0 % (170.7 u, mol). EXAMPLE 4 A mixture of 0.0005 mol of curcumin and 0.001 mol of D-glucose was taken in a 100 ml two necked round or flat bottomed flask along with 40-60 ml of di-isopropyl ether in presence of 50% (w/w D-glucose) amyloglucosidase and 0.4 ml of lOmM disodium hydrogen phosphate buffer pH 7.0 and refluxed with stirring on a magnetic stirrer with a hot plate for a period of 72h. The solvent was distilled off and the reaction mixture held in a water bath for 5 - 10 min to denature the enzyme. About 10- 15 ml of water was added and the whole reaction mixture was evaporated to dryness. The dry mass was repeatedly extracted with dichloromethane to remove unreacted curcumin. The dried residue consisting of curcumin glycoside and unreacted glucose was subjected to HPLC analysis to determine the extent of conversion. Conversion yield was found to be 22.6 % (226.2µ mol). EXAMPLE 5 A mixture of 0.0005 mol of curcumin and 0.001 mol of D-glucose was taken in a 100 ml two necked round or flat bottomed flask along with 40-60 ml of di-isopropyl ether in presence of 50% (w/w D-glucose) amyloglucosidase and 0.6 ml of lOmM acetate buffer pH 4.0 and refluxed with stirring on a magnetic stirrer with a hot plate for a period of 72h. The solvent was distilled off and the reaction mixture held in a water bath for 5 - 10 min to denature the enzyme. About 10-15 ml of water was added and the whole reaction mixture was evaporated to dryness. The dry mass was repeatedly extracted with dichloromethane to remove unreacted curcumin. The dried residue consisting of curcumin glycoside and unreacted glucose was subjected to HPLC analysis to determine the extent of conversion. Conversion yield was found to be 44.4 % ( 443.7 u, mol). EXAMPLE 6 A mixture of 0.0005 mol of curcumin and 0.001 mol of D-glucose was taken in a 100 ml two necked round or flat bottomed flask along with 40-60 ml of di-isopropyl ether in presence of 50% (w/w D-glucose) amyloglucosidase and 0.8 ml of l0mM acetate buffer pH 4.0 and refluxed with stirring on a magnetic stirrer with a hot plate for a period of 72h. The solvent was distilled off and the reaction mixture held in a water bath for 5 - 10 min to denature the enzyme. About 10- 15 ml of water was added and the whole reaction mixture was evaporated to dryness. The dry mass was repeatedly extracted with dichloromethane to remove unreacted curcumin. The dried residue consisting of curcumin glycoside and unreacted glucose was subjected to HPLC analysis to determine the extent of conversion. Conversion yield was found to be 39.6 % ( 395.8 µ mol). EXAMPLE 7 A mixture of 0.0005 mol of curcumin and 0.001 mol of D-glucose was taken in a 100 ml two necked round or flat bottomed flask along with 40-60 ml of di-isopropyl ether in presence of 50% (w/w D-glucose) amyloglucosidase and 1.0 ml of l0mM acetate buffer pH 4.0 and refluxed with stirring on a magnetic stirrer with a hot plate for a period of 72h. The solvent was distilled off and the reaction mixture held in a water bath for 5 - 10 min to denature the enzyme. About 10-15 ml of water was added and the whole reaction mixture was evaporated to dryness. The dry mass was repeatedly extracted with dichloromethane to remove unreacted curcumin. The dried residue consisting of curcumin glycoside and unreacted glucose was subjected to HPLC analysis to determine the extent of conversion. Conversion yield was found to be 47.5 % (475.4 u mol). Table 1. Conversion yields of cucrcumin glucosides. (Table Removed) The main advantages of the invention are: 1. This method gives higher yields than those obtained using microbial cultures. 2. Incubation period up to only 2-3 days is required. 3. The aglycon employed can be a sterically hindered phenol like curcumin. 4. The experiments by this method can be conducted at any scale using a single experimental set up. 5. The curcumin glucoside formed is water soluble and can be used as a food colorant. Both α and ß - glycosides are formed. In case of D-glucose the proportion of the a- D-glucoside was found to be more than that ofthe ß- glucoside. We Claim: 1) An enzymatic process for the preparation of curcumin glycoside which comprises: (a) digesting a mixture of carbohydrates and curcumin with the enzyme glucosidase, under reflux conditions in a non-polar solvents, for a period of 2-5 days at pH ranging from 4.0 - 8.0 and at a temperature in the range of 50°C - 75°C in order to obtain reaction product having curcumin glycoside ; (b) distilling the product obtained in step (a) in order to remove the solvent; (c) holding the reaction mixture in a water bath at a temperature in the range of 80-100 deg C for a period of 5-10 minutes in order to inactivate the enzyme glucosidase; (d) adding water to the product obtained in step (c ) followed by evaporation of the water to obtain a dry mass having curcumin glycoside and un-reacted glucose ;. (e) removing un-reacted glucose by repeatedly extracting curcumin glycoside with dichloromethane by a known method to obtain the desired product. 2) An enzymatic process as claimed in claim 1, wherein carbohydrates used are monsaccharides and/or disaccharides. 3) An enzymatic process as claimed in claims 1 and 2, wherein monosaccharides used are selected from the group consisting of D-glucose, d-fructose, D-galactose, D-mannose, D-arabinose, Robose and deoxy-ribose. 4) An enzymatic process as claimed in claims 1-3, wherein disaccharides used are selected from the group consisting of lactose, maltose, trehalose. 5) An enzymatic process as claimed in claims 1-4, wherein non-polar solvent used is selected from n-octane, di-isopropyl ether, chloroform, hexane, ethylmethyl ketone and petroleum ether having boiling points in the range 50°C - 75°C . 6) An enzymatic process as claimed in claims 1-5, wherein non-polar solvent used is at the concentration in the range of 10 - 80% (by weight of mono or disaccharides used). 7) An enzymatic process as claimed in claims 1-6, wherein the pH used is maintained by using 10 mM acetate buffer. 8) An enzymatic process as claimed in claims 1-7, wherein the reflux condiotion used is provided by stirring in a two necked round- or flat- bottomed flask fitted with a cold water circulated condenser. 9) An enzymatic process as claimed in claims 1-8, wherein glucosidase enzyme used is selected from the group consisting of amyloglucosidase, p-glucosidase and other glycosidases. 10) An enzymatic process as claimed in claims 1-9, wherein yield of the curcumin glycoside obtained is in the range of 45-50%. 11) An enzymatic process as claimed in claims 1-10, wherein curcumin glucoside obtained has both alpha and beta form. 12) An enzymatic process as claimed in claims 1-10, wherein curcumin glucoside obtained is soluble in water to the extent of 0.066%. 13) An enzymatic process as claimed in claims 1-10, wherein curcumin glucoside obtained has total color of about 2.7 in the aqueous solution. 14) An enzymatic method for the preparation of curcumin glycoside substantially as herein described with reference to the examples accompanying this specification.

Documents:

756-DEL-2005-Abstract-(09-03-2011).pdf

756-del-2005-abstract.pdf

756-DEL-2005-Claims-(09-03-2011).pdf

756-del-2005-claims.pdf

756-DEL-2005-Correspondence-Others-(09-03-2011).pdf

756-del-2005-correspondence-others.pdf

756-DEL-2005-Description (Complete)-(09-03-2011).pdf

756-del-2005-description (complete).pdf

756-del-2005-description (provisional).pdf

756-DEL-2005-Form-1-(09-03-2011).pdf

756-del-2005-form-1.pdf

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756-del-2005-form-2.pdf

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756-DEL-2005-Form-5-(09-03-2011).pdf

756-del-2005-form-5.pdf