Novel method for extraction of Oxyresveratrol from Artocarpus hirsutus

Abstract

Disclosed is a novel method for extraction of Oxyresveratrol from Artocarpus hirsutus.

Claims

1. A novel method for extraction of Oxyresveratrol from Artocarpus hirsutus, said method comprising steps of: a) Cutting, drying the wood of A. hirsutus and pulverising to coarse powder; b) Extracting powdered material from step a with hot ethanol (9 volumes) completely c) Filtering to separate the ethanol extract from step b; d) Concentrating the extract from step c under vacuum at 50-55 C. to thick paste; e) Drying the extract from step d completely in vacuum tray dryer at 65-70 C. to get powder; f) Dissolving the powder from step e in ethanol (2 volumes); g) Addition of the ethanolic extract from step f slowly into 10 volumes of water under continuous stirring; h) Separating the insolubles obtained from step g to get clear layer by filtration; i) Washing the clear layer from step h with chloroform (1 volume) and discard the chloroform layer j) Extracting the clear layer from step i with ethyl acetate (1 volume) k) Concentrating the ethyl acetate layer from step j under vacuum at 50-55 C. to thick paste; l) Drying the concentrated extract from step k to get powder in vacuum tray dryer at 65-70 C.; m) Dissolving the powder from step 1 in water (4 volumes) and heating upto 80-90 C. for 8 h under stirring; n) Cool the water layer from step m to 10-15 C. for 8 h and allowed for crystallization; o) Filtering the crystals obtained from step n; p) Drying the crystals from step o in a vacuum at 70-75 C.; q) Characterizing the crystals from step p as Oxyresveratrol from its .sup.1H and .sup.13C NMR spectra as well as from its LCMS spectrum as represented in STR#1 ##STR00002##

Description

BRIEF DESCRIPTION OF THE DRAWINGS (FIGS. 1 and 2)

[0011] FIG. 1 shows .sup.1H-NMR (DMSO-d.sub.6, 300 MHz): 9.72 (1H, s, 5-OH), 9.54 (1H, s, OH), 9.32 (2H, s, OH), 7.33 (1H, d, J=8.7 Hz, H-6), 7.14 (1H, d, J=16.5 Hz, H-), 6.76 (1H, d, J=16.5 Hz, H-), 6.34 (2H, d, J=2.1 Hz, H-2 and H-6), 6.30 (1H, d, J=2.4 Hz, H-3), 6.24 (1H, dd, J=8.4 and 2.1 Hz, H-5), 6.06 (1H, t, J=2.1 Hz, H-4).

[0012] FIG. 2 shows .sup.13C NMR of Oxyresveratrol (DMSO-d.sub.6, 75 MHz): 158.75 (C-3 and 5), 158.37 (C-4), 156.30 (C-2), 140.36 (C-1), 127.60 (C-6), 124.97 (C-), 123.57 (C-), 115.61 (C-1), 107.60 (C-5), 104.34 (C-2 and 6), 102.87 (C-4), 101.70 (C-3).

[0013] FIGS. 3a and 3b shows Liquid Chromatography Mass Spectrometry (LCMS) of compound Oxyresveratrol obtained from A. hirsutus

[0014] FIG. 4 shows High Performance Liquid Chromatography (HPLC) of compound Oxyresveratrol obtained from A. hirsutus

DETAILED DESCRIPTION OF THE MOST PRFFRED EMBODIMENT

[0015] In the most preferred embodiment, the present invention discloses a novel method for extraction of Oxyresveratrol from Artocarpus hirsutus, said method comprising steps of: [0016] a) Cutting, drying the wood of A. hirsutus and pulverising to coarse powder; [0017] b) Extracting powdered material from step a with hot ethanol (9 volumes) completely [0018] c) Filtering to separate the ethanol extract from step b; [0019] d) Concentrating the extract from step c under vacuum at 50-55 C. to thick paste; [0020] e) Drying the extract from step d completely in vacuum tray dryer at 65-70 C. to get powder; [0021] f) Dissolving the powder from step e in ethanol (2 volumes); [0022] g) Addition of the ethanolic extract from step f slowly into 10 volumes of water under continuous stirring; [0023] h) Separating the insolubles obtained from step g to get clear layer by filtration; [0024] i) Washing the clear layer from step h with chloroform (1 volume) and discard the chloroform layer [0025] j) Extracting the clear layer from step i with ethyl acetate (1 volume) [0026] k) Concentrating the ethyl acetate layer from step j under vacuum at 50-55 C. to thick paste; [0027] l) Drying the concentrated extract from step k to get powder in vacuum tray dryer at 65-70 C.; [0028] m) Dissolving the powder from step 1 in water (4 volumes) and heating upto 80-90 C. for 8 h under stirring; [0029] n) Cool the water layer from step in to 10-15 C. for 8 h and allowed for crystallization; [0030] o) Filtering the crystals obtained from step n; [0031] p) Drying the crystals from step o in a vacuum at 70-75 C.; [0032] Characterizing the crystals from step p as Oxyresveratrol from its .sup.1H and .sup.13C NMR spectra as well as from its LCMS spectrum as represented in STR#1

##STR00001##

[0033] Other features and advantages of the present invention will become apparent from the following more detailed description, taken in conjunction with the accompanying images, which illustrate, by way of example, the principle of the invention.

EXAMPLE

[0034] Preparation of the Extract

[0035] Artocarpus hirsutus wood was collected and cut into small pieces, dried in shade. The dried material was pulverised to a coarse powder and stored in an air tight container. The powdered material was extracted completely with soaked volume of hot ethanol (3 volumes3 times). The ethanolic extracts were separated by filtration and collected in a clean container. The combined extracts were concentrated to thick paste under vacuum at 50-55 C. and dried in vacuum tray drier to get powder. The powdered extract was collected and stored at room temperature in air tight container.

[0036] Isolation and Characterization of Active Compound

[0037] The powdered ethanolic extract from A. hirsutus wood was redissolved in a small amount (2 volumes) of ethanol and poured into 10 volumes of water. The material was filtered to separate the water soluble and water insoluble fraction. The water soluble fraction was then extracted with ethyl acetate (1 volume) and the ethyl acetate layer was dried completely to get the powder. The powder obtained from ethyl acetate fraction was then poured into water (4 volumes) and stirred at 80-90 C. for 8 h. After cooling at room temperature, the solution was filtered and the crystallised solid material was dried under vacuum at 65 -70 C. This filtered crystallised material was characterized as Oxyresveratrol from its .sup.1H and .sup.13C NMR spectra as well as from its LCMS spectrum and was comparable with the reported values. The isolated Oxyresveratrol shows the following values: C.sub.14H.sub.12O.sub.4. m. p.: 191-194 C. Color: pale brown fine powder, APCI-MS m/z 245.00 (M+H.sup.+) and 245.05 (M-H.sup.) (C.sub.14H.sub.12O.sub.4 requires 244.2426). Purity of the compound was further verified by HPLC.

[0038] While the invention has been described with reference to a preferred embodiment, it is to be clearly understood by those skilled in the art that the invention is not limited thereto. Rather, the scope of the invention is to be interpreted only in conjunction with the appended claims.