JP3943082B2 - Substance having molecular structure of 8- (c-β-D-glucopyranosyl) -7,3 ', 4'-trihydroxyflavone, separation method thereof and drug composition using the same - Google Patents

Description

[0001]
[Industrial application fields]
The present invention relates to a novel compound, 8- (c-β-D-glucopyranosyl) -7,3 ′, 4′-trihydroxyflavone. The present invention also relates to a method for separating the novel compound 8- (c-β-D-glucopyranosyl) -7,3 ′, 4′-trihydroxyflavone from Pterocarpus marsupium .
[0002]
[Prior art]
Pterocarpus marsupium Roxb (Leguminosae), known as the Indian Kinoki or Bijasar, is well known in the central hills of India and the Indian Peninsula (Appendix 1). The leaves, flowers and resin extracts have been used to treat diarrhea, toothache, high fever, urinary tract infections and skin infections (Document 2). Bark extracts have long been considered effective in the treatment of diabetes (Appendix 3). According to the report of Chakravarty, the hypoglycemic activity factor is (−)-epicatechin, and its effect is due to regeneration of pancreatic beta cells (Reference 4). However, this allegation has been questioned by Kolb et al. (Document 5) and Sheehan et al. (Document 6). At present, further studies are considered necessary before considering (-)-epicatechin as a potential anti-diabetic agent for human clinical research.
[0003]
Indian System of Medicine doctors have the view that heartwood is more useful in the treatment of diabetics than cypress bark. Only the heartwood is clearly red, and when immersed in water, the water is colored red with blue-green fluorescence. It is also a claim that this is suitable for use as an antidiabetic drug. The hypoglycemic effect of water or alcoholic extracts from cypress heartwood has been experimentally proven (Documents 7 and 8) and has also been demonstrated in clinical studies (Documents 9 and 10). Mushroom heartwood contains many phenols. Although chemical research on quinoki heartwood dates back to 1946, early research on this drug was fragmented (Appendix 11).
[0004]
Previously reported studies on this plant disclosed the following chemical components:
[0005]
1. From the ether extract of cypress heartwood, isoflavonoid glycol 4,4'-dihydroxy-α-methylhydrobenzoin (Mar. 12) named Marusupol, a derivative of benzofuranone and Carpusin 2,4 ′, 6-Trihydroxy-4-methoxybenzo (b) furan 3 (2H) -1 (Appendix 13), named 1,3-bis (Propterol, a 2-propanol derivative 4-Hydroxyphenyl) propan-2-ol (Document 14), 1- (2,4-dihydroxyphenyl) -3- (4-hydroxyphenyl) propan-2-ol, named Propterol B (Document 15) 6-Hydroxy-7-O-methyl-3- (3-hydroxy-4-O-methyl benzyl) chroman-4-1 (Document 1) ) Has been obtained.
[0006]
2. From the ethyl acetate-dissolved component of the heartwood alcohol extract, petrospin β, 2 ′, 4,4 ′,-tetrahydroxy-3 ′ (c-β-D-glucopyranoside) dihydrochalcone (Document 17), Malspinol ( Marsupinol (Document 18), 5,4'-dimethoxy-8-methylisoflavone-7-O-α-L-rhamnopyranoside, retusin-O-β-D-glucopyranoside and irisolidin-7-O-α-L -Rhamnopyranoside (Reference 19), 5,7'-dihydroxy-6-methoxy-7-O-α-L-rhamnopyranoside (Reference 20).
[0007]
3. From the ethyl acetate extract of defatted heartwood, a benzofuranone derivative, 2,6-dihydroxy-2- (p-hydroxylbenzyl) -4-methoxy-3 (2H) -benzofuranone (named Marspin) (Document 21), Ptero Stilbin, (2S) -hydroxyflavone, isoliquiritigenin, liquiritigenin, 7,4'-dihydroxyflavone, 5-deoxycamphorol and 3,7,4'-trihydroxyflavone (Document 22), 2 Two glycosides, 8-C-β-D-glucopyranosyl-3,7,4'-trihydroxy, 3,7,3 ', 4'-tetrahydroxyflavone and 3'-C-β-D-glucopyranosyl -Α-Hydroxy dihydrochalcone (Document 23) is obtained.
[0008]
4). Serine-4 (15) -ene-1β, 11-diol, β-eudesmol, erythrodiol-3-monoacetate and pterostilbene (Document 24) can be obtained from gasoline extract of cypress root. 4,6,4'-Trihydroxyaurone 6-O-rhamnopyranoside and 4,6,4'-trihydroxy-7-methylaurone-4-O-rhamnopyranoside (Reference 25) Have (-)-Epicatechin (Document 26) is obtained from the ethanol extract of the bark of the mushroom.
[0009]
However, these prior arts do not provide details on the biological activity associated with such chemical components. Also, these prior arts only disclose the preparation of ether extract, ethyl acetate extract and ethyl acetate soluble components of alcohol extract, and the method of preparing the extract of Kinoki heartwood with water and any chemical components from the extract. No attempt to separate is disclosed.
[0010]
[Problems to be solved by the invention]
Therefore, the main object of the present invention is to prepare an aqueous extract of cypress heartwood and to obtain chemical components from the extract. Another object of the present invention is to study an aqueous extract of cypress heartwood to obtain biologically active ingredients useful in the treatment of diabetes.
[0011]
[Means for Solving the Problems]
The above and other objects of the present invention are achieved by preparing an n-butanol soluble component of an aqueous extract of cypress heartwood and further isolating a new biologically active component therefrom. Accordingly, the present invention provides a novel compound, 8- (C-β-glucopyranosyl) -7,3 ′, 4′-trihydroxyflavone {8- (C-β-D-glucopyranosyl) -7,3 ′, 4 '-trihydroxyflavone} is provided.
[0012]
The present invention also provides a method comprising the following steps for separating 8- (C-β-glucopyranosyl) -7,3 ′, 4′-trihydroxyflavone. (A) plant, Pterocarpus marsupium heartwood is powdered, (b) extracted from the powdered plant using a protic solvent, (c) the extract is concentrated to a minimum volume, and with different organic solvents Separate to remove non-polar components and (d) separate 8- (C-β-glucopyranosyl) -7,3 ′, 4′-trihydroxyflavone from the residue.
[0013]
In one embodiment of the invention, the protic solvent used in the preparation of the extract in step (b) is selected from the group consisting of water, methanol, ethanol, propanol, butanol and mixtures thereof. Furthermore, in an embodiment of the invention, the polar solvent used to extract into the water layer is selected from ethyl acetate, propanol, butanol. In another embodiment of the present invention, the organic solvent used to remove non-polar components in step (c) is selected from the group consisting of hexane, petroleum ether and chloroform. In other embodiments of the present invention, the chromatographic method used to separate 8- (C-β-glucopyranosyl) -7,3 ′, 4′-trihydroxyflavone is medium pressure liquid chromatography (MPLC), Selected from high performance liquid chromatography (HPLC), flash chromatography.
[0014]
The invention also effective amounts of drug in a carrier acceptable as a drug 8- (C-beta-glucopyranosyl) -7,3 ', it relates to a drug composition containing 4'-trihydroxy hula Von. In one embodiment of the invention, the amount of 8- (C-β-glucopyranosyl) -7,3 ′, 4′-trihydroxyflavone in the drug composition is from 0.5 to 1 kg of patient body weight. The range is 10 mg.
[0015]
The present invention is also 8 for the preparation of pharmaceutical composition for the treatment of diabetes (C-beta-glucopyranosyl) -7,3 ', relates to the use of 4'-trihydroxy hula Von.
[0016]
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides a method for separating 8- (C-β-glucopyranosyl) -7,3 ′, 4′-trihydroxyflavone, which method comprises the following steps.
(A) Powdering the heartwood of Pterocarpus marsupium,
(B) extracting the powdered plant material using a protic solvent;
(C) Concentrate the aqueous extract to a minimum volume, separate with an organic solvent of increasing polarity to remove non-polar components, extract into an aqueous layer with a polar solvent, remove the solvent to obtain a residue,
(D) Separating 8- (C-β-glucopyranosyl) -7,3 ′, 4′-trihydroxyflavone from the residue.
[0017]
The solvent used to prepare the extract is water, methanol, ethanol, propanol, butanol, or mixtures thereof. The organic solvent used in step (c) to remove non-polar components is selected from the group consisting of hexane, petroleum ether and chloroform. The polar solvent used for extraction into the aqueous layer is selected from ethyl acetate, propanol or butanol. The chromatography used for the separation of 8- (C-β-glucopyranosyl) -7,3 ′, 4′-trihydroxyflavone may be medium pressure liquid chromatography, flash chromatography or the like. The elution solvent required in medium pressure liquid chromatography is pumped through a column by a pump, and in flash chromatography, the solvent is pumped by air pressure. The compound has the molecular formula C ₂₁ H ₂₀ O ₁₀ (m / z 433 [M + 1] by FAB mass spectrometry). This result was supported by ¹³ C nuclear magnetic resonance and DEPT spectra.
[0018]
Compound 8- (C-β-glucopyranosyl) -7,3 ', 4'-trihydroxyflavone is isolated from n-butanol soluble component of water leachate from cypress heartwood and has anti- diabetic activity in humans and animals Show. The research that has been conducted in this technical field is about ethyl acetate extract components of ethyl acetate extract and alcohol extract, and there is no disclosure in the prior art about the compounds.
[0019]
The method for separating the active element from the mushroom consists of separating the powdered heartwood water extract using various organic solvents containing 1 to 6 carbon atoms in the molecule. 8- (C-β-Glucopyranosyl) -7,3 ′, 4′-trihydroxyflavone can be converted from polar components to medium pressure liquid chromatography (MPLC), high performance liquid chromatography (HPLC) or silica gel (230-400 mesh). It is isolated by applying modern chromatography techniques such as flash chromatography used and exhibits hypoglycemic activity.
[0020]
The compound 8- (C-β-glucopyranosyl) -7,3 ′, 4′-trihydroxyflavone has been evaluated for low diabetic activity in Wistarrat fasted for 18 hours. A hypoglycemic effect has been recorded in all wrinkles at a prescription of 10 mg / kg (body weight). From an average of 92 mg / 100 ml of blood before administration to an average of 68 mg / 100 ml of blood, the average drop is recorded as 24 mg / 100 ml of blood. In comparison, the average drop in hypoglycemic agent employed in the control test in the study was 23 mg / 100 ml blood.
[0021]
The compound has been proven to be phenol by the phosphomolybdic acid test (blue) and ferric chloride reaction (green). It is also recognized as a flavon from the reaction of the Shinoda test. The presence of hydroxyl, carbonyl and phenyl nuclei is indicated by infrared absorption at 3228, 1615, 1554, 1448 and 1422 cm- ¹ . The ultraviolet absorption spectrum of this compound shows maximum absorption at λ _max ^MeOH 219, 238, 260, 320, 358 nm, and moves deeply to 219, 238, 267, 320, 367 nm in the presence of NaOAc. This observation suggests that there is a free hydroxyl group located at C-7.
[0022]
^{The 1} H nuclear magnetic resonance spectrum (200 MHz, dimethyl sulfoxide-d ₆ ) is assumed to be due to steric high density due to glucosyl and the B ring, but the signal shows broadening in the aromatic region. Spectral examination reveals a single signal of δ6.98 (1H), which is characteristic of protons at the C-3 position of flavones. The double proton signal of δ8.28 (1H, d, J = 2.1 Hz) is shifted by a low magnetic field due to the proximity effect of C = O, but δ6.95 (1H, d, J = 8.3 Hz). Ortho-coupled to the dual signal. This ortho bond is due to the C-5 and C-6 protons, indicating that only these two protons belong to the A ring and C-8 is contained in the glycosyl group. δ 7.84 (1H, br, J = 2.1 Hz), 7.97 (1H, brdd, J = 2.1, 8.7 Hz), and 6.99 (1H, d, J = 8.7 Hz) The proton signal is derived from the B ring proton. Furthermore, ¹ H and ¹³ C nuclear magnetic resonance spectra are due to glucose constituents. C—C bonds were demonstrated by ¹ H and ¹³ C heteronuclear correlations between the anomeric proton of δ 5.16 and the double carbon of δ 79.3 in the characteristic region of glucoside substituted C-. Furthermore, the binding constant of the signal resulting from the anomeric proton of glucopyranoside (J = 9.5 Hz) indicated that the glucoside link is in the β configuration. Thus, the above analysis led to the conclusion that the structure is 8- (C-β-glucopyranosyl) -7,3 ′, 4′-trihydroxyflavone.
[0023]
Details of the present invention will be described below with reference to examples, but the examples do not constitute limitations on the technical scope of the present invention.
[0024]
Example 1
A powder (1 kg) of mushroom heartwood was extracted with 80% ethanol water (3 × 3 l) for 48 hours. The concentrated product after extraction was separated with hexane, chloroform, propanol and butanol in order. For the polar extract, hexane, chloroform, methanol, and ethanol were sequentially used, and medium pressure liquid chromatography using silica gel (100 to 200 mesh) was performed. The active compound was purified by repeated medium pressure liquid chromatography and flash chromatography on silica gel (230-400 mesh) using trichloromethyl-methanol (19: 1) as a solvent, and 8- (C-β-glucopyranosyl) -7 , 3 ′, 4′-trihydroxyflavone (yield 0.046%), melting point 202-204 ° C., [α] _D ¹⁹ + 25.6 ° (methanol, c, 0.5).
[0025]
Example 2
Mushroom heartwood was extracted with warm water for 4 × 4 hours. The extract concentrate was dissolved and separated sequentially with hexane, chloroform, propanol and butanol. 8- (C-β-glucopyranosyl) -7,3 ′, 4′-trihydroxy was obtained by flash chromatography on silica gel (100 to 200 mesh) using hexane, chloroform, ethyl acetate and methanol as solvent systems from the obtained polar extract. Obtained flavon-rich ingredients. The component was repeatedly chromatographed on silica gel (230-400 mesh) using EtOAc-MeOH (19.5: 0.5) as a solvent to obtain the molecular formula 8- (C-β-glucopyranosyl) -7,3 ′, 4′- Trihydroxyflavone (yield 0.049%), melting point 202-204 ° C., [α] _D ¹⁹ + 25.6 ° (methanol, c, 0.5) was obtained.
[0026]
Example 3
Butterberries were boiled with water until the water volume was reduced to 1/4 (16 times). Filtration, concentration and sequential separation using hexane, chloroform, ethyl acetate, propanol and n-butanol. The obtained polar extract was subjected to column chromatography on silica gel (60 to 120 mesh) using hexane, chloroform, ethyl acetate and methanol as a solvent system to obtain 8- (C-β-glucopyranosyl) -7,3 ′, 4′- A component rich in trihydroxyflavone was obtained. The 8- (C-β-glucopyranosyl) -7,3 ′, 4′-trihydroxyflavone-rich component was mixed with silica gel (100-200 mesh) using a mixed solvent of ethyl acetate-acetone (8: 2). Repeated column chromatography 8- (C-β-glucopyranosyl) -7,3 ′, 4′-trihydroxyflavone (0.051% yield), mp 202-204 ° C., [α] _D ¹⁹ + 25.6 ° (Methanol, c, 0.5) was obtained.
[0027]
【The invention's effect】
1. The resulting compound 8- (C-beta-glucopyranosyl) -7,3 ', 4'-trihydroxy hula Von is a compound of new molecular structures having anti-diabetic activity.
The method for separating 2.8- (C-β-glucopyranosyl) -7,3 ′, 4′-trihydroxyflavone is relatively simple.
[0028]
【Reference document】
1.Jain, SKMedicinal Plants, National Bank trust, New Delhi, 1968, p. 116
2. Chopra, RN, Chopra, IC , Handa, KL and Kapur, LD Indigenous Drugs of India, 2 nd Ed., Dhar, UN and Sons Private Limited, Calcutta, 1958, p. 522
3.Kirtikar, KR and Basu, BDIndian Medicinal Plants, 2 nd Ed. Edited by Blatter, E., Cailes, JF and Mhaskar, KS Sinbh and Singh, Delhi. India, 1975, p. 2135
4.Chakravarthy, BK, Gupta, S. and Gode, KD, Lancet, 1982, 272 (cited references)
5.Kolb, H., Kiesel, U., Grenlich, B. and Bosch, JVD Lancrt, 1982, 1303
6.Sheehan, EW, Zemaitis, MA, Slatkin, DJ and Schiff, Jr. PL Journal of Natural Products, 1983, 46, 232
7.Shah, DS Indian Journal of Medical Research, 1967,55, 166 (cited references)
8.Gupta, SS Indian Journal of Medical Research, 1963,51, 716
9.Sepha, GC and Bose, SNJ Ind. Med.Assoc. 1956, 27, 383
10.Kedar, P. and Chkrabarti, CH, Maharastra Med. J., 1981, 28, 165
11.Bhargava, PN, Proc. Ind. Acad. Sci., 1946, 24A, 496
12.Rao, AVS, Mathew, J., Phytochemistry, 1982, 21, 1837
13.Mathew, J. and Rao, AVS Phytochemistry, 1983, 22, 794
14.Rao, AVS, Mathew, J. and Shankaran, AVB Phytochemistry, 1984, 23, 897
15.Mathew, J., Rao, AVS and Rambhav, S. Current Science, 1984, 53, 576
16.Jain, SC, Sharma, SK, Kumar, R., Rajwansh, VK and Babu, VR Phytochemistry, 1997, 44, 765
17.Adinarayana, D., Syamsundar, KV, Seligmann, O. and Wagner, H.
Z. Naturforsch. 1982, 37C, 145
18.Trivedi, JJ Indian J. Phys. Pharmacol. 1997, 15, 51
19.Mitra, J. and Joshi, T. Phytochemistry, 1982, 21, 2429
20.Mitra, J. and Joshi, T. Phytochemistry, 1983, 22, 2326
21.Maurya, R., Ray, AB, Duah, FK, Slatkin, DJ and Schiff, PLJr. Heterocycles 1982, 19, 2103
22.Maurya, R., Ray, AB, Duah, FK, Slatkin, DJ and Schiff, PLJr.J.Nat.Prod.1984, 47, 179
23. Bezuidenhoudt, BCB, Brandt, EV and Ferreira, EV
Phytochemistry, 1987, 26, 531
24.Adinarayana, D. and Syamasundar, KV Phytochemistry, 1982, 22, 1082
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26. Chakravarthy, BK and Gode, KD Planta Medica 1985, 56

Claims (11)

8- (C-beta-glucopyranosyl) -7,3 ', 4'-trihydroxy hula von Na Ru things quality.   2. The substance according to claim 1, wherein said substance is isolated from Pterocarpus marsupium. 8- (C-beta-glucopyranosyl) -7,3 ', a method of separating 4'-trihydroxy hula von ing ones quality,
(A) Powdering the heartwood of plant mushrooms,
(B) preparing a pulverized plant raw material extract using a protic solvent,
(C) Concentrate the extract to a minimum volume, remove non-polar components and separate using different organic solvents that increase polarity, extract to aqueous layer with polar solvent, remove solvent to obtain residue ,
(D) A separation method comprising the above steps, wherein 8- (C-β-glucopyranosyl) -7,3 ′, 4′-trihydroxyflavone is separated from the residue.   4. The separation method according to claim 3, wherein the protic solvent used in the step (b) is selected from water, methanol, ethanol, propanol, butanol and a mixed solution thereof.   The separation method according to claim 3, wherein the organic solvent used for removing the nonpolar component in the step (c) is selected from hexane, petroleum ether and chloroform.   The separation method according to claim 3, wherein the polar solvent used for extraction into the aqueous layer is selected from ethyl acetate, propanol and butanol.   The method used to separate the 8- (C-β-glucopyranosyl) -7,3 ′, 4′-trihydroxyflavone is from medium pressure liquid chromatography (MPLC), high performance liquid chromatography (HPLC) and flash chromatography. 4. The separation method according to claim 3, wherein the separation method is selected. Drug composition characterized by comprising a pharmaceutically effective amount of 8- (C-beta-glucopyranosyl) -7,3 ', 4'-trihydroxy hula Von and a pharmaceutically acceptable carrier. The amount of 8- (C-β-glucopyranosyl) -7,3 ′, 4′-trihydroxyflavone contained in the drug composition is in the range of 0.5 to 10 mg per 1 kg of the patient's body weight. pharmaceutical composition of claim 8 wherein. Is characterized by using in the preparation of pharmaceutical composition for the treatment of diabetes 8- (C-beta-glucopyranosyl) -7,3 ', 4'-trihydroxy Fra Von Using. The amount of 8- (C-β-glucopyranosyl) -7,3 ′, 4′-trihydroxyflavone used in the preparation of the drug composition is in the range of 0.5-10 mg / kg patient body weight. The method for using 8- (C-β-glucopyranosyl) -7,3 ′, 4′-trihydroxyflavone according to claim 10 .