CN101544679B

CN101544679B - Method for extracting ursolic acid from vegetable Dian clove

Info

Publication number: CN101544679B
Application number: CN2008100494044A
Authority: CN
Inventors: 康文艺; 李昌勤; 王天晓; 魏金凤; 王金梅; 宋艳丽; 姬志强
Original assignee: Henan University
Current assignee: Henan University
Priority date: 2008-03-25
Filing date: 2008-03-25
Publication date: 2011-06-08
Anticipated expiration: 2028-03-25
Also published as: CN101544679A

Abstract

The invention relates to a method for extracting ursolic acid from vegetable Dian clove. The method comprises the following steps that: (1) the dried Dian clove is picked up, crushed and undergoes repeated cold soaking by 10L of 95 percent ethanol for three times, seven days for each time, and the ethanol is filtered, decompressed and reclaimed to obtain a concentrated extractive; (2) the obtained ethanol extractive is tested; and (3) the extract obtained from the step(2) is subjected to Sephadex LH-20 gel column chromatography and is separated to obtain the finished product by taking methanol as an eluant and compare with the standard product. The separation materials used by the method include macroporous adsorptive resin and gel, so that the separation materials have stable physical-chemical properties, are not dissolved in acids, alkalis and organic dissolvants, have good selectivity on organic matters, and are not affected by existence of organic salts and strong-ion low-moleculecompounds; and the separation materials are easily eluted and regenerated, thereby facilitating the cyclic utilization.

Description

A kind of method of from vegetable Dian clove, extracting ursolic acid

Technical field

The present invention relates to a kind of method of from plant, extracting ursolic acid, relate in particular to a kind of from the madder wort Dian clove method of extraction separation ursolic acid.

Background technology

Ursolic acid (ursolic acid, UA), the another name ursonic acid, very wide in distributed in nature, be present in leaf, the fruit of ericad black bearberry, select the leaf of gloomy ginseng vegetable wool paulownia, the fruit of madder wort cape jasmine is in the plants such as leaf of gentianaceae plant humidogene flower bud, oleaceae plant glossy privet.Ursolic acid has biological effect widely, and nineteen ninety is classified as one of most promising tumor prevention medicine by Japan.It has significantly stable and cooling effect to central nervous system; External all have anti-microbial activity to G+ and G-bacterium and yeast; The effect of anti-diabetic, antiulcer agent and blood fat reducing is arranged; Multiple carcinogenic, short cancer thing there is resistant function; Multiple malignant cell there is the growth of inhibition effect; Ursolic acid and derivative thereof have the activity of inhibition to virus; In addition, can induce the F9 teratocarcinoma cell to become endoderm cell and angiogenesis inhibitor.Because ursolic acid is extensive in distributed in nature, is present in a lot of plants, and good biological activity is arranged.Generally, ursolic acid is by the chemical process synthetic, adopts the synthetic big and difficult treatment of " three wastes " treatment capacity that has of chemical process, easily environment is produced and pollute, and cost is higher.Up to the present, about the report of extraction separation ursolic acid from other plant has a lot, but all be from oleaceae plant and cuckoo medicine section plant, to extract mostly, obtaining ursolic acid from the madder wort Dian clove does not have report as yet.

Summary of the invention

The object of the present invention is to provide a kind of from the madder wort Dian clove method of extraction separation ursolic acid, its extracting method is simple, cost is low, productive rate is high.

To achieve these goals, technical scheme of the present invention has adopted a kind of method of extracting ursolic acid from vegetable Dian clove, and may further comprise the steps: (1) gets dry Dian clove, pulverizing, is 95% ethanol 10L repetition cold soaking 3 times with concentration, each 7 days, filter, decompression recycling ethanol gets concentrated solution;

(2) the ethanol concentrated solution that obtains is tested;

(3) enriched material that step (2) is obtained carries out Sephadex LH-20 gel filtration chromatography, is eluent with methyl alcohol, and standard control separates the pure product that obtain.

The step that described step (2) is tested the ethanol concentrated solution is: ethanol extract is disperseed with 10% ethanol water (volume ratio), is 1: 30 according to medicinal extract and D101 macroporous resin weight ratio; Will disperse 10% aqueous ethanolic solution of medicinal extract add resin column, water-ethanol solvent gradient elution, 60% ethanol water section concentrate enriched material.

The step that described step (2) is tested the ethanol concentrated solution is: spissated extract is dispersed in the 2L water, uses ethyl acetate extraction three times; Ethyl acetate extract carries out 200-300 order silica gel medium pressure column chromatography, with sherwood oil-acetone solvent (volume ratio is 30: 1) wash-out, detects with the thin-layer chromatography reference standard, merges the position of this compound, and reduction vaporization gets enriched material.

Dian clove of the present invention (Luculia pinciana Hook.) is a Rubiaceae Dian clove platymiscium, is distributed in the Himalayas to Yunnan Province of China and Guangxi, has higher gardens, pharmaceutical use.Because Dian clove plant type grace is with luxuriant foliage and spreading branches in leafy profusion, emerald green light, the florescence is long, and titbit is intensive and in great numbers, and pattern is gorgeous and fragrant, at present mainly as ornamencal flower and tree.But it also has a long history as Chinese medicinal materials among the people.According to record: Dingxiangye FOLIUM SYRINGAE, promptly in the family in the basin person of planting be also, its bitter, suffering, slightly warm in nature; Fragrance is gone into lung, ends on lung cold cough or hemoptysis or the phlegm and is with blood.About the pharmaceutical use of Dian clove platymiscium, Wu Zhengyi chief editor " Xinhua's book on Chinese herbal medicine outline " is documented: root has promoting blood flow to regulate menstruation, anti-inflammatory analgetic, buck functionality.Can be used for menoxenia, rheumatalgia, wound, migraine, nervous etc., external application can be controlled venomous snake bite; Flowers and fruits have relieving cough and reducing sputum function, can be used for Whooping cough, chronic bronchitis, pulmonary tuberculosis; Leaf has the detumescence function, can apply wound and swell and ache.The Dian clove raw material sources are abundant, and the cost that extracts ursolic acid is low.The parting material that uses in the extracting method of the present invention has macroporous adsorbent resin and gel, not only physico-chemical property is stable for they, is insoluble to acid, alkali and organic solvent, better to the organism selectivity, as not existed by inorganic salts and strong ion low molecular compound influence; And be easy to wash-out regeneration, be of value to recycled for multiple times.

Embodiment

Embodiment 1

Get dry Dian clove 20kg, pulverize, with 95% industrial alcohol 10L cold soaking 3 times, each 7 days, filter, decompression recycling ethanol gets concentrated solution, spissated extract is dispersed in the 2L water, uses ethyl acetate successively, propyl carbinol respectively extracts three times, ethyl acetate extract carries out 200-300 order silica gel medium pressure column chromatography, with sherwood oil-acetone solvent (volume ratio is 30: 1) wash-out, detect with the thin-layer chromatography reference standard, merge the position of this compound, carry out Sephadex LH-20 gel filtration chromatography with methanol-eluted fractions, obtain the pure product of this compound.

Embodiment 2

The pre-treatment of macroporous resin: D101 resin dress post, soak 2h with 5%NaOH after, with 2 column volumes of flow velocity wash-out of 4 column volumes per hour, be washed till pH value neutrality with distilled water again; Add alcohol immersion 1h, with 3.5 column volumes of flow velocity wash-out of 2 column volumes per hour, being washed till with distilled water does not at last have the alcohol flavor.The preparation of sample solution: get dry Dian clove 20kg, after the pulverizing, with 95% industrial alcohol cold soaking 3 times, each 7 days, filter, decompression recycling ethanol gets concentrated solution, adds water to suitable concentration according to 1: 30 (medicinal extract and macroporous resin weight ratio); Sample solution is added resin column, the water-ethanol solvent gradient elution, 60% ethanol water section is condensed into medicinal extract, carries out Sephadex LH-20 gel filtration chromatography, is eluent with methyl alcohol, and standard control separates the pure product that obtain.

The ursolic acid that adopts method of the present invention to extract, its proterties is: white powder.Molecular formula: C ₃₀H ₄₈O ₃, molecular weight: 456.

The instrument material: fusing point is measured with the micro-fusing point instrument of XRC-1 type that Sichuan scientific instrument factory produces, and thermometer is not proofreaied and correct; Optically-active is measured with the SPEA-300 instrument; Infrared at Bio-Rad FTS-135 infrared measurement; Ultraviolet is measured on UV-210A ultraviolet spectroscopy instrument; 1H, 13C NMR collection of illustrative plates is by the Brukeram-400MHz nmr determination.TMS is interior mark; The two dimensional NMR collection of illustrative plates is by the DRX-500MHz nmr determination; Mass spectrum is measured by VG.AUTOSpec-3000 type mass spectrograph.

Condition determination and result: Mp:305-308 ℃; Optical Rotat-ion:[α] _d ²⁰-8.8 (c0.25, CHCl ₃).EIMS?m/z(％)：456(M ⁺，20)，410(8)，248(100)，233(30)，213(75)，175(33)，131(30)，119(40)，105(45)，91(35)。 ¹H?NMR(400MHz，C ₅D ₅N)δ：3.38(1H，dd，J＝11.3，3.4Hz，H-3)，5.57(1H，brs，H-12)，2.42(1H，d，J＝11.32Hz，H-18)，0.67(3H，s，H-23)，0.63(3H，s，H-24)，0.84(3H，s，H-25)，0.81(3H，s，H-26)，1.03(3H，d，J＝5.9Hz，H-29)，1.01(3H，d，J＝6.1Hz，H-30). ¹³CNMR(125MHz，δin?ppm，pyridine-d ₅)δ：39.2(C-1)，29.1(C-2)，77.9(C-3)，39.8(C-4)，55.6(C-5)，18.6(C-6，32.6(C-7)，37.0(C-8)，53.3(C-9)，37.0(C-10)，23.1(C-11)，125.4(C-12)，139.04(C-13)，41.6(C-14)，27.9(C-15)，42.3(C-16)，47.8(C-17)，39.3(C-18)，39.2(C-19)，33.4(C-20)，30.9(C-21)，33.0(C-22)，24.7(C-23)，16.3(C-24)，15.5(C-25)，17.2(C-26)，23.7(C-27)，179.7(C-28)，28.5(C-29)，24.7(C-30)。

It should be noted last that: above embodiment is the unrestricted technical scheme of the present invention in order to explanation only, although the present invention is had been described in detail with reference to the foregoing description, those of ordinary skill in the art is to be understood that: still can make amendment or be equal to replacement the present invention, and not breaking away from any modification or partial replacement of the spirit and scope of the present invention, it all should be encompassed in the middle of the claim scope of the present invention.

Claims

1. method of from vegetable Dian clove, extracting ursolic acid, it is characterized in that: may further comprise the steps: (1) gets dry Dian clove, pulverize, be that 95% ethanol 10L repeats cold soaking 3 times with concentration, each 7 days, filtration, decompression recycling ethanol gets concentrated extract; (2) ethanol extract that obtains is tested; (3) enriched material that step (2) is obtained carries out Sephadex LH-20 gel filtration chromatography, is eluent with methyl alcohol, and standard control separates the pure product that obtain; Wherein, the step that described step (2) is tested ethanol extract is: ethanol extract is disperseed with 10% ethanol water (volume ratio), is 1: 30 according to medicinal extract and D101 macroporous resin weight ratio; Will disperse 10% aqueous ethanolic solution of medicinal extract add resin column, water-ethanol solvent gradient elution, 60% ethanol water section concentrate enriched material; Perhaps be: spissated extract is dispersed in the 2L water, uses ethyl acetate extraction three times; Ethyl acetate extract carries out 200-300 order silica gel medium pressure column chromatography, is 30: 1 sherwood oil one acetone solvent wash-out with volume ratio, detects with the thin-layer chromatography reference standard, merges the position of this compound, and reduction vaporization gets enriched material.