The preparation method of high-purity momordica glycoside V and purposes
Technical field
The present invention relates to a kind of preparation method of momordica glycoside V, especially prepare the preparation method of purity greater than 98% momordica glycoside V.
Background technology
Grosvenor Momordica, (Momordica grosvenori swingle), the fruit that belongs to Curcurbitaceae herbaceous perennial vine plant, it is the southern endemic plant of China, mainly originate in backlands district, osmanthus, Guangxi, some subtropical zones on the south the Changjiang river also have wild on a small quantity, but are best with the geographic Grosvenor Momordica in Guilin.The past Grosvenor Momordica is for a long time as Chinese medicine and beverage dry fruit and eat, Grosvenor Momordica has effect, the clinical diseases such as pulmonary tuberculosis, asthma, gastritis, Whooping cough, chronic and acute tracheitis and acute and chronic tonsillitis for the treatment of such as give birth to rule, the pyrolysis heat that disappears, relieving cough and reducing sputum, cool blood moistening lung, bring high blood pressure down of quenching the thirst.People not only study as sweet ignorant agent the strong sweet ignorant material momordica glycoside V in the Grosvenor Momordica in recent years, and its pharmacology is also begun to pay attention to.
Open source literature has been reported the extracting method of many Momordia grosvenori aglycones, as solvent extration, resin adsorption method, microwave extraction method, micro-porous filter, report as open source literature: 1.[autograph] Study on extraction [author] Li Yan group Wang Ce [periodical name] the research and development of natural products .1995 of Grosvenor Momordica saponin, 7 (4) .[digests] from Grosvenor Momordica, extracted the saponin of strong sweet taste with health role.Experimental results show that Ca (OH) the 2nd, a kind of good finings, D280, D290, D61 and three kinds of homemade ion-exchange resins of D151 have good decoloring ability, and the AB08 polymeric adsorbent can separate this saponin effectively.With water is the yield height of the saponin yield of solvent than aqueous ethanolic solution, and quantity of solvent is suitable with heavy 6 times of raw material.Ca (OH)
2Make finings than alum, AlCl
3Effective, and can not bring very big saponin loss, clarification should at room temperature be carried out.Strong alkali resin D290 and D280 are than the good decolorizing effect of acidic resins, and D280 is than the good decolorizing effect of D290.Should operate down at 25 ℃ from handing over decolouring.The operation of A8-8 polymeric adsorbent absorption Grosvenor Momordica saponin should be under the room temperature about 20 ℃ with the operated in flow rate of SV2.Can make the desorb from the AB-8 polymeric adsorbent effectively of arhat saponin with 50% aqueous ethanolic solution.2.[autograph] technical study [author] Li Jun that the Grosvenor Momordica glucoside extracted with Orthogonal Method, Lu Cheng, the chemistry world .1999 of Guangxi Normal University's [periodical name], 40 (2) .-92-94[digests] adopt orthogonal experimental design that the extraction using alcohol technology of Grosvenor Momordica glucoside in the Grosvenor Momordica has been carried out systematic study, preferred processing parameter, the result shows; With 30 times of 30% ethanol that raw material is heavy, extracting 3h under 75-80 ℃ of slight boiling condition is top condition.3.[autograph] refining research [author] Liu Zhongdong of mogroside (V), Zhengzhou Grain College [periodical name] ion-exchange and absorption .1999,15 (4) .[digests] utilize domestic resin to study the process for purification of mogroside (V), studied concentration and pH to the influence of process for refining and physics, chemistry and the spectral quality of product.4. process for extracting rubusoside of fruit of Gorsvenor Momordica, Chinese patent: application (patent) number: 02128065.7 application (patent right) people: Hongye Green Vegetable Product LLC, Guilin address: the QiXing, Guilin City, GuangXi Zhuang Autonomous nationality District district executes No. 6 summaries in home road: the present invention is with the Grosvenor Momordica fragmentation, add ethanolic soln, under 30~60 ℃ of temperature, soaked 5~15 hours.Filter to get filtrate, temperature is reduced to 20~35 ℃, and filtrate is sent into three kinds of membrane separation assemblies through pressure pump order under 0.01~0.2MPa operating pressure; The first step is held back and is isolated spissated Grosvenor Momordica fiber, protein, pectin and impurity, is used to make organic fertilizer; The second stage is spissated Momordica-Glycosides solution, through vacuum-drying, gets the Momordica-Glycosides product; The third stage is spissated momordica grosvenori polysaccharide and trace element, is the Grosvenor Momordica massecuite; The residual filtrate recycling use.5. method Chinese patent that from Grosvenor Momordica, extracts Momordica-Glycosides: application (patent) number: 03117430.2, application (patent right) people: Guilin Laiyin Biological Product Co., Ltd address: No. 18 summaries in Xiangjiang Rd., Xing'an County, Guilin, Guangxi Zhuang Autonomous Region: the present invention relates to a kind of method of from Grosvenor Momordica, extracting Momordica-Glycosides, be to be raw material with the fresh Fructus Momordicae, with water is extraction solvent, adopting micro-filtration, ultrafiltration, nanofiltration is main extraction process, and wherein microfiltration membrane adopts the millipore filtration in 0.05 μ m~0.3 μ m aperture; It is 20000~80000 hollow fiber ultrafiltration membrane that ultra-filtration membrane adopts the relative molecular weight that dams; It is that 100~400 nanofiltration membrane or RO reverse osmosis equipment concentrate that the relative molecular weight that dams is adopted in nanofiltration.Because present method is to operate under cryogenic condition, thereby product color degree, solvability, clarity and taste are better, in addition, membrane separation technique is high-precision molecular screening, avoided the loss of activeconstituents, energy consumption and cost have been reduced, product yield and quality have been improved, and the micro-filtration of the integrated membrane sepn of present method, ultrafiltration, nanofiltration are raw material with the fresh fruit, are extraction solvent with water, form advanced separation purifying technique flow process, the separation of finishing Momordica-Glycosides reaches and concentrates, and has simplified production technique, has shortened the production cycle.6. title: the method application (patent) of extraction separation multiple components number from Grosvenor Momordica: 02113548.7 application (patent right) people: location, He Wei level land: eight Li Jie economic and technological development zone, Guilin City, Guangxi Zhuang Autonomous Region Guilin strength company summary: a kind of from Grosvenor Momordica the method for extraction separation multiple components, be that water obtains extracting solution with the broken Grosvenor Momordica of buck processing afterwards again, handle through Fatty Alcohol(C12-C14 and C12-C18) again and obtain throw out (I) and lysate (II), throw out (I) through the resin anion(R.A) post again combining fat alcohol processing purifying obtain momordica grosvenori polysaccharide.Lysate (II) is through polyamide resin column, and the effluent liquid from post is handled purifying through macroporous resin column combining fat alcohol again and obtained Momordica-Glycosides.By the composition that polyamide resin column adsorbs, concentrate drying promptly obtains grosvenor momordica flavonoid after alkaline fat alcohol washes out.Three kinds are extracted all suitable healthcare products and the foodstuff additive done of composition.7.[autograph] microwave-assisted extracts [mechanism] the South China Science ﹠ Engineering University food and the biotechnology institutes such as the refined Li Lin in research [author] multitude sea of Grosvenor Momordica saponin, [periodical name] Food science .2003,24 (2) .-92-95[keywords] to extract Grosvenor Momordica saponin Grosvenor Momordica processing condition medical, edibles [digest] be raw material with the dried fructus momordicae to microwave-assisted, under the microwave radiation condition, with water is solvent extraction Grosvenor Momordica saponin, investigated microwave power, microwave irradiation time, solid-to-liquid ratio, extraction time, extract the influence of factors on extraction rate such as progression, the optimum extraction process condition of determining is: microwave power is 638W, and microwave irradiation time is 25min, and solid-to-liquid ratio is 1: 30, the water-bath extraction time is 2h, and extraction progression is secondary.The extraction yield of Grosvenor Momordica saponin is 90.35% with this understanding.8.[autograph] macroporous adsorbent resin extracts [mechanism] the South China Science ﹠ Engineering University food and the biotechnology institutes such as the refined Li Lin in research [author] multitude sea of Grosvenor Momordica saponin, [periodical name] foodstuffs industry science and technology .2003,24 (2) .-19-21[digests] studied the method that macroporous adsorbent resin extracts the Grosvenor Momordica saponin, concentration, pH and the salt ionic concentration of screening, Grosvenor Momordica saponin solution that comprises resin determined suitable absorption and desorption condition to the influence of adsorption process and the selection of strippant.9.[autograph] application [author] Zhu Xiaoyun of microwave technology in the fresh momordica glucoside extracts, He Chaowen [mechanism] Guilin strength whole food company limited, Guangxi University's biotechnology and sugar industry engineering college [periodical name] Guangxi light industry .2002 (2) .[digest] adopt orthogonal experiment to investigate microwave technology to extract Grosvenor Momordica in the Momordica-Glycosides technology and feed intake thing liquor ratio, microwave output power, extraction time, optimize the preferred plan of microwave extraction to the influence of extraction efficiency.With this scheme is experimental group, is control group with conventional water-boiling method, carries out parallel laboratory test.The result shows: the efficient of microwave extraction Momordica-Glycosides obviously is better than conventional water-boiling method.Be a kind of save time, economize can, new extracting method easy and simple to handle.10.[autograph] high performance liquid chromatography prepares Momordica grosvenori mogroside V standard substance [author] Yu Lijuan, Chen Quanbin, the auspicious brightness of justice, Yang Ruiyun, Zhang Yizheng [mechanism] Guangxi Normal University resource and environment is learned system, Sichuan University's molecular biology and biotechnology key lab [periodical name] chromatogram .2003 (7) .[digest] for furtheing investigate the pharmacological effect of functional component in the Grosvenor Momordica, further develop the effective constituent in the Grosvenor Momordica, the aqueous extract of Grosvenor Momordica fresh fruit is separated with the AB-8 polymeric adsorbent, behind the D-280 ion-exchange resin decolorization, utilize half preparative high-performance liquid chromatographic method to obtain triterpenoid saponin compounds Momordica grosvenori mogroside V standard substance, purity reaches 98.5%.Chromatographic column is Alltech Econosphere NH
2Post; Moving phase is acetonitrile-water (volume ratio is 68: 32) solution, flow velocity 5mL/min; Detect wavelength 203nm; 40 ℃ of column temperatures.That this method has is easy and simple to handle, favorable reproducibility, product purity advantages of higher.
Above-mentioned these methods have been discussed the extraction separation of mogroside (glucoside) from different perspectives, and the separation purity that has is higher, and method is simple, but the certain operations complex process is also arranged, can't suitability for industrialized production.And it is not because the polarity of mogroside is big, and easily separated with pigment.Preparation for high-purity momordica glycoside, there is half preparative high-performance liquid chromatographic method to separate at present, though the purity of the momordica glycoside V that this method separation obtains is very high, but used instrument is relatively more expensive and produce composition also than higher, is not suitable for the production of a large amount of purity greater than 90% momordica glycoside V.
Summary of the invention
In order to overcome the deficiency that existing momordica glycoside V purity is low, yield is low or production cost is high, the invention provides a kind of preparation method of high-purity momordica glycoside V, this production method can not only improve momordica glycoside V purity, and can reduce production costs.
The technical solution adopted for the present invention to solve the technical problems is:
(1) earlier Grosvenor Momordica is pulverized, with the alcohol heating reflux extraction 8-18h of 20%-70%, leaching extracting solution, the dregs of a decoction add 30%~60% alcohol heating reflux extraction 4-8h again, and united extraction liquid also is evaporated to paste, obtains A;
(2) A is separated with D101 type macroporous resin column, first deionized water and 20%~30% ethanol are eluted to colourless respectively, again with 30%~50% ethanol elution agent wash-out and collect its elutriant, are evaporated to the medicinal extract shape, must B;
(3) in normal phase silicagel column separated, utilizing chloroform-methanol-water (60: 40: 1~60: 40: 10) was eluent, made collected elutriant ratio be easier to reclaim; Separate at reverse phase silica gel C-18 post, utilize acetone-water to be eluent according to 25: 75~35: 65, used solvent toxicity is lower, uses the acetone of general industrial level to get final product, so production cost is lower.
It is nh 2 column that above-described HPLC detects chromatographic column; Moving phase: EtCN: H2O=75: 25~60: 40; Detect wavelength: 203nm; Flow velocity: 0.6ml/min~1ml/min.
The invention has the beneficial effects as follows: separate obtaining the mogroside standard substance by aforesaid method, can not only improve momordica glycoside V purity, and can reduce production costs, method is simple, good product purity.This provides convenience for studying monomeric activity of mogroside V and drug disposition process, also provides beneficial reference for preparing other Grosvenor Momordica Triterpene Glycosides class standard product.Simultaneously, measure the content of mogroside V in fruit, have great importance for the production of quality, the purchase of instructing Grosvenor Momordica and the mogroside product of determining the Grosvenor Momordica quality.
High-purity momordica glycoside V of the present invention can also be as senior sweeting agent except can be used as standard substance.The high sweet taste substance of contained sugariness is a kind of desirable natural sweeteners of low calory.
Embodiment
Embodiment one: (1). and get Grosvenor Momordica 5kg, pulverize, with 20% ethanol 5000ml heating and refluxing extraction 10h, leaching extracting solution, the dregs of a decoction add 60% ethanol 4000ml heating and refluxing extraction 6h again, and united extraction liquid also is evaporated to paste, obtains A.
(2). A is separated with D101 type macroporous resin column (the about 15cm of internal diameter, D101 type macroporous resin 5kg), and first deionized water and 20% ethanol are eluted to colourless respectively, again with 35% ethanol elution agent wash-out and collect its elutriant, are evaporated to the medicinal extract shape, must B.
(3). with B normal phase silicagel column (the about 10cm of internal diameter, silica gel 4kg, dry column-packing, sample on the dry method) separates, with chloroform-methanol-water (60: 40: 1) is eluent, collects the elutriant of the spot of Rf=0.23 (stationary phase is positive phase-plate, and developping agent is propyl carbinol-acetic acid-water=4: 1: 1), concentrating under reduced pressure obtains C.
(4). with C reverse phase silica gel C-18 post (the about 5cm of internal diameter, reverse phase silica gel C-180.5kg, wet method dress post, sample on the wet method) separates, acetone-water (25: 75) is an eluent, collects the elutriant of the spot of Rf=0.23 (positive phase-plate, propyl carbinol-acetic acid-water=4: 1: 1), concentrating under reduced pressure obtains the momordica glycoside V between 85%~98%.
Embodiment two: (1). and get Grosvenor Momordica 4kg, pulverize, with 30% ethanol 5000ml heating and refluxing extraction 16h, leaching extracting solution, the dregs of a decoction add 35% ethanol 5000ml heating and refluxing extraction 6h again, and united extraction liquid also is evaporated to paste, obtains A.
(2). A is separated with D101 type macroporous resin column (the about 15cm of internal diameter, D101 type macroporous resin 5kg), and first deionized water and 25% ethanol are eluted to colourless respectively, again with 40% ethanol elution agent wash-out and collect its elutriant, are evaporated to the medicinal extract shape, must B.
(3). with B normal phase silicagel column (the about 10cm of internal diameter, silica gel 4kg, dry column-packing, sample on the dry method) separates, with chloroform-methanol-water (60: 40: 5) is eluent, collects the elutriant of the spot of Rf=0.23 (stationary phase is positive phase-plate, and developping agent is propyl carbinol-acetic acid-water=4: 1: 1), concentrating under reduced pressure obtains C.
(4). with C reverse phase silica gel C-18 post (the about 5cm of internal diameter, reverse phase silica gel C-18 0.5kg, wet method dress post, sample on the wet method) separates, acetone-water (30: 70) is an eluent, collects the elutriant of the spot of Rf=0.23 (positive phase-plate, propyl carbinol-acetic acid-water=4: 1: 1), concentrating under reduced pressure obtains the momordica glycoside V between 85%~98%.
Embodiment three: (1). and get Grosvenor Momordica 5kg, pulverize, with 65% ethanol 5000ml heating and refluxing extraction 10h, leaching extracting solution, the dregs of a decoction add 55% ethanol 5000ml heating and refluxing extraction 5h again, and united extraction liquid also is evaporated to paste, obtains A.
(2). A is separated with D101 type macroporous resin column (the about 15cm of internal diameter, D101 type macroporous resin 5kg), and first deionized water and 30% ethanol are eluted to colourless respectively, again with 50% ethanol elution agent wash-out and collect its elutriant, are evaporated to the medicinal extract shape, must B.
(3). with B normal phase silicagel column (the about 10cm of internal diameter, silica gel 4kg, dry column-packing, sample on the dry method) separates, with chloroform-methanol-water (60: 40: 3) is eluent, collects the elutriant of the spot of Rf=0.23 (stationary phase is positive phase-plate, and developping agent is propyl carbinol-acetic acid-water=4: 1: 1), concentrating under reduced pressure obtains C.
(4). with C reverse phase silica gel C-18 post (the about 5cm of internal diameter, reverse phase silica gel C-18 0.5kg, wet method dress post, sample on the wet method) separates, acetone-water (30: 70) is an eluent, collect the elutriant of the spot of Rf=0.23 (positive phase-plate, propyl carbinol-acetic acid-water=4: 1: 1), and utilize HPLC to detect (chromatographic column: nh 2 column collected each part elutriant; Moving phase: EtCN: H
2O=75: 25; Detect wavelength: 203nm; Flow velocity: 1ml/min), merge the identical and purity of retention time greater than 98% momordica glycoside V component, concentrating under reduced pressure obtains purity at the momordica glycoside V more than 98%.
Embodiment four: (1). and get Grosvenor Momordica 4kg, pulverize, with 45% ethanol 5000ml heating and refluxing extraction 7h, leaching extracting solution, the dregs of a decoction add 65% ethanol 5000ml heating and refluxing extraction 7h again, and united extraction liquid also is evaporated to paste, obtains A.
(2). A is separated with D101 type macroporous resin column (the about 15cm of internal diameter, D101 type macroporous resin 5kg), and first deionized water and 20% ethanol are eluted to colourless respectively, again with 40% ethanol elution agent wash-out and collect its elutriant, are evaporated to the medicinal extract shape, must B.
(3). with B normal phase silicagel column (the about 10cm of internal diameter, silica gel 5kg, dry column-packing, sample on the dry method) separates, with chloroform-methanol-water (60: 40: 2) is eluent, collects the elutriant of the spot of Rf=0.23 (stationary phase is positive phase-plate, and developping agent is propyl carbinol-acetic acid-water=4: 1: 1), concentrating under reduced pressure obtains C.
(4). with C reverse phase silica gel C-18 post (the about 5cm of internal diameter, reverse phase silica gel C-18 0.5kg, wet method dress post, sample on the wet method) separates, acetone-water (27: 73) is an eluent, collect the elutriant of the spot of Rf=0.23 (positive phase-plate, propyl carbinol-acetic acid-water=4: 1: 1), and utilize HPLC to detect (chromatographic column: nh 2 column collected each part elutriant; Moving phase: EtCN: H
2O=60: 40; Detect wavelength: 203nm; Flow velocity: 0.6ml/min), merge the identical and purity of retention time greater than 98% momordica glycoside V component, concentrating under reduced pressure obtains purity at the momordica glycoside V more than 98%.