CN101481398B - Method for preparing high-purity 5-hydroxy-lamiophlomiol A glycoside and lamiophlomiol A glycoside extract from lamiophlomiol at the same time - Google Patents
Method for preparing high-purity 5-hydroxy-lamiophlomiol A glycoside and lamiophlomiol A glycoside extract from lamiophlomiol at the same time Download PDFInfo
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Abstract
The invention provides a method for simultaneously preparing high-purity sesamoside extract and a high-purity phlorigidoside C extract. In the method, lamiophlomis rotate is taken as a raw material, warmly immersed and extracted with ethanol solution, condensed, and then sequentially purified by a polyamide column and macroporous resin, and finally purified by a liquid phase chromatographic preparation system to obtain the high-purity sesamoside extract and the high-purity phlorigidoside with the concentration respectively reaching 98.85% and 99.37% which meets the quality requirements for reference substance used for national content determination. The method saves a silicagel column chromatography step, is easily operated, significantly reduces the loss of the sesamoside and the phlorigedoside C, and is suitable for industrialized large-scale production.
Description
Technical field
The invention belongs to medical technical field, specifically relate to a kind of from the plant Root of Common Lamiophlomis separation and purification simultaneously as the method for the monomeric compound sesamoside and the phlorigidoside C of reference substance.
Background technology
Root of Common Lamiophlomis [lamiophlomis rotata (benth.) kudo] is national folk herbal medicine such as Tibetan, illiteracy, Nahsi, has effects such as hemostasis, analgesia, promoting blood circulation and removing blood stasis, Azelaic Acid, strengthening immunity.Just on the books in the Tibetanmedicine masterpiece Four-Volume Medical Code of China before more than 1,000 year and " brilliant pearl book on Chinese herbal medicine ".Root of Common Lamiophlomis grows in high mountain steppe, river shoal of height above sea level 2700～4500m etc. and locates.Main product in Tibet, provinces and regions such as Qinghai, Yunnan, Sichuan, Gansu, the Tibetan medicine uses it for osteomyelitis, joint grasserie, fractures, gets injured by a fall, bullet wound etc.
Chinese Pharmacopoeia one one of version in 2005 has been included the Root of Common Lamiophlomis medicinal material, but wherein as the assay item of the key index of quality of medicinal material control, adopts the content of measuring luteolin in the medicinal material.Flavones ingredient has many-sided physiologically active, but whether the flavonoid in the Root of Common Lamiophlomis is that the main pharmacy composition in its hemostasis, analgesic awaits further to study.There are some researches show: the secoiridoid constituents in the Root of Common Lamiophlomis is its hemostasis, analgesic activity main pharmacodynamics composition, referring to the positive equality people of merchant, and PLA's Acta Pharmaceutica Sinica, Vol 21 (2005), and 272～274.Iridoid glycoside in the Root of Common Lamiophlomis comprises the compound of at least 26 kinds of similar, and wherein sesamoside and phlorigidoside C are 2 higher iridoid glycoside constituents of content in the Root of Common Lamiophlomis.
The Root of Common Lamiophlomis over-ground part is used alcohol reflux, and gained ethanol stream medicinal extract is used acetone extraction, and acetone extract is through the polymeric amide chromatography; Water elution part through silica gel column chromatography repeatedly, separates having obtained sesamoside, referring to people such as Zhang Chengzhong again; Herbal medicine, Vol 23 (1992), 509-510.This method is used the method separation and purification high polarity iridoid glycosides ingredient s esamoside of conventional silica gel column chromatography, and separating effect is not good, and product purity is low, and tends to exist bigger loss; The Root of Common Lamiophlomis root is used 95% ethanol and 50% ethanol ultrasonic extraction successively, and 50% ethanol extraction is used the D-1400 macroporous resin purification, and 10% ethanol elution part is through purification by silica gel column chromatography; Again through ODS-A reverse phase filler purifying; Sesamoside and phlorigidoside C have been prepared, referring to people such as Jun-Jie Tan, Helv.Chim.Acta; Vol 90 (2007), 143-148.There is following limitation in this method: the iridoid glycosides compositional polarity is bigger, has good water-solubility, and they also have very big solubleness in alcohol-water composition mixed solvent.Research shows: 95% ethanol is also better to the extraction effect of only iridoid glycoside, and its dried cream yield can reach 13%, and the content of iridoid glycosides ingredient s esamoside and phlorigidoside C can reach 4.7% and 3.1% respectively in the dried cream.Therefore earlier with 95% extraction using alcohol Root of Common Lamiophlomis medicinal material, can cause the greater loss of sesamoside and phlorigidoside C.
Chinese patent CN 1660163A discloses a kind of method of from Root of Common Lamiophlomis, extracting total iridoid glycoside, and it is raw material that this method adopts Root of Common Lamiophlomis, gets 20-70 ℃ of warm lixiviate with the 0-95% ethanolic soln; Extracting solution is concentrated into the medicinal extract shape, adds 10-15 times of 30-90 ℃ of water dissolution, puts cold; Filter, the weeding of grease soluble components gets extracting solution, and extracting solution adds polyamide column; Merge macroporous resin column on effluent and the water lotion, the ethanol elution macroporous resin column of 10-95% obtains total iridoid glycoside.The total iridoid glycoside that this method obtains is for forming complicated mixture system, and whether report does not contain sesamoside and phlorigidoside C.
Sesamoside also has discovery in other plant: the Root of Younghusband Jerusalemsage herb, and to pulverize the back and use 95% extraction using alcohol, extracting solution is reclaimed in underpressure distillation, gets crude extract.Crude extract is after treatment respectively with sherwood oil, chloroform, ETHYLE ACETATE and n-butanol extraction, and the n-butanol extraction part has then obtained compound sesamoside respectively through silica gel column chromatography, referring to people such as Gao Yongli, and Chinese medicinal materials, Vol 30 (2007), 1239-1242; Take from right exsiccant phlomis umbrosa root, the alcohol heating reflux with 95% after pulverizing extracts 3 times, each 6h.The extracting solution concentrating under reduced pressure gets medicinal extract, after medicinal extract adds the water suspendible; Extract with sherwood oil, vinyl acetic monomer, propyl carbinol respectively, the ethyl acetate extraction thing is through silicagel column, and gel permeation chromatography separates repeatedly; Preparation HPLC purifying obtains compound sesamoside, referring to people such as Liu Pu; Acta Pharmaceutica Sinica, Vol 42 (2007), 401-404; Radish bark of ash root meal is used 95% alcohol reflux, each 2h, and united extraction liquid, concentrating under reduced pressure gets medicinal extract.Medicinal extract through D101 type absorption with macroporous adsorbent resin, is used 10%, 40%, 95% ethanol elution with water dissolution and after diluting respectively.40% ethanol elution thing medicinal extract is through the decompression silica gel column chromatography, and chloroform/methanol gradient elution, gained each several part separate with preparative high-performance liquid chromatographic through silica gel column chromatography repeatedly again; Divide to obtain compound sesamoside, referring to people such as remained shock happiness, CHINA JOURNAL OF CHINESE MATERIA MEDICA; Vol 31 (2006), 656-658; Phlomis umbrosa underground part medicinal powder; Earlier with the lixiviate 4 times under slight boiling condition of 95% ethanol; Behind the concentrating under reduced pressure extractive substance suspended in water and extract respectively with ETHYLE ACETATE and propyl carbinol successively; N-butyl alcohol extract is used the chloroform/methanol gradient elution through silica gel column chromatography, and TCL detects and merges into 9 flow points (Fr1-9).Fr-3 is through silica gel column chromatography, and the chloroform/methanol gradient elution obtains sesamoside, referring to people such as Yang Yonglis, and Lanzhou University's journal, Vol 40 (2004), 67-71.
Above preparation technology adopts phytochemical research method, and extraction and the purifying process of sesamoside and phlorigidoside C are not all done further investigation, and research level also is confined to laboratory scale, is not suitable for suitability for industrialized production.Repeat aforesaid method, sesamoside that separation obtains and the purity of phlorigidoside C can not be up to state standards on inspection.Simultaneously, there are not the sesamoside of National standard and phlorigidoside C reference substance selling on the market.
On the basis of forefathers' research; The contriver is to the deficiency that at present should technology exists; Explore through research repeatedly, it is simple to have invented a kind of technology finally, the feasible method from Root of Common Lamiophlomis over-ground part while separation and purification sesamoside and phlorigidoside C in the industry.The chemical structural formula of sesamoside and phlorigidoside C is following:
Summary of the invention
The purpose of this invention is to provide a kind of method that purifying can be used as the sesamoside and the phlorigidoside C of reference substance of from Root of Common Lamiophlomis, extracting simultaneously.
The present invention realizes through following technical scheme:
(1) extract: the Root of Common Lamiophlomis meal, add 6-12 doubly, the ethanol of 50-80%, 50-80 ℃ of temperature soaked, extract 3 times, each 1-3 hour, united extraction liquid, concentrating under reduced pressure reclaims ethanol, gets liquid concentrator.
(2) polyamide purifying: the liquid concentrator that step (1) is obtained adds on the polyamide column, uses the 3-5BV water elution, collects upper prop effluent and water elution liquid, gets mixed solution.
(3) macroporous resin purification: the mixed solution that step (2) is obtained adds on the macroporous resin column, use the 5-10BV water elution earlier, uses the ethanol elution of 3-5BV 30-80% again, collects alcohol eluen, be evaporated to dried, must bullion.
(4) liquid chromatography preparation system purifying: the bullion that step (3) is obtained is through liquid chromatography preparation system RP-C18 column purification, and methanol-water (20-35%) wash-out detects wavelength 237nm; Collecting RT respectively is stream part of 9-17min and 11-19min; Concentrating under reduced pressure, lyophilize promptly gets.
Liquid chromatography preparation system purifying of the present invention comprises medium-pressure or high pressure liquid chromatography preparation system.
Medium-pressure or high pressure liquid chromatography preparation system of the present invention adopts the RP-C18 post.
The invention has the advantages that:
(1) sesamoside and the phlorigidoside C that prepare of this method, purity has reached 98.85% and 99.37% respectively.This method is to be suggested first, uses this method products therefrom to reach the specification of quality of national assay with reference substance first;
(2) this method adopts medium-pressure or high pressure liquid chromatography preparation system, has saved the step of silica gel column chromatography, and is simple to operate, significantly reduced the loss of sesamoside and phlorigedoside C;
(3) this method can separation and purification simultaneously obtain sesamoside and two kinds of materials of phlorigidoside C from Root of Common Lamiophlomis, and this technology has been practiced thrift cost greatly, is fit to industrialized production.
Attach: the carbon spectrum of sesamoside and plorigidoside C, hydrogen spectrum data such as following table:
The carbon spectrum data of sesamoside and plorigidoside C
The hydrogen spectrum data of sesamoside and plorigidoside C
Description of drawings
Fig. 1 is the performance liquid purity test figure of Sesamoside of the present invention, detects through performance liquid, and Sesamoside purity reaches 99.85%.
Fig. 2 is the performance liquid purity test figure of Phlorigidoside C of the present invention, detects through performance liquid, and Phlorigidoside purity reaches 99.37%.
Embodiment
Embodiment one
(1) extract: Root of Common Lamiophlomis meal 100g, add 6 times of amounts, 50% ethanol, 50 ℃ of temperature are soaked, extract 3 times, each 1 hour, united extraction liquid, concentrating under reduced pressure reclaims ethanol, and liquid concentrator filters, and must specific density is 1.05 liquid concentrator 300ml.
(2) polyamide purifying: liquid concentrator is added on the polyamide column, use the 3BV water elution, collect upper prop effluent and water elution liquid, get mixed solution 600ml.
(3) macroporous resin purification: mixed solution adds on the AB-8 macroporous resin column, use the 5BV water elution earlier, uses the ethanol elution of 3BV 30% again, collects alcohol eluen, be evaporated to dried, must crude extract 10.3g.
(4) liquid chromatography preparation system purifying: crude extract is through medium pressure liquid chromatography preparation system RP-C18 column purification, and methanol-water (25%) wash-out detects wavelength 237nm; Collecting RT respectively is stream part of 14min and 16min; Concentrating under reduced pressure, lyophilize promptly gets sesamoside; 312mg and phlorigidoside C, 285mg.
Embodiment two
(1) extract: Root of Common Lamiophlomis meal 100g, add 8 times of amounts, 60% ethanol, 70 ℃ of temperature are soaked, extract 3 times, each 1 hour, united extraction liquid, concentrating under reduced pressure reclaims ethanol, must specific density is 1.05 liquid concentrator 300ml.
(2) polyamide purifying: liquid concentrator is added on the polyamide column, use the 5BV water elution, collect upper prop effluent and water elution liquid, get mixed solution 800ml.
(3) macroporous resin purification: mixed solution adds on the XAD-1600 macroporous resin column, use the 8BV water elution earlier, uses the ethanol elution of 5BV 70% again, collects alcohol eluen, be evaporated to dried, must crude extract 11.9g.
(4) liquid chromatography preparation system purifying: crude extract is through medium pressure liquid chromatography preparation system RP-C18 column purification, and methanol-water (20%) wash-out detects wavelength 237nm; Collecting RT respectively is stream part of 11min and 13min; Concentrating under reduced pressure, lyophilize promptly gets sesamoside; 345mg and phlorigidoside C, 296mg.
Embodiment three
(1) extract: Root of Common Lamiophlomis meal 100g, add 10 times of amounts, 80% ethanol, 80 ℃ of temperature are soaked, extract 3 times, each 2 hours, united extraction liquid, concentrating under reduced pressure reclaims ethanol, must specific density is 1.10 liquid concentrator 140ml.
(2) polyamide purifying: crude extract 1 is added on the polyamide column, use the 5BV water elution, collect upper prop effluent and water elution liquid, get mixed solution 640ml.
(3) macroporous resin purification: liquid concentrator adds on the XAD-16 macroporous resin column, use the 10BV water elution earlier, uses the ethanol elution of 4BV 80% again, collects alcohol eluen, be evaporated to dried, must crude extract 11.9g.
(4) liquid chromatography preparation system purifying: crude extract is through medium pressure liquid chromatography preparation system RP-C18 column purification, and methanol-water (35%) wash-out detects wavelength 237nm; Collecting RT respectively is stream part of 6min and 9min; Concentrating under reduced pressure, lyophilize promptly gets sesamoside; 345mg and phlorigidoside C, 296mg.
Embodiment four
(1) extract: Root of Common Lamiophlomis meal 100g, add 8 times of amounts, 60% ethanol, 70 ℃ of temperature are soaked, extract 3 times, each 1 hour, united extraction liquid, concentrating under reduced pressure reclaims ethanol, must specific density is 1.05 liquid concentrator 300ml.
(2) polyamide purifying: liquid concentrator is added on the polyamide column, use the 5BV water elution, collect upper prop effluent and water elution liquid, get mixed solution 800ml.
(3) macroporous resin purification: mixed solution adds on the D101 macroporous resin column, use the 5BV water elution earlier, uses the ethanol elution of 4BV70% again, collects alcohol eluen, be evaporated to dried, must crude extract 10.8g.
(4) liquid chromatography preparation system purifying: crude extract is through HPLC preparation system RP-C18 column purification, and methanol-water (20%) wash-out detects wavelength 237nm; Collecting RT respectively is stream part of 6min and 9min; Concentrating under reduced pressure, lyophilize promptly gets sesamoside; 365mg and phlorigidoside C, 294mg.
Claims (3)
1. method that from Root of Common Lamiophlomis, prepares 98.85% purity sesamoside and 99.37% purity phlorigidosideC extract simultaneously may further comprise the steps:
(1) extract: the Root of Common Lamiophlomis meal, add the ethanol of 6-12 times of 50-80%, 50-80 ℃ of warm lixiviate got 3 times, each 1-3 hour, united extraction liquid, concentrating under reduced pressure reclaims ethanol, liquid concentrator;
(2) polyamide purifying: the liquid concentrator that step (1) is obtained adds on the polyamide column, uses the 3-5BV water elution, collects upper prop effluent and water elution liquid, gets mixed solution;
(3) macroporous resin purification: the mixed solution that step (2) is obtained adds on the macroporous resin column, use the 5-10BV water elution earlier, uses the ethanol elution of 3-5BV 30-80% again, collects alcohol eluen, be evaporated to dried, must bullion;
(4) liquid chromatography preparation system purifying: the bullion that step (3) is obtained is through liquid chromatography preparation system purifying, carries out gradient elution with the methanol-water of 20-35%, detects wavelength 237nm; Collecting RT respectively is stream part of 9-17min and 11-19min; Concentrating under reduced pressure, lyophilize promptly gets.
2. the method for preparing 98.85% purity sesamoside and 99.37% purity phlorigidosideC extract simultaneously as claimed in claim 1 is characterized in that: the liquid chromatography preparation system that (4) step wherein adopts comprises medium-pressure or high pressure liquid chromatography preparation system.
3. the method for preparing 98.85% purity sesamoside and 99.37% purity phlorigidosideC extract simultaneously as claimed in claim 2 is characterized in that: described medium-pressure or high pressure liquid chromatography preparation system adopts the RP-C18 post.
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| CN 200810182239 CN101481398B (en) | 2009-02-23 | 2009-02-23 | Method for preparing high-purity 5-hydroxy-lamiophlomiol A glycoside and lamiophlomiol A glycoside extract from lamiophlomiol at the same time |
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| CN106589009B (en) * | 2016-12-08 | 2019-05-21 | 中国科学院西北高原生物研究所 | Phlorigidoside C natural products standard items preparation method and applications in lamiophlomis rotata |
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