CN1903239B - Method for extraction and separation of rhizome of chuanixong - Google Patents

Method for extraction and separation of rhizome of chuanixong Download PDF

Info

Publication number
CN1903239B
CN1903239B CN2005100122743A CN200510012274A CN1903239B CN 1903239 B CN1903239 B CN 1903239B CN 2005100122743 A CN2005100122743 A CN 2005100122743A CN 200510012274 A CN200510012274 A CN 200510012274A CN 1903239 B CN1903239 B CN 1903239B
Authority
CN
China
Prior art keywords
component
mobile phase
collection time
elutriant
phase
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Expired - Fee Related
Application number
CN2005100122743A
Other languages
Chinese (zh)
Other versions
CN1903239A (en
Inventor
程翼宇
贺庆
王毅
王学伟
李云飞
水文波
胡兴江
葛志伟
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Tasly Pharmaceutical Group Co Ltd
Original Assignee
Tianjin Tasly Pharmaceutical Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Tianjin Tasly Pharmaceutical Co Ltd filed Critical Tianjin Tasly Pharmaceutical Co Ltd
Priority to CN2005100122743A priority Critical patent/CN1903239B/en
Publication of CN1903239A publication Critical patent/CN1903239A/en
Application granted granted Critical
Publication of CN1903239B publication Critical patent/CN1903239B/en
Expired - Fee Related legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Landscapes

  • Medicines Containing Plant Substances (AREA)

Abstract

A standardized extracting and separating method for extracting the chemical components from a Chinese-medicinal material to maximal extent and further separating them to obtain the chemical components with different polarities is disclosed. To be specific, the Chuan-xiong rhizome is divided into several chemical components according to their polarities. Each component contains only several main compounds. Said components can constitute a component library used for medicine screening to find out novel medicines.

Description

A kind of extraction and separation method of Ligusticum wallichii medicinal material
Technical field
The invention belongs to field of medicaments, particularly relate to Ligusticum wallichii extraction, separation criterionization.
Background technology
At present, in the world, herbal medicine all has certain market, along with people increasing and the aging of population to the health requirements level of understanding, sub-health stateization, people thirst for back to nature more, utilize the high pharmacological agent of pure natural degree, prevent some chemical synthetic drugs cann't be solved problem, so the application of natural plant exceeds the background of its original traditional national culture.From natural drug, seek the little and inexpensive medicine of side effect and become the target that countries in the world pharmaceutical manufacturer is chased.The European Community has carried out unified legislation to herbal medicine, state herbal medicine status such as Canada and Australia have legalized, United States Government has also drafted the plant amedica management method, the compound mixed preparation that begins to accept natural drug is as curative, and these provide good international environment for Chinese medicine enters international medical market as curative.On the other hand, along with the quickening of global economic integration progress, particularly China becomes a full member of WTO, and Chinese Medicine market incorporates the breadth and depth of international medical big market and will further aggravate.Face the enormous impact of Asian countries's traditional medicine product such as the cut-throat competition of powerful transnational medical group and Japan, Korea S, India, Thailand and European countries' plant amedicas such as Germany, France, numerous products that China's traditional Chinese medicine produces are owing to still can not meet the standard of international medical market and requirement and being kept outside of the door.
The stdn that Chinese medicinal materials extracts and the stdn of Chinese medicinal preparation method are that Chinese patent medicine moves towards the world market or really realizes big industrial key link, also are that the scientific research personnel of Chinese Medicine worker and foreign study natural plant makes great efforts the direction studied always.Extracting method about Chinese medicine is the emphasis of field of Chinese medicines research at present, the scientific research personnel focuses on the more effective constituent which kind of method of employing could obtain with the sight of research always from Chinese medicinal materials, therefore the situation that present Chinese medicinal materials extracts is: also different at its extraction of effective components of different Chinese medicinal materialss, and adopting different extracting method to need the extraction that a large amount of different extraction equipment are used to support Chinese medicinal materials at different Chinese medicinal materialss, this is unsuitable for the stdn that Chinese medicinal materials extracts.
Ligusticum wallichii is the dry rhizome of samphire Ligusticum wallichii Ligusticum chuanxiong Hort..The traditional Chinese medical science thinks that it distinguishes the flavor of hot, and is warm in nature, returns liver, courage, pericardium channel.Have blood-activating and qi-promoting, wind-expelling pain-stopping is applicable to menoxenia, through closing dysmenorrhoea ， Disorder lump in the abdomen stomachache, the shouting pain of the chest side of body, tumbling and swelling, headache, rheumatic arthralgia.Modern study shows that main rhizome contains volatile oil, alkaloid, phenolic constituent, lactone, forulic acid in the Ligusticum wallichii.Rhizome contains volatile oil about 1%.Identify fuel-displaced in composition have 40 kinds, account for 93.64% of volatile oil, wherein principal constituent be that Z-ligustilide (ligustilide) accounts for 58%, 3-fourth husband lactone (3-butylphthalide) 5.29% and sabinene (sabinene) 6.08%.Contained lactone compound in the rhizome, except that above-mentioned two kinds of mentioning, still contain butylene husband lactone (butylidene phthalide), cnidium lactone (sankyunolide), neocnidilide (neocnidilide), 4-hydroxyl-3-fourth husband lactone (4-hydroxy-3-butyl phthalide), chuanxingol (chuanxingol), two Z-ligustilide (2,2 '-diligustilide), and 3-butyl-3,6,7-trihydroxy--4,5,6,7-tetrahydrochysene phthalide etc.Nitrogenous compound has trimethylamine hydrochloride, choline hydrochloride, the different bright acyl of L--L-Xie Ansuan acid anhydride (L-isobutyl-L-valineanhydride), L-valyl-L-Xie Ansuan acid anhydride, 1-ethanoyl-β-Ka quinoline, 1-β-propylene acetate-7-aldehyde radical-β-Ka quinoline, uridylic, VITAMIN B4 and gland and adenosine.Acidity or phenoloid have 4-hydroxy 3-methoxybenzene ethene, 1-hydroxyl-1-(3-methoxyl group-4-hydroxybenzene)-ethane, 4-hydroxy-benzoic acid, coffic acid, vanillic acid, forulic acid (ferulic acid), sedanoic acid (sadanic acid), rhubarb yellow (chrysophic acid), palmitinic acid, Vanillin and linolic acid.In addition, the Ligusticum wallichii rhizome still contains neutral oil, and its composition is 15,16,17, the octadecanoic acid ethyl ester, different 17, isooctadecane acetoacetic ester and different methyl margarate.Other contains 5,5 '-furil ether (bis-5,5 '-formylfurfur ether), spainulenol (spathulenol), β-Gu Zaichun, sucrose and a kind of fat glycerides.
How to determine a kind of extraction, separation criterion method, the active constituents of medicine in the Ligusticum wallichii not only can be extracted fully, can also further separate the active constituents of medicine that is obtained by separation method.The establishment of this extraction, separation criterionization is that present Chinese medicine is realized modern key factor.
Summary of the invention
The object of the present invention is to provide a kind of Ligusticum wallichii extraction, separation criterionization.Exactly Rhizoma Chuanxiong extract is divided into fractions specifically, each component comprises several main compound, has constituted a medicinal material component pool by these medicinal material components.Can carry out drug screening to the medicinal material component pool, thereby find new drug.
For modernization, the stdn that realizes that Ligusticum wallichii extracts, the inventor is by a large amount of tests, the extraction of effective components of present Ligusticum wallichii is concluded and put in order, with seek a kind of can standardized extracting method, promptly under this extraction conditions, adopt this standard extraction methods that Ligusticum wallichii is extracted, can obtain the effective constituent in the Chinese medicine to greatest extent.
Ligusticum wallichii of the present invention extracts, separation criterionization is that the inventor passes through test, and after Chinese medicinal materials adopted different extracting method to compare simply together, the resulting Ligusticum wallichii that is suitable for suitability for industrialized production extracted, separation criterionization.
The present invention can implement through the following steps:
(1) extraction process: take by weighing the Ligusticum wallichii medicinal material,, add ethyl acetate and ethanol then its grinding and sieving, the heating extract extracting solution fr.1.In the dregs of a decoction, add ethanol, the heating extract extracting solution fr.2.In the dregs of a decoction, add entry at last, the heating extract extracting solution fr.3.
(2) separating technology: after extracting solution fr.1 is condensed into medicinal extract, use silica gel mixed sample, adopt normal phase silicagel column that it is separated, at first use sherwood oil and ethyl acetate as moving phase, get elutriant fr.4, change chloroform and methyl alcohol then, get elutriant fr.5 as moving phase, use methyl alcohol as moving phase at last, get elutriant fr.6.After extracting solution fr.2 is condensed into medicinal extract, with sample on the dissolve with methanol, adopt the ODS-C18 post that it is separated, the methyl alcohol of at first using lower concentration is as moving phase, get elutriant fr.7, the methyl alcohol that changes intermediate concentration then gets elutriant fr.8 as moving phase, use pure methanol solution as moving phase at last, get elutriant fr.9.
(3) preparation separating technology: continue to separate fr.5 and the fr.8 that obtains in the previous process with preparative liquid chromatography.Chromatographic column is semipreparative column (Lichrospher C18; 10.0mm * 250mm, 5 μ m), moving phase is water and acetonitrile, gradient elution is collected cut in the corresponding time period, obtains to have passed through further isolating each component.
Preferred the present invention can implement through the following steps:
(1) extraction process: take by weighing the Ligusticum wallichii medicinal material, with its grinding and sieving, adding 5～12 times of amount volume ratios then is 1: 1 ethyl acetate and ethanol, and reflux 0.5～3 hour is extracted 1～3 time, and merging filtrate gets extracting solution fr.1.Add 5～12 times of amount 70% ethanol in the dregs of a decoction, reflux 0.5～3 hour is extracted 1～3 time, and merging filtrate gets extracting solution fr.2.Add entry in the dregs of a decoction at last, reflux 0.5～3 hour is extracted 1～3 time, and merging filtrate gets extracting solution fr.3.
(2) separating technology: after extracting solution fr.1 is condensed into medicinal extract, use silica gel mixed sample, adopt normal phase silicagel column that it is separated, be that 50: 1 sherwood oil and ethyl acetate are as moving phase at first with volume ratio, elutriant fr.4, change volume ratio then and be 10: 1 chloroform and methyl alcohol as moving phase, elutriant fr.5, use methyl alcohol as moving phase at last, get elutriant fr.6.After extracting solution fr.2 is condensed into medicinal extract, with sample on 2～10% dissolve with methanol, adopt the ODS-C18 post that it is separated, at first use 2～10% methyl alcohol as moving phase, get elutriant fr.7, change 40～80% methyl alcohol then, get elutriant fr.8 as moving phase, use 100% methanol solution as moving phase at last, get elutriant fr.9.
(3) preparation separating technology: continue to separate fr.5 and the fr.8 that obtains in the previous process with preparative liquid chromatography.Chromatographic column is semipreparative column (Lichrospher C18; 10.0mm * 250mm, 5 μ m), moving phase is water and acetonitrile, and gradient elution, flow velocity are 3ml/min, and column temperature is a room temperature, collects cut in the corresponding time period, obtains through further isolating each component.
Best the present invention can implement through the following steps:
(1) extraction process: take by weighing the Ligusticum wallichii medicinal material, with its grinding and sieving, adding 8 times of amount volume ratios then is 1: 1 ethyl acetate and ethanol, and reflux 1 hour is extracted 2 times, and merging filtrate gets extracting solution fr.1; Add 8 times of amount 70% ethanol in the dregs of a decoction, reflux 1 hour is extracted 2 times, and merging filtrate gets extracting solution fr.2; Add entry in the dregs of a decoction at last, reflux 1 hour is extracted 2 times, and merging filtrate gets extracting solution fr.3;
(2) separating technology: fr.1 is condensed into medicinal extract with extracting solution, use silica gel mixed sample, adopt normal phase silicagel column that it is separated, be that 50: 1 sherwood oil and ethyl acetate are as moving phase at first with volume ratio, elutriant fr.4, change volume ratio then and be 10: 1 chloroform and methyl alcohol as moving phase, elutriant fr.5, use methyl alcohol as moving phase at last, get elutriant fr.6; Fr.2 is condensed into medicinal extract with extracting solution, with sample on 5% dissolve with methanol, adopt the ODS-C18 post that it is separated, at first use 5% methyl alcohol as moving phase, get elutriant fr.7, change 60% methyl alcohol then, get elutriant fr.8 as moving phase, use 100% methanol solution as moving phase at last, get elutriant fr.9;
(3) preparation separating technology: use preparative liquid chromatography from fr.5 and fr.8;
The separation condition of fr.5: chromatographic column is semipreparative column (the Lichrospher C18 of Chinese nation; 10.0mm * 250mm, 5 μ m); Adopt gradient elution, moving phase is water (A) and acetonitrile (B), and flow velocity is 3ml/min, and column temperature is a room temperature, and gradient is as follows:
During 0min, mobile phase A is 75% water, and Mobile phase B is 25% acetonitrile solution;
During 40min, mobile phase A is 5% water, and Mobile phase B is 95% acetonitrile solution;
During 50min, mobile phase A is 5% water, and Mobile phase B is 95% acetonitrile solution;
The fr.5 separating resulting: the component and the corresponding collection time that are obtained by the preparative liquid chromatography separation are as follows:
Component fr.51, collection time 3.0-10.0min;
Component fr.52, collection time 10.0-16.2min;
Component fr.53, collection time 16.2-24.0min;
Component fr.54, collection time 24.0-27.8min;
Component fr.55, collection time 27.8-31.8min;
Component fr.56, collection time 31.8-38.0min;
Component fr.57, collection time 38.0-43.0min;
Component fr.58, collection time 43.0-50.0min;
The fr.8 separation condition: chromatographic column is Agilent preparative column (Zorbax SB-C18; 21.2mm * 250mm); Adopt gradient elution, moving phase is 0.3% aqueous acetic acid (A) and 0.3% acetate acetonitrile solution (B), and flow velocity is 10ml/min, and column temperature is a room temperature, and gradient is as follows:
During 0min, mobile phase A is 95% 0.3% aqueous acetic acid, and Mobile phase B is 5% 0.3% acetate acetonitrile solution;
During 8min, mobile phase A is 80% 0.3% aqueous acetic acid, and Mobile phase B is 20% 0.3% acetate acetonitrile solution;
During 38min, mobile phase A is 50% 0.3% aqueous acetic acid, and Mobile phase B is 50% 0.3% acetate acetonitrile solution;
During 48min, mobile phase A is 5% 0.3% aqueous acetic acid, and Mobile phase B is 95% 0.3% acetate acetonitrile solution;
During 55min, mobile phase A is 5% 0.3% aqueous acetic acid, and Mobile phase B is 95% 0.3% acetate acetonitrile solution;
The fr.8 separating resulting: the component and the corresponding collection time that are obtained by the preparative liquid chromatography separation are as follows:
Component fr.81, collection time 4.0-19.8min;
Component fr.82, collection time 19.8-26.0min;
Component fr.83, collection time 26.0-35.0min;
Component fr.84, collection time 35.0-42.0min;
Component fr.85, collection time 42.0-44.0min;
Component fr.86, collection time 44.0-47.0min;
Component fr.87, collection time 47.0-55.0min.
In order to obtain concrete Ligusticum wallichii effective constituent, best extraction and separation method of the present invention is:
(1) extraction process: take by weighing Ligusticum wallichii medicinal material 250g, with its grinding and sieving, adding 8 times of amount volume ratios then is 1: 1 ethyl acetate and ethanol, and reflux 1 hour is extracted 2 times, and merging filtrate gets extracting solution fr.1.Add 8 times of amount 70% ethanol in the dregs of a decoction, reflux 1 hour is extracted 2 times, and merging filtrate gets extracting solution fr.2.Add entry in the dregs of a decoction at last, reflux 1 hour is extracted 2 times, and merging filtrate gets extracting solution fr.3.
(2) separating technology: extracting solution fr.1 is concentrated 51.8g medicinal extract, get fr.1 medicinal extract 10.2g silica gel mixed sample, adopt normal phase silicagel column that it is separated, at first with volume ratio be 50: 1 sherwood oil and ethyl acetate as moving phase, elutriant fr.4, change volume ratio then and be 10: 1 chloroform and methyl alcohol as moving phase, get elutriant fr.5, concentrate the 2.5g sample, use methyl alcohol as moving phase at last, elutriant fr.6.With extracting solution fr.2 concentrate 88.7g medicinal extract, get 10.0g medicinal extract with sample on 5% dissolve with methanol, adopt the ODS-C18 post that it is separated, at first use 5% methyl alcohol as moving phase, get elutriant fr.7, change 60% methyl alcohol then as moving phase, get elutriant fr.8, concentrate the 5.6g sample, use 100% methanol solution as moving phase at last, elutriant fr.9.
(3) preparation separating technology: in order from step (2), to obtain fr.5 and the more concrete effective constituent of fr.8 in the Rhizoma Chuanxiong extract, continue to separate fr.5 and fr.8 among the present invention with preparative liquid chromatography.
The separation condition of fr.5: chromatographic column is semipreparative column (the Lichrospher C18 of Chinese nation; 10.0mm * 250mm, 5 μ m); Adopt gradient elution, moving phase is water (A) and acetonitrile (B), and flow velocity is 3ml/min, and column temperature is a room temperature, and gradient is as follows:
During 0min, mobile phase A is 75% water, and Mobile phase B is 25% acetonitrile solution;
During 40min, mobile phase A is 5% water, and Mobile phase B is 95% acetonitrile solution;
During 50min, mobile phase A is 5% water, and Mobile phase B is 95% acetonitrile solution;
The fr.5 separating resulting: get component fr.5 2.0g altogether, use the dissolve with ethanol sample introduction, the component and the corresponding collection time that are obtained by the preparative liquid chromatography separation are as follows:
Component fr.51, collection time 3.0-10.0min, 220mg;
Component fr.52, collection time 10.0-16.2min, 117mg;
Component fr.53, collection time 16.2-24.0min, 136mg;
Component fr.54, collection time 24.0-27.8min, 221mg;
Component fr.55, collection time 27.8-31.8min, 175mg;
Component fr.56, collection time 31.8-38.0min, 107mg;
Component fr.57, collection time 38.0-43.0min, 328mg;
Component fr.58, collection time 43.0-50.0min, 177mg;
The fr.8 separation condition: chromatographic column is Agilent preparative column (Zorbax SB-C18; 21.2mm * 250mm); Adopt gradient elution, moving phase is water (A) and acetonitrile (B), and flow velocity is 10ml/min, and column temperature is a room temperature, and gradient is as follows:
During 0min, mobile phase A is 95% 0.3% aqueous acetic acid, and Mobile phase B is 5% 0.3% acetate acetonitrile solution;
During 8min, mobile phase A is 80% 0.3% aqueous acetic acid, and Mobile phase B is 20% 0.3% acetate acetonitrile solution;
During 38min, mobile phase A is 50% 0.3% aqueous acetic acid, and Mobile phase B is 50% 0.3% acetate acetonitrile solution;
During 48min, mobile phase A is 5% 0.3% aqueous acetic acid, and Mobile phase B is 95% 0.3% acetate acetonitrile solution;
During 55min, mobile phase A is 5% 0.3% aqueous acetic acid, and Mobile phase B is 95% 0.3% acetate acetonitrile solution;
The fr.8 separating resulting: get component fr.8 2.0g altogether, the dissolve with methanol sample introduction with 20%, the component and the corresponding collection time that are obtained by the preparative liquid chromatography separation are as follows:
Component fr.81, collection time 4.0-19.8min, 182mg;
Component fr.82, collection time 19.8-26.0min, 200mg;
Component fr.83, collection time 26.0-35.0min, 106mg;
Component fr.84, collection time 35.0-42.0min, 141mg;
Component fr.85, collection time 42.0-44.0min, 39mg;
Component fr.86, collection time 44.0-47.0min, 87mg;
Component fr.87, collection time 47.0-55.0min, 107mg.
Among the present invention, contained compound sees Table 1 in each component of Ligusticum wallichii, and its Analysis and Identification method is referring to embodiment three.
Contained compound in each component of table 1. Ligusticum wallichii
Figure G05112274320050818D000071
Above Ligusticum wallichii, extraction solution can increase or reduce when industrialization is extracted according to corresponding ratio, as scale operation can be unit with kilogram or with the ton, small-scale production can be unit with the gram also, and weight can increase or reduce, but its weight proportion constant rate.
The extraction that the present invention set up, separation criterionization go for any Chinese medicinal materials in the present Chinese medicine, for example can be used for asafoetide, tarragon, st-yrax, Semen Platycladi, turtle shell, betel nut, peppermint, Semen Ricini, Hold, Psoralea corylifolia, Root of Indigowoad, the Bi roots of grass, crotons, Root of Medicinal Indian mulberry, Rhizoma Menispermi, Root of coastal Glehnia, bletilla, Root of Chinese Pulsatilla, the root of herbaceous peony, the root of Dahurain angelica, Rhizoma Typhonii, Rhizome of Lalang Grass, gingko, Cynanchum glaucescens, White Hyacinth Bean, radix ampelopsis, Root-bark of Densefruit Pittany, radix cynanchi atrati, Herba Lobeliae Chinensis, Herba Scutellariae Barbatae, the tuber of pinellia, lily, borneol, radix bupleuri, cicada slough, Papermulberry Fruit, Changshan, bark of tree of heaven, Squama Manis, Herba Andrographis, Semen Phaseoli, Red Halloysite, the radix paeoniae rubrathe, rhizoma atractylodis, Siberian Cocklour Fruit, agalloch eaglewood, Stringy Stonecrop Herb, Leafy twigs of Oriental Arborvitae, radix aconiti agrestis, Folium Aconiti Kusnezoffii, in one's early teens, tsaoko, Unibract Fritillary Bulb, Root of Medicinal Cyathula, monkshood, Ligusticum wallichii, Szechwan Chinaberry Fruit, Semen Plantaginis, Semen Sojae Preparatum, Radix Codonopsis, Herba Lophatheri, the bark of eucommia, levisticum, Arisaema with Bile, setose thistle, Pericarpium Arecae, Tree Peony Bark, plaster of paris, Peel of Chinese Waxgourd, Cordyceps sinensis, earthworm, the Fruit of Belveder, Root-bark of Chinese Wolfberry, Radix Rehmanniae Preparata, Humifuse Euphorbia Herb, garden burnet, Radix Angelicae Sinensis, Rush, cloves, sword bean, Leaf of Indigowoad, date, rheum officinale, Small Centipeda Herb, curcuma zedoary, catechu, Chinese torreyanut, duckweed, Bi separates, raspberry, Buddha's hand, monkshood, Poria cocos, the root of fangji, windproof, sennae, honey, gekko, cassia twig, the root of kudzu vine, Jehol Ligusticum Rhizome, Rhizoma Alpiniae Officinarum, Rhizome of Fortune's Drynaria, rice sprout, Flower of Buerger Pipewort, rhizoma cibotii, wolfberry fruit, Folium Ilicis Cornutae, yncaria stem with hooks, rhizoma nardostachyos, Radix Glycyrrhizae, the root of gansui, Snakegourd Fruit, Snakegourd Seed, Snakegourd Peel, Stem of Manshurian Dutchmanspipe, rhizoma zingiberis, baked ginger, dried lacquer, Lappula echinata, the Radix Astragali, sealwort, marine alga, sophora flower, Kadsura Pepper Stem, lotus leaf, the root of large-flowered skullcap, the coptis, golden cypress, Chinese prickly ash, Tuber Fleeceflower Root, giant knotweed, Rhizoma Picrorhizae, the bark of official magnolia, Pummelo Peel, Hemp Seed, Silktree Albizzia Bark, Flower of Silktree Albizzia, Radix Knoxiae, safflower, Semen Sesami Nigrum, chrysanthemum, stiff silkworm, balloonflower root, tangerine seed, turmeric, Yunnan Caulis Spatholobi, Flos Celosiae Cristatae, Root of Hairystalk Tinospora, Inula lineariifolia Turcz., Wild Buckwheat Rhizome, Longhairy Antenoron Herb, Japanese Honeysuckle, dalbergia wood, schizonepeta, semen allii tuberosi, Semen Cassiae, Flos Farfarae, the myrobalan, Semen Armeniacae Amarum, kuh-seng, Cortex Meliae, Semen Raphani, stone-like omphalia, Flos Campsis, Pyrola Herb, Radix Rhapontici seu Radix Echinopsis, play both sides of the street, lotus seeds, Stem of confederate-jasmine, aloe, reed rhizome, pointed at both ends, Shinyleaf Pricklyash Root, the capsule of weeping forsythia, glossy ganoderma, Folium Apocyni Veneti, Grosvenor Momordica, Semen Litchi, rough gentian, longan aril, Herba Erodii, Yerbadetajo Herb, Chinese ephedra, Simpleleaf Shrub Chastetree Fruit, Rhododendron dauricum, Oleum Rhododendri Daurici, Pale Butterflybush Flower, plum blossom, Chinese ephedra, the tuber of dwarf lilyturf, Fructus Hordei Germinatus, Rose, Root of Medicinal Changium, vervain, Virginia snakeroot, nux vomica, Semen Strychni Pulveratum, Lasiosphaera fenzlii, purslane, pawpaw, the banksia rose, the scouring rush, Root of Upright Ladybell, Glossy Privet Fruit, Great Burdock Achene, the root of bidentate achyranthes, rhizoma nelumbinis, cattail pollen, taraxacum, Loquat Leaf, eupatorium, Tumeric, fringed pink, the bark of ash, adder-wort, Gorgon fruit, notopterygium root, Slender Dutchmanspipe Root, Stem of Orientoine, rascal, Semen Celosiae, sweet wormwood, indigo naturalis, madder, the Semen Pharbitidis, the root of purple-flowered peucedanum, Rhizome of Obscured Homalomena, Semen Euphorbiae, bark of ash, Hedge Prinsepia Nut, Semen Myristicae, Herba Cistanches, Chinese cassia tree, genseng, the Ginseng Leaf, Rhizome of Grass leaf Sweelflag, cynomorium songaricum, mulberry leaf, Phytolacca acinosa, mulberry fruit, Fructus Cnidii, White Mulberry Root-bark, ramulus mori, ramulus mori, loranthus parasiticus, Wilsom Buckeye Seed, Spina Date Seed, blackberry lily, Styrax, Semen Astragali Complanati, the Rangooncreeper Fruit, the calyx and receptacle of a persimmon, fructus amomi, Radix Sophorae Tonkinensis, skunk bush, Chinese yam, Pseudobulbus Cremastrae Seu Pleiones, hawthorn, field thistle, rattletop, Cornu Bubali, Pulvis Cornus Bubali Concentratus, pyrrosia lingua, Sea-ear Shell, the stem of noble dendrobium, pomegranate rind, ginger, Vegetable Sponge of Luffa, Ligusticum wallichii, rhizoma sparganic, Semen Lepidii (Semen Descurainiae), South Dodder Seed Chinese Dodder Seed, peach kernel, the stem pith of the rice-paper plant, santal, Snakegourd Root, Tabasheer, Rhizoma Arisaematis, rhizoma Gastrodiae, Root of Muskroot-like Semiaquilegia, Root of Heterophylly Faalsestarwort, Golden Larch Bark, Rhizome of Glabrous Greenbrier, Medcinal Evodia Fruit, Root of Sixpetal Clematis, Semen Vaccariae, Wu Chia Pee, shizandra berry, Turkey-galls, Concha Arcae, the root of three-nerved spicebush, dark plum, Rhizome of Decumbent Corydalis, teasel root, Inula Flower Herba Siegesbeckiae, Longstamen Onion Bulb, Spica Prunellae, the flower bud of lily magnolia, Root-bark of Chinese Silkvine, rhizoma cyperi, citron, elscholtiza, sweet fennel, thizoma curculiginis, Herba Agrimoniae, radix scrophulariae, Weathered Sodium Sulfate, Radix Panacis Quinquefolii, Crinis Carbonisatus, dragon's blood, Radix Cynanchi Paniculati, Herba Epimedii, Motherwort Herb, Ginkgo Leaf, Semen Coicis, Starwort Root, polygala root, lilac daphne, Semen Pruni, root tuber of aromatic turmeric, Herba Houttuyniae, oriental wormwood, kosam seeds, Flos Rosae Chinensis, radix polygonati officinalis, Yanhusuo, Thunberg Fritillary Bulb, Fructus Gleditsiae Abnormalis, perilla seed, aster, Philippine Violet Herb, Asian puccoon, umbellate pore furgus, Spine of Chinese Honeylocust, the wind-weed, the Herba Lycopi, rhizoma alismatis, nacre, the dried immature fruit of citron orange, cape jasmine.
Any form on the pharmaceutics be can make according to the Ligusticum wallichii effective constituent that the inventive method obtained, injection and oral preparations comprised.Wherein injection comprises injection liquid, drip liquid, powder injection; Oral preparations comprises granule, tablet, electuary, powder, oral liquid, sugar coated tablet, film coated tablet, enteric coated tablet, capsule, hard capsule, soft capsule, sucks agent, granule, pill, paste, sublimed preparation, sprays, pill, disintegrating agent, orally disintegrating tablet, micropill etc.
To those skilled in the art, technology contents disclosed according to the present invention, those skilled in the art will very clear other embodiment of the present invention, and the embodiment of the invention is only as example.Under the situation of not violating purport of the present invention and scope; can carry out various changes and improvements to the present invention; for example selected solution can be other lower alcohols or other organic solvents, but as long as use extracting method of the present invention, all within protection domain of the present invention.
Description of drawings
Provide Ligusticum wallichii finger printing, water-soluble each the component ultraviolet spectrogram of Ligusticum wallichii, water-soluble each the component mass spectrum of Ligusticum wallichii, fat-soluble each the component ultraviolet spectrogram of Ligusticum wallichii, fat-soluble each the component mass spectrum of Ligusticum wallichii below, be intended to further specify the present invention, but the present invention is not construed as limiting.
Fig. 1 Ligusticum wallichii finger printing
Water-soluble each the component uv-spectrogram of Fig. 2 Ligusticum wallichii
Water-soluble each the component mass spectrum of Fig. 3 Ligusticum wallichii
Fat-soluble each the component uv-spectrogram of Fig. 4 Ligusticum wallichii
Fat-soluble each the component mass spectrum of Fig. 5 Ligusticum wallichii
Embodiment
Embodiment one
(1) extraction process: take by weighing 250g Ligusticum wallichii medicinal material,, add ethyl acetate and ethanol then its grinding and sieving, the heating extract extracting solution fr.1.In the dregs of a decoction, add ethanol, the heating extract extracting solution fr.2.In the dregs of a decoction, add entry at last, the heating extract extracting solution fr.3.
(2) separating technology: with extracting solution fr.1 concentrate 49.6g medicinal extract, get fr.1 medicinal extract 10.2g silica gel mixed sample, adopt normal phase silicagel column that it is separated, at first use sherwood oil and ethyl acetate as moving phase, get elutriant fr.4, change chloroform and methyl alcohol then as moving phase, get elutriant fr.5, concentrate the 2.2g sample, use methyl alcohol as moving phase at last, elutriant fr.6.With extracting solution fr.2 concentrate 85.6g medicinal extract, get sample on the 10.0g medicinal extract dissolve with methanol, adopt the ODS-C18 post that it is separated, the methyl alcohol of at first using lower concentration gets elutriant fr.7 as moving phase, and the methyl alcohol that changes intermediate concentration then is as moving phase, get elutriant fr.8, concentrate the 5.0g sample, use pure methanol solution as moving phase at last, elutriant fr.9.
(3) preparation separating technology: continue to separate fr.5 and the fr.8 that obtains in the previous process with preparative liquid chromatography.Chromatographic column is semipreparative column (Lichrospher C18; 10.0mm * 250mm, 5 μ m), moving phase is water and acetonitrile, gradient elution is collected cut in the corresponding time period, obtains to have passed through further isolating each component.
The fr.5 separating resulting: get component fr.5 2.0g altogether, use the dissolve with ethanol sample introduction, the component and the corresponding collection time that are obtained by the preparative liquid chromatography separation are as follows:
Component fr.51, collection time 3.0-10.0min, 217mg;
Component fr.52, collection time 10.0-16.2min, 112mg;
Component fr.53, collection time 16.2-24.0min, 129mg;
Component fr.54, collection time 24.0-27.8min, 214mg;
Component fr.55, collection time 27.8-31.8min, 168mg;
Component fr.56, collection time 31.8-38.0min, 101mg;
Component fr.57, collection time 38.0-43.0min, 318mg;
Component fr.58, collection time 43.0-50.0min, 169mg;
The fr.8 separating resulting: get component fr.8 2.0g altogether, the dissolve with methanol sample introduction with 20%, the component and the corresponding collection time that are obtained by the preparative liquid chromatography separation are as follows:
Component fr.81, collection time 4.0-19.8min, 177mg;
Component fr.82, collection time 19.8-26.0min, 195mg;
Component fr.83, collection time 26.0-35.0min, 98mg;
Component fr.84, collection time 35.0-42.0min, 133mg;
Component fr.85, collection time 42.0-44.0min, 35mg;
Component fr.86, collection time 44.0-47.0min, 81mg;
Component fr.87, collection time 47.0-55.0min, 100mg.
Embodiment two
(1) extraction process: take by weighing 500g Ligusticum wallichii medicinal material, with its grinding and sieving, adding 5～12 times of amount volume ratios then is 1: 1 ethyl acetate and ethanol, and reflux 0.5～3 hour is extracted 1～3 time, and merging filtrate gets extracting solution fr.1.Add 5～12 times of amount 70% ethanol in the dregs of a decoction, reflux 0.5～3 hour is extracted 1～3 time, and merging filtrate gets extracting solution fr.2.Add entry in the dregs of a decoction at last, reflux 0.5～3 hour is extracted 1～3 time, and merging filtrate gets extracting solution fr.3.
(2) separating technology: with extracting solution fr.1 concentrate 99.8g medicinal extract, get fr.1 medicinal extract 20g silica gel mixed sample, adopt normal phase silicagel column that it is separated, at first with volume ratio be 50: 1 sherwood oil and ethyl acetate as moving phase, elutriant fr.4, change volume ratio then and be 10: 1 chloroform and methyl alcohol as moving phase, get elutriant fr.5, concentrate the 4.8g sample, use methyl alcohol as moving phase at last, elutriant fr.6.With extracting solution fr.2 concentrate 171.3g medicinal extract, get 20.0g medicinal extract with sample on 2～10% dissolve with methanol, adopt the ODS-C18 post that it is separated, at first use 2～10% methyl alcohol as moving phase, get elutriant fr.7, change 40～80% methyl alcohol then as moving phase, get elutriant fr.8, concentrate the 9.9g sample, use 100% methanol solution as moving phase at last, elutriant fr.9.
(3) preparation separating technology: continue to separate fr.5 and the fr.8 that obtains in the previous process with preparative liquid chromatography.Chromatographic column is semipreparative column (Lichrospher C18; 10.0mm * 250mm, 5 μ m), moving phase is water and acetonitrile, and gradient elution, flow velocity are 3ml/min, and column temperature is a room temperature, collects cut in the corresponding time period, obtains through further isolating each component.
The fr.5 separating resulting: get component fr.5 4.0g altogether, use the dissolve with ethanol sample introduction, the component and the corresponding collection time that are obtained by the preparative liquid chromatography separation are as follows:
Component fr.51, collection time 3.0-10.0min, 428mg;
Component fr.52, collection time 10.0-16.2min, 226mg;
Component fr.53, collection time 16.2-24.0min, 265mg;
Component fr.54, collection time 24.0-27.8min, 424mg;
Component fr.55, collection time 27.8-31.8min, 341mg;
Component fr.56, collection time 31.8-38.0min, 203mg;
Component fr.57, collection time 38.0-43.0min, 647mg;
Component fr.58, collection time 43.0-50.0min, 344mg;
The fr.8 separating resulting: get component fr.8 4.0g altogether, the dissolve with methanol sample introduction with 20%, the component and the corresponding collection time that are obtained by the preparative liquid chromatography separation are as follows:
Component fr.81, collection time 4.0-19.8min, 355mg;
Component fr.82, collection time 19.8-26.0min, 384mg;
Component fr.83, collection time 26.0-35.0min, 201mg;
Component fr.84, collection time 35.0-42.0min, 272mg;
Component fr.85, collection time 42.0-44.0min, 73mg;
Component fr.86, collection time 44.0-47.0min, 161mg;
Component fr.87, collection time 47.0-55.0min, 200mg.
Embodiment three
One, the extraction separation of Ligusticum wallichii medicinal material
(1) extraction process: take by weighing Ligusticum wallichii medicinal material 250g, with its grinding and sieving, adding 8 times of amount volume ratios then is 1: 1 ethyl acetate and ethanol, and reflux 1 hour is extracted 2 times, and merging filtrate gets extracting solution fr.1.Add 8 times of amount 70% ethanol in the dregs of a decoction, reflux 1 hour is extracted 2 times, and merging filtrate gets extracting solution fr.2.Add entry in the dregs of a decoction at last, reflux 1 hour is extracted 2 times, and merging filtrate gets extracting solution fr.3.
(2) separating technology: with extracting solution fr.1 concentrate 51.8g medicinal extract, get fr.1 medicinal extract 10.2g silica gel mixed sample, adopt normal phase silicagel column that it is separated, at first with volume ratio be 50: 1 sherwood oil and ethyl acetate as moving phase, elutriant fr.4, change volume ratio then and be 10: 1 chloroform and methyl alcohol as moving phase, get elutriant fr.5, concentrate the 2.5g sample, use methyl alcohol as moving phase at last, elutriant fr.6.With extracting solution fr.2 concentrate 88.7g medicinal extract, get 10.0g medicinal extract with sample on 5% dissolve with methanol, adopt the ODS-C18 post that it is separated, at first use 5% methyl alcohol as moving phase, get elutriant fr.7, change 60% methyl alcohol then as moving phase, get elutriant fr.8, concentrate the 5.6g sample, use 100% methanol solution as moving phase at last, elutriant fr.9.
(3) preparation separating technology: in order from step (2), to obtain fr.5 and the more concrete effective constituent of fr.8 in the Rhizoma Chuanxiong extract, continue to separate fr.5 and fr.8 among the present invention with preparative liquid chromatography.
The separation condition of fr.5: chromatographic column is semipreparative column (the Lichrospher C18 of Chinese nation; 10.0mm * 250mm, 5 μ m); Adopt gradient elution, moving phase is water (A) and acetonitrile (B), and flow velocity is 3ml/min, and column temperature is a room temperature, and gradient is as follows:
During 0min, mobile phase A is 75% water, and Mobile phase B is 25% acetonitrile solution;
During 40min, mobile phase A is 5% water, and Mobile phase B is 95% acetonitrile solution;
During 50min, mobile phase A is 5% water, and Mobile phase B is 95% acetonitrile solution;
The fr.5 separating resulting: get component fr.5 2.0g altogether, use the dissolve with ethanol sample introduction, the component and the corresponding collection time that are obtained by the preparative liquid chromatography separation are as follows:
Component fr.51, collection time 3.0-10.0min, 220mg;
Component fr.52, collection time 10.0-16.2min, 117mg;
Component fr.53, collection time 16.2-24.0min, 136mg;
Component fr.54, collection time 24.0-27.8min, 221mg;
Component fr.55, collection time 27.8-31.8min, 175mg;
Component fr.56, collection time 31.8-38.0min, 107mg;
Component fr.57, collection time 38.0-43.0min, 328mg;
Component fr.58, collection time 43.0-50.0min, 177mg;
The fr.8 separation condition: chromatographic column is Agilent preparative column (Zorbax SB-C18; 21.2mm * 250mm); Adopt gradient elution, moving phase is water (A) and acetonitrile (B), and flow velocity is 10ml/min, and column temperature is a room temperature, and gradient is as follows:
During 0min, mobile phase A is 95% 0.3% aqueous acetic acid, and Mobile phase B is 5% 0.3% acetate acetonitrile solution;
During 8min, mobile phase A is 80% 0.3% aqueous acetic acid, and Mobile phase B is 20% 0.3% acetate acetonitrile solution;
During 38min, mobile phase A is 50% 0.3% aqueous acetic acid, and Mobile phase B is 50% 0.3% acetate acetonitrile solution;
During 48min, mobile phase A is 5% 0.3% aqueous acetic acid, and Mobile phase B is 95% 0.3% acetate acetonitrile solution;
During 55min, mobile phase A is 5% 0.3% aqueous acetic acid, and Mobile phase B is 95% 0.3% acetate acetonitrile solution;
The fr.8 separating resulting: get component fr.8 2.0g altogether, the dissolve with methanol sample introduction with 20%, the component and the corresponding collection time that are obtained by the preparative liquid chromatography separation are as follows:
Component fr.81, collection time 4.0-19.8min, 182mg;
Component fr.82, collection time 19.8-26.0min, 200mg;
Component fr.83, collection time 26.0-35.0min, 106mg;
Component fr.84, collection time 35.0-42.0min, 141mg;
Component fr.85, collection time 42.0-44.0min, 39mg;
Component fr.86, collection time 44.0-47.0min, 87mg;
Component fr.87, collection time 47.0-55.0min, 107mg.
Two, analyze
2.1 reagent and instrument
Agilent 1100 high performance liquid chromatography-GC-MS (U.S. Agilent company) fit over line vacuum de-aerator, quarternary low pressure pump, automatic sampler, column oven, DAD detector, 1946D electron spray(ES) level Four bar mass detector, Chemstation chromatographic working station; R-200 type Rotary Evaporators (Switzerland BUCHI company); Flying pigeon board TGL-16G high speed tabletop centrifuge (Anting Scientific Instrument Factory, Shanghai); METTLER-AE240 type electronic balance (plum Teller-Tuo benefit Instr Ltd.).
Acetonitrile is chromatographically pure reagent (MERCK), and water is WAHAHA pure water (Hangzhou WAHAHA group), and acetate is analytical pure (Hangzhou chemical reagent work).
2.2 analytical procedure
Sample source: Ligusticum wallichii component fr.51 1.25mg, fr.52 1.15mg, fr.53 1.23mg, fr.54 1.11mg, fr.551.40mg, fr.56 1.38mg, fr.57 1.25mg, fr.58 1.46mg.
Specimen preparation: sample 1ml dissolve with ethanol, ultrasonic, centrifugal (10000r/min), standby.
Chromatographic condition: chromatographic column Agilent Zorbax SB-C 18Post (4.6mm * 150mm, 5 μ m); Adopt gradient elution, mobile phase A is 0.2% glacial acetic acid aqueous solution mutually, and Mobile phase B is mutually for containing the acetonitrile solution of 0.2% Glacial acetic acid; The gradient elution program is as follows: in the time of 0 minute, mobile phase A is that 90% 0.2% glacial acetic acid aqueous solution, Mobile phase B are the acetonitrile solution of 10% 0.2% Glacial acetic acid; In the time of 10 minutes, mobile phase A is that 50% 0.2% glacial acetic acid aqueous solution, Mobile phase B are the acetonitrile solution of 50% 0.2% Glacial acetic acid; In the time of 30 minutes, mobile phase A is that 5% 0.2% glacial acetic acid aqueous solution, Mobile phase B are the acetonitrile solution of 95% 0.2% Glacial acetic acid; Flow velocity 0.5mL/min; 30 ℃ of column temperatures; DAD detects in the interscan of 190-400nm scope; Sample size 5 μ L.
Mass spectrum condition: negative ions scan pattern, sweep limit 100-2000; Dry gas (N 2Stream speed 10L/min, 350 ℃ of atomization temperatures, capillary voltage 3500V, cracking voltage 100V.
ELSD condition: nitrogen 1.4L/min, drift tube temperature: 100 ℃.
Sample source: Ligusticum wallichii component fr.81 1.11mg, fr.82 1.43mg, fr.83 1.19mg, fr.84 1.09mg, fr.851.16mg, fr.86 1.10mg, fr.87 1.15mg.
Specimen preparation: sample is with the dissolving of 1ml 50% methanol-water, and is ultrasonic, and centrifugal (10000r/min) is standby.
Chromatographic condition: chromatographic column Agilent Zorbax SB-C 18Post (4.6mm * 150mm, 5 μ m); Adopt gradient elution, mobile phase A is 0.2% glacial acetic acid aqueous solution mutually, and Mobile phase B is mutually for containing the acetonitrile solution of 0.2% Glacial acetic acid; The gradient elution program is as follows: in the time of 0 minute, mobile phase A is that 95% 0.2% glacial acetic acid aqueous solution, Mobile phase B are the acetonitrile solution of 5% 0.2% Glacial acetic acid; In the time of 10 minutes, mobile phase A is that 70% 0.2% glacial acetic acid aqueous solution, Mobile phase B are the acetonitrile solution of 30% 0.2% Glacial acetic acid; In the time of 30 minutes, mobile phase A is that 50% 0.2% glacial acetic acid aqueous solution, Mobile phase B are the acetonitrile solution of 50% 0.2% Glacial acetic acid; Flow velocity 0.5mL/min; 30 ℃ of column temperatures; DAD detects in the interscan of 190-400nm scope; Sample size 5 μ L.
Mass spectrum condition: negative ions scan pattern, sweep limit 100-2000; Dry gas (N 2Stream speed 10L/min, 350 ℃ of atomization temperatures, capillary voltage 3500V, cracking voltage 100V.
ELSD condition: nitrogen 1.8L/min, drift tube temperature: 105 ℃.
2.3 analytical results
Analytical results: Fig. 2 is water-soluble each the component uv-spectrogram of Ligusticum wallichii, and Fig. 3 is water-soluble each the component mass spectrum of Ligusticum wallichii, and Fig. 4 is fat-soluble each the component uv-spectrogram of Ligusticum wallichii, and Fig. 5 is fat-soluble each the component mass spectrum of Ligusticum wallichii.
The qualification result of compound sees Table 2 in the Ligusticum wallichii component.
Table 2. Ligusticum wallichii component composition qualification result
Figure G05112274320050818D000151
Reference:
1、Ru?Yan，Song-Lin?Li，Hoi-Sing?Chung，Yun-Kau?Tam，Ge?Lin.Simultaneous?quantification?of12?bioactive?components?of?Ligusticum?chuanxiong?Hort.by?high-performance?liquidchromatography.Journal?of?Pharmaceutical?and?Biomedical?Analysis?37(2005)87-95
2, Liang Mingjin, He Lang dashes, Li Yongmao. research of Ligusticum wallichii efficient part gas chromatography and mass spectromentry and fingerprint map analyzing.The mass spectrum journal.Vol.25?No.3?2004。151-154
Three, Ligusticum wallichii component medicine efficacy screening experiment
3.1 pharmacological model: neonatal rat myocardial cell hypoxic-ischemic model
After former generation myocardial cell cultivated birth SD suckling mouse execution in 1～3 day, it was dirty to core, and is cut into 1mm after PBS liquid cleans 3About fragment.With 0.125% trypsin Sigma, USA)+0.05% (Gibco is USA) in 37 ℃ of digestion 3～4 times for collagenase II.The differential adherent method was separated into fibrocyte after 1.5 hours, and cell suspension is transferred to (every hole about 4 * 10 in 48 orifice plates 5Cell).(Gibco, (Falcon, USA) cell of cultivating 3～6 days supplies medicine efficacy screening USA) to add 10% foetal calf serum through DMEM.Screen and replaced with serum-free medium in preceding 1 day.Cultivate 3～6 days myocardial cell, PBS washing back adds the pastille nutrient solution.Place 95%N 2And 5%CO 2Anoxic is 6 hours in the incubator.Then at CO 2Reoxygenation is 3 hours in the incubator.Get cell conditioned medium liquid and measure LDH content.
Dosage regimen is extracted the Rhizoma Chuanxiong extract that obtains and is made into 10 with sugar-free Hanks liquid -4G/mL test liquid, fat-soluble component can add a small amount of DMSO hydrotropy (the DMSO final concentration is controlled at below 0.2%).The final concentration of each component in nutrient solution is controlled at 10 -5G/mL.
Serum lactic dehydrogenase content can be represented the cell injury degree in the serum lactic dehydrogenase assay cell culture fluid.The serum lactic dehydrogenase content that bleeds in the supernatant liquor is high more, shows that cell injury is also remarkable.The determination of lactate dehydrogenase test kit builds up bio-engineering research institute available from Nanjing.Measuring method is: get 30 μ l cell conditioned medium liquid, add 50 μ l matrix damping fluids and 10 μ l lactic dehydrogenase enzyme cofactors, 37 ℃ vibrated 15 minutes, added 50 μ l 2,4 dinitrophenyl hydrazines again, and 37 ℃ vibrated 15 minutes.Parallel blank.Extract reaction solution 50 μ l and add 200 μ l 0.4mol/L sodium hydroxide solutions, leave standstill 3～4 minutes after, measure photometric quantity with microplate reader in 450nm.
Drug effect all data is as a result represented with the mean value variance.Adopt monolateral variance analysis and T check carrying out statistical analysis.Compare with model group, P＜0.05 is thought significantly effectively.Medicine efficacy screening the results are shown in Table 3.According to myocardial cell's serum lactic dehydrogenase (LDH) The selection result, Ligusticum wallichii component fr.51, fr.52, fr.53, fr.81 has the highly significant effect to reducing LDH release.
Table 3. myocardial cell The selection result
3.2 huve cell anoxic reoxygenation model
The healthy newborn umbilical cord is got in former generation huve cell cultivation, cleans the interior residual bloodstain of vein blood vessel with 30～50ml Hanks liquid.Look the 0.1% collagenase I that umbilical cord length adds respective amount, change incubator digestion 15～20 minutes over to.Subsequently Digestive system in the umbilical cord is carefully collected in the beaker, and with Hanks liquid with beaker rinse twice, collect in the lump and be sub-packed in centrifuge tube in the beaker, centrifugal 10 minutes of 900rpm.Supernatant liquor is removed in suction, with the abundant dissolution precipitation of M199 nutrient solution (contain 10% foetal calf serum, 100U/ml is two anti-, 0.15mg/ml Heparin, 10 μ M VC).The gained cell suspension is sub-packed in 5cm 2Culturing bottle places in the incubator and cultivates.Changed in second day and adopt the M199 nutrient solution that contains 30 μ g/ml ECGS, changed a nutrient solution and be paved with the bottom surface in per afterwards three days until cell.Used cell is former generation endotheliocyte.The culturing bottle inner cell is seeded to 96 orifice plates before the modeling and cultivated one day, used cell concentration is 3.5～40,000/hole.During modeling, inhale and abandon old M199 nutrient solution, adding pastille does not have phenol red M199 nutrient solution, and every hole total amount is 100 μ l.Place 95%N 2And 5%CO 2Anoxic is 12 hours in the incubator, then at CO 2Reoxygenation is 2 hours in the incubator.Get cell conditioned medium liquid and measure LDH content and NO content.
Dosage regimen is extracted the Rhizoma Chuanxiong extract that obtains and is made into 10 with sugar-free Hanks liquid -4G/mL test liquid, fat-soluble component can add a small amount of DMSO hydrotropy (the DMSO final concentration is controlled at below 0.2%).The final concentration of each component in no phenol red M199 nutrient solution is controlled at 10 -5G/mL.
The NO assay adopts Greiss reagent method to measure, and gets 90 μ l cell conditioned medium liquid, adds equivalent Greiss reagent, leaves standstill under the room temperature 10 minutes, measures absorbance with microplate reader in 550nm.
Drug effect all data is as a result represented with the mean value variance.Adopt monolateral variance analysis and T check carrying out statistical analysis.Compare with model group, P＜0.05 is thought significantly effectively.Medicine efficacy screening the results are shown in Table 4.7 components of Ligusticum wallichii have the effect that suppresses NO overexpression behind endotheliocyte anoxic-reoxygenation, and fr.53, fr.54, fr.55, fr.81 are significantly effective.
Table 4. endotheliocyte The selection result
Figure G05112274320050818D000171
Above test-results explanation, the Chinese medicinal materials effective constituent that adopts Chinese medicinal materials stdn extracting method of the present invention to obtain can be as the preparation medicine.

Claims (4)

  1. A Ligusticum wallichii extract, the separation criterion method, comprise the steps:
    (1) extraction process: take by weighing the Ligusticum wallichii medicinal material, with its grinding and sieving, adding 5～12 times of amount volume ratios then is 1: 1 ethyl acetate and ethanol, and reflux 0.5～3 hour is extracted 1～3 time, and merging filtrate gets extracting solution fr.1; Add 5～12 times of amount 70% ethanol in the dregs of a decoction, reflux 0.5～3 hour is extracted 1～3 time, and merging filtrate gets extracting solution fr.2; Add entry in the dregs of a decoction at last, reflux 0.5～3 hour is extracted 1～3 time, and merging filtrate gets extracting solution fr.3;
    (2) separating technology: after extracting solution fr.1 is condensed into medicinal extract, use silica gel mixed sample, adopt normal phase silicagel column that it is separated, be that 50: 1 sherwood oil and ethyl acetate are as moving phase at first with volume ratio, elutriant fr.4, change volume ratio then and be 10: 1 chloroform and methyl alcohol as moving phase, elutriant fr.5, use methyl alcohol as moving phase at last, get elutriant fr.6; After extracting solution fr.2 is condensed into medicinal extract, with sample on 2～10% dissolve with methanol, adopt the ODS-C18 post that it is separated, at first use 2～10% methyl alcohol as moving phase, get elutriant fr.7, change 40～80% methyl alcohol then, get elutriant fr.8 as moving phase, use 100% methanol solution as moving phase at last, get elutriant fr.9;
    (3) preparation separating technology: separate fr.5 and fr.8 with preparative liquid chromatography; Chromatographic column is a Lichrospher C18 semipreparative column, and big or small 10.0mm * 250mm, particle diameter are 5 μ m, moving phase is water and acetonitrile, and gradient elution, flow velocity are 3ml/min, column temperature is a room temperature, collects cut in the corresponding time period, obtains through further isolating each component.
  2. 2. Ligusticum wallichii as claimed in claim 1 extracts, the separation criterion method, comprises the steps:
    (1) extraction process: take by weighing the Ligusticum wallichii medicinal material, with its grinding and sieving, adding 8 times of amount volume ratios then is 1: 1 ethyl acetate and ethanol, and reflux 1 hour is extracted 2 times, and merging filtrate gets extracting solution fr.1; Add 8 times of amount 70% ethanol in the dregs of a decoction, reflux 1 hour is extracted 2 times, and merging filtrate gets extracting solution fr.2; Add entry in the dregs of a decoction at last, reflux 1 hour is extracted 2 times, and merging filtrate gets extracting solution fr.3;
    (2) separating technology: fr.1 is condensed into medicinal extract with extracting solution, use silica gel mixed sample, adopt normal phase silicagel column that it is separated, be that 50: 1 sherwood oil and ethyl acetate are as moving phase at first with volume ratio, elutriant fr.4, change volume ratio then and be 10: 1 chloroform and methyl alcohol as moving phase, elutriant fr.5, use methyl alcohol as moving phase at last, get elutriant fr.6; Fr.2 is condensed into medicinal extract with extracting solution, with sample on 5% dissolve with methanol, adopt the ODS-C18 post that it is separated, at first use 5% methyl alcohol as moving phase, get elutriant fr.7, change 60% methyl alcohol then, get elutriant fr.8 as moving phase, use 100% methanol solution as moving phase at last, get elutriant fr.9;
    (3) preparation separating technology: separate fr.5 and fr.8 with preparative liquid chromatography;
    The separation condition of fr.5: chromatographic column is a Lichrospher C18 Chinese nation semipreparative column, and big or small 10.0mm * 250mm, particle diameter are 5 μ m; Adopt gradient elution, mobile phase A is that water and Mobile phase B are acetonitrile, and flow velocity is 3ml/min, and column temperature is a room temperature, and gradient is as follows:
    During 0min, mobile phase A is 75% water, and Mobile phase B is 25% acetonitrile solution;
    During 40min, mobile phase A is 5% water, and Mobile phase B is 95% acetonitrile solution;
    During 50min, mobile phase A is 5% water, and Mobile phase B is 95% acetonitrile solution;
    The fr.5 separating resulting: the component and the corresponding collection time that are obtained by the preparative liquid chromatography separation are as follows:
    Component fr.51, collection time 3.0-10.0min;
    Component fr.52, collection time 10.0-16.2min;
    Component fr.53, collection time 16.2-24.0min;
    Component fr.54, collection time 24.0-27.8min;
    Component fr.55, collection time 27.8-31.8min;
    Component fr.56, collection time 31.8-38.0min;
    Component fr.57, collection time 38.0-43.0min;
    Component fr.58, collection time 43.0-50.0min;
    The fr.8 separation condition: chromatographic column is a Zorbax SB-C18 board Agilent preparative column, big or small 21.2mm * 250mm; Adopt gradient elution, mobile phase A is that 0.3% aqueous acetic acid and Mobile phase B are 0.3% acetate acetonitrile solution, and flow velocity is 10ml/min, and column temperature is a room temperature, and gradient is as follows:
    During 0min, mobile phase A is 95% 0.3% aqueous acetic acid, and Mobile phase B is 5% 0.3% acetate acetonitrile solution;
    During 8min, mobile phase A is 80% 0.3% aqueous acetic acid, and Mobile phase B is 20% 0.3% acetate acetonitrile solution;
    During 38min, mobile phase A is 50% 0.3% aqueous acetic acid, and Mobile phase B is 50% 0.3% acetate acetonitrile solution;
    During 48min, mobile phase A is 5% 0.3% aqueous acetic acid, and Mobile phase B is 95% 0.3% acetate acetonitrile solution;
    During 55min, mobile phase A is 5% 0.3% aqueous acetic acid, and Mobile phase B is 95% 0.3% acetate acetonitrile solution;
    The fr.8 separating resulting: the component and the corresponding collection time that are obtained by the preparative liquid chromatography separation are as follows:
    Component fr.81, collection time 4.0-19.8min;
    Component fr.82, collection time 19.8-26.0min;
    Component fr.83, collection time 26.0-35.0min;
    Component fr.84, collection time 35.0-42.0min;
    Component fr.85, collection time 42.0-44.0min;
    Component fr.86, collection time 44.0-47.0min;
    Component fr.87, collection time 47.0-55.0min.
  3. 3. Ligusticum wallichii as claimed in claim 2 extracts, the separation criterion method, it is characterized in that:
    Main compound is Vanillin among the component fr.51, Ferulic acid;
    Main compound is Senkyunolide I or Senkyunolide H among the component fr.52;
    Main compound is Neocnidilide among the component fr.53, (E)-form, 3,4-Methyl ene, 5-Meether
    Or 3-(3,4,5-Trihydroxyphenyl)-2-propenoic acid;
    Main compound is 3-Butylidene-1 (3H)-isobenzofuranone among the component fr.54;
    Main compound is Riligustilide among the component fr.56, Levistolide A,
    9-octadecadienic?acid?methyl?ester；
    Main compound is 9-octadecadienic acid methyl ester among the component fr.57.
  4. 4. Ligusticum wallichii as claimed in claim 2 extracts, the separation criterion method, it is characterized in that:
    Main compound is Ferulic acid among the component fr.84;
    Main compound is Senkyunolide I or Senkyunolide H among the component fr.85.
CN2005100122743A 2005-07-29 2005-07-29 Method for extraction and separation of rhizome of chuanixong Expired - Fee Related CN1903239B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN2005100122743A CN1903239B (en) 2005-07-29 2005-07-29 Method for extraction and separation of rhizome of chuanixong

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN2005100122743A CN1903239B (en) 2005-07-29 2005-07-29 Method for extraction and separation of rhizome of chuanixong

Publications (2)

Publication Number Publication Date
CN1903239A CN1903239A (en) 2007-01-31
CN1903239B true CN1903239B (en) 2011-01-12

Family

ID=37672699

Family Applications (1)

Application Number Title Priority Date Filing Date
CN2005100122743A Expired - Fee Related CN1903239B (en) 2005-07-29 2005-07-29 Method for extraction and separation of rhizome of chuanixong

Country Status (1)

Country Link
CN (1) CN1903239B (en)

Families Citing this family (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101347479B (en) * 2007-07-20 2011-10-05 天津天士力制药股份有限公司 Effective component of acanthopanax and preparation and use thereof
CN101229355B (en) * 2008-01-30 2012-01-11 苟于洋 Rheumatism soup
CN101550081B (en) * 2008-04-02 2014-03-19 天士力制药集团股份有限公司 Effective components of Cacumen Platycladi and preparation method thereof
CN101550072B (en) * 2008-04-02 2014-03-19 天士力制药集团股份有限公司 Effective component of cogongrass rhizome and preparation method thereof
CN110274963B (en) * 2018-03-13 2022-10-18 天士力医药集团股份有限公司 Fingerprint detection method for diabetes
CN110441433A (en) * 2019-08-27 2019-11-12 山东省兽药质量检验所（山东省畜产品质量检测中心） A kind of ten black scattered UPLC-PDA detection method
CN110850019B (en) * 2019-11-29 2022-02-18 南京南医堂健康产业发展有限公司 Method for detecting fingerprint of Minggu Shuqing drink
CN110787200B (en) * 2019-12-17 2021-08-24 成都师范学院 Method for microwave synergistic extraction of ligusticum wallichii effective substance
CN113917031B (en) * 2021-10-12 2022-06-07 广东雷允上药业有限公司 Method for determining blood concentration of lung-tonifying and blood-activating capsule in vivo by UHPLC-MS/MS method

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
杨光明，等.川芎药材指纹图谱研究与产地比较（Ⅰ）.《南京中医药大学学报（自然科学版）》.2002,第18卷(第3期),172－174. *
梁明金,等.川芎有效部位气相色谱_质谱研究与指纹图谱分析.《质谱学报》.2004,第25卷(第3期),150-154. *

Also Published As

Publication number Publication date
CN1903239A (en) 2007-01-31

Similar Documents

Publication Publication Date Title
CN1903239B (en) Method for extraction and separation of rhizome of chuanixong
CN1903250B (en) Method for extraction and separation of red rooted salvia
Ohsugi et al. Active-oxygen scavenging activity of traditional nourishing-tonic herbal medicines and active constituents of Rhodiola sacra
Huang et al. Ethnopharmacology, phytochemistry, and pharmacology of Cornus officinalis Sieb. et Zucc
He et al. Evaluation of antidiabetic potential of selected traditional Chinese medicines in STZ-induced diabetic mice
CN101073587B (en) Method for separating and extracting Chinese Thorowax Root
Azietaku et al. A review of the ethnopharmacology, phytochemistry and pharmacology of Notopterygium incisum
Kong et al. Inhibition of xanthine oxidase by some Chinese medicinal plants used to treat gout
CN1903241B (en) Method for extraction and separation of pseudo-ginseng
Zhang et al. Botany, traditional use, phytochemistry, pharmacology, quality control, and authentication of Radix Gentianae Macrophyllae-A traditional medicine: A review
CN101073592B (en) Method for separating and extracting Milkvetch Root
CN101073606B (en) Method for separating and extracting white Peony Root
WO2009117953A1 (en) Pharmaceutical composition having antidepressive, antianxious action and preparation method thereof
Siril Pharmacognostic studies on Indian madder (Rubia cordifolia L.)
US20150352168A1 (en) Pharmaceutical Composition for Treating Headache, and Preparation Method Thereof
Ma et al. An assessment of traditional Uighur medicine in current Xinjiang region (China)
CN101073623B (en) Method for separating and extracting cattail pollen
Busia Fundamentals of herbal medicine: Major plant families, analytical methods, materia medica
CN1903259B (en) Method for extraction and separation of moutan bark
CN104288245B (en) Anti-aging and constitutional pharmaceutical composition and preparation method thereof and detection method
Li et al. Glehnia littoralis Fr. Schmidtex Miq.: a systematic review on ethnopharmacology, chemical composition, pharmacology and quality control
Sahoo et al. Experimental and clinical trial investigations of phyto-extracts, phyto-chemicals and phyto-formulations against oral lichen planus: A systematic review
Singh et al. Antianxiety investigations of Centaurea behen Linn. and Elaeocarpus ganitrus Roxb
Bhadoriya et al. Diuretic activity of extract of Salvia officinalis L
CN102526305A (en) Chinese medicinal capsules for treating prostatitis and prostatic hyperplasia and preparation method for Chinese medicinal capsules

Legal Events

Date Code Title Description
C06 Publication
PB01 Publication
C41 Transfer of patent application or patent right or utility model
TA01 Transfer of patent application right

Effective date of registration: 20070511

Address after: Hedong District of Beichen Puji road in Tianjin No. 2 city of the modern Chinese Medicine

Applicant after: Tianjin Tianshili Pharmaceutical Co., Ltd.

Address before: Beichen Xinyi Road white Tianjin Liaohe Road No. 1

Applicant before: Tianshili Modern Traditional Chinese Medicine Research & Devleopment Co., Ltd.,

C10 Entry into substantive examination
SE01 Entry into force of request for substantive examination
C14 Grant of patent or utility model
GR01 Patent grant
C56 Change in the name or address of the patentee

Owner name: TASLY PHARMACEUTICAL GROUP CO., LTD.

Free format text: FORMER NAME: TIANJIN TIANSHILI PHARMACEUTICAL CO., LTD.

CP01 Change in the name or title of a patent holder

Address after: 300402 Tianjin City Hedong District of Beichen Puji Road No. 2 city of the modern Chinese Medicine

Patentee after: Tasly Pharmaceutical Group Co., Ltd.

Address before: 300402 Tianjin City Hedong District of Beichen Puji Road No. 2 city of the modern Chinese Medicine

Patentee before: Tianjin Tianshili Pharmaceutical Co., Ltd.

CP01 Change in the name or title of a patent holder
CP01 Change in the name or title of a patent holder

Address after: 300402 Tianjin City Hedong District of Beichen Puji Road No. 2 city of the modern Chinese Medicine

Patentee after: Tasly Pharmaceutical Group Limited by Share Ltd

Address before: 300402 Tianjin City Hedong District of Beichen Puji Road No. 2 city of the modern Chinese Medicine

Patentee before: Tasly Pharmaceutical Group Co., Ltd.

CF01 Termination of patent right due to non-payment of annual fee
CF01 Termination of patent right due to non-payment of annual fee

Granted publication date: 20110112

Termination date: 20190729