CN1903239A - Method for extraction and separation of rhizome of chuanixong - Google Patents
Method for extraction and separation of rhizome of chuanixong Download PDFInfo
- Publication number
- CN1903239A CN1903239A CN 200510012274 CN200510012274A CN1903239A CN 1903239 A CN1903239 A CN 1903239A CN 200510012274 CN200510012274 CN 200510012274 CN 200510012274 A CN200510012274 A CN 200510012274A CN 1903239 A CN1903239 A CN 1903239A
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- methanol
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- 230000032683 aging Effects 0.000 description 1
- 150000001298 alcohols Chemical class 0.000 description 1
- 229930013930 alkaloid Natural products 0.000 description 1
- 150000003797 alkaloid derivatives Chemical class 0.000 description 1
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- 235000004883 caffeic acid Nutrition 0.000 description 1
- 229940074360 caffeic acid Drugs 0.000 description 1
- OSGAYBCDTDRGGQ-UHFFFAOYSA-L calcium sulfate Chemical compound [Ca+2].[O-]S([O-])(=O)=O OSGAYBCDTDRGGQ-UHFFFAOYSA-L 0.000 description 1
- 239000009670 cang er zi wan Substances 0.000 description 1
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- SGMZJAMFUVOLNK-UHFFFAOYSA-M choline chloride Chemical compound [Cl-].C[N+](C)(C)CCO SGMZJAMFUVOLNK-UHFFFAOYSA-M 0.000 description 1
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- 229960002424 collagenase Drugs 0.000 description 1
- 210000002249 digestive system Anatomy 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 239000008298 dragée Substances 0.000 description 1
- 238000007877 drug screening Methods 0.000 description 1
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- OIZQSWBUTZRVTR-UHFFFAOYSA-N ethene 2-methoxyphenol Chemical group OC1=C(C=CC=C1)OC.C=C OIZQSWBUTZRVTR-UHFFFAOYSA-N 0.000 description 1
- XYIBRDXRRQCHLP-UHFFFAOYSA-N ethyl acetoacetate Chemical compound CCOC(=O)CC(C)=O XYIBRDXRRQCHLP-UHFFFAOYSA-N 0.000 description 1
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- IPCSVZSSVZVIGE-UHFFFAOYSA-N hexadecanoic acid Chemical compound CCCCCCCCCCCCCCCC(O)=O IPCSVZSSVZVIGE-UHFFFAOYSA-N 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 229940056582 human hair preparation Drugs 0.000 description 1
- 230000010354 integration Effects 0.000 description 1
- NLYAJNPCOHFWQQ-UHFFFAOYSA-N kaolin Chemical compound O.O.O=[Al]O[Si](=O)O[Si](=O)O[Al]=O NLYAJNPCOHFWQQ-UHFFFAOYSA-N 0.000 description 1
- 238000011031 large-scale manufacturing process Methods 0.000 description 1
- OYHQOLUKZRVURQ-IXWMQOLASA-N linoleic acid Natural products CCCCC\C=C/C\C=C\CCCCCCCC(O)=O OYHQOLUKZRVURQ-IXWMQOLASA-N 0.000 description 1
- 235000020778 linoleic acid Nutrition 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 238000007726 management method Methods 0.000 description 1
- GBMDVOWEEQVZKZ-UHFFFAOYSA-N methanol;hydrate Chemical compound O.OC GBMDVOWEEQVZKZ-UHFFFAOYSA-N 0.000 description 1
- HUEBIMLTDXKIPR-UHFFFAOYSA-N methyl heptadecanoate Chemical compound CCCCCCCCCCCCCCCCC(=O)OC HUEBIMLTDXKIPR-UHFFFAOYSA-N 0.000 description 1
- VYQNWZOUAUKGHI-UHFFFAOYSA-N monobenzone Chemical compound C1=CC(O)=CC=C1OCC1=CC=CC=C1 VYQNWZOUAUKGHI-UHFFFAOYSA-N 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 239000002547 new drug Substances 0.000 description 1
- 150000002885 octadecanoids Chemical class 0.000 description 1
- 239000003921 oil Substances 0.000 description 1
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- 239000003960 organic solvent Substances 0.000 description 1
- 230000002018 overexpression Effects 0.000 description 1
- 210000003516 pericardium Anatomy 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N phenol group Chemical group C1(=CC=CC=C1)O ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 1
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- YQUVCSBJEUQKSH-UHFFFAOYSA-N protochatechuic acid Natural products OC(=O)C1=CC=C(O)C(O)=C1 YQUVCSBJEUQKSH-UHFFFAOYSA-N 0.000 description 1
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- KZJWDPNRJALLNS-VJSFXXLFSA-N sitosterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CC[C@@H](CC)C(C)C)[C@@]1(C)CC2 KZJWDPNRJALLNS-VJSFXXLFSA-N 0.000 description 1
- UAJTZZNRJCKXJN-UHFFFAOYSA-M sodium;2-dodecoxy-2-oxoethanesulfonate Chemical compound [Na+].CCCCCCCCCCCCOC(=O)CS([O-])(=O)=O UAJTZZNRJCKXJN-UHFFFAOYSA-M 0.000 description 1
- 239000007901 soft capsule Substances 0.000 description 1
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- 125000000383 tetramethylene group Chemical group [H]C([H])([*:1])C([H])([H])C([H])([H])C([H])([H])[*:2] 0.000 description 1
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- SZYJELPVAFJOGJ-UHFFFAOYSA-N trimethylamine hydrochloride Chemical compound Cl.CN(C)C SZYJELPVAFJOGJ-UHFFFAOYSA-N 0.000 description 1
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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
Technical field
The invention belongs to field of medicaments, particularly relate to Rhizoma Chuanxiong extraction, separation criterionization.
Background technology
At present, in the world, Chinese 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 Drug therapy 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 medical herbs, state medical herbs status such as Canada and Australia have legalized, U.S. government has also drafted the plant amedica management method, the compound recipe mix 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 keen competition of powerful transnational medical group and Japan, Korea S, India, Thailand and European countries' plant amedica such as Germany, France, numerous products that China's 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 standardization that Chinese crude drug extracts and the standardization of Chinese medicinal preparation method are that Chinese patent medicine moves towards the international 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 ingredient which kind of method of employing could obtain with the sight of research always from Chinese crude drug, therefore the situation that present Chinese crude drug extracts is: also different at its extraction of effective components of different Chinese crude drugs, and adopting different extracting method to need the extraction that a large amount of different extraction equipment are used to support Chinese crude drug at different Chinese crude drugs, this is unsuitable for the standardization that Chinese crude drug extracts.
Rhizoma Chuanxiong is the dry rhizome of samphire Rhizoma Chuanxiong Ligusticum chuanxiong Hort..The traditional Chinese medical science is thought, its acrid in the mouth is warm in nature, returns liver, gallbladder, pericardium channel.Have blood-activating and qi-promoting, wind-expelling pain-stopping is applicable to menoxenia, amenorrhea dysmenorrhea ， mass in the abdomen stomachache, the twinge of the breast side of body, tumbling and swelling, headache, rheumatic arthralgia.Modern study shows that main rhizome contains volatile oil, alkaloid, phenolic constituent, lactone, ferulic acid in the Rhizoma Chuanxiong.Rhizome contains volatile oil about 1%.Identify fuel-displaced in composition have 40 kinds, account for 93.64% of volatile oil, wherein main constituent be that 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), neocindilide (neocnidilide), 4-hydroxyl-3-fourth husband lactone (4-hydroxy-3-butyl phthalide), chuanxingol (chuanxingol), two ligustilide (2,2 '-diligustilide), and 3-butyl-3,6,7-trihydroxy-4,5,6,7-tetrahydrochysene Phthalide etc.Nitrogen-containing compound has trimethylamine hydrochloride, choline hydrochloride, the different bright acyl of L--L-valine acid anhydride (L-isobutyl-L-valineanhydride), L-valyl-L-valine acid anhydride, 1-acetyl group-β-Ka quinoline, 1-β-propylene acetic acid-7-aldehyde radical-β-Ka quinoline, uracil, adenine and gland and adenosine.Acidity or phenoloid have 4-hydroxy 3-methoxybenzene ethylene, 1-hydroxyl-1-(3-methoxyl group-4-hydroxy benzenes)-ethane, 4-hydroxy benzoic acid, caffeic acid, vanillic acid, ferulic acid (ferulic acid), sedanoic acid (sadanic acid), chrysophanic acid (chrysophic acid), Palmic acid, vanillin and linoleic acid.In addition, the Rhizoma Chuanxiong rhizome still contains neutral oil, and its composition is 15,16,17, the octadecanoid acid ethyl ester, different 17, isooctadecane acetoacetic ester and different methyl margarate.Other contains 5,5 '-furil ether (bis-5,5 '-formylfurfur ether), spathulenol (spathulenol), cupreol, sucrose and a kind of fat glycerides.
How to determine a kind of extraction, separation criterion method, the active constituents of medicine in the Rhizoma Chuanxiong 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 Rhizoma Chuanxiong extraction, separation criterionization.Exactly Rhizoma Chuanxiong extract is divided into fractions specifically, each component comprises several main compound, has constituted a medical material component pool by these medical material components.Can carry out drug screening to the medical material component pool, thereby find new drug.
For modernization, the standardization that realizes that Rhizoma Chuanxiong extracts, the inventor is by a large amount of tests, the extraction of effective components of present Rhizoma Chuanxiong 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 Rhizoma Chuanxiong is extracted, can obtain the effective ingredient in the Chinese medicine to greatest extent.
Rhizoma Chuanxiong of the present invention extracts, separation criterionization is that the inventor passes through test, and after Chinese crude drug adopted different extracting method to compare simply together, the resulting Rhizoma Chuanxiong 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 Rhizoma Chuanxiong medical material, with its grinding and sieving, add ethyl acetate and ethanol then, heating extraction gets extracting solution fr.1.Add ethanol in medicinal residues, heating extraction gets extracting solution fr.2.Add entry in medicinal residues at last, heating extraction gets extracting solution fr.3.
(2) separating technology: after extracting solution fr.1 is condensed into extractum, use silica gel mixed sample, adopt normal phase silicagel column that it is separated, at first use petroleum ether and ethyl acetate as mobile phase, get eluent fr.4, change chloroform and methanol then, get eluent fr.5 as mobile phase, use methanol as mobile phase at last, get eluent fr.6.After extracting solution fr.2 is condensed into extractum, with sample on the dissolve with methanol, adopt the ODS-C18 post that it is separated, the methanol of at first using low concentration is as mobile phase, get eluent fr.7, the methanol that changes intermediate concentration then gets eluent fr.8 as mobile phase, use pure methanol solution as mobile phase at last, get eluent 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 semi-preparative column (Lichrospher C18; 10.0mm * 250mm, 5 μ m), mobile phase is water and acetonitrile, gradient elution is collected fraction 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 Rhizoma Chuanxiong medical 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 medicinal residues, reflux 0.5～3 hour is extracted 1～3 time, and merging filtrate gets extracting solution fr.2.Add entry in medicinal residues 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 extractum, use silica gel mixed sample, adopt normal phase silicagel column that it is separated, be that 50: 1 petroleum ether and ethyl acetate are as mobile phase at first with volume ratio, eluent fr.4, change volume ratio then and be 10: 1 chloroform and methanol as mobile phase, eluent fr.5, use methanol as mobile phase at last, get eluent fr.6.After extracting solution fr.2 is condensed into extractum, with sample on 2～10% dissolve with methanol, adopt the ODS-C18 post that it is separated, at first use 2～10% methanol as mobile phase, get eluent fr.7, change 40～80% methanol then, get eluent fr.8 as mobile phase, use 100% methanol solution as mobile phase at last, get eluent 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 semi-preparative column (Lichrospher C18; 10.0mm * 250mm, 5 μ m), mobile phase is water and acetonitrile, and gradient elution, flow velocity are 3ml/min, and column temperature is a room temperature, collects fraction 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 Rhizoma Chuanxiong medical 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 medicinal residues, reflux 1 hour is extracted 2 times, and merging filtrate gets extracting solution fr.2; Add entry in medicinal residues 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 extractum with extracting solution, use silica gel mixed sample, adopt normal phase silicagel column that it is separated, be that 50: 1 petroleum ether and ethyl acetate are as mobile phase at first with volume ratio, eluent fr.4, change volume ratio then and be 10: 1 chloroform and methanol as mobile phase, eluent fr.5, use methanol as mobile phase at last, get eluent fr.6; Fr.2 is condensed into extractum with extracting solution, with sample on 5% dissolve with methanol, adopt the ODS-C18 post that it is separated, at first use 5% methanol as mobile phase, get eluent fr.7, change 60% methanol then, get eluent fr.8 as mobile phase, use 100% methanol solution as mobile phase at last, get eluent 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 semi-preparative column (the Lichrospher C18 of Chinese nation; 10.0mm * 250mm, 5 μ m); Adopt gradient elution, mobile 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 acquisition time that are obtained by the preparative liquid chromatography separation are as follows:
Component fr.51, acquisition time 3.0-10.0min;
Component fr.52, acquisition time 10.0-16.2min;
Component fr.53, acquisition time 16.2-24.0min;
Component fr.54, acquisition time 24.0-27.8min;
Component fr.55, acquisition time 27.8-31.8min;
Component fr.56, acquisition time 31.8-38.0min;
Component fr.57, acquisition time 38.0-43.0min;
Component fr.58, acquisition 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, mobile 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 acquisition time that are obtained by the preparative liquid chromatography separation are as follows:
Component fr.81, acquisition time 4.0-19.8min;
Component fr.82, acquisition time 19.8-26.0min;
Component fr.83, acquisition time 26.0-35.0min;
Component fr.84, acquisition time 35.0-42.0min;
Component fr.85, acquisition time 42.0-44.0min;
Component fr.86, acquisition time 44.0-47.0min;
Component fr.87, acquisition time 47.0-55.0min.
In order to obtain concrete Rhizoma Chuanxiong effective ingredient, best extraction separation method of the present invention is:
(1) extraction process: take by weighing Rhizoma Chuanxiong medical 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 medicinal residues, reflux 1 hour is extracted 2 times, and merging filtrate gets extracting solution fr.2.Add entry in medicinal residues 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 extractum, get fr.1 extractum 10.2g silica gel mixed sample, adopt normal phase silicagel column that it is separated, at first with volume ratio be 50: 1 petroleum ether and ethyl acetate as mobile phase, eluent fr.4, change volume ratio then and be 10: 1 chloroform and methanol as mobile phase, get eluent fr.5, concentrate the 2.5g sample, use methanol as mobile phase at last, eluent fr.6.With extracting solution fr.2 concentrate 88.7g extractum, get 10.0g extractum with sample on 5% dissolve with methanol, adopt the ODS-C18 post that it is separated, at first use 5% methanol as mobile phase, get eluent fr.7, change 60% methanol then as mobile phase, get eluent fr.8, concentrate the 5.6g sample, use 100% methanol solution as mobile phase at last, eluent fr.9.
(3) preparation separating technology: in order from step (2), to obtain fr.5 and the more concrete effective ingredient 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 semi-preparative column (the Lichrospher C18 of Chinese nation; 10.0mm * 250mm, 5 μ m); Adopt gradient elution, mobile 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 acquisition time that are obtained by the preparative liquid chromatography separation are as follows:
Component fr.51, acquisition time 3.0-10.0min, 220mg;
Component fr.52, acquisition time 10.0-16.2min, 117mg;
Component fr.53, acquisition time 16.2-24.0min, 136mg;
Component fr.54, acquisition time 24.0-27.8min, 221mg;
Component fr.55, acquisition time 27.8-31.8min, 175mg;
Component fr.56, acquisition time 31.8-38.0min, 107mg;
Component fr.57, acquisition time 38.0-43.0min, 328mg;
Component fr.58, acquisition 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, mobile 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 acquisition time that are obtained by the preparative liquid chromatography separation are as follows:
Component fr.81, acquisition time 4.0-19.8min, 182mg;
Component fr.82, acquisition time 19.8-26.0min, 200mg;
Component fr.83, acquisition time 26.0-35.0min, 106mg;
Component fr.84, acquisition time 35.0-42.0min, 141mg;
Component fr.85, acquisition time 42.0-44.0min, 39mg;
Component fr.86, acquisition time 44.0-47.0min, 87mg;
Component fr.87, acquisition time 47.0-55.0min, 107mg.
Among the present invention, contained chemical compound sees Table 1 in each component of Rhizoma Chuanxiong, and its Analysis and Identification method is referring to embodiment three.
Contained chemical compound in each component of table 1. Rhizoma Chuanxiong
| The Rhizoma Chuanxiong component | Compounds identified |
| fr.51 fr.52 fr.53 fr.54 fr.56 fr.57 fr.84 fr.85 | Vanillin, Ferulic acid; Senkyunolide I or Senkyunolide H; Neocnidilide, (E)-form, 3,4-Methylene, 5-Meether or 3-(3,4,5-Trihydroxyphenyl)-2-propenoic acid; 3-Butylidene-1 (3H)-isobenzofuranone; Riligustilide, Levistolide A, 9-octadecadienic acid methyl ester; 9-octadecadienic acid methyl ester; Ferulic acid; Senkyunolide I or Senkyunolide H. |
Above Rhizoma Chuanxiong, extraction solution can increase or reduce when industrialization is extracted according to corresponding ratio, as large-scale production 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 goes for any Chinese crude drug in the present Chinese medicine, for example can be used for Resina Ferulae, Folium Artemisiae Argyi, Benzoinum, Semen Platycladi, Carapax Trionycis, Semen Arecae, Herba Menthae, Semen Ricini, holds, Fructus Psoraleae, Radix Isatidis, Fructus Piperis Longi, Fructus Crotonis, Radix Morindae Officinalis, Rhizoma Menispermi, Radix Glehniae, Pseudobulbus Bletillae (Rhizoma Bletillae), the Radix Pulsatillae, the Radix Paeoniae Alba, the Radix Angelicae Dahuricae, Rhizoma Typhonii, Rhizoma Imperatae, Semen Ginkgo, Rhizoma Cynanchi Stauntonii, Semen Lablab Album, Radix Ampelopsis, Cortex Dictamni, Radix Cynanchi Atrati, Herba Lobeliae Chinensis, Herba Scutellariae Barbatae, the Rhizoma Pinelliae, Bulbus Lilii, Borneolum Syntheticum, Radix Bupleuri, Periostracum Cicadae, Fructus Broussonetiae, Radix Dichroae, Cortex Ailanthi, Squama Manis, Herba Andrographis, Semen Phaseoli, Halloysitum Rubrum, Radix Paeoniae Rubra, Rhizoma Atractylodis, Fructus Xanthii, Lignum Aquilariae Resinatum, Herba Sedi, Cacumen Platycladi, Radix Aconiti Kusnezoffii, Folium Aconiti Kusnezoffii, Semen Alpiniae Katsumadai, Fructus Tsaoko, Bulbus Fritillariae Cirrhosae, Radix Cyathulae, Radix Aconiti, Rhizoma Chuanxiong, Fructus Toosendan, Semen Plantaginis, Semen Sojae Preparatum, Radix Codonopsis, Herba Lophatheri, the Cortex Eucommiae, Radix Angelicae Pubescentis, Arisaema Cum Bile, Radix Cirsii Japonici, Pericarpium Arecae, Cortex Moutan, Gypsum Fibrosum Preparatum, Exocarpium Benincasae, Cordyceps, Pheretima, the Fructus Kochiae, Cortex Lycii, Radix Rehmanniae Preparata, Herba Euphorbiae Humifusae, Radix Sanguisorbae, Radix Angelicae Sinensis, Medulla Junci, Flos Caryophylli, Semen Canavaliae, Folium Isatidis, Fructus Jujubae, Radix Et Rhizoma Rhei, Herba Centipedae, Rhizoma Curcumae, catechu, Semen Torreyae, Herba Spirodelae, Rhizoma Dioscoreae Septemlobae, Fructus Rubi, Fructus Citri Sarcodactylis, Radix Aconiti Lateralis Preparata, Poria, Radix Stephaniae Tetrandrae, Radix Saposhnikoviae, Folium Sennae, Mel, Gecko, Ramulus Cinnamomi, Radix Puerariae, Rhizoma Ligustici, Rhizoma Alpiniae Officinarum, Rhizoma Drynariae, Fructus Setariae Germinatus, Flos Eriocauli, Rhizoma Cibotii, Fructus Lycii, Folium Ilicis Cornutae, Ramulus Uncariae Cum Uncis, Radix Et Rhizoma Nardostachyos, Radix Glycyrrhizae, Radix Kansui, Fructus Trichosanthis, Semen Trichosanthis, Pericarpium Trichosanthis, Caulis Aristolochiae Manshuriensis, Rhizoma Zingiberis, Rhizoma Zingiberis Preparatum, Resina Toxicodendri, Fructus Carpesii, the Radix Astragali, Rhizoma Polygonati, Sargassum, Flos Sophorae, Caulis Piperis Kadsurae, Folium Nelumbinis, Radix Scutellariae, Rhizoma Coptidis, Cortex Phellodendri, Pericarpium Zanthoxyli, Radix Polygoni Multiflori, Rhizoma Polygoni Cuspidati, Rhizoma Picrorhizae, Cortex Magnoliae Officinalis, Exocarpium Citri Grandis, Fructus Cannabis, Cortex Albiziae, Flos Albiziae, Radix Knoxiae, Flos Carthami, Semen Sesami Nigrum, Flos Chrysanthemi, Bombyx Batryticatus, Radix Platycodonis, Semen Citri Reticulatae, Rhizoma Curcumae Longae, Caulis Spatholobi, Flos Celosiae Cristatae, Radix Tinosporae, Herba Inulae, Rhizoma Fagopyri Dibotryis, Herba Lysimachiae, Flos Lonicerae, Lignum Dalbergiae Odoriferae, Herba Schizonepetae, Semen Allii Tuberosi, Semen Cassiae, Flos Farfarae, the Fructus Chebulae, Semen Armeniacae Amarum, Radix Sophorae Flavescentis, Cortex Meliae, Semen Raphani, Omphalia, Flos Campsis, Herba Pyrolae, Radix Rhapontici, Fructus Liquidambaris, Semen Nelumbinis, Caulis Trachelospermi, Aloe, Rhizoma Phragmitis, Rhizoma Anemones Raddeanae, Radix Zanthoxyli, Fructus Forsythiae, Ganoderma, Folium Apocyni Veneti, Fructus Momordicae, Semen Litchi, Radix Gentianae, Arillus Longan, Herba Erodii, Herba Ecliptae, Herba Ephedrae, Fructus Viticis, Folium Rhododendri Daurici, Oleum Rhododendri Daurici, Flos Buddlejae, Flos Mume, Herba Ephedrae, Radix Ophiopogonis, Fructus Hordei Germinatus, Flos Rosae Rugosae, Radix Changii, Herba Verbenae, Fructus Aristolochiae, Semen Strychni, Semen Strychni Pulveratum, Lasiosphaera Seu Calvatia, Herba Portulacae, Fructus Chaenomelis, the Radix Aucklandiae, the Herba Equiseti Hiemalis, Radix Adenophorae, Fructus Ligustri Lucidi, Fructus Arctii, Radix Achyranthis Bidentatae, Nodus Nelumbinis Rhizomatis, Pollen Typhae, Herba Taraxaci, Folium Eriobotryae, Herba Eupatorii, Rhizoma Wenyujin Concisum, Herba Dianthi, Cortex Fraxini, Rhizoma Bistortae, Semen Euryales, Rhizoma Et Radix Notopterygii, Radix Aristolochiae, Caulis Sinomenii, Pericarpium Citri Reticulatae Viride, Semen Celosiae, Herba Artemisiae Annuae, Indigo Naturalis, Radix Rubiae, Semen Pharbitidis, Radix Peucedani, Rhizoma Homalomenae, Semen Euphorbiae, Radix Gentianae Macrophyllae, Nux Prinsepiae, Semen Myristicae, Herba Cistanches, Cortex Cinnamomi, Radix Ginseng, Folium Ginseng, Rhizoma Acori Graminei, Herba Cynomorii, Folium Mori, Radix Phytolaccae, Fructus Mori, Fructus Cnidii, Cortex Mori, Ramulus Mori, Ramulus Mori, Herba Taxilli, Semen Aesculi, Semen Ziziphi Spinosae, Rhizoma Belamcandae, Styrax, Semen Astragali Complanati, Fructus Quisqualis, Calyx Kaki, Fructus Amomi, Radix Sophorae Tonkinensis, Fructus Corni, Rhizoma Dioscoreae, Pseudobulbus Cremastrae Seu Pleiones, Fructus Crataegi, Herba Cirsii, Rhizoma Cimicifugae, Cornu Bubali, Pulvis Cornus Bubali Concentratus, Folium Pyrrosiae, Concha Haliotidis, Herba Dendrobii, Pericarpium Granati, Rhizoma Zingiberis Recens, Retinervus Luffae Fructus, Rhizoma Chuanxiong, rhizoma sparganic, Semen Lepidii (Semen Descurainiae), Semen Cuscutae, Semen Persicae, Medulla Tetrapanacis, Lignum Santali Albi, Radix Trichosanthis, Concretio Silicea Bambusae, Rhizoma Arisaematis, Rhizoma Gastrodiae, Radix Semiaquilegiae, Radix Pseudostellariae, Cortex Pseudolaricis, Rhizoma Smilacis Glabrae, Fructus Evodiae, Radix Clematidis, Semen Vaccariae, Cortex Acanthopancis, Fructus Schisandrae Chinensis, Galla Chinensis, Concha Arcae, the Radix Linderae, Fructus Mume, Rhizoma Corydalis Decumbentis, Radix Dipsaci, Flos Inulae Herba Siegesbeckiae, Bulbus Allii Macrostemonis, Spica Prunellae, Flos Magnoliae, Cortex Periplocae, Rhizoma Cyperi, Fructus Citri, Herba Moslae, Fructus Foeniculi, Rhizoma Curculiginis, Herba Agrimoniae, Radix Scrophulariae, Matrii Sulfas Exsiccatus, Radix Panacis Quinquefolii, Crinis Carbonisatus, Sanguis Draxonis, Radix Cynanchi Paniculati, Herba Epimedii, Herba Leonuri, Folium Ginkgo, Semen Coicis, Radix Stellariae, Radix Polygalae, Flos Genkwa, Semen Pruni, Radix Curcumae, Herba Houttuyniae, Herba Artemisiae Scopariae, Fructus Bruceae, Flos Rosae Chinensis, Rhizoma Polygonati Odorati, Rhizoma Corydalis, Bulbus Fritillariae Thunbergii, Fructus Gleditsiae Abnormalis, Fructus Perillae, Radix Asteris, Herba Violae, Radix Arnebiae (Radix Lithospermi), Polyporus, Spina Gleditsiae, the Rhizoma Anemarrhenae, the Herba Lycopi, Rhizoma Alismatis, Concha Margaritifera, Fructus Aurantii Immaturus, Fructus Gardeniae.
Any form on the pharmaceutics be can make according to the Rhizoma Chuanxiong effective ingredient that the inventive method obtained, injection and oral formulations comprised.Wherein injection comprises injection, drip liquid, injectable powder; Oral formulations 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, unguentum, sublimed preparation, spray, drop pill, disintegrating agent, oral cavity disintegration 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 Rhizoma Chuanxiong finger printing, each component ultraviolet spectrogram of Rhizoma Chuanxiong water solublity, each component mass spectrum of Rhizoma Chuanxiong water solublity, fat-soluble each the component ultraviolet spectrogram of Rhizoma Chuanxiong, fat-soluble each the component mass spectrum of Rhizoma Chuanxiong below, be intended to further specify the present invention, but the present invention is not construed as limiting.
Fig. 1 Rhizoma Chuanxiong finger printing
Each component uv-spectrogram of Fig. 2 Rhizoma Chuanxiong water solublity
Each component mass spectrum of Fig. 3 Rhizoma Chuanxiong water solublity
Fat-soluble each the component uv-spectrogram of Fig. 4 Rhizoma Chuanxiong
Fat-soluble each the component mass spectrum of Fig. 5 Rhizoma Chuanxiong
The specific embodiment
Embodiment one
(1) extraction process: take by weighing 250g Rhizoma Chuanxiong medical material, with its grinding and sieving, add ethyl acetate and ethanol then, heating extraction gets extracting solution fr.1.Add ethanol in medicinal residues, heating extraction gets extracting solution fr.2.Add entry in medicinal residues at last, heating extraction gets extracting solution fr.3.
(2) separating technology: with extracting solution fr.1 concentrate 49.6g extractum, get fr.1 extractum 10.2g silica gel mixed sample, adopt normal phase silicagel column that it is separated, at first use petroleum ether and ethyl acetate as mobile phase, get eluent fr.4, change chloroform and methanol then as mobile phase, get eluent fr.5, concentrate the 2.2g sample, use methanol as mobile phase at last, eluent fr.6.With extracting solution fr.2 concentrate 85.6g extractum, get sample on the 10.0g extractum dissolve with methanol, adopt the ODS-C18 post that it is separated, the methanol of at first using low concentration gets eluent fr.7 as mobile phase, and the methanol that changes intermediate concentration then is as mobile phase, get eluent fr.8, concentrate the 5.0g sample, use pure methanol solution as mobile phase at last, eluent 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 semi-preparative column (Lichrospher C18; 10.0mm * 250mm, 5 μ m), mobile phase is water and acetonitrile, gradient elution is collected fraction 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 acquisition time that are obtained by the preparative liquid chromatography separation are as follows:
Component fr.51, acquisition time 3.0-10.0min, 217mg;
Component fr.52, acquisition time 10.0-16.2min, 112mg;
Component fr.53, acquisition time 16.2-24.0min, 129mg;
Component fr.54, acquisition time 24.0-27.8min, 214mg;
Component fr.55, acquisition time 27.8-31.8min, 168mg;
Component fr.56, acquisition time 31.8-38.0min, 101mg;
Component fr.57, acquisition time 38.0-43.0min, 318mg;
Component fr.58, acquisition 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 acquisition time that are obtained by the preparative liquid chromatography separation are as follows:
Component fr.81, acquisition time 4.0-19.8min, 177mg;
Component fr.82, acquisition time 19.8-26.0min, 195mg;
Component fr.83, acquisition time 26.0-35.0min, 98mg;
Component fr.84, acquisition time 35.0-42.0min, 133mg;
Component fr.85, acquisition time 42.0-44.0min, 35mg;
Component fr.86, acquisition time 44.0-47.0min, 81mg;
Component fr.87, acquisition time 47.0-55.0min, 100mg.
Embodiment two
(1) extraction process: take by weighing 500g Rhizoma Chuanxiong medical 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 medicinal residues, reflux 0.5～3 hour is extracted 1～3 time, and merging filtrate gets extracting solution fr.2.Add entry in medicinal residues 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 extractum, get fr.1 extractum 20g silica gel mixed sample, adopt normal phase silicagel column that it is separated, at first with volume ratio be 50: 1 petroleum ether and ethyl acetate as mobile phase, eluent fr.4, change volume ratio then and be 10: 1 chloroform and methanol as mobile phase, get eluent fr.5, concentrate the 4.8g sample, use methanol as mobile phase at last, eluent fr.6.With extracting solution fr.2 concentrate 171.3g extractum, get 20.0g extractum with sample on 2～10% dissolve with methanol, adopt the ODS-C18 post that it is separated, at first use 2～10% methanol as mobile phase, get eluent fr.7, change 40～80% methanol then as mobile phase, get eluent fr.8, concentrate the 9.9g sample, use 100% methanol solution as mobile phase at last, eluent 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 semi-preparative column (Lichrospher C18; 10.0mm * 250mm, 5 μ m), mobile phase is water and acetonitrile, and gradient elution, flow velocity are 3ml/min, and column temperature is a room temperature, collects fraction 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 acquisition time that are obtained by the preparative liquid chromatography separation are as follows:
Component fr.51, acquisition time 3.0-10.0min, 428mg;
Component fr.52, acquisition time 10.0-16.2min, 226mg;
Component fr.53, acquisition time 16.2-24.0min, 265mg;
Component fr.54, acquisition time 24.0-27.8min, 424mg;
Component fr.55, acquisition time 27.8-31.8min, 341mg;
Component fr.56, acquisition time 31.8-38.0min, 203mg;
Component fr.57, acquisition time 38.0-43.0min, 647mg;
Component fr.58, acquisition 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 acquisition time that are obtained by the preparative liquid chromatography separation are as follows:
Component fr.81, acquisition time 4.0-19.8min, 355mg;
Component fr.82, acquisition time 19.8-26.0min, 384mg;
Component fr.83, acquisition time 26.0-35.0min, 201mg;
Component fr.84, acquisition time 35.0-42.0min, 272mg;
Component fr.85, acquisition time 42.0-44.0min, 73mg;
Component fr.86, acquisition time 44.0-47.0min, 161mg;
Component fr.87, acquisition time 47.0-55.0min, 200mg.
Embodiment three
One, the extraction separation of Rhizoma Chuanxiong medical material
(1) extraction process: take by weighing Rhizoma Chuanxiong medical 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 medicinal residues, reflux 1 hour is extracted 2 times, and merging filtrate gets extracting solution fr.2.Add entry in medicinal residues 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 extractum, get fr.1 extractum 10.2g silica gel mixed sample, adopt normal phase silicagel column that it is separated, at first with volume ratio be 50: 1 petroleum ether and ethyl acetate as mobile phase, eluent fr.4, change volume ratio then and be 10: 1 chloroform and methanol as mobile phase, get eluent fr.5, concentrate the 2.5g sample, use methanol as mobile phase at last, eluent fr.6.With extracting solution fr.2 concentrate 88.7g extractum, get 10.0g extractum with sample on 5% dissolve with methanol, adopt the ODS-C18 post that it is separated, at first use 5% methanol as mobile phase, get eluent fr.7, change 60% methanol then as mobile phase, get eluent fr.8, concentrate the 5.6g sample, use 100% methanol solution as mobile phase at last, eluent fr.9.
(3) preparation separating technology: in order from step (2), to obtain fr.5 and the more concrete effective ingredient 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 semi-preparative column (the Lichrospher C18 of Chinese nation; 10.0mm * 250mm, 5 μ m); Adopt gradient elution, mobile 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 acquisition time that are obtained by the preparative liquid chromatography separation are as follows:
Component fr.51, acquisition time 3.0-10.0min, 220mg;
Component fr.52, acquisition time 10.0-16.2min, 117mg;
Component fr.53, acquisition time 16.2-24.0min, 136mg;
Component fr.54, acquisition time 24.0-27.8min, 221mg;
Component fr.55, acquisition time 27.8-31.8min, 175mg;
Component fr.56, acquisition time 31.8-38.0min, 107mg;
Component fr.57, acquisition time 38.0-43.0min, 328mg;
Component fr.58, acquisition 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, mobile 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 acquisition time that are obtained by the preparative liquid chromatography separation are as follows:
Component fr.81, acquisition time 4.0-19.8min, 182mg;
Component fr.82, acquisition time 19.8-26.0min, 200mg;
Component fr.83, acquisition time 26.0-35.0min, 106mg;
Component fr.84, acquisition time 35.0-42.0min, 141mg;
Component fr.85, acquisition time 42.0-44.0min, 39mg;
Component fr.86, acquisition time 44.0-47.0min, 87mg;
Component fr.87, acquisition 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 degasser, quarternary low pressure pump, automatic sampler, column oven, DAD detector, 1946D electron spray level Four bar mass detector, Chemstation chromatographic work 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 (prunus mume (sieb.) sieb.et zucc. Teller-Tuo benefit Instr Ltd.).
Acetonitrile is chromatographically pure reagent (MERCK), and water is WAHAHA pure water (Hangzhou WAHAHA group), and acetic acid is analytical pure (Hangzhou chemical reagent work).
2.2 analytical method
Sample source: Rhizoma Chuanxiong 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.
Sample 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 limits 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: Rhizoma Chuanxiong 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.
Sample 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 limits 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 analysis result
Analysis result: Fig. 2 is each component uv-spectrogram of Rhizoma Chuanxiong water solublity, and Fig. 3 is each component mass spectrum of Rhizoma Chuanxiong water solublity, and Fig. 4 is fat-soluble each the component uv-spectrogram of Rhizoma Chuanxiong, and Fig. 5 is fat-soluble each the component mass spectrum of Rhizoma Chuanxiong.
The qualification result of chemical compound sees Table 2 in the Rhizoma Chuanxiong component.
Table 2. Rhizoma Chuanxiong component composition qualification result
| Component | Chromatographic retention (min) | Uv-absorption maximum wavelength (nm) | [M-H] - | Chemical compound | Remarks |
| fr.51 | 9.5 11.2 12.6 15.0 | 206，218， 262，296 218，246， 292，322 274 218，268 | 151 193 223 329 | Vanillin Ferulic acid Senkyunolide I；Senkyunolide H | Document 1 document 1 document 1 |
| fr.52 | |||||
| fr.53 | |||||
| 15.4 | 214，270， 324 | 193 | neocnidilide | Document 1 | |
| 16.4 | 206，218， 270，335 | 221 | 3-(3，4，5-Trihydroxyphenyl)-2-propenoic acid；(E)-form，3，4-Methylene， 5-Me ether | Natural product database D NP | |
| fr.54 | 11.2 | 218，246， 292，322 | 193 | Ferulic acid | |
| 17.9 | 222 | 205 | |||
| 18.5 | 218，270， 296，318 | 203 | 3-Butylidene-1(3H)-isobenzofuranone | Natural product database D NP | |
| fr.55 | 21.5 | 206，280 | 313 | ||
| 21.8 | 206，280 | 313 | |||
| fr.56 | 23.9 | 222，282， 326 | 265 | ||
| 27.0 | 224 | 380 | Riligustilide | Document 1 | |
| 29.2 | 224，282 | 380 | Levistolide A | Document 1 | |
| 29.9 | 226，276 | 295 | 9-octadecadienic acid methyl ester | Document 2 |
| fr.57 | 26.3 | 222，276 | 295 | 9-octadecadienic acid methyl ester | Document 2 |
| fr.81 fr.82 fr.83 fr.84 fr.85 fr.86 | 4.0 9.7 12.6 18.5 | 260，314 216，246， 295，326 214，296， 322 218，246， 292，322 | 677 353 179 193 | Ferulic acid | Document 1 |
| 19.4 | 218，246， 296，328 | 257 | |||
| 19.8 | 218，246， 295，326 | 257 | |||
| 23.7 | 278 | 223 | Senkyunolide I；Senkyunolide H | Document 1 | |
| 25.1 | 218，276 | 431 | |||
| 29.6 | 220，290， 322 | 577 | |||
| fr.87 | 29.7 | 220，290， 322 | 577 |
List of references:
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 Rhizoma Chuanxiong effective site gas chromatography and mass spectromentry and fingerprint map analyzing.The mass spectrum journal.Vol.25 No.3 2004。151-154
Three, Rhizoma Chuanxiong component medicine efficacy screening experiment
3.1 pharmacological model: neonatal rat myocardial cell hypoxic-ischemic model
After former generation, myocardial cell was cultivated birth SD neonatal rat 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% hyclone 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 culture fluid.Place 95%N
2And 5%CO
2Anoxia 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, liposoluble constituent 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 culture fluid is controlled at 10
-5G/mL.
Lactic acid dehydrogenase content can be represented the cell injury degree in the lactic acid dehydrogenase assay cell culture fluid.The lactic acid dehydrogenase content that bleeds in the supernatant 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.Assay method is: get 30 μ l cell conditioned medium liquid, add 50 μ l substrate buffer 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 shading value 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 lactic acid dehydrogenase (LDH) The selection result, Rhizoma Chuanxiong 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
| Component | Mean | Std | P1 | P2 |
| Fr.51 fr.52 fr.53 fr.81 | 0.18 0.208 0.199 0.17 0.198 0.2 | 0.006 0.041 0.01 0.008 0.033 0.019 | 0.020921 0.025454 0.098942 0.000842 0.128979 | 0.020309 0.019721 0.04731 0.020214 0.128979 |
3.2 huve cell anoxia 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 is removed in suction, with the abundant dissolution precipitation of M199 culture fluid (contain 10% hyclone, 100U/ml is two anti-, 0.15mg/ml Heparin, 10 μ M VC).Gained cell suspension is sub-packed in 5cm
2Culture bottle places in the incubator and cultivates.Changed in second day and adopt the M199 culture fluid that contains 30 μ g/ml ECGS, changed a culture fluid and be paved with the bottom surface in per afterwards three days until cell.Used cell is former generation endotheliocyte.The culture 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 culture fluid, adding pastille does not have phenol red M199 culture fluid, and every hole total amount is 100 μ l.Place 95%N
2And 5%CO
2Anoxia 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, liposoluble constituent 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 culture fluid 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 Rhizoma Chuanxiong have the effect that suppresses NO overexpression behind endotheliocyte anoxia-reoxygenation, and fr.53, fr.54, fr.55, fr.81 are significantly effective.
Table 4. endotheliocyte The selection result
| Rhizoma Chuanxiong | Mean | Std | P |
| Fr.53 fr.54 fr.55 fr.81 model group | 0.061 0.062 0.062 0.060 0.070 | 0.001 0.001 0.001 0.004 0.005 | 0.035 ** 0.043 ** 0.049 ** 0.013 ** |
Above result of the test explanation, the Chinese crude drug effective ingredient that adopts Chinese crude drug standardization extracting method of the present invention to obtain can be as the preparation medicine.
Claims (5)
1. Rhizoma Chuanxiong extraction, separation criterion method may further comprise the steps:
(1) extraction process: take by weighing the Rhizoma Chuanxiong medical material, with its grinding and sieving, add ethyl acetate and ethanol then, heating extraction gets extracting solution fr.1; Add ethanol in medicinal residues, heating extraction gets extracting solution fr.2; Add entry in medicinal residues at last, heating extraction gets extracting solution fr.3;
(2) separating technology: after extracting solution fr.1 is condensed into extractum, use silica gel mixed sample, adopt normal phase silicagel column that it is separated, at first use petroleum ether and ethyl acetate as mobile phase, get eluent fr.4, change chloroform and methanol then, get eluent fr.5 as mobile phase, use methanol as mobile phase at last, get eluent fr.6; After extracting solution fr.2 is condensed into extractum, with sample on the dissolve with methanol, adopt the ODS-C18 post that it is separated, the methanol of at first using low concentration is as mobile phase, get eluent fr.7, the methanol that changes intermediate concentration then gets eluent fr.8 as mobile phase, use pure methanol solution as mobile phase at last, get eluent fr.9;
(3) preparation separating technology: separate fr.5 and fr.8 with preparative liquid chromatography; Chromatographic column is a semi-preparative column, and mobile phase is water and acetonitrile, and gradient elution is collected fraction in the corresponding time period, obtains to have passed through further isolating each component.
2. Rhizoma Chuanxiong as claimed in claim 1 extracts, the separation criterion method, comprises the steps:
(1) extraction process: take by weighing the Rhizoma Chuanxiong medical 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%7 alcohol in medicinal residues, reflux 0.5～3 hour is extracted 1～3 time, and merging filtrate gets extracting solution fr.2; Add entry in medicinal residues 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 extractum, use silica gel mixed sample, adopt normal phase silicagel column that it is separated, be that 50: 1 petroleum ether and ethyl acetate are as mobile phase at first with volume ratio, eluent fr.4, change volume ratio then and be 10: 1 chloroform and methanol as mobile phase, eluent fr.5, use methanol as mobile phase at last, get eluent fr.6; After extracting solution fr.2 is condensed into extractum, with sample on 2～10% dissolve with methanol, adopt the ODS-C18 post that it is separated, at first use 2～10% methanol as mobile phase, get eluent fr.7, change 40～80% methanol then, get eluent fr.8 as mobile phase, use 100% methanol solution as mobile phase at last, get eluent fr.9;
(3) preparation separating technology: separate fr.5 and fr.8 with preparative liquid chromatography; Chromatographic column is semi-preparative column (LichrospherC18; 10.0mm * 250mm, 5 μ m), mobile phase is water and acetonitrile, and gradient elution, flow velocity are 3ml/min, and column temperature is a room temperature, collects fraction in the corresponding time period, obtains through further isolating each component.
3. Rhizoma Chuanxiong as claimed in claim 2 extracts, the separation criterion method, comprises the steps:
(1) extraction process: take by weighing the Rhizoma Chuanxiong medical 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 medicinal residues, reflux 1 hour is extracted 2 times, and merging filtrate gets extracting solution fr.2; Add entry in medicinal residues 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 extractum with extracting solution, use silica gel mixed sample, adopt normal phase silicagel column that it is separated, be that 50: 1 petroleum ether and ethyl acetate are as mobile phase at first with volume ratio, eluent fr.4, change volume ratio then and be 10: 1 chloroform and methanol as mobile phase, eluent fr.5, use methanol as mobile phase at last, get eluent fr.6; Fr.2 is condensed into extractum with extracting solution, with sample on 5% dissolve with methanol, adopt the ODS-C18 post that it is separated, at first use 5% methanol as mobile phase, get eluent fr.7, change 60% methanol then, get eluent fr.8 as mobile phase, use 100% methanol solution as mobile phase at last, get eluent 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 semi-preparative column (the Lichrospher C18 of Chinese nation; 10.0mm * 250mm, 5 μ m); Adopt gradient elution, mobile 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 acquisition time that are obtained by the preparative liquid chromatography separation are as follows:
Component fr.51, acquisition time 3.0-10.0min;
Component fr.52, acquisition time 10.0-16.2min;
Component fr.53, acquisition time 16.2-24.0min;
Component fr.54, acquisition time 24.0-27.8min;
Component fr.55, acquisition time 27.8-31.8min;
Component fr.56, acquisition time 31.8-38.0min;
Component fr.57, acquisition time 38.0-43.0min;
Component fr.58, acquisition 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, mobile 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 acquisition time that are obtained by the preparative liquid chromatography separation are as follows:
Component fr.81, acquisition time 4.0-19.8min;
Component fr.82, acquisition time 19.8-26.0min;
Component fr.83, acquisition time 26.0-35.0min;
Component fr.84, acquisition time 35.0-42.0min;
Component fr.85, acquisition time 42.0-44.0min;
Component fr.86, acquisition time 44.0-47.0min;
Component fr.87, acquisition time 47.0-55.0min.
4. Rhizoma Chuanxiong as claimed in claim 3 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-Methylene, 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.
5. Rhizoma Chuanxiong as claimed in claim 3 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.
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