CN101073587A - Method for separating and extracting Chinese Thorowax Root - Google Patents
Method for separating and extracting Chinese Thorowax Root Download PDFInfo
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- CN101073587A CN101073587A CN 200610013743 CN200610013743A CN101073587A CN 101073587 A CN101073587 A CN 101073587A CN 200610013743 CN200610013743 CN 200610013743 CN 200610013743 A CN200610013743 A CN 200610013743A CN 101073587 A CN101073587 A CN 101073587A
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Abstract
The invention is concerned with the standard extraction and isolation method of Bupleurum. It can form a medicinal materials component store according to different polarity levels of the Bupleurum divide into several groups, which each group contents few types of compound. It can use to discover new medicine by conducting drug screening from the component store. The method can reduce the period of medicine extraction and isolation, and reduce the consumption of manpower and material resources, and it suits for most types of medicine.
Description
Technical field
The invention belongs to field of medicaments, particularly relate to Radix Bupleuri 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, the problem of utilize the high Drug therapy of pure natural degree, preventing some chemical synthetic drugs cann't be solved, 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.
Radix Bupleuri is Umbelliferae (Umbelliferae) Bupleurum (Bupleurum) plant.There are 200 kinds of Bupleurum plants in the whole world, and China has reported 42 kinds, and 17 mutation and 7 distortion (Cheng Biqiang, analogy is learned thrifty. Yunnan tropical and subtropical zone spice berry. and Kunming: publishing house of Yunnan University, 1995.69-73)." People's Republic of China's allusion quotation " (nineteen ninety-five version) regulation Radix Bupleuri Bupleurum chineseDC. and Radix Bupleuri Scorzonerifolii B.scorzonerifolium Willd. are medicinal Radix Bupleuri.The former main product in Liaoning, ground such as Gansu, Hebei, Henan, Shandong, latter's main product in Hubei, ground such as Sichuan, Jiangsu, Anhui.Main Ingredients and Appearance is a saikoside in the Radix Bupleuri root, and next contains plant sterol, adonite, and a small amount of volatile oil, polysaccharide; Aerial parts mainly contains flavonoid, small amounts of soap glycoside, lignanoids, Coumarins composition.Saikoside has physiologically actives such as analgesia, calmness, antiinflammatory; Find that at present the Radix Bupleuri polysaccharide has effects such as antiinflammatory, blood fat reducing, raise immunity and antiviral, stronger in addition antiulcer action (Zhang Yongwen. the structure of Radix Bupleuri pectin polysaccharide and pharmacology activity research progress J. foreign medical science Chinese medicine fascicle, 1996,18 (4): 20-23.), autoimmune diseases such as hepatitis and SLE, nephritis and rheumatoid are had important clinic value.Flavonoid has the effect of enhancing capillary function etc.
How to determine a kind of extraction, separation criterion method, the active constituents of medicine in the Radix Bupleuri 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 Radix Bupleuri extraction, separation criterionization.Exactly Radix Bupleuri 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 Radix Bupleuri extracts, the inventor is by a large amount of tests, the extraction of effective components of present Radix Bupleuri 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 Radix Bupleuri is extracted, can obtain the effective ingredient in the Chinese medicine to greatest extent.
Radix Bupleuri 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 Radix Bupleuri 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 Radix Bupleuri 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 a 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.
Preferably, the present invention can implement through the following steps:
(1) extraction process: take by weighing the Radix Bupleuri 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 preparative column (Zorbax SB-C18; 21.2mm * 250mm), mobile phase is water and acetonitrile, and gradient elution, flow velocity are 10ml/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 Radix Bupleuri 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 70% 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: in order from step (2), to obtain fr.5 and the more concrete effective ingredient of fr.8 in the Radix Bupleuri 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 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 90% water, and Mobile phase B is 10% acetonitrile solution;
During 40min, mobile phase A is 25% water, and Mobile phase B is 75% acetonitrile solution;
During 45min, 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 3.0-7.0min;
Component fr.52, acquisition time 7.0-14.9min;
Component fr.53, acquisition time 14.9-21.0min;
Component fr.54, acquisition time 21.0-26.8min;
Component fr.55, acquisition time 26.8-32.2min;
Component fr.56, acquisition time 32.2-41.0min;
Component fr.57, acquisition time 41.0-47.0min;
Component fr.58, acquisition time 47.0-50.0min;
The fr.8 separation condition: chromatographic column is Agilent preparative column (Zorbax SB-C18; 21.2mm * 250mm); Adopt gradient elution, mobile phase is 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 98% aqueous solution, and Mobile phase B is 2% acetonitrile solution;
During 10min, mobile phase A is 95% aqueous solution, and Mobile phase B is 5% acetonitrile solution;
During 15min, mobile phase A is 85% aqueous solution, and Mobile phase B is 15% acetonitrile solution;
During 35min, mobile phase A is 50% aqueous solution, and Mobile phase B is 50% acetonitrile solution;
During 45min, mobile phase A is 5% aqueous solution, and Mobile phase B is 95% 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 4.0-7.0min;
Component fr.82, acquisition time 7.0-9.0min;
Component fr.83, acquisition time 9.0-17.8min;
Component fr.84, acquisition time 17.8-24.0min;
Component fr.85, acquisition time 24.0-33.3min;
Component fr.86, acquisition time 33.3-38.0min;
Component fr.87, acquisition time 38.0-45.0min;
In order to obtain concrete Radix Bupleuri effective ingredient, best extraction separation method of the present invention is:
(1) extraction process: take by weighing Radix Bupleuri 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 36.6g extractum, use 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 6.3g sample, use methanol as mobile phase at last, eluent fr.6.With extracting solution fr.2 concentrate 29.3g extractum, get 3.0g 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 70% methanol then as mobile phase, get eluent fr.8, concentrate the 2.8g 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 Radix Bupleuri 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 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 90% water, and Mobile phase B is 10% acetonitrile solution;
During 40min, mobile phase A is 25% water, and Mobile phase B is 75% acetonitrile solution;
During 45min, 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 1.1g 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 3.0-7.0min; 23mg;
Component fr.52, acquisition time 7.0-14.9min; 35mg
Component fr.53, acquisition time 14.9-21.0min; 27mg
Component fr.54, acquisition time 21.0-26.8min; 22mg
Component fr.55, acquisition time 26.8-32.2min; 20mg
Component fr.56, acquisition time 32.2-41.0min; 50mg
Component fr.57, acquisition time 41.0-47.0min; 108mg
Component fr.58, acquisition time 47.0-50.0min; 31mg
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 98% aqueous solution, and Mobile phase B is 2% acetonitrile solution;
During 10min, mobile phase A is 95% aqueous solution, and Mobile phase B is 5% acetonitrile solution;
During 15min, mobile phase A is 85% aqueous solution, and Mobile phase B is 15% acetonitrile solution;
During 35min, mobile phase A is 50% aqueous solution, and Mobile phase B is 50% acetonitrile solution;
During 45min, mobile phase A is 5% aqueous solution, and Mobile phase B is 95% acetonitrile solution;
The fr.8 separating resulting: get component fr.8 1.6g 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 4.0-7.0min; 560mg;
Component fr.82, acquisition time 7.0-9.0min; 10mg;
Component fr.83, acquisition time 9.0-17.8min; 90mg;
Component fr.84, acquisition time 17.8-24.0min; 86mg;
Component fr.85, acquisition time 24.0-33.3min; 91mg;
Component fr.86, acquisition time 33.3-38.0min; 16mg;
Component fr.87, acquisition time 38.0-45.0min; 15mg.
Among the present invention, contained chemical compound sees Table 1 in each component of Radix Bupleuri, and its Analysis and Identification method is referring to embodiment three.
Contained chemical compound in each component of table 1. Radix Bupleuri
| The Radix Bupleuri component | Compounds identified |
| fr.51 | 2，9-Heptadecadiene-4，6-diyn-1-ol，8CI；(all-Z)-form； |
| fr.52 | Massoialactone or dibenzoburan |
| fr.55 | (E, E)-farnesylacetone or podocarp-12-en-14-one or hexahydrofaresylacetone |
| fr.56 | 2,8,10-Heptadecatriene-4,6-diyn-1-ol; (E, E, E)-and form, Ac and decyl decanate; |
| fr.57 | Octadecenoic acid methyl ester |
| fr.82 | 3,3 ', 4,4 '-Tetrahydroxylign-7-en-9,9 '-olide; (R, E (Z))-form, 3 ', 4 '-Methylene, 4-Me ether and Guayarol; 3 '-Me ether and 2,9-Heptadecadiene-4,6-diyne-1,8-diol, 8CI; (2Z, 8S, 9Z)-form; 3,5,7,9-Tridecatetraene; (3E, 5Z, 7Z, 9E)-form |
| fr.83 | agaruspirol |
| fr.84 | 2-Phenylethanol；O-[b-D-Glucopyranosyl-(1→2)-b-D- glucopyranoside] |
| fr.85 | 2-Phenylethanol；O-[b-D-Glucopyranosyl-(1→2)-b-D- glucopyranoside] |
| fr.86 | 13，28-Epoxy-11-oleanene-3，16，23-triol；(3b，13b，16a(b))-form， 3-O-[b-D-Glucopyranosyl-(1→3)-b-D-fucopyranosidc] |
| fr.87 | (E, E)-farnesylacetone or podocarp-12-en-14-one or hexahydrofaresylacetone and 13,28-Epoxy-11-oleanene-3,16,23-triol; (3b, 13b, 16a (b))-form, 3-O-[b-D-Glucopyranosyl-(1 → 3)-b-D-fucopyranoside] |
Above Radix Bupleuri, 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 Herba Polygoni Avicularis, 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, 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, Radix Salviae Miltiorrhizae, 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, Herba Taraxaci, Pollen Typhae, 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, Radix Notoginseng, 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 Radix Bupleuri 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 the Radix Bupleuri finger printing below, each component ultraviolet spectrogram of Radix Bupleuri water solublity, each component mass spectrum of Radix Bupleuri water solublity, fat-soluble each the component ultraviolet spectrogram of Radix Bupleuri, fat-soluble each the component mass spectrum of Radix Bupleuri are intended to further specify the present invention, but the present invention are not construed as limiting.
Fig. 1 Radix Bupleuri finger printing
Each component ultraviolet spectrogram of Fig. 2 Radix Bupleuri water solublity
Each component mass spectrum of Fig. 3 Radix Bupleuri water solublity
Fat-soluble each the component ultraviolet spectrogram of Fig. 4 Radix Bupleuri
Fat-soluble each the component mass spectrum of Fig. 5 Radix Bupleuri
The specific embodiment
Below further specify the present invention by the specific embodiment, be not construed as limiting the invention in any form.
Embodiment one
(1) extraction process: take by weighing Radix Bupleuri medical material 250g, with its grinding and sieving, adding 5 times of amount volume ratios then is 1: 1 ethyl acetate and ethanol, and reflux 2 hours is extracted 3 times, and merging filtrate gets extracting solution fr.1.Add 10 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 1.5 hours is extracted 2 times, and merging filtrate gets extracting solution fr.3.
(2) separating technology: with extracting solution fr.1 concentrate 35.6g extractum, use 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 5.8g sample, use methanol as mobile phase at last, eluent fr.6.With extracting solution fr.2 concentrate 26.3g extractum, get 2.6g 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 70% methanol then as mobile phase, get eluent fr.8, concentrate the 2.6g 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 Radix Bupleuri 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 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 90% water, and Mobile phase B is 10% acetonitrile solution;
During 40min, mobile phase A is 25% water, and Mobile phase B is 75% acetonitrile solution;
During 45min, 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 1.2g 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 3.0-7.0min; 22mg;
Component fr.52, acquisition time 7.0-14.9min; 36mg
Component fr.53, acquisition time 14.9-21.0min; 25mg
Component fr.54, acquisition time 21.0-26.8min; 20mg
Component fr.55, acquisition time 26.8-32.2min; 19mg
Component fr.56, acquisition time 32.2-41.0min; 46mg
Component fr.57, acquisition time 41.0-47.0min; 100mg
Component fr.58, acquisition time 47.0-50.0min; 33mg
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 98% aqueous solution, and Mobile phase B is 2% acetonitrile solution;
During 10min, mobile phase A is 95% aqueous solution, and Mobile phase B is 5% acetonitrile solution;
During 15min, mobile phase A is 85% aqueous solution, and Mobile phase B is 15% acetonitrile solution;
During 35min, mobile phase A is 50% aqueous solution, and Mobile phase B is 50% acetonitrile solution;
During 45min, mobile phase A is 5% aqueous solution, and Mobile phase B is 95% acetonitrile solution;
The fr.8 separating resulting: get component fr.8 1.6g 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 4.0-7.0min; 530mg
Component fr.82, acquisition time 7.0-9.0min; 12mg
Component fr.83, acquisition time 9.0-17.8min; 92mg
Component fr.84, acquisition time 17.8-24.0min; 88mg
Component fr.85, acquisition time 24.0-33.3min; 90mg
Component fr.86, acquisition time 33.3-38.0min; 16mg
Component fr.87, acquisition time 38.0-45.0min; 16mg
Embodiment two
(1) extraction process: take by weighing Radix Bupleuri medical material 500g, with its grinding and sieving, adding 10 times of amount volume ratios then is 1: 1 ethyl acetate and ethanol, and reflux 3 hours is extracted 2 times, and merging filtrate gets extracting solution fr.1.Add 8 times of amount 70% ethanol in medicinal residues, reflux 2 hours is extracted 3 times, and merging filtrate gets extracting solution fr.2.Add entry in medicinal residues at last, reflux 2.5 hours is extracted 2 times, and merging filtrate gets extracting solution fr.3.
(2) separating technology: with extracting solution fr.1 concentrate 70.6g extractum, use 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 5.3g sample, use methanol as mobile phase at last, eluent fr.6.With extracting solution fr.2 concentrate 27.3g extractum, get 3.0g 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 70% methanol then as mobile phase, get eluent fr.8, concentrate the 2.8g 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 Radix Bupleuri 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 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 90% water, and Mobile phase B is 10% acetonitrile solution;
During 40min, mobile phase A is 25% water, and Mobile phase B is 75% acetonitrile solution;
During 45min, 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 2.4g 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 3.0-7.0min; 43mg
Component fr.52, acquisition time 7.0-14.9min; 65mg
Component fr.53, acquisition time 14.9-21.0min; 47mg
Component fr.54, acquisition time 21.0-26.8min; 42mg
Component fr.55, acquisition time 26.8-32.2min; 40mg
Component fr.56, acquisition time 32.2-41.0min; 90mg
Component fr.57, acquisition time 41.0-47.0min; 208mg
Component fr.58, acquisition time 47.0-50.0min; 51mg
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 98% aqueous solution, and Mobile phase B is 2% acetonitrile solution;
During 10min, mobile phase A is 95% aqueous solution, and Mobile phase B is 5% acetonitrile solution;
During 15min, mobile phase A is 85% aqueous solution, and Mobile phase B is 15% acetonitrile solution;
During 35min, mobile phase A is 50% aqueous solution, and Mobile phase B is 50% acetonitrile solution;
During 45min, mobile phase A is 5% aqueous solution, and Mobile phase B is 95% acetonitrile solution;
The fr.8 separating resulting: get component fr.8 2.7g 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 4.0-7.0min; 760mg
Component fr.82, acquisition time 7.0-9.0min; 19mg
Component fr.83, acquisition time 9.0-17.8min; 160mg
Component fr.84, acquisition time 17.8-24.0min; 156mg
Component fr.85, acquisition time 24.0-33.3min; 171mg
Component fr.86, acquisition time 33.3-38.0min; 26mg
Component fr.87, acquisition time 38.0-45.0min; 25mg.
Embodiment three
One, the extraction separation of Radix Bupleuri medical material
(1) extraction process: take by weighing Radix Bupleuri 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 36.6g extractum, use 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 6.3g sample, use methanol as mobile phase at last, eluent fr.6.With extracting solution fr.2 concentrate 29.3g extractum, get 3.0g 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 70% methanol then as mobile phase, get eluent fr.8, concentrate the 2.8g 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 Radix Bupleuri 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 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 90% water, and Mobile phase B is 10% acetonitrile solution;
During 40min, mobile phase A is 25% water, and Mobile phase B is 75% acetonitrile solution;
During 45min, 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 1.2g 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 3.0-7.0min; 23mg;
Component fr.52, acquisition time 7.0-14.9min; 35mg
Component fr.53, acquisition time 14.9-21.0min; 27mg
Component fr.54, acquisition time 21.0-26.8min; 22mg
Component fr.55, acquisition time 26.8-32.2min; 20mg
Component fr.56, acquisition time 32.2-41.0min; 50mg
Component fr.57, acquisition time 41.0-47.0min; 108mg
Component fr.58, acquisition time 47.0-50.0min; 31mg
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 98% aqueous solution, and Mobile phase B is 2% acetonitrile solution;
During 10min, mobile phase A is 95% aqueous solution, and Mobile phase B is 5% acetonitrile solution;
During 15min, mobile phase A is 85% aqueous solution, and Mobile phase B is 15% acetonitrile solution;
During 35min, mobile phase A is 50% aqueous solution, and Mobile phase B is 50% acetonitrile solution;
During 45min, mobile phase A is 5% aqueous solution, and Mobile phase B is 95% acetonitrile solution;
The fr.8 separating resulting: get component fr.8 1.6g 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 4.0-7.0min; 560mg;
Component fr.82, acquisition time 7.0-9.0min; 10mg;
Component fr.83, acquisition time 9.0-17.8min; 90mg;
Component fr.84, acquisition time 17.8-24.0min; 86mg;
Component fr.85, acquisition time 24.0-33.3min; 91mg;
Component fr.86, acquisition time 33.3-38.0min; 16mg;
Component fr.87, acquisition time 38.0-45.0min; 15mg.
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: Radix Bupleuri component fr.51 1.37mg, fr.52 1.06mg, fr.53 1.05mg, fr.54 1.40mg, fr.551.20mg, fr.56 1.05mg, fr.57 1.00mg, fr.58 1.22mg.
Sample preparation: sample 1ml dissolve with ethanol, ultrasonic, centrifugal (10000r/min), standby.
The HPLC condition: chromatographic column is Agilent Zorbax SB-C18 type chromatographic column (4.6mm * 150mm, 5 μ m), carries out gradient elution with binary mobile phase.Mobile phase A is 0.2%HAc-H
2O, Mobile phase B is 0.2%HAc-CH
3CN; Linear gradient is: 0min, 10%B; 10min, 50%B; 30min, 95%B.Flow velocity is 0.5mL/min; Column temperature is 30 ℃; 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 1.41/min, drift tube temperature: 100 ℃.
Sample source: Radix Bupleuri component fr.81 1.30mg, fr.82 1.46mg, fr.83 1.20mg, fr.84 1.20mg, fr.851.09mg, fr.86 0.95mg, fr.87 1.30mg.
Sample preparation: sample is with the dissolving of 1ml 50% methanol-water, and is ultrasonic, and centrifugal (10000r/min) is standby.
The HPLC condition: chromatographic column is Agilent Zorbax SB-C18 type chromatographic column (4.6mm * 150mm, 5 μ m), carries out gradient elution with binary mobile phase.Mobile phase A is 0.01%HAc-H
2O, Mobile phase B is 0.01%HAc-CH
3CN; Linear gradient is: 0min, 5%B; 5min, 20%B; 30min, 50%B; 35min, 50%B.Flow velocity is 0.5mL/min; Column temperature is 30 ℃; 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 ultraviolet spectrogram of Radix Bupleuri water solublity, and Fig. 3 is each component mass spectrum of Radix Bupleuri water solublity, and Fig. 4 is fat-soluble each the component ultraviolet spectrogram of Radix Bupleuri, and Fig. 5 is fat-soluble each the component mass spectrum of Radix Bupleuri.
Each compound identification result in the Radix Bupleuri component
[1-3]See Table 2.
Each component qualification result of table 2 Radix Bupleuri
| Component | Rt(min) | [M-H] - | Maximum UV | Chemical compound | Molecular formula or structural formula | Remarks |
| Fr.51 | 4.12 | 243 | 194，262 | 2，9- Heptadecadiene- 4，6-diyn-1-ol，8CI； (all-Z)-form | C 17H 24O | DNP |
| Fr.52 | 8.77 | 415 | 194，242，274 | |||
| 9.39 | 167 | 204，218，260 ，290 | Massoialactone or dibenzoburan | C 10H 16O 2Or C 12H 8O 1 | Document 2 | |
| 9.87 | 321 | 202，260 | ||||
| Fr.53 | 11.87 | 187 | 202，258 | |||
| 13.9 | 171 | 208 | ||||
| 16.0 | 169 | 222 | ||||
| Fr.54 | 14.9 | 329 | 230 | |||
| 15.36 | 171 | 230，268 | ||||
| Fr.55 | 15.13 | 261 | 200，230，282 | (E, E)-farnesylacetone or podocarp-12-en-14-one or hexahydrofaresyla cetone | C 18H 30O 1Or C 17H 26O 2 | Document 2 |
| 15.76 | 269 | 214，276，322 | ||||
| 17.87 | 677 | 233，278 | ||||
| Fr.56 | 18.87 | 283 | 202，274 | 2，8，10- Heptadecatriene- 4，6-diyn-1-ol； (E，E，E)-form，Ac | C 19H 24O 2 | DNP |
| 19.63 | 311 | 214，273，316 | decyl decanate | C 20H 40O 2 | Document 2 | |
| 21.34 | 313 | 252 | ||||
| 21.74 | 313 | 232，290，338 |
| Fr.57 | 26.2 | 295 | 234 | Octadecenoic acid methyl ester | Document 1 | |
| Fr.58 | 13.98 | 821 | 252 | |||
| Fr.82 | 5.8 | 282 | 252，278 | |||
| 10.52 | 353 | 296，324 | 3，3′，4，4′- Tetrahydroxylign- 7-en-9，9′-olide； (R，E(Z))-form， 3′，4′-Methylene，4- Me ether | C 20H 18O 6 | DNP | |
| 11.8 | 371 | 194，230 | Guayarol；3′-Me ether | C 21H 24O 6 | DNP | |
| 12.26 | 259 | 200，218，260 ，292 | 2，9- Heptadecadiene- 4，6-diyne-1，8-diol， 8CI；(2Z，8S，9Z)- form | C 17H 24O 2 | DNP | |
| 13.39 | 289 | 212，246，296 | ||||
| 14.15 | 175 | 206 | 3，5，7，9- Tridecatetraene； (3E，5Z，7Z，9E)- form | C 13H 20 | DNP | |
| Fr.83 | 8.75 | 326，539 | 204，260 | |||
| 11.46 | 221 | 202，260，304 | agaruspirol | Document 1 | ||
| 13.44 | 289 | 210，244，292 | ||||
| Fr.84 | 11.43 | 463 | 202，268 | |||
| 15.73 | 445 | 278，316 | 2-Phenylethanol； O-[b-D- Glucopyranosyl-(1 →2)-b-D- glucopyranoside] | C 20H 30O 11 | DNP | |
| 17.69 | 459 | 274，312 | ||||
| 18.67 | 459 | 274 | ||||
| Fr.85 | 15.69 | 445 | 278，316 | 2-Phenylethanol； O-[b-D- Glucopyranosyl-(1 →2)-b-D- glucopyranoside] | C 20H 30O 11 | DNP |
| 17.69 | 459 | 274，312 | ||||
| 18.68 | 459 | 274 | ||||
| Fr.86 | 23.53 | 925 | 198，280 | |||
| 24.66 | 329 | 232，280 | ||||
| 27.29 | 779 | 252 | 13，28-Epoxy-11- oleanene-3，16，23- triol； (3b，13b，16a(b))- form，3-O-[b-D- Glucopyranosyl-(1 →3)-b-D- fucopyranoside] | C 42H 68O 13 | DNP |
| Fr.87 | 24.23 | 261 | 200，230，280 | (E, E)-farnesylacetone or podocarp-12-en-14-one or hexahydrofaresyla cetone | C 18H 30O 1 | Document 2 |
| 24.65 | 329 | 287，330 | ||||
| 25.68 | 269 | 216，276，322 | ||||
| 27.3 | 779 | 252 | 13，28-Epoxy-11- oleanene-3，16，23- triol； (3b，13b，16a(b))- form，3-O-[b-D- Glucopyranosyl-(1 →3)-b-D- fucopyranoside] | C 42H 68O 13 | DNP |
1. document 1: Meng Qing, Guo Xiaoling, Feng Yifan, Chen Gengfu, beam Hamming. Radix Bupleuri supercritical carbon dioxide extraction chemical constituent gas phase-mass spectrometry analysis; 2005 VOL.16 NO.1;
2. document 2: Li Ying etc. the chemical constitution study of Yinchuan Radix Bupleuri; China's wild plant resource, fourth phase nineteen ninety-five;
(3.DNP Dictionary of Natural Products): natural product data base.
Claims (5)
1. Radix Bupleuri extraction, separation criterion method may further comprise the steps:
(1) extraction process: take by weighing the Radix Bupleuri 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 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. Radix Bupleuri as claimed in claim 1 extracts, the separation criterion method, comprises the steps:
(1) extraction process: take by weighing the Radix Bupleuri 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: separate fr.5 and fr.8 with preparative liquid chromatography; Chromatographic column is preparative column (Zorbax SB-C18; 21.2mm * 250mm), mobile phase is water and acetonitrile, and gradient elution, flow velocity are 10ml/min, and column temperature is a room temperature, collects fraction in the corresponding time period, obtains through further isolating each component.
3. Radix Bupleuri as claimed in claim 2 extracts, the separation criterion method, comprises the steps:
(1) extraction process: take by weighing the Radix Bupleuri 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 70% 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 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 90% water, and Mobile phase B is 10% acetonitrile solution;
During 40min, mobile phase A is 25% water, and Mobile phase B is 75% acetonitrile solution;
During 45min, 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 3.0-7.0min;
Component fr.52, acquisition time 7.0-14.9min;
Component fr.53, acquisition time 14.9-21.0min;
Component fr.54, acquisition time 21.0-26.8min;
Component fr.55, acquisition time 26.8-32.2min;
Component fr.56, acquisition time 32.2-41.0min;
Component fr.57, acquisition time 41.0-47.0min;
Component fr.58, acquisition time 47.0-50.0min;
The fr.8 separation condition: chromatographic column is Agilent preparative column (Zorbax SB-C18; 21.2mm * 250mm); Adopt gradient elution, mobile phase is 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 98% aqueous solution, and Mobile phase B is 2% acetonitrile solution;
During 10min, mobile phase A is 95% aqueous solution, and Mobile phase B is 5% acetonitrile solution;
During 15min, mobile phase A is 85% aqueous solution, and Mobile phase B is 15% acetonitrile solution;
During 35min, mobile phase A is 50% aqueous solution, and Mobile phase B is 50% acetonitrile solution;
During 45min, mobile phase A is 5% aqueous solution, and Mobile phase B is 95% 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 4.0-7.0min;
Component fr.82, acquisition time 7.0-9.0min;
Component fr.83, acquisition time 9.0-17.8min;
Component fr.84, acquisition time 17.8-24.0min;
Component fr.85, acquisition time 24.0-33.3min;
Component fr.86, acquisition time 33.3-38.0min;
Component fr.87, acquisition time 38.0-45.0min.
4. Radix Bupleuri as claimed in claim 3 extracts, the separation criterion method, it is characterized in that:
Main compound is 2 among the component fr.51,9-Heptadecadiene-4,6-diyn-1-ol, 8CI; (all-Z) form;
Main compound is Massoialactone or dibenzoburan among the component fr.52;
Among the component fr.55 main compound be (E, E)-farnesylacetone or podocarp-12-en-14-one or hexahydrofaresylacetone;
Main compound is 2,8 among the component fr.56,10-Heptadecatriene-4,6-diyn-1-ol; (E, E, E)-and form, Ac and decyl decanate;
Main compound is an octadecenoic acid methyl ester among the component fr.57.
5. Radix Bupleuri as claimed in claim 3 extracts, the separation criterion method, it is characterized in that:
Main compound is 3,3 ', 4 among the component fr.82,4 '-Tetrahydroxylign-7-en-9,9 '-olide;
(R, E (Z))-form, 3 ', 4 '-Methylene, 4-Me ether and Guayarol; 3 '-Me ether and
2，9-Heptadecadiene-4，6-diyne-l，8-diol，8CI；(2Z，8S，9Z)-form；3，5，7，9-Tridecatetraene；(3E，5Z，7Z，9E)-form；
Main compound is agaruspirol among the component fr.83;
Main compound is 2-Phenylethanol among the component fr.84; O-[b-D-Glucopyranosyl-(1 → 2)-b-D-glucopyranoside];
Main compound is 2-Phenylethanol among the component fr.85; O-[b-D-Glucopyranosyl-(1 → 2)-b-D-glucopyranoside];
Main compound is 13 among the component fr.86,28-Epoxy-11-oleanene-3,16,23-triol;
(3b，13b，16a(b))-form，3-O-[b-D-Glucopyranosyl-(1→3)-b-D-fucopyranoside]；
Among the component fr.87 main compound be (E, E)-farnesylacetone or podocarp-12-en-14-one or hexahydrofaresylacetone and 13,28-Epoxy-11-oleanene-3,16,23-triol;
(3b，13b，16a(b))-form，3-O-[b-D-Glucopyranosy]-(1→3)-b-D-fucopyranoside]。
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