CN1903250A - Method for extraction and separation of red rooted salvia - Google Patents
Method for extraction and separation of red rooted salvia Download PDFInfo
- Publication number
- CN1903250A CN1903250A CN 200510012277 CN200510012277A CN1903250A CN 1903250 A CN1903250 A CN 1903250A CN 200510012277 CN200510012277 CN 200510012277 CN 200510012277 A CN200510012277 A CN 200510012277A CN 1903250 A CN1903250 A CN 1903250A
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- mobile phase
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- methanol
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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 red sage root 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 Radix Salviae Miltiorrhizae 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.
Radix Salviae Miltiorrhizae, another name blood is taken root, red, Herba Wedeliae Wallichii, Arisaema balansae Engl..Radix Salviae Miltiorrhizae is from plant Salvia miltiorrhiza rhizome.Radix Salviae Miltiorrhizae is used as medicine and begins to be stated from the Shennong's Herbal of Han dynasty.The traditional Chinese medical science is thought, its bitter in the mouth cold nature has promoting blood circulation to restore menstrual flow, an effect of the silt pain relieving of dispelling, the relieving restlessness that clears away heart-fire, removing heat from blood eliminating carbuncle, is applicable to the treatment menoxenia, coronary heart disease, arteriosclerosis, hyperlipidemia, angina pectoris, dysmenorrhea, amenorrhea, metrorrhagia, leukorrhagia, traumatic injury blood stasis, insomnia, hepatitis B, neurasthenia, the schistosomicide hepatosplenomegaly, diseases such as arthralgia, particularly gynecological, internal medicine and external wounds card belongs to the blood stasis hot person that holds concurrently and uses always.Modern study shows, mainly contain Tanshinone I, IIA, IIB (Tanshinone I in the Radix Salviae Miltiorrhizae, IIA, IIB), iso tanshinone I, IIA (Isotanshinone I, IIA), cryptotanshinone (Cryptotanshinone), different cryptotanshinone (Isocryptotanshinone), methyltanshinone (Methyltanshinone), hydroxyl TANSHINONES etc.
How to determine a kind of extraction, separation criterion method, the active constituents of medicine in the Radix Salviae Miltiorrhizae 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 Salviae Miltiorrhizae extraction, separation criterionization.Exactly Radix Salviae Miltiorrhizae 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 Salviae Miltiorrhizae extracts, the inventor is by a large amount of tests, the extraction of effective components of present Radix Salviae Miltiorrhizae 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 Salviae Miltiorrhizae is extracted, can obtain the effective ingredient in the Chinese medicine to greatest extent.
Radix Salviae Miltiorrhizae 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 Salviae Miltiorrhizae 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 red rooted salvia, 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 red rooted salvia, 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 red rooted salvia, 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: in order from step (2), to obtain fr.5 and the more concrete effective ingredient of fr.8 in the Radix Salviae Miltiorrhizae 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 50% water, and Mobile phase B is 50% acetonitrile solution;
During 35min, mobile phase A is 20% water, and Mobile phase B is 80% acetonitrile solution;
During 45min, mobile phase A is 5% water, and Mobile phase B is 95% acetonitrile solution;
During 55min, 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 2.0-5.6min;
Component fr.52, acquisition time 5.6-14.0min;
Component fr.53, acquisition time 14.0-18.2min;
Component fr.54, acquisition time 18.2-27.1min;
Component fr.55, acquisition time 27.1-30.3min;
Component fr.56, acquisition time 30.3-35.2min;
Component fr.57, acquisition time 35.2-38.4min;
Component fr.58, acquisition time 38.4-41.9min;
Component fr.59, acquisition time 41.9-46.2min;
Component fr.59, acquisition time 46.2-54.5min.
The fr.8 separation condition: chromatographic column is Agilent preparative column (Zorbax SB-C18; 21.2mm * 250mm); Adopt gradient elution, mobile phase is 0.1%HAc aqueous solution (A) and 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 85% 0.1%HAc aqueous solution, and Mobile phase B is 15% acetonitrile solution;
During 10in, mobile phase A is 79% 0.1%HAc aqueous solution, and Mobile phase B is 21% acetonitrile solution;
During 15min, mobile phase A is 69% 0.1%HAc aqueous solution, and Mobile phase B is 31% acetonitrile solution;
During 30min, mobile phase A is 63% 0.1%HAc aqueous solution, and Mobile phase B is 37% acetonitrile solution;
During 35min, mobile phase A is 15% 0.1%HAc aqueous solution, and Mobile phase B is 85% acetonitrile solution;
During 45min, mobile phase A is 15% 0.1%HAc aqueous solution, and Mobile phase B is 85% 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 2.0-12.1min;
Component fr.82, acquisition time 12.1-17.5min;
Component fr.83, acquisition time 17.5-23.6min;
Component fr.84, acquisition time 23.6-27.5min;
Component fr.85, acquisition time 27.5-36.0min;
Component fr.86, acquisition time 36.0-45min.
In order to obtain concrete effective component in red sage, best extraction separation method of the present invention is:
(1) extraction process: take by weighing red rooted salvia 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 17.2g 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 2.5g sample, use methanol as mobile phase at last, eluent fr.6.With extracting solution fr.2 concentrate 138.3g extractum, get 20g 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 2g 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 Salviae Miltiorrhizae 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 50% water, and Mobile phase B is 50% acetonitrile solution;
During 35min, mobile phase A is 20% water, and Mobile phase B is 80% acetonitrile solution;
During 45min, mobile phase A is 5% water, and Mobile phase B is 95% acetonitrile solution;
During 55min, 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 2.0-5.6min, 49.3mg;
Component fr.52, acquisition time 5.6-14.0min, 28.0mg;
Component fr.53, acquisition time 14.0-18.2min, 7.3mg;
Component fr.54, acquisition time 18.2-27.1min, 11.5mg;
Component fr.55, acquisition time 27.1-30.3min, 5.0mg;
Component fr.56, acquisition time 30.3-35.2min, 6.6mg;
Component fr.57, acquisition time 35.2-38.4min, 8.0mg;
Component fr.58, acquisition time 38.4-41.9min, 6.2mg;
Component fr.59, acquisition time 41.9-46.2min, 6.1mg;
Component fr.59, acquisition time 46.2-54.5min, 28.3mg.
The fr.8 separation condition: chromatographic column is Agilent preparative column (Zorbax SB-C18; 21.2mm * 250mm); Adopt gradient elution, mobile phase is 0.1%HAc aqueous solution (A) and 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 85% 0.1%HAc aqueous solution, and Mobile phase B is 15% acetonitrile solution;
During 10min, mobile phase A is 79% 0.1%HAc aqueous solution, and Mobile phase B is 21% acetonitrile solution;
During 15min, mobile phase A is 69% 0.1%HAc aqueous solution, and Mobile phase B is 31% acetonitrile solution;
During 30min, mobile phase A is 63% 0.1%HAc aqueous solution, and Mobile phase B is 37% acetonitrile solution;
During 35min, mobile phase A is 15% 0.1%HAc aqueous solution, and Mobile phase B is 85% acetonitrile solution;
During 45min, mobile phase A is 15% 0.1%HAc aqueous solution, and Mobile phase B is 85% acetonitrile solution;
The fr.8 separating resulting: get component fr.8 1.5g 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 2.0-12.1min, 9.8mg;
Component fr.82, acquisition time 12.1-17.5min, 29.7mg;
Component fr.83, acquisition time 17.5-23.6min, 618.7mg;
Component fr.84, acquisition time 23.6-27.5min, 351.0mg;
Component fr.85, acquisition time 27.5-36.0min, 84.0mg;
Component fr.86, acquisition time 36.0-45min, 33.5mg.
Among the present invention, contained chemical compound sees Table 1 in each component of Radix Salviae Miltiorrhizae, and its Analysis and Identification method is referring to embodiment three.
Contained chemical compound in each component of table 1. Radix Salviae Miltiorrhizae
| Danshen component | Compounds identified |
| fr.52 fr.53 fr.54 fr.55 fr.57 fr.59 fr.81 fr.82 fr.83 fr.84 fr.86 | Neocryptotanshinone; Paeoniflorin; Trijuganone C, Dihydrotanshinone I; Dihydrotanshinone I; 6,7-Dihydroxy-8,13-abietadiene-11,12-dione, (6a, 7b)-form, 6-Deoxy; Danshenxinkun B or Methylenedihydrotanshinquinone, 1,2-Didehydro or Nortanshinone or Tanshinone I, 1,2,15,16-Tetrahydro; Linthospermic acid, Salvianolic acid B; Protocatechuic aldehyde, Linthospermic acid; Salvianolic acid B, Salvianolic acid E or Salvianolic acid B isomer; Salvianolic acid B, Salvianolic acid E or Salvianolic acid B isomer; Neocryptotanshinone; |
Above Radix Salviae Miltiorrhizae, 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, 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 effective component in red sage 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 Salviae Miltiorrhizae finger printing below, each component ultraviolet spectrogram of salvia-soluble, each component mass spectrum of salvia-soluble, fat-soluble each the component ultraviolet spectrogram of Radix Salviae Miltiorrhizae, fat-soluble each the component mass spectrum of Radix Salviae Miltiorrhizae are intended to further specify the present invention, but the present invention are not construed as limiting.
Fig. 1 Radix Salviae Miltiorrhizae finger printing
Each component uv-spectrogram of Fig. 2 salvia-soluble
Each component mass spectrum of Fig. 3 salvia-soluble
Fat-soluble each the component uv-spectrogram of Fig. 4 Radix Salviae Miltiorrhizae
Fat-soluble each the component mass spectrum of Fig. 5 Radix Salviae Miltiorrhizae
The specific embodiment
Embodiment one
(1) extraction process: take by weighing red rooted salvia 250g, 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 16.8g 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 as mobile phase, get eluent fr.5, concentrate the 2.3g sample, use methanol as mobile phase at last, eluent fr.6.With extracting solution fr.2 concentrate 133.6g extractum, get 20g 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 is as mobile phase, eluent fr.8, concentrate the 1.88g sample.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.
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 2.0-5.6min, 47.1mg;
Component fr.52, acquisition time 5.6-14.0min, 26.8mg;
Component fr.53, acquisition time 14.0-18.2min, 6.5mg;
Component fr.54, acquisition time 18.2-27.1min, 10.2mg;
Component fr.55, acquisition time 27.1-30.3min, 4.8mg;
Component fr.56, acquisition time 30.3-35.2min, 6.1mg;
Component fr.57, acquisition time 35.2-38.4min, 7.6mg;
Component fr.58, acquisition time 38.4-41.9min, 5.9mg;
Component fr.59, acquisition time 41.9-46.2min, 5.7mg;
Component fr.59, acquisition time 46.2-54.5min, 26.6mg.
The fr.8 separating resulting: get component fr.8 1.5g 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 2.0-12.1min, 9.1mg;
Component fr.82, acquisition time 12.1-17.5min, 28.6mg;
Component fr.83, acquisition time 17.5-23.6min, 610.3mg;
Component fr.84, acquisition time 23.6-27.5min, 348.5mg;
Component fr.85, acquisition time 27.5-36.0min, 81.8mg.
Component fr.86, acquisition time 36.0-45min, 32.4mg.
Embodiment two
(1) extraction process: take by weighing red rooted salvia 500g, 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 33.6g 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 4.6g sample, use methanol as mobile phase at last, eluent fr.6.With extracting solution fr.2 concentrate 268.4g extractum, get 40g 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 3.88g 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 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 2.0-5.6min, 96.6mg;
Component fr.52, acquisition time 5.6-14.0min, 54.3mg;
Component fr.53, acquisition time 14.0-18.2min, 13.2mg;
Component fr.54, acquisition time 18.2-27.1min, 20.9mg;
Component fr.55, acquisition time 27.1-30.3min, 9.3mg;
Component fr.56, acquisition time 30.3-35.2min, 11.9mg;
Component fr.57, acquisition time 35.2-38.4min, 15.5mg;
Component fr.58, acquisition time 38.4-41.9min, 11.0mg;
Component fr.59, acquisition time 41.9-46.2min, 11.3mg;
Component fr.59, acquisition time 46.2-54.5min, 54.4mg.
The fr.8 separating resulting: get component fr.8 3.0g 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 2.0-12.1min, 17.8mg;
Component fr.82, acquisition time 12.1-17.5min, 58.6mg;
Component fr.83, acquisition time 17.5-23.6min, 1229.5mg;
Component fr.84, acquisition time 23.6-27.5min, 697.3mg;
Component fr.85, acquisition time 27.5-36.0min, 160.9mg.
Component fr.86, acquisition time 36.0-45min, 63.7mg.
Embodiment three
One, the extraction separation of red rooted salvia
(1) extraction process: take by weighing red rooted salvia 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 17.2g 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 2.5g sample, use methanol as mobile phase at last, eluent fr.6.With extracting solution fr.2 concentrate 138.3g extractum, get 20g 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 2g 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 Salviae Miltiorrhizae 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 50% water, and Mobile phase B is 50% acetonitrile solution;
During 35min, mobile phase A is 20% water, and Mobile phase B is 80% acetonitrile solution;
During 45min, mobile phase A is 5% water, and Mobile phase B is 95% acetonitrile solution;
During 55min, 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 2.0-5.6min, 49.3mg;
Component fr.52, acquisition time 5.6-14.0min, 28.0mg;
Component fr.53, acquisition time 14.0-18.2min, 7.3mg;
Component fr.54, acquisition time 18.2-27.1min, 11.5mg;
Component fr.55, acquisition time 27.1-30.3min, 5.0mg;
Component fr.56, acquisition time 30.3-35.2min, 6.6mg;
Component fr.57, acquisition time 35.2-38.4min, 8.0mg;
Component fr.58, acquisition time 38.4-41.9min, 6.2mg;
Component fr.59, acquisition time 41.9-46.2min, 6.1mg;
Component fr.59, acquisition time 46.2-54.5min, 28.3mg.
The fr.8 separation condition: chromatographic column is Agilent preparative column (Zorbax SB-C18; 21.2mm * 250mm); Adopt gradient elution, mobile phase is 0.1% aqueous acetic acid (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 85% 0.1%HAc aqueous solution, and Mobile phase B is 15% acetonitrile solution;
During 10min, mobile phase A is 79% 0.1%HAc aqueous solution, and Mobile phase B is 21% acetonitrile solution;
During 15min, mobile phase A is 69% 0.1%HAc aqueous solution, and Mobile phase B is 31% acetonitrile solution;
During 30min, mobile phase A is 63% 0.1%HAc aqueous solution, and Mobile phase B is 37% acetonitrile solution;
During 35min, mobile phase A is 15% 0.1%HAc aqueous solution, and Mobile phase B is 85% acetonitrile solution;
During 45min, mobile phase A is 15% 0.1%HAc aqueous solution, and Mobile phase B is 85% acetonitrile solution;
The fr.8 separating resulting: get component fr.8 1.5g 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 2.0-12.1min, 9.8mg;
Component fr.82, acquisition time 12.1-17.5min, 29.7mg;
Component fr.83, acquisition time 17.5-23.6min, 618.7mg;
Component fr.84, acquisition time 23.6-27.5min, 351.0mg;
Component fr.85, acquisition time 27.5-36.0min, 84.0mg.
Component fr.86, acquisition time 36.0-45min, 33.5mg.
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: Danshen component fr.51 1.35mg, fr.52 1.33mg, fr.53 1.29mg, fr.54 1.34mg, fr.551.32mg, fr.56 1.31mg, fr.57 1.30mg, fr.58 1.32mg, fr.59 1.38mg, fr.510 1.27mg.
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 30% 0.2% glacial acetic acid aqueous solution, Mobile phase B are the acetonitrile solution of 70% 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; It is long to detect the wavelength all-wave; 30 ℃ of column temperatures; 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: Danshen component fr.81 1.38mg, fr.82 1.29mg, fr.83 1.32mg, fr.84 1.36mg, fr.851.39mg, fr.86 1.38mg.
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 80% 0.2% glacial acetic acid aqueous solution, Mobile phase B are the acetonitrile solution of 20% 0.2% glacial acetic acid; In the time of 15 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 20 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; It is long to detect the wavelength all-wave; 30 ℃ of column temperatures; 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 salvia-soluble, and Fig. 3 is each component mass spectrum of salvia-soluble, and Fig. 4 is fat-soluble each the component uv-spectrogram of Radix Salviae Miltiorrhizae, and Fig. 5 is fat-soluble each the component mass spectrum of Radix Salviae Miltiorrhizae.
Each compound identification result in the Danshen component
[1-5]See Table 2.
Table 2. Danshen component compound identification result
| Component | Chromatographic retention (min) | Molecular weight | [M+H] +/[M-H] - | Uv-absorption maximum wavelength UV (nm) | Chemical compound |
| fr.51 fr.52 fr.53 fr.54 fr.55 fr.56 fr.57 fr.58 fr.59 fr.510 fr.81 fr.82 fr.83 fr.84 fr.85 fr.86 | 14.80 16.14 14.62 16.36 16.80 18.03 18.03 17.96 21.28 19.13 9.87 10.02 6.45 9.87 10.02 10.55 10.02 10.55 9.87 10.02 12.66 10.36 | 328 314 328 340 298 278 278 296 296 280 538 718 138 538 718 718 718 718 538 718 552 565 | +：329，351 +：315，337 +：285，311，329 -：313，278 +：341，363，703 +：299，321，619 +：279，579 +：279，579 +：297，615 +：297，615 +：281，583 -：279，579 -：537，559 -：717，739 -：137，275 -：537，559 -：358，537 +：717，1077 -：457，1077 -：358，537 +：717，1077 -：457，1077 -：537，559 -：358，537 +：717，1077 -：551，1103 -：565，663 | 200 210，262，310 210，250，310 230，260 220，250，270 220，240，290 220，240，290 220，280 228，286 232，290，308 230，254，280 230，280，312 232，290，308 230，254，280 280，330 230，254，280 280，330 232，290，308 230，254，280 230，254，280，320 254，280 | Neocryptotanshinone Trijuganone C Dihydrotanshinone I Dihydrotanshinone I Cryptotanshinone Danshenxinkun B or Methylenedihydrotanshin quinone or Miltirone; 1,2-Didehydro or Nortanshinone or Tanshinone I; 1; 2; 15,16-Tetrahydro Linthospermic acid Salvianolic acid B Protocatechuic aldehyde Linthospermic acid Salvianolic acid B Salvianolic acid E or Salvianolic acid B isomer Salvianolic acid B Salvianolic acid E or Salvianolic acid B isomer Linthospermic acid Salvianolic acid B |
| 12.80 | 314 | -：313 | 254，280 | Neocryptotanshinone |
List of references:
1, DNP (native compound data base).
2、L.N.Li，Salvianolic acids and related compounds，Medicinal and Aromatic Plants-IndustrialProfiles，14(2000)，81-89。
3、C.B.Ai，L.N.Li，Salvianolic acids D and E：two new depsides from Salvia miltiorrihiza.PlantaMedica，58(1992)，197-199。
4、D.G.kang，H.C.Oh，E.J.Sohn，et al.Lithospermic acid B isolated from Salvia miltiorrihizaameliorates ischemia/reperfusion-induced renal injury in rats.Life Sciences，75(2004)，1801-1816。
5, girth is new, and Luo Houwei, red plumage are just military, salvia-soluble The Chemical Constituents, China Medicine University's journal, 30 (1999), 411-416.
Three, Danshen 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 Radix Salviae Miltiorrhizae 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, Danshen component fr.8, fr.82, fr.84, fr.86 has the highly significant effect to reducing LDH release.
Table 3. myocardial cell The selection result
| Component | Mean | Std | P1 | P2 |
| Fr.5 fr.51 fr.52 fr.53 fr.54 fr.55 fr.56 fr.57 fr.58 fr.59 fr.510 fr.8 fr.81 fr.82 fr.83 fr.84 fr.85 fr.86 | 0.368 0.38775 0.356 0.377 0.366 0.39325 0.41975 0.3765 0.406 0.4515 0.372 0.29125 0.3305 0.26675 0.305 0.33875 0.366 0.2325 0.42825 0.379 | 0.03122 0.052999 0.044952 0.142854 0.034312 0.064459 0.111491 0.037969 0.091924 0.026514 0.113155 0.026986 0.061997 0.044507 0.096038 0.03351 0.007483 0.04658 0.057279 0.016432 | 0.057118 0.169648 0.047212 0.265092 0.055755 0.223961 0.448276 0.091379 0.059282 0.244545 0.204601 0.002471 0.029882 0.002158 0.03484 0.017851 0.037282 0.000912 0.074711 | 0.277924 0.381576 0.186804 0.489355 0.259922 0.341642 0.248388 0.453876 0.100191 0.001755 0.00072 0.0906 0.001609 0.111584 0.037196 0.099966 0.000512 0.074711 |
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 Radix Salviae Miltiorrhizae 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.
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 4.According to endotheliocyte LDH (lactic acid dehydrogenase) The selection result, Radix Salviae Miltiorrhizae fr.84 has the highly significant effect to reducing LDH release, and Radix Salviae Miltiorrhizae fr.51 produces effect.
Table 4. endotheliocyte LDH The selection result
| Radix Salviae Miltiorrhizae | Mean | Std | P |
| Fr.51 fr.84 model group | 0.089 0.110 0.122 0.125 | 0.032 0.007 0.006 0.005 | 0.087 0.003 |
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. Radix Salviae Miltiorrhizae extraction, separation criterion method may further comprise the steps:
(1) extraction process: take by weighing red rooted salvia, 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. Radix Salviae Miltiorrhizae as claimed in claim 1 extracts, the separation criterion method, comprises the steps:
(1) extraction process: take by weighing red rooted salvia, 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 ff.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: use preparative liquid chromatography from fr.5 and fr.8; 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.
3. Radix Salviae Miltiorrhizae as claimed in claim 2 extracts, the separation criterion method, comprises the steps:
(1) extraction process: take by weighing red rooted salvia, 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 50% water, and Mobile phase B is 50% acetonitrile solution;
During 35min, mobile phase A is 20% water, and Mobile phase B is 80% acetonitrile solution;
During 45min, mobile phase A is 5% water, and Mobile phase B is 95% acetonitrile solution;
During 55min, 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 2.0-5.6min;
Component fr.52, acquisition time 5.6-14.0min;
Component fr.53, acquisition time 14.0-18.2min;
Component fr.54, acquisition time 18.2-27.1min;
Component fr.55, acquisition time 27.1-30.3min;
Component fr.56, acquisition time 30.3-35.2min;
Component fr.57, acquisition time 35.2-38.4min;
Component fr.58, acquisition time 38.4-41.9min;
Component fr.59, acquisition time 41.9-46.2min;
Component fr.510, acquisition time 46.2-54.5min;
The fr.8 separation condition: chromatographic column is Agilent preparative column (Zorbax SB-C18; 21.2mm * 250mm); Adopt gradient elution, mobile phase is 0.1%HAc aqueous solution (A) and 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 85% 0.1%Hac aqueous solution, and Mobile phase B is 15% acetonitrile solution;
During 10min, mobile phase A is 79% 0.1%Hac aqueous solution, and Mobile phase B is 21% acetonitrile solution;
During 15min, mobile phase A is 69% 0.1%Hac aqueous solution, and Mobile phase B is 31% acetonitrile solution;
During 30min, mobile phase A is 63% 0.1%Hac aqueous solution, and Mobile phase B is 37% acetonitrile solution;
During 35min, mobile phase A is 15% 0.1%Hac aqueous solution, and Mobile phase B is 85% acetonitrile solution;
During 45min, mobile phase A is 15% 0.1%Hac aqueous solution, and Mobile phase B is 85% 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 2.0-12.1min;
Component fr.82, acquisition time 12.1-17.5min;
Component fr.83, acquisition time 17.5-23.6min;
Component fr.84, acquisition time 23.6-27.5min;
Component fr.85, acquisition time 27.5-36.0min;
Component fr.86, acquisition time 36.0-45min.
4. Radix Salviae Miltiorrhizae as claimed in claim 3 extracts, the separation criterion method, it is characterized in that:
Main compound is Neocryptotanshinone among the component fr.52;
Main compound is Paeoniflorin among the component fr.53;
Main compound is Trijuganone C among the component fr.54, Dihydrotanshinone I;
Main compound is Dihydrotanshinone I among the component fr.55;
Main compound is 6 among the component fr.57,7-Dihydroxy-8, and 13-abietadiene-11,12-dione, (6a, 7b)-form, 6-Deoxy;
Main compound is 1,2,15 among the component fr.59,16-Tetrahydro; 1,2-Didehydro or Nortanshinone or Tanshinone I, Danshenxinkun B or Methylenedihydrotanshinquinone.
5. Radix Salviae Miltiorrhizae as claimed in claim 3 extracts, the separation criterion method, it is characterized in that:
Main compound is Linthospermic acid among the component fr.81, Salvianolic acid B;
Main compound is Protocatechuic aldehyde among the component fr.82, Linthospermic acid;
Main compound is Salvianolic acid B among the component fr.83, Salvianolic acid E or Salvianolic acid B isomer;
Main compound is Salvianolic acid B among the component fr.84, Salvianolic acid E or Salvianolic acid B isomer;
Main compound is Neocryptotanshinone among the component fr.86.
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