CN1903250B - Method for extraction and separation of red rooted salvia - Google Patents

Method for extraction and separation of red rooted salvia Download PDF

Info

Publication number
CN1903250B
CN1903250B CN2005100122777A CN200510012277A CN1903250B CN 1903250 B CN1903250 B CN 1903250B CN 2005100122777 A CN2005100122777 A CN 2005100122777A CN 200510012277 A CN200510012277 A CN 200510012277A CN 1903250 B CN1903250 B CN 1903250B
Authority
CN
China
Prior art keywords
component
mobile phase
acquisition time
solution
methanol
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Expired - Fee Related
Application number
CN2005100122777A
Other languages
Chinese (zh)
Other versions
CN1903250A (en
Inventor
程翼宇
贺庆
王毅
王学伟
李云飞
胡兴江
葛志伟
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Tasly Pharmaceutical Group Co Ltd
Original Assignee
Tianjin Tasly Pharmaceutical Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Tianjin Tasly Pharmaceutical Co Ltd filed Critical Tianjin Tasly Pharmaceutical Co Ltd
Priority to CN2005100122777A priority Critical patent/CN1903250B/en
Publication of CN1903250A publication Critical patent/CN1903250A/en
Application granted granted Critical
Publication of CN1903250B publication Critical patent/CN1903250B/en
Expired - Fee Related legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Landscapes

  • Medicines Containing Plant Substances (AREA)

Abstract

A standardized extracting and separating method for extracting the chemical components from a Chinese-medicinal material to maximal extent and further separating them to obtain the chemical components with different polarities is disclosed. To be specific, the 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

A kind of extraction separation method of red rooted salvia
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, and Along with people's increases and the aging of population 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 gets into 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 Asian countries's traditional medicine product such as keen competition and Japan, Korea S, India, Thailand of powerful transnational medical group and the enormous impact of European countries' plant amedica such as Germany, France, numerous products that China's Chinese medicine produces since the standard that still can not meet international medical market with requirement and quilt is kept outside of the door.
The standardization that Chinese crude drug extracts is that Chinese patent medicine moves towards international market or the big industrial key link of real realization with the standardization of Chinese medicinal preparation method, also is that the scientific research personnel of Chinese Medicine worker and foreign study natural plant makes great efforts the direction studied always.Method for distilling 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 to different its extraction of effective components of Chinese crude drug; And adopting different method for distilling to need the extraction that a large amount of different extraction equipment are used to support Chinese crude drug to different Chinese crude drugs, this is inappropriate 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; The 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 confirm 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 through 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 passes through lot of test; The extraction of effective components of present Radix Salviae Miltiorrhizae is concluded and put in order; With seek a kind of can standardized method for distilling, 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.
The Radix Salviae Miltiorrhizae that the present invention adopted extracts, separation criterionization is that the inventor passes through test, and after Chinese crude drug adopted different method for distilling 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 step:
(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.In medicinal residues, add ethanol, heating extraction gets extracting solution fr.2.In medicinal residues, add entry 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,, adopt the ODS-C18 post that it is separated with appearance on the dissolve with methanol; 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 step:
(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.In medicinal residues, add 5～12 times of amount 70% ethanol, reflux 0.5～3 hour is extracted 1～3 time, and merging filtrate gets extracting solution fr.2.In medicinal residues, add entry 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; At first using volume ratio is that 50: 1 petroleum ether and ethyl acetate is as mobile phase; Eluent fr.4, the chloroform and the methanol that change volume ratio then and be 10: 1 is as mobile phase, must eluent fr.5; Use methanol as mobile phase at last, get eluent fr.6.After extracting solution fr.2 is condensed into extractum,, adopt the ODS-C18 post that it is separated with appearance on 2～10% dissolve with methanol; 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 step:
(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.In medicinal residues, add 8 times of amount 70% ethanol, reflux 1 hour is extracted 2 times, and merging filtrate gets extracting solution fr.2.In medicinal residues, add entry 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, uses silica gel mixed sample, adopts normal phase silicagel column that it is separated; At first using volume ratio is that 50: 1 petroleum ether and ethyl acetate is as mobile phase; Eluent fr.4, the chloroform and the methanol that change volume ratio then and be 10: 1 is as mobile phase, must eluent fr.5; Use methanol as mobile phase at last, get eluent fr.6.Fr.2 is condensed into extractum with extracting solution, with appearance on 5% dissolve with methanol, adopts 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 following:
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 following:
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 following:
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 following:
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.In medicinal residues, add 8 times of amount 70% ethanol, reflux 1 hour is extracted 2 times, and merging filtrate gets extracting solution fr.2.In medicinal residues, add entry 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, the employing normal phase silicagel column separates it; At first use volume ratio be 50: 1 petroleum ether and ethyl acetate as mobile phase, eluent fr.4, the chloroform and the methanol that change volume ratio then and be 10: 1 are 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 appearance on 5% dissolve with methanol, employing ODS-C18 post separates it; 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 following:
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 following:
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 following:
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 following:
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 is seen 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
Above Radix Salviae Miltiorrhizae, extraction solution can increase or reduce when industrialization is extracted according to corresponding ratio; Like 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.
Established by the present invention extraction, separation standards can be applied in any of the current Chinese herbal medicine, for example, can be used for ferulic, leaves, benzoin, Bozi, turtle shell, betel nuts, mint, castor bean,
Figure 200510012277710000210003_0
build, psoralen , Radix, Piper longum, Croton, Morinda, Menispermi, littoralis, white, and, bulbul, white peony root, angelica, white tuber, cogongrass rhizome, ginkgo, white front, white beans, white Lian, fresh white skin , Bai Wei, lobelia, barbata, Breit, lily, borneol, Bupleurum, cicada, mulberry real son, Changshan, piles of skins, pangolin, Andrographis, red bean, halloysite, red peony root, herb, fruit, aloes, sarmentosum, Oriental Arborvitae, Aconitum, Aconitum leaves, grass cardamom, strawberry, Fritillaria, Cyathula, Radix, Chuanxiong, Toosendan, Plantago, SSP, Codonopsis, simplxs leaf, Eucommia, independent living, bile Southern Star, Daji, Arecae, moutan, calcined gypsum, melon skin, Cordyceps sinensis, earthworm, Kochia scoparia, Digupi, Rehmannia, humifusa, Burnet, angelica, rushes, cloves, beans, Folium, jujube, rhubarb, centipeda, Curcuma, catechu, pistachio nuts, duckweed, castor seed solution, raspberry, bergamot, monkshood, Poria, Stephania, wind, senna , honey, Gecko, Gui, arrowroot, Ligusticum, galangal, Drynaria, Guya, Eriocaulon, Woodwardia, medlar, holly leaves, Uncaria, nard, licorice, euphorbia, Trichosanthes , Trichosanthes son, Trichosanthes skin, Guan Mu Tong, ginger, roasted ginger, dried paint, Lappula, Astragalus, Huang Jing, seaweed, SJ, the sea breeze rattan, lotus leaf, Scutellaria, Coptis, Phellodendron, pepper, Polygonum, Polygonum cuspidatum, picrorhizae, Magnolia, Citrus grandis, hemp seed, Albizia, Albizia julibrissin, red Euphorbia, safflower, black sesame, chrysanthemum, silkworm, bellflower, Ju, turmeric, Millettla, cockscomb, gold fruit Lam, Kim boiling grass, golden buckwheat, loosestrife, honeysuckle, dalbergia, Nepeta, chive seed, cassia, Coltsfoot, myrobalan, bitter almond, Sophora, Neem bark, radish seed, Lei Wan, Campsis, Pyrolae grass, Loulu, Passepartout, lotus seeds, contact Ishido, aloe vera, reed rhizome, two sharp, two needles, forsythia, Ganoderma lucidum, Apocynum leaves, Mangosteen, litchi, gentian, longan, Geranium, Eclipta, ephedra, Vitex, Rhododendron, mountain oil, Buddleia, plum, ephedra, Radix, malt, roses, smyrnioides, verbena, Aristolochia, Strychnos, MA vomica powder, puffballs, purslane, papaya, wood, wood thieves, Adenophorae, Ligustrum lucidum, burdock, Achyranthes, Oujie, Puhuang, dandelion leaves, Perrin, turmeric tablets, Dianthus, Qinpi, Polygonum, Gorgon, Notopterygium, green wood, acutum, peel, seeds, Artemisia annua, indigo, madder, Pharbitidis, Peucedanum, Homalomena, daughter son, Gentiana, Rui Ren, nutmeg, Cistanche , cinnamon, ginseng, ginseng leaf, Shichangpu, Cynomorium, mulberry, commercial land, mulberry, Cnidium, Morus alba, mulberry, mulberry, mistletoe, Aesculi, Semen, Shegan, Styrax, sand Yuan son, so that a gentleman, Shi Di, Amomum, tonkinensis, dogwood, Chinese yam, indica, hawthorn, thistle, black cohosh, buffalo horn, buffalo horn concentrated powder, Shi Wei, abalone, Dendrobium, pomegranate , ginger, loofah, Panax, triangular, Tinglizi, dodder seed, walnuts, through the grass, sandalwood, TCS, Tianzhu Huang, Araceae, Tianma, day ambrette, heterophylla, soil wattle bark, soil Fuling, Evodia, Clematis, Vaccaria, Wujiapi, Schisandra, gall, corrugated son, aggregata, ebony, Xiatianwu, Dipsacus, Inula, Siegesbeckiae, scallion white, Prunella, magnolia, Periplocae, Cyperus, citron, Elsholtzia, cumin, Curculigoside, Agrimony, Scrophulariaceae, Xuan Ming powder, ginseng, blood over charcoal, blood, Xu Changqing, Epimedium, motherwort, ginkgo biloba, Coix, silver Bupleurum, Polygala, Daphne, Yuli, turmeric, Houttuynia, capillaris, javanica, rose, Polygonatum, Corydalis, Fritillaria, Gleditsiae, perilla, Aster, Viola, comfrey, Polyporus , herb, Anemarrhena, Zeeland, Alisma, mother of pearl, citrus aurantium, gardenia.
Any form on the pharmaceutics be can process 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 an example.Under the situation of not violating purport of the present invention and scope; Can carry out various changes and improvement to the present invention; The solution of for example being selected for use can be other lower alcohols or other organic solvents, but as long as use method for distilling 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 constituted restriction.
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.In medicinal residues, add ethanol, heating extraction gets extracting solution fr.2.In medicinal residues, add entry 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, the employing normal phase silicagel column separates it; 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 appearance 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 following:
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 following:
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.In medicinal residues, add 5～12 times of amount 70% ethanol, reflux 0.5～3 hour is extracted 1～3 time, and merging filtrate gets extracting solution fr.2.In medicinal residues, add entry 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, the employing normal phase silicagel column separates it; At first use volume ratio be 50: 1 petroleum ether and ethyl acetate as mobile phase, eluent fr.4, the chloroform and the methanol that change volume ratio then and be 10: 1 are 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 appearance on 2～10% dissolve with methanol, employing ODS-C18 post separates it; 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 following:
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 following:
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.In medicinal residues, add 8 times of amount 70% ethanol, reflux 1 hour is extracted 2 times, and merging filtrate gets extracting solution fr.2.In medicinal residues, add entry 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, the employing normal phase silicagel column separates it; At first use volume ratio be 50: 1 petroleum ether and ethyl acetate as mobile phase, eluent fr.4, the chloroform and the methanol that change volume ratio then and be 10: 1 are 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 appearance on 5% dissolve with methanol, employing ODS-C18 post separates it; 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 following:
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 following:
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 following:
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 following:
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 HPLC-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 is used the 1ml dissolve with ethanol, and is ultrasonic, and centrifugal (10000r/min) is subsequent use.
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 following: 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 subsequent use.
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 following: 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
Figure G05112277720050818D000141
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 after PBS liquid cleans, is cut into 1mm 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 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, the cell of USA) 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.Get reactant liquor 50 μ l and add 200 μ l 0.4mol/L sodium hydroxide solutions, leave standstill 3～4 minutes after, measure shading value with ELIASA in 450nm.
Drug effect all data is as a result represented with the mean variance.Adopt monolateral variance analysis and T check carrying out statistical analysis.Compare with model group, P＜0.05 is thought significantly effectively.The medicine efficacy screening result sees 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
3.2 huve cell anoxia reoxygenation model
The healthy newborn umbilical cord is got in former generation huve cell cultivation, cleans the residual bloodstain in the 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.Get reactant liquor 50 μ l and add 200 μ l 0.4mol/L sodium hydroxide solutions, leave standstill 3～4 minutes after, measure shading value with ELIASA in 450nm.
Drug effect all data is as a result represented with the mean variance.Adopt monolateral variance analysis and T check carrying out statistical analysis.Compare with model group, P＜0.05 is thought significantly effectively.The medicine efficacy screening result sees 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
Above result of the test explanation, the Chinese crude drug effective ingredient that adopts Chinese crude drug standardization method for distilling of the present invention to obtain can be as the preparation medicine.

Claims (3)

1. Radix Salviae Miltiorrhizae extraction, separation method may further comprise 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; In medicinal residues, add 8 times of amount 70% ethanol, reflux 1 hour is extracted 2 times, and merging filtrate gets extracting solution fr.2; In medicinal residues, add entry 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, uses silica gel mixed sample, adopts normal phase silicagel column that it is separated; At first using volume ratio is that 50: 1 petroleum ether and ethyl acetate is as mobile phase; Eluent fr.4, the chloroform and the methanol that change volume ratio then and be 10: 1 is as mobile phase, must eluent fr.5; Use methanol as mobile phase at last, get eluent fr.6; Fr.2 is condensed into extractum with extracting solution, with appearance on 5% dissolve with methanol, adopts 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: leave fr.5 and fr.8 with preparative liquid chromatography;
The separation condition of fr.5: chromatographic column is the semi-preparative column 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 following:
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 following:
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 following:
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 following:
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.
2. Radix Salviae Miltiorrhizae as claimed in claim 1 extracts, separation 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.
3. Radix Salviae Miltiorrhizae as claimed in claim 1 extracts, separation 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.
CN2005100122777A 2005-07-29 2005-07-29 Method for extraction and separation of red rooted salvia Expired - Fee Related CN1903250B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN2005100122777A CN1903250B (en) 2005-07-29 2005-07-29 Method for extraction and separation of red rooted salvia

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN2005100122777A CN1903250B (en) 2005-07-29 2005-07-29 Method for extraction and separation of red rooted salvia

Publications (2)

Publication Number Publication Date
CN1903250A CN1903250A (en) 2007-01-31
CN1903250B true CN1903250B (en) 2012-02-22

Family

ID=37672710

Family Applications (1)

Application Number Title Priority Date Filing Date
CN2005100122777A Expired - Fee Related CN1903250B (en) 2005-07-29 2005-07-29 Method for extraction and separation of red rooted salvia

Country Status (1)

Country Link
CN (1) CN1903250B (en)

Families Citing this family (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101347533B (en) * 2007-07-20 2013-05-22 天士力制药集团股份有限公司 Effective component of gamene and preparation and use thereof
CN101347534B (en) * 2007-07-20 2012-06-06 天津天士力制药股份有限公司 Effective component of gamene and preparation and use thereof
CN101347508B (en) * 2007-07-20 2012-02-15 天津天士力制药股份有限公司 Effective component of Sinomenium acutum and preparation thereof
CN101347509B (en) * 2007-07-20 2012-05-09 天津天士力制药股份有限公司 Effective component of Sinomenium acutum and preparation thereof
CN101347518B (en) * 2007-07-20 2012-06-27 天津天士力制药股份有限公司 Effective component of red paeonia and preparation and use thereof
CN101347521B (en) * 2007-07-20 2013-07-10 天士力制药集团股份有限公司 Effective component of Clematis chinensis and preparation and use thereof
CN101428083B (en) * 2007-11-06 2012-06-06 天津天士力制药股份有限公司 Effective component of cortex dictamni, preparation method and use thereof
CN101428062B (en) * 2007-11-06 2012-03-28 天津天士力制药股份有限公司 Effective component of schisandra chinensis, preparation method and use thereof
CN101428071B (en) * 2007-11-06 2012-02-22 天津天士力制药股份有限公司 Effective component of pulsatilla, preparation method and use thereof
CN101433612B (en) * 2007-11-12 2012-03-07 天津天士力制药股份有限公司 Effective component of Chinese dittany bark as well as preparation method and use thereof
CN101433613B (en) * 2007-11-13 2012-05-09 天津天士力制药股份有限公司 Effective component of Chinese dittany bark as well as preparation method and use thereof
CN101507769B (en) * 2008-02-15 2012-07-04 天津天士力制药股份有限公司 Active component of cortex dictamni and preparation method and use thereof
CN105395680B (en) * 2015-12-29 2019-09-17 余洋 It is a kind of to treat neurasthenic Chinese medicine composition and its preparation method and application
CN113009035B (en) * 2021-03-04 2022-08-02 福建卫生职业技术学院 Method for identifying pharmacodynamic components of traditional Chinese medicine based on knocking out target components and application
CN113933438B (en) * 2021-11-11 2023-11-10 威海华洋药业有限公司 Content determination method and preparation method of astragalus membranaceus cough-relieving oral liquid

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
刘国生.新疆丹参根中的化学成分研究.国外医学中医中药分册21 3.1999,21(3),49-50.
刘国生.新疆丹参根中的化学成分研究.国外医学中医中药分册21 3.1999,21(3),49-50. *
岑颖州等.丹参化学成分的研究.暨南大学学报（自然科学版）14 3.1993,14(3),55-60.
岑颖州等.丹参化学成分的研究.暨南大学学报(自然科学版)14 3.1993,14(3),55-60. *

Also Published As

Publication number Publication date
CN1903250A (en) 2007-01-31

Similar Documents

Publication Publication Date Title
CN1903250B (en) Method for extraction and separation of red rooted salvia
CN101073587B (en) Method for separating and extracting Chinese Thorowax Root
Ohsugi et al. Active-oxygen scavenging activity of traditional nourishing-tonic herbal medicines and active constituents of Rhodiola sacra
Chen et al. Ophiopogon japonicus—A phytochemical, ethnomedicinal and pharmacological review
Yang et al. Phytochemical analysis of traditional Chinese medicine using liquid chromatography coupled with mass spectrometry
Li et al. Chemical markers for the quality control of herbal medicines: an overview
He et al. Evaluation of antidiabetic potential of selected traditional Chinese medicines in STZ-induced diabetic mice
Wang et al. Compatibility art of traditional Chinese medicine: from the perspective of herb pairs
Qi et al. Anti-diabetic agents from natural products—an update from 2004 to 2009
Li et al. Natural medicines used in the traditional Chinese medical system for therapy of diabetes mellitus
Chen et al. Cytotoxicity and antihyperglycemic effect of minor constituents from Rhizoma Coptis in HepG2 cells
Zhang et al. Botany, traditional use, phytochemistry, pharmacology, quality control, and authentication of Radix Gentianae Macrophyllae-A traditional medicine: A review
Wan-Ying et al. TCM-based new drug discovery and development in China
CN101073606B (en) Method for separating and extracting white Peony Root
CN1903241B (en) Method for extraction and separation of pseudo-ginseng
CN1903239B (en) Method for extraction and separation of rhizome of chuanixong
Yang et al. Advances in understanding of health‐promoting benefits of medicine and food homology using analysis of gut microbiota and metabolomics
Ren et al. Research progress of natural medicine Astragalus mongholicus Bunge in treatment of myocardial fibrosis
Mazzio et al. In vitro screening of tumoricidal properties of international medicinal herbs: part II
CN101073623B (en) Method for separating and extracting cattail pollen
CN104288245B (en) Anti-aging and constitutional pharmaceutical composition and preparation method thereof and detection method
Yang et al. Ethnopharmacology, phytochemistry, and pharmacology of the genus glehnia: a systematic review
Li et al. Glehnia littoralis Fr. Schmidtex Miq.: a systematic review on ethnopharmacology, chemical composition, pharmacology and quality control
Hou et al. Salt processing: A unique and classic technology for Chinese medicine processing
CN1903279B (en) Method for extraction and separation of Immature Bitter orange

Legal Events

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

Effective date of registration: 20070511

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

Applicant after: TIANJIN TASLY PHARMACEUTICAL Co.,Ltd.

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

Applicant before: TIANJIN TASLY NATURAL MEDICINE

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

Owner name: TASLY PHARMACEUTICAL GROUP CO., LTD.

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

CP01 Change in the name or title of a patent holder

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

Patentee after: TASLY PHARMACEUTICAL GROUP Co.,Ltd.

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

Patentee before: TIANJIN TASLY PHARMACEUTICAL Co.,Ltd.

CP01 Change in the name or title of a patent holder

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

Patentee after: TASLY PHARMACEUTICAL GROUP Co.,Ltd.

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

Patentee before: TASLY PHARMACEUTICAL GROUP Co.,Ltd.

CP01 Change in the name or title of a patent holder
CF01 Termination of patent right due to non-payment of annual fee

Granted publication date: 20120222

Termination date: 20210729

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