CN101073606B - Method for separating and extracting white Peony Root - Google Patents
Method for separating and extracting white Peony Root Download PDFInfo
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- CN101073606B CN101073606B CN2006100137419A CN200610013741A CN101073606B CN 101073606 B CN101073606 B CN 101073606B CN 2006100137419 A CN2006100137419 A CN 2006100137419A CN 200610013741 A CN200610013741 A CN 200610013741A CN 101073606 B CN101073606 B CN 101073606B
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Abstract
The invention is concerned with the standard extraction and isolation method of Paeonia lactiflora Pallas. It can form a medicinal materials component store according to different polarity levels of the Paeonia lactiflora Pallas divide into several groups, which each group contents few types of compound. It can use to discoverage new medicine by conducting drug screening from the component store.The method can reduce the period of medicine extraction and isolation and reduce the consumption of manpower and material resources, and it suits for most types of medicine.
Description
Technical field
The invention belongs to field of medicaments, particularly relate to Radix Paeoniae Alba extraction, separation criterion method.
Background technology
At present, in the world, Chinese herbal medicine all has certain market, along with people increasing and the aging of population to the health requirements level of understanding, sub-health stateization, people thirst for back to nature more, the problem of utilize the high Drug therapy of pure natural degree, preventing some chemical synthetic drugs cann't be solved, so the application of natural plant exceeds the background of its original traditional national culture.From natural drug, seek the little and inexpensive medicine of side effect and become the target that countries in the world pharmaceutical manufacturer is chased.The European Community has carried out unified legislation to medical herbs, state medical herbs status such as Canada and Australia have legalized, U.S. government has also drafted the plant amedica management method, the compound recipe mix preparation that begins to accept natural drug is as curative, and these provide good international environment for Chinese medicine enters international medical market as curative.On the other hand, along with the quickening of global economic integration progress, particularly China becomes a full member of WTO, and Chinese Medicine market incorporates the breadth and depth of international medical big market and will further aggravate.Face the enormous impact of Asian countries's traditional medicine product such as the keen competition of powerful transnational medical group and Japan, Korea S, India, Thailand and European countries' plant amedica such as Germany, France, numerous products that China's Chinese medicine produces are owing to still can not meet the standard of international medical market and requirement and being kept outside of the door.
The standardization that Chinese crude drug extracts and the standardization of Chinese medicinal preparation method are that Chinese patent medicine moves towards the international market or really realizes big industrial key link, also are that the scientific research personnel of Chinese Medicine worker and foreign study natural plant makes great efforts the direction studied always.Extracting method about Chinese medicine is the emphasis of field of Chinese medicines research at present, the scientific research personnel focuses on the more effective ingredient which kind of method of employing could obtain with the sight of research always from Chinese crude drug, therefore the situation that present Chinese crude drug extracts is: also different at its extraction of effective components of different Chinese crude drugs, and adopting different extracting method to need the extraction that a large amount of different extraction equipment are used to support Chinese crude drug at different Chinese crude drugs, this is unsuitable for the standardization that Chinese crude drug extracts.
The Radix Paeoniae Alba (Pasonia lactiflora Pall) be cohosh go crust dry root, cold nature, bitter in the mouth acid.There are nourishing blood to suppress the hyperactive liver, slow middle pain relieving, yin fluid astringing to receive the function of antiperspirant.China's breast abdomen pain over the hypochondriac region, dysentery stomachache, spontaneous perspiration, fever due to yin deficiency, menoxenia, metrorrhagia, leukorrhagia of being used for the treatment of among the people.Modern study shows, mainly contains peoniflorin (paeoniflorin) compounds in the Radix Paeoniae Alba, also contains benzoic acid, cupreol, 1,2,3,4, the yellow times acyl tannin of 6-, times tannin, and gallotannin, gallic acid, progallin A, compositions such as d-catechin.
How to determine a kind of extraction, separation criterion method, the active constituents of medicine in the Radix Paeoniae Alba not only can be extracted fully, can also further separate the active constituents of medicine that is obtained by separation method 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 Paeoniae Alba extraction, separation criterionization.Exactly Radix Paeoniae Alba 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 the Radix Paeoniae Alba is extracted, the inventor is by a large amount of tests, the extraction of effective components of the present Radix Paeoniae Alba 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 the Radix Paeoniae Alba is extracted, can obtain the effective ingredient in the Chinese medicine to greatest extent.
The Radix Paeoniae Alba of the present invention is extracted, 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 Paeoniae Alba that is suitable for suitability for industrialized production was extracted, separation criterionization.
The present invention can implement through the following steps:
(1) extraction process: take by weighing white Peony Root, 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 the fr.8 (fr.5 is owing to only containing a few chemical compound, so do not need further separation) that obtains in the previous process with preparative liquid chromatography.Chromatographic column is a preparative column, and mobile phase is water and acetonitrile, and gradient elution is collected fraction in the corresponding time period, obtains to have passed through further isolating each component.
Preferably, the present invention can implement through the following steps:
(1) extraction process: take by weighing white Peony Root, with its grinding and sieving, adding 5～12 times of amount volume ratios then is 7: 3 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 7: 3 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 the fr.8 that obtains in the previous process with preparative liquid chromatography.Chromatographic column is preparative column (Zorbax SB-C18; 21.2mm * 250mm), mobile phase is water and acetonitrile, and gradient elution, flow velocity are 10ml/min, and column temperature is a room temperature, collects fraction in the corresponding time period, obtains through further isolating each component.
Best the present invention can implement through the following steps:
(1) extraction process: take by weighing white Peony Root, with its grinding and sieving, adding 8 times of amount volume ratios then is 7: 3 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 7: 3 chloroform and methanol as mobile phase, eluent fr.5, use methanol as mobile phase at last, get eluent fr.6.Fr.2 is condensed into extractum with extracting solution, with sample on 5% dissolve with methanol, adopt the ODS-C18 post that it is separated, at first use 5% methanol as mobile phase, get eluent fr.7, change 70% methanol then, get eluent fr.8 as mobile phase, use 100% methanol solution as mobile phase at last, get eluent fr.9.
(3) preparation separating technology: in order from step (2), to obtain the more concrete effective ingredient of fr.8 in the Radix Paeoniae Alba extract, continue to separate fr.8 among the present invention with preparative liquid chromatography.
The fr.8 separation condition: chromatographic column is Agilent preparative column (Zorbax SB-C18; 21.2mm * 250mm); Adopt gradient elution, mobile phase is water (A) and acetonitrile (B), and flow velocity is 10ml/min, and column temperature is a room temperature, and gradient is as follows:
During 0min, mobile phase A is 85% water, and Mobile phase B is 15% acetonitrile solution;
During 5min, mobile phase A is 82% water, and Mobile phase B is 18% acetonitrile solution;
During 20min, mobile phase A is 70% water, and Mobile phase B is 30% acetonitrile solution;
During 40min, mobile phase A is 65% water, and Mobile phase B is 35% acetonitrile solution;
During 45min, mobile phase A is 5% water, and Mobile phase B is 95% acetonitrile solution;
The fr.8 separating resulting: the component and the corresponding acquisition time that are obtained by the preparative liquid chromatography separation are as follows:
Component fr.81, acquisition time 4.0-14.0min;
Component fr.82, acquisition time 14.0-18.1min;
Component fr.83, acquisition time 18.1-20.0min;
Component fr.84, acquisition time 20.0-25.5min;
Component fr.85, acquisition time 25.5-28.9min;
Component fr.86, acquisition time 28.9-31.3min;
Component fr.87, acquisition time 31.3-38.0min;
Component fr.88, acquisition time 38.0-45.0min.
In order to obtain concrete effective component of paeonia lactiflora, best extraction separation method of the present invention is:
(1) extraction process: take by weighing white Peony Root 250g, with its grinding and sieving, adding 8 times of amount volume ratios then is 7: 3 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 16.8g 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 7: 3 chloroform and methanol as mobile phase, get eluent fr.5, concentrate the 1.8g sample, use methanol as mobile phase at last, eluent fr.6.With extracting solution fr.2 concentrate 66.8g extractum, get 2.8g extractum with sample on 5% dissolve with methanol, adopt the ODS-C18 post that it is separated, at first use 5% methanol as mobile phase, get eluent fr.7, change 70% methanol then as mobile phase, get eluent fr.8, concentrate the 1.06g sample, use 100% methanol solution as mobile phase at last, eluent fr.9.
(3) preparation separating technology: in order from step (2), to obtain the more concrete effective ingredient of fr.8 in the Radix Paeoniae Alba extract, continue to separate fr.8 among the present invention with preparative liquid chromatography.
The fr.8 separation condition: chromatographic column is Agilent preparative column (Zorbax SB-C18; 21.2mm * 250mm); Adopt gradient elution, mobile phase is water (A) and acetonitrile (B), and flow velocity is 10ml/min, and column temperature is a room temperature, and gradient is as follows:
During 0min, mobile phase A is 85% water, and Mobile phase B is 15% acetonitrile solution;
During 5min, mobile phase A is 82% water, and Mobile phase B is 18% acetonitrile solution;
During 20min, mobile phase A is 70% water, and Mobile phase B is 30% acetonitrile solution;
During 40min, mobile phase A is 65% water, and Mobile phase B is 35% acetonitrile solution;
During 45min, mobile phase A is 5% water, and Mobile phase B is 95% acetonitrile solution;
The fr.8 separating resulting: get component fr.8 1.06g altogether, the dissolve with methanol sample introduction with 20%, the component and the corresponding acquisition time that are obtained by the preparative liquid chromatography separation are as follows:
Component fr.81, acquisition time 4.0-14.0min, 136mg;
Component fr.82, acquisition time 14.0-18.1min, 141mg;
Component fr.83, acquisition time 18.1-20.0min, 269mg;
Component fr.84, acquisition time 20.0-25.5min, 105mg;
Component fr.85, acquisition time 25.5-28.9min, 67mg;
Component fr.86, acquisition time 28.9-31.3min, 34mg;
Component fr.87, acquisition time 31.3-38.0min, 30mg;
Component fr.88, acquisition time 38.0-45.0min, 13mg.
Among the present invention, contained chemical compound sees Table 1 in each component of the Radix Paeoniae Alba, and the concrete analysis authentication method is seen embodiment three.
Contained chemical compound in each component of table 1 Radix Paeoniae Alba
The above Radix Paeoniae Alba, extraction solution can increase or reduce when industrialization is extracted according to corresponding ratio, as large-scale production can be unit with kilogram or with the ton, small-scale production can be unit with the gram also, and weight can increase or reduce, but its weight proportion constant rate.
The extraction that the present invention set up, separation criterionization goes for any Chinese crude drug in the present Chinese medicine, for example can be used for Resina Ferulae, Folium Artemisiae Argyi, Benzoinum, Semen Platycladi, Carapax Trionycis, Semen Arecae, Herba Menthae, Semen Ricini Herba Polygoni Avicularis, Fructus Psoraleae, Radix Isatidis, Fructus Piperis Longi, Fructus Crotonis, Radix Morindae Officinalis, Rhizoma Menispermi, Radix Glehniae, Pseudobulbus Bletillae (Rhizoma Bletillae), the Radix Pulsatillae, the Radix 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 OphiopogonisFructus 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 of paeonia lactiflora 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 each component ultraviolet spectrogram of the Radix Paeoniae Alba below, each component ultraviolet spectrogram of Radix Paeoniae Alba water solublity, each component mass spectrum of Radix Paeoniae Alba water solublity are intended to further specify the present invention, but the present invention are not construed as limiting.
Each component ultraviolet spectrogram of Fig. 1 Radix Paeoniae Alba
Each component ultraviolet spectrogram of Fig. 2 Radix Paeoniae Alba water solublity
Each component mass spectrum of Fig. 3 Radix Paeoniae Alba water solublity
The specific embodiment
Below further specify the present invention by the specific embodiment, be not construed as limiting the invention in any form.
Embodiment one
(1) extraction process: take by weighing white Peony Root 250g, with its grinding and sieving, adding 10 times of amount volume ratios then is 7: 3 ethyl acetate and ethanol, and reflux 3 hours is extracted 3 times, and merging filtrate gets extracting solution fr.1.Add 6 times of amount 70% ethanol in medicinal residues, reflux 2 hours is extracted 2 times, and merging filtrate gets extracting solution fr.2.Add entry in medicinal residues at last, reflux 1.5 hours is extracted 2 times, and merging filtrate gets extracting solution fr.3.
(2) separating technology: with extracting solution fr.1 concentrate 16.8g 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 7: 3 chloroform and methanol as mobile phase, get eluent fr.5, concentrate the 1.6g sample, use methanol as mobile phase at last, eluent fr.6.With extracting solution fr.2 concentrate 56.6g extractum, get 2.8g extractum with sample on 5% dissolve with methanol, adopt the ODS-C18 post that it is separated, at first use 5% methanol as mobile phase, get eluent fr.7, change 70% methanol then as mobile phase, get eluent fr.8, concentrate the 1.00g sample, use 100% methanol solution as mobile phase at last, eluent fr.9.
(3) preparation separating technology: continue to separate the fr.8 that obtains in the previous process with preparative liquid chromatography.Chromatographic column is Agilent preparative column (Zorbax SB-C18; 21.2mm * 250mm), adopting gradient elution, mobile phase is water (A) and acetonitrile (B), and flow velocity is 10ml/min, and column temperature is a room temperature, and gradient is as follows:
During 0min, mobile phase A is 85% water, and Mobile phase B is 15% acetonitrile solution;
During 5min, mobile phase A is 82% water, and Mobile phase B is 18% acetonitrile solution;
During 20min, mobile phase A is 70% water, and Mobile phase B is 30% acetonitrile solution;
During 40min, mobile phase A is 65% water, and Mobile phase B is 35% acetonitrile solution;
During 45min, mobile phase A is 5% water, and Mobile phase B is 95% acetonitrile solution;
The fr.8 separating resulting: get component fr.8 0.90g altogether, the dissolve with methanol sample introduction with 20%, the component and the corresponding acquisition time that are obtained by the preparative liquid chromatography separation are as follows:
Component fr.81, acquisition time 4.0-14.0min, 126mg;
Component fr.82, acquisition time 14.0-18.1min, 132mg;
Component fr.83, acquisition time 18.1-20.0min, 229mg;
Component fr.84, acquisition time 20.0-25.5min, 125mg;
Component fr.85, acquisition time 25.5-28.9min, 57mg;
Component fr.86, acquisition time 28.9-31.3min, 26mg;
Component fr.87, acquisition time 31.3-38.0min, 28mg;
Component fr.88, acquisition time 38.0-45.0min, 20mg.
Embodiment two
(1) extraction process: take by weighing white Peony Root 500g, with its grinding and sieving, adding 12 times of amount volume ratios then is 7: 3 ethyl acetate and ethanol, and reflux 1 hour is extracted 3 times, and merging filtrate gets extracting solution fr.1.Add 6 times of amount 70% ethanol in medicinal residues, reflux 3 hours is extracted 1 time, and merging filtrate gets extracting solution fr.2.Add entry in medicinal residues at last, reflux 2 hours is extracted 2 times, and merging filtrate gets extracting solution fr.3.
(2) separating technology: with extracting solution fr.1 concentrate 21.8g 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 7: 3 chloroform and methanol as mobile phase, get eluent fr.5, concentrate the 2.8g sample, use methanol as mobile phase at last, eluent fr.6.With extracting solution fr.2 concentrate 116.8g extractum, get 2.8g extractum with sample on 5% dissolve with methanol, adopt the ODS-C18 post that it is separated, at first use 5% methanol as mobile phase, get eluent fr.7, change 70% methanol then as mobile phase, get eluent fr.8, concentrate the 1.11g sample, use 100% methanol solution as mobile phase at last, eluent fr.9.
(3) preparation separating technology: continue to separate the fr.8 that obtains in the previous process with preparative liquid chromatography.Chromatographic column is Agilent preparative column (Zorbax SB-C18; 21.2mm * 250mm), adopting gradient elution, mobile phase is water (A) and acetonitrile (B), and flow velocity is 10ml/min, and column temperature is a room temperature, and gradient is as follows:
During 0min, mobile phase A is 85% water, and Mobile phase B is 15% acetonitrile solution;
During 5min, mobile phase A is 82% water, and Mobile phase B is 18% acetonitrile solution;
During 20min, mobile phase A is 70% water, and Mobile phase B is 30% acetonitrile solution;
During 40min, mobile phase A is 65% water, and Mobile phase B is 35% acetonitrile solution;
During 45min, mobile phase A is 5% water, and Mobile phase B is 95% acetonitrile solution;
The fr.8 separating resulting: get component fr.8 1.06g altogether, the dissolve with methanol sample introduction with 20%, the component and the corresponding acquisition time that are obtained by the preparative liquid chromatography separation are as follows:
Component fr.81, acquisition time 4.0-14.0min, 126mg;
Component fr.82, acquisition time 14.0-18.1min, 111mg:
Component fr.83, acquisition time 18.1-20.0min, 239mg;
Component fr.84, acquisition time 20.0-25.5min, 95mg;
Component fr.85, acquisition time 25.5-28.9min, 57mg;
Component fr.86, acquisition time 28.9-31.3min, 26mg;
Component fr.87, acquisition time 31.3-38.0min, 22mg;
Component fr.88, acquisition time 38.0-45.0min, 12mg.
Embodiment three
One, the extraction separation of white Peony Root
(1) extraction process: take by weighing white Peony Root 250g, with its grinding and sieving, adding 8 times of amount volume ratios then is 7: 3 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 16.8g 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 7: 3 chloroform and methanol as mobile phase, get eluent fr.5, concentrate the 1.8g sample, use methanol as mobile phase at last, eluent fr.6.With extracting solution fr.2 concentrate 66.8g extractum, get 2.8g extractum with sample on 5% dissolve with methanol, adopt the ODS-C18 post that it is separated, at first use 5% methanol as mobile phase, get eluent fr.7, change 70% methanol then as mobile phase, get eluent fr.8, concentrate the 1.06g sample, use 100% methanol solution as mobile phase at last, eluent fr.9.
(3) preparation separating technology: in order from step (2), to obtain the more concrete effective ingredient of fr.8 in the Radix Paeoniae Alba extract, continue to separate fr.8 among the present invention with preparative liquid chromatography.
The fr.8 separation condition: chromatographic column is Agilent preparative column (Zorbax SB-C18; 21.2mm * 250mm); Adopt gradient elution, mobile phase is water (A) and acetonitrile (B), and flow velocity is 10ml/min, and column temperature is a room temperature, and gradient is as follows:
During 0min, mobile phase A is 85% water, and Mobile phase B is 15% acetonitrile solution;
During 5min, mobile phase A is 82% water, and Mobile phase B is 18% acetonitrile solution;
During 20min, mobile phase A is 70% water, and Mobile phase B is 30% acetonitrile solution;
During 40min, mobile phase A is 65% water, and Mobile phase B is 35% acetonitrile solution;
During 45min, mobile phase A is 5% water, and Mobile phase B is 95% acetonitrile solution;
The fr.8 separating resulting: get component fr.8 1.06g altogether, the dissolve with methanol sample introduction with 20%, the component and the corresponding acquisition time that are obtained by the preparative liquid chromatography separation are as follows:
Component fr.81, acquisition time 4.0-14.0min, 136mg;
Component fr.82, acquisition time 14.0-18.1min, 141mg;
Component fr.83, acquisition time 18.1-20.0min, 269mg;
Component fr.84, acquisition time 20.0-25.5min, 105mg;
Component fr.85, acquisition time 25.5-28.9min, 67mg;
Component fr.86, acquisition time 28.9-31.3min, 34mg;
Component fr.87, acquisition time 31.3-38.0min, 30mg;
Component fr.88, acquisition time 38.0-45.0min, 13mg.
Two. analyze
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. analytical method
Sample source: Radix Paeoniae Alba component fr.5 40mg.
Sample preparation: sample 1ml dissolve with ethanol, ultrasonic, centrifugal (10000r/min), standby.
The HPLC condition: chromatographic column is Agilent Zorbax SB-C18 type chromatographic column (4.6mm * 150mm, 5 μ m), carries out gradient elution with binary mobile phase.Mobile phase A is 0.2%HAc-H
2O, Mobile phase B is 0.2%HAc-CH
3CN; Linear gradient is: 0min, 10%B; 10min, 50%B; 30min, 95%B.Flow velocity is 0.5mL/min; Column temperature is 30 ℃; DAD detects in the interscan of 190-400nm scope; Sample size 5 μ L.
Mass spectrum condition: negative ions scan pattern, sweep limits 100～2000; Dry gas (N
2Stream speed 10L/min, 350 ℃ of atomization temperatures, capillary voltage 3500V, cracking voltage 100V.
ELSD condition: nitrogen 1.4L/min, drift tube temperature: 100 ℃
Sample source: Radix Paeoniae Alba component fr.81 136mg, fr.82 141mg, fr.83 269mg, fr.84 105mg, fr.8567mg, fr.86 34mg, fr.87 30mg, fr.88 13mg.
Sample preparation: sample is with the dissolving of 1ml 50% methanol-water, and is ultrasonic, and centrifugal (10000r/min) is standby.The HPLC condition: chromatographic column is Agilent Zorbax SB-C18 type chromatographic column (4.6mm * 150mm, 5 μ m), carries out gradient elution with binary mobile phase.Mobile phase A is 0.2%HAc-H
2O, Mobile phase B is 0.2%HAc-CH
3CN; Linear gradient is: 0min, 10%B; 10min, 30%B; 30min, 50%B.Flow velocity is 0.5mL/min; Column temperature is 30 ℃; DAD detects in the interscan of 190-400nm scope; Sample size 5 μ L.
Mass spectrum condition: negative ions scan pattern, sweep limits 100～2000; Dry gas (N
2Stream speed 10L/min, 350 ℃ of atomization temperatures, capillary voltage 3500V, cracking voltage 100V.
ELSD condition: nitrogen 1.8L/min, drift tube temperature: 105 ℃
3. analysis result
Analysis result: Fig. 2 is each component uv-spectrogram of Radix Paeoniae Alba water solublity, and Fig. 3 is each component mass spectrum of Radix Paeoniae Alba water solublity.
Each compound identification the results are shown in Table 2 in the Radix Paeoniae Alba component.
Table 2. Radix Paeoniae Alba component composition qualification result
Annotate: DNP (Dictionary of Natural Products) natural product data base;
Document 1: the few magnificent thesis for the doctorate of Wu, the chemical constitution study of five kinds of medicinal plants and purple powder born of the same parents bolete, Kunming Inst. of Botany, Chinese Academy of Sciences's calendar year 2001 December subject major name: phytochemistry.
Claims (3)
- The Radix Paeoniae Alba extract, separation method, comprise the steps:(1) extraction process: take by weighing white Peony Root, with its grinding and sieving, adding 5～12 times of amount volume ratios then is 7: 3 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 7: 3 chloroform and methanol as mobile phase, eluent f r.5, use methanol as mobile phase at last, get eluent fr.6; After extracting solution fr.2 is condensed into extractum, with sample on 2～10% dissolve with methanol, adopt the ODS-C18 post that it is separated, at first use 2～10% methanol as mobile phase, get eluent fr.7, change 40～80% methanol then, get eluent fr.8 as mobile phase, use 100% methanol solution as mobile phase at last, get eluent fr.9;(3) preparation separating technology: separate fr.8 with preparative liquid chromatography; Chromatographic column is preparative column (Zorbax SB-C18; 21.2mm * 250mm), mobile phase is water and acetonitrile, and gradient elution, flow velocity are 10ml/min, and column temperature is a room temperature, collects fraction in the corresponding time period, obtains through further isolating each component.
- 2. the Radix Paeoniae Alba as claimed in claim 1 is extracted, separation method, may further comprise the steps:(1) extraction process: take by weighing white Peony Root, with its grinding and sieving, adding 8 times of amount volume ratios then is 7: 3 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: 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 7: 3 chloroform and methanol as mobile phase, eluent fr.5, use methanol as mobile phase at last, get eluent fr.6; Fr.2 is condensed into extractum with extracting solution, with sample on 5% dissolve with methanol, adopt the ODS-C18 post that it is separated, at first use 5% methanol as mobile phase, get eluent fr.7, change 70% methanol then, get eluent fr.8 as mobile phase, use 100% methanol solution as mobile phase at last, get eluent fr.9;(3) preparation separation industries: separate fr.8 with preparative liquid chromatography;The separation condition of fr.8: chromatographic column is preparative column (Zorbax SB-C18; 21.2mm * 250mm), adopting gradient elution, mobile phase is water (A) and acetonitrile (B), and flow velocity is 10ml/min, and column temperature is a room temperature, and gradient is as follows:During 0min, mobile phase A is 85% water, and Mobile phase B is 15% acetonitrile solution;During 5min, mobile phase A is 82% water, and Mobile phase B is 18% acetonitrile solution;During 20min, mobile phase A is 70% water, and Mobile phase B is 30% acetonitrile solution;During 40min, mobile phase A is 65% water, and Mobile phase B is 35% acetonitrile solution;During 45min, mobile phase A is 5% water, and Mobile phase B is 95% acetonitrile solution;The fr.8 separating resulting: the component and the corresponding acquisition time that are obtained by the preparative liquid chromatography separation are as follows:Component fr.81, acquisition time 4.0-14.0min;Component fr.82, acquisition time 14.0-18.1min;Component fr.83, acquisition time 18.1-20.0min;Component fr.84, acquisition time 20.0-25.5min;Component fr.85, acquisition time 25.5-28.9min;Component fr.86, acquisition time 28.9-31.3min;Component fr.87, acquisition time 31.3-38.0min;Component fr.88, acquisition time 38.0-45.0min.
- 3. the Radix Paeoniae Alba as claimed in claim 2 is extracted, separation method, it is characterized in that:Main compound is Albiflorin among the component fr.82;Main compound is Paeoniflorin among the component fr.83;Main compound is 1,2,3,4 among the component fr.84,6-Pentagalloylglucose;Main compound is Digalloyl-1 among the component fr.85,2,3, and 4-tetra-O-galloyl-D-glucopyranose, Galloyl-Paeoniflorin;Main compound is Methyl gallate among the component fr.86, Albiflorin,1，2，3，4，6-Pentagalloylglucose；Main compound is Methyl gallate among the component fr.87,1,2,3,4, and 6-Pentagalloylglucose;Main compound is 1,2,3,4 among the component fr.88,6-Pentagalloylglucose, Paeonidaninol a orb.
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| CN101474264B (en) * | 2009-01-22 | 2011-09-21 | 浙江大学 | Effective component of white peony root as well as preparation method and use thereof |
| CN102336792A (en) * | 2010-07-22 | 2012-02-01 | 辽宁科技大学 | Three-zone simulated moving bed chromatography method for separating and purifying paeoniflorin |
| CN102258587B (en) * | 2011-03-10 | 2013-06-12 | 浙江大学 | Preparation method and application of red paeony root active ingredient |
| CN102125609B (en) * | 2011-03-10 | 2013-03-13 | 浙江大学 | Red paeony root active ingredient with myocardial protection effect and preparation method thereof |
| CN102119984B (en) * | 2011-03-13 | 2013-03-13 | 浙江大学 | Preparation method and application of carthamus tinctorius L. active ingredient containing hydroxysafflor yellow A |
| CN102998386A (en) * | 2012-11-22 | 2013-03-27 | 宁波立华制药有限公司 | Method for measuring radix paeoniae alba diglucoside in radix paeoniae alba callus by utilizing high performance liquid chromatography |
| CN104725448A (en) * | 2013-12-19 | 2015-06-24 | 北京农学院 | Albiflorin extracting method |
| CN104478971A (en) * | 2014-11-24 | 2015-04-01 | 天津理工大学 | Preparation method of high purity paeoniflorin |
| CN108707172A (en) * | 2018-06-27 | 2018-10-26 | 吉林大学珠海学院 | A kind of preparation method of high-purity Paeoniflorin |
| CN109557233B (en) * | 2019-01-29 | 2020-08-18 | 浙江中医药大学 | Method for determining content of multi-index components in white paeony root extracting solution |
| CN111662348B (en) * | 2020-06-11 | 2022-03-08 | 湖南甜蔓生物科技有限公司 | Method for extracting paeoniflorin and albiflorin from paeonia lactiflora |
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