CN1903259B - Method for extraction and separation of moutan bark - Google Patents

Method for extraction and separation of moutan bark Download PDF

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CN1903259B
CN1903259B CN2005100122758A CN200510012275A CN1903259B CN 1903259 B CN1903259 B CN 1903259B CN 2005100122758 A CN2005100122758 A CN 2005100122758A CN 200510012275 A CN200510012275 A CN 200510012275A CN 1903259 B CN1903259 B CN 1903259B
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CN1903259A (en
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程翼宇
贺庆
王毅
王学伟
李云飞
葛志伟
胡兴江
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Tasly Pharmaceutical Group Co Ltd
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Tianjin Tasly Pharmaceutical Co Ltd
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Abstract

A standardized extracting and separating method for extracting the chemical components from a Chinese-medicinal material to maximal extent and further separating them to obtain the chemical components with different polarities is disclosed. To be specific, the paeonol 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 and separation method of root bark of tree peony medicinal material
Technical field
The invention belongs to field of medicaments, particularly relate to root bark of tree peony extraction, separation method.
Background technology
At present, in the world, herbal medicine all has certain market, along with people increasing and the aging of population to the health requirements level of understanding, sub-health stateization, people thirst for back to nature more, utilize the high pharmacological agent 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 herbal medicine, state herbal medicine status such as Canada and Australia have legalized, United States Government has also drafted the plant amedica management method, the compound mixed 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 cut-throat competition of powerful transnational medical group and Japan, Korea S, India, Thailand and European countries' plant amedicas such as Germany, France, numerous products that China's traditional 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 stdn that Chinese medicinal materials extracts and the stdn of Chinese medicinal preparation method are that Chinese patent medicine moves towards the world 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 constituent which kind of method of employing could obtain with the sight of research always from Chinese medicinal materials, therefore the situation that present Chinese medicinal materials extracts is: extraction, separation method at different its effective constituent of Chinese medicinal materials are also different, and the extraction of adopting different extraction, separation methods to need a large amount of different extractions and separating device to be used to support Chinese medicinal materials at different Chinese medicinal materials, separate, this is unsuitable for, and Chinese medicinal materials extracts, isolating stdn.
The Tree Peony Bark another name root bark of tree peony, unpeeled CORTEX MOUTAN, wooden Chinese herbaceous peony, Luoyang flower are the root skin of Paeoniaceae plant tree peony.What tree peony was written into ancient books and records the earliest as medicine is Shennong's Herbal.The mechanism that bright Ni Zhumo " book on Chinese herbal medicine converges and says " controls the gas regulating blood condition to tree peony has been done comparatively detailed elaboration: " Tree Peony Bark clears away heart-fire, and supports kidney, and liver, sharp envelope, and control four through blood system volt fire, the gas medicine is also in the blood.It is kind that to control woman's blood vessels extravesated blood in obstructed and postpartum more than.Control bleeding from five sense organs or subcutaneous tissue, haematemesis, uterine bleeding, pouring blood again, fall and pounce on hemostasis, all blood for sick, system can be controlled it.Cover its gas perfume (or spice), perfume (or spice) can sharp gas and promoting circulation of blood; Its bitter, hardship can the therapeutic methods to keep the adverse qi flowing downward and are stopped blooding; It is cool in nature, and cold can be with blood and green blood; Its flavor is hot again, and suffering can push away Chen Xueer and cause new blood also.So Zhen Quanfang controls woman's blood, Yin Re and with withered, lumbar vertebrae pain, hot polydipsia heavily added the root bark of tree peony with four things and tested most night.”
Modern study indicates that Tree Peony Bark has clearing heat and cooling blood, the effect of promoting blood circulation to remove blood stasis.Be used for the treatment of the febrile disease pathogenic heat attacking blood system in febrile diseases, send out spot, tell nosebleed, the pyreticosis later stage, hot Fu Yin divided heating, deficiency of Yin osteopyrexia and fever, and amenorrhea due to stagnation of blood, dysmenorrhoea, carbuncle sore tumefacting virus falls and pounces on the pain of injury, diseases such as rheumatic fever numbness.The main chemical compositions of Tree Peony Bark is paeonol, paeoniflorin, OP, Oxypaeoniflorin, Benzoylpaeoniflorin and benzoyl Oxypaeoniflorin, volatile oil, and phenylformic acid, plant sterol, sucrose, glucose, pectinose etc.And paeoniflorin, OP, Benzoylpaeoniflorin are main effective constituent.
How to determine a kind of extraction, separation criterion method, the active constituents of medicine in the Tree Peony Bark not only can be extracted fully, can also further separate the active constituents of medicine that is obtained by separation method.The establishment of this extraction, separation criterionization is that present Chinese medicine is realized modern key factor.
Summary of the invention
The object of the present invention is to provide the extraction of a kind of root bark of tree peony mark, separation criterion method.Exactly Cortex Moutan extract is divided into fractions specifically, each component comprises several main compound, has constituted a medicinal material component pool by these medicinal material components.Can carry out drug screening to the medicinal material component pool, thereby find new drug.
In order to realize root bark of tree peony extraction, isolating modernization, stdn, the inventor is by a large amount of tests, extraction, separation method to the effective constituent of the present root bark of tree peony are concluded and are put in order, with seek a kind of can standardized extracting method, promptly under this extraction conditions, adopt this extraction standardized method that the root bark of tree peony is extracted, can obtain the effective constituent in the Chinese medicine to greatest extent; By the separation criterion method, the active constituents of medicine that extracts is further separated.
The root bark of tree peony of the present invention extracts, the separation criterion method is that the inventor passes through test, after Chinese medicinal materials adopts different extracting method to compare simply together, and the resulting Tree Peony Bark extraction separation standardized method that is suitable for suitability for industrialized production.
The present invention can implement through the following steps:
(1) extraction process: take by weighing root bark of tree peony medicinal material,, add ethyl acetate and ethanol then its grinding and sieving, the heating extract extracting solution fr.1.In the dregs of a decoction, add ethanol, the heating extract extracting solution fr.2.In the dregs of a decoction, add entry at last, the heating extract extracting solution fr.3.
(2) separating technology: after extracting solution fr.1 is condensed into medicinal extract, use silica gel mixed sample, adopt normal phase silicagel column that it is separated, at first use sherwood oil and ethyl acetate as moving phase, get elutriant fr.4, change chloroform and methyl alcohol then, get elutriant fr.5 as moving phase, use methyl alcohol as moving phase at last, get elutriant fr.6.After extracting solution fr.2 is condensed into medicinal extract, with sample on the dissolve with methanol, adopt the ODS-C18 post that it is separated, the methyl alcohol of at first using lower concentration is as moving phase, get elutriant fr.7, the methyl alcohol that changes intermediate concentration then gets elutriant fr.8 as moving phase, use pure methanol solution as moving phase at last, get elutriant 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 semipreparative column (Lichrospher C18; 10.0mm * 250mm, 5 μ m), moving phase is water and acetonitrile, gradient elution is collected cut in the corresponding time period, obtains to have passed through further isolating each component.
Preferred the present invention can implement through the following steps:
(1) extraction process: take by weighing root bark of tree peony medicinal material, with its grinding and sieving, adding 5～12 times of amount volume ratios then is 1: 1 ethyl acetate and ethanol, and reflux 0.5～3 hour is extracted 1～3 time, and merging filtrate gets extracting solution fr.1.Add 5～12 times of amount 70% ethanol in the dregs of a decoction, reflux 0.5～3 hour is extracted 1～3 time, and merging filtrate gets extracting solution fr.2.Add entry in the dregs of a decoction 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 medicinal extract, use silica gel mixed sample, adopt normal phase silicagel column that it is separated, be that 50: 1 sherwood oil and ethyl acetate are as moving phase at first with volume ratio, elutriant fr.4, change volume ratio then and be 10: 1 chloroform and methyl alcohol as moving phase, elutriant fr.5, use methyl alcohol as moving phase at last, get elutriant fr.6.After extracting solution fr.2 is condensed into medicinal extract, with sample on 2～10% dissolve with methanol, adopt the ODS-C18 post that it is separated, at first use 2～10% methyl alcohol as moving phase, get elutriant fr.7, change 40～80% methyl alcohol then, get elutriant fr.8 as moving phase, use 100% methanol solution as moving phase at last, get elutriant 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 semipreparative column (Lichrospher C18; 10.0mm * 250mm, 5 μ m), moving phase is water and acetonitrile, and gradient elution, flow velocity are 3ml/min, and column temperature is a room temperature, collects cut 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 root bark of tree peony medicinal material, with its grinding and sieving, adding 8 times of amount volume ratios then is 1: 1 ethyl acetate and ethanol, and reflux 1 hour is extracted 2 times, and merging filtrate gets extracting solution fr.1.Add 8 times of amount 70% ethanol in the dregs of a decoction, reflux 1 hour is extracted 2 times, and merging filtrate gets extracting solution fr.2.Add entry in the dregs of a decoction 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 medicinal extract with extracting solution, use silica gel mixed sample, adopt normal phase silicagel column that it is separated, be that 50: 1 sherwood oil and ethyl acetate are as moving phase at first with volume ratio, elutriant fr.4, change volume ratio then and be 10: 1 chloroform and methyl alcohol as moving phase, elutriant fr.5, use methyl alcohol as moving phase at last, get elutriant fr.6.Fr.2 is condensed into medicinal extract with extracting solution, with sample on 5% dissolve with methanol, adopt the ODS-C18 post that it is separated, at first use 5% methyl alcohol as moving phase, get elutriant fr.7, change 60% methyl alcohol then, get elutriant fr.8 as moving phase, use 100% methanol solution as moving phase at last, get elutriant fr.9.
(3) preparation separating technology: in order from step (2), to obtain fr.5 and the more concrete effective constituent of fr.8 in the Cortex Moutan extract, continue to separate fr.5 and fr.8 among the present invention with preparative liquid chromatography.
The fr.5 separation condition: chromatographic column is semipreparative column (the Lichrospher C18 of Chinese nation; 10.0mm * 250mm, 5 μ m), moving phase is water (A) and acetonitrile (B), and flow velocity is 3ml/min, and column temperature is a room temperature.The gradient elution program is as follows:
In the time of 0 minute, mobile phase A is that 80% the aqueous solution, Mobile phase B are 20% acetonitrile solution;
In the time of 40 minutes, mobile phase A is that 20% the aqueous solution, Mobile phase B are 80% acetonitrile solution;
In the time of 50 minutes, mobile phase A is that 5% the aqueous solution, Mobile phase B are 95% acetonitrile solution.
The fr.5 separating resulting: the component and the corresponding collection time that are obtained by the preparative liquid chromatography separation are as follows: collection in 4.0～7.6 minutes obtains component fr.51,7.6 collected and obtain component fr.52 in～9.7 minutes, 9.7 collected and obtain component fr.53 in～14.2 minutes, 14.2 collected and obtain component fr.54 in～18.4 minutes, 21.8 collected and obtain component fr.55 in～26.5 minutes, 21.8 collected and obtain component fr.56 in～26.5 minutes, 26.5 collected and obtain component fr.57 in～30.3 minutes, 30.3 collected and obtain component fr.58 in～35.5 minutes, collection in 35.5～45 minutes obtains component fr.59.
The fr.8 separation condition: chromatographic column is semipreparative column (the Lichrospher C18 of Chinese nation; 10.0mm * 250mm, 5 μ m), moving phase is water (A) and acetonitrile (B), and flow velocity is 3ml/min, and column temperature is a room temperature.The gradient elution program is as follows:
In the time of 0 minute, mobile phase A is that 90% the aqueous solution, Mobile phase B are 10% acetonitrile solution;
In the time of 8 minutes, mobile phase A is that 81% the aqueous solution, Mobile phase B are 19% acetonitrile solution;
In the time of 60 minutes, mobile phase A is that 65% the aqueous solution, Mobile phase B are 35% acetonitrile solution;
In the time of 70 minutes, mobile phase A is that 95% the aqueous solution, Mobile phase B are 5% acetonitrile solution.
The fr.8 separating resulting: the component and the corresponding collection time that are obtained by the preparative liquid chromatography separation are as follows: collection in 4.0～7.7 minutes obtains component fr.81,7.7 collected and obtain component fr.82 in～10.7 minutes, 10.7 collected and obtain component fr.83 in～15.8 minutes, 15.8 collected and obtain component fr.84 in～18.8 minutes, 18.8 collected and obtain component fr.85 in～23.2 minutes, 23.2 collected and obtain component fr.86 in～27.4 minutes, 27.4 collected and obtain component fr.87 in～33.1 minutes, 33.1 collected and obtain component fr.88 in～40.2 minutes, 40.2 collected and obtain component fr.89 in～44.4 minutes, 44.4 collected and obtain component fr.810 in～48.5 minutes, 48.5 collected and obtain component fr.811 in～58 minutes, collected in 58～67 minutes and obtain component fr.812, collection in 67～75 minutes obtains component fr.813.
In order to obtain concrete root bark of tree peony effective constituent, best extraction of the present invention, separation method are:
(1) extraction process: take by weighing root bark of tree peony medicinal material 250g, with its grinding and sieving, adding 8 times of amount volume ratios then is 1: 1 ethyl acetate and ethanol, and reflux 1 hour is extracted 2 times, and merging filtrate gets extracting solution fr.1.Add 8 times of amount 70% ethanol in the dregs of a decoction, reflux 1 hour is extracted 2 times, and merging filtrate gets extracting solution fr.2.Add entry in the dregs of a decoction 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 22g medicinal extract, use silica gel mixed sample, adopt normal phase silicagel column that it is separated, at first with volume ratio be 50: 1 sherwood oil and ethyl acetate as moving phase, elutriant fr.4, change volume ratio then and be 10: 1 chloroform and methyl alcohol as moving phase, get elutriant fr.5, concentrate the 6.2g sample, use methyl alcohol as moving phase at last, elutriant fr.6.With extracting solution fr.2 concentrate 55g medicinal extract, with sample on 5% dissolve with methanol, adopt the ODS-C18 post that it is separated, at first use 5% methyl alcohol as moving phase, get elutriant fr.7, change 60% methyl alcohol then as moving phase, get elutriant fr.8, concentrate the 2.6g sample, use 100% methanol solution as moving phase at last, elutriant fr.9.
(3) preparation separating technology: in order from step (2), to obtain fr.5 and the more concrete effective constituent of fr.8 in the Cortex Moutan extract, continue to separate fr.5 and fr.8 among the present invention with preparative liquid chromatography.
The fr.5 separation condition: chromatographic column is semipreparative column (the Lichrospher C18 of Chinese nation; 10.0mm * 250mm, 5 μ m), moving phase is water (A) and acetonitrile (B), and flow velocity is 3ml/min, and column temperature is a room temperature.The gradient elution program is as follows:
In the time of 0 minute, mobile phase A is that 80% the aqueous solution, Mobile phase B are 20% acetonitrile solution;
In the time of 40 minutes, mobile phase A is that 20% the aqueous solution, Mobile phase B are 80% acetonitrile solution;
In the time of 50 minutes, mobile phase A is that 5% the aqueous solution, Mobile phase B are 95% acetonitrile solution.
Fr.5 separating resulting: get component fr.5 1.4g, dissolve with ethanol sample introduction with 10%, the component and the corresponding collection time that are obtained by the preparative liquid chromatography separation are as follows: collection in 4.0～7.6 minutes obtains component fr.51 0.16g, 7.6 collected and obtain component fr.52 0.11g in～9.7 minutes, 9.7 collected and obtain component fr.53 0.32g in～14.2 minutes, 14.2 collected and obtain component fr.540.13g in～18.4 minutes, 21.8 collected and obtain component fr.55 0.21g in～26.5 minutes, 21.8 collected and obtain component fr.56 0.10g in～26.5 minutes, 26.5 collected and obtain component fr.57 0.2g in～30.3 minutes, 30.3 collected and obtain component fr.58 0.09g in～35.5 minutes, collection in 35.5～45 minutes obtains component fr.59 0.07g.
The fr.8 separation condition: chromatographic column is semipreparative column (the Lichrospher C18 of Chinese nation; 10.0mm * 250mm, 5 μ m), moving phase is water (A) and acetonitrile (B), and flow velocity is 3ml/min, and column temperature is a room temperature.The gradient elution program is as follows:
In the time of 0 minute, mobile phase A is that 90% the aqueous solution, Mobile phase B are 10% acetonitrile solution;
In the time of 8 minutes, mobile phase A is that 81% the aqueous solution, Mobile phase B are 19% acetonitrile solution;
In the time of 60 minutes, mobile phase A is that 65% the aqueous solution, Mobile phase B are 35% acetonitrile solution;
In the time of 70 minutes, mobile phase A is that 95% the aqueous solution, Mobile phase B are 5% acetonitrile solution.
Fr.8 separating resulting: get fr.82.6g, dissolve with methanol sample introduction with 20%, the component and the corresponding collection time that are obtained by the preparative liquid chromatography separation are as follows: collection in 4.0～7.7 minutes obtains component fr.81 0.05g, 7.7 collected and obtain component fr.82 0.12g in～10.7 minutes, 10.7 collected and obtain component fr.83 0.18g in～15.8 minutes, 15.8 collected and obtain component fr.840.28g in～18.8 minutes, 18.8 collected and obtain component fr.85 0.18g in～23.2 minutes, 23.2 collected and obtain component fr.86 0.23g in～27.4 minutes, 27.4 collected and obtain component fr.87 0.18g in～33.1 minutes, 33.1 collected and obtain component fr.88 0.16g in～40.2 minutes, 40.2 collected and obtain component fr.89 0.1g in～44.4 minutes, 44.4 collected and obtain component fr.810 0.12g in～48.5 minutes, 48.5 collected and obtain component fr.811 0.17g in～58 minutes, collected in 58～67 minutes and obtain component fr.812 0.06g, collection in 67～75 minutes obtains component fr.8130.15g.
Among the present invention, contained compound sees Table 1 in each component of the root bark of tree peony, and its Analysis and Identification method is referring to embodiment three.
Contained compound in each component of table 1. root bark of tree peony
Figure G05112275820050818D000061
The above root bark of tree peony extracts solution can be increased or reduce when industrialization is extracted according to corresponding ratio, as scale operation 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 stdn extraction and separation method that the present invention set up goes for the most of Chinese medicinal materials in the present Chinese medicine, for example can be used for asafoetide, tarragon, st-yrax, Semen Platycladi, turtle shell, betel nut, peppermint, Semen Ricini,
Figure 200510012275810000210003_0
Hold, Psoralea corylifolia, Root of Indigowoad, the Bi roots of grass, crotons, Root of Medicinal Indian mulberry, Rhizoma Menispermi, Root of coastal Glehnia, bletilla, Root of Chinese Pulsatilla, the root of herbaceous peony, the root of Dahurain angelica, Rhizoma Typhonii, Rhizome of Lalang Grass, gingko, Cynanchum glaucescens, White Hyacinth Bean, radix ampelopsis, Root-bark of Densefruit Pittany, radix cynanchi atrati, Herba Lobeliae Chinensis, Herba Scutellariae Barbatae, the tuber of pinellia, lily, borneol, radix bupleuri, cicada slough, Papermulberry Fruit, Changshan, bark of tree of heaven, Squama Manis, Herba Andrographis, Semen Phaseoli, Red Halloysite, the radix paeoniae rubrathe, rhizoma atractylodis, Siberian Cocklour Fruit, agalloch eaglewood, Stringy Stonecrop Herb, Leafy twigs of Oriental Arborvitae, radix aconiti agrestis, Folium Aconiti Kusnezoffii, in one's early teens, tsaoko, Unibract Fritillary Bulb, Root of Medicinal Cyathula, monkshood, Ligusticum wallichii, Szechwan Chinaberry Fruit, Semen Plantaginis, Semen Sojae Preparatum, Radix Codonopsis, Herba Lophatheri, the bark of eucommia, levisticum, Arisaema with Bile, setose thistle, Pericarpium Arecae, the red sage root, plaster of paris, Peel of Chinese Waxgourd, Cordyceps sinensis, earthworm, the Fruit of Belveder, Root-bark of Chinese Wolfberry, Radix Rehmanniae Preparata, Humifuse Euphorbia Herb, garden burnet, Radix Angelicae Sinensis, Rush, cloves, sword bean, Leaf of Indigowoad, date, rheum officinale, Small Centipeda Herb, curcuma zedoary, catechu, Chinese torreyanut, duckweed, Bi separates, raspberry, Buddha's hand, monkshood, Poria cocos, the root of fangji, windproof, sennae, honey, gekko, cassia twig, the root of kudzu vine, Jehol Ligusticum Rhizome, Rhizoma Alpiniae Officinarum, Rhizome of Fortune's Drynaria, rice sprout, Flower of Buerger Pipewort, rhizoma cibotii, wolfberry fruit, Folium Ilicis Cornutae, yncaria stem with hooks, rhizoma nardostachyos, Radix Glycyrrhizae, the root of gansui, Snakegourd Fruit, Snakegourd Seed, Snakegourd Peel, Stem of Manshurian Dutchmanspipe, rhizoma zingiberis, baked ginger, dried lacquer, Lappula echinata, the Radix Astragali, sealwort, marine alga, sophora flower, Kadsura Pepper Stem, lotus leaf, the root of large-flowered skullcap, the coptis, golden cypress, Chinese prickly ash, Tuber Fleeceflower Root, giant knotweed, Rhizoma Picrorhizae, the bark of official magnolia, Pummelo Peel, Hemp Seed, Silktree Albizzia Bark, Flower of Silktree Albizzia, Radix Knoxiae, safflower, Semen Sesami Nigrum, chrysanthemum, stiff silkworm, balloonflower root, tangerine seed, turmeric, Yunnan Caulis Spatholobi, Flos Celosiae Cristatae, Root of Hairystalk Tinospora, Inula lineariifolia Turcz., Wild Buckwheat Rhizome, Longhairy Antenoron Herb, Japanese Honeysuckle, dalbergia wood, schizonepeta, semen allii tuberosi, Semen Cassiae, Flos Farfarae, the myrobalan, Semen Armeniacae Amarum, kuh-seng, Cortex Meliae, Semen Raphani, stone-like omphalia, Flos Campsis, Pyrola Herb, Radix Rhapontici seu Radix Echinopsis, play both sides of the street, lotus seeds, Stem of confederate-jasmine, aloe, reed rhizome, pointed at both ends, Shinyleaf Pricklyash Root, the capsule of weeping forsythia, glossy ganoderma, Folium Apocyni Veneti, Grosvenor Momordica, Semen Litchi, rough gentian, longan aril, Herba Erodii, Yerbadetajo Herb, Chinese ephedra, Simpleleaf Shrub Chastetree Fruit, Rhododendron dauricum, Oleum Rhododendri Daurici, Pale Butterflybush Flower, plum blossom, Chinese ephedra, the tuber of dwarf lilyturf, Fructus Hordei Germinatus, Rose, Root of Medicinal Changium, vervain, Virginia snakeroot, nux vomica, Semen Strychni Pulveratum, Lasiosphaera fenzlii, purslane, pawpaw, the banksia rose, the scouring rush, Root of Upright Ladybell, Glossy Privet Fruit, Great Burdock Achene, the root of bidentate achyranthes, rhizoma nelumbinis, cattail pollen, taraxacum, Loquat Leaf, eupatorium, Tumeric, fringed pink, the bark of ash, adder-wort, Gorgon fruit, notopterygium root, Slender Dutchmanspipe Root, Stem of Orientoine, rascal, Semen Celosiae, sweet wormwood, indigo naturalis, madder, the Semen Pharbitidis, the root of purple-flowered peucedanum, Rhizome of Obscured Homalomena, Semen Euphorbiae, bark of ash, Hedge Prinsepia Nut, Semen Myristicae, Herba Cistanches, Chinese cassia tree, genseng, the Ginseng Leaf, Rhizome of Grass leaf Sweelflag, cynomorium songaricum, mulberry leaf, Phytolacca acinosa, mulberry fruit, Fructus Cnidii, White Mulberry Root-bark, ramulus mori, ramulus mori, loranthus parasiticus, Wilsom Buckeye Seed, Spina Date Seed, blackberry lily, Styrax, Semen Astragali Complanati, the Rangooncreeper Fruit, the calyx and receptacle of a persimmon, fructus amomi, Radix Sophorae Tonkinensis, skunk bush, Chinese yam, Pseudobulbus Cremastrae Seu Pleiones, hawthorn, field thistle, rattletop, Cornu Bubali, Pulvis Cornus Bubali Concentratus, pyrrosia lingua, Sea-ear Shell, the stem of noble dendrobium, pomegranate rind, ginger, Vegetable Sponge of Luffa, pseudo-ginseng, rhizoma sparganic, Semen Lepidii (Semen Descurainiae), South Dodder Seed Chinese Dodder Seed, peach kernel, the stem pith of the rice-paper plant, santal, Snakegourd Root, Tabasheer, Rhizoma Arisaematis, rhizoma Gastrodiae, Root of Muskroot-like Semiaquilegia, Root of Heterophylly Faalsestarwort, Golden Larch Bark, Rhizome of Glabrous Greenbrier, Medcinal Evodia Fruit, Root of Sixpetal Clematis, Semen Vaccariae, Wu Chia Pee, shizandra berry, Turkey-galls, Concha Arcae, the root of three-nerved spicebush, dark plum, Rhizome of Decumbent Corydalis, teasel root, Inula Flower Herba Siegesbeckiae, Longstamen Onion Bulb, Spica Prunellae, the flower bud of lily magnolia, Root-bark of Chinese Silkvine, rhizoma cyperi, citron, elscholtiza, sweet fennel, thizoma curculiginis, Herba Agrimoniae, radix scrophulariae, Weathered Sodium Sulfate, Radix Panacis Quinquefolii, Crinis Carbonisatus, dragon's blood, Radix Cynanchi Paniculati, Herba Epimedii, Motherwort Herb, Ginkgo Leaf, Semen Coicis, Starwort Root, polygala root, lilac daphne, Semen Pruni, root tuber of aromatic turmeric, Herba Houttuyniae, oriental wormwood, kosam seeds, Flos Rosae Chinensis, radix polygonati officinalis, Yanhusuo, Thunberg Fritillary Bulb, Fructus Gleditsiae Abnormalis, perilla seed, aster, Philippine Violet Herb, Asian puccoon, umbellate pore furgus, Spine of Chinese Honeylocust, the wind-weed, the Herba Lycopi, rhizoma alismatis, nacre, the dried immature fruit of citron orange, cape jasmine.
Any form on the pharmaceutics be can make according to the root bark of tree peony effective constituent that the inventive method obtained, injection and oral preparations comprised.Wherein injection comprises injection liquid, drip liquid, powder injection; Oral preparations 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, paste, sublimed preparation, sprays, pill, disintegrating agent, orally disintegrating 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 root bark of tree peony finger printing below, water-soluble each the component ultraviolet spectrogram of the root bark of tree peony, water-soluble each the component mass spectrum of the root bark of tree peony, fat-soluble each the component ultraviolet spectrogram of the root bark of tree peony, fat-soluble each the component mass spectrum of the root bark of tree peony are intended to further specify the present invention, but the present invention are not construed as limiting.
Fig. 1 root bark of tree peony finger printing
Water-soluble each the component uv-spectrogram of Fig. 2 root bark of tree peony
Water-soluble each the component mass spectrum of Fig. 3 root bark of tree peony
Fat-soluble each the component uv-spectrogram of Fig. 4 root bark of tree peony
Fat-soluble each the component mass spectrum of Fig. 5 root bark of tree peony
Embodiment
Embodiment one
The extraction of the root bark of tree peony with separate
(1) extraction process: take by weighing 250g root bark of tree peony medicinal material,, add ethyl acetate and ethanol then its grinding and sieving, the heating extract extracting solution fr.1.In the dregs of a decoction, add ethanol, the heating extract extracting solution fr.2.In the dregs of a decoction, add entry at last, the heating extract extracting solution fr.3.
(2) separating technology: with extracting solution fr.1 concentrate 20g medicinal extract, use silica gel mixed sample, adopt normal phase silicagel column that it is separated, at first use sherwood oil and ethyl acetate as moving phase, get elutriant fr.4, change chloroform and methyl alcohol then as moving phase, get elutriant fr.5, concentrate the 5.9g sample, use methyl alcohol as moving phase at last, elutriant fr.6.With extracting solution fr.2 concentrate 51g medicinal extract, with sample on the dissolve with methanol, adopt the ODS-C18 post that it is separated, the methyl alcohol of at first using lower concentration gets elutriant fr.7 as moving phase, and the methyl alcohol that changes intermediate concentration then is as moving phase, get elutriant fr.8, concentrate the 2.2g sample, use pure methanol solution as moving phase at last, elutriant 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 semipreparative column (Lichrospher C18; 10.0mm * 250mm, 5 μ m), moving phase is water and acetonitrile, gradient elution is collected cut in the corresponding time period, obtains to have passed through further isolating each component.
Fr.5 separating resulting: get component fr.5 1.5g, dissolve with ethanol sample introduction with 10%, the component and the corresponding collection time that are obtained by the preparative liquid chromatography separation are as follows: collection in 4.0～7.6 minutes obtains component fr.51 0.11g, 7.6 collected and obtain component fr.52 0.09g in～9.7 minutes, 9.7 collected and obtain component fr.53 0.29g in～14.2 minutes, 14.2 collected and obtain component fr.540.11g in～18.4 minutes, 21.8 collected and obtain component fr.55 0.19g in～26.5 minutes, 21.8 collected and obtain component fr.56 0.09g in～26.5 minutes, 26.5 collected and obtain component fr.57 0.2g in～30.3 minutes, 30.3 collected and obtain component fr.58 0.08g in～35.5 minutes, collection in 35.5～45 minutes obtains component fr.59 0.05g.
Fr.8 separating resulting: get fr.8 2.5g, dissolve with methanol sample introduction with 20%, the component and the corresponding collection time that are obtained by the preparative liquid chromatography separation are as follows: collection in 4.0～7.7 minutes obtains component fr.81 0.04g, 7.7 collected and obtain component fr.82 0.10g in～10.7 minutes, 10.7 collected and obtain component fr.83 0.16g in～15.8 minutes, 15.8 collected and obtain component fr.840.26g in～18.8 minutes, 18.8 collected and obtain component fr.85 0.17g in～23.2 minutes, 23.2 collected and obtain component fr.86 0.21g in～27.4 minutes, 27.4 collected and obtain component fr.87 0.15g in～33.1 minutes, 33.1 collected and obtain component fr.88 0.12g in～40.2 minutes, 40.2 collected and obtain component fr.89 0.09g in～44.4 minutes, 44.4 collected and obtain component fr.810 0.11g in～48.5 minutes, 48.5 collected and obtain component fr.811 0.13g in～58 minutes, collected in 58～67 minutes and obtain component fr.812 0.04g, collection in 67～75 minutes obtains component fr.813 0.12g.
Embodiment two
The extraction of the root bark of tree peony with separate
(1) extraction process: take by weighing 500g root bark of tree peony medicinal material, with its grinding and sieving, adding 5～12 times of amount volume ratios then is 1: 1 ethyl acetate and ethanol, and reflux 0.5～3 hour is extracted 1～3 time, and merging filtrate gets extracting solution fr.1.Add 5～12 times of amount 70% ethanol in the dregs of a decoction, reflux 0.5～3 hour is extracted 1～3 time, and merging filtrate gets extracting solution fr.2.Add entry in the dregs of a decoction 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 41g medicinal extract, use silica gel mixed sample, adopt normal phase silicagel column that it is separated, at first with volume ratio be 50: 1 sherwood oil and ethyl acetate as moving phase, elutriant fr.4, change volume ratio then and be 10: 1 chloroform and methyl alcohol as moving phase, get elutriant fr.5, concentrate the 11.8g sample, use methyl alcohol as moving phase at last, elutriant fr.6.With extracting solution fr.2 concentrate 108g medicinal extract, with sample on 2～10% dissolve with methanol, adopt the ODS-C18 post that it is separated, at first use 2～10% methyl alcohol as moving phase, get elutriant fr.7, change 40～80% methyl alcohol then as moving phase, get elutriant fr.8, concentrate the 4.8g sample, use 100% methanol solution as moving phase at last, elutriant 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 semipreparative column (Lichrospher C18; 10.0mm * 250mm, 5 μ m), moving phase is water and acetonitrile, and gradient elution, flow velocity are 3ml/min, and column temperature is a room temperature, collects cut in the corresponding time period, obtains through further isolating each component.
Fr.5 separating resulting: get component fr.53g, dissolve with ethanol sample introduction with 10%, the component and the corresponding collection time that are obtained by the preparative liquid chromatography separation are as follows: collection in 4.0～7.6 minutes obtains component fr.51 0.29g, 7.6 collected and obtain component fr.52 0.21g in～9.7 minutes, 9.7 collected and obtain component fr.53 0.62g in～14.2 minutes, 14.2 collected and obtain component fr.540.23g in～18.4 minutes, 21.8 collected and obtain component fr.55 0.4g in～26.5 minutes, 21.8 collected and obtain component fr.56 0.17g in～26.5 minutes, 26.5 collected and obtain component fr.57 0.37g in～30.3 minutes, 30.3 collected and obtain component fr.58 0.17g in～35.5 minutes, collection in 35.5～45 minutes obtains component fr.59 0.12g.
Fr.8 separating resulting: get fr.85g, dissolve with methanol sample introduction with 20%, the component and the corresponding collection time that are obtained by the preparative liquid chromatography separation are as follows: collection in 4.0～7.7 minutes obtains component fr.81 0.09g, 7.7 collected and obtain component fr.82 0.22g in～10.7 minutes, 10.7 collected and obtain component fr.83 0.33g in～15.8 minutes, 15.8 collected and obtain component fr.840.53g in～18.8 minutes, 18.8 collected and obtain component fr.85 0.31g in～23.2 minutes, 23.2 collected and obtain component fr.86 0.42g in～27.4 minutes, 27.4 collected and obtain component fr.87 0.31g in～33.1 minutes, 33.1 collected and obtain component fr.88 0.31g in～40.2 minutes, 40.2 collected and obtain component fr.89 0.17g in～44.4 minutes, 44.4 collected and obtain component fr.810 0.22g in～48.5 minutes, 48.5 collected and obtain component fr.811 0.32g in～58 minutes, collected in 58～67 minutes and obtain component ff.812 0.1g, collection in 67～75 minutes obtains component fr.8130.28g.
Embodiment three
One. the extraction of the root bark of tree peony with separate
Accurately take by weighing root bark of tree peony medicinal material 250g, with its grinding and sieving, adding 8 times of amount volume ratios then is 1: 1 ethyl acetate and ethanol, and reflux 1 hour is extracted 2 times, and merging filtrate gets extracting solution fr.1.Add 8 times of amount 70% ethanol in the dregs of a decoction, reflux 1 hour is extracted 2 times, and merging filtrate gets extracting solution fr.2.Add entry in the dregs of a decoction at last, reflux 1 hour is extracted 2 times, and merging filtrate gets extracting solution fr.3.
With extracting solution fr.1 concentrate 22g medicinal extract, use silica gel mixed sample, adopt normal phase silicagel column that it is separated, at first with volume ratio be 50: 1 sherwood oil and ethyl acetate as moving phase, elutriant fr.4, change volume ratio then and be 10: 1 chloroform and methyl alcohol as moving phase, get elutriant fr.5, concentrate the 6.2g sample, use methyl alcohol as moving phase at last, elutriant fr.6.With extracting solution fr.2 concentrate 55g medicinal extract, with sample on 5% dissolve with methanol, adopt the ODS-C18 post that it is separated, at first use 5% methyl alcohol as moving phase, get elutriant fr.7, change 60% methyl alcohol then as moving phase, get elutriant fr.8, concentrate the 2.6g sample, use 100% methanol solution as moving phase at last, elutriant fr.9.
Continue to separate fr.5 and fr.8 with preparative liquid chromatography.
The fr.5 separation condition: chromatographic column is semipreparative column (the Lichrospher C18 of Chinese nation; 10.0mm * 250mm, 5 μ m), moving phase is water (A) and acetonitrile (B), and flow velocity is 3ml/min, and column temperature is a room temperature.The gradient elution program is as follows:
In the time of 0 minute, mobile phase A is that 80% the aqueous solution, Mobile phase B are 20% acetonitrile solution;
In the time of 40 minutes, mobile phase A is that 20% the aqueous solution, Mobile phase B are 80% acetonitrile solution;
In the time of 50 minutes, mobile phase A is that 5% the aqueous solution, Mobile phase B are 95% acetonitrile solution.
Fr.5 separating resulting: get component fr.5 1.4g, dissolve with ethanol sample introduction with 10%, the component and the corresponding collection time that are obtained by the preparative liquid chromatography separation are as follows: collection in 4.0～7.6 minutes obtains component fr.51 0.16g, 7.6 collected and obtain component fr.52 0.11g in～9.7 minutes, 9.7 collected and obtain component fr.53 0.32g in～14.2 minutes, 14.2 collected and obtain component fr.540.13g in～18.4 minutes, 21.8 collected and obtain component fr.55 0.21g in～26.5 minutes, 21.8 collected and obtain component fr.56 0.10g in～26.5 minutes, 26.5 collected and obtain component fr.57 0.2g in～30.3 minutes, 30.3 collected and obtain component fr.58 0.09g in～35.5 minutes, collection in 35.5～45 minutes obtains component fr.59 0.07g.
The fr.8 separation condition: chromatographic column is semipreparative column (the Lichrospher C18 of Chinese nation; 10.0mm * 250mm, 5 μ m), moving phase is water (A) and acetonitrile (B), and flow velocity is 3ml/min, and column temperature is a room temperature.The gradient elution program is as follows:
In the time of 0 minute, mobile phase A is that 90% the aqueous solution, Mobile phase B are 10% acetonitrile solution;
In the time of 8 minutes, mobile phase A is that 81% the aqueous solution, Mobile phase B are 19% acetonitrile solution;
In the time of 60 minutes, mobile phase A is that 65% the aqueous solution, Mobile phase B are 35% acetonitrile solution;
In the time of 70 minutes, mobile phase A is that 95% the aqueous solution, Mobile phase B are 5% acetonitrile solution.
Fr.8 separating resulting: get fr.8 2.6g, dissolve with methanol sample introduction with 20%, the component and the corresponding collection time that are obtained by the preparative liquid chromatography separation are as follows: collection in 4.0～7.7 minutes obtains component fr.81 0.05g, 7.7 collected and obtain component fr.82 0.12g in～10.7 minutes, 10.7 collected and obtain component fr.83 0.18g in～15.8 minutes, 15.8 collected and obtain component fr.840.28g in～18.8 minutes, 18.8 collected and obtain component fr.85 0.18g in～23.2 minutes, 23.2 collected and obtain component fr.86 0.23g in～27.4 minutes, 27.4 collected and obtain component fr.87 0.18g in～33.1 minutes, 33.1 collected and obtain component fr.88 0.16g in～40.2 minutes, 40.2 collected and obtain component fr.89 0.1g in～44.4 minutes, 44.4 collected and obtain component fr.810 0.12g in～48.5 minutes, 48.5 collected and obtain component fr.811 0.17g in～58 minutes, collected in 58～67 minutes and obtain component fr.812 0.06g, collection in 67～75 minutes obtains component fr.8130.15g.
Two. analyze
2.1 instrument and reagent
Agilent 1100 high performance liquid chromatography-GC-MS (U.S. Agilent company) fit over line vacuum de-aerator, quarternary low pressure pump, automatic sampler, column oven, DAD detector, 1946D electron spray(ES) level Four bar mass detector, Chemstation chromatographic working 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 (plum Teller-Tuo benefit Instr Ltd.).
Acetonitrile is chromatographically pure reagent (Merck), and water is WAHAHA pure water (Hangzhou WAHAHA group).
2.2 analytical procedure
Sample source: tree-peony root bark component fr.51 1.75mg, fr.52 1.76mg, fr.53 1.78mg, fr.54 1.74mg, fr.551.69mg, fr.56 1.72mg, fr.57 1.84mg, fr.58 1.88mg, fr.59 1.22mg.
Specimen preparation: sample is used the 1ml dissolve with ethanol respectively, and is ultrasonic, and centrifugal (10000r/min) is standby.
Chromatographic condition: chromatographic column Agilent Zorbax SB-C18 post (4.6mm * 150mm, 5 μ m); Adopt gradient elution, mobile phase A is 0.2% glacial acetic acid aqueous solution mutually, and Mobile phase B is mutually for containing the acetonitrile solution of 0.2% Glacial acetic acid; The gradient elution program is as follows: in the time of 0 minute, mobile phase A is that 90% 0.2% glacial acetic acid aqueous solution, Mobile phase B are 10% 0.2% Glacial acetic acid acetonitrile solution; In the time of 10 minutes, mobile phase A is that 50% 0.2% glacial acetic acid aqueous solution, Mobile phase B are 50% 0.2% Glacial acetic acid acetonitrile solution; In the time of 30 minutes, mobile phase A is that 5% 0.2% glacial acetic acid aqueous solution, Mobile phase B are 95% 0.2% Glacial acetic acid acetonitrile solution; In the time of 35 minutes, mobile phase A is that 5% 0.2% glacial acetic acid aqueous solution, Mobile phase B are 95% 0.2% Glacial acetic acid acetonitrile solution.Flow velocity 0.5mL/min; It is long to detect the wavelength all-wave; 30 ℃ of column temperatures; Sample size is 5 μ l.
Mass spectrum condition: negative ions scan pattern, sweep limit 100～2000; Dry gas (N 2Stream speed 10L/min, 350 ℃ of atomization temperatures, capillary voltage 3500V, cracking voltage 100V.
Sample source: tree-peony root bark component fr.81 1.88mg, fr.82 1.83mg, fr.83 1.90mg, fr.84 1.86mg, fr.851.88mg, fr.86 1.78mg, fr.87 1.85mg, fr.88 1.88mg, fr.89 1.81mg, ff.810 1.75mg, fr.8111.79mg, fr.812 1.94mg.
Specimen preparation: sample is with the dissolving of 1ml 50% methanol-water, and is ultrasonic, and centrifugal (10000r/min) is standby.
Chromatographic condition: chromatographic column Agilent Zorbax SB-C 18Post (4.6mm * 150mm, 5 μ m); Adopt gradient elution, mobile phase A is 0.2% glacial acetic acid aqueous solution mutually, and Mobile phase B is mutually for containing the acetonitrile solution of 0.2% Glacial acetic acid; The gradient elution program is as follows: in the time of 0 minute, mobile phase A is that 90% 0.2% glacial acetic acid aqueous solution, Mobile phase B are 10% 0.2% Glacial acetic acid acetonitrile solution; In the time of 20 minutes, mobile phase A is that 70% 0.2% glacial acetic acid aqueous solution, Mobile phase B are 30% 0.2% Glacial acetic acid acetonitrile solution; In the time of 30 minutes, mobile phase A is that 50% 0.2% glacial acetic acid aqueous solution, Mobile phase B are 50% 0.2% Glacial acetic acid acetonitrile solution; In the time of 35 minutes, mobile phase A is that 50% 0.2% glacial acetic acid aqueous solution, Mobile phase B are 50% 0.2% Glacial acetic acid acetonitrile solution.Flow velocity 0.5mL/min; It is long to detect the wavelength all-wave; 30 ℃ of column temperatures; Sample size is 5 μ l.
Mass spectrum condition: negative ions scan pattern, sweep limit 100～2000; Dry gas (N 2Stream speed 10L/min, 350 ℃ of atomization temperatures, capillary voltage 3500V, cracking voltage 100V.
2.3 analytical results
Analytical results: Fig. 2 is water-soluble each the component uv-spectrogram of the root bark of tree peony, and Fig. 3 is water-soluble each the component mass spectrum of the root bark of tree peony, and Fig. 4 is fat-soluble each the component uv-spectrogram of the root bark of tree peony, and Fig. 5 is fat-soluble each the component mass spectrum of the root bark of tree peony.
The qualification result of compound in the tree-peony root bark component [1-12]See Table 2.
Table 2. tree-peony root bark component compound identification result
Tree-peony root bark component Chromatographic retention (min) [M-H]- Ultraviolet maximum absorption wavelength (nm) Compound
fr.51 5.01 169 215，280 Gallic Acid (gallic acid) [1]
9.5 479 230 Paeoniflorin (peoniflorin) [1]
fr.52 5.01 169 215，280 Gallic Acid (gallic acid) [1]
9.5 479 230 Paeoniflorin (peoniflorin) [1]
fr.53 9.5 479 230 Paeoniflorin (peoniflorin) [1]
fr.54 9.5 479 230 Paeoniflorin (peoniflorin) [1]
10.8 631 215，280 Galloyl-Paeoniflorin (gallic acid peoniflorin) [2]
11.6 615 215，280 Mudanpioside h (paeonoside h) [3]
fr.55 12.0 599 230 Mudanpioside c (paeonoside c) [4]
12.3 599 230 Benzoyloxypaeoniflorin (Benzoyloxypaeoniflorin) [4] [5]
fr.56 13.5 583 230 Benzoylpaeoniflorin (benzoylpaeoniflorin) [1] [4] [6] [7] [8]
fr.81 4.4 493 215，280 Unknown
4.6 463 215，280 Mudanoside b (paeonoside b) [4]
fr.82 4.3 493 215，280 Paeonidanin[9]
4.4 493 215，280 Unknown
4.6 463 215，275 Mudanoside b (paeonoside b) [4]
fr.83 9.8 494 254 Oxypaeoniflorin (Hydroxy peoniflorin) [4] [10] [11]
fr.84 9.8 494 254 Oxypaeoniflorin (Hydroxy peoniflorin) [4] [10] [11]
fr.85 14.8 479 230 Paeoniflorin (peoniflorin) [1]
fr.86 15.7 787 215，280 1,2,3,6-tretra-O-gaLloly-β-D-glucose (four gallic acid glucose) [12]
16.3 787 215，280 1,2,3,6-tretra-O-gaLloly-β-D-glucose (four gallic acid glucose) [12]
17.1 611 215，280 Suffruticoside A or Suffruticoside B or Suffruticoside C or Suffruticoside D[2]
fr.87 18.3 939 215，280 1,2,3,4,6-Pentagalloylglucose (five gallic acid peoniflorins) [12]
fr.88 20.1 1091 215，280 Digalloyl-1,2,3,4-tetra-O-galloyl--D-glucopyranose six gallic acid peoniflorins [12]
21.0 1091 215，280 Digalloyl-1,2,3,4-tetra-O-galloyl--D-glucopyranose (six gallic acid peoniflorins) [12]
fr.89 18.3 939 215，275 1,2,3,4,6-Pentagalloylglucose (five gallic acid peoniflorins) [12]
21.0 1091 215，280 Digalloyl-1,2,3,4-tetra-O-galloyl--D-glucopyranose (six gallic acid peoniflorins) [12]
fr.810 25.2 599 230 Mudanpioside c (paeonoside c) [4]
26.1 599 230 Benzoyloxypaeoniflorin (Benzoyloxypaeoniflorin) [4] [5]
fr.811 26.1 599 230 Benzoyloxypaeoniflorin (Benzoyloxypaeoniflorin) [4] [5]
fr.812 29.7 583 230 Benzoylpaeoniflorin (benzoylpaeoniflorin) [1] [4] [6] [7] [8]
Reference:
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[6]Kosato?H.，Arichi?S.，Kubo?M.，Matsuda?H.，Kimura?Y.，Kitagawa?I.，Yoshikawa?M.，Wakanyaku?Shinpojium，1984，14，86。
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Three, pharmacological evaluation
3.1 pharmacological model: neonatal rat myocardial cell hypoxic-ischemic model
After former generation myocardial cell cultivated birth SD suckling mouse execution in 1～3 day, it was dirty to core, and is cut into the fragment about 1mm3 after PBS liquid cleans.With 0.125% trypsin Sigma, USA)+0.05% (Gibco is USA) in 37 ℃ of digestion 3～4 times for collagenase II.The differential adherent method was separated into fibrocyte after 1.5 hours, and cell suspension is transferred to (about 4 * 105 cells in every hole) in 48 orifice plates.(Gibco, (Falcon, USA) cell of cultivating 3-6 days supplies medicine efficacy screening USA) to add 10% foetal calf serum 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 nutrient solution.Place 95%N 2And 5%CO 2Anoxic 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 tree-peony root bark component that obtains and is made into 10 with sugar-free Hanks liquid -4G/mL test liquid, fat-soluble component 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 nutrient solution is controlled at 10 -5G/mL.
Serum lactic dehydrogenase content can be represented the cell injury degree in the serum lactic dehydrogenase assay cell culture fluid.The serum lactic dehydrogenase content that bleeds in the supernatant liquor 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.Measuring method is: get 30 μ l cell conditioned medium liquid, add 50 μ l matrix damping fluids and 10 μ l lactic dehydrogenase enzyme cofactors, 37 ℃ vibrated 15 minutes, added 50 μ l 2,4 dinitrophenyl hydrazines again, and 37 ℃ vibrated 15 minutes.Parallel blank.Extract reaction solution 50 μ l and add 200 μ l 0.4mol/L sodium hydroxide solutions, leave standstill 3～4 minutes after, measure photometric quantity with microplate reader in 450nm.
Drug effect result
All data are represented with the mean value variance.Adopt monolateral variance analysis and T check carrying out statistical analysis.Compare with model group, P＜0.05 is thought significantly effectively.Medicine efficacy screening the results are shown in Table 3 and table 4.According to myocardial cell's serum lactic dehydrogenase (LDH) The selection result, root bark of tree peony fr.51, fr.52, fr.53, fr.54, fr.56, fr.57, fr.58, fr.81, fr.83, fr.810 has the highly significant effect to reducing LDH release.
Table 3. myocardial cell The selection result
Figure G05112275820050818D000151
Table 4. myocardial cell screens structure
Figure G05112275820050818D000161
3.2 huve cell anoxic reoxygenation model
The healthy newborn umbilical cord is got in former generation huve cell cultivation, cleans the interior residual bloodstain of vein blood vessel with 30～50mlHanks 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 liquor is removed in suction, with the abundant dissolution precipitation of M199 nutrient solution (contain 10% foetal calf serum, 100U/ml is two anti-, 0.15mg/ml Heparin, 10 μ M VC).The gained cell suspension is sub-packed in 5cm 2Culturing bottle places in the incubator and cultivates.Changed in second day and adopt the M199 nutrient solution that contains 30 μ g/ml ECGS, changed a nutrient solution and be paved with the bottom surface in per afterwards three days until cell.Used cell is former generation endotheliocyte.The culturing 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 nutrient solution, adding pastille does not have phenol red M199 nutrient solution, and every hole total amount is 100 μ l.Place 95%N 2And 5%CO 2Anoxic 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 tree-peony root bark component that obtains and is made into 10 with sugar-free Hanks liquid -4G/mL test liquid, fat-soluble component 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 nutrient solution is controlled at 10 -5G/mL.
Serum lactic dehydrogenase content can be represented the cell injury degree in the serum lactic dehydrogenase assay cell culture fluid.The serum lactic dehydrogenase content that bleeds in the supernatant liquor 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.Measuring method is: get 30 μ l cell conditioned medium liquid, add 50 μ l matrix damping fluids and 10 μ l lactic dehydrogenase enzyme cofactors, 37 ℃ vibrated 15 minutes, added 50 μ l 2,4 dinitrophenyl hydrazines again, and 37 ℃ vibrated 15 minutes.Parallel blank.Extract reaction solution 50 μ l and add 200 μ l 0.4mol/L sodium hydroxide solutions, leave standstill 3～4 minutes after, measure photometric quantity with microplate reader in 450nm.
The NO assay adopts Greiss reagent method to measure, and gets 90 μ l cell conditioned medium liquid, adds equivalent Greiss reagent, leaves standstill under the room temperature 10 minutes, measures absorbance with microplate reader in 550nm.
Drug effect all data is as a result represented with the mean value variance.Adopt monolateral variance analysis and T check carrying out statistical analysis.Compare with model group, P＜0.05 is thought significantly effectively.Medicine efficacy screening the results are shown in Table 5 and table 6.According to endotheliocyte NO The selection result, root bark of tree peony fr.811 has the highly significant effect to improving NO release, and fr.56 has unusual effect.According to endotheliocyte LDH (serum lactic dehydrogenase) The selection result, root bark of tree peony fr.58 has the highly significant effect to reducing LDH release, and fr.52, fr.53, fr.54, fr.56, fr.57 have unusual effect.
Table 5. endotheliocyte NO The selection result
Figure G05112275820050818D000171
Table 6. endotheliocyte LDH The selection result
Figure G05112275820050818D000172
Above test-results explanation, the Chinese medicinal materials effective constituent that adopts Chinese medicinal materials stdn extracting method of the present invention to obtain can be as the preparation medicine.

Claims (3)

1. root bark of tree peony extraction, separation method comprise the steps:
(1) extraction process: take by weighing root bark of tree peony medicinal material, with its grinding and sieving, adding 8 times of amount volume ratios then is 1: 1 ethyl acetate and ethanol, and reflux 1 hour is extracted 2 times, and merging filtrate gets extracting solution fr.1; Add 8 times of amount 70% ethanol in the dregs of a decoction, reflux 1 hour is extracted 2 times, and merging filtrate gets extracting solution fr.2; Add entry in the dregs of a decoction 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 medicinal extract with extracting solution, use silica gel mixed sample, adopt normal phase silicagel column that it is separated, be that 50: 1 sherwood oil and ethyl acetate are as moving phase at first with volume ratio, elutriant fr.4, change volume ratio then and be 10: 1 chloroform and methyl alcohol as moving phase, elutriant fr.5, use methyl alcohol as moving phase at last, get elutriant fr.6; Fr.2 is condensed into medicinal extract with extracting solution, with sample on 5% dissolve with methanol, adopt the ODS-C18 post that it is separated, at first use 5% methyl alcohol as moving phase, get elutriant fr.7, change 60% methyl alcohol then, get elutriant fr.8 as moving phase, use 100% methanol solution as moving phase at last, get elutriant fr.9;
(3) preparation separating technology: continue to separate fr.5 and fr.8 with preparative liquid chromatography;
The fr.5 separation condition: chromatographic column is the semipreparative column Lichrospher C18 of Chinese nation; 10.0mm * 250mm, 5 μ m, mobile phase A is that water and B are acetonitrile, and flow velocity is 3ml/min, and column temperature is a room temperature, and the gradient elution program is as follows:
In the time of 0 minute, mobile phase A is that 80% the aqueous solution, Mobile phase B are 20% acetonitrile solution;
In the time of 40 minutes, mobile phase A is that 20% the aqueous solution, Mobile phase B are 80% acetonitrile solution;
In the time of 50 minutes, mobile phase A is that 5% the aqueous solution, Mobile phase B are 95% acetonitrile solution;
The fr.5 separating resulting: the component and the corresponding collection time that are obtained by the preparative liquid chromatography separation are as follows:
4.0 collected and obtain component fr.51 in～7.6 minutes, 7.6 collected and obtain component fr.52 in～9.7 minutes, 9.7 collected and obtain component fr.53 in～14.2 minutes, 14.2 collected and obtain component fr.54 in～18.4 minutes, 21.8 collected and obtain component fr.55 in～26.5 minutes, collection in 21.8～26.5 minutes obtains component fr.56, collection in 26.5～30.3 minutes obtains component fr.57,30.3 collected and obtain component fr.58 in～35.5 minutes, collection in 35.5～45 minutes obtains component fr.59;
The fr.8 separation condition: chromatographic column is the semipreparative column Lichrospher C18 of Chinese nation; 10.0mm * 250mm, 5 μ m, mobile phase A is that water and B are acetonitrile, and flow velocity is 3ml/min, and column temperature is a room temperature, and the gradient elution program is as follows:
In the time of 0 minute, mobile phase A is that 90% the aqueous solution, Mobile phase B are 10% acetonitrile solution;
In the time of 8 minutes, mobile phase A is that 81% the aqueous solution, Mobile phase B are 19% acetonitrile solution;
In the time of 60 minutes, mobile phase A is that 65% the aqueous solution, Mobile phase B are 35% acetonitrile solution;
In the time of 70 minutes, mobile phase A is that 95% the aqueous solution, Mobile phase B are 5% acetonitrile solution;
The fr.8 separating resulting: the component and the corresponding collection time that are obtained by the preparative liquid chromatography separation are as follows: collection in 4.0～7.7 minutes obtains component fr.81,7.7 collected and obtain component fr.82 in～10.7 minutes, 10.7 collected and obtain component fr.83 in～15.8 minutes, 15.8 collected and obtain component fr.84 in～18.8 minutes, 18.8 collected and obtain component fr.85 in～23.2 minutes, 23.2 collected and obtain component fr.86 in～27.4 minutes, 27.4 collected and obtain component fr.87 in～33.1 minutes, 33.1 collected and obtain component fr.88 in～40.2 minutes, 40.2 collected and obtain component fr.89 in～44.4 minutes, 44.4 collected and obtain component fr.810 in～48.5 minutes, 48.5 collected and obtain component fr.811 in～58 minutes, collected in 58～67 minutes and obtain component fr.812, collection in 67～75 minutes obtains component fr.813.
2. the root bark of tree peony as claimed in claim 1 extracts, separation method, it is characterized in that:
Main compound is a peoniflorin among the component fr.51, gallic acid;
Main compound is a peoniflorin among the component fr.52, gallic acid;
Main compound is a peoniflorin among the component fr.53;
Main compound is the gallic acid peoniflorin among the component fr.54, paeonoside h, peoniflorin;
Main compound is paeonoside c among the component fr.55, Benzoyloxypaeoniflorin;
Main compound is a benzoylpaeoniflorin among the component fr.56.
3. the root bark of tree peony as claimed in claim 1 extracts, separation method, it is characterized in that:
Main compound is paeonoside b among the component fr.81;
Main compound is paeonoside b among the component fr.82;
Main compound is a Hydroxy peoniflorin among the component fr.83;
Main compound is a Hydroxy peoniflorin among the component fr.84;
Main compound is a peoniflorin among the component fr.85;
Main compound is four gallic acid glucose among the component fr.86, Nutgalls acyloxylation peoniflorin a or Nutgalls acyloxylation peoniflorin b or Nutgalls acyloxylation peoniflorin c or Nutgalls acyloxylation peoniflorin d;
Main compound is the gallic acid peoniflorin among the component fr.87;
Main compound is six gallic acid peoniflorins among the component fr.88;
Main compound is five gallic acid peoniflorins among the component fr.89, six gallic acid peoniflorins;
Main compound is paeonoside c among the component fr.810, Benzoyloxypaeoniflorin;
Main compound is a Benzoyloxypaeoniflorin among the component fr.811;
Main compound is a benzoylpaeoniflorin among the component fr.812.
CN2005100122758A 2005-07-29 2005-07-29 Method for extraction and separation of moutan bark Expired - Fee Related CN1903259B (en)

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