CN102190693B - Method for preparing hyperin from Dogbane leaves - Google Patents
Method for preparing hyperin from Dogbane leaves Download PDFInfo
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- CN102190693B CN102190693B CN 201110075635 CN201110075635A CN102190693B CN 102190693 B CN102190693 B CN 102190693B CN 201110075635 CN201110075635 CN 201110075635 CN 201110075635 A CN201110075635 A CN 201110075635A CN 102190693 B CN102190693 B CN 102190693B
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Abstract
The invention discloses a method for preparing hyperin from Dogbane leaves, which comprises the following steps: firstly. determining the optimum extraction temperature, a separation material and the concentration of an eluting agent; secondly, regulating the pH value, and extracting with a solvent having high hyperin dissolving and extracting power, thus obtaining a liquid extract; thirdly, heating at 80-85 DEG C under reflux of ethanol or methanol, and extracting; and finally, standing and crystallizing at a preferable cooling temperature, thus effectively separating the hyperin and isoquercitrin which are two flavonoid glycoside components having similar properties. In the invention, the hyperin is further purified by preparing a liquid phase; and a moving phase is examined and determined by methodology, and is subjected to targeted and efficient separation and purification based on the absorption wavelength of the hyperin, thus obtaining the hyperin having purity up to 90-99%. Thehyperin conforms to the requirements for Class A active pharmaceutical ingredients.
Description
Technical field
The present invention relates to a kind of method that from herbal medicine, prepares bulk drug, be specifically related to a kind of from Folium Apocyni Veneti extraction separation obtain the preparation method of high purity Quercetin 3-galactoside.
Background technology
Folium Apocyni Veneti is the dry leave of apocynaceae plant kendir, mainly contains compositions such as flavonoid, tannin, low molecular organic acids class, long chain fatty acid ester, alcohols, steroidal class in the Folium Apocyni Veneti.Compendium of Material Medica put down in writing its have according to one's conscience throb with fear, only dizzy, disappear and breathe heavily the effect of cough-relieving, cardiac stimulant diuresis.Modern study shows, multiple pharmacological effect such as that Folium Apocyni Veneti has is hypotensive, reducing blood-fat, antidepressant, calmness, diuresis.
Quercetin 3-galactoside (Quercetin-3-O-β-D-galactopyranoside) and isoquercitrin etc. are flavonoid glycoside composition in the Folium Apocyni Veneti, and modern pharmacological research shows that Quercetin 3-galactoside has analgesia, protection cardio-cerebrovascular and antidepressant isoreactivity.The method of separating and purifying flavone is more in the prior art, mainly contain chromatography, but all there are very big difference in the flavones kind that contains in the different plants and structure, thereby chemical property there are differences, apply mechanically general extracting and purifying method in extracting purge process, the purity of the flavones that obtains is not high, as reaching very high purity, its separation efficiency is lower, and cost is higher; The present method of extracting the Quercetin 3-galactoside of purification of high-purity at Folium Apocyni Veneti is reported seldom, and complex chemical composition in the Folium Apocyni Veneti, especially the Quercetin 3-galactoside that contains in the Folium Apocyni Veneti is close with isoquercitrin character, very difficult in the extraction separation process, and the distinctive chemical structure type of Quercetin 3-galactoside is because its activity has the value of utilization by force very much, therefore in order to take full advantage of the Folium Apocyni Veneti resource, meet the need of market, the necessary cover of developing extracts purification efficiency height, cost is low and Quercetin 3-galactoside purity is high preparation method.
Summary of the invention:
Goal of the invention: the objective of the invention is provides the preparation method of Quercetin 3-galactoside in a kind of Folium Apocyni Veneti in order to solve the deficiencies in the prior art.
Technical scheme: in order to realize above purpose, the preparation method of Quercetin 3-galactoside in the Folium Apocyni Veneti provided by the invention specifically may further comprise the steps:
(1) get Folium Apocyni Veneti medicinal material selection, clean, put into extractor after the drying, add the hydrophilic solvent of 5 ~ 10 times of amounts, reflux 1 ~ 3 time is filtered, united extraction liquid, concentrates, and gets the Folium Apocyni Veneti extracting solution, and is standby;
(2) get Folium Apocyni Veneti extracting solution that step (1) obtains and add ethanol and make concentration of ethanol reach 75% ~ 85%, leave standstill, precipitate, leach solution, concentrate, the precipitation solution that gets the Folium Apocyni Veneti extracting solution is stand-by;
(3) get macroporous resin column on the Folium Apocyni Veneti precipitation solution that step (2) obtains, water and concentration are 10% ethanol elution respectively earlier, be 50% ~ 70% ethanol elution again with concentration, the thin-layer chromatography inspection is known, is collected 50% ~ 70% ethanol eluate, concentrates, obtain the crude extract of Quercetin 3-galactoside in the Folium Apocyni Veneti, standby;
(4) get Quercetin 3-galactoside crude extract that step (3) obtains with ethyl acetate or n-butanol extraction, get extraction liquid, concentrate and obtain extract, add hydrophilic solvent then, heating and refluxing extraction 3 ~ 5 times, each 1 ~ 2 hour, filtered while hot, filtrate decompression concentrates, 4 ℃ of refrigeration crystallizations, with hydrophilic solvent washing, drying, namely get purity and be 90% ~ 96% Quercetin 3-galactoside.
The preparation method of Quercetin 3-galactoside in the above-described Folium Apocyni Veneti; the temperature of step (1) and the described heating and refluxing extraction of step (4) is 80 ~ 85 ℃; this operation steps continues to reflux 2 hours with 80 ~ 85 ℃ in the heating and refluxing extraction process; can guarantee that the extraction of flavonoid glycoside compound Quercetin 3-galactoside can not destroy the active structure of Quercetin 3-galactoside more completely simultaneously; this shows through constituent structure research and pharmacologically active clearly: the structure of Quercetin 3-galactoside can change under 110 ℃ of left and right sides high temperature; look into youngster's ketoside as being become by original flavonoid glycoside; thereby active the reduction, so the present invention can guarantee the Quercetin 3-galactoside stability of structure with regard to the temperature that strict control is extracted when extraction and purifying Quercetin 3-galactoside.And step (1) and the described hydrophilic solvent of step (4) are the ethanol of methyl alcohol or concentration 10 ~ 100%.
As preferred version, the Quercetin 3-galactoside extracting solution adds behind the ethanol of high density that concentration of ethanol is 80% in the Quercetin 3-galactoside extracting solution in the step (2), the purpose of this step operation is in order to remove macromolecular cpd such as the compositions such as tannin, polysaccharide in the Quercetin 3-galactoside extracting solution, be convenient to the purifying process of back, can improve purification efficiency greatly.
Above-described preparation method, as preferred version, macroporous adsorbent resin described in the step (3) is nonpolar macroporous adsorption resin, as more excellent scheme, described nonpolar macroporous adsorption resin adopts D101, AB-8 or HPD100 type macroporous adsorbent resin, the present invention is by the macroporous resin of orthogonal experiment screening opposed polarity different model, different concns (10%, 20%, 40%, 50%, 60%, 70%, 80%) ethanol elution agent, content with Quercetin 3-galactoside is index, experimental result shows with nonpolar macroporous adsorption resin D101, AB-8 or HPD100 type macroporous adsorbent resin impurity-eliminating effect are relatively good, and experimental result shows that concentration is that 10% ethanol can the big slightly impurity of the most of polarity of wash-out, but a little less than the elutive power to the Quercetin 3-galactoside composition, and that concentration is 50% ~ 70% ethanol is best to the elutive power of Quercetin 3-galactoside, substantially can wash-out all Quercetin 3-galactoside compositions of absorption, but the increase along with alcohol concn, though can go out Quercetin 3-galactoside by wash-out, but also can elute some other impurity such as chlorophyll together simultaneously, make the purity drop of Quercetin 3-galactoside, so the sorbing material that the present invention determines the best through the orthogonal experiment The selection result is D101, AB-8 or HPD100 type macroporous adsorbent resin and eluent are that concentration is 50% ~ 70% ethanol; And step (3) can wash away the big water-soluble impurity of most of polarity with the pure water rinsing macroporous resin earlier, is conducive to follow-up purifying.The present invention with 50% ~ 70% ethanol elution flavones ingredient the time with thin-layer chromatography as inspection knowledge means, used thin layer plate is the polymeric amide thin layer plate, developping agent is that ethyl acetate and the acetone of 6:4 is formed by volume ratio, developer is 10% aluminum chloride, and under the 365nm ultraviolet lamp, inspect, merge stream part of containing the Quercetin 3-galactoside composition, the stream part of removing other composition, therefore the purpose of separating is strong.
Above-described preparation method, as preferred version, step (4) is regulated the pH value to 3 of Quercetin 3-galactoside crude extract solution earlier with hydrochloric acid before with ethyl acetate or n-butanol extraction, the Quercetin 3-galactoside compound is the polyphenol hydroxyl flavonoid glycoside compound, the present invention is through the screening of different pH values, when can making targetedly with the Quercetin 3-galactoside sedimentation of 4 phenolic hydroxyl groups, the pH value to 3 of transferring Quercetin 3-galactoside crude extract solution with acid assembles, and then with solvent purpose extraction is arranged, make Quercetin 3-galactoside commentaries on classics rapidly and efficiently be dissolved in extraction solvent, can improve extraction efficiency more than 3 times, but acidity again can not be too strong, because acidity is too strong, the easy hydrolysis of the glycosidic bond of Quercetin 3-galactoside flavonoid glycoside compound becomes aglycon and semi-lactosi, and therefore, the present invention detects by a large amount of experiment screenings and in conjunction with thin-layer chromatography and high performance liquid chromatography, determine that best pH value is 3, under this pH value condition, be dissolved in extraction solvent thereby both can impel Quercetin 3-galactoside to assemble quick commentaries on classics of sedimentation, and the glycosidic bond of Quercetin 3-galactoside can hydrolysis.
Above-described preparation method, the present invention investigates different solvents to Quercetin 3-galactoside flavonoid glycoside composition extracting power, The selection result shows that the solubleness of Quercetin 3-galactoside constituents in ethyl acetate or propyl carbinol is bigger, therefore as preferred version, get behind the Quercetin 3-galactoside crude extract that step (4) obtains again with ethyl acetate or n-butanol extraction, can effectively extract Quercetin 3-galactoside flavonoid glycoside compound in the Quercetin 3-galactoside crude extract, thereby be separated with other impurity, further improve the purity of Quercetin 3-galactoside.
The preparation method of Quercetin 3-galactoside in the above-described Folium Apocyni Veneti, as preferred version, ethyl acetate or n-butanol extraction concentrate and obtain extract, add ethanol or methyl alcohol then, 80 ~ 85 ℃ of heating and refluxing extraction 3 ~ 5 times, each 1 ~ 2 hour, filtered while hot, filtrate decompression concentrates, 4 ℃ of refrigeration crystallizations, with ethanol or methanol wash, drying gets final product to such an extent that purity is 90% ~ 96% Quercetin 3-galactoside.The present invention on the basis of ethyl acetate or n-butyl alcohol extract according to structure and the chemical property of Quercetin 3-galactoside, adopt ethanol or methyl alcohol refluxing extraction under 80 ~ 85 ℃ of suitable temps can further Quercetin 3-galactoside fully be extracted for 3 ~ 5 times again, and then in preferred 4 ℃ of coolings down, Quercetin 3-galactoside can well crystallization, thereby can the very effective Quercetin 3-galactoside that character is close and two kinds of flavonoid glycoside compositions of isoquercitrin separately, thereby can obtain the Quercetin 3-galactoside of purity 90% ~ 96%.
The preparation method of Quercetin 3-galactoside in the another kind of Folium Apocyni Veneti provided by the invention is characterized in that may further comprise the steps:
(1) get Folium Apocyni Veneti medicinal material selection, clean, put into extractor after the drying, add the hydrophilic solvent of 5 ~ 10 times of amounts, reflux 1 ~ 3 time is filtered, united extraction liquid, concentrates, and gets the Folium Apocyni Veneti extracting solution, and is standby;
(2) get Folium Apocyni Veneti extracting solution that step (1) obtains and add ethanol and make concentration of ethanol reach 75% ~ 85%, leave standstill, precipitate, leach solution, concentrate, the precipitation solution that gets the Folium Apocyni Veneti extracting solution is stand-by;
(3) get macroporous resin column on the Folium Apocyni Veneti precipitation solution that step (2) obtains, water and concentration are 10% ethanol elution respectively earlier, be 50% ~ 70% ethanol elution again with concentration, the thin-layer chromatography inspection is known, is collected 50% ~ 70% ethanol eluate, concentrates, obtain the crude extract of Quercetin 3-galactoside in the Folium Apocyni Veneti, standby;
(4) get Quercetin 3-galactoside crude extract that step (3) obtains with ethyl acetate or n-butanol extraction, get extraction liquid, concentrate and obtain extract, add hydrophilic solvent then, heating and refluxing extraction 3 ~ 5 times, each 1 ~ 2 hour, filtered while hot, filtrate decompression concentrates, and 4 ℃ of refrigeration crystallizations are washed with hydrophilic solvent, dry, Quercetin 3-galactoside, adopt the preparation liquid phase to be further purified then, obtain purity and be 98% ~ 99% Quercetin 3-galactoside.
The preparation method of Quercetin 3-galactoside in the above-described Folium Apocyni Veneti, the temperature of step (1) and the described heating and refluxing extraction of step (4) is 80 ~ 85 ℃, step (1) and the described hydrophilic solvent of step (4) they are the ethanol of methyl alcohol or concentration 10 ~ 100%.
As preferred version, the Quercetin 3-galactoside extracting solution adds behind the ethanol of high density that concentration of ethanol is 80% in the Quercetin 3-galactoside extracting solution in the step (2), the purpose of this step operation is in order to remove macromolecular cpd such as the compositions such as tannin, polysaccharide in the Quercetin 3-galactoside extracting solution, be convenient to the purifying process of back, can improve purification efficiency greatly.
Above-described preparation method, as preferred version, the macroporous adsorbent resin described in the step (3) is nonpolar macroporous adsorption resin, as more excellent scheme, described nonpolar macroporous adsorption resin adopts D101, AB-8 or HPD100 type macroporous adsorbent resin.
As preferred version, the preparation method of Quercetin 3-galactoside in the above-described Folium Apocyni Veneti, wherein the chromatographic condition of the described preparation liquid phase of step (4) is: chromatographic column is the anti-phase C18 post of Agilent, moving phase is that (condition of gradient elution is methyl alcohol-0.5% phosphate aqueous solution gradient elution: 0 to 30 minute, the content of methyl alcohol rises to 80% by 5% content that rises to 40%, 30 to 60 minute methyl alcohol by 40%), detecting wavelength is 360 nm, flow velocity is 5ml/min, and detector is diode-array detector.
The present invention continues by preparation liquid phase process purifying Quercetin 3-galactoside, its chromatographic condition is: chromatographic column is selected the anti-phase C18 post of Agilent for use, moving phase is investigated with methyl alcohol-0.5% phosphate aqueous solution gradient elution through methodology, as preferably, the condition of gradient elution is: 0 to 30 minute, the content of methyl alcohol rises to 50% by 5%, the content of 30 to 60 minutes methyl alcohol rises to 88% by 50%, under this moving phase condition, the present invention utilizes diode-array detector, detects the Quercetin 3-galactoside peak under the 360nm wavelength, and purpose is arranged, collect stream part targetedly, can obtain purity and be 96 ~ 99% Quercetin 3-galactoside.
Beneficial effect: the preparation method of Quercetin 3-galactoside compares with prior art and has the following advantages in the Folium Apocyni Veneti provided by the invention:
(1) preparation method provided by the invention is workable, extraction separation efficient height, separate the Quercetin 3-galactoside purity height that obtains and can reach more than 90 ~ 99%, meet the requirement of a class bulk drug, and consuming little energy among the preparation technology, production cost is low, environmental protection, can realize industrialized production.
(2) preparation method of Quercetin 3-galactoside in the Folium Apocyni Veneti provided by the invention, characteristics according to the contained chemical ingredients of kendir medicinal material, than the investigation of system, extract temperature to the influence of Quercetin 3-galactoside quality, determine that optimum temps is 80 ~ 85 ℃, the result can not change when guaranteeing that Quercetin 3-galactoside has big extraction efficiency; The present invention adopts macroporous resin purification enrichment total flavones earlier in sepn process, determine best macroporous resin material, especially aspect eluent, high spot reviews the elutive power of Different concentrations of alcohol, determine elution processes, earlier with 10% ethanol removal of impurities, use again the total flavones elutive power by force to 50 ~ 70% ethanol elutions a little less than the impurity elutive power, can at utmost obtain the high total flavones of purity, purification efficiency is improved, the present invention regulates to adopt at Quercetin 3-galactoside dissolution extraction ability strong ethyl acetate or propyl carbinol after the pH value again and extracts then, after obtaining extraction liquid, the present invention adopts ethanol or methyl alcohol 80 ~ 85 ℃ of heating and refluxing extraction again, and then under preferred cooling temperature, place crystallization, can the very effective Quercetin 3-galactoside that character is close and two kinds of flavonoid glycoside compositions of isoquercitrin separately, reach 90% ~ 96% Quercetin 3-galactoside thereby can obtain purity.The present invention also adopts preparation liquid phase purifying Quercetin 3-galactoside in addition, determines moving phase with methodological study, and according to the Quercetin 3-galactoside absorbing wavelength, carries out separation and purification targetedly, high efficiency, can obtain purity and reach 96 ~ 99% Quercetin 3-galactoside.
Embodiment
According to following embodiment, the present invention may be better understood.Yet, those skilled in the art will readily understand that the described concrete material proportion of embodiment, processing condition and result thereof only are used for explanation the present invention, and should also can not limit the present invention described in detail in claims.
Embodiment 1
The preparation method of Quercetin 3-galactoside in the Folium Apocyni Veneti specifically may further comprise the steps:
(1) get Folium Apocyni Veneti medicinal material selection, clean, put into extractor after the drying, add the methyl alcohol of 10 times of amounts, 85 ℃ of reflux 2 times, filtered in each 2 hours, united extraction liquid, concentrate, get the Folium Apocyni Veneti extracting solution, standby;
(2) get Folium Apocyni Veneti extracting solution that step (1) obtains and add ethanol and make concentration of ethanol reach 80%, leave standstill, precipitate, leach solution, concentrate, the precipitation solution that gets the Folium Apocyni Veneti extracting solution is stand-by;
(3) get D101 type macroporous resin column on the Folium Apocyni Veneti precipitation solution that step (2) obtains, at first water washes 5 ~ 10 column volumes, use 1 ~ 3 column volume of 10% ethanol elution then, use 3 ~ 5 column volumes of 60% ethanol elution again, the thin-layer chromatography inspection is known, and collects 60% ethanol elution, decompression and solvent recovery under 60 ℃ of conditions, concentrate, get the Folium Apocyni Veneti crude extract, standby;
(4) get Quercetin 3-galactoside crude extract that step (3) obtains with water saturated n-butanol extraction 10 times, combining extraction liquid, decompression and solvent recovery, vacuum-drying gets the Folium Apocyni Veneti n-butyl alcohol extract; Add 8 times of amount 95% ethanol then, 80 ℃ of heating and refluxing extraction 3 times, each 1 hour, filtered while hot, filtrate decompression reclaims solvent, 4 ℃ of refrigerations 12 hours, places crystallization then, uses 95% washing with alcohol then, drains; Add 5 times of amount 95% ethanol again 80 ℃ of refluxing extraction 2 times, each 1 hour, filtered while hot, filtrate decompression reclaims solvent, place refrigeration 12 hours for 4 ℃, the leaching crystallization is with 95% washing with alcohol, drying, high performance liquid chromatography detect, and obtain purity and be 95% Quercetin 3-galactoside.
Embodiment 2
The preparation method of Quercetin 3-galactoside in the Folium Apocyni Veneti specifically may further comprise the steps:
(1) get Folium Apocyni Veneti medicinal material selection, clean, put into extractor after the drying, add 80% ethanol of 8 times of amounts, 85 ℃ of reflux 3 times, filtered in each 1.5 hours, united extraction liquid, concentrate, get the Folium Apocyni Veneti extracting solution, standby;
(2) get Folium Apocyni Veneti extracting solution that step (1) obtains and add ethanol and make concentration of ethanol reach 85%, leave standstill, precipitate, leach solution, concentrate, the precipitation solution that gets the Folium Apocyni Veneti extracting solution is stand-by;
(3) get AB-8 type macroporous resin column on the Folium Apocyni Veneti precipitation solution that step (2) obtains, at first water washes 5 ~ 10 column volumes, use 1 ~ 3 column volume of 20% ethanol elution then, use 3 ~ 5 column volumes of 55% ethanol elution again, the thin-layer chromatography inspection is known, and collects 55% ethanol elution, decompression and solvent recovery under 60 ℃ of conditions, concentrate, get the Folium Apocyni Veneti crude extract, standby;
(4) get the Quercetin 3-galactoside crude extract that step (3) obtains and transfer pH=3 with dilute hydrochloric acid, use ethyl acetate extraction again 8 times, combining extraction liquid, decompression and solvent recovery, vacuum-drying gets the Folium Apocyni Veneti acetic acid ethyl ester extract; Add 6 times of amount methyl alcohol then, 80 ℃ of heating and refluxing extraction 3 times, each 1 hour, filtered while hot, filtrate decompression reclaims solvent, 4 ℃ of refrigerations 12 hours, places crystallization then, uses methanol wash then, drains; Add 5 times of amount methyl alcohol again 80 ℃ of refluxing extraction 3 times, each 1 hour, filtered while hot, filtrate decompression reclaims solvent, places refrigeration 12 hours for 4 ℃, the leaching crystallization, with methanol wash, drying, high performance liquid chromatography detects, and obtains purity and be 95.8% Quercetin 3-galactoside.
Embodiment 3
The preparation method of Quercetin 3-galactoside in the Folium Apocyni Veneti specifically may further comprise the steps:
(1) get Folium Apocyni Veneti medicinal material selection, clean, put into extractor after the drying, add 50% ethanol of 10 times of amounts, 85 ℃ of reflux 2 times, filtered in each 3 hours, united extraction liquid, concentrate, get the Folium Apocyni Veneti extracting solution, standby;
(2) get Folium Apocyni Veneti extracting solution that step (1) obtains and add ethanol and make concentration of ethanol reach 75%, leave standstill, precipitate, leach solution, concentrate, the precipitation solution that gets the Folium Apocyni Veneti extracting solution is stand-by;
(3) get HPD100 type macroporous resin column on the Folium Apocyni Veneti precipitation solution that step (2) obtains, at first water washes 5 ~ 10 column volumes, use 1 ~ 3 column volume of 10% ethanol elution then, use 3 ~ 5 column volumes of 50 ~ 70% ethanol elutions again, the thin-layer chromatography inspection is known, and collects 60% ethanol elution, decompression and solvent recovery under 60 ℃ of conditions, concentrate, get the Folium Apocyni Veneti crude extract, standby;
(4) get the Quercetin 3-galactoside crude extract usefulness that step (3) obtains, transfer pH=3 with dilute hydrochloric acid, adopt water saturated n-butanol extraction again 10 times, combining extraction liquid, decompression and solvent recovery, vacuum-drying gets the Folium Apocyni Veneti n-butyl alcohol extract; Add 10 times of amount 80% methyl alcohol then, 80 ℃ of heating and refluxing extraction 3 times, each 1 hour, filtered while hot, filtrate decompression reclaims solvent, 4 ℃ of refrigerations 12 hours, places crystallization then, uses methanol wash then, drains; Add 5 times of amount 80% methyl alcohol again 80 ℃ of refluxing extraction 2 times, each 1 hour, filtered while hot, filtrate decompression reclaims solvent, place refrigeration 12 hours for 4 ℃, the leaching crystallization is with methanol wash, drying, high performance liquid chromatography detect, and obtain purity and be 96.2% Quercetin 3-galactoside.
Embodiment 4
The preparation method of Quercetin 3-galactoside in the Folium Apocyni Veneti specifically may further comprise the steps:
(1) get Folium Apocyni Veneti medicinal material selection, clean, put into extractor after the drying, add 60% ethanol of 10 times of amounts, 85 ℃ of reflux 2 times, filtered in each 3 hours, united extraction liquid, concentrate, get the Folium Apocyni Veneti extracting solution, standby;
(2) get Folium Apocyni Veneti extracting solution that step (1) obtains and add ethanol and make concentration of ethanol reach 80%, leave standstill, precipitate, leach solution, concentrate, the precipitation solution that gets the Folium Apocyni Veneti extracting solution is stand-by;
(3) get D101 type macroporous resin column on the Folium Apocyni Veneti precipitation solution that step (2) obtains, at first water washes 5 ~ 10 column volumes, use 1 ~ 3 column volume of 10% ethanol elution then, use 3 ~ 5 column volumes of 50 ~ 70% ethanol elutions again, the thin-layer chromatography inspection is known, and collects 60% ethanol elution, decompression and solvent recovery under 60 ℃ of conditions, concentrate, get the Folium Apocyni Veneti crude extract, standby;
(4) get the Quercetin 3-galactoside crude extract usefulness that step (3) obtains, transfer pH=3 with dilute hydrochloric acid, adopt water saturated n-butanol extraction again 8 times, combining extraction liquid, decompression and solvent recovery, vacuum-drying gets the Folium Apocyni Veneti n-butyl alcohol extract; Add 10 times of amount ethanol then, 80 ℃ of heating and refluxing extraction 3 times, each 1 hour, filtered while hot, filtrate decompression reclaims solvent, 4 ℃ of refrigerations 12 hours, places crystallization then, uses methanol wash then, drains; Add 6 times of amount ethanol again 80 ℃ of refluxing extraction 2 times, each 1 hour, filtered while hot, filtrate decompression reclaims solvent, place refrigeration 12 hours for 4 ℃, the leaching crystallization is with methanol wash, dry, preparation liquid phase purifying is gone up in methyl alcohol dilution back, obtains purity and be 98.6% Quercetin 3-galactoside, and its chromatographic condition is: chromatographic column is the anti-phase C18 post of Agilent, moving phase is that (condition of gradient elution is methyl alcohol-0.5% phosphate aqueous solution gradient elution: 0 to 30 minute, the content of methyl alcohol rises to 80% by 5% content that rises to 40%, 30 to 60 minute methyl alcohol by 40%), the detection wavelength is 360nm, flow velocity is 5ml/min, and detector is diode-array detector.
Embodiment 5
The preparation method of Quercetin 3-galactoside in the Folium Apocyni Veneti specifically may further comprise the steps:
(1) get Folium Apocyni Veneti medicinal material selection, clean, put into extractor after the drying, add the methyl alcohol of 10 times of amounts, 85 ℃ of reflux 2 times, filtered in each 2 hours, united extraction liquid, concentrate, get the Folium Apocyni Veneti extracting solution, standby;
(2) get Folium Apocyni Veneti extracting solution that step (1) obtains and add ethanol and make concentration of ethanol reach 80%, leave standstill, precipitate, leach solution, concentrate, the precipitation solution that gets the Folium Apocyni Veneti extracting solution is stand-by;
(3) get D101 type macroporous resin column on the Folium Apocyni Veneti precipitation solution that step (2) obtains, at first water washes 5 ~ 10 column volumes, use 2 ~ 3 column volumes of 10% ethanol elution then, use 4 ~ 5 column volumes of 60% ethanol elution again, the thin-layer chromatography inspection is known, and collects 60% ethanol elution, decompression and solvent recovery under 60 ℃ of conditions, concentrate, get the Folium Apocyni Veneti crude extract, standby;
(4) get the Quercetin 3-galactoside crude extract that step (3) obtains, transfer pH=3 with dilute hydrochloric acid, use water saturated n-butanol extraction 12 times again, combining extraction liquid, decompression and solvent recovery, vacuum-drying gets the Folium Apocyni Veneti n-butyl alcohol extract; Add 8 times of amount 90% ethanol then, 80 ℃ of heating and refluxing extraction 3 times, each 1 hour, filtered while hot, filtrate decompression reclaims solvent, 4 ℃ of refrigerations 12 hours, places crystallization then, uses 90% washing with alcohol then, drains; Add 6 times of amount 95% ethanol again 80 ℃ of refluxing extraction 2 times, each 1 hour, filtered while hot, filtrate decompression reclaims solvent, place refrigeration 12 hours for 4 ℃, the leaching crystallization, with 95% washing with alcohol, drying is with methanol wash, dry, preparation liquid phase purifying is gone up in methyl alcohol dilution back, obtains purity and be 99.1% Quercetin 3-galactoside, and its chromatographic condition is: chromatographic column is the anti-phase C18 post of Agilent, moving phase is that (condition of gradient elution is methyl alcohol-0.5% phosphate aqueous solution gradient elution: 0 to 30 minute, the content of methyl alcohol rises to 80% by 5% content that rises to 40%, 30 to 60 minute methyl alcohol by 40%), detecting wavelength is 360 nm, flow velocity is 5ml/min, and detector is diode-array detector.
The preparation method that provides of invention, technological design is reasonable, and is simple to operate, extraction efficiency is high, be fit to mass preparation, and the Quercetin 3-galactoside purity for preparing simultaneously can reach 90 ~ 99%, meets the requirement of a class bulk drug.
Above embodiment only is explanation technical conceive of the present invention and characteristics; its purpose is to allow the people that is familiar with this technology understand content of the present invention and is implemented; can not limit protection scope of the present invention with this; all equivalences that spirit is done according to the present invention change or modify, and all should be encompassed in protection scope of the present invention.
Claims (1)
1. the preparation method of Quercetin 3-galactoside in the Folium Apocyni Veneti is characterized in that may further comprise the steps:
(1) get Folium Apocyni Veneti medicinal material selection, clean, put into extractor after the drying, add the methyl alcohol of 5 ~ 10 times of amounts or the ethanol of concentration 10 ~ 100%, 80 ~ 85 ℃ of reflux 1 ~ 3 time are filtered, united extraction liquid, concentrate, and get the Folium Apocyni Veneti extracting solution, and are standby;
(2) get Folium Apocyni Veneti extracting solution that step (1) obtains and add ethanol and make concentration of ethanol reach 80%, leave standstill, precipitate, leach solution, concentrate, the precipitation solution that gets the Folium Apocyni Veneti extracting solution is stand-by;
(3) get D101, AB-8 or HPD100 type macroporous adsorbent resin on the Folium Apocyni Veneti precipitation solution that step (2) obtains, water and concentration are 10% ethanol elution respectively earlier, be 50% ~ 70% ethanol elution again with concentration, the thin-layer chromatography inspection is known, is collected 50% ~ 70% ethanol eluate, concentrates, obtain the crude extract of Quercetin 3-galactoside in the Folium Apocyni Veneti, standby;
(4) get the Quercetin 3-galactoside crude extract that step (3) obtains, transfer pH=3 with dilute hydrochloric acid, then with ethyl acetate or n-butanol extraction, get extraction liquid, concentrate and obtain extract, the ethanol that adds methyl alcohol or concentration 10 ~ 100% then, 80 ~ 85 ℃ of heating and refluxing extraction 3 ~ 5 times, each 1 ~ 2 hour, filtered while hot, filtrate decompression concentrates, and 4 ℃ of refrigeration crystallizations are with the washing with alcohol of methyl alcohol or concentration 10 ~ 100%, dry, Quercetin 3-galactoside, adopt the preparation liquid phase to be further purified then, obtain purity and be 96% ~ 99% Quercetin 3-galactoside;
The chromatographic condition of the described preparation liquid phase of above step (4) is: chromatographic column is the anti-phase C18 post of Agilent, and moving phase is methyl alcohol-0.5% phosphate aqueous solution gradient elution, and the detection wavelength is 360nm, and flow velocity is 5ml/min, and detector is diode-array detector.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201110075635 CN102190693B (en) | 2011-03-29 | 2011-03-29 | Method for preparing hyperin from Dogbane leaves |
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| CN102399252A (en) * | 2011-12-15 | 2012-04-04 | 成都普思生物科技有限公司 | Preparation method of cowherb seed flavonoid glycoside monomer |
| CN102924537B (en) * | 2012-10-30 | 2015-02-25 | 中国科学院烟台海岸带研究所 | Method for preparing hyperoside and isoquercitrin simultaneously from dogbane leaves |
| CN103169788B (en) * | 2013-04-16 | 2014-07-23 | 南京中医药大学 | Chinese date leaf extractive and application thereof in preparation of medicament or health food for preventing and treating liver injury |
| CN115669470A (en) * | 2020-12-11 | 2023-02-03 | 北京林业大学 | Application of AemYB30 and/or AeUF3GaT1 in improving seting rate of Abelmoschus manihot |
| CN118217318A (en) * | 2024-01-30 | 2024-06-21 | 山东中医药大学 | Apocynum venetum leaf extract and preparation method and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1493578A (en) * | 2002-10-29 | 2004-05-05 | 中国人民解放军北京军医学院 | Preparation method of golden peach glycoside and its new use in medicine |
| CN101386634A (en) * | 2008-09-05 | 2009-03-18 | 周春晓 | Hyperin extraction method and preparation and use thereof |
| CN101690739A (en) * | 2009-06-30 | 2010-04-07 | 长春师范学院 | Preparation method of dogbane leaf extractive |
| CN101891785A (en) * | 2010-08-16 | 2010-11-24 | 江西山香药业有限公司 | Extraction method of hyperin and application thereof in medicament preparation |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1493578A (en) * | 2002-10-29 | 2004-05-05 | 中国人民解放军北京军医学院 | Preparation method of golden peach glycoside and its new use in medicine |
| CN101386634A (en) * | 2008-09-05 | 2009-03-18 | 周春晓 | Hyperin extraction method and preparation and use thereof |
| CN101690739A (en) * | 2009-06-30 | 2010-04-07 | 长春师范学院 | Preparation method of dogbane leaf extractive |
| CN101891785A (en) * | 2010-08-16 | 2010-11-24 | 江西山香药业有限公司 | Extraction method of hyperin and application thereof in medicament preparation |
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