CN103655658A - Technology for extracting xanthin in plains coreopsis and application of extract thereof - Google Patents
Technology for extracting xanthin in plains coreopsis and application of extract thereof Download PDFInfo
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Abstract
The invention discloses a technology for extracting xanthin in plains coreopsis. The technology comprises the steps of soaking, extracting and concentrating. The xanthin obtained by the technology for extracting xanthin in plains coreopsis provided by the invention is good in water solubility, the application field of the xanthin is expanded, and the water-soluble xanthin is easier to be absorbed by a human body when being applied to a food, a health-care product or a drug. Furthermore, compared with the extraction rate of the prior art, the extraction rate of the xanthin obtained by the technology for extracting the xanthin in the plains coreopsis provided by the invention is effectively improved, and can be up to 40%. A spontaneously hypertensive rat is lavaged by the extract of the technology, so that the blood pressure can be significantly reduced, and the effect can be generated by reducing the levels of endothelin and angiotensin II in the blood.
Description
Technical field
The present invention relates to a kind of extraction process of xanthin.
Background technology
Dichromatism Coreopsis basalis is the dry herb of feverfew dichromatism Coreopsis basalis (Coreopsis tinctoria Nutt.), for rufous base portion outer ring is yellow or flavous ligulate flower, for Tickseed annual or life in 2 years, have another name called dichromatism Coreopsis basalis, fluxweed.Begin to be loaded in the book on Chinese herbal medicine outline > > of < < Xinhua, there is the effect of heat-clearing and toxic substances removing, removing dampness dysentery relieving, cure mainly the humidifier fevers such as conjunctival congestion and swelling pain, dysentery due to damp-heat, dysentery.This plant originates in North America, American Indian of North America is for (lists of references: Foster such as stomachache, diarrhoea, hemorrhage and emetic, S., Duke, J.A., 1990.A Field Guide to Medicinal Plants.Eastern and Central North America.Houghton Mifflin Co.).In Portugal, among the people with the water that every day, 2 glasss of dichromatism golden pheasant Flos Chrysanthemis soaked, for controlling diabetes.
Its flower of hotan area employing among the people is for prevention and cure of cardiovascular disease; Modern pharmacology research shows that dichromatism Coreopsis basalis has antiinflammatory, improves activity (list of references: Dias, T., the Mota-Filipe of glucose tolerance, H., Liu, B., Jones, Houghton, P.J., Paulo, A., 2010.Recovery of oral glucose tolerance by Wistar rats after treatment with Coreopsis tinctoria infusion.Phytotherapy Research24,699-705.).
A Yi tells human relations etc. and dichromatism Coreopsis basalis chemical composition is carried out to preliminary experiment result shows that dichromatism Coreopsis basalis contains the class materials such as flavone, coumarin, polyphenol, saponin, organic acid, polysaccharide, aminoacid.By GC/MS, identify that dichromatism Coreopsis basalis volatilization main body of oil is limonene (63.5%), 3-carene (7.03%), β-to Umbrella ellagic acid (5.23%), zingiberene (2.45%) etc.The rutin of take is measured in dichromatism Coreopsis basalis general flavone content between 13-20% as reference substance UV-VIS spectrophotometry; The glucose of take is 13-27% as contrast adopts phenol sulfuric acid process to measure total sugar.The trace element that ICP method is measured in dichromatism Coreopsis basalis has Cu, Mn, Zn, Ni, Fe, Al etc.; Contain 17 seed amino acids such as serine, threonine; With HPLC method, measure dichromatism Coreopsis basalis Content of Chlorogenic Acid approximately 0.5%.
Nineteen fifty-seven, Japanese scholars identified the composition of xanthin pigment in dichromatism Coreopsis basalis, wherein contain cyanidin-O-heteroside (cyanidin-3-glucoside), from the outer ring yl moiety of dichromatism golden pheasant Flos Chrysanthemi, obtain three compound marein (marein, 4'-glucosidoxy-2', 3', 3,4-tetrahydroxychalcone, (I)), coreopsin (maritimein, 6-glucosidoxy-7,3', 4'-trihydroxyaurone, (VI)) and Flavanomarein(III).
In literary composition, also mention and in dichromatism Coreopsis basalis, contain caffeoylquinic acids and chlorogenic acid (list of references: MASAMSIH IMOKORIY.Anthochlor Pigments of Coreopsis tinctoria.CONTRIBUTION FROM THE BOTANICAINLS TITUTE, FACULOTFY S CIENCE, UNIVERSITY OF TOKYO.1957.vol.79:214-220.).
The chemical composition with field dichromatism Coreopsis basalis has been studied by Tu Pengfei seminar of College of Pharmacy, Beijing Univ in 2005, chemical composition to dichromatism golden pheasant Flos Chrysanthemi has been carried out compared with systematic research, therefrom separation obtains 30 compounds, use spectroscopic technique to identify wherein 24 compound structures, comprising: 14 flavone compounds, 4 organic acid compounds, 4 poly-alkyne-glycoside compounds and 2 phytosterin compounds.(table 1)
Compound (list of references: Zhang Yuan, Tu Pengfei in table 1. dichromatism Coreopsis basalis.The chemical composition of dichromatism Coreopsis basalis and Tamarix ramosissima and bioactivity research. Peking University's thesis for the doctorate .)
Within 2010, Portugal scholar is studied chemical composition and biological activity, after extracting, obtains 13 compounds and structure is confirmed with ethyl acetate.And adopt HPLC method to carry out quantitative analysis to marein, Flavanomarein, chlorogenic acid and dicaffeoylquinic acid in the different extracts of dichromatism Coreopsis basalis.
Active component in dichromatism golden pheasant Flos Chrysanthemi is flavone compound, is mainly the structure of chalcone and flavanone.Japanese scholars is referred to as xanthin (Anthochlor Pigments), this flavonoids acts on the different phase of glucose metabolism, Portugal scholar find marein-and the action target spot of flavanone compound be impaired pancreatic cell, recover the secretion (list of references: Teresa Diasa.The flavonoid-rich fraction of Coreopsis tinctoria promotes glucose tolerance regain through pancreatic function recovery in streptozotocin-induced glucose-intolerant rats.Journal of Ethnopharmacology132 (2010) 483-490.) of insulin.
Summary of the invention
The technical problem to be solved in the present invention is to overcome existing defect, provides a kind of extraction ratio high, and extraction process is simple, and the extraction process of xanthin in the xanthin good water solubility of extracting, the dichromatism Coreopsis basalis that nontoxic, purity is high.
Object of the present invention is carried out specific implementation by the following technical programs:
In dichromatism Coreopsis basalis, an extraction process for xanthin, comprises the steps:
1) soak: by the fragmentation of dichromatism Coreopsis basalis dried floral, by purity, be 42%-68% alcohol solution dipping 8-17 hour afterwards;
2) extract: 1-5 hour that the mixed material in step 1) is refluxed at 43-67 ℃, filter to obtain extracting solution for the first time, then continue to add 42%-68% alcoholic solution in filter cake, at 43-67 ℃, extract 1-2 time, each 1-5 hour;
3) concentrated: said extracted liquid is merged, adopt secondary concentration technique, be specially and control material at 40-70 ℃, twice flash distillation or first concentrating under reduced pressure again flash distillation once, obtain final concentrated extracting solution, obtain lyophilized powder after lyophilization.
Further,
When twice flash distillation of described secondary concentration process using, concrete operations are as follows:
A. the repeatedly extracting solution once concentration: by step 2) merges, and by Multistage flash evaporator, concentrates, and controlling solution temperature is 40-60 ℃, and flow velocity is 50-200ml/min, and vacuum is 0.02-2MPa, concentrated solution for the first time;
B. secondary concentration: will concentrated solution to be by flash distillation instrument for the first time, control solution temperature is 50-70 ℃, flow velocity is 50-200ml/min, obtains final concentrated extracting solution.
When the first concentrating under reduced pressure of described secondary concentration process using flash distillation technique once again, concrete operations are as follows:
A. the repeatedly extracting solution once concentration: by step 2) merges, concentrating under reduced pressure, and temperature of charge is controlled at 40-70 ℃, concentrated 7-9 hour under vacuum 0.02-2MPa;
B. secondary concentration: concentrated solution concentrates in Multistage flash evaporator for the first time, and solution temperature is controlled at 50-70 ℃, vacuum 0.02-2MPa.
Preferably, in described step 1), the mass ratio of described dichromatism Coreopsis basalis dried floral and alcoholic solution is 1:2-20; The purity of described alcoholic solution is 50%-60%.Soak 12 hours.
Preferably, described step 2) in, the purity of the alcoholic solution at every turn adding is 50-60%, the quality of the alcoholic solution that addition adds with step 1) equates.Each temperature of charge of purifying is 55 ℃.
More excellent, the first concentrating under reduced pressure of secondary concentration process using flash distillation technique once again in described step 3), the alcohol solution dipping that is 55% by purity in described step 1) 12 hours; Described step 2) in, the temperature of charge extracting is for the first time 55 ℃, and reflux, extract, 2.5 hours is filtered to obtain extracting solution for the first time, and it is 55% ethanol dope that gained filter cake continues to add purity, and temperature of charge is reflux, extract, 2.5 hours at 55 ℃; Described step 1) and step 2) in add alcoholic solution quality be 15 times of dichromatism Coreopsis basalis dried floral quality.
Dichromatism Coreopsis basalis alcohol extract is in the application for the treatment of spontaneously hypertensive.
Beneficial effect of the present invention:
The good water solubility of the extraction process gained xanthin of xanthin in dichromatism Coreopsis basalis provided by the invention, has expanded the application of xanthin, and while being applied to food, health product or medicine, water miscible xanthin is easier to absorption of human body; And in dichromatism Coreopsis basalis provided by the invention, the extraction ratio of the extraction process gained xanthin of xanthin has obtained effective lifting compared with the extraction ratio of prior art, can reach 40%; The present invention adopts second alcohol and water to extract as solvent, and the process of flash distillation evaporates all ethanol thoroughly, and gained xanthin purity is high, nontoxic, can safety for food, health product and medicine field; In addition, technique of the present invention is simple and easy to control, with low cost.The present invention further develops for dichromatism Coreopsis basalis extractive of general flavone from now on, carries out health product or new drug and declares, careful on selective extraction solvent, and requires process safety, easy.Final purpose is to obtain a kind of easyly, efficient, and production cost is low, and the technique that extraction ratio is high will guarantee that extract is nontoxic, the features such as good water solubility.Therefore the present invention has selected ethanol for extracting solvent.
At present the report for work ethanol extraction technique of middle dichromatism Coreopsis basalis total flavones of document only limits to laboratory process, not attempting amplificationization produces, and general flavone content is only up to 25%, extraction time is long, under high temperature, active component is had to destruction, and the present invention is through amplifying pilot experiment gained, extraction ratio and general flavone content are all higher than the laboratory result of reporting at present, the content of application protection is the production technology after multiple batches of pilot plant test, extract for animal pharmacodynamic experiment is also the product of pilot scale, because product is stablized, high conformity, ensured the reliability of the pharmacodynamic experiment result obtaining.
In addition, the thickening temperature adopting in technique, extraction time and last drying mode, the structure that all ensures as much as possible flavone compound is not wherein destroyed, therefore the screening of this pilot process is to be differentiated in conjunction with take rutin and extraction process is evaluated as the method that reference substance UV measures total flavones by HPLC finger printing, thin layer, be not that the single employing of traditional method be take the UV that rutin is reference substance and measured total flavones, so technique is more secure to the extraction of flavone compound.
To sum up, in dichromatism Coreopsis basalis provided by the invention, the extraction process of xanthin can be real is applied to suitability for industrialized production, for the commercial value of xanthin in dichromatism Coreopsis basalis has been opened the new situation.
Accompanying drawing explanation
Accompanying drawing is used to provide a further understanding of the present invention, and forms a part for description, for explaining the present invention, is not construed as limiting the invention together with embodiments of the present invention.In the accompanying drawings:
Fig. 1 is the effects process chart in embodiment 1;
Fig. 2 is the thin layer discriminating figure of the laboratory extract in embodiment 1, and wherein, a is the figure that inspects under 366nm, and b is the figure that inspects under visible ray;
Fig. 3 is the thin-layer chromatogram to the dichromatism Coreopsis basalis xanthin extract extracting under technique of the present invention in embodiment 6, wherein, and A--laboratory extract, first extract of B--, C--second batch extract, the 3rd batch of extract of D--, the 3rd batch of extract of E--;
Fig. 4 is the thin-layer chromatogram of three batches of pilot extraction things in embodiment 7, and wherein, 1 for mixed mark, is chlorogenic acid, marein and cyanidin-3-O-glucoside from top to bottom according to this, and 2,3,4 are respectively three batches of samples;
Fig. 5 is dichromatism Coreopsis basalis pilot extraction thing HPLC finger printing in embodiment 7, wherein, I is the finger printing that 280nm wavelength detects, II is the finger printing that 350nm wavelength detects, A is mixed mark, be followed successively by 1--chlorogenic acid, 2--Flavanomarein, 3--coreopsin, 4--marein, B is that 20121212 lot number samples, C are that 20121215 lot number samples, D are 20121217 lot number samples;
Fig. 6 is the need testing solution HPLC feature spectrogram of dichromatism golden pheasant Flos Chrysanthemi in embodiment 7, and wherein, detection wavelength is 280nm, and peak 2 is chlorogenic acid, and peak 3 is Flavanomarein, and peak 5 is coreopsin, and peak 7 is marein.
The specific embodiment
Below in conjunction with accompanying drawing, the preferred embodiments of the present invention are described, should be appreciated that preferred embodiment described herein, only for description and interpretation the present invention, is not intended to limit the present invention.
Related having to instrument in following examples: multi-functional extraction thickener group (DC-NSG-30, upper Haidah journey experimental facilities company limited); Multistage flash evaporation instrument (TMF-320A), dichromatism Coreopsis basalis is provided by the Kunlun Xue Juxue chrysanthemum company.
Embodiment 1:
Laboratory extraction process and quality
1, craft screening
Extraction process to flavone compound in dichromatism Coreopsis basalis is investigated by the technology path of accompanying drawing 1.
The final technique of screening is: take each 100g of dichromatism Coreopsis basalis (pulverizing 60 mesh sieves), 55% ethanol that adds 18BV, soak after 3h, 55 ℃ of reflux, extract, 2.5h, filter, filter cake adds after 55% ethanol of 18BV directly in 55 ℃ of reflux, extract, 2.5h, filters, merge filtrate twice, concentrating under reduced pressure is also settled to 500mL.(number of applying for a patent is: 201210270752.0, the date of application is on August 1st, 2012)
Get extracting solution and carry out lyophilization, extract yield is that 50%(200g dry flower obtains 100g lyophilized powder).
2, thin layer chromatography is differentiated
By definite laboratory process, extract three parts of dichromatism Coreopsis basalis flavone extracts, carry out lyophilization.Get respectively lyophilized powder powder 0.5g, put in 10ml measuring bottle, add water standardize solution, obtain the need testing solution of 50mg/mL.Get respectively 1 μ L need testing solution, semi-automatic point sample instrument is put on the efficient plate of same silica gel G, the solvent of toluene-ethyl acetate-formic acid (9:7:3) launches, take out, dry, spray successively 2-amino-ethyl xenyl borate and PEG400 and develop the color, under ultraviolet light 366nm and visible ray, inspect (accompanying drawing 2).
The color of speckle, number and R on thin layer figure
fbe worth identically, all contain marein (R
f0.09), chlorogenic acid (R
f0.4), Cy-3-G (R
f=0.04) and okanin (R
f0.73), illustrate that three batches of extract components are consistent, laboratory extraction process is stable.
3, assay
Above-mentioned need testing solution is measured by the chlorogenic acid in quality standard and Determination of Total Flavonoids method, and chlorogenic acid and general flavone content in xanthin extract are listed table 2 in.
Chlorogenic acid and the general flavone content of table 2. laboratory extract
In laboratory extract, general flavone content, lower than estimated value, is because extracting solution is long at laboratory concentration time, causes content to decline.
Embodiment 2:
1. soak: Coreopsis basalis dried floral 2kg, does not need to shatter, by 55% ethanol 30kg soaked overnight (15 hours).
2. extract: 60 ℃ of heating-up temperatures, 55 ℃ of temperature of charge, reflux 2 hours, filters to obtain the about 20kg of extracting solution for the first time; Material continues to add 55% ethanol 30kg, extracts 2 hours, filters to obtain the about 30kg of extracting solution, merges extracted twice liquid.
3. concentrated: extracting solution concentrates by Multistage flash evaporator, and parameters is 90 ℃ of preheating one temperature, 105 ℃ of preheating two temperature, 90 ℃ of flash vessel temperature, 50 ℃ of solution temperatures, flow velocity 100ml/min, vacuum-0.07mpa, obtains once concentration liquid 20kg.
4. secondary concentration: 20kg once concentration liquid is by flash distillation instrument, flow velocity 80ml/min, 55 ℃ of solution temperatures, final concentrated solution 10kg, totally 12 hours.
Embodiment 3:
1. soak: Coreopsis basalis dried floral 2kg, by 55% ethanol 30kg soaked overnight (12 hours), soak sampling.
2. extract: 60 ℃ of heating-up temperatures, 55 ℃ of temperature of charge, reflux 2.5 hours, filters to obtain the about 20kg of extracting solution for the first time; Extracting solution sampling for the first time.Material continues to add 55% ethanol 30kg, extracts 2 hours, filters to obtain the about 30kg of extracting solution, extracting solution sampling for the second time.
3. concentrated: to merge extracted twice liquid, to concentrating under reduced pressure in concentration tank, 55 ℃ of temperature of charge, vacuum 0.07-0.08Mpa, the about 8h of concentration time.Obtain concentrated solution A13kg(and stay 5kg)
4. the contrast of two kinds of method for concentration:
Get concentrated solution 8kg and concentrate by Multistage flash evaporator, one 90 ℃ of preheatings, 2 105 ℃ of preheatings, 105 ℃ of flash vessels, 60 ℃ of solution, vacuum 0.07Mpa, final concentrated solution B5kg.
Embodiment 4:
Extraction process 3,, concrete operations are:
1. soak: Coreopsis basalis dried floral 2kg, by 55% ethanol 30kg soaked overnight (12 hours).
2. extract: 60 ℃ of heating-up temperatures, 55 ℃ of temperature of charge, reflux 2.5 hours, filters to obtain extracting solution for the first time, and material continues to add 55% ethanol 30kg, extracts 2.5 hours, filters to obtain extracting solution.
3. concentrated: to merge extracted twice liquid, to concentrating under reduced pressure in concentration tank, 55 ℃ of temperature of charge, vacuum 0.07-0.08Mpa, the about 8h of concentration time.Obtain concentrated solution and concentrate (one 90 ℃ of preheatings, 2 105 ℃ of preheatings, 105 ℃ of flash vessels, 60 ℃ of solution, vacuum 0.07Mpa) by Multistage flash evaporator, final concentrated solution 20121011 is 8kg.
Embodiment 5:
Extraction process 4, concrete operations are:
1. soak: Coreopsis basalis dried floral 2kg, by 55% ethanol 30kg soaked overnight (12 hours).
2. extract: 60 ℃ of heating-up temperatures, 55 ℃ of temperature of charge, reflux 2.5 hours, filters to obtain extracting solution for the first time, and material continues to add 55% ethanol 30kg, extracts twice, and each 2.5 hours, filter to obtain extracting solution, extracting solution keeps sample for the third time.
3. concentrated: to merge three times extracting solution, to concentrating under reduced pressure in concentration tank, 55 ℃ of temperature of charge, vacuum 0.07-0.08Mpa, the about 8h of concentration time.Obtain concentrated solution and concentrate (one 90 ℃ of preheatings, 2 105 ℃ of preheatings, 105 ℃ of flash vessels, 60 ℃ of solution, vacuum 0.07Mpa) by Multistage flash evaporator, final concentrated solution 20121014 is 10kg.
Embodiment 6:
The extraction of substance component analysis of tetra-batches of extraction processes of embodiment 2-5
1, flour extraction
Get extracting solution 100ml and carry out lyophilization, obtain lyophilized powder 10.4g, calculated yield is that 50%(is that 2kg dry flower obtains 1kg lyophilized powder)
2, the content of chlorogenic acid and total flavones
The analytical method of setting up according to seminar detects four batches of product Content of Chlorogenic Acids and general flavone content, and result is listed in table 3.
Table 3. dichromatism golden pheasant Flos Chrysanthemi pilot extraction thing Content of Chlorogenic Acid and flavones content
3, thin layer chromatography is differentiated
Get respectively laboratory and extract sample and each 1 μ l of different process dichromatism Coreopsis basalis pilot extraction sample, with semi-automatic point sample instrument, put on the efficient plate of same silica gel G; Take toluene: the solution of ethyl acetate: formic acid=9:7:3 launches as developing solvent; After launching completely, plate is taken out and is dried, then spray 2-APB successively, PEG400 develops the color; In ultraviolet light 366nm(accompanying drawing 3), wherein, A--laboratory extract, first extract of B--, C--second batch extract, the 3rd batch of extract of D--, the 3rd batch of extract of E--.
4, conclusion
In second batch extracting solution, concentrated solution B is that many full flashing is concentrated on the basis of concentrated solution A.Concentrated solution A by 8kg, by flash distillation, is condensed into 5kg.In theory, the chlorogenic acid total amount of concentrated solution B should be about 40g, and total flavones total amount should be about 170g.After actual measurement, find not loss.Therefore visible flash evaporation technology can not cause damage to chlorogenic acid and total flavones.
First is compared with the 3rd batch, and first technique is carried out twice flash distillation, and the 3rd batch is ethanol concentrating under reduced pressure, successively flash distillation.In result, see, the chlorogenic acid of twice and general flavone content difference are little.
According to the result of thin layer discriminating and assay, confirm that the chemical composition in different process pilot extraction thing is all consistent with laboratory extract.But chlorogenic acid and general flavone content in different process pilot extraction thing are variant.If simple, consider it is total flavones yield, technique 4(20121014 batch) at technique 3(20121011 batch) by extraction time increase once, total flavones is the highest, but increasing degree is not very high, from follow-up amplification, produces and considers, technique 4 will increase cost.Consider, finally select technique 3 for best pilot extraction technique.
Pilot product extracting solution Content of Chlorogenic Acid and total flavones are all higher.After getting pilot scale concentrated solution and carrying out lyophilizing, gained flour extraction is consistent with test chamber, and flavones content is also consistent, but chlorogenic acid content doubles
Embodiment 7:
Confirmatory experiment
According to the 3rd batch of extraction process in embodiment 4, carry out three batches of demonstration tests, lot number is 20121212,20121215,20121217, and inventory is 2kg, and final fluid extract is about 10L, lyophilization, obtaining lyophilized powder is 1014g, 1279g and 1056g according to this.Three batches of lyophilized powders are carried out to quality analysis.
1, thin layer is differentiated
Get respectively hybrid standard product 2 μ l and three crowdes of dichromatism Coreopsis basalis pilot extraction test sample 1 μ l, semi-automatic point sample is on the efficient plate of same silica gel G, toluene-ethyl acetate-formic acid=9:7:3 solution of take launches as developing solvent, after launching completely, plate is taken out and to be dried, then spray 2-APB successively, PEG400 develops the color; Under ultraviolet light 366nm, inspect (referring to accompanying drawing 4,1, for mixed mark, be chlorogenic acid, marein and cyanidin-3-O-glucoside according to this, 2,3,4 are respectively three batches of samples) from top to bottom, the speckle number of three batches of extracts, color and Rf value are consistent.
2, assay
By three batches of pilot scale checking dichromatism coreopsis extract test samples, the method that seminar sets up is measured its Content of Chlorogenic Acid and general flavone content, and result is listed table 4 in.
Table 4. dichromatism coreopsis extract lyophilized powder Content of Chlorogenic Acid and determination of total flavonoids result
3, finger printing
Chromatographic condition Shim-pack VP-ODS chromatographic column (250mm * 4.6mm, 5 μ m); Flow velocity 1.0ml/min; Detect wavelength 280nm and 350nm; Column temperature: 37 ℃; Sample size 10 μ l; Mobile phase A: 1000ml water adds 20ml glacial acetic acid and adds 55ml acetonitrile; Mobile phase B: acetonitrile, gradient elution program is as table 5.
Table 5. gradient elution program
Get respectively three batches of need testing solutions and mixed each 10 μ L sample introductions of mark, record chromatogram (Fig. 5), I is the finger printing that 280nm wavelength detects, II is the finger printing that 350nm wavelength detects, A is mixed mark, be followed successively by 1--chlorogenic acid, 2--Flavanomarein, 3--coreopsin, 4--marein, B is that 20121212 lot number samples, C are that 20121215 lot number samples, D are 20121217 lot number samples.
4, conclusion
According to document and pharmacodynamic experiment result, active component in dichromatism Coreopsis basalis is flavone compound, on laboratory process basis, take general flavone content and finger printing as index, carry out pilot process optimization, after determining optimised process, verify three batches, after lyophilization, obtain dichromatism Coreopsis basalis xanthin lyophilized powder, its Content of Chlorogenic Acid is 1%, general flavone content is 40%, pass through assay, thin layer is differentiated and finger printing, show that pilot process is stable, finger printing comparison with dichromatism Coreopsis basalis medical material, wherein main compound does not change (referring to accompanying drawing 6: detect wavelength 280nm, peak 2 is chlorogenic acid, peak 3 is Flavanomarein, peak 5 is coreopsin, peak 7 is marein).
Embodiment 8:
Application experiment with the dichromatism Coreopsis basalis alcohol extract of extraction process gained of the present invention in medicine
Object: observe the effect of Xinjiang dichromatism Coreopsis basalis alcohol extraction to spontaneous hypertensive rat blood pressure and myocardial remodelling, promote it further to develop.Method: intervene to respectively the dichromatism Coreopsis basalis ethanol extract and positive drug captopril, the Cortex Eucommiae plain presser that obtain with pilot scale 4 weeks the spontaneous hypertensive rat of blood pressure stabilization, adopt non-invasive blood pressure measuring to observe its action effect to blood pressure; Application of radiation immunization is measured the variation of its blood plasma angiotensin I, Endothelin and calcitonin gene-related peptide; The variation that application euzymelinked immunosorbent assay (ELISA) is measured malonaldehyde (MDA), glutathion peroxidase (GSH-PX), inducible nitric oxide synthase (iNOS) content in rat blood serum.
1 material:
1.1 experiment materials: Xinjiang dichromatism Coreopsis basalis ethanol extract pilot scale lyophilized powder.
1.2 laboratory animals:
Male spontaneous hypertensive rat (SHR),, buys from Beijing Vital River Experimental Animals Technology Co., Ltd. by 40; Male Wistar rat,, buys from Beijing Vital River Experimental Animals Technology Co., Ltd. the quality certification number: SCXK (capital) 2012-0001 by 8; Feeding environment illumination 12 hours/day, 21 ± 2 ℃ of temperature, humidity 45-55%.
1.3 experiment reagents:
Captopril tablets: Sino-U.S. Shanghai Shi Guibao pharmaceutical Co. Ltd, specification: 12.5mg lot number: 1211031;
Cortex Eucommiae plain presser: Guizhou San Rentang pharmaceutcal corporation, Ltd, specification: 300mg lot number: 20121206;
Glutathion peroxidase enzyme linked immunological kit: Bioengineering Research Institute's specification is built up in Nanjing: 96T lot number: 201305;
Malonaldehyde enzyme linked immunological kit: Bioengineering Research Institute's specification is built up in Nanjing: 96T lot number: 201305; Inducible nitric oxide synthase enzyme linked immunological kit: Bioengineering Research Institute's specification is built up in Nanjing: 96T lot number: 201305;
Calcitonin-gene-related peptide radioimmunoassay kits: Beijing North biotechnology research institute specification: 100 pipe lot numbers: 130620;
Endothelin radioimmunoassay kits: Beijing North biotechnology research institute specification: 100 pipe lot numbers: 130420; AngiotensinⅡ radioimmunoassay kits: Beijing North biotechnology research institute specification: 100 pipe lot numbers: 130420;
1.4 experimental apparatus:
BP-98A non-invasive blood pressure measuring, Beijing Ruan Long Bioisystech Co., Ltd;
Microplate reader, Austria, model: Infinite F50;
Considerable low-temperature centrifuge: Hunan, Changsha instrument centrifuge instrument company limited model: TDL-5M;
Refrigerated centrifuge: Zhong Jia branch company of Keda Innovation Co., Ltd model: KDC-2046;
Analytical balance: prunus mume (sieb.) sieb.et zucc. Teller-Tuo Lede model: AB204-N;
Electronic balance: Zhongshan city Heng Xin Electronics Co., Ltd.;
Constant-temperature incubation case: SANYO GS electrical equipment
γ radiation immunity arithmometer: Zhong Jia branch company of Keda Innovation Co., Ltd model: GC-2016;
2 methods
2.1 animal grouping and administrations:
The SHR rat adaptability that be 11 weeks age in week was fed after 1 week, within continuous two days, measure rat blood pressure, the blood pressure of two days is got to average, then according to each rat blood pressure value equilibrium, be divided at random 6 groups, be respectively: (6 of model group (6), Cortex Eucommiae sheet groups, 187.5mg/kg), Captopril group is (7,4mg/kg), low dose group is (7,0.1-0.8g/kg), middle dosage group is (7,0.4-1.2g/kg), high dose group is (7,0.8-2.4g/kg), 8 Wistar rats are blank group.
Each organizes feeding normal diet, freely intakes.Each is organized rat every morning filling and feeds once, and gavage volume is 1ml/100g.Gastric infusion is 4 weeks continuously.
2.2 observation items, detection index and method:
Observe behavior, the feed drinking-water situation of respectively organizing rat every day, record weekly body weight once.Within every 5 days, adopt non-invasive blood pressure measuring Measure blood pressure once, all carry out in the afternoon blood pressure measurement.During operation, first rat is put in muff and heated 5min left and right, then continuous measurement is 5-6 time, averages as the pressure value of this rat.
Intervene after 4 weeks the pentobarbital sodium solution intraperitoneal injection of anesthesia of 2.5 ﹪.Abdominal aortic blood (3 parts, a anticoagulant adds 8-hydroxyl azoles quinoline and dimercaptopropanol, BAL, a anticoagulant does not add 8-hydroxyl azoles quinoline and dimercaptopropanol, BAL, the 3rd part of not anticoagulant), separated plasma and serum ,-80 ℃ of preservations.
The content of angiotensin (Ang) II in employing measured by radioimmunoassay blood plasma, endothelin level (ET), calcitonin-gene-related peptide.Application euzymelinked immunosorbent assay (ELISA) is measured malonaldehyde (MDA), glutathion peroxidase (GSH-PX), inducible nitric oxide synthase (iNOS) content in rat blood serum.
Isolating cardiac, heart cold saline lavation by taking out, cuts off atrium and right ventricular free wall along atrioventricular ring, filter paper suck dry moisture, using remaining interventricular septum and left ventricular free wall as left ventricular mass, the ratio of left ventricular mass and body weight is as left ventricular mass index.Separated kidney and liver organization.Weigh and calculate organ index.
Each index requires to operate in strict accordance with test kit description.(in Table 6-9) radioimmunoassay kits carries out in Nuclear Medicine Department of No.1 Hospital Attached to Xinjiang Medical Univ..
Table 6 enzyme linked immunological kit operating procedure
Table 7 angiotensinⅡ radioimmunoassay kits operating procedure
Table 8ET radioimmunoassay kits operating procedure
Table 9CGRP radioimmunoassay kits operating procedure
2.3 statistical analysis methods
Adopt SPSS17.0 software kit to process data.Result with
represent, measurement data uses the variance analysis of one factor analysis of variance and repeated measure to compare, and with P<0.05, has statistical significance.
3 results
3.1 impacts of Xinjiang dichromatism Coreopsis basalis ethanol extract on SHR rat blood pressure
Statistical result showed, on blood pressure, impact has statistical significance (F=103.7 to time factor, P<0.01), on blood pressure, impact has statistical significance (F=959.6 to grouping factor, P<0.01) and the reciprocal action of grouping and time factor also there is statistical significance (F=8.2, P<0.01).
Before administration, compare with blank group, model group blood pressure is apparently higher than blank group (P<0.01); And other respectively organize rat blood pressure and the equal no difference of science of statistics of model group (P>0.05).Intervene after 5 days, compare with model group, Captopril group blood pressure obviously decline (P<0.01); The high, medium and low dosage group of dichromatism Coreopsis basalis ethanol extract and positive drug Cortex Eucommiae sheet group blood pressure and model group no difference of science of statistics (P>0.05).Intervene after 10 days, Captopril group, the middle and high dosage group of dichromatism Coreopsis basalis ethanol extract rat blood pressure have obvious decline compared with model group, have statistical significance (P<0.01); Positive drug Cortex Eucommiae sheet group blood pressure also has decline (P<0.05); And dichromatism Coreopsis basalis ethanol extract low dose group blood pressure drops not obvious (P>0.05).Intervene after 15 days, in positive drug Captopril group, the middle and high dosage group of dichromatism Coreopsis basalis ethanol extract, rat blood pressure is decreased significantly compared with model group, has statistical significance (P<0.01); Positive drug Cortex Eucommiae sheet group blood pressure also has decline (P<0.05); And dichromatism Coreopsis basalis ethanol extract low dose group blood pressure drops not obvious (P>0.05).Since the 20th day to experiment, finish, each intervention group rat blood pressure continuous decrease, compares and all have obvious decline (P<0.01), and at experimental session, model group rat blood pressure is continually and steadily at certain level with model group.(in Table 9)
Table 10: the impact of Xinjiang dichromatism Coreopsis basalis ethanol extract on SHR rat blood pressure
Note: compare " * " with model group and represent P<0.05, " * * " represents P<0.01; Compare " ◆ " with blank group and represent P<0.05, " ◆ ◆ " represent P<0.01
3.2 impacts of Xinjiang dichromatism Coreopsis basalis ethanol extract to SHR rat left ventricle index (LVMI, mg/g) and each organ index
Intervene after 4 weeks, compare with blank group, the left ventricle index of model group rat obviously raises (P<0.01), and positive drug Captopril group and dichromatism Coreopsis basalis ethanol extract high dose group rat left ventricle index are decreased significantly (P<0.01) compared with model group; In dichromatism Coreopsis basalis ethanol extract, dosage group rat left ventricle index also declines to some extent, and decline also has statistical significance (P<0.05); And though positive drug Cortex Eucommiae sheet group and dichromatism Coreopsis basalis ethanol extract low dose group rat left ventricle index have decline, not statistically significant (P>0.05).(in Table 2-2)
Compare model group rat heart index obviously raise (P<0.01) with blank group; Positive drug Captopril group rat heart index obviously reduces (P<0.01) compared with model group; The cardiac index of dichromatism Coreopsis basalis ethanol extract high dose group rat is compared with model group, has significant difference (P<0.05); Positive drug Cortex Eucommiae sheet group, dichromatism Coreopsis basalis ethanol extract low dose group and middle dosage group and model group no difference of science of statistics.(in Table 2-2)
Compare with blank group, model group rat liver exponential sum renal index all raises to some extent, but only has liver index to have significant difference (P<0.01), and renal index difference not significantly (P>0.05); Other each intervention group rat liver exponential sum renal indexes all decline to some extent compared with model group, but no difference of science of statistics, and also in dichromatism Coreopsis basalis ethanol extract, dosage rat fall is maximum.(in Table 10)
Table 11: the impact of Xinjiang dichromatism Coreopsis basalis ethanol extract on SHR rat LVMI and each organ index
Note: compare " * " with model group and represent P<0.05, " * * " represents P<0.01; Compare " ◆ " with blank group and represent P<0.05, " ◆ ◆ " represent P<0.01
The impact of 3.3 Xinjiang dichromatism Coreopsis basalis ethanol extract on Endothelin (ET), angiotensin (A II), calcitonin-gene-related peptide (CGRP) content in SHR rat plasma
Intervene after 4 weeks, compare with blank group, the content of A II significantly raise (P<0.01) in model group rat plasma.Compare with model group, in other intervention group rat plasmas, the content of A II obviously reduces, and has remarkable statistical significance (P<0.01).(in Table 2-3)
Compare with blank group, in model group rat blood serum, the content of ET obviously raises, and has statistical significance (P<0.01).Compare with model group, in positive drug Captopril group rat plasma, the content of ET significantly declines, and has statistical significance (P<0.01).In dichromatism Coreopsis basalis ethanol extract high dose group rat plasma, the content of ET declines to some extent, has statistical significance (P<0.05).And the content of ET declines not obviously in positive drug Cortex Eucommiae sheet group, low, the middle dosage group of dichromatism Coreopsis basalis ethanol extract rat plasma, do not there is statistical significance (P>0.05).(in Table 2-3)
In model group rat blood serum, the content of CGRP is few compared with blank group, due to the large cause of standard deviation, declines and does not have statistical significance (P>0.05).But do not there is statistical significance (P>0.05).Compare with model group, in other intervention group rat plasmas, the content of CGRP is all in rising trend, and dichromatism Coreopsis basalis ethanol extract has dosage correlation to the impact of CGRP content, but does not have statistical significance (P<0.05).(in Table 11)
Table 12: the impact of Xinjiang dichromatism Coreopsis basalis ethanol extract on ET, A II, CGRP content in SHR rat plasma
Note: compare " * " with model group and represent P<0.05, " * * " represents P<0.01; Compare " ◆ " with blank group and represent P<0.05, " ◆ ◆ " represent P<0.01
The impact of 3.4 Xinjiang dichromatism Coreopsis basalis ethanol extract on malonaldehyde (MDA), glutathion peroxidase (GSH-PX), inducible nitric oxide synthase (iNOS) content in SHR rat blood serum
Intervene after 4 weeks, compare with blank group, the content of MDA significantly raise (P<0.01) in model group rat blood serum.Positive drug Captopril group is compared MDA content with model group obviously reduces (P<0.01), and dichromatism Coreopsis basalis alcohol extraction object height, middle dosage group are compared MDA content with model group obviously reduces (P<0.01).And positive drug Cortex Eucommiae sheet group is compared with model group with dichromatism Coreopsis basalis low dose group, though decrease, no difference of science of statistics (P>0.05).(in Table 12)
In model group rat blood serum, more blank group of the content of GSH-PX is low, and has statistical significance (P<0.05).Compare with model group, in each intervention group rat blood serum, the content of GSH-PX all raises to some extent, and dichromatism Coreopsis basalis ethanol extract respectively organizes rising amplitude and have dosage correlation, but does not have statistical significance (P>0.05).(in Table 2-4)
In model group rat blood serum, more blank group of the content of iNOS significantly increases, and has statistical significance (P<0.01).In positive drug Captopril group and dichromatism Coreopsis basalis alcohol extraction high dose group rat blood serum, the content of iNOS is starkly lower than model group, and has statistical significance (P<0.01).And the content of iNOS in Cortex Eucommiae sheet group, low, the middle dosage group of dichromatism Coreopsis basalis ethanol extract rat blood serum also decreases, but do not there is statistical significance (P>0.05).(in Table 12)
Table 13: the impact of Xinjiang dichromatism Coreopsis basalis ethanol extract on MDA, GSH-PX, iNOS in SHR rat blood serum
Note: compare " * " with model group and represent P<0.05, " * * " represents P<0.01; Compare " ◆ " with blank group and represent P<0.05, " ◆ ◆ " represent P<0.01
Result: intervene after 4 weeks, each intervention group rat blood pressure all has decline, and positive drug Captopril group, dichromatism Coreopsis basalis ethanol extract high dose group and middle dosage group rat blood pressure obviously decline; Dichromatism Coreopsis basalis alcohol extraction object height, middle dosage and captopril can obviously reduce the content of ET and A II in rat plasma, also can increase the content of CGRP in blood plasma; Dichromatism Coreopsis basalis alcohol extraction object height and captopril can obviously reduce the content of MDA and iNOS in rat blood serum, increase the content of GSH-PX in serum; Dichromatism Coreopsis basalis alcohol extraction object height and captopril also can reduce rat left ventricle exponential sum cardiac index simultaneously.
Conclusion: dichromatism Coreopsis basalis ethanol extract has certain effect to spontaneous hypertensive rat, can improve spontaneous hypertensive rat vivo oxidation stress level and vascular endothelial function.
The foregoing is only the preferred embodiments of the present invention, be not limited to the present invention, although the present invention is had been described in detail with reference to previous embodiment, for a person skilled in the art, its technical scheme that still can record aforementioned each embodiment is modified, or part technical characterictic is wherein equal to replacement.Within the spirit and principles in the present invention all, any modification of doing, be equal to replacement, improvement etc., within all should being included in protection scope of the present invention.
Claims (9)
1. an extraction process for xanthin in dichromatism Coreopsis basalis, is characterized in that: comprise the steps:
1) soak: by the fragmentation of dichromatism Coreopsis basalis dried floral, by purity, be 42%-68% alcohol solution dipping 8-17 hour afterwards;
2) extract: 1-5 hour that the mixed material in step 1) is refluxed at 43-67 ℃, filter to obtain extracting solution for the first time, then continue to add 42%-68% alcoholic solution in filter cake, at 43-67 ℃, extract 1-2 time, each 1-5 hour;
3) concentrated: said extracted liquid is merged, adopt secondary concentration technique, be specially and control material at 40-70 ℃, twice flash distillation or first concentrating under reduced pressure again flash distillation once, obtain final concentrated extracting solution, obtain lyophilized powder after lyophilization.
2. the extraction process of xanthin in dichromatism Coreopsis basalis according to claim 1, is characterized in that: twice flash distillation of described secondary concentration process using, and concrete operations are as follows:
A. the repeatedly extracting solution once concentration: by step 2) merges, and by Multistage flash evaporator, concentrates, and controlling solution temperature is 40-60 ℃, and flow velocity is 50-200ml/min, and vacuum is 0.02-2MPa, concentrated solution for the first time;
B. secondary concentration: will concentrated solution to be by flash distillation instrument for the first time, control solution temperature is 50-70 ℃, flow velocity is 50-200ml/min, obtains final concentrated extracting solution.
3. the extraction process of xanthin in dichromatism Coreopsis basalis according to claim 1, is characterized in that: the first concentrating under reduced pressure of described secondary concentration process using is flash distillation technique once again, and concrete operations are as follows:
A. the repeatedly extracting solution once concentration: by step 2) merges, concentrating under reduced pressure, and temperature of charge is controlled at 40-70 ℃, concentrated 7-9 hour under vacuum 0.02-2MPa;
B. secondary concentration: concentrated solution concentrates in Multistage flash evaporator for the first time, and solution temperature is controlled at 50-70 ℃, vacuum 0.02-2MPa.
4. according to the extraction process of xanthin in the dichromatism Coreopsis basalis described in claim 1-3 any one, it is characterized in that: in described step 1), the mass ratio of described dichromatism Coreopsis basalis dried floral and alcoholic solution is 1:2-20; The purity of described alcoholic solution is 50%-60%.
5. according to the extraction process of xanthin in the dichromatism Coreopsis basalis described in claim 1-3 any one, it is characterized in that: in described step 1), soak 12 hours.
6. according to the extraction process of xanthin in the dichromatism Coreopsis basalis described in claim 1-3 any one, it is characterized in that: described step 2), the purity of the alcoholic solution at every turn adding is 50-60%, and the quality of the alcoholic solution that addition adds with step 1) equates.
7. according to the extraction process of xanthin in the dichromatism Coreopsis basalis described in claim 1-3 any one, it is characterized in that: described step 2), each temperature of charge of purifying is 55 ℃.
8. the extraction process of xanthin in dichromatism Coreopsis basalis according to claim 3, is characterized in that: the alcohol solution dipping that is 55% by purity in described step 1) 12 hours; Described step 2) in, the temperature of charge extracting is for the first time 55 ℃, and reflux, extract, 2.5 hours is filtered to obtain extracting solution for the first time, and it is 55% ethanol dope that gained filter cake continues to add purity, and temperature of charge is reflux, extract, 2.5 hours at 55 ℃; Described step 1) and step 2) in add alcoholic solution quality be 15 times of dichromatism Coreopsis basalis dried floral quality.
9. dichromatism Coreopsis basalis alcohol extract according to claim 1 is in the application for the treatment of spontaneously hypertensive.
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