CN1634324A - Dahurian rhododendron leaf extract and extracting method thereof - Google Patents
Dahurian rhododendron leaf extract and extracting method thereof Download PDFInfo
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Abstract
The invention relates to the extracting process for Dahurian rhododendron leaf extract and extracting method, wherein the extract comprises, flavones 30-90% (measured by using hyperin as the representing compostion), hyperin compound 6.3-42%, farrerol 0.4-8%, quercetin compound 0.7-7%, the extraction method comprises employing polyamide resin separating column, clarifying the liquid with clearing agent and purifying.
Description
Technical field
The present invention relates to a kind of Rhododendron dahuricum extract and extracting method thereof, a kind of Flavonoid substances that particularly extracts in the Folium Rhododendri Daurici and the extracting method of this flavonoid class material.
Background technology
Folium Rhododendri Daurici is the dried leaves (Rhododendron dauricum L) of ericad Folium Cuculus polioephalus, claims to reach son perfume, Rhododendron simsii Planch., Folium Rhododendri Mucronulati etc. again.Be the natural drug very widely of pharmacological action simply, the effect that the Chinese medicine Folium Rhododendri Daurici has cough-relieving, eliminates the phlegm, relievings asthma is used for the treatment of acute and chronic bronchitis, asthma.Its active ingredient is a Flavonoid substances.In the Folium Rhododendri Daurici product of listing at present, the preparation that is used as medicine with Folium Rhododendri Daurici crude drug or crude extract is arranged, as " Oleum Rhododendri Daurici drop pill ", " antitussive and antiasthmatic syrup ".The compound preparation that is used as medicine with Folium Rhododendri Daurici has " the sudden and violent red capsule of a kind of reed mentioned in ancient books " (main component: Folium Rhododendri Daurici, Radix Scutellariae, Cortex Syringae).
Chinese medicine normally is made up of the multiclass composition simply, such as saccharide, amino acids, protide, Coumarins, flavonoid, tannin class, alkaloids, terpenoid and steroidal class or the like.Conventional extracting method (as with decocting or ethanol extraction) can extract its major part, but the not necessarily all composition classifications of its real activated material, and usually only be wherein a group composition onset.Therefore obtain a kind ofly as far as possible invalid components being removed, and, be the emphasis and the difficult point of Chinese medicine modern study the high-purity extract that effective ingredient keeps as far as possible.To the further refining purification of its effective ingredient is that Chinese medicine is realized modern inexorable trend, and does not see the report that the high-purity Rhododendron dahuricum extract is arranged in domestic and international disclosed document and the patent.
Active ingredient in the Folium Rhododendri Daurici is a Flavonoid substances as previously mentioned.Flavonoid substances, belong to flavonoid, flavanol compound, flavanoid, flavanone alcohols, anthocyan, two benzene pyrrones, osajin, chalcones, dihydrochalcone-type on the chemical constitution, the combination of one or more of glycosides that the monoploid of the chemical compound of aurones class, homoisoflavone class or polymer compounds and they and monosaccharide or oligosaccharide form or other forms of derivant.At present Flavonoid substances is often only characterized its character with " general flavone content " this more upper notion.To profound level, such as in the Flavonoid substances be which active ingredient is worked actually, understanding never such as these content effectively part, do not make definite sign.The mensuration of general flavone content normally adopts a kind of Main Ingredients and Appearance that wherein contains as representative, adopts a kind of general detection method that a certain big constituents in the extract is all calculated by the molecular weight of representing composition; Therefore the general flavone content that records is a kind of relative value, and it is chosen the influence of the molecular weight, detection method etc. of representation compound.Therefore, only Flavonoid substances is characterized with " general flavone content ", be difficult to reflect different Flavonoid substances (as, Flavonoid substances that from different plants, extracts or the Flavonoid substances that from same kind of plant, extracts with distinct methods) true characteristics, its concrete flavone compound that comprises of different Flavonoid substances (even general flavone content is identical or close) has a great difference, and the pharmacology that it reacted, the property of medicine have huge difference.The extracting method difference, even same kind of plant, the Flavonoid substances of gained is also different.Therefore, from a certain plant, extract the Flavonoid substances that obtains with a kind of extracting method that is different from prior art fully, angle from profound level such as concrete formation, content or its physical-chemical parameters of Flavonoid substances, be exactly a kind of novel substance, can not belong to " Flavonoid substances " big class because of it and deny that it is a novel substance.
The flavone compound that is present in the Folium Rhododendri Daurici plant that prior art has identified has: hyperin, isohyperoside, farrerol, 8-demethylfarrerol, Quercetin, avicularin, kaempferol, myricetin, gossypetin, taxifolin etc.The extract (Flavonoid substances) that each new extracting method obtains is owing to comprise different flavone compounds, different content, and there are very big-difference in its pharmacology, the property of medicine, all are a kind of new materials.
The disclosed Chinese patent relevant with Rhododendron dahuricum extract relates generally to Folium Rhododendri Daurici crude drug or the application of crude extract aspect medicine, comprise following patent: " lozenge for curing chronic bronchitis and preparation method thereof " (CN1265907A), " rhododendron mariesii injection and preparation method thereof " (CN1265906A), " dahurian rhododendron leaf oil aerosol and preparation method thereof " (CN1097301A), " sudden and violent red capsule against cough of a kind of reed mentioned in ancient books and preparation method thereof " (CN1165014A) etc.What above-mentioned technology related to is Folium Rhododendri Daurici crude drug or crude extract, and its content is all less than 10% (w/w), and is different with the feature of extract of the present invention.
The Folium Rhododendri Daurici total flavonoids method for preparing extractive mainly still is alcohol deposition method behind decocting method, the decocting, alcohol extracting method, ethanol extract from water precipitation etc. at present, its paste-forming rate is higher (generally more than 20%, minimum also more than 15%), particularly the detached dowel material therefor select for use improper, be difficult to the saccharide in the Rhododendron dahuricum extract, tannin class, amino acids, and the partial invalidity flavones ingredient remove, further increased paste-forming rate; These extracts often have that hygroscopicity is big, dose is big and the unconspicuous shortcoming of drug effect, and the extract that therefore existing extracting method obtains mostly is crude extract; Here the quality (as 3g) of the final dry extract for preparing of the dry quality of medicinal material of the paste-forming rate unit of being meant (as the 100g medical material).Paste-forming rate is more little, shows that the refining degree of extract is high more; But have only paste-forming rate not all right, because if the extract of preparation does not contain active component, then paste-forming rate is more little, and possible pharmacologically active can be more little.
Summary of the invention
The present invention is directed to the high-purity extract that can't definitely be characterized at present, thereby the Folium Rhododendri Daurici problem of all being used as medicine with crude drug or crude extract, provide a kind of definite sign, at the Folium Rhododendri Daurici that outstanding role is pharmaceutically arranged (high-purity) extract.
The present invention also at the extracting method paste-forming rate height of existing Rhododendron dahuricum extract, extract obtained be the problem of crude extract all, a kind of extracting method of high-purity Rhododendron dahuricum extract is provided.
The present invention adopts following technical scheme to realize: a kind of Rhododendron dahuricum extract, and (1) as representing composition that this extract is measured, general flavone content 30% in the extract with hyperin---and 90%; (2) in the extract, the content 6.3% of hyperin chemical compound---42%; (3) in the extract, the content 0.4% of farrerol chemical compound---8%; (4) in the extract, the content 0.7% of Quercetin chemical compound---7%.
Rhododendron dahuricum extract of the present invention is a kind of novel substance that extracts from Folium Rhododendri Daurici first with brand-new extraction method, with the content of general flavone content, hyperin chemical compound, farrerol chemical compound and Quercetin chemical compound it is carried out definite sign.At present, the Flavonoid substances that characterizes with above-mentioned characteristic manner is not seen the report of pertinent literature.
Adopt in the Folium Rhododendri Daurici the higher and middle flavone compound of molecular weight---hyperin---of content as representing composition (molecular weight is 466), adopt known aluminum chloride (AlCl
3) and potassium acetate (KAc) as the general developer of flavone, the 400-420nm place on the UV, visible light spectrophotometer measures, and avoided like this because the difference to the extract definition that the content assaying method disunity causes.Owing to the Chinese crude drug extract is changed by the Chinese crude drug place of production, collecting season and extracting method different have certain variation, after Rhododendron dahuricum extract was repeatedly measured, general flavone content was 30% in the Folium Rhododendri Daurici total flavonoids extract of the present invention---90%.Above-mentioned content assaying method can be measured out with the flavone of different parent nucleus (aglycon).
Extractive of general flavone of the present invention can further adopt conventional plant chemical method purification, for example various column chromatographies (decompression column chromatography, polydextran gel, reversed phase column chromatography, preparation property high performance liquid chromatogram etc.), these methods are self-explantory for those skilled in that art.Therefore extract of the present invention has defined four indexs, no matter other monomeric compound content how, so long as extract from the plant Folium Rhododendri Daurici, and four indices is in above-mentioned scope, and then this extract is in protection scope of the present invention.
Above-mentioned content assaying method can be measured out with the flavone of different parent nucleus (aglycon).Studies show that further hyperin, farrerol and Quercetin have the typical pharmacology feature of Folium Rhododendri Daurici.
The Rhododendron dahuricum extract that the present invention characterized is a kind of novel substance that extracts from Folium Rhododendri Daurici first, and not only refining degree height, and kept the pharmacologically active of most of Folium Rhododendri Daurici has cough-relieving, relievings asthma, expectorant effect etc.And dose is much smaller than Folium Rhododendri Daurici runic thing, its pharmacology, the property of medicine have been compared remarkable difference with Crude extract of Rhododendron dahuricum, characteristics with modern Chinese medicine " dosage is little, toxicity is little, side effect is little, " " efficient, quick-acting, long-acting " have satisfied field of medicaments for a long time to demand efficient, the high-purity Rhododendron dahuricum extract.Therefore Rhododendron dahuricum extract provided by the invention has high value at field of medicaments, can be used in treatment and prevents diseases such as acute and chronic bronchitis, asthma.
Rhododendron dahuricum extract of the present invention is a kind of highly purified extract, and has significant pharmacologically active; The feature of extract that adopted suitable index definition.Why the advanced preparation of Chinese medicine is difficult to exploitation, is difficult to accomplish taking convenience, easy to carry, and its key is exactly that dosing is excessive, is difficult to be prepared into advanced preparation.Highly purified Rhododendron dahuricum extract of the present invention has purposes widely in this regard, has good drug development prospect, for the further exploitation of extract is laid a good foundation.Extract of the present invention has significant cough-relieving, effect such as relieving asthma, eliminate the phlegm, being expected on the one hand provides a kind of new treatment to select to the patient who suffers from these diseases, for more effectively utilizing the Folium Rhododendri Daurici plant resources, the application of widening the Folium Rhododendri Daurici plant is also significant on the other hand.
Rhododendron dahuricum extract pharmacodynamics of the present invention detects
(1) the extract antitussive action of Folium Rhododendri Daurici total flavonoids experiment
Experimental technique: Kunming kind white mice, be divided into matched group, extract group and crude extract group, extract group and the (administration 3 days of crude extract group gastric infusion, every day 1 time), 1h after last administration places inverted 500ml beaker respectively with three groups of mices, in put a cotton balls, draw ammonia (25%～28%) 0.2ml with the 1ml syringe, inject cotton balls, be inverted beaker rapidly.Cough number of times in cough latent period of observed and recorded mice and the 2min.
Experimental result: (1) is compared with the blank group, and the cough latent period of extract administration group obviously prolongs, in the 2min
The cough number of times obviously reduces.
(2) compare with the crude extract group, the dose of extract administration group significantly reduces and cough suppressing effect is better than slightly
Put forward the thing group.
Group dosage number of animals incubation period (s) cough number of times (inferior/2min)
Blank group-10 18.60 ± 5.71 26.13 ± 3.91
Extract 67mg/kg 10 35.48 ± 7.86 14.78 ± 5.07
Crude extract 600mg/kg 10 21.36 ± 9.96 22.50 ± 10.47
(2) the extract phlegm-dispelling functions of Folium Rhododendri Daurici total flavonoids experiment
Experimental technique: Kunming kind white mice, gastric infusion (administration 3 days, every day 1 time), 0.5h after last administration, by lumbar injection 0.25% phenol red liquid 0.5ml, injection back 30min takes off cervical vertebra and puts to death, and faces upward the position and is fixed on the operation plate, cuts off neck center skin, separate trachea, No. 7 syringe needles that under larynx head polished insert about 3mm in the trachea, after fixing with the toe-in bundle, draw 5%NaHCO with the 1cm syringe
30.5ml, by syringe needle lavation respiratory tract 3 times (not stopping) back and forth at every turn, irrigating solution is injected the 5ml volumetric flask, repeat 3 times, use 5%NaHCO
3Standardize solution is surveyed its ultraviolet absorptivity value at the 546nm place, calculates its concentration value according to standard curve.
Experimental result: (1) is compared with the blank group, and the respiratory tract phenol red concentration of extract administration treated animal significantly raises.
(2) compare with the crude extract group, the dose of extract administration group significantly reduces and expectorant effect is better than slightly
Put forward the thing group.
Group dosage number of animals phenol red concentration (μ g/ml)
Blank group-10 1.794 ± 0.270
Extract 67mg/kg 10 2.863 ± 0.607
Crude extract 600mg/kg 10 2.142 ± 0.312
(3) the extract antiasthmatic effect of Folium Rhododendri Daurici total flavonoids experiment
Experimental technique: Cavia porcellus, gastric infusion (administration 7 days, every day 1 time), 1h after last administration tests Cavia porcellus respectively according to order before and after the administration and draws and breathe heavily incubation period, surpasses 360 seconds persons, is designated as 360 seconds, and the number of animals of twitching takes place in record, relatively changes before and after the medication.
Experimental result: (1) is compared with the blank group, and significant prolongation incubation period is breathed heavily in drawing of extract administration treated animal.
(2) compare with the crude extract group, the dose of extract administration group significantly reduces and the effect of relievining asthma is better than slightly
Put forward the thing group.
Group dosage number of animals is drawn and is breathed heavily incubation period (s)
Blank group-8 66.50 ± 7.73
Extract 11.5mg/kg 8 80.50 ± 8.50
Crude extract 300mg/kg 8 70.24 ± 7.78
The extracting method of Rhododendron dahuricum extract, comprise that detached dowel separates in the preparation, upper prop liquid of Folium Rhododendri Daurici upper prop liquid, to eluting, the eluent of detached dowel concentrate, dry; Detached dowel adopts polyamide, and after upper prop liquid adsorbed by polyamide, first water or weight concentration were the solvent wash of 5%-25%, and eluent is abandoned or adopted; The reuse weight concentration is 40～90% solvent elution, there is not the flavone reaction to eluent and aluminum chloride, eluent is standby, and wherein the used solvent of eluting is one or more aqueous solutions with the arbitrary proportion combination of rudimentary alcohol, ketone, ester (as ethanol, methanol, acetone, ethyl acetate).The present invention is preferred polyamide is as column material, can with the saccharide in the Folium Rhododendri Daurici upper prop liquid, tannin class, amino acids, and composition such as partial invalidity flavones ingredient remove (being that polyamide does not adsorb these materials), and keep effectively Flavonoid substances (this part of reservation is current not fully realizing) of effective ingredient and part.Therefore, extracting method of the present invention makes the paste-forming rate of extract be reduced (below 3%) (enrichment factor is improved).Choosing and concentration of eluent directly influences contained component of final extract and content, determines promptly whether extracting method can obtain the extract of aforementioned characteristic; The determining of eluent that the present invention is used and concentration and polyamide resin column draws on a large amount of experiment basis; compare with existing extracting method and to have outstanding substantive distinguishing features; and bring marked improvement (be embodied in and reduced paste-forming rate, obtained the present invention's highly purified extract required for protection).
Folium Rhododendri Daurici upper prop liquid contains the granule insoluble matter of a large amount of colloidal substances, suspension etc. usually after leaving standstill, these materials can seriously stop up resin column, influence the life-span of resin column; Secondly contain a large amount of strong adsorbability materials in Folium Rhododendri Daurici upper prop liquid, polyamide is bigger to the absorption affinity of this type of material, should remove in advance, otherwise also can influence life-span of resin column.For this reason, the present invention is before upper prop liquid upper prop, add in upper prop liquid that clarifier is clarified, purification process, clarifier be in wherein a kind of solution of chitin kind, protide, ZTC clarifier or chitin kind, protide, the ZTC clarifier any two with 1-2: the solution that the 1-2 ratio is made, chitin kind, protide, ZTC clarifier separately or the consumption of two kinds of combinations account for the 0.6%-2.4% (g/g) of Folium Rhododendri Daurici consumption.The clarifier consumption does not reach the purpose of clarification, purification less, and the clarifier consumption too much can cause the loss of required active ingredient.
Because the complexity of upper prop liquid, a kind of clarifier usually can not reach clarifying effect preferably, needs to unite use more; But adopt wherein any also can be with upper prop liquid clarification, just effect is slightly different, influence is little.Therefore the invention reside in to adopt clarifier that Folium Rhododendri Daurici upper prop liquid is carried out the upper prop pre-treatment.Protide clarifier among the present invention is meant gelatin.Chitin kind clarifier among the present invention is meant chitin, or is the clarifier that the various derivants (as chitosan, also claiming chitosan) of raw material are made with the chitin.The ZTC clarifier is the existing product of public offering on the market, and it comprises A component and B component.
The polyamide that the present invention adopts can be the resin of various particle size ranges, and the resin that also can be various grain shapes is as Powdered, graininess and spherical resin.
Water or weight concentration are the solvent wash of 5%-25%, mainly are to remove the more weak glycoside of excess sample solution, non-adsorbable saccharide, amino acids, absorption affinity and some micromolecule phenolic substances etc., and eluent is abandoned or adopted; Directly operating weight concentration is 40～90% solvent elution then, does not have the flavone reaction until eluent and aluminum chloride; Eluent concentrates, and concentrated solution is carried out drying, can get extractive of general flavone; Water or weight concentration are that the solvent wash of 5%-25% excessively can be washed required active ingredient off, washing is not enough can not non-adsorbable saccharide, amino acids, absorption affinity is more weak glycoside and some micromolecule phenolic substances etc. fully wash off, will influence paste-forming rate.The present invention further optimizes extracting method, is the solvent wash of 5%-25% with the water or the weight concentration of 1-5 times of resin column volume.
Water among the present invention can be the pure water that does not add any material, also can be add suitable modifier as, a spot of acid, alkali or salt, these all are the known shared methods in this area; Weight concentration is that the solvent of 5%-25% is meant among the present invention, is one or more combination of the rudimentary alcohol, ketone, ester etc. of solvent with water, as ethanol, methanol, acetone, ethyl acetate; Also can add suitable modifier as, a spot of acid, alkali or salt; Weight concentration is that the solvent of 40%-90% is meant among the present invention, is one or more combination of the rudimentary alcohol, ketone, ester etc. of solvent with water, as ethanol, methanol, acetone, ethyl acetate; Also can add suitable modifier as, a spot of acid, alkali or salt.Preferred water of the present invention is the remove impurity solvent, and the ethanol of 50%-85% is eluant.
Eluent among the present invention after purified adopts and concentrates, behind the dry technology, can obtain highly purified extract.Wherein concentrate and to adopt methods such as concentrating under reduced pressure, thin film concentration; Drying can adopt technology such as normal pressure oven dry, drying under reduced pressure, spray drying and lyophilization.These methods all are the public methods in this area.
Upper prop liquid preparation of the present invention, adopt public method well known in the art, available moisture or not aqueous methanol, ethanol, acetone, the mixture as solvent of one or more arbitrary proportions of ethyl acetate, n-butyl alcohol, normal hexane or analog, or extract by these solvents and acid, the alkali acidity or the basic solvent that are made into, can pass through methods extractions such as reflux, percolation, supersound extraction, microwave extraction or high pressure extract.The preferred 25-85% alcohol reflux of the present invention.
Can obtain Rhododendron dahuricum extract of the present invention with extracting method of the present invention.Adopt polyamide as separation means, can remove a large amount of impurity, thereby make effective ingredient enrichment, paste-forming rate low, remove impurity and concentrated two procedures have been finished simultaneously, compare with traditional method, production cycle of this method shortens, and product stability strengthens, and can remove the toxic substance in the medical material and the poisonous metal or the heavy metal element of pollution.The preferred clarifier of extracting method of the present invention carries out the pre-treatment of upper prop liquid, can obviously prolong the life-span of detached dowel.
Description of drawings
Fig. 1 is embodiment 1 a hyperin HPLC collection of illustrative plates;
Fig. 2 is embodiment 1 a farrerol HPLC collection of illustrative plates;
Fig. 3 is embodiment 1 a Quercetin HPLC collection of illustrative plates;
Fig. 4 is embodiment 2 hyperin HPLC collection of illustrative plates;
Fig. 5 is embodiment 2 farrerol HPLC collection of illustrative plates;
Fig. 6 is embodiment 2 Quercetin HPLC collection of illustrative plates;
Fig. 7 is embodiment 3 hyperin HPLC collection of illustrative plates;
Fig. 8 is embodiment 3 farrerol HPLC collection of illustrative plates;
Fig. 9 is embodiment 3 Quercetin HPLC collection of illustrative plates;
The specific embodiment
Following specific embodiment is that the present invention is described in detail more, but scope of the present invention is not constituted any restriction.
The preparation of embodiment 1. Rhododendron dahuricum extracts
1. extract
Get Folium Rhododendri Daurici crude drug (dry stem and leaf 300g), pulverize, under 85 ℃-100 ℃ temperature, with the 40% ethanol water reflux, extract, 3 times (3000ml * 3) of 3000ml, each 1hr, merge extractive liquid,, standing over night under the room temperature, filter, go precipitation, filtrate decompression concentrate (55 ℃, 0.08-0.1MPa), to cumulative volume 1800ml, concentrated solution adds 1% aqueous gelatin solution 540ml (be equivalent to 5.4 gram gelatin, can be made into the aqueous gelatin solution of other low concentration, as long as gelatin is fully dissolved), stir, room temperature leaves standstill 4-8hr, and sucking filtration discards residue, supernatant is crossed polyamide resin column (the about 400ml of column volume that handles well, flow velocity 400ml/hr), with the colour developing of aluminum chloride 95% alcoholic solution, whether adsorb saturated with the spray of polyamide thin layer with the conclusive evidence resin column; Subsequently successively with 1200ml water, the 50% ethanol water eluting of 1500ml, the about 400ml/hr of elution flow rate.Collect 50% ethanol water and concentrating under reduced pressure (55 ℃ 0.08-0.1MPa) are done near; Behind the water bath method, 75 ℃ of oven dried 24-72hr must be brown to the yellowish-brown powder, and Rhododendron dahuricum extract is about 6.0g (water content≤5%).
2. content detection:
(1) mensuration of hyperin:
Chromatographic condition: immobile phase ODS post (4.6mm I.D. * 150mm, 5 μ m); Mobile phase: methanol: 0.5% phosphate aqueous solution (transferring PH with triethylamine is 3) (45: 55); Flow velocity: 0.8ml/min; Detect wavelength: 355nm; Sample size: 10-20 μ l, gained HPLC collection of illustrative plates such as Fig. 1 show that wherein retention time (min) is the hyperin that is of 12-13.The hyperin content of Rhododendron dahuricum extract (example 1) is 16.6%.
(2) mensuration of farrerol:
Chromatographic condition: immobile phase ODS post (4.6mm I.D. * 150mm, 5 μ m), mobile phase: methanol: water (60: 40), flow velocity: 1ml/min; Detect wavelength: 296nm; Sample size: 10-20 μ l, gained HPLC collection of illustrative plates such as Fig. 2 show that wherein retention time (min) is the farrerol that is of 16-17.The farrerol content of Rhododendron dahuricum extract (example 1) is 1.1%.
(3) mensuration of Quercetin:
Chromatographic condition: immobile phase ODS post (4.6mm I.D. * 150mm, 5 μ m), mobile phase: methanol: water: glacial acetic acid (50: 50: 2), flow velocity: 1ml/min; Detect wavelength: 370nm; Sample size: 10-20 μ l, gained HPLC collection of illustrative plates such as Fig. 3 show that wherein retention time (min) is the Quercetin that is of 10-11.The quercetin content of Rhododendron dahuricum extract (example 1) is 3.8%.
(4) content of total flavone is measured
The reference substance solution preparation:
It is an amount of that precision takes by weighing hyperin reference substance (120 ℃ of following drying under reduced pressure are to constant weight), puts in the 100ml volumetric flask, and add 60% alcoholic solution and make dissolving in right amount, and rare to scale, shake up.Get the solution that contains anhydrous hyperin 60 μ g among every 1ml.
The need testing solution preparation:
It is an amount of that precision takes by weighing dry this product (embodiment 1), places the 100ml measuring bottle, adds 60% dissolve with ethanol solution, is settled to scale, shakes up, standby.
Assay method: get reference substance gradient concentration solution and need testing solution, put in the 10ml volumetric flask, through AlCl3 and KAc colour developing, behind the 60% ethanol standardize solution, press spectrophotography (2000 editions appendix IVA of Chinese Pharmacopoeia), measure absorbance at the 418nm place, the total flavones content of material is 48.0%.
The preparation of embodiment 2. Rhododendron dahuricum extracts
1. extract
Get the place of production, collecting season is different from the Folium Rhododendri Daurici crude drug (dry stem and leaf 300g) of example 1, pulverize, under 85 ℃-100 ℃ temperature, with the 60% ethanol water reflux, extract, 3 times (3000ml * 3) of 3000ml, each 1hr, merge extractive liquid,, standing over night under the room temperature, filter, go precipitation, filtrate decompression concentrate (55 ℃, 0.08-0.1MPa), to cumulative volume 1800ml, concentrated solution adds 1% chitosan solution 270ml, stirs, and adds 1% aqueous gelatin solution 270ml again, stir, room temperature leaves standstill 4-8hr, and sucking filtration discards residue, supernatant is crossed polyamide resin column (the about 400ml of column volume that handles well, flow velocity 400ml/hr), with the colour developing of aluminum chloride 95% alcoholic solution, whether adsorb saturated with the spray of polyamide thin layer with the conclusive evidence resin column; Subsequently successively with 1500ml10% ethanol, the 60% aqueous acetone solution eluting of 1500ml, the about 400ml/hr of elution flow rate.Collect 60% aqueous acetone solution and concentrating under reduced pressure (40 ℃ 0.08-0.1MPa) are done near; Behind the water bath method, 75 ℃ of oven dried 24-72hr must be brown to the yellowish-brown powder, and Rhododendron dahuricum extract is about 4.8g (water content≤5%).
2. content detection:
(1) mensuration of hyperin:
Chromatographic condition: immobile phase ODS post (4.6mm I.D. * 150mm, 5 μ m); Mobile phase: methanol: 0.5% phosphate aqueous solution (transferring PH with triethylamine is 3) (45: 55); Flow velocity: 0.8ml/min; Detect wavelength: 355nm; Sample size: 10-20 μ l, gained HPLC collection of illustrative plates as shown in Figure 4, wherein retention time (min) be 12-13 for hyperin.The hyperin content of Rhododendron dahuricum extract (example 2) is 20.8%.
(2) mensuration of farrerol:
Chromatographic condition: immobile phase ODS post (4.6mm I.D. * 150mm, 5 μ m), mobile phase: methanol: water (60: 40), flow velocity: 1ml/min; Detect wavelength: 296nm; Sample size: 10-20 μ l, gained HPLC collection of illustrative plates such as Fig. 5 show that wherein retention time (min) is the farrerol that is of 16-17.The farrerol content of Rhododendron dahuricum extract (example 2) is 2.4%.
(3) mensuration of Quercetin:
Chromatographic condition: immobile phase ODS post (4.6mm I.D. * 150mm, 5 μ m), mobile phase: methanol: water: glacial acetic acid (50: 50: 2), flow velocity: 1ml/min; Detect wavelength: 370nm; Sample size: 10-20 μ l, gained HPLC collection of illustrative plates such as Fig. 6 show that wherein retention time (min) is the Quercetin that is of 10-11.The quercetin content of Rhododendron dahuricum extract (example 2) is 4.75%.
(4) content of total flavone is measured
The reference substance solution preparation:
It is an amount of that precision takes by weighing hyperin reference substance (120 ℃ of following drying under reduced pressure are to constant weight), puts in the 100ml volumetric flask, and add 60% alcoholic solution and make dissolving in right amount, and rare to scale, shake up.Get the solution that contains anhydrous hyperin 60 μ g among every 1ml.
The need testing solution preparation:
It is an amount of that precision takes by weighing dry this product (embodiment 2), places the 100ml measuring bottle, adds 60% dissolve with ethanol solution, is settled to scale, shakes up, standby.
Assay method: get reference substance gradient concentration solution and need testing solution, put in the 10ml volumetric flask, through AlCl
3With the KAc colour developing, behind the 60% ethanol standardize solution, press spectrophotography (2000 editions appendix IVA of Chinese Pharmacopoeia), measure absorbance at the 418nm place, the total flavones content of material is 60.5%.
The preparation of embodiment 3. Rhododendron dahuricum extracts
1. extract
Get the place of production, collecting season is different from example 1,2 Folium Rhododendri Daurici crude drug (dry stem and leaf 300g), pulverize, under 85 ℃-100 ℃ temperature, with the 60% ethanol water reflux, extract, 3 times (3000ml * 3) of 3000ml, each 1hr, merge extractive liquid,, standing over night under the room temperature, filter, go precipitation, filtrate decompression concentrate (55 ℃, 0.08-0.1MPa), to cumulative volume 1800ml, concentrated solution adds 1%ZTC1 type clarifier B component 216ml, stirs, and adds 1%ZTC type clarifier A component 108ml again, stir, room temperature leaves standstill 4-8hr, and sucking filtration discards residue, supernatant is crossed polyamide resin column (the about 400ml of column volume that handles well, flow velocity 400ml/hr), with the colour developing of aluminum chloride 95% alcoholic solution, whether adsorb saturated with the spray of polyamide thin layer with the conclusive evidence resin column; Subsequently successively with 900ml15% ethanol, the 70% methanol aqueous solution eluting of 1500ml, the about 400ml/hr of elution flow rate.Collect 70% methanol aqueous solution and concentrating under reduced pressure (45 ℃ 0.08-0.1MPa) are done near; Behind the water bath method, 75 ℃ of oven dried 24-72hr must be brown to the yellowish-brown powder, and Rhododendron dahuricum extract is about 3.6g (water content≤5%).
2. content detection:
(1) mensuration of hyperin:
Chromatographic condition: immobile phase ODS post (4.6mm I.D. * 150mm, 5 μ m); Mobile phase: methanol: 0.5% phosphate aqueous solution (transferring PH with triethylamine is 3) (45: 55); Flow velocity: 0.8ml/min; Detect wavelength: 355nm; Sample size: 10-20 μ l, gained HPLC collection of illustrative plates such as Fig. 7 show that wherein retention time (min) is the hyperin that is of 12-13.The hyperin content of Rhododendron dahuricum extract (example 3) is 32.7%.
(2) mensuration of farrerol:
Chromatographic condition: immobile phase ODS post (4.6mm I.D. * 150mm, 5 μ m), mobile phase: methanol: water (60: 40), flow velocity: 1ml/min; Detect wavelength: 296nm; Sample size: 10-20 μ l, gained HPLC collection of illustrative plates such as Fig. 8 show that wherein retention time (min) is the farrerol that is of 16-17.The farrerol content of Rhododendron dahuricum extract (example 3) is 5.83%.
(3) mensuration of Quercetin:
Chromatographic condition: immobile phase ODS post (4.6mm I.D. * 150mm, 5 μ m), mobile phase: methanol: water: glacial acetic acid (50: 50: 2), flow velocity: 1ml/min; Detect wavelength: 370nm; Sample size: 10-20 μ l, gained HPLC collection of illustrative plates such as Fig. 9 show that wherein retention time (min) is the Quercetin that is of 10-11.The quercetin content of Rhododendron dahuricum extract (example 3) is 6.33%.
(4) content of total flavone is measured
The reference substance solution preparation:
It is an amount of that precision takes by weighing hyperin reference substance (120 ℃ of following drying under reduced pressure are to constant weight), puts in the 100ml volumetric flask, and add 60% alcoholic solution and make dissolving in right amount, and rare to scale, shake up.Get the solution that contains anhydrous hyperin 60 μ g among every 1ml.
The need testing solution preparation:
It is an amount of that precision takes by weighing dry this product (embodiment 3), places the 100ml measuring bottle, adds 60% dissolve with ethanol solution, is settled to scale, shakes up, standby.
Assay method: get reference substance gradient concentration solution and need testing solution, put in the 10ml volumetric flask, through AlCl3 and KAc colour developing, behind the 60% ethanol standardize solution, press spectrophotography (2000 editions appendix IVA of Chinese Pharmacopoeia), measure absorbance at the 418nm place, the total flavones content of material is 79.6%.
Claims (5)
1, a kind of Rhododendron dahuricum extract is characterized by:
(1) with hyperin as representing composition that this extract is measured, general flavone content 30% in the extract---90%;
(2) in the extract, the content 6.3% of hyperin chemical compound---42%;
(3) in the extract, the content 0.4% of farrerol chemical compound---8%;
(4) in the extract, the content 0.7% of Quercetin chemical compound---7%.
2, a kind of extracting method of Rhododendron dahuricum extract as claimed in claim 1, comprise that detached dowel separates in the preparation, upper prop liquid of Folium Rhododendri Daurici upper prop liquid, to eluting, the eluent of detached dowel concentrate, dry; It is characterized by: detached dowel adopts polyamide; After upper prop liquid adsorbed by polyamide, first water or weight concentration were the solvent wash of 5%-25%, and eluent is abandoned or adopted; The reuse weight concentration is 40～90% solvent elution, does not have the flavone reaction to eluent and aluminum chloride, and eluent is standby, and wherein the used solvent of eluting is one or more aqueous solutions with the arbitrary proportion combination of rudimentary alcohol, ketone, ester.
3, extracting method as claimed in claim 2 is characterized by: water or weight concentration with 1-5 times of resin column volume are the solvent wash of 5%-25%.
4, as claim 2 or 3 described extracting method, it is characterized by: preferred water is the remove impurity solvent, and the ethanol of 50%-85% is eluant.
5, as claim 2 or 3 described extracting method, it is characterized by: before upper prop liquid upper prop, add in upper prop liquid that clarifier is clarified, purification process, clarifier be in wherein a kind of solution of chitin kind, protide, ZTC clarifier or chitin kind, protide, the ZTC clarifier any two with 1-2: the solution that the 1-2 ratio is made, chitin kind, protide, ZTC clarifier separately or the consumption of two kinds of combinations account for the 0.6%-2.4% (g/g) of Folium Rhododendri Daurici consumption.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104311615A (en) * | 2014-09-30 | 2015-01-28 | 西北师范大学 | Method for extracting and separating hyperoside and gossypetin-3-O-beta-D-galactoside from rhododendron przewalskii maxim. leaves |
| CN108404011A (en) * | 2018-05-10 | 2018-08-17 | 王芬 | A kind of haze removing heat from the lung to relieve cough Chinese medicine composition and preparation method thereof |
| CN115192624A (en) * | 2022-05-23 | 2022-10-18 | 成都杨天万应制药有限公司 | Extraction process and application of rhododendron Liangshan |
-
2004
- 2004-11-18 CN CNB2004100645506A patent/CN100333732C/en not_active Expired - Fee Related
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104311615A (en) * | 2014-09-30 | 2015-01-28 | 西北师范大学 | Method for extracting and separating hyperoside and gossypetin-3-O-beta-D-galactoside from rhododendron przewalskii maxim. leaves |
| CN108404011A (en) * | 2018-05-10 | 2018-08-17 | 王芬 | A kind of haze removing heat from the lung to relieve cough Chinese medicine composition and preparation method thereof |
| CN115192624A (en) * | 2022-05-23 | 2022-10-18 | 成都杨天万应制药有限公司 | Extraction process and application of rhododendron Liangshan |
| CN115192624B (en) * | 2022-05-23 | 2023-10-13 | 成都杨天万应制药有限公司 | Extraction process and application of rhododendron simsii |
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