CN101357147B - Extract of dogbane leaf with finger print and preparation and analytical method thereof - Google Patents
Extract of dogbane leaf with finger print and preparation and analytical method thereof Download PDFInfo
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Abstract
A dogbane leaf extract with a fingerprint contains 50-60% of the total flavone based on hyperoside, wherein, the content of the hyperoside is 10-17%, the content of isoquercitrin is 10-16%, the contents of the isoquercitrin and kaempferol are respectively 25-35% and 3.5-4.5% after hydrolysis, the content of proanthocyanidin is 15-25%, and the extract has a definite fingerprint of the total flavone. The preparation method adopts low-polar or middle-polar macroporous absorbent resin to prepare a crude product of the extract from a concentrated solution of an extracting solution of the dogbane leaf extract, the content of the total flavone in the crude product is 40-44%, and the extract with the definite fingerprint is obtained by refining the crude product with the mixture of ethanol and ethyl acetate, and a corresponding analysis method, especially an assay method for the proanthocyanidin, is established. The extract meets the standard requirements of five oral bulk drugs, and the preparation method of the extract is convenient in operation and available in industrialization.
Description
One, technical field
The present invention relates to a kind of active substance that in Chinese herbal medicine, extracts and method for distilling thereof; Being particularly related to extract that mainly contains the flavonoid active substance and preparation method thereof, exactly is a kind of extract and preparation and the analytical method that finger printing arranged of a kind of extraction from Kindir leaf.
Two, background technology
China's Folium Apocyni Veneti resource has three kinds: Kindir leaf (Apocynum venetum L.) is claimed ambary, abutilon poacynum hendersonii woodson leaf [Poacynum hendersonni (Hook.f.) Wodson] and abutilon purpura Folium Apocyni Veneti [Poacynum pictum (Schrenk) Baill] again; Be referred to as Folium Apocyni Veneti; Because of belonging to not congener; Its flavone component also has different, does not have comparability each other.
According to bibliographical information Folium Apocyni Veneti (Apocynum venetum L.) contain flavone and glucosides, triterpene and sterol, lignanoid, coumarin, organic acid and ester, cyclic alcohol, anthocyanidin, tannin-, constituents surplus saccharide and aminoacid etc. 10.Pharmacological research shows that flavones ingredient is one of main active classification wherein, and chemical research shows that the Folium Apocyni Veneti flavones ingredient is with hyperin, isoquercitrin, Trifolin, 6 " 0-acetyl group hyperin, astragaloside, 6 "-0-acetyl group isoquercitrin, 6 " 0-acetyl group astragaloside,, component content such as Quercetin and kaempferol is higher.With regard to regard to a kind of Folium Apocyni Veneti, the extract that different method for distilling obtains is because contained flavone compound kind is different, content is also different, and there are very big-difference in its pharmacology, the property of medicine.In other words, the extract that Different Extraction Method obtains all is the different combinations thing, all is a kind of new compositions in other words, though all be referred to as extract.
Extract the Folium Apocyni Veneti active component at present following several method arranged:
1, first water or alcohol extraction are suspended from the extract mixture behind the recovery solvent in the water, use chloroform, ethyl acetate, n-butanol extraction respectively, are divided into several extraction parts.Reuse silicagel column or polydextran gel column chromatography, separate into the one pack system material.This method is applicable to the research of laboratory to single flavone component, also is not suitable for the preparation of total flavones.
2, application number 02110431.X discloses a kind of " extraction separation method of effective part from dogbane "; The Herba Apocyni veneti extracting solution passes through D101 after treatment; HPD100, the HPD600 macroporous adsorptive resins adopts water elution to water elution liquid colourless earlier; And with 20-39% ethanol water eluting, the ethanol water eluting of reuse 40-70%.The extractum that the eluent of collection 40-70% concentrates back formation is effective site; General flavone content is 50-60%, and the shortcoming of this technical scheme is: the effective site of failing clearly to be provided which flavone component is made up of, and is 50-60% though the effective site general flavone content is provided; But not clear and definite content assaying method and reference substance; As everyone knows, be different with different assay method of a kind of sample or the different resulting content results of reference substance, such as NaNO
2-Al (NO
3)
3The content that-NaOH method is measured is apparently higher than AlCl
3Method; The content that the reference substance rutin is measured is apparently higher than the hyperin reference substance; Because of Kindir leaf (except the ambary of Xinjiang) rutin content is 0.07%, hyperin content is 0.35%, so general flavone content can only adopt AlCl in Kindir leaf and the effective site
3Method, reference substance can only adopt hyperin.
3, application number 200410064551.0 discloses " a kind of Herba Apocyni veneti extract and method for distilling thereof ", gets Folium Apocyni Veneti 300g, measures 40% ethanol water reflux, extract, 3 times, each 0.5hr with 10 times; Merge extractive liquid,, deposition is gone in hold over night, filtration under the room temperature, and filtrate decompression is concentrated into cumulative volume 900mL; Concentrated solution adds the acetic acid solution 180mL of 1% chitosan, stirs 2min, and room temperature leaves standstill 2-4hr, sucking filtration; Discard residue, supernatant is crossed the polyamide resin column of handling well, subsequently with 600mL water; After using hydrochloric acid to transfer pH to be 3, carry out the eluting remove impurity, then with 70% ethanol water eluting to there not being AlCl
3Reaction is collected 70% ethanol water and is evaporated near doing, and behind the water bath method, 80 ℃ of oven dried get yellowish-brown to yellow powder, are the high-purity extract of Herba Apocyni veneti, and paste-forming rate is 1.8%.The hyperin content of the high-purity extract of Herba Apocyni veneti is 17.8%, total flavones (in hyperin) content is 42.8%, the quercetin content after the hydrolysis is 36.7%.The existing problem of this technical scheme is: though the quercetin content after hyperin content, general flavone content, the hydrolysis is provided; Which but can not by flavone component form by clear and definite extract; Can not intactly reflect this extract quality; Total flavones (in hyperin) content is 42.8%, does not reach five types of crude drug standard general flavone contents and be 50% requirement.
4, the disclosing of application number 200510123187.5 a kind of " Kindir leaf total flavone extract, its preparation and application ".Its preparation method is: filtrating is through polyamide column chromatography, and at first water washes 8-12 column volumes, reuse variable concentrations alcohol-water solution stepwise elution, Fractional Collections, spissated method; Though this method can get the high-purity total flavones; But because of all needing alkali-acid regeneration behind the each chromatography of polyamide column, so this method cost is high; Man-hour is long, is not suitable for the oral five types of crude drug of preparation.
Three, summary of the invention
It is not enough to the present invention is directed to above-mentioned prior art, and aiming to provide a kind ofly has fixedly that component is the dogbane leaf extractive of finger printing, and technical problem to be solved is to make up corresponding preparation method and analytical method.
The Folium Apocyni Veneti that the present invention claims is meant Kindir leaf (Apocynum venetum L.).Described dogbane leaf extractive is exactly in Kindir leaf (Apocynum venetum L.), to extract the extract that obtains.
The dogbane leaf extractive that finger printing is arranged that the present invention is alleged is meant and contains the total flavones finger printing in this extract, it is characterized in that general flavone content counts 50-60% (percentage by weight, down with) with hyperin; Wherein hyperin content is 10-17%, and isoquercitrin content is 10-16%, and the content that the content of total-flavonoid aglycone is measured Quercetin and kaempferol after in acid hydrolysis is respectively 25-35% and 3.5-4.5%; Proanthocyanidin content is counted 15-25% with the chlorination anthocyanidin.
Described total flavones finger printing is except that 1 hyperin, 2 isoquercitrins; Also have 3 Trifolin, 46 " O-acetyl group hyperin, 5 astragaloside, 66 "-O-acetyl group isoquercitrin, 76 " O-acetyl group astragaloside, 8 unknown peaks, 9 Quercetins, 10 kaempferols, as shown in Figure 1.
The method for preparing of said extracted thing is to be raw material with exsiccant Folium Apocyni Veneti coarse powder, comprises extraction, concentrates, separates and refining; Described extraction is that coarse powder is used at least 8 times of amounts (weight/volume w/v) 70-80% (percent by volume v/v, down together) alcoholic solution reflux, extract, at least twice, obtains ethanol extract; When ethanol extract is evaporated to the 1/4-1/6 volume, cools off, leave standstill, remove by filter deposition; Obtain concentrated solution, concentrated solution separates with low pole or middle polarity macroporous resin adsorption, successively reuse 50-75% ethanol elution behind water and 20% ethanol elution; Collect the 50-75% ethanol elution, obtain the extract bullion after the concentrating under reduced pressure drying; Described making with extra care is that the extract bullion is made with extra care; Here it is, and the extract bullion is used at least 10 times of amount (w/v) ethanol and ethyl acetate mixed solvent reflux, extract, at least twice; The volume ratio of ethanol and ethyl acetate is 1:5-10; Filter, obtain refining liquid, refining liquid obtains the dogbane leaf extractive that general flavone content (in hyperin) is 50-60% (percentage by weight w/w) after decompression precipitation, drying.
Described low pole or middle polarity macroporous resin are selected from the macroporous resin of polystyrene type or the preparation of crosslinked polypropylene nitrile, are macroporous resins such as AB-8 type or DM301 type like model.
Preferred alcohol and ethyl acetate volume ratio are the mixed solvent of 1:6-8 when refining, further preferred volume ratio 1:7; Perhaps adopting methanol and ethyl acetate volume ratio is the mixed solvent of 1:5-8.
Concrete preparation process is:
A, exsiccant Folium Apocyni Veneti is ground into the crude drug coarse powder,, extracts 2-3 time altogether with 75% ethanol water heating and refluxing extraction 1-2 hour that 8-10 doubly measures (g/ml);
B, extracting solution is evaporated to 1/5 volume being lower than under 60 ℃ the condition, leaves standstill filtration in 12 hours under the room temperature;
C, filtrating are through low pole or Semi-polarity macroporous adsorbent resin column chromatography, and the macroporous adsorbent resin consumption is 2.5 times of amounts (W/W) of crude drug
Preferred AB-8, low pole and Semi-polarity macroporous adsorbent resins such as DM301;
D. at first water washes residual solution and other impurity in 8-10 column volume flush awaies of macroporous adsorbent resin resin, with 20% ethanol elution to eluent and FeCl
3Inkless green reaction is with the flush away tannin, and 2.5 or 3 column volumes of reuse 50-75% ethanol or 50-75% methanol-eluted fractions are collected the 50-75% ethanol or the 50-75% meoh eluate of 0.5-2.5 or 0.5-3 column volume; This eluent is being lower than concentrating under reduced pressure under 60 ℃ of conditions, and vacuum drying gets by weight percentage that general flavone content reaches 40%-44% dogbane leaf extractive bullion;
Making with extra care of e, extract bullion; Doubly measure the ethyl acetate of (g/ml) with 10-20: ethanol (V/V) (5:1-10:1) or ethyl acetate: methanol (V/V) is (5:1-10:1). preferred ethyl acetate: ethanol (V/V) (6:1-8:1), reflux, extract, 2-3 time, 1-2 hour at every turn; Filter; Merge extractive liquid,, in 60 ℃ of decompression and solvent recoveries, 80 ℃ of drying under reduced pressure get by weight percentage that general flavone content reaches 50%-60% dogbane leaf extractive.
The analytical method of each component is following in the extract:
1, the assay determination of general flavone content is to be reference substance with the hyperin, adopts AlCl3-HAc-NaAc complexation colorimetry to measure.
2, main component hyperin, isoquercitrin analysis on Content are measured in the total flavones, are reference substance with hyperin and isoquercitrin, adopt high effective liquid chromatography for measuring, and chromatographiccondition is:
(1) chromatographic column: adopt ODS-C18 chromatographic column 150mmx6mm, 5 μ m; (2) mobile phase: water-acetonitrile-phosphoric acid (825:175:1); (3) detecting wavelength is 365nm; (4) column temperature is 27 ℃; (5) flow velocity 1.0ml/min.
3, total-flavonoid aglycone assay, Quercetin and kaempferol content after the hydrolysis of employing high effective liquid chromatography for measuring:
Get the about 0.05g of dried extract, accurate claim fixed, with 80% dissolve with methanol and be settled to 25ml; Get 2ml and be diluted to 10ml, add hydrochloric acid 1ml, put in 90 ℃ of water-baths reflux 60 minutes with 80% methanol; Take out, cooling is transferred in the 25ml measuring bottle immediately; Add 80% methanol and be diluted to scale, shake up, promptly get.Use Quercetin and kaempferol to be reference substance, the HPLC method is measured, and chromatographiccondition is:
(1) chromatographic column: adopt ODS-C18 post (Tianjin, island 150 * 6.0mm); (2) mobile phase: methanol-0.4% (concentration expressed in percentage by volume) phosphoric acid solution (volume ratio 45:40); (3) wavelength: 360nm; (4) column temperature: 30 ℃; (5) flow velocity 1.0ml/min.
4, proanthocyanidin content, adopt n-butyl alcohol one salt acid system to measure:
Get the about 0.1g of extract, accurate claim surely, dissolve, add hydrochloric acid 10ml and water 5ml, put in the water-bath reflux 80 minutes with 70% ethanol 40ml; Take out, cold after, filter, residue with 70% ethanol elution to colourless, filtrating and washing liquid merging; Accurately add 70% ethanol to 250ml,, get this solution 50ml, put in the round-bottomed flask, be evaporated to about 3ml;, concentrated solution moves into separatory funnel, at the bottom of the garden flask respectively water 10ml wash twice with water 5ml, washing liquid immigration separatory funnel; The solution that merges is finally got with the 15ml n-butyl alcohol, and coextraction 3 times merges butanol extraction liquid; Accurately add n-butyl alcohol to 100ml,, measure with spectrophotometer, measure the absorbance of n-butyl alcohol liquid at 545nm.
Use the Ax500/75xm formula, calculate the percentage composition of proanthocyanidin (in the chlorination anthocyanidin).
In the formula: A: sample n-butyl alcohol liquid is at the absorbance at 545nm place;
75: the chlorination anthocyanidin is at the absorptance at 545nm place;
M: the sample amount of taking by weighing (g).
5, adopt following chromatographiccondition to measure the finger printing of Folium Apocyni Veneti total flavones, as shown in Figure 1.
(1) chromatographic column: adopt ODS-C18 chromatographic column 150mmx4.6mm, 5 μ m;
(2) mobile phase adopts linear gradient elution, and wherein A is acetonitrile one methanol V/V (10:1) mutually, and B is 0.4% phosphoric acid solution mutually.
Eluent gradient:
Time (min) A (%) B (%)
0-32 16 84
32-35 27 73
35-52 27 73
52-53 16 84
53-60 16 84
(3) detecting wavelength is 360nm: (4) column temperature is 27 ℃; (5) flow velocity 1.0ml/min.
Compared with present technology, beneficial effect of the present invention is embodied in:
1, the present invention adopts low pole or semipolar macroporous adsorbent resin from Kindir leaf, to extract enrichment total flavones bullion earlier, and reuse ethyl acetate and ethanol are made with extra care by the amount proportioning of (5:1-10:1), and is simple to operate, is easy to suitability for industrialized production.General flavone content meets the standard of oral five types of crude drug in hyperin >=50%.
2, the present invention adopts HPLC; Can carry out clear and definite qualitative and quantitative to the flavone component of hyperin, isoquercitrin; Also can carry out clear and definite qualitative and quantitative to total-flavonoid aglycone Quercetin after the hydrolysis and kaempferol; And set up the proanthocyanidin content assaying method, also set up total flavones finger printing in the dogbane leaf extractive in addition.
Four, description of drawings
Fig. 1 is total flavones HPLC collection of illustrative plates in Kindir leaf (the Apocynum venetum L.) extract of the inventive method preparation; Also be the finger printing of total flavones, among the figure: 1 for hyperin, 2 for isoquercitrin, 3 for Trifolin, 4 be 6 " O-acetyl group hyperin, 5 for astragaloside, 6 be 6 "-O-acetyl group isoquercitrin, 7 is 6, and " O-acetyl group astragaloside, 8 is kaempferol for Quercetin, 10 for unknown peak, 9.
This collection of illustrative plates can be to the constituent of Folium Apocyni Veneti total flavones: hyperin, isoquercitrin, Trifolin, 6 " O-acetyl group hyperin, astragaloside, 6 "-" O-acetyl group astragaloside, Quercetin, kaempferol flavone component carry out clear and definite qualitative and quantitative O-acetyl group isoquercitrin, 6.
Five, the specific embodiment
Through the specific embodiment the inventive method is described further below.
(1) about the preparation of Kindir leaf extract
Embodiment 1:
Get Kindir leaf crude drug coarse powder 100g; Adding 800ml concentration is 75% ethanol water heating and refluxing extraction 2 hours, extracts merge extractive liquid, altogether 3 times; Extracting solution is evaporated to 1/5 volume being lower than under 60 ℃ the condition, leaves standstill filtration in 12 hours under the room temperature; Through 250g AB-8 low pole macroporous adsorption resin chromatography post is housed, at first water is with 8 column volumes of 2BV/h flow velocity flushing with 1 column volume (BV)/h flow velocity for filtrating, with 20% ethanol elution extremely and FeCl
3Inkless green reaction, reuse 50% ethanol are collected 50% ethanol elution of 0.5-2.5 column volume with 2.5 column volumes of 1BV/h flow velocity eluting; This eluent is being lower than concentrating under reduced pressure under 60 ℃ of conditions, and vacuum drying gets the dogbane leaf extractive bullion, is 42.0% in hyperin bullion general flavone content.
Making with extra care of extract bullion: get bullion 1g, add 10ml ethyl acetate by volume: ethanol 5:1 reflux, extract, 2 times, each 2 hours; Filter; Merge extractive liquid,, in 60 ℃ of decompression and solvent recoveries, 80 ℃ of drying under reduced pressure get dogbane leaf extractive; Recovery rate is 2.1%, and these article general flavone content counts 50.0% with hyperin.Outward appearance yellowish-brown powder.
Embodiment 2:
Get Kindir leaf crude drug coarse powder 100g; Adding 1000ml concentration is 75% ethanol water heating and refluxing extraction 2 hours, extracts merge extractive liquid, altogether 3 times; Extracting solution is evaporated to 1/5 volume being lower than under 60 ℃ the condition, leaves standstill filtration in 12 hours under the room temperature; Filtrating with the 1BV/h flow velocity through 250g AB-8 low pole macroporous adsorption resin chromatography post is housed,, at first water is with 10 column volumes of 2BV/h flow velocity flushing, with 20% ethanol elution extremely and FeCl
3Inkless green reaction, reuse 50% ethanol are collected 50% ethanol elution of 0.5-2.5 column volume with 2.5 column volumes of 1BV/h flow velocity eluting; This eluent is being lower than concentrating under reduced pressure under 60 ℃ of conditions, and vacuum drying gets the dogbane leaf extractive bullion, is 44.0% in general flavone content in the bullion of hyperin.
Making with extra care of extract bullion: get extract bullion 1g, add 10ml ethyl acetate by volume: ethanol is 6:1 reflux, extract, 3 times, each 2 hours; Filter; Merge extractive liquid,, in 60 ℃ of decompression and solvent recoveries, 80 ℃ of drying under reduced pressure get dogbane leaf extractive; Recovery rate is 2.0%, and these article are 56.0% in the hyperin general flavone content.Outward appearance yellowish-brown powder.
Embodiment 3:
Get Kindir leaf crude drug coarse powder 100g; Add 800ml concentration and be 75% ethanol water heating and refluxing extraction 2 hours, and extracted merge extractive liquid, altogether 3 times; Extracting solution is evaporated to 1/5 volume being lower than under 60 ℃ the condition, leaves standstill filtration in 12 hours under the room temperature; Filtrating with the 1BV/h flow velocity through 250g AB-8 low pole macroporous adsorption resin chromatography post is housed,, at first water is with 8 column volumes of 2BV/h flow velocity flushing, with 20% ethanol elution extremely and FeCl
3Inkless green reaction, reuse 75% ethanol are collected 75% ethanol elution of 0.5-3 column volume with 3 column volumes of 1BV/h flow velocity eluting; This eluent is being lower than concentrating under reduced pressure under 60 ℃ of conditions, and vacuum drying gets the dogbane leaf extractive bullion, is 42.0% in general flavone content in the hyperin extract bullion.
Making with extra care of extract bullion: get extract bullion 1g, add 10ml ethyl acetate by volume: ethanol is 7:1 reflux, extract, 2 times, each 2 hours; Filter; Merge extractive liquid,, in 60 ℃ of decompression and solvent recoveries, 80 ℃ of drying under reduced pressure get dogbane leaf extractive; Recovery rate is 2.10%, and these article are 51.0% in the hyperin general flavone content.Outward appearance yellowish-brown powder.
Embodiment 4:
Get Kindir leaf crude drug coarse powder 100g; Add 1000ml concentration and be 75% ethanol water heating and refluxing extraction 2 hours, and extracted merge extractive liquid, altogether 3 times; Extracting solution is evaporated to 1/5 volume being lower than under 60 ℃ the condition, leaves standstill filtration in 12 hours under the room temperature; Filtrating with the 1BV/h flow velocity through 250g AB-8 low pole macroporous adsorption resin chromatography post is housed,, at first water is with 10 column volumes of 2BV/h flow velocity flushing, with 20% ethanol elution extremely and FeCl
3Inkless green reaction, reuse 75% ethanol are collected 75% ethanol elution of 3 column volumes with 3 column volumes of 1BV/h flow velocity eluting; This eluent is being lower than concentrating under reduced pressure under 60 ℃ of conditions, and vacuum drying gets the dogbane leaf extractive bullion, is 41.0% in general flavone content in the hyperin bullion.
Making with extra care of extract bullion: get extract bullion 1g, add 20ml ethyl acetate by volume: ethanol is 8:1 reflux, extract, 3 times, each 1.5 hours; Filter; Merge extractive liquid,, in 60 ℃ of decompression and solvent recoveries, 80 ℃ of drying under reduced pressure get dogbane leaf extractive; Recovery rate is 1.90%, and these article are 55.0% in the hyperin general flavone content.Outward appearance yellowish-brown powder.
Embodiment 5:
Get Kindir leaf crude drug coarse powder 100g; Add 1000ml concentration and be 75% ethanol water heating and refluxing extraction 2 hours, and extracted merge extractive liquid, altogether 3 times; Extracting solution is evaporated to 1/5 volume being lower than under 60 ℃ the condition, leaves standstill filtration in 12 hours under the room temperature; Through 250g DM301 Semi-polarity macroporous adsorption resin chromatography post is housed, at first water is with 8 column volumes of 2BV/h flow velocity flushing with the 1BV/h flow velocity for filtrating, with 20% ethanol elution extremely and FeCl
3Inkless green reaction, reuse 75% ethanol are collected 75% ethanol elution of 0.5-2.5 column volume with 2.5 column volumes of 1BV/h flow velocity eluting; This eluent is being lower than concentrating under reduced pressure under 60 ℃ of conditions, and vacuum drying gets the dogbane leaf extractive bullion, is 41.0% in general flavone content in the hyperin bullion.
Making with extra care of extract bullion: get extract bullion 1g, add 10ml and be ethyl acetate by volume: ethanol is 6:1 reflux, extract, 2 times, each 2 hours; Filter; Merge extractive liquid,, in 60 ℃ of decompression and solvent recoveries, 80 ℃ of drying under reduced pressure get dogbane leaf extractive; Recovery rate is 1.92%, and these article are 51.0% in the hyperin general flavone content.Outward appearance yellowish-brown powder.
(2), about the total-flavonoid aglycone Quercetin after total flavones, the hydrolysis in the Kindir leaf extract and kaempferol, main constituent hyperin, isoquercitrin, the assay of proanthocyanidin and the determining fingerprint pattern of total flavones:
1, after the dogbane leaf extractive drying, general flavone content is reference substance with the hyperin, adopts AlCl
3-HAc-NaAc complexation colorimetric method for determining, its general flavone content is seen table 1
2, total-flavonoid aglycone assay, Quercetin and kaempferol content after the hydrolysis of employing high effective liquid chromatography for measuring:
Get the about 0.05g of dried dogbane leaf extractive, accurate claim fixed, with 80% dissolve with methanol and be settled to 25ml; Get 2ml and be diluted to 10ml, add hydrochloric acid 1ml, put in 90 ℃ of water-baths reflux 60 minutes with 80% methanol; Take out, cooling is transferred in the 25ml measuring bottle immediately; Add 80% methanol and be diluted to scale, shake up, promptly get.Use Quercetin and kaempferol to be reference substance, the HPLC method is measured, and its content is seen table 1, and chromatographiccondition is:
Chromatographic column: adopt ODS-C18 post (Tianjin, island 150 * 6.0mm);
Mobile phase: methanol-0.4% phosphoric acid solution (45:40);
Detect wavelength: 360nm; Column temperature: 30 ℃; Flow velocity 1.0ml/min.
3, after the Folium Apocyni Veneti total flavones drying, main constituent: hyperin, isoquercitrin all adopt high effective liquid chromatography for measuring, and its content is seen table 1, and chromatographiccondition is:
Chromatographic column: adopt ODS-C18 chromatographic column 150mmx6mm, 5 μ m;
Mobile phase: water-acetonitrile-phosphoric acid (825:175:1);
The detection wavelength is 365nm; Column temperature is 27 ℃; Flow velocity 1.0ml/min.
4, former total anthocyanidin content, adopt n-butyl alcohol one salt acid system to measure:
Get the about 0.1g of dried dogbane leaf extractive, accurate claim surely, dissolve, add hydrochloric acid 10ml and water 5ml, put in the water-bath reflux 80 minutes with 70% ethanol 40ml; Take out, cold after, filter, residue with 70% ethanol elution to colourless, filtrating and washing liquid merging; Accurately add 70% ethanol to 250ml,, get this solution 50ml, put in the round-bottomed flask, be evaporated to about 3ml;, concentrated solution moves into separatory funnel, at the bottom of the garden flask respectively water 10ml wash twice with water 5ml, washing liquid immigration separatory funnel, the solution of merging; Finally get with the 15ml n-butyl alcohol, coextraction 3 times merges butanol extraction liquid, accurately adds n-butyl alcohol to 100ml, measures the absorbance of n-butyl alcohol liquid at 545nm.
Use the Ax500/75xm formula, calculate the percentage composition of proanthocyanidin (in the chlorination anthocyanidin), its content is seen table 1.
Table 1 is the content of total-flavonoid aglycone behind the total flavones, hydrolysis in the dogbane leaf extractive, hyperin, isoquercitrin and proanthocyanidin
| Embodiment | General flavone content % | Total-flavonoid aglycone content % Quercetin, kaempferol | Hyperin content % | Isoquercitrin content % | Proanthocyanidin content % (in the chlorination anthocyanidin) |
| 12345 | 50.056.051.055.051.0 | 27.5 3.630.7 4.227.9 3.730.1 4.027.7 3.6 | 14.115.714.715.013.7 | 13.214.813.514.012.8 | 20.719.120.319.220.2 |
5, adopt following chromatographiccondition to measure the finger printing of Folium Apocyni Veneti total flavones:
Chromatographic column: adopt ODS-C18 chromatographic column 150mmx4.6mm, 5 μ m;
Mobile phase adopts linear gradient elution, and wherein A is acetonitrile one methanol (10:1) mutually, and B is 0.4% phosphoric acid solution mutually. eluent gradient;
Time (min) A (%) B (%)
0-32 16 84
32-35 27 73
35-52 27 73
52-53 16 84
53-60 16 84
The detection wavelength is that 360nm, column temperature are 27 ℃, flow velocity 1.0ml/min.
The finger printing of the Folium Apocyni Veneti total flavones that is obtained for the present embodiment method shown in Figure 1
Among Fig. 1,1 for hyperin, 2 for isoquercitrin, 3 for Trifolin, 4 be 6 " O-acetyl group hyperin, 5 for astragaloside, 6 be 6 "-O-acetyl group isoquercitrin, 7 is 6, and " O-acetyl group astragaloside, 8 is kaempferol for Quercetin, 10 for unknown peak, 9.
Claims (6)
1. preparation method of dogbane leaf extractive that finger printing is arranged; Spreading out leaf (Apocynum venetum L.) coarse powder with Apocynum is raw material; Comprise extraction, concentrate, separate and make with extra care; It is characterized in that: described extraction is the raw material coarse powder with at least 8 times of amounts (w/v) 70-80v/v% alcohol reflux at least twice, obtains ethanol extract, cools off when ethanol extract is evaporated to the 1/4-1/6 volume, leaves standstill, separates and obtain concentrated solution; Concentrated solution separates with AB-8 type or DM301 type macroporous resin adsorption; Reuse 50-75v/v% ethanol elution behind water and the 20v/v% ethanol elution is collected the 50-75v/v% ethanol elution successively, obtains the extract bullion after decompression precipitation, the drying; Said making with extra care is that the extract bullion extracts at least twice with at least 10 times of amount (w/v) ethanol and ethyl acetate mixed solvent; The volume ratio 1 of ethanol and ethyl acetate: 5-10; Separate obtaining refining liquid, the total flavones amount that refining liquid obtains in hyperin after decompression precipitation, drying is the extract of 50-60wt%.
2. method for preparing according to claim 1 is characterized in that: the volume ratio of mixed solvent ethanol and ethyl acetate is 1: 6-8.
3. method for preparing according to claim 2 is characterized in that: the volume ratio of mixed solvent ethanol and ethyl acetate is 1: 7.
4. method for preparing according to claim 1 is characterized in that: described ethanol and ethyl acetate mixed solvent replace with methanol and ethyl acetate mixed solvent, and methanol and ethyl acetate volume ratio are 1: 5-8.
5. the dogbane leaf extractive that finger printing is arranged by the described method preparation of claim 1 is the extract that in Apocynum Folium Apocyni Veneti (Apocynum venetum L.), extracts, and it is characterized in that:
(1) general flavone content is counted 50-60wt% with hyperin, and wherein hyperin content is 10-17wt%, and isoquercitrin content is 10-16wt%;
(2) Quercetin and the kaempferol content of total-flavonoid aglycone content after in acid hydrolysis is respectively 25-35wt% and 3.5-4.5wt%;
(3) proanthocyanidin content is counted 15-25wt% with chlorination anthocyanidin content;
(4) in the total flavones finger printing 1,1 hyperin, 2 isoquercitrins, 3 Trifolin, 46 " O-acetyl group hyperin, 5 astragaloside, 66 "-O-acetyl group isoquercitrin, 76 " O-acetyl group astragaloside, 8 unknown peaks, 9 Quercetins, 10 kaempferols;
(5), adopt following chromatographiccondition to measure Folium Apocyni Veneti total flavones finger printing
(i) chromatographic column: adopt ODS-C18 chromatographic column 150mm x 4.6mm, 5 μ m;
(ii) mobile phase adopts linear gradient elution, and wherein A is acetonitrile one methanol mutually, and wherein V/V ratio is 10: 1, and B is 0.4% phosphoric acid solution mutually;
Eluent gradient:
(iii) detecting wavelength is 360nm: (iv) column temperature is 27 ℃; (v) flow velocity 1.0ml/min.
6. by proanthocyanidin analytical method contained in the described extract of claim 5, use spectrophotometer method, it is characterized in that: adopt hydrochloric acid hydrolysis-n-butanol extraction to handle the back and measure the extract absorbance, concrete grammar is: get extract 0.1g; With 70% ethanol 40ml dissolving, add hydrochloric acid 10ml and water 5ml, put in the water-bath reflux 80 minutes, the taking-up cooled and filtered; To colourless, filtrating and washing liquid merge residue, add 70% ethanol to 250ml, get this solution 50ml with 70% ethanol elution; Put in the round-bottomed flask, be evaporated to 3ml, concentrated solution moves into separatory funnel, and round-bottomed flask water 10ml and water 5ml is respectively washed twice; Washing liquid moves into separatory funnel, and the solution of merging is used the 15ml n-butanol extraction, coextraction 3 times; Merge butanol extraction liquid, add n-butyl alcohol, measure the absorbance of n-butyl alcohol liquid at 545nm to 100ml;
Calculate the proanthocyanidin percentage composition that obtains in the chlorination anthocyanidin with A x 500/75x m formula,
In the formula: A: sample n-butyl alcohol liquid is at the absorbance at 545nm place,
75: the chlorination anthocyanidin is at the absorptance at 545nm place,
M: the sample amount of taking by weighing (g).
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| CN106018652B (en) * | 2016-07-28 | 2018-06-15 | 江西省农业科学院农产品质量安全与标准研究所 | The assay method of Determination of Hyperoside in a kind of plant dried flower or cured leaf |
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| CN109021045A (en) * | 2018-07-13 | 2018-12-18 | 鲁东大学 | The method for separating hemp glycosides, Yi Quercetin skin glycosides and chlorogenic acid simultaneously in largeleaf poacynum leaf leaf |
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