CN106248856A - The discrimination method of effective ingredient in a kind of gingko leaf preparation - Google Patents
The discrimination method of effective ingredient in a kind of gingko leaf preparation Download PDFInfo
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Abstract
The invention discloses the discrimination method of effective ingredient in a kind of gingko leaf preparation, comprise the steps: the preparation of (1) test sample: taking gingko leaf preparation to be measured appropriate, add n-butanol extraction, filtration, filtrate is evaporated, residue adds ethanol makes dissolving, as need testing solution；(2) preparation of reference substance: take Folium Ginkgo control formulation appropriate, same to step (1), prepare reference substance solution；(3) thin layer chromatography: draw need testing solution and reference substance solution each 1～10 μ l, point sample is in same silica gel G plate respectively, launch with the mixture of ethyl acetate, isopropanol, first alcohol and water, take out in thin plate, air and dry, spray with 1～5% aluminum chloride ethanol solution, it is placed in 365nm ultra-violet lamp fluorescence viewing, observes speckle.The discrimination method that the present invention provides, the good separating effect of each effective ingredient, clear spot, Rf value are moderate, it is possible to accurately differentiate each effective ingredient, beneficially qualitative analysis and quality control.
Description
Technical field
The present invention relates to pharmaceutical analysis technical field, be specifically related to the discriminating side of effective ingredient in a kind of gingko leaf preparation
Method, main by the effective ingredient in thin layer chromatography qualitative analysis gingko leaf preparation, particularly analyze flavone compound.
Background technology
Semen Ginkgo (Ginkgo Biloba) is one the most ancient in the most existing current seed plant, is also to review
Unique survivor of Semen Ginkgo family during the Permian Period before more than 200,000,000 years.Folium Ginkgo plant medicament extract Chinese and much country
All being used for Drug therapy, its medical value is the highest, has antagonism platelet activating factor, removes free radical, antiinflammatory, anti-mistake
Quick, expand blood vessel, protect cardiovascular and cerebrovascular vessel, improve peripheral blood circulation, reduce the multiple pharmacology such as serum cholesterol and anticancer adjuvant
Learn activity.Flavonoid glycoside and terpene lactone in Folium Ginkgo are the main components producing above-mentioned pharmacological activity, are also that Folium Ginkgo carries
Take the principle active component in thing and quality control index.
One of principle active component in Folium Ginkgo is flavone compound, including at least 14 kinds of different compounds, example
Such as flavonol, flavone, flavanone and bisflavone etc., in these compounds, flavonoid glycoside and flavonol glycosides include kaempferide, Cortex querci dentatae
Element and isorhamnetin and glucose or rhamnose conjugate (general designation total flavonoids), be the silver being usually used in therapeutic purposes on market
In Folium Pruni preparation (such asDeng) the most significant composition, experiment shows flavonoid glycoside and Huang
Keto-alcohol glycosides is strong antioxidant, can the cell that causes due to age ageing of scavenging activated oxygen thus avoid and tissue damage
Wound, they can have a strong impact on brain function, including memory and attention, list of references such as: J.Pincemail et al., La
Presse Medicale Vol.15(1996),1475-1479；J.Robak et al., BiochemPharmacol Vol.37
(1988),837-841；J.Kleijnen Yu P.Knipschild Ginkgo biloba (Drug Profiles), the
Lancet340:1136(1992)。
At present, clinical or commercially available all kinds of gingko leaf preparations (include but not limited to capsule, injection and other drug effects
Compound preparation etc. associated with composition) in, principal agent composition is Folium Ginkgo or its extract, is also by total flavonoids and terpene lactone
Quality control is carried out as effective ingredient.
The method of quality control of existing gingko leaf preparation, the standard with reference to Chinese Pharmacopoeia performs (to refer to 2015 editions " middle traditional Chinese medicines
Allusion quotation " one, P1491-1493), the indentification by TLC step of total flavonoids is: point sample amount: 3 μ l, fixing phase: containing 4% vinegar
The silica gel G plate that carboxymethylcellulose sodium solution is binding agent of acid sodium, developing solvent: ethyl acetate-butanone-formic acid-water (5:3:1:
1), developer: 3% aluminum chloride ethanol solution, it is placed under ultra-violet lamp (365nm) and inspects its fluorescence, inspect result and see Fig. 1.
The method is disadvantageous in that: between speckle, boundary obscures, and hangover becomes linear, inferior separating effect is described, is unfavorable for differentiating,
It is difficult to effective ingredient in gingko leaf preparation is carried out qualitative analysis and quality control.
Liu Ruiming, Guo Li etc., " in Chinese patent medicine, Folium Ginkgo thin layer chromatography quickly differentiates " one literary composition (time treasure's traditional Chinese medical science traditional Chinese medicines,
The 4th phase of volume 10 in 1999, P265) the middle a kind of method proposing quick discriminating Folium Ginkgo, the discriminating step of its thin layer chromatography is:
Point sample amount: 10 μ l, fixing phase: silica gel G plate, developing solvent: n-Butanol acetic acid-water (4:1:5), it is placed under ultra-violet lamp (365nm)
Inspect its fluorescence, inspect result and see that (in Fig. 2, sequence number 1 refers to multiple bladder to Fig. 2, and sequence number 2 refers to that Folium Ginkgo control medicinal material, sequence number 3 refer to Semen Ginkgo
Leaf capsule).The method is disadvantageous in that: each effective ingredient in Folium Ginkgo extract can not be separated by the method,
Result of inspecting after some plate only manifests principal spot, only shows that product contains Folium Ginkgo extract, and discrimination is poor.
CN1508542A discloses the Chinese invention patent of entitled " compositions extracted from gingko biloba leaves and preparation method and application ", relates to
A kind of method of the effective ingredient in qualification and quantitative analysis gingko leaf preparation, the discriminating step of the thin layer chromatography of its flavone is:
Point sample amount: 10 μ l, fixing phase: silica gel G plate, developing solvent: acetic ether-methanoic acid-acetic acid-water (100:11:11:26), developer:
1% aluminum chloride ethanol solution, is placed under ultra-violet lamp (365nm) and inspects its fluorescence (refer to description and implement 5 parts).The party
Method is disadvantageous in that: in case study on implementation, only word description is inspected result and shown 8 yellow spottings, and there is no collection of illustrative plates proves,
The accuracy identified cannot be judged.
CN1939392A discloses the Chinese invention patent of entitled " method of quality control of a kind of compound Chinese medicinal preparation ", relates to
And the differential test method to total flavonoids, the discriminating step of employing thin layer chromatography is: point sample amount: 1～30 μ l, fixing phase:
0.1～8% silica gel G plate that carboxymethylcellulose sodium solution is binding agent of sodium acetate, developing solvent: ethyl acetate/Ethyl formate-
Butanone/acetone-methanol/alcohol-water=(1-20): (0.5-10): (0.1-5): (0.1-5), developer: 0.5～10% trichlorine
Change aluminum ethanol solution, be placed under ultra-violet lamp (365nm) inspect its fluorescence (refer to weigh 4 Step d, power 5 Step d and enforcement
Example 6 part).The method is disadvantageous in that: knowable to the developing solvent the selection result of embodiment 6, only when using fixing phase:
The carboxymethylcellulose sodium solution of 0.4% sodium acetate is the silica gel G plate of binding agent, developing solvent: ethyl acetate-butanone-methanol-water
(5:3:1:1), time, the separating effect of total flavonoids is clear, but there is the defect of edge effect.
CN1772013A discloses the Chinese invention patent of entitled " method of quality control of ginkgo-dipyridamine injection ", relates to
And the discrimination method of Flavonoid substances in Folium Ginkgo injection, the discriminating step of its thin layer chromatography is: point sample amount: 5～20 μ l, Gu
Determine phase: polyamide film, developing solvent: dichloromethane-glacial acetic acid-50～99% ethanol=(1～10): (1～6): (0.2～5),
Developer: 0.1～20% aluminum chloride ethanol solution, is placed under ultra-violet lamp (365nm) and inspects its fluorescence and (refer to description real
Execute example 1 part).The method is disadvantageous in that: selecting due to the difference of material polarity of developing solvent, can make at lamellae
The concentration at two edges ratio, middle low, causes Rf value improper, and impact exhibition analysis result is unfavorable for qualitative analysis.
In sum, the discrimination method of effective ingredient in the gingko leaf preparation of Chinese Pharmacopoeia defined how is improved, it is provided that
A kind of suitable discrimination method, especially selects a kind of suitably developing solvent, it is possible to accurately all kinds of in discriminating gingko leaf preparation have
Effect composition (flavonoid), it is achieved the qualitative analysis to effective ingredient, is the technical barriers not yet captured of those skilled in the art.
Summary of the invention
Present invention aim at providing the discrimination method of effective ingredient in a kind of gingko leaf preparation, improve existing thin
Layer chromatography condition so that flavonoids effective constituent can sharp separation, precisely differentiate that and method favorable reproducibility solves existing
The drawbacks described above that technology exists.
The technical solution adopted in the present invention is specific as follows:
In a kind of gingko leaf preparation, the discrimination method of effective ingredient, comprises the steps:
(1) preparation of test sample: taking gingko leaf preparation to be measured appropriate, add n-butanol extraction, filtration, filtrate is evaporated, residue
Add ethanol and make dissolving, as need testing solution；
(2) preparation of reference substance: taking Folium Ginkgo control formulation appropriate, add n-butanol extraction, filtration, filtrate is evaporated, residue
Add ethanol and make dissolving, as reference substance solution；
(3) thin layer chromatography: draw need testing solution and reference substance solution each 1～10 μ l, point sample is in same silicon respectively
Glue G plate, launches with the mixture of ethyl acetate, isopropanol, first alcohol and water, takes out in thin plate, air and dry, and spray is with 1～5% 3
Aluminum chloride ethanol solution, is placed in 365nm ultra-violet lamp fluorescence viewing, observes speckle；
Wherein, silica gel G plate is the silica gel g thin-layer plate with the carboxymethylcellulose sodium solution containing 4% sodium acetate as binding agent,
In developing solvent, the volume ratio of each component is ethyl acetate-isopropanol-methanol-water=(1～4): (6～9): (0.5～1): (1～
2)。
Preferably, in step (1) and step (2), adding n-butyl alcohol 15ml, warm macerating 15 minutes also shakes constantly, lets cool, mistake
Filter, filtrate is evaporated, and residue adds ethanol 2ml makes dissolving.
Preferably, in step (3), need testing solution, the point sample amount of reference substance solution are 3 μ l.
Preferably, in step (3), developer is 3% aluminum chloride ethanol solution.
Preferably, in step (3), each component of developing solvent and volume ratio are ethyl acetate-isopropanol-methanol-water=2:
6:1:1。
The principle using indentification by TLC gingko leaf preparation effective ingredient in the present invention is: according to component (the most each effectively
Composition, lower same) different with the adsorption coefficient in developing solvent at adsorbent, lamellae adsorbs many times, desorption, then
Absorption, then desorption, the translational speed onboard that adsorption coefficient is big is slow, and the translational speed onboard that adsorption coefficient is little is fast,
Differential migration is produced for being separated on lamellae.Therefore adsorbent determines whether to test successfully with the selection of developing solvent often
Key factor.
Specific in the present invention, owing to adsorbent is usually silica gel G plate, the probability replacing selection is relatively low, and developing solvent
Selection relatively big on the impact such as separating effect, disengaging time, spot shape, therefore select suitable developing solvent the most crucial.
It is known that during thin layer chromatography, the determination of chromatographic condition, chromatographic results judge between right and wrong the most important,
And the quality of chromatograph speckle is the key judging chromatographic results.Inappropriate developing solvent is selected to there will be hangover, edge effect, ratio
The situations such as shifting value is improper, the judgement of severe jamming chromatographic results, affect accuracy and reliability that sample differentiates.Such as launch
The pH value of agent can affect acid, alkali composition form of dissociating wherein in sample liquid, and the material polarity of multi-form is different, at lamellae
The speed of upper movement is the most different, easily causes conditions of streaking.The most such as when using mixed solvent to launch, polarity is more weak and boiling point
Relatively low developing solvent is more volatile in lamellae two edge, cause concentration at lamellae two edge ratio middle low, plate
The ratio of upper developing solvent is inconsistent (i.e. Rf value is improper), and polarity changes, and exhibition analysis result easily produces edge effect.
Therefore, inventor, when selecting developing solvent, is the polarity according to component, the activity of adsorbent, the polarity of developing solvent
The relativeness of this three selects, and fights for adsorbent surface active center by component molecular with developing solvent molecule and obtains
Separate；On the basis of taking into full account various influence factor, through repeatedly creative experimental exploring, just filter out the applicable present invention
The exclusive developing solvent of discrimination method, has innovative value.
It should be added that gingko leaf preparation effective ingredient involved in the present invention refers to flavone compound, tool
Body relates to Quercetin, kaempferide, isorhamnetin these three composition, and these three composition is also that academic circles at present judges pharmacologically active
Common recognition composition.
In a word, in the gingko leaf preparation that the present invention provides, the discrimination method of effective ingredient, improves the thin layer of prior art
Chromatographic condition, screens appropriate developing solvent so that the effective ingredient of gingko leaf preparation is good separating effect, speckle in discrimination process
Clearly, Rf value is moderate, it is possible to accurately differentiate each effective ingredient, beneficially qualitative analysis and quality control；And this discriminating side
The favorable reproducibility of method, highly versatile, the effective ingredient being applicable to commercially available all kinds of gingko leaf preparations differentiates, using value is big.
Accompanying drawing explanation
1, Fig. 1 is that the thin layer chromatography using Chinese Pharmacopoeia method inspects result figure.
2, Fig. 2 is that the thin layer chromatography using document " in Chinese patent medicine, Folium Ginkgo thin layer chromatography quickly differentiates " method inspects result
Figure.
3, Fig. 3 is that embodiment 1 uses the thin layer chromatography of the inventive method to inspect result figure.
Detailed description of the invention
The discrimination method of embodiment 1 gingko leaf preparation effective ingredient
(1) preparation of test sample: take gingko leaf preparation to be measured appropriate (being approximately equivalent to total flavonoids 48mg), add n-butyl alcohol
15ml extracts, and warm macerating 15 minutes also shakes constantly, lets cool, and filters, and filtrate is evaporated, and residue adds ethanol 2ml makes dissolving, as examination
Product solution；
(2) preparation of reference substance: take Folium Ginkgo control formulation appropriate (about 0.2mg), adds n-butyl alcohol 15ml and extracts, warm macerating 15
Minute and shake constantly, let cool, filter, filtrate is evaporated, and residue adds ethanol 2ml makes dissolving, as reference substance solution；
(3) thin layer chromatography: draw need testing solution and each 3 μ l of reference substance solution, distinguishes point sample in same with containing 4%
The carboxymethylcellulose sodium solution of sodium acetate is the silica gel g thin-layer plate of binding agent, with ethyl acetate, isopropanol, first alcohol and water
Mixture launches, each component of developing solvent and volume ratio: ethyl acetate-isopropanol-methanol-water=2:6:1:1, taking-up thin plate,
Drying in air, spray, with 3% aluminum chloride ethanol solution, is placed in 365nm ultra-violet lamp fluorescence viewing, observes speckle.
Inspect result and see Fig. 3, clearly show 3 speckles, correspond to Quercetin, kaempferide, isorhamnetin respectively.
The discrimination method of embodiment 2 gingko leaf preparation effective ingredient
In the present embodiment, the condition of thin layer chromatography makes into: point sample amount is 10 μ l, and developing solvent is ethyl acetate-isopropyl
Alcohol-methanol-water=4:9:0.5:2, developer is 5% aluminum chloride ethanol solution, and remaining differentiates that step is with reference to embodiment 1.
Inspect result and clearly show 3 speckles.
The discrimination method of embodiment 3 gingko leaf preparation effective ingredient
In the present embodiment, the condition of thin layer chromatography makes into: point sample amount is 1 μ l, and developing solvent is ethyl acetate-isopropyl
Alcohol-methanol-water=1:6:1:1, developer is 1% aluminum chloride ethanol solution, and remaining differentiates that step is with reference to embodiment 1.
Inspect result and clearly show 3 speckles.
The screening of embodiment 4 developing solvent
Due to the Quercetin of gingko leaf preparation effective ingredient, kaempferide, isorhamnetin physicochemical property different, and pole
The impact of sex differernce for a plate, separate very key, add negative interference that may be present, usual terms is difficult to reach accurate
The requirement differentiated, carries out the investigation of developing solvent influence factor for this inventor according to similar compatibility principle, with screening suitably exhibition
Open agent.
The discrimination method that investigation method provides with reference to the present invention is carried out, and only replaces various developing solvent, and other experiment conditions are not
Becoming, the selection result see table shown.
| Sequence number | Condition | Phenomenon |
| 1 | Ethyl acetate-butanone-formic acid-water=5:3:1:1 | Between speckle, boundary obscures, hangover, becomes linear |
| 2 | Acetate-methanol-water-normal hexane=10:2:1:5 | Between speckle, boundary obscures, hangover, becomes linear |
| 3 | Ethyl acetate acetic acid-normal hexane=5:3:1 | Reference substance is expanded to forward position |
| 4 | Ethyl acetate-isopropanol-methanol-water=2:6:1:1 | It is the most clear to separate, and Rf value is the most moderate, negative noiseless |
| 5 | Ethyl acetate-isopropanol-methanol-water=4:9:0.5:2 | Being separated clearly, Rf value is moderate, negative noiseless |
| 6 | Ethyl acetate-isopropanol-methanol-water=1:6:1:1 | Being separated clearly, Rf value is moderate, negative noiseless |
In above-mentioned list, the corresponding Chinese Pharmacopoeia of sequence number 1, sequence number 4-6 correspondence embodiment of the present invention 1-3, it is known that: when select with
On the silica gel g thin-layer plate that carboxymethylcellulose sodium solution is binding agent containing 4% sodium acetate, with ethyl acetate-isopropanol-first
When alcohol-water=2:6:1:1 is developing solvent, launching best results, the now separation of three kinds of effective ingredient is the most clear, and Rf value is the suitableeest
In, negative noiseless.
Claims (5)
1. a discrimination method for effective ingredient in gingko leaf preparation, comprises the steps:
(1) preparation of test sample: taking gingko leaf preparation to be measured appropriate, add n-butanol extraction, filtration, filtrate is evaporated, and residue adds second
Alcohol makes dissolving, as need testing solution；
(2) preparation of reference substance: taking Folium Ginkgo control formulation appropriate, add n-butanol extraction, filtration, filtrate is evaporated, and residue adds second
Alcohol makes dissolving, as reference substance solution；
(3) thin layer chromatography: draw need testing solution and reference substance solution each 1～10 μ l, point sample is in same silica gel G respectively
Plate, launches with the mixture of ethyl acetate, isopropanol, first alcohol and water, takes out in thin plate, air and dries, and sprays with 1～5% trichlorine
Change aluminum ethanol solution, be placed in 365nm ultra-violet lamp fluorescence viewing, observe speckle；
Described silica gel G plate is the silica gel g thin-layer plate with the carboxymethylcellulose sodium solution containing 4% sodium acetate as binding agent, institute
In the developing solvent stated, the volume ratio of each component is ethyl acetate-isopropanol-methanol-water=(1～4): (6～9): (0.5～1):
(1～2).
The discrimination method of effective ingredient in gingko leaf preparation the most according to claim 1, it is characterised in that described step
(1) and in step (2), adding n-butyl alcohol 15ml, warm macerating 15 minutes also shakes constantly, lets cool, and filters, and filtrate is evaporated, and residue adds second
Alcohol 2ml makes dissolving.
The discrimination method of effective ingredient in gingko leaf preparation the most according to claim 1, it is characterised in that described step
(3), in, need testing solution, the point sample amount of reference substance solution are 3 μ l.
The discrimination method of effective ingredient in gingko leaf preparation the most according to claim 1, it is characterised in that described step
(3), in, developer is 3% aluminum chloride ethanol solution.
The discrimination method of effective ingredient in gingko leaf preparation the most according to claim 1, it is characterised in that described step
(3), in, each component of developing solvent and volume ratio are ethyl acetate-isopropanol-methanol-water=2:6:1:1.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112903844A (en) * | 2021-01-19 | 2021-06-04 | 皖西学院 | Detection method for content of pteridophytin in fiddlehead |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112903844A (en) * | 2021-01-19 | 2021-06-04 | 皖西学院 | Detection method for content of pteridophytin in fiddlehead |
| CN112903844B (en) * | 2021-01-19 | 2022-12-16 | 皖西学院 | Detection method for content of pteridonin in pteridium aquilinum |
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| C06 | Publication | ||
| PB01 | Publication | ||
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| GR01 | Patent grant | ||
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| CB03 | Change of inventor or designer information |
Inventor after: Chen Guanhua Inventor after: Xu Penghe Inventor after: Jin Xiao Inventor after: Lu Zhenyu Inventor after: Chen Lingfang Inventor after: Yao Liping Inventor after: Xu Xiuhui Inventor after: Song Yuanzheng Inventor after: Jiang Guochao Inventor after: Song Dandan Inventor after: Xu Rong Inventor after: He Yu Inventor before: Xu Chunling Inventor before: Xu Penghe Inventor before: Jin Xiao Inventor before: Xu Xiuhui Inventor before: Yao Liping Inventor before: Song Yuanzheng Inventor before: Chen Lingfang Inventor before: Jiang Guochao Inventor before: Song Dandan Inventor before: Xu Rong Inventor before: He Yu |
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| CB03 | Change of inventor or designer information |