CN103323558A - Ginkgo biloba preparation analysis sample rapid preparation method based on solid phase extraction technology - Google Patents
Ginkgo biloba preparation analysis sample rapid preparation method based on solid phase extraction technology Download PDFInfo
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- CN103323558A CN103323558A CN2013102985982A CN201310298598A CN103323558A CN 103323558 A CN103323558 A CN 103323558A CN 2013102985982 A CN2013102985982 A CN 2013102985982A CN 201310298598 A CN201310298598 A CN 201310298598A CN 103323558 A CN103323558 A CN 103323558A
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- gingko leaf
- kaempferol
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- 244000194101 Ginkgo biloba Species 0.000 title claims abstract description 47
- 238000002414 normal-phase solid-phase extraction Methods 0.000 title claims description 18
- 238000005516 engineering process Methods 0.000 title claims description 17
- 235000008100 Ginkgo biloba Nutrition 0.000 title abstract description 12
- 238000004458 analytical method Methods 0.000 title abstract description 11
- 238000000605 extraction Methods 0.000 claims abstract description 25
- 239000000463 material Substances 0.000 claims abstract description 19
- 150000002596 lactones Chemical class 0.000 claims abstract description 12
- 229930003935 flavonoid Natural products 0.000 claims abstract description 7
- 150000002215 flavonoids Chemical class 0.000 claims abstract description 7
- 235000017173 flavonoids Nutrition 0.000 claims abstract description 7
- 239000007790 solid phase Substances 0.000 claims abstract description 7
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- 235000008777 kaempferol Nutrition 0.000 claims description 23
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- 235000005875 quercetin Nutrition 0.000 claims description 15
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Abstract
The invention belongs to the field of traditional Chinese medicinal preparation analysis and relates to a rapid extraction and preparation method of a plurality of effective components in a ginkgo biloba preparation. According to the invention, aginkgo biloba preparation is crushed and then undergoes sieving, a certain amount of a ginkgo biloba preparation sample is uniformly mixed with a solid-phase dispersant, the mixed materials are uniformly placed into a syringe with the bottom packed with a microporous membrane and undergo elution, and flavonoid and lactone components in the ginkgo biloba preparation can be extracted. In combination with the HPLC-DAD-ELSD hyphenated technique, the method provided by the invention can be used to rapidly and accurately determine contents of 16 effective components, rapidly reflect the quality of the ginkgo biloba preparation and raise production efficiency. The method provided by the invention is simple and accurate, and has advantages of small amount of samples, high extraction efficiency, short extraction time, good reappearance and high recovery rate.
Description
Technical field
The present invention relates to the rapid extraction preparation method of effective constituent in the multiple gingko leaf preparation, belong to the analysising drug form of Chinese materia medica field.
Technical background
Gingko leaf preparation is to treat at present cardiovascular and cerebrovascular disease natural drug effective and commonly used.Statistics shows, the annual sales amount of world market ginkgo leaf goods is up to 55,000,000,000 yuan, China's gingko leaf preparation annual sales amount was from 600,000,000 yuan of 12,000,000,000 yuan of developing into 2012 in 2000, become at present the leading kind of cardio-cerebrovascular autonomic drug, more than 100 of the ginkgo agents of the present approved different size of China, related preparations is widely used in China.
Gingko leaf preparation in, all record in state's pharmacopeia such as U.S., Europe.China, the U.S., European Pharmacopoeia standard are measured flavonoids and lactone composition respectively, wherein flavones ingredient is all to measure total-flavonoid aglycone content behind the acid hydrolysis flavonol glycosides, and be converted to total flavonoids content, the content of measuring flavone aglycone after the acid hydrolysis only can react the total amount of flavones ingredient, is difficult to embody the difference between heterogeneity.The lactone composition is all by detecting after the step process such as evaporate to dryness and methyl alcohol redissolution after ultrasonic extraction, the ethyl acetate extraction, the existence of a large amount of auxiliary materials makes the sample preparation process more loaded down with trivial details, the sample preparation methods time and effort consuming, use aborning and be difficult in time obtain accurately sample quality information, affect production efficiency, used simultaneously too much organic reagent also to cause to a certain degree pollution.Set up a kind of more effectively and rapidly analytic sample preparation method, significant for the quality of express-analysis gingko leaf preparation.
Matrix dispersion solid phase extractions (MSPD, matrix solid-phase dispersion) is the Barker professor of U.S. Louisiana state university proposed and gave theoretical explanation in 1989 a kind of quick sample treatment technology.Its principle is to have the carrier solid phase extractions material of multiple polymers to grind with sample with being coated with stain, obtains the potpourri of leather hard and it is filled post as filler, then uses different eluent solvent pillars, and various determinands are eluted.Its advantage is to have concentrated the processes such as sample homogenize in traditional sample pre-treatments, extraction, purification, does not need repeatedly to extract, concentrates, the operation steps such as constant volume, has avoided the loss in the sample preparation process.MSPD is applicable to the retention analysis of medicine, and it is a kind of simple efficient actual extraction and purification methods, is applicable to the extraction and cleaning of various molecular structures and polarity residues of pesticides, has improved analysis speed, has reduced reagent dosage, has been suitable for automated analysis.The method is usually used in using less in the Chinese medicine preparation constituent analysis in the sample pre-treatments of the experiments such as medicine impurity analysis, Detecting Pesticide and Analysis of Heavy Metal at present.
But HPLC-DAD-ELSD coupling technique Fast synchronization detects the chemical composition that has or not uv absorption, use in recent years comparatively ripe in China, each large scientific research institutions and colleges and universities delivered relative literature, and its good stability, the characteristics that application is strong make it become the conventional equipment of generally acknowledged development of Chinese herb products." use in the Chinese pharmacopoeia, empirical tests the method has good practicality to the method, possesses versatility and the universality of popularization at 2010 editions.
Ginkgo agent mainly adopts traditional water bath reflux method to extract sample aborning at present, and carry out respectively the assay of flavones and lactone, if can set up a kind of fast preparation method of sample for analysis, simplify the sample extraction step, organic reagent uses when reducing sample extraction, realize flavones and the synchronous quantitative test of lactone, fast timely monitoring product production overall process quality in the time of can helping enterprise to produce, can find in time that the problematic sample of quality further processes, enhance productivity, reduce production costs.
Summary of the invention
The object of the present invention is to provide a kind of rapid extraction preparation method based on effective constituent in the gingko leaf preparation of solid phase extractions technology, solved that traditional analysis sample preparation methods extraction time is long, solvent load is large, the loss of effective constituent preparation process is many, can not in time reflect the problem such as testing sample quality.That the method has is efficient, quick, easy and simple to handle, organic solvent consumption less and collection extract, purify, separate a step and finish, farthest avoided the loss of sample.
The present invention carries out sample pre-treatments by the matrix solid-phase dispersion extraction method for the commercially available gingko leaf preparation of part, selects flavones and lactone in the preparation to investigate index as method, compares with conventional sample treatment.Described compound is respectively: Quercetin3-O-2,6-dirhamnosylglucoside, Kaempferol 3-O-2 ", 6 " dirhamnosylglucoside, Quercetin 3-O-rutinoside, Kaempferol 3-O-rutinoside, lsorhamnetin 3-O-rutinoside, the glucosylrhamnosid of Quercetin 3-O-2 " (6 "-p-coumaroyl), Kaempferol 3-O-2 " glucosylrhamnoside; the glucosylrhamnoside of Kaempferol 3-O-2 " (6 "-p-coumaroyl); Quercetin; Kaempferol; Isorhamnetin; Ginkgolide A, Ginkgolide B, Ginkgolide C, Ginkgolide J, Bilobalide.
The inventive method comprises following steps:
With the gingko leaf preparation grinding and sieving, get a certain amount of gingko leaf preparation sample and solid phase dispersion agent, place mortar, add a certain amount of deionized water, fully grind, gingko leaf preparation powder and column chromatography material are mixed, the mixed material bottom of evenly packing into is lined with in the syringe of miillpore filter, pad a small amount of absorbent cotton in upper end after the compacting.Add first a certain amount of deionized water wash-out, eluent discards, add subsequently a certain amount of mixed organic solvents wash-out, collect a certain amount of eluent, measure Quercetin 3-O-2 in the extract with efficient liquid phase chromatographic analysis behind the constant volume "; 6 "-dirhamnosylglucoside, Kaempferol 3-O-2 ", 6 " dirhamnosylglucoside, Quercetin 3-O-rutinoside, Kaempferol 3-O-rutinoside, Isorhamnetin 3-O-rutinoside, the glucosylrhamnosid of Quercetin3-O-2 " (6 "-p-coumaroyl), Kaempferol 3-O-2 " glucosylrhamnoside; the glucosylrhamnoside of Kaempferol3-O-2 " (6 "-p-coumaroyl); Quercetin; Kaempferol; lsorhamnetin; Ginkgolide A, Ginkgolide B, Ginkgolide C, Ginkgolide J, the content of Bilobalide effective constituent.
In the above-mentioned gingko leaf preparation analytic sample fast preparation method based on the solid phase extractions technology, after pulverizing, gingko leaf preparation needs after sieve to guarantee that certain uniformity coefficient, sample volume are 0.1-0.5g, pulverizing, to cross 100 mesh sieves.
In the above-mentioned gingko leaf preparation analytic sample fast preparation method based on the solid phase extractions technology, the solid phase dispersion agent need add q.s, guaranteeing that the gingko leaf preparation powder is uniformly dispersed, and can with solid phase dispersion agent abundant adsorption and de-adsorption at short notice, the solid phase dispersion agent is C
18Column chromatography material, addition are 2-10g, and material particle size is the 80-100 order.
In the above-mentioned gingko leaf preparation analytic sample fast preparation method based on the solid phase extractions technology, the bottom is lined with the syringe of miillpore filter for guaranteeing that the gained eluent can directly advance high performance liquid chromatograph and detect, and filter membrane is organic phase 0.22 μ m.
In the above-mentioned gingko leaf preparation analytic sample fast preparation method based on the solid phase extractions technology, the mixed organic solvents eluent can affect kind and the content of effective constituent in the extract, used eluent is the mixed solution that water and methyl alcohol form, and consumption is 5-15mL, and methanol content is 50%-100%.
In the above-mentioned gingko leaf preparation analytic sample fast preparation method based on the solid phase extractions technology, the eluant, eluent flow velocity also can affect final extraction effect, and the eluant, eluent flow velocity of this method is 0.5-1mL/min.
The present invention extracts the method for flavones and lactone effective constituent in the gingko leaf preparation, and the method is easy, accurate, amount of samples is few, and extraction efficiency is high, extraction time is short, favorable reproducibility, the recovery are high.This method combines with the HPLC-DAD-ELSD coupling technique, can be used in the Accurate Determining gingko leaf preparation content of 16 kinds of flavones and lactone composition, objectively detects more fast the quality of gingko leaf preparation comprehensively, can help enterprise to enhance productivity.
Description of drawings
Fig. 1 is based on the rapid extraction preparation method of effective constituent in the gingko leaf preparation of solid phase extractions technology and 16 kinds of effective constituent chromatograms of conventional Ultrasound extraction method gained sample extracting solution.
Fig. 2 is that 3 different manufacturers gingko leaf preparations are used 16 kinds of effective constituent chromatograms based on rapid extraction preparation method's gained sample extracting solution of effective constituent in the gingko leaf preparation of solid phase extractions technology.
Embodiment
Embodiment 1: take Bilobanone ester dispersible tablets as object, adopt rapid extraction preparation method and conventional Ultrasound extraction method extraction efficiency based on effective constituent in the gingko leaf preparation of solid phase extractions technology to compare.
Instrument and material:
Instrument: Agilent 1100 high performance liquid chromatographs (comprising G1315B diode array detector and the coupling of All-tech evaporative light-scattering detector), chromatographic column is Agilent C
18Chromatographic column (250 * 4.6mm, 5.0Micron80A).
Material: Bilobanone ester dispersible tablets (Jiangsu Shenlong Pharmaceutical Co., Ltd., lot number: 111215) acetonitrile (Merck, chromatographically pure), formic acid (Merck, chromatographically pure), deionized water is made by Millipore Direct-Q pure water generator, Quercetin 3-O-2 "; 6 "-dirhamnosylglucoside, Kaempferol 3-O-2 "; 6 "-dirhamnosylglucoside, Quercetin 3-O-rutinoside, Kaempferol 3-O-rutinoside, Isorhamnetin 3-O-rutinoside, the glucosylrhamnosid of Quercetin 3-O-2 " (6 "-p-coumaroyl), Kaempferol 3-O-2 " glucosylrhamnoside; Kaempferol 3-O-2 " (6 "-p-coumaroyl) glucosylrhamnoside is made by oneself by the laboratory, warp
1H NMR and
13C NMR carries out Structural Identification, high performance liquid chromatography detect purity all greater than 98%, Quercetin, Kaempferol, Isorhamnetin, GinkgolideA, Ginkgolide B, Ginkgolide C, Ginkgolide J, Bilobalide reference substance available from Nat'l Pharmaceutical ﹠ Biological Products Control Institute.
Sample pre-treatments:
Conventional method: Bilobanone ester dispersible tablets is pulverized rear 100 mesh sieves of crossing, get approximately 1g of powder, accurately weighed, put in the apparatus,Soxhlet's, added the methenyl choloride refluxing extraction 2 hours, and discarded methenyl choloride liquid, the dregs of a decoction volatilize, adding methanol eddy extracted 4 hours, be transferred in the 50ml measuring bottle after being concentrated into smaller size smaller, and add methyl alcohol to the scale constant volume, shake up, cross 0.22 μ m miillpore filter, and get final product.
Rapid extraction preparation method based on effective constituent in the gingko leaf preparation of solid phase extractions technology: Bilobanone ester dispersible tablets is pulverized rear 100 mesh sieves of crossing, get approximately 0.2g of powder, add the C of 5-10g
18The column chromatography material places mortar, adds the 0.5-2mL deionized water, fully grinds 5 minutes, and the mixed material bottom of evenly packing into is lined with in the syringe of miillpore filter pad a small amount of absorbent cotton in upper end after the compacting.Add first 5-15mL deionized water wash-out with the 0.5-1mL/min flow velocity, eluent discards, and adds subsequently 50-100% methanol solvate wash-out, quantitative collection 10mL eluent, and get final product.
Above-mentioned two kinds of method gained sample extracting solutions are detected Quercetin 3-O-2 in the gingko leaf preparation extract synchronously with HPLC-DAD-ELSD coupling method "; 6 "-dirhamnosylglucoside, Kaempferol 3-O-2 "; 6 "-dirhamnosylglucoside, Quercetin 3-O-rutinoside, Kaempferol3-O-rutinoside, Isorhamnetin3-O-rutinoside, the glucosylrhamnosid of Quercetin 3-O-2 " (6 "-p-coumaroyl), Kaempferol 3-O-2 " glucosylrhamnoside; the glucosylrhamnoside of Kaempferol 3-O-2 " (6 "-p-coumaroyl); Quercetin; Kaempferol; Isorhamnetin; Ginkgolide A, Ginkgolide B, Ginkgolide C, Ginkgolide J, the content of Bilobalide effective constituent (such as Fig. 1), found that two kinds of methods relatively have no notable difference, point out new method and classic method to compare extraction effect identical, but extraction time, solvent-oil ratio, the aspects such as amount of samples have clear improvement.
Embodiment 2: take 3 kinds of gingko leaf preparations as object, adopt the rapid extraction preparation method based on effective constituent in the gingko leaf preparation of solid phase extractions technology to analyze flavonoids and lactone component content in the gingko leaf preparation.
Instrument is with embodiment 1
Material: Bilobanone ester dispersible tablets (Jiangsu Shenlong Pharmaceutical Co., Ltd., lot number: 111215), ginkgo biloba p.e sheet (the large pharmaceutical factory of German Weil-McLain doctor Shu Pei, lot number: 1350311), (Jiangsu Feima Pharmaceutical Co., Ltd., lot number: 110201) all the other materials are with embodiment 1 for Folium Ginkgo.
Sample pre-treatments:
Bilobanone ester dispersible tablets, ginkgo biloba p.e sheet and Folium Ginkgo are pulverized respectively rear mistake 100 mesh sieves, respectively get approximately 0.2g of powder, add respectively the C of 5-10g
18The column chromatography material places mortar, adds the 0.5-2mL deionized water, fully grinds 5 minutes, and the mixed material bottom of evenly packing into is lined with in the syringe of miillpore filter pad a small amount of absorbent cotton in upper end after the compacting.Add first 5-15mL deionized water wash-out with the 0.5-1mL/min flow velocity, eluent discards, and adds subsequently 50-100% methanol solvate wash-out, quantitative collection 10mL eluent, and get final product.
Above-mentioned 3 different manufacturers gingko leaf preparation sample extracting solutions are detected Quercetin3-O-2 in the gingko leaf preparation extract synchronously with HPLC-DAD-ELSD coupling method "; 6 "-dirhamnosylglucoside, Kaempferol 3-O-2 "; 6 "-dirhamnosylglucoside, Quercetin 3-O-rutinoside, Kaempferol 3-O-rutinoside, Isorhamnetin 3-O-rutinoside, the glucosylrhamnosid of Quercetin 3-O-2 " (6 "-p-coumaroyl), Kaempferol 3-O-2 " glucosylrhamnoside; the glucosylrhamnoside of Kaempferol 3-O-2 " (6 "-p-coumaroyl); Quercetin; Kaempferol; Isorhamnetin; GinkgolideA, Ginkgolide B, Ginkgolide C, Ginkgolide J, the content of Bilobalide effective constituent (such as Fig. 2), found that using the method can extract flavonoids in the different manufacturers gingko leaf preparation and lactone composition preferably, can obtain active constituent content information in the sample fast.
Claims (2)
1. gingko leaf preparation analytic sample fast preparation method based on the solid phase extractions technology, it is characterized in that, utilize the solid phase extractions technology, with extraction, the purification of flavonoids in the gingko leaf preparation and lactone effective constituent, separate a step and finish, it comprises the steps:
With the gingko leaf preparation grinding and sieving, get a certain amount of gingko leaf preparation sample and a certain amount of solid phase dispersion agent, place mortar, add a certain amount of deionized water, fully grind, gingko leaf preparation powder and column chromatography material are mixed, the mixed material bottom of evenly packing into is lined with in the syringe of miillpore filter, pad a small amount of absorbent cotton in upper end after the compacting.Add first a certain amount of deionized water wash-out, eluent discards, and adds subsequently a certain amount of mixed organic solvents wash-out, collects a certain amount of eluent, measures the content of effective constituent in the extract behind the constant volume with efficient liquid phase chromatographic analysis.
Described sample volume is 0.1-0.5g, crosses 100 mesh sieves after pulverizing.
Described solid phase dispersion agent is C
18Column chromatography material, consumption are 2-10g, and material particle size is the 80-100 order.
It is organic phase 0.22 μ m that the syringe that described bottom is lined with miillpore filter is selected filter membrane.
Described mixed organic solvents is the mixed solution that water and methyl alcohol form, and consumption is 5-15mL, and methanol content is 50%-100%.
Described eluant, eluent flow velocity is 0.5-1mL/min.
2. according to the described gingko leaf preparation analytic sample fast preparation method based on the solid phase extractions technology of claims, it is characterized in that, the representation compound of flavonoids and lactone composition is in the described gingko leaf preparation: Quercetin 3-O-2 "; 6 "-dirhamnosylglucoside, Kaempferol 3-O-2 ", 6 " dirhamnosylglucoside, Quercetin 3-O-rutinoside, Kaempferol3-O-rutinoside, Isorhamnetin 3-O-rutinoside, the glucosylrhamnosid of Quercetin 3-O-2 " (6 "-p-coumaroyl), Kaempferol 3-O-2 " glucosylrhamnoside; the glucosylrhamnoside of Kaempferol 3-O-2 " (6 "-p-coumaroyl); Quercetin; Kaempferol; Isorhamnetin; Ginkgolide A, Ginkgolide B, Ginkgolide C, Ginkgolide J, Bilobalide.
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| CN108816194A (en) * | 2018-06-15 | 2018-11-16 | 东北林业大学 | Utilize the mesoporous silicon composite of metal organic framework-matrix solid-phase dispersion technique separation and concentration Quercetin method |
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| CN103638071A (en) * | 2013-12-23 | 2014-03-19 | 湖南科技学院 | Efficient production method of gingko medicinal extract |
| CN106279315A (en) * | 2015-05-15 | 2017-01-04 | 北京华润高科天然药物有限公司 | One prepares Quercetin-3-O-2 ' ', the method for 6 ' '-two rhamanopyranosyl glucosides from Folium Ginkgo extract |
| CN106279317A (en) * | 2015-05-15 | 2017-01-04 | 北京华润高科天然药物有限公司 | A kind of method preparing Quercetin-3-o-glucoside from Folium Ginkgo extract |
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| CN105753920A (en) * | 2016-02-19 | 2016-07-13 | 浙江大学 | Method for extracting alpha-glucosidase inhibitor from litchi pulp |
| CN105753920B (en) * | 2016-02-19 | 2018-06-15 | 浙江大学 | A kind of method that alpha-glucosidase restrainer is extracted from litchi pulp |
| CN108816194A (en) * | 2018-06-15 | 2018-11-16 | 东北林业大学 | Utilize the mesoporous silicon composite of metal organic framework-matrix solid-phase dispersion technique separation and concentration Quercetin method |
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