CN101549021B

CN101549021B - Method for testing quality of perfoliate knotweed medicinal materials

Info

Publication number: CN101549021B
Application number: CN2009103028652A
Authority: CN
Inventors: 周欣; 陈华国; 赵超; 赵鸿宾
Original assignee: Guizhou Education University
Current assignee: Guizhou Education University
Priority date: 2009-06-03
Filing date: 2009-06-03
Publication date: 2011-11-30
Anticipated expiration: 2029-06-03
Also published as: CN101549021A

Abstract

The invention discloses a method for testing quality of perfoliate knotweed medicinal material, which includes part or whole of description, identification, examination and content measurement. The aforementioned identification includes thin-layered chromatography of taking quercetin reference substance and/or quercetin-3-O-beta-D-n butyl glucuronic acid reference substance as contrast, the content measurement measures the content of quercetin in medicinal materials, which is indicated as a high performance liquid chromatography of taking quercetin as contrast and the organic facies : aqueous phase as 10-40 : 90-60. Compared to the existing technology, the invention perfects the quality detecting standard of perfoliate knotweed medicinal materials, recuperates the deficiency of the existing quality detecting technology, which makes this quality detecting technology more scientific and reasonable, and ensures its security and validity in clinical application.

Description

The detection method of polygonum perfoliatum medicinal material

Technical field:

The present invention relates to a kind of quality determining method of polygonum perfoliatum medicinal material, belong to the technical field of medicinal material being carried out quality control.

Background technology:

Chinese medicine that China is traditional and preparation thereof lack the tight quality standard and the detection means of science mostly, be difficult to effectively control its inherent quality, can not guarantee the safe, effective of medication, also not meet the requirement of international medical market, seriously restrict the development of China's Chinese medicine industry.Strengthening the Chinese crude drug quality controling research is Chinese medicine standardization, standardized key issue.Have only the quality control of science that Chinese crude drug is carried out, could guarantee traditional Chinese medicine quality, realize " safety, effective, stable, controlled " of Chinese medicine.

Polygonum perfoliatum is the dry herb of polygonaceae arsesmart polygonum perfoliatum Polygonum perfoliatum Linn.; Be born in mountain valley, the bushes or by the ditch; Main product in Guizhou, Jiangsu, Zhejiang, Fujian, Jiangxi, Guangdong, Guangxi, Sichuan, Hunan.Tap when bloom summer, dry.Another name: the white grass in river, Herba Lespedezae Cuneatae, pears head thorn, snake are only waited.Polygonum perfoliatum is the conventional Chinese medicine material, also is seedling medicine commonly used.Nature,taste and action with cure mainly: acid; Bitter; Property is put down; Have clearing heat and detoxicating; Inducing diuresis and reducing edema; The effect of diffusing stasis of blood hemostasis is usually used in treating the furunculosis carbuncle and swells; Erysipelas; The paralysis cheek; Mastitis; The Ting ear; Tonsillitis; Cold, fever; The cough with lung heat; Pertussis; Scrofula; Haemorrhoids; The lymphogranuloma inguinale bubo; Rush down dysentery; Jaundice; Tympanites; Oedema; Stranguria with turbid discharge; Band down; Malaria; Wind fire cute conjunctivitis; Treating swelling and pain by traumatic injury; Spit blood; Have blood in stool; Snake bite and insect sting.As everyone knows, polygonum perfoliatum medicinal ingredient complexity mainly comprises following a few class: alkaloid, bitter principle, benzo chromogen ketone, terpene, flavones, coumarin, lignanoid, steroid, volatile oil and organic acid.Studies show that the number of chemical composition of polygonum perfoliatum has various concrete pharmacological actions.But its method of quality control bibliographical information seldom, is that the thin-layer chromatography Study on Identification has been carried out in contrast with the caffeic acid in " Guizhou Province's Chinese crude drug, national quality of medicinal material standard " version in 2003 only.Therefore, for the clinical practice that makes polygonum perfoliatum is safer, effective, science, reasonable, must strengthen its quality control.

Summary of the invention:

The objective of the invention is to: the quality determining method that a kind of polygonum perfoliatum medicinal material is provided.The present invention carries out quantitative examination by the content to Quercetin in the polygonum perfoliatum and serves as that thin-layer chromatography research is carried out in contrast with Quercetin and Quercetin-3-O-β-positive butyl ester of D-glucuronic acid, the perfect quality inspection standard of polygonum perfoliatum medicinal material, remedied the deficiency of existing quality detection technology, the quality detection technology that makes the polygonum perfoliatum medicinal material is science, rationally more.

Quality determining method of the present invention comprise in proterties, discriminating, inspection, the assay project partly or entirely, described discriminating comprise with the Quercetin reference substance be contrast, with methenyl choloride: ethyl acetate: methyl alcohol: formic acid=2～10: 1～3: 0.5～1.5: 0.2～0.7 be developping agent thin-layered chromatography and/or with Quercetin-positive butyl ester reference substance of 3-O-β-D-glucuronic acid be contrast, with methyl alcohol: formic acid: water=7～11: 0.2～0.8: 0.2～0.6 is the thin-layered chromatography of developping agent; Assay is the assay to contained Quercetin in the medicinal material, the content assaying method of Quercetin be with the Quercetin reference substance be contrast, with organic phase: water=10～40: 90～60 high performance liquid chromatography.

Concrete thin-layer chromatography discrimination method is:

(1) get polygonum perfoliatum medicinal material 2g,, filter with 70～90% alcohol reflux, 1～2h, the filtrate evaporate to dryness, residue is with methyl alcohol 1～2mL dissolving, as need testing solution; It is an amount of that precision takes by weighing the Quercetin reference substance, makes 0.1～0.5mgml with methyl alcohol ^-1Solution, product solution in contrast; According to " each 2～5 μ L of need testing solution and Quercetin reference substance solution are drawn in the test of an appendix VIB of Chinese pharmacopoeia thin-layered chromatography, put in same silica G respectively ₂₅₄On the thin layer plate, with methenyl choloride: ethyl acetate: methyl alcohol: formic acid=2～10: 1～3: 0.5～1.5: 0.2～0.7 is developping agent, launch, take out, dry, spray is put under the 254nm uviol lamp and is inspected with 1%～2% aluminium choride ethanolic solution, in the test sample chromatogram, with the corresponding position of Quercetin reference substance chromatogram on show the same color spot;

(2) get polygonum perfoliatum medicinal material 2g,, filter with 70%～90% alcohol reflux, 1～2h, the filtrate evaporate to dryness, residue is with methyl alcohol 1～2mL dissolving, as need testing solution; It is an amount of that precision takes by weighing Quercetin-positive butyl ester reference substance of 3-O-β-D-glucuronic acid, makes 0.1～0.5mgml with methyl alcohol ^-1Solution, product solution in contrast; According to " appendix a VIB of Chinese pharmacopoeia thin-layered chromatography test, draw each 2～5 μ L of need testing solution and Quercetin-positive butyl ester reference substance solution of 3-O-β-D-glucuronic acid, put respectively on same polyamide thin layer plate, with methyl alcohol: formic acid: water=7～11: 0.2～0.8: 0.2～0.6 is developping agent, launch, take out, dry, spray is with 1%～2% aluminium choride ethanolic solution, put under the 254nm uviol lamp and inspect, in the test sample chromatogram, with Quercetin-3-O-β-corresponding position of the positive butyl ester reference substance of D-glucuronic acid chromatogram on show the same color spot.

Concrete quercetin content assay method is: according to " an appendix VI of Chinese pharmacopoeia D high effective liquid chromatography for measuring:

Chromatographic condition and system suitability test: with the octadecyl silane is filling agent; With organic phase: water=10～40: 90～60 is moving phase; Column temperature is 25～35 ℃; The detection wavelength is 340～370nm; Number of theoretical plate calculates by the Quercetin peak should be not less than 3000;

The preparation of reference substance solution: it is an amount of that precision takes by weighing the Quercetin reference substance, adds methyl alcohol and make the solution that every 1mL contains 0.01～0.05mg, promptly;

The preparation of need testing solution: get powder 1g in the polygonum perfoliatum medicinal material, the accurate title, decide, and puts in the tool plug conical flask, accurate 3～7% hydrochloric acid ethanol solutions, the 20～50mL that adds claims to decide weight, heating and refluxing extraction 3～6 hours, put coldly, claim again to decide weight, supply the weight that subtracts mistake with absolute ethyl alcohol, shake up, filter, get subsequent filtrate 1～5mL, water-bath volatilizes, and residue adds dissolve with methanol, is settled to 10mL, shake up, filter, filtrate is as need testing solution;

Determination method: accurate respectively reference substance solution and each 5～20 μ L of need testing solution of drawing, inject liquid chromatograph, measure, promptly;

Polygonum perfoliatum is pressed dry product and is calculated, and contains Quercetin C ₁₅H ₁₉O ₇Weight must not be less than 0.20%.

Above-described organic phase is acetonitrile or methyl alcohol, and described water is formic acid, acetate or the phosphate aqueous solution of mass concentration 0.001%～5%.

Organic phase in the moving phase is preferably acetonitrile, and water is preferably mass concentration 0.02% phosphoric acid solution, and the volume ratio of organic phase and water is preferably 30: 70.

The most comprehensive quality determining method of the present invention comprises following project:

Proterties: stem slightly is square, and corner angle are arranged, multi-branched, and diameter reaches 0.2cm, and surperficial aubergine, pale brown look or yellow green have delavay raspberry herb on the corner angle, and memorandum is expanded, the long 2～6cm of internode, section fibrous, yellow-white has marrow or hollow; The leaf alternate has long handle, and peltate and given birth to; The many shrinkages of blade are nearly equilateral triangle after the expansion, celadon is to rufous, and lower surface vein and petiole all have down the hook of giving birth to thorn; Ochrea is wrapped on the stipes or comes off; Short spike top is given birth to or is born in the top axil, the bract circle, spend little, many atrophys or come off; Gas is little, and stem is lightly seasoned, the acid of leaf flavor;

Differentiate: (1) stem square section: epidermis is a row prothenchyma (of wood), includes the rufous material; Cortex is thin, 3～5 row cells; Tender stem pericyclic fiber Shu Lianxu becomes circular layer, and old stem is cut off into interrupted circular layer, cell wall thickness, lignify by ray; The old stem tool of bast bast fiber, the wall thickness lignify, cambium layer is obvious; Xylem vessel is big, single or 3～5 in groups; The marrow cell is big, the hollow that has; Old stem is at the visible most calcium oxalate cluster crystals of cortex, bast, ray and marrow, and tender stem is then rare or do not have;

(2) surface of leaf is seen: the irregular polygon of epicuticle cell, the nearly straight or microbend of anticline; It has the secretory cell of similar round, diameter 65 μ m down; The glandular hairs minority, head 28 cells, handle is short; The wavy bending of lower epidermis cell anticline; Flat shaft type of pore or inequality, glandular hairs are many slightly; It is unicellular that nonglandular hair mostly is; Master pulse and leaf margin are dredged and are given birth to the hook-shaped thorn of being made up of multiple row rhomboid or rectangle cell; Mesophyll cell contains calcium oxalate cluster crystal, diameter 17-62 μ m;

(3) get polygonum perfoliatum medicinal material 2g,, filter with 70～90% alcohol reflux, 1～2h, the filtrate evaporate to dryness, residue is with methyl alcohol 1～2mL dissolving, as need testing solution; It is an amount of that precision takes by weighing the Quercetin reference substance, makes 0.1～0.5mgml with methyl alcohol ^-1Solution, product solution in contrast; According to " each 2～5 μ L of need testing solution and Quercetin reference substance solution are drawn in the test of an appendix VIB of Chinese pharmacopoeia thin-layered chromatography, put in same silica G respectively ₂₅₄On the thin layer plate, with methenyl choloride: ethyl acetate: methyl alcohol: formic acid=2～10: 1～3: 0.5～1.5: 0.2～0.7 is developping agent, launch, take out, dry, spray is put under the 254nm uviol lamp and is inspected with 1%～2% aluminium choride ethanolic solution, in the test sample chromatogram, with the corresponding position of Quercetin reference substance chromatogram on show the same color spot;

(4) get polygonum perfoliatum medicinal material 2g,, filter with 70%～90% alcohol reflux, 1～2h, the filtrate evaporate to dryness, residue is with methyl alcohol 1～2mL dissolving, as need testing solution; It is an amount of that precision takes by weighing Quercetin-positive butyl ester reference substance of 3-O-β-D-glucuronic acid, makes 0.1～0.5mgml with methyl alcohol ^-1Solution, product solution in contrast; According to " appendix a VIB of Chinese pharmacopoeia thin-layered chromatography test, draw each 2～5 μ L of need testing solution and Quercetin-positive butyl ester reference substance solution of 3-O-β-D-glucuronic acid, put respectively on same polyamide thin layer plate, with methyl alcohol: formic acid: water=7～11: 0.2～0.8: 0.2～0.6 is developping agent, launch, take out, dry, spray is with 1%～2% aluminium choride ethanolic solution, put under the 254nm uviol lamp and inspect, in the test sample chromatogram, with Quercetin-3-O-β-corresponding position of the positive butyl ester reference substance of D-glucuronic acid chromatogram on show the same color spot

Check: (1) moisture is according to " an appendix IX of the Chinese pharmacopoeia H first method aquametry is measured, and must not surpass 14.0%;

(2) total ash is according to " an appendix IX of Chinese pharmacopoeia K measures, and must not surpass 10.0%;

(3) extract: water-soluble extractives is according to " cold-maceration under an appendix X of the Chinese pharmacopoeia A water-soluble extractives determination method item is measured, and must not be less than 13.0%;

Ethanol soluble extractives is according to " cold-maceration under an appendix X of the Chinese pharmacopoeia A ethanol soluble extractives determination method item is measured, and ethanol must not be less than 11.0% as solvent;

Assay: according to " an appendix VI of Chinese pharmacopoeia D high effective liquid chromatography for measuring:

Preferred quality determining method comprises following project:

(3) get polygonum perfoliatum medicinal material 2g,, filter with 90% alcohol reflux 2h, the filtrate evaporate to dryness, residue is with methyl alcohol 1mL dissolving, as need testing solution; It is an amount of that precision takes by weighing the Quercetin reference substance, makes 0.1mgml with methyl alcohol ^-1Solution, product solution in contrast; According to " appendix a VIB of Chinese pharmacopoeia thin-layered chromatography test, draw each 5 μ L of need testing solution and Quercetin reference substance solution, put respectively on same silica G 254 thin layer plates, with methenyl choloride: ethyl acetate: methyl alcohol: formic acid=8: 2: 1: 0.5 is developping agent, launches, take out, dry, spray is put under the 254nm uviol lamp and is inspected with 1% aluminium choride ethanolic solution, in the test sample chromatogram, with the corresponding position of Quercetin reference substance chromatogram on show the same color spot;

(4) get polygonum perfoliatum medicinal material 2g,, filter with 90% alcohol reflux 2h, the filtrate evaporate to dryness, residue is with methyl alcohol 1mL dissolving, as need testing solution; It is an amount of that precision takes by weighing Quercetin-positive butyl ester reference substance of 3-O-β-D-glucuronic acid, makes 0.1mgml with methyl alcohol ^-1Solution, product solution in contrast; According to " appendix a VIB of Chinese pharmacopoeia thin-layered chromatography test, draw each 5 μ L of need testing solution and Quercetin-positive butyl ester reference substance solution of 3-O-β-D-glucuronic acid, put respectively on same polyamide thin layer plate, with methyl alcohol: formic acid: water=9: 0.5: 0.4 is developping agent, launch, take out, dry, spray is with 1% aluminium choride ethanolic solution, put under the 254nm uviol lamp and inspect, in the test sample chromatogram, with Quercetin-3-O-β-corresponding position of the positive butyl ester reference substance of D-glucuronic acid chromatogram on show the same color spot;

Chromatographic condition and system suitability test: with the octadecyl silane is filling agent; With acetonitrile: 0.02% phosphoric acid solution=30: 70 is a moving phase; Column temperature is 25 ℃; The detection wavelength is 370nm; Number of theoretical plate calculates by the Quercetin peak should be not less than 3000;

The preparation of reference substance solution: it is an amount of that precision takes by weighing the Quercetin reference substance, adds methyl alcohol and make the solution that every 1mL contains 0.02mg, promptly;

The preparation of need testing solution: get powder 1g in the polygonum perfoliatum medicinal material, the accurate title, decide, and puts in the tool plug conical flask, the accurate 5% hydrochloric acid ethanol solution 25mL that adds claims to decide weight, heating and refluxing extraction 4 hours, put coldly, claim again to decide weight, supply the weight that subtracts mistake with absolute ethyl alcohol, shake up, filter, get subsequent filtrate 2mL, water-bath volatilizes, and residue adds dissolve with methanol, is settled to 10mL, shake up, filter, filtrate is as need testing solution;

Determination method: accurate respectively reference substance solution and each 20 μ L of need testing solution of drawing, inject liquid chromatograph, measure, promptly;

Polygonum perfoliatum (Polygonum perfoliatum L.) medicinal material records in " Guizhou Province's Chinese crude drug, national quality of medicinal material standard " version in 2003, this standard is described the proterties of polygonum perfoliatum, the microscopical identification of cauline leaf, adopting caffeic acid simultaneously is reference substance, has set up the thin-layer chromatography discrimination method of polygonum perfoliatum.These methods can not satisfy the needs of polygonum perfoliatum application development all from angle foundation qualitatively, and for this reason, the inventor has carried out Study of Lifting to its quality standard.

Find by consulting the domestic and foreign literature data: the chemical constitution study report of polygonum perfoliatum seldom, active component is indeterminate, for this reason, the inventor has carried out systematic research to the chemical constitution of polygonum perfoliatum, and result of study shows: flavone compound is its main chemical compositions.Anti-oxidant, removing oxygen radical effect that flavone compound has; Regulate the cardiovascular system effect; Anti inflammatory immunity and the anti-ageing effect of waiting for a long time.For more effective control quality of medicinal material,, adopt high performance liquid chromatography to carry out assay, and assay has been carried out methodological study according to the characteristics of one of the contained principal ingredient of polygonum perfoliatum Quercetin.

Below be the inventor to the quality determining method of polygonum perfoliatum experimentize research and preferred process:

One, inspection item

1. moisture is measured according to aquametry (" an appendix IX of Chinese pharmacopoeia H first method).The inventor has measured the content of moisture in the polygonum perfoliatum medicinal material in 20 different places of production, the results are shown in Table 1:

The polygonum perfoliatum medicinal material determination of moisture of the different place of production of table 1 is table (n=3) as a result

The place of production	Moisture (%)	The place of production	Moisture (%)
				Flower small stream Gao Po	13.5	Manjusri township, Guangshan County, Henan Province	12.7
The expensive road 63Km (in the shade) that abides by	12.9	Long for a long time	10.5
				The expensive road 63Km (facing south) that abides by	11.5	Longli paddy pin	12.6
Herd-boy Guan Daxing field	13.4	The steep mountain valley with clumps of trees and bamboo of waterside town	11.0
				Guizhou along the river	10.3	In the national college of education in the south of Guizhou Province campus	12.3
Expensive abiding by under the 64km bridge of road	9.8	Tongren, the Jiangkou, moral is prosperous	11.2
				Newly add the cattle farm, stockaded village	11.6	Persimmon flower level ground, Xi Feng county	11.6
The Zhenfeng, Guizhou	12.3	The Qianxi County	12.4
				Slight slope is closed in Kweiyang not cloth	12.1	The national Hou Shan of college of education in the south of Guizhou Province	11.3
Expensive abiding by on the 64km bridge of road	11.9	The Chenxi, Hunan	12.9

The moisture mean value of each place of production polygonum perfoliatum medicinal material is: 11.9%, and mxm. is 13.5%.Because the areal variation in the different places of production, tentative moisture must not surpass 14.0%, lists it in quality inspection standard.

Ash content according to the ash determination method " an appendix IX of Chinese pharmacopoeia K measures, and the inventor has measured the content of ash content in the polygonum perfoliatum medicinal material in 20 different places of production, the results are shown in Table 2:

The polygonum perfoliatum medicinal material ash content test of the different place of production of table 2 is table (n=3) as a result

The place of production	Ash content (%)	The place of production	Ash content (%)
				Flower small stream Gao Po	7.8	Manjusri township, Guangshan County, Henan Province	8.2
The expensive road 63Km (in the shade) that abides by	8.7	Long for a long time	7.8
				The expensive road 63Km (facing south) that abides by	8.7	Longli paddy pin	6.8
Herd-boy Guan Daxing field	7.9	The steep mountain valley with clumps of trees and bamboo of waterside town	7.4
				Guizhou along the river	6.6	In the national college of education in the south of Guizhou Province campus	8.6
Expensive abiding by under the 64km bridge of road	7.8	Tongren, the Jiangkou, moral is prosperous	8.4
				Newly add the cattle farm, stockaded village	8.8	Persimmon flower level ground, Xi Feng county	8.2
The Zhenfeng, Guizhou	8.3	The Qianxi County	8.6
				Slight slope is closed in Kweiyang not cloth	7.6	The national Hou Shan of college of education in the south of Guizhou Province	8.3
Expensive abiding by on the 64km bridge of road	7.9	The Chenxi, Hunan	8.7

The ash content mean value of each place of production polygonum perfoliatum medicinal material is: 8.06%, and mxm. is 8.8%.Because the areal variation in the different places of production, tentative ash content must not surpass 10.0%, lists it in quality inspection standard.

3. extract is measured the water-soluble extractives and the ethanol soluble extractives of polygonum perfoliatum respectively according to the cold-maceration under the determination of extractives method item (" an appendix X of Chinese pharmacopoeia A), and measurement result is as follows:

(1) water-soluble extractives

Measured the polygonum perfoliatum medicinal material in 20 different places of production respectively according to the cold-maceration under the water-soluble extractives determination method item (" an appendix X of Chinese pharmacopoeia A), measurement result sees Table 3:

The different place of production of table 3 polygonum perfoliatum medicinal material water-soluble extractives measurement result table (n=3)

The place of production	Extract (%)	The place of production	Extract (%)
				Flower small stream Gao Po	13.7	Manjusri township, Guangshan County, Henan Province	13.6
The expensive road 63Km (in the shade) that abides by	16.2	Long for a long time	14.4
				The expensive road 53Km (facing south) that abides by	15.4	Longli paddy pin	19.1
Herd-boy Guan Daxing field	19.0	The steep mountain valley with clumps of trees and bamboo of waterside town	18.9
				Guizhou along the river	20.1	In the national college of education in the south of Guizhou Province campus	18.3
Expensive abiding by under the 64km bridge of road	13.4	Tongren, the Jiangkou, moral is prosperous	16.4
				Newly add the cattle farm, stockaded village	21.6	Persimmon flower level ground, Xi Feng county	12.2
The Zhenfeng, Guizhou	17.9	The Qianxi County	17.9
				Slight slope is closed in Kweiyang not cloth	18.0	The national Hou Shan of college of education in the south of Guizhou Province	14.2
Expensive abiding by on the 64km bridge of road	18.4	The Chenxi, Hunan	13.5

The water-soluble extractives content mean value of each place of production polygonum perfoliatum medicinal material is: 16.61%, because the areal variation in the different places of production, by mean value 20% the minimum of floating downward as water-soluble extractives, that is: 16.61% * (1-20%)=13.29% ≈ 13.0% lists it in quality inspection standard.

(2) ethanol soluble extractives

According to the cold-maceration under the ethanol soluble extractives determination method item (" an appendix X of Chinese pharmacopoeia A), as solvent, measured the polygonum perfoliatum medicinal material in 20 different places of production with ethanol respectively, measurement result sees Table 4:

The different place of production of table 4 polygonum perfoliatum medicinal material ethanol soluble extractives measurement result table (n=3)

The place of production	Extract (%)	The place of production	Extract (%)
				Flower small stream Gao Po	6.6	Manjusri township, Guangshan County, Henan Province	8.4
The expensive road 53Km (in the shade) that abides by	10.3	Long for a long time	8.9
				The expensive road 53Km (facing south) that abides by	14.3	Longli paddy pin	12.6
Herd-boy Guan Daxing field	12.9	The steep mountain valley with clumps of trees and bamboo of waterside town	10.63
				Guizhou along the river	12.5	In the national college of education in the south of Guizhou Province campus	13.1
Expensive abiding by under the 64km bridge of road	22.7	Tongren, the Jiangkou, moral is prosperous	8.2
				Newly add the cattle farm, stockaded village	27.6	Persimmon flower level ground, Xi Feng county	4.1
The Zhenfeng, Guizhou	23.9	The Qianxi County	9.0
				Slight slope is closed in Kweiyang not cloth	25.2	The national Hou Shan of college of education in the south of Guizhou Province	6.1
Expensive abiding by on the 64km bridge of road	26.6	The Chenxi, Hunan	7.3

The ethanol soluble extractives content mean value of each place of production polygonum perfoliatum medicinal material is: 13.56%, because the areal variation in the different places of production, by mean value 20% the minimum of floating downward as ethanol soluble extractives, that is: 13.56% * (1-20%)=10.85% ≈ 11.0% lists it in quality inspection standard.

Two, the foundation of thin-layer chromatography discrimination method

1. reagent

Tlc silica gel G ₂₅₄, analyze pure (Haiyang Chemical Plant, Qingdao); Polyamide thin layer chromatography board (Anhui Wan Xi silicon source material factory); Quantitative kapillary (Drummond Scientific Co.USA.); Quercetin and Quercetin-3-O-β-positive butyl ester of D-glucuronic acid (self-control) by the wave spectrum analysis technology (IR, ¹H-NMR, ¹³C-NMR, MS) analyze, identify its structure; It is pure that methyl alcohol, chloroform, ethyl acetate etc. are analysis.

2. experiment material

Collected 20 batch samples altogether, from Guizhou Hua Xi, herd-boy Guan Daxing field, Guizhou along the river, ground such as Zhenfeng, Guizhou, Manjusri township, Guangshan County, Henan Province, Chenxi, Hunan, be accredited as Polygonum perfoliatumL. through the He Shunzhi researcher of Guiyang College of Traditional Chinese Medicine.

3. method and result

3.1 method is got polygonum perfoliatum medicinal material (No. three sieves) 2g, with 90% alcohol reflux 2h, filters, and the filtrate evaporate to dryness, residue is with methyl alcohol 1mL dissolving, as need testing solution.In addition precision takes by weighing Quercetin and Quercetin-positive butyl ester reference substance of 3-O-β-D-glucuronic acid is an amount of respectively, makes 0.1mgml with methyl alcohol respectively ^-1Solution, product solution in contrast.

According to thin-layered chromatography (" appendix VIB of Chinese pharmacopoeia version in 2005) test, draw each 5 μ L of need testing solution and Quercetin reference substance solution, put in same silica G respectively ₂₅₄On the thin layer plate, with methenyl choloride-ethyl acetate-methyl alcohol-formic acid (8: 2: 1: 0.5) be developping agent, launch, take out, dry, spray is put under the uviol lamp (254nm) and is inspected with 1% aluminium choride ethanolic solution, in the test sample chromatogram, with the corresponding position of Quercetin reference substance chromatogram on show the same color spot.

According to thin-layered chromatography (" appendix VIB of Chinese pharmacopoeia version in 2005) test, draw each 5 μ L of need testing solution and Quercetin-positive butyl ester reference substance solution of 3-O-β-D-glucuronic acid, put respectively on same polyamide thin layer plate, with methyl alcohol-formic acid-water (9: 0.5: 0.4) is developping agent, launch, take out, dry, spray is with 1% aluminium choride ethanolic solution, put under the uviol lamp (254nm) and inspect, in the test sample chromatogram, with Quercetin-3-O-β-corresponding position of the positive butyl ester reference substance of D-glucuronic acid chromatogram on show the same color spot.

3.2 as a result the test sample chromatogram with reference substance chromatogram relevant position on show the spot of same color.The polygonum perfoliatum medicinal material in different places of production difference to some extent on thin-layer chromatography simultaneously.

Three, the foundation of quercetin content assay method

1. instrument and reagent

High performance liquid chromatograph: Aglient 1100 high performance liquid chromatographs (quaternary pump, DAD detecting device, automatic sampler); CTO-10Asvp column oven (U.S.); 100,000/balance (Mei Teletuo benefit).

Chromatogram acetonitrile (Tianjin section close europeanized reagent development centre); Water (redistilled water faces and uses preceding preparation); Hydrochloric acid, phosphoric acid etc. (analyzing alcohol); (the Chinese biological goods are identified institute, lot number: 110257-200201) to the Quercetin reference substance; Polygonum perfoliatum medicinal material (20 different places of production).

2. chromatographic condition

Chromatographic column: Hypersil ODS (4.6 * 250mm) posts, acetonitrile-0.02% phosphoric acid (30: 70) is moving phase, flow velocity is 1mlmin ^-1, detect wavelength 370nm, 25 ℃ of column temperatures.

3. the selection of extraction conditions

(1) extracts choice of Solvent

Get with 3 parts of a collection of polygonum perfoliatum (Qianxi County, Guizhou) medicinal materials, every part of 1g, the accurate title, decide, put in the 50mL tool plug conical flask, accurate respectively absolute ethyl alcohol (containing 5% hydrochloric acid), 50% methyl alcohol (containing 5% hydrochloric acid), each 25mL of methyl alcohol (containing 5% hydrochloric acid) of adding claims to decide weight, ultrasonic Extraction 30min supplies the weight that subtracts mistake with institute's solubilizer respectively, shakes up, filter, get subsequent filtrate 2mL, water-bath volatilizes, residue adds dissolve with methanol, is settled to 10mL, shakes up, filter, promptly.

Measure by above-mentioned content assaying method, calculate, the results are shown in Table 5.

Table 5 extracts choice of Solvent

Extract solvent	Quercetin content (%)	Relative percentage (%)
			Methyl alcohol	0.037	77.08
50% methyl alcohol	0.012	25.00
			Absolute ethyl alcohol	0.048	100.00

As shown in Table 5, the extraction effect of various different solvents is compared, and the absolute ethyl alcohol effect is best, and methyl alcohol takes second place, and 50% methyl alcohol is the poorest, so select absolute ethyl alcohol as extracting solvent.

(2) extracting method examination

Method one: ultrasonic Extraction is got with a collection of polygonum perfoliatum (Qianxi County, Guizhou) medicinal material 1g, and accurate the title decides, and puts in the 50mL tool plug conical flask, accurate absolute ethyl alcohol (the containing 5% hydrochloric acid) 25mL that adds, claim to decide weight, ultrasonic Extraction 2 times, each 30min filters, merging filtrate concentrates, and is settled to 25mL with absolute ethyl alcohol, shake up, filter, get subsequent filtrate 2mL, water-bath volatilizes, and residue adds dissolve with methanol, is settled to 10mL, shake up, filter, promptly.

Method two: refluxing extraction is got with a collection of polygonum perfoliatum (Qianxi County, Guizhou) medicinal material 1g, and accurate the title decides, and puts in the 50mL tool plug conical flask, accurate absolute ethyl alcohol (the containing 5% hydrochloric acid) 25mL that adds claims to decide weight, refluxing extraction 3 hours, supply the weight that subtracts mistake with absolute ethyl alcohol, shake up, filter, get subsequent filtrate 2mL, water-bath volatilizes, and residue adds dissolve with methanol, be settled to 10mL, shake up, filter, promptly.

Measure by above-mentioned content assaying method, calculate, the results are shown in Table 6.

The selection of table 6 extracting method

Extracting method	Quercetin content (%)	Relative percentage (%)
			Ultrasonic Extraction	0.051	20.40％
Refluxing extraction	0.25	100.00

As shown in Table 6, the refluxing extraction effect obviously is better than ultrasonic Extraction, so adopt refluxing extraction as extracting method.

(3) examination of return time

Get with 5 parts of a collection of polygonum perfoliatum (Qianxi County, Guizhou) medicinal materials, every part of 1g, the accurate title, decide, put in the 50mL tool plug conical flask, accurate absolute ethyl alcohol (the containing 5% hydrochloric acid) 25mL that adds claims to decide weight, refluxing extraction is 2,3,4,5,6 hours respectively, supplies the weight that subtracts mistake with absolute ethyl alcohol, shakes up, filter, get subsequent filtrate 2mL, water-bath volatilizes, residue adds dissolve with methanol, is settled to 10mL, shakes up, filter, promptly.

Measure by above-mentioned content assaying method, calculate, the results are shown in Table 7.

The examination of table 7 return time is table as a result

Extraction time (hour)	Quercetin content (%)	Relative percentage (%)
			2	0.121	46.15
3	0.163	61.54
			4	0.260	98.85
5	0.261	99.24
			6	0.263	100.00

As shown in Table 7, refluxing extraction can be extracted the Quercetin in the polygonum perfoliatum fully in 4 hours.

(4) extract the examination of solvent acidity

Get with 4 parts of a collection of polygonum perfoliatum (Qianxi County, Guizhou) medicinal materials, every part of 1g, the accurate title, decide, put in the 50mL tool plug conical flask, (hydrochloric concentration is respectively accurate respectively adding absolute ethyl alcohol 25mL in the absolute ethyl alcohol: 4%, 5%, 6%, 7%), claim to decide weight, refluxing extraction 4 hours is supplied the weight that subtracts mistake with absolute ethyl alcohol, shakes up, filter, get subsequent filtrate 2mL, water-bath volatilizes, residue adds dissolve with methanol, is settled to 10mL, shakes up, filter, promptly.

Measure by above-mentioned content assaying method, calculate, the results are shown in Table 8.

Table 8 extracts solvent acidity examination table as a result

Extract solvent acidity (%)	Quercetin content (%)	Relative percentage (%)
			4	0.217	82.19
5	0.261	99.23
			6	0.260	98.86
7	0.263	100

By table 8 result as can be known, the hydrochloric acid absolute ethyl alcohol of selection 5.0% is as extracting solvent, and extraction effect is similar to 6.0% and 7.0% hydrochloric acid absolute ethyl alcohol, does not have notable difference, considers actual conditions, and the hydrochloric acid absolute ethyl alcohol of selection 5.0% is as extracting solvent.

(5) investigation of medicinal material granularity

Get with 4 parts of a collection of polygonum perfoliatum (Qianxi County, Guizhou) medicinal materials, be ground into meal respectively, middle powder, fine powder, fine powder takes by weighing the about 1g of medicinal powder of all size, accurate respectively absolute ethyl alcohol (the containing 5% hydrochloric acid) 25mL that adds, claim to decide weight, refluxing extraction 4 hours is supplied the weight that subtracts mistake with absolute ethyl alcohol, shakes up, filter, get subsequent filtrate 2mL, water-bath volatilizes, and residue adds dissolve with methanol, be settled to 10mL, shake up, filter, be prepared into need testing solution, measure the content of Quercetin according to chromatographic condition of the present invention, the result shows, the content of measuring Quercetin wherein during the polygonum perfoliatum pulverizing medicinal materials is become behind the powder is the most suitable, and test findings sees Table 9.

Table 9 medicinal material granularity examination test findings table

The medicinal material granularity	Quercetin content (%)	Relative percentage (%)
			Meal	0.247	92.51
Middle powder	0.265	99.25
			Fine powder	0.264	98.87
Fine powder	0.267	100.00

4. system suitability test difference precision is drawn each 20 μ L of Quercetin reference substance solution, polygonum perfoliatum medicinal material need testing solution and blank reagent solution, inject liquid chromatograph, measure, the retention time of Quercetin is about 11.7 minutes, the degree of separation of Quercetin and other component peaks is greater than 1.5 under this test condition, negative noiseless, number of theoretical plate all is higher than 3000 by the Quercetin peak, so the regulation number of theoretical plate is not less than 3000 by the Quercetin peak.

5. detect being chosen on the liquid chromatograph of wavelength, carry out all-wave scanning in 200～400nm scope, the result shows: the maximum absorption wavelength of Quercetin is 370nm, so selected 370nm is the detection wavelength of Quercetin in the polygonum perfoliatum.

6. the purity test of Quercetin calculates through area normalization method, and its purity is 99.78%.

7. the investigation precision of linear relationship takes by weighing the Quercetin reference substance 10.26mg that is dried to constant weight through phosphorus pentoxide and puts in the 100mL volumetric flask, adds dissolve with methanol and is diluted to scale, shakes up, and makes the reference substance stock solution that every 1mL contains Quercetin 0.1026mg.Accurate respectively this reference substance stock solution of absorption 0.5mL, 1.0mL, 1.5mL, 2.0mL, 2.5mL, 3.0mL, 3.5mL, shake up to scale with methanol constant volume in the 10mL volumetric flask, make serial reference substance solution.Accurate respectively each the 20 μ L of this reference substance series solution that draw, inject high performance liquid chromatograph, measure peak area by chromatographic condition of the present invention, the record chromatogram the results are shown in Table 10, and is horizontal ordinate with the sample size X (μ g) of reference substance, peak area value Y is an ordinate, the drawing standard curve, the result shows that Quercetin is good in 0.1026～0.7182 μ g scope internal linear relation.

The examination of table 10 Quercetin linear relationship is table as a result

Sample size (μ g)	Peak area
		0.1026	265.9
0.2052	645.4
		0.3078	1095.8
0.4104	1473.6
		0.5130	1859.2
0.6156	2261.8
		0.7182	2589.6

Regression equation: y=3817.60x-110.86, g=0.9998.

The equation of crossing initial point through match is: y=3601.50x, related coefficient g=0.9997.

With two formulas in the peak area substitution of a sample, relative deviation is 0.14% as a result, can think that intercept is zero, and available one point external standard method calculates content, and Quercetin has good linear relationship in 0.1026～0.7182 μ g scope.

8. precision test precision is measured Quercetin reference substance solution (concentration 0.4104mg/mL) 20uL, repeats sample introduction 6 times, and the record chromatogram the results are shown in table 11, and its RSD is 0.05%, illustrates that this method has good precision.

Table 11 Quercetin precision experimental result

9. replica test is got with each 1g of powder in a collection of polygonum perfoliatum (Qianxi County, the Guizhou) medicinal material, totally 6 parts, prepares test liquid by the preparation method of need testing solution in the content assaying method of the present invention.Accurate each the 20 μ L of need testing solution that draw inject high performance liquid chromatograph, measure Quercetin peak area integrated value by chromatographic condition of the present invention, calculate content, ask relative standard deviation.The result shows that the reappearance of the method mensuration Quercetin is good, RSD=1.89%.The results are shown in Table 12.

Table 12 reproducible test results table

10. stability test is got with powder 1g in a collection of polygonum perfoliatum (Qianxi County, the Guizhou) medicinal material, preparation method by need testing solution in the content assaying method of the present invention prepares test liquid, at room temperature press table 13 stipulated time sample introduction, measure the Quercetin peak area, ask relative standard deviation.The result shows that Quercetin is basicly stable in 36 hours at least, RSD=1.2%.The results are shown in Table 13.

Table 13 Quercetin stability experiment is table as a result

11. accuracy test adopts the application of sample recovery test, get the same batch sample (Qianxi County, Guizhou of the replica test of known content, the Quercetin average content is 0.264%, be 2.64mg/g) 9 parts, precision takes by weighing each about 0.5g, 1～3 part adds Quercetin reference substance solution (0.50mg/mL) 2.0mL respectively, 4～6 parts add Quercetin reference substance solution (0.50mg/mL) 2.6mL respectively, 7～9 parts add Quercetin reference substance solution (0.50mg/mL) 3.2mL respectively, are prepared into need testing solution by the preparation method of need testing solution, according to above-mentioned chromatographic condition, sample introduction, measure, the record chromatogram calculates content, asks relative standard deviation.The result shows that the method has average recovery preferably, the results are shown in Table 14.

Table 14 recovery test is table as a result

Sequence number	Sample weighing (g)	Theoretical content (mg)	Addition (mg)	Measured value (mg)	The recovery (%)	Average recovery rate (%)	RSD(％)
								1	0.5012	1.32	1.00	2.28	96.00	?	?
2	0.4997	1.32	1.00	2.27	97.00	96.33	0.6
								3	0.5021	1.33	1.00	2.29	96.00	?	?
4	0.5036	1.33	1.30	2.59	96.92	?	?
								5	0.4986	1.32	1.30	2.61	99.23	97.43	1.6
6	0.5051	1.33	1.30	2.58	96.15	?	?
								7	0.5026	1.33	1.60	2.90	98.12	?	?
8	0.4973	1.31	1.60	2.86	96.88	97.29	0.7
								9	0.5062	1.34	1.60	2.89	96.87	?	?

12. serviceability test

Get with powder 1g in a collection of polygonum perfoliatum (Qianxi County, the Guizhou) medicinal material, totally 3 parts, handle sample by the method for the invention, get need testing solution, standby, carry out the contrast examination research of following chromatographic condition: moving phase composition and ratio variation, the comparison of different chromatographic column, different column temperature comparison, different in flow rate compare, difference detects wavelength ratio, and examination the results are shown in Table 15-19.

Table 15 moving phase is formed and ratio changes examination table as a result

Moving phase is formed	The moving phase ratio	Degree of separation	Appearance time (min)	Quercetin content (%)
					Acetonitrile-0.02 phosphoric acid solution	25∶75	＞1.5	22.7	0.263
Acetonitrile-0.02 phosphoric acid solution	30∶70	＞1.5	11.3	0.265
					Acetonitrile-0.02 phosphoric acid solution	35∶65	＞1.5	7.3	0.261

The different chromatographic columns of table 16 are relatively examined or check table as a result

Chromatographic column	Degree of separation	Appearance time (min)	Quercetin content (%)
				Hypersil?ODS(4.6×250mm)	＞1.5	?10.3	0.263
Eclipse?XDB-C8(4.6×150mm)	＞1.5	?6.1	0.265
				ZORBAX?C18(4.6×250mm)	＞1.5	?11.7	0.261

The different column temperatures of table 17 are relatively examined or check table as a result

Column temperature (℃)	Degree of separation	Appearance time (min)	Quercetin content (%)
				25	＞1.5	?11.3	0.264
30	＞1.5	?10.8	0.265
				35	＞1.5	?10.4	0.261

[0147]Table 18 different in flow rate is relatively examined or check table as a result

Flow velocity (mLmin-1)	Degree of separation	Appearance time (min)	Quercetin content (%)
				0.8	＞1.5	13.9	0.263
1.0	＞1.5	11.3	0.264
				1.2	＞1.5	9.5	0.261

The different wavelength ratio that detect of table 19 are examined or check table as a result

Detect wavelength nm	Degree of separation	Appearance time (min)	Quercetin content (%)
				365	＞1.5	11.4	0.261
370	＞1.5	11.4	0.263
				375	＞1.5	11.4	0.264

13. sample determination prepares test sample and reference substance solution by the method for the invention, sample introduction writes down chromatogram respectively, calculates the content of Quercetin, the results are shown in Table 20.

Quercetin content measurement result table (n=2) in the table 2020 batch medicinal material

Sample	Quercetin content (%)	Sample	Quercetin content (%)
				Flower small stream Gao Po	0.552	Manjusri township, Guangshan County, Henan Province	0.158
Guizhou is all spared	0.339	Long for a long time	0.224
				The expensive road 63Km (facing south) that abides by	0.264	Longli paddy pin	0.171
Herd-boy Guan Daxing field	0.353	The steep mountain valley with clumps of trees and bamboo of waterside town	0.183
				Guizhou along the river	0.194	The south of Guizhou Province	0.191
Expensive abiding by under the 64km bridge of road	0.258	Tongren	0.181
				Newly add the cattle farm, stockaded village	0.351	Persimmon flower level ground, Xi Feng county	0.256
The Zhenfeng, Guizhou	0.196	The Qianxi County	0.264
				Slight slope is closed in Kweiyang not cloth	0.257	The Hunan Huaihua	0.203
The Guiding County, Guizhou	0.252	The Chenxi, Hunan	0.148

Compared with prior art, the present invention by setting up Quercetin in the polygonum perfoliatum medicinal material content assaying method and be the thin-layer chromatography discrimination method of contrast with Quercetin and Quercetin-3-O-β-positive butyl ester of D-glucuronic acid, the perfect quality inspection standard of polygonum perfoliatum medicinal material, remedied the deficiency of existing quality detection technology, the quality detection technology that makes the polygonum perfoliatum medicinal material is science, rationally more; The precision height of quality determining method of the present invention, favorable reproducibility, good stability, recovery height, measurement result is accurate, can effectively control the quality of polygonum perfoliatum medicinal material, thereby guarantees the security and the validity of its clinical practice.

Embodiment:

Further specify the present invention by the following examples, but not as limitation of the present invention.

Embodiments of the invention 1: the quality determining method of polygonum perfoliatum medicinal material comprises following project:

Proterties: stem slightly is square, and corner angle are arranged, multi-branched, and diameter reaches 0.2cm, and surperficial aubergine, pale brown look or yellow green have delavay raspberry herb on the corner angle, and memorandum is expanded, the long 2～6cm of internode, section fibrous, yellow-white has marrow or hollow; The leaf alternate has long handle, and peltate and given birth to; The many shrinkages of blade are nearly equilateral triangle after the expansion, celadon is to rufous, and lower surface vein and petiole all have down the hook of giving birth to thorn; Ochrea is wrapped on the stipes or comes off; Short spike top is given birth to or is born in the top axil, the bract circle, spend little, many atrophys or come off; Gas is little, and stem is lightly seasoned, the acid of leaf flavor.

Differentiate: (1) stem square section: epidermis is a row prothenchyma (of wood), includes the rufous material; Cortex is thin, 3～5 row cells; Tender stem pericyclic fiber Shu Lianxu becomes circular layer, and old stem is cut off into interrupted circular layer, cell wall thickness, lignify by ray; The old stem tool of bast bast fiber, the wall thickness lignify, cambium layer is obvious; Xylem vessel is big, single or 3～5 in groups; The marrow cell is big, the hollow that has; Old stem is at the visible most calcium oxalate cluster crystals of cortex, bast, ray and marrow, and tender stem is then rare or do not have.

(2) surface of leaf is seen: the irregular polygon of epicuticle cell, the nearly straight or microbend of anticline; It has the secretory cell of similar round, diameter 65 μ m down; The glandular hairs minority, head 2-8 cell, handle is short; The wavy bending of lower epidermis cell anticline; Flat shaft type of pore or inequality, glandular hairs are many slightly; It is unicellular that nonglandular hair mostly is; Master pulse and leaf margin are dredged and are given birth to the hook-shaped thorn of being made up of multiple row rhomboid or rectangle cell; Mesophyll cell contains calcium oxalate cluster crystal, diameter 17-62 μ m.

(3) get polygonum perfoliatum medicinal material 2g,, filter with 90% alcohol reflux 2h, the filtrate evaporate to dryness, residue is with methyl alcohol 1mL dissolving, as need testing solution; It is an amount of that precision takes by weighing the Quercetin reference substance, makes 0.1mgml with methyl alcohol ^-1Solution, product solution in contrast; According to " appendix a VIB of Chinese pharmacopoeia version in 2005 thin-layered chromatography test, draw each 5 μ L of need testing solution and Quercetin reference substance solution, put respectively on same silica G 254 thin layer plates, with methenyl choloride: ethyl acetate: methyl alcohol: formic acid=8: 2: 1: 0.5 is developping agent, launches, take out, dry, spray is put under the 254nm uviol lamp and is inspected with 1% aluminium choride ethanolic solution, in the test sample chromatogram, with the corresponding position of Quercetin reference substance chromatogram on show the same color spot.

(4) get polygonum perfoliatum medicinal material 2g,, filter with 90% alcohol reflux 2h, the filtrate evaporate to dryness, residue is with methyl alcohol 1mL dissolving, as need testing solution; It is an amount of that precision takes by weighing Quercetin-positive butyl ester reference substance of 3-O-β-D-glucuronic acid, makes 0.1mgml with methyl alcohol ^-1Solution, product solution in contrast; According to " appendix a VIB of Chinese pharmacopoeia version in 2005 thin-layered chromatography test, draw each 5 μ L of need testing solution and Quercetin-positive butyl ester reference substance solution of 3-O-β-D-glucuronic acid, put respectively on same polyamide thin layer plate, with methyl alcohol: formic acid: water=9: 0.5: 0.4 is developping agent, launch, take out, dry, spray is with 1% aluminium choride ethanolic solution, put under the 254nm uviol lamp and inspect, in the test sample chromatogram, with Quercetin-3-O-β-corresponding position of the positive butyl ester reference substance of D-glucuronic acid chromatogram on show the same color spot.

Check: (1) moisture is according to " an appendix IX of the Chinese pharmacopoeia H first method aquametry is measured, and must not surpass 14.0%.

(2) total ash is according to " an appendix IX of Chinese pharmacopoeia K measures, and must not surpass 10.0%.

Ethanol soluble extractives is according to " cold-maceration under an appendix X of the Chinese pharmacopoeia A ethanol soluble extractives determination method item is measured, and ethanol must not be less than 11.0% as solvent.

Assay: according to " an appendix VI of Chinese pharmacopoeia version in 2005 D high effective liquid chromatography for measuring:

Chromatographic condition and system suitability test: with the octadecyl silane is filling agent; With acetonitrile: 0.02% (mass concentration) phosphoric acid solution=30: 70 is a moving phase; Column temperature is 25 ℃; The detection wavelength is 370nm; Number of theoretical plate calculates by the Quercetin peak should be not less than 3000.

The preparation of reference substance solution: it is an amount of that precision takes by weighing the Quercetin reference substance, adds methyl alcohol and make the solution that every 1mL contains 0.02mg, promptly.

The preparation of need testing solution: get powder 1g in the polygonum perfoliatum medicinal material, the accurate title, decide, and puts in the tool plug conical flask, the accurate 5% hydrochloric acid ethanol solution 25mL that adds claims to decide weight, heating and refluxing extraction 4 hours, put coldly, claim again to decide weight, supply the weight that subtracts mistake with absolute ethyl alcohol, shake up, filter, get subsequent filtrate 2mL, water-bath volatilizes, and residue adds dissolve with methanol, is settled to 10mL, shake up, filter, filtrate is as need testing solution.

Determination method: accurate respectively reference substance solution and each 20 μ L of need testing solution of drawing, inject liquid chromatograph, measure, promptly.

Polygonum perfoliatum is pressed dry product and is calculated, and contains Quercetin (C ₁₅H ₁₉O ₇) weight must not be less than 0.20%.

Embodiments of the invention 2: the quality determining method of polygonum perfoliatum medicinal material can only contain following project:

(2) get polygonum perfoliatum medicinal material 2g,, filter with 80% alcohol reflux 1h, the filtrate evaporate to dryness, residue is with methyl alcohol 2mL dissolving, as need testing solution; It is an amount of that precision takes by weighing the Quercetin reference substance, makes 0.3mgml with methyl alcohol ^-1Solution, product solution in contrast; According to " appendix a VIB of Chinese pharmacopoeia version in 2005 thin-layered chromatography test, draw each 2 μ L of need testing solution and Quercetin reference substance solution, put respectively on same silica G 254 thin layer plates, with methenyl choloride: ethyl acetate: methyl alcohol: formic acid=10: 3: 1.5: 0.7 is developping agent, launches, take out, dry, spray is put under the 254nm uviol lamp and is inspected with 2% aluminium choride ethanolic solution, in the test sample chromatogram, with the corresponding position of Quercetin reference substance chromatogram on show the same color spot.

(2) extract: water-soluble extractives is according to " cold-maceration under an appendix X of the Chinese pharmacopoeia A water-soluble extractives determination method item is measured, and must not be less than 13.0%;

Chromatographic condition and system suitability test: with the octadecyl silane is filling agent; With acetonitrile: 0.01% (mass concentration) acetic acid solution=35: 65 is a moving phase; Column temperature is 30 ℃; The detection wavelength is 350nm; Number of theoretical plate calculates by the Quercetin peak should be not less than 3000.

The preparation of reference substance solution: it is an amount of that precision takes by weighing the Quercetin reference substance, adds methyl alcohol and make the solution that every 1mL contains 0.01mg, promptly.

The preparation of need testing solution: get powder 1g in the polygonum perfoliatum medicinal material, the accurate title, decide, and puts in the tool plug conical flask, the accurate 4% hydrochloric acid ethanol solution 35mL that adds claims to decide weight, heating and refluxing extraction 3 hours, put coldly, claim again to decide weight, supply the weight that subtracts mistake with absolute ethyl alcohol, shake up, filter, get subsequent filtrate 3mL, water-bath volatilizes, and residue adds dissolve with methanol, is settled to 10mL, shake up, filter, filtrate is as need testing solution.

Determination method: accurate respectively reference substance solution and each 8 μ L of need testing solution of drawing, inject liquid chromatograph, measure, promptly.

Embodiments of the invention 3: the quality determining method of polygonum perfoliatum medicinal material can only contain following project:

Differentiate: the surface of (1) leaf is seen: the irregular polygon of epicuticle cell, the nearly straight or microbend of anticline; It has the secretory cell of similar round, diameter 65 μ m down; The glandular hairs minority, head 2-8 cell, handle is short; The wavy bending of lower epidermis cell anticline; Flat shaft type of pore or inequality, glandular hairs are many slightly; It is unicellular that nonglandular hair mostly is; Master pulse and leaf margin are dredged and are given birth to the hook-shaped thorn of being made up of multiple row rhomboid or rectangle cell; Mesophyll cell contains calcium oxalate cluster crystal, diameter 17-62 μ m.

(2) get polygonum perfoliatum medicinal material 2g,, filter with 80% alcohol reflux 1h, the filtrate evaporate to dryness, residue is with methyl alcohol 2mL dissolving, as need testing solution; It is an amount of that precision takes by weighing Quercetin-positive butyl ester reference substance of 3-O-β-D-glucuronic acid, makes 0.3mgml with methyl alcohol ^-1Solution, product solution in contrast; According to " appendix a VIB of Chinese pharmacopoeia version in 2005 thin-layered chromatography test, draw each 2 μ L of need testing solution and Quercetin-positive butyl ester reference substance solution of 3-O-β-D-glucuronic acid, put respectively on same polyamide thin layer plate, with methyl alcohol: formic acid: water=11: 0.8: 0.6 is developping agent, launch, take out, dry, spray is with 2% aluminium choride ethanolic solution, put under the 254nm uviol lamp and inspect, in the test sample chromatogram, with Quercetin-3-O-β-corresponding position of the positive butyl ester reference substance of D-glucuronic acid chromatogram on show the same color spot.

Chromatographic condition and system suitability test: with the octadecyl silane is filling agent; With methyl alcohol: 0.03% (mass concentration) formic acid solution=20: 80 is a moving phase; Column temperature is 35 ℃; The detection wavelength is 360nm; Number of theoretical plate calculates by the Quercetin peak should be not less than 3000.

The preparation of reference substance solution: it is an amount of that precision takes by weighing the Quercetin reference substance, adds methyl alcohol and make the solution that every 1mL contains 0.03mg, promptly.

The preparation of need testing solution: get powder 1g in the polygonum perfoliatum medicinal material, the accurate title, decide, and puts in the tool plug conical flask, the accurate 6% hydrochloric acid ethanol solution 45mL that adds claims to decide weight, heating and refluxing extraction 5 hours, put coldly, claim again to decide weight, supply the weight that subtracts mistake with absolute ethyl alcohol, shake up, filter, get subsequent filtrate 4mL, water-bath volatilizes, and residue adds dissolve with methanol, is settled to 10mL, shake up, filter, filtrate is as need testing solution.

Determination method: accurate respectively reference substance solution and each 15 μ L of need testing solution of drawing, inject liquid chromatograph, measure, promptly.

Embodiments of the invention 4: the quality determining method of polygonum perfoliatum medicinal material can contain following project:

Differentiate: (1) gets polygonum perfoliatum medicinal material 2g, with 70% alcohol reflux 1.5h, filter, and the filtrate evaporate to dryness, residue is with methyl alcohol 1.5mL dissolving, as need testing solution; It is an amount of that precision takes by weighing the Quercetin reference substance, makes 0.5mgml with methyl alcohol ^-1Solution, product solution in contrast; According to " appendix a VIB of Chinese pharmacopoeia thin-layered chromatography test, draw each 5 μ L of need testing solution and Quercetin reference substance solution, put respectively on same silica G 254 thin layer plates, with methenyl choloride: ethyl acetate: methyl alcohol: formic acid=3: 1: 0.5: 0.2 is developping agent, launches, take out, dry, spray is put under the 254nm uviol lamp and is inspected with 1% aluminium choride ethanolic solution, in the test sample chromatogram, with the corresponding position of Quercetin reference substance chromatogram on show the same color spot.

(2) get polygonum perfoliatum medicinal material 2g,, filter with 70% alcohol reflux 1.5h, the filtrate evaporate to dryness, residue is with methyl alcohol 1.5mL dissolving, as need testing solution; It is an amount of that precision takes by weighing Quercetin-positive butyl ester reference substance of 3-O-β-D-glucuronic acid, makes 0.5mgml with methyl alcohol ^-1Solution, product solution in contrast; According to " appendix a VIB of Chinese pharmacopoeia thin-layered chromatography test, draw each 5 μ L of need testing solution and Quercetin-positive butyl ester reference substance solution of 3-O-β-D-glucuronic acid, put respectively on same polyamide thin layer plate, with methyl alcohol: formic acid: water=7: 0.2: 0.2 is developping agent, launch, take out, dry, spray is with 1% aluminium choride ethanolic solution, put under the 254nm uviol lamp and inspect, in the test sample chromatogram, with Quercetin-3-O-β-corresponding position of the positive butyl ester reference substance of D-glucuronic acid chromatogram on show the same color spot.

Check: extract: water-soluble extractives is according to " cold-maceration under an appendix X of the Chinese pharmacopoeia A water-soluble extractives determination method item is measured, and must not be less than 13.0%;

Chromatographic condition and system suitability test: with the octadecyl silane is filling agent; With acetonitrile: 0.05% (mass concentration) phosphoric acid solution=25: 75 is a moving phase; Column temperature is 25 ℃; The detection wavelength is 340nm; Number of theoretical plate calculates by the Quercetin peak should be not less than 3000.

The preparation of reference substance solution: it is an amount of that precision takes by weighing the Quercetin reference substance, adds methyl alcohol and make the solution that every 1mL contains 0.05mg, promptly.

The preparation of need testing solution: get powder 1g in the polygonum perfoliatum medicinal material, the accurate title, decide, and puts in the tool plug conical flask, the accurate 3% hydrochloric acid ethanol solution 30mL that adds claims to decide weight, heating and refluxing extraction 6 hours, put coldly, claim again to decide weight, supply the weight that subtracts mistake with absolute ethyl alcohol, shake up, filter, get subsequent filtrate 5mL, water-bath volatilizes, and residue adds dissolve with methanol, is settled to 10mL, shake up, filter, filtrate is as need testing solution.

Determination method: accurate respectively reference substance solution and each 10 μ L of need testing solution of drawing, inject liquid chromatograph, measure, promptly.

Claims

1. the detection method of a polygonum perfoliatum medicinal material, described detection method comprises proterties, discriminating, inspection and assay project, it is characterized in that: described discriminating comprise with the Quercetin reference substance be contrast, with methenyl choloride: ethyl acetate: methyl alcohol: formic acid=2～10: 1～3: 0.5～1.5: 0.2～0.7 be developping agent thin-layered chromatography and/or with Quercetin-positive butyl ester reference substance of 3-O-β-D-glucuronic acid be contrast, with methyl alcohol: formic acid: water=7～11: 0.2～0.8: 0.2～0.6 is the thin-layered chromatography of developping agent; Assay is the assay to contained Quercetin in the medicinal material, the content assaying method of Quercetin be with the Quercetin reference substance be contrast, with organic phase: water=10～40: 90～60 high performance liquid chromatography; Concrete thin-layer chromatography discrimination method is:

(1) get polygonum perfoliatum medicinal material 2g,, filter with 70～90% alcohol reflux, 1～2h, the filtrate evaporate to dryness, residue is with methyl alcohol 1～2mL dissolving, as need testing solution; It is an amount of that precision takes by weighing the Quercetin reference substance, makes 0.1～0.5mgml with methyl alcohol ^-1Solution, product solution in contrast; According to " each 2～5 μ L of need testing solution and Quercetin reference substance solution are drawn in the test of an appendix VI of Chinese pharmacopoeia B thin-layered chromatography, put in same silica G respectively ₂₅₄On the thin layer plate, with methenyl choloride: ethyl acetate: methyl alcohol: formic acid=2～10: 1～3: 0.5～1.5: 0.2～0.7 is developping agent, launch, take out, dry, spray is put under the 254nm uviol lamp and is inspected with 1%～2% aluminium choride ethanolic solution, in the test sample chromatogram, with the corresponding position of Quercetin reference substance chromatogram on show the same color spot;

(2) get polygonum perfoliatum medicinal material 2g,, filter with 70%～90% alcohol reflux, 1～2h, the filtrate evaporate to dryness, residue is with methyl alcohol 1～2mL dissolving, as need testing solution; It is an amount of that precision takes by weighing Quercetin-positive butyl ester reference substance of 3-O-β-D-glucuronic acid, makes 0.1～0.5mgml with methyl alcohol ^-1Solution, product solution in contrast; According to " appendix a VI of Chinese pharmacopoeia B thin-layered chromatography test, draw each 2～5 μ L of need testing solution and Quercetin-positive butyl ester reference substance solution of 3-O-β-D-glucuronic acid, put respectively on same polyamide thin layer plate, with methyl alcohol: formic acid: water=7～11: 0.2～0.8: 0.2～0.6 is developping agent, launch, take out, dry, spray is with 1%～2% aluminium choride ethanolic solution, put under the 254nm uviol lamp and inspect, in the test sample chromatogram, with Quercetin-3-O-β-corresponding position of the positive butyl ester reference substance of D-glucuronic acid chromatogram on show the same color spot;

Chromatographic condition and system suitability test: with the octadecyl silane is filling agent; With organic phase: water=10～40: 90～60 is moving phase; Column temperature is 25～35 ℃; The detection wavelength is 340～370nm; Number of theoretical plate calculates by the Quercetin peak should be not less than 3000; Described organic phase is acetonitrile or methyl alcohol, and described water is formic acid, acetate or the phosphate aqueous solution of mass concentration 0.001%～5%;

2. according to the detection method of the described polygonum perfoliatum medicinal material of claim 1, it is characterized in that: the organic phase in the moving phase is an acetonitrile, and water is mass concentration 0.02% phosphoric acid solution, and the volume ratio of organic phase and water is 30: 70.

3. according to the detection method of the described polygonum perfoliatum medicinal material of claim 1, it is characterized in that: described detection method comprises following project:

(2) surface of leaf is seen: the irregular polygon of epicuticle cell, the nearly straight or microbend of anticline; It has the secretory cell of similar round, diameter 65 μ m down; The glandular hairs minority, head 2-8 cell, handle is short; The wavy bending of lower epidermis cell anticline; Flat shaft type of pore or inequality, glandular hairs are many slightly; It is unicellular that nonglandular hair mostly is; Master pulse and leaf margin are dredged and are given birth to the hook-shaped thorn of being made up of multiple row rhomboid or rectangle cell; Mesophyll cell contains calcium oxalate cluster crystal, diameter 17-62 μ m;

(3) get polygonum perfoliatum medicinal material 2g,, filter with 70～90% alcohol reflux, 1～2h, the filtrate evaporate to dryness, residue is with methyl alcohol 1～2mL dissolving, as need testing solution; It is an amount of that precision takes by weighing the Quercetin reference substance, makes 0.1～0.5mgml with methyl alcohol ^-1Solution, product solution in contrast; According to " each 2～5 μ L of need testing solution and Quercetin reference substance solution are drawn in the test of an appendix VI of Chinese pharmacopoeia B thin-layered chromatography, put in same silica G respectively ₂₅₄On the thin layer plate, with methenyl choloride: ethyl acetate: methyl alcohol: formic acid=2～10: 1～3: 0.5～1.5: 0.2～0.7 is developping agent, launch, take out, dry, spray is put under the 254nm uviol lamp and is inspected with 1%～2% aluminium choride ethanolic solution, in the test sample chromatogram, with the corresponding position of Quercetin reference substance chromatogram on show the same color spot;

(4) get polygonum perfoliatum medicinal material 2g,, filter with 70%～90% alcohol reflux, 1～2h, the filtrate evaporate to dryness, residue is with methyl alcohol 1～2mL dissolving, as need testing solution; It is an amount of that precision takes by weighing Quercetin-positive butyl ester reference substance of 3-O-β-D-glucuronic acid, makes 0.1～0.5mgml with methyl alcohol ^-1Solution, product solution in contrast; According to " appendix a VI of Chinese pharmacopoeia B thin-layered chromatography test, draw each 2～5 μ L of need testing solution and Quercetin-positive butyl ester reference substance solution of 3-O-β-D-glucuronic acid, put respectively on same polyamide thin layer plate, with methyl alcohol: formic acid: water=7～11: 0.2～0.8: 0.2～0.6 is developping agent, launch, take out, dry, spray is with 1%～2% aluminium choride ethanolic solution, put under the 254nm uviol lamp and inspect, in the test sample chromatogram, with Quercetin-3-O-β-corresponding position of the positive butyl ester reference substance of D-glucuronic acid chromatogram on show the same color spot;

4. according to the detection method of claim 2 or 3 described polygonum perfoliatum medicinal materials, it is characterized in that: described detection method comprises following project:

(3) get polygonum perfoliatum medicinal material 2g,, filter with 90% alcohol reflux 2h, the filtrate evaporate to dryness, residue is with methyl alcohol 1mL dissolving, as need testing solution; It is an amount of that precision takes by weighing the Quercetin reference substance, makes 0.1mgml with methyl alcohol ^-1Solution, product solution in contrast; According to " appendix a VI of Chinese pharmacopoeia B thin-layered chromatography test, draw each 5 μ L of need testing solution and Quercetin reference substance solution, put respectively on same silica G 254 thin layer plates, with methenyl choloride: ethyl acetate: methyl alcohol: formic acid=8: 2: 1: 0.5 is developping agent, launches, take out, dry, spray is put under the 254nm uviol lamp and is inspected with 1% aluminium choride ethanolic solution, in the test sample chromatogram, with the corresponding position of Quercetin reference substance chromatogram on show the same color spot;

(4) get polygonum perfoliatum medicinal material 2g,, filter with 90% alcohol reflux 2h, the filtrate evaporate to dryness, residue is with methyl alcohol 1mL dissolving, as need testing solution; It is an amount of that precision takes by weighing Quercetin-positive butyl ester reference substance of 3-O-β-D-glucuronic acid, makes 0.1mgml with methyl alcohol ^-1Solution, product solution in contrast; According to " appendix a VI of Chinese pharmacopoeia B thin-layered chromatography test, draw each 5 μ L of need testing solution and Quercetin-positive butyl ester reference substance solution of 3-O-β-D-glucuronic acid, put respectively on same polyamide thin layer plate, with methyl alcohol: formic acid: water=9: 0.5: 0.4 is developping agent, launch, take out, dry, spray is with 1% aluminium choride ethanolic solution, put under the 254nm uviol lamp and inspect, in the test sample chromatogram, with Quercetin-3-O-β-corresponding position of the positive butyl ester reference substance of D-glucuronic acid chromatogram on show the same color spot;