CN104459011A - Detecting method for leucoderma treatment tablet containing eight traditional Chinese medicines - Google Patents
Detecting method for leucoderma treatment tablet containing eight traditional Chinese medicines Download PDFInfo
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Abstract
The invention provides a detecting method for a leucoderma treatment tablet containing eight traditional Chinese medicines. The detecting method comprises the following steps: (I) performing an identifying method of fructus psoraleae, astragalus and salviae miltiorrhizae in the leucoderma treatment tablet containing eight traditional Chinese medicines as follows: (1) performing microscopic identification on the fructus psoraleae in the leucoderma treatment tablet containing eight traditional Chinese medicines; (2) performing thin-layer identification on the astragalus in the leucoderma treatment tablet containing eight traditional Chinese medicines; and (3) performing thin-layer identification on the salviae miltiorrhizae in the leucoderma treatment tablet containing eight traditional Chinese medicines; (II) performing efficient liquid-phase chromatographic detection on psoralen and isopsoralen of fructus psoraleae in the leucoderma treatment tablet containing eight traditional Chinese medicines, wherein the total amount of the psoralen and isopsoralen is not lower than 0.30mg. The detecting method disclosed by the invention is stable and reliable, and strong in specifity and scientificity, can be used for effectively monitoring the quality of the leucoderma treatment tablet containing eight traditional Chinese medicines, and has a relatively great application value.
Description
Technical field
The present invention relates to compound Chinese medicinal preparation, be specifically related to the detection method for the treatment of leukodermic Chinese medicine eight taste filler sheet.
Background technology
Leucoderma is a kind of posteriority leukoderma, and current research confirms, because patients with vitiligo ultraviolet protection ability is weak, the incidence of disease of cutaneum carcinoma is higher than normal person a lot, and also can bring out various diseases, the traditional Chinese medical science is thought simultaneously, leucoderma is mainly not enough because of native endowment, fear impairing kidney, depressed rage impairing the liver, with the passing of time consume impairment of yin blood, make liver kidney blood and essence asthenia, multiple diseases caused by external factors ailment said due to cold or exposure, obstruction of meridian, make local stagnation of QI-blood, cause space between skin and muscles qi-blood disharmony, skin must not moisten support caused by.The relevant leucoderma disease of modern medicine because of and pathogenesis theory numerous, theory of autoimmunity, melanocyte autoclasia theory, neurochemistry factor theory, the melanocyte growth factor is had to lack theory, inherent cause etc., wherein, melanocyte generation shortage and autoclasia theory are occupied an leading position.
Chinese medicine eight taste filler sheet side is made up of the Radix Astragali, puncture vine, Ligusticum wallichii, Radix Angelicae Sinensis, the red sage root, Psoralea corylifolia, safflower, rhizoma cyperi 8 taste Chinese crude drug.Wherein Radix Angelicae Sinensis, the soft liver of the Radix Astragali are nourished blood, strengthening the spleen and replenishing qi, mend the foundation of acquired constitution to fill source of generating QI and blood; Psoralea corylifolia enriching kidney essence, " yi and Kui are derived from the same origin, liver kidney is ruled together "; Ligusticum wallichii, safflower, the red sage root promoting flow of qi and blood circulation; Tribulus terrestris, rhizoma cyperi dispelling wind are eliminating evil with dredging collateral, play the regulation of qi and blood altogether, effect promoting blood circulation and removing blood stasis.All medicines share can the regulation of qi and blood, promoting blood circulation and removing blood stasis, tonifies the liver and kidney, and dispels the wind loose evil.
Because herbal mixture eight taste filler sheet has eight taste Chinese medicines, complicated component is various, usually all mutual interference can be brought in the detection of tcm product, therefore, to judging that this medicine Chinese traditional medicine is after being prepared into product, judge that the technique of product is whether reasonable, effective constituent whether enrichment in product, whether the quality of final products is stablized, and setting up a quality standard that is rational, that embody product quality and detection level is very crucial technology.
Summary of the invention
Technical matters to be solved by this invention is the detection method of research and design Chinese medicine eight taste filler sheet, by the chromatogram in modern times and the analytical technology such as micro-, select reasonably separation, the method extracted and means, effectively control its active component as index, new foundation controls the standard of product quality.
The present invention adopts thin-layer chromatography to carry out comprehensive thin-layer chromatography (TLC) qualitative examination to the eight taste medicinal material Radixs Astragali, the red sage root, Radix Angelicae Sinensis, stir-baked SEMEN PSORALEAE with salt solution, stir-baked FRUCTUS TRIBULI, safflower etc. in this Chinese medicine preparation, assay research has been carried out to monarch drug in a prescription Psoralea corylifolia, wherein the Radix Astragali, the red sage root, stir-baked SEMEN PSORALEAE with salt solution pass through the detection method of this compound product preferably, obtain comparatively satisfied thin layer chromatogram analysis and the method for quantitatively determining of Psoralea corylifolia.Modern medicine is thought, containing organic acid and other various trace elements in the red sage root, Radix Angelicae Sinensis, therefore has the effect of blood circulation promoting and enriching; The main chemical compositions contained in the Radix Astragali is flavonoids, saponins and polysaccharide etc., has the effect of enhanced machine body immunity function and copingability.Stir-baked SEMEN PSORALEAE with salt solution contains furocoumarin class material, is improved skin to ultraviolet-sensitive effect, the formation of stimulation melanin cell, also has stronger activation to the activity of tyrosinase.Already confirmed, the activity of tyrosinase is directly relevant with melanin content, raises the activity of tyrosinase, accelerates melanogenic medicine and can improve leukodermic result for the treatment of.The general name of 6,7 or 7,8 that furocoumarin (Furocoumarine) the is Coumarins compounds also closed with furyl.Psoralen (Psoralene) molecular formula is C
11h
6o
3, Isopsoralen (Angelicin), molecular formula is C
11h
6o
3, be furocoumarin main representative material.So it is representative that the present invention selectes psoralen and isopsorapen two kinds of active components, as index composition, control the quality of product.The invention provides the detection method of psoralen and isopsorapen assay in the discrimination method of Psoralea corylifolia, the Radix Astragali, the red sage root in Chinese medicine eight taste filler sheet and Psoralea corylifolia.
The invention provides a kind of detection method of Chinese medicine eight taste filler sheet, the method comprises the following steps:
(1) discrimination method of Psoralea corylifolia, the Radix Astragali and the red sage root in Chinese medicine eight taste filler sheet:
(1) the Psoralea corylifolia microscopical characters in Chinese medicine eight taste filler sheet, in micro-characteristic pattern, is shown in Fig. 1
(2) TLC distinguish of the Radix Astragali in Chinese medicine eight taste filler sheet, in feature spectrogram, is shown in Fig. 2
(3) TLC distinguish of the red sage root in Chinese medicine eight taste filler sheet, in feature spectrogram, is shown in Fig. 3
(2) the high performance liquid chromatography content detection of the psoralen and isopsorapen of Psoralea corylifolia in Chinese medicine eight taste filler sheet: every sheet content is with psoralen (C
11h
6o
3) and Isopsoralen (C
11h
6o
3) calculation of total, be not less than 0.30mg, see Fig. 5
Particularly, the discrimination method of Psoralea corylifolia, the Radix Astragali and the red sage root in Chinese medicine eight taste filler sheet of the present invention comprises the following steps:
(1) the present invention's Psoralea corylifolia of adopting thin layer chromatography to differentiate in eight taste filler sheets
Get eight taste filler sheets appropriate, porphyrize, takes 2g, adds ethyl acetate or methyl alcohol, ultrasonic or hot reflux, and filter, filtrate evaporate to dryness, residue adds ethyl acetate or methyl alcohol makes dissolving, as need testing solution.Separately get Psoralea corylifolia control medicinal material, be made in the same way of control medicinal material solution.Test according to thin-layered chromatography (Chinese Pharmacopoeia version in 2010 VI B), draw above-mentioned control medicinal material solution, need testing solution, put respectively on same silica gel g thin-layer plate, with n-hexane-ethyl acetate (4:1) or petroleum ether-ethyl acetate (4:1) for developping agent, launch, take out, dry, under putting ultraviolet lamp (365nm) or spray with 10% sodium hydrate methanol solution put ultraviolet lamp (365nm) under inspect.In test sample chromatogram, with on control medicinal material solution relevant position, show same color spot.
(2) employing thin layer chromatography differentiates the Radix Astragali in eight taste filler sheets
Because of one of main ingredient that the Radix Astragali is in we, its main active is Astragaloside IV, and the present invention selects Astragaloside IV product in contrast, differentiates the quality of this product.
Get eight taste filler sheets appropriate, porphyrize, takes 2g, add water or appropriate amount of ethanol, put in water-bath and add hot reflux or ultrasonic, let cool, get supernatant, use ether jolting, washing, discard ether solution, then extract for several times with water saturated normal butyl alcohol jolting, merge n-butanol extracting liquid, wash with the ammonia solution of equivalent, then the water washing using the normal butyl alcohol of equivalent saturated is for several times, normal butyl alcohol liquid evaporate to dryness, residue adds methyl alcohol 1ml makes dissolving, centrifugal, gets supernatant as need testing solution.Separately get Radix Astragali control medicinal material appropriate, be made in the same way of control medicinal material solution.Get Astragaloside IV reference substance, add methyl alcohol and make in right amount, in contrast product solution.Test according to thin-layered chromatography (Chinese Pharmacopoeia version in 2010 annex VI B), draw above-mentioned three kinds of solution appropriate, put respectively on same silica gel g thin-layer plate, with ethyl acetate, alcohol and water (8:2:1) or (methenyl choloride-methanol-water (12:5:3) is for developping agent, launch, take out, dry, direct heating or the ethanol solution of sulfuric acid sprayed with 10%, 105 DEG C are heated to clear spot.In test sample chromatogram, on the position corresponding to control medicinal material chromatogram, the principal spot of aobvious same color and fluorescence principal spot.
(3) employing thin layer chromatography differentiates the red sage root in eight taste filler sheets
Get eight taste filler sheets appropriate, porphyrize, takes 2g, add water or appropriate amount of ethanol, put in water-bath and add hot reflux or ultrasonic dissolution, add hydrochloric acid solution appropriate, mixing, centrifugal, get supernatant, add equivalent extracted by ether 2 times, merge extract, centrifugal layering, draw ether extracted liquid, volatilize, make dissolving in right amount with absolute ethyl alcohol, as need testing solution.Separately get red sage root control medicinal material appropriate, be made in the same way of control medicinal material solution.Test according to thin-layered chromatography (Chinese Pharmacopoeia version in 2010 VI B), draw above-mentioned two kinds of solution appropriate, put respectively on same silica gel g thin-layer plate, with ethyl acetate-methenyl choloride-benzene-formic acid (6:5:3:1) or methenyl choloride-acetone-formic acid (8:2:1) for developping agent, launch, take out, dry, spray the ferric trichloride ethanolic solution with 10%, 105 DEG C are heated to clear spot.In test sample chromatogram, with on control medicinal material solution relevant position, show identical colored speckles.
(2) high performance liquid chromatography is adopted quantitatively to detect the psoralen and isopsorapen content of Psoralea corylifolia in eight taste filler sheets.
Because the main active in Psoralea corylifolia is psoralen (C
11h
6o
3) and Isopsoralen (C
11h
6o
3), therefore, select psoralen and isopsorapen as the standard items quantitatively detecting Psoralea corylifolia.
The psoralen and isopsorapen assay of Psoralea corylifolia:
Chromatographic condition and system suitability: take octadecylsilane chemically bonded silica as filling agent; With methanol-water (50:50) or (55:45) for mobile phase; Determined wavelength is 246nm.Number of theoretical plate calculates should be not less than 3000 by Psoralea corylifolia peak.
The preparation of reference substance solution: be taken at drying under reduced pressure (vacuum tightness-0.1Mpa, temperature 60 C) the psoralen and isopsorapen reference substance of 12-24 hour appropriate, accurately weighed, add methyl alcohol and make every 1ml respectively containing the solution of 15-20 μ g, to obtain final product.
The preparation of need testing solution: get eight taste filler sheet 20, fine ground, accurately weighed 2.0g, puts in apparatus,Soxhlet's or in round-bottomed flask, adds methyl alcohol or ethanol 80-120ml, adds hot reflux 2-5 hour; Or soaked overnight, then add hot reflux 2-5 hour, let cool, be transferred in 100ml volumetric flask, add methanol constant volume to scale, shake up, filter, get subsequent filtrate, obtain test sample.
Determination method: accurate absorption reference substance solution and need testing solution each 10-20 μ l respectively, injection liquid chromatography, measures.
Every sheet eight taste filler sheet contains Psoralea corylifolia, with psoralen (C
11h
6o
3) and Isopsoralen (C
11h
6o
3) calculation of total, be not less than 0.30mg.
The preparation method of its preferred need testing solution is: get eight taste filler sheet 20, and fine ground, accurately weighed 2.0g, puts in apparatus,Soxhlet's, adds methyl alcohol 80ml, adds hot reflux 4 hours; Let cool, be transferred in 100ml volumetric flask, add methanol constant volume to scale, shake up, filter, get subsequent filtrate, obtain test sample.
In the Chinese medicine Engage of standard epoch, the quality of product is the life of product, and the standard having product is then have gordian technique.The invention provides the detection method of the quality standard of eight taste filler flake products, through the methodological study of science, the method optimized is reliable and stable, selectivity and science strong, effectively can detect the Related Component of herbal mixture eight taste filler sheet, for user provides the product of ensuring the quality of products, there is larger using value.
Accompanying drawing explanation
The thin-layer chromatogram of Psoralea corylifolia in Fig. 1, embodiment 1 eight taste filler sheet, in figure: from left to right, spot sequence, negative blank (salt deficiency Psoralea corylifolia) the 2-stir-baked SEMEN PSORALEAE with salt solution of 1-positive single medicinal material 3-4-5-eight taste filler sheet sample;
The thin-layer chromatogram of the Radix Astragali in Fig. 2, embodiment 1 eight taste filler sheet, in figure: from left to right, spot sequence, 1-negative blank (lacking the Radix Astragali) 2-Astragaloside IV 3-Radix Astragali control medicinal material 4-5-6-eight taste filler sheet sample; Left figure is inspection under daylight, and right figure inspects under uviol lamp 365nm;
The thin-layer chromatogram of the red sage root in Fig. 3, embodiment 1 eight taste filler sheet, in figure: from left to right, spot sequence, 1-negative blank (lacking the red sage root) 2-red sage root control medicinal material 3-4-5-eight taste filler sheet sample;
The high-efficient liquid phase chromatogram of Fig. 4, embodiment 4 psoralen (front), Isopsoralen (afterwards) reference substance;
The high-efficient liquid phase chromatogram of Psoralea corylifolia-psoralen, Isopsoralen in Fig. 5, embodiment 4 eight taste filler sheet sample;
High-efficient liquid phase chromatogram in Fig. 6, embodiment 4 blank sample (lacking Psoralea corylifolia).
Embodiment
The eight taste filler flake products that following examples use are produced by Shanghai Baisuixing Pharmaceutical Co., Ltd.
Embodiment 1, eight taste filler sheet detection experiment
(1) in thin-layer chromatography chromatography, Psoralea corylifolia is differentiated: get eight taste filler sheet 10, porphyrize, takes 2g, adds ethyl acetate 20ml, ultrasonic process 15 minutes, and filter, filtrate evaporate to dryness, residue adds ethyl acetate 1ml makes dissolving, as need testing solution.Separately get Psoralea corylifolia control medicinal material (Chinese medicine biological standardization institute) 0.5g, be made in the same way of control medicinal material solution.Test according to thin-layered chromatography (Chinese Pharmacopoeia version in 2010 VI B), draw above-mentioned control medicinal material solution 5 μ l, test sample 10 μ l, put respectively on same silica gel g thin-layer plate, with normal hexane-ethyl acetate (4:1V/V) for developping agent, launch, take out, dry, spray under putting ultraviolet lamp (365nm) with the sodium hydrate methanol solution of 10% and inspect.In test sample chromatogram, with on control medicinal material solution relevant position, show same color spot.Psoralea corylifolia microscopical characters in eight taste filler sheets, in micro-characteristic pattern, is shown in Fig. 1.
(2) get this product 10, porphyrize, takes 2g, and add water 50ml, put in water-bath and add hot reflux 30 minutes, let cool, centrifugal, get supernatant, extract 2 times with ether jolting, each 50ml, discards ether solution, extract 2 times, each 50ml with water saturated normal butyl alcohol jolting again, merge n-butanol extracting liquid, wash with the ammonia solution of equivalent, then use the water washing 2 times that the normal butyl alcohol of equivalent is saturated, normal butyl alcohol liquid evaporate to dryness, residue adds methyl alcohol 1ml makes dissolving, centrifugal, gets supernatant as need testing solution.(Chinese medicine biological standardization institute 1g, is made in the same way of control medicinal material solution separately to get Radix Astragali control medicinal material.Get Astragaloside IV reference substance (Chinese medicine biological standardization institute), add methyl alcohol and make the solution of every 1ml containing 0.5mg, product solution in contrast.Test according to thin-layered chromatography (Chinese Pharmacopoeia version in 2010 annex VI B), draw each 5 μ l of above-mentioned three kinds of solution, put respectively on same silica gel g thin-layer plate, with ethyl acetate, alcohol and water (8:2:1V/V) for developping agent, launch, take out, dry, spray the ethanol solution of sulfuric acid with 10%, 105 DEG C are heated to clear spot.In test sample chromatogram, on the position corresponding to control medicinal material chromatogram, the principal spot of aobvious same color and fluorescence principal spot.In eight taste filler sheets, the TLC distinguish of the Radix Astragali, in feature spectrogram, is shown in Fig. 2.
(3) get this product 10, porphyrize, takes 2g, use 20ml water, ultrasonic process is dissolved for 30 minutes, adds 0.1mol/l hydrochloric acid solution 10ml, mixing, centrifugal 10 minutes, gets supernatant, add equivalent extracted by ether 2 times, merge extract, centrifugal layering, draw ether extracted liquid, volatilize, make dissolving, as need testing solution with absolute ethyl alcohol 1ml.Separately get red sage root control medicinal material (Chinese medicine biological standardization institute) 0.5g, be made in the same way of control medicinal material solution.Test according to thin-layered chromatography (Chinese Pharmacopoeia version in 2010 VI B), draw each 5 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, with ethyl acetate-methenyl choloride-benzene-formic acid (6:5:3:1V/V) for developping agent, launch, take out, dry, spray the ferric trichloride ethanolic solution with 10%, 105 DEG C are heated to clear spot.In test sample chromatogram, with on control medicinal material solution relevant position, show identical colored speckles.In eight taste filler sheets, the TLC distinguish of the red sage root, in feature spectrogram, is shown in Fig. 3.
(4) the psoralen and isopsorapen content detection of Psoralea corylifolia:
Chromatographic condition and system suitability: take octadecylsilane chemically bonded silica as filling agent; With methanol-water (50:50) for mobile phase; Determined wavelength is 246nm.Number of theoretical plate calculates should be not less than 3000 by Psoralea corylifolia peak.
The preparation of reference substance solution: be taken at the drying under reduced pressure psoralen of 12 hours (Chinese medicine biological standardization institute) and Isopsoralen (Chinese medicine biological standardization institute) reference substance, accurately weighed, add methyl alcohol and make every 1ml respectively containing the solution of 15 μ g, to obtain final product.
The preparation of need testing solution: get eight tastes and stop white tiles 20, fine ground, claim 2.5g, accurately weighed, put in apparatus,Soxhlet's, add methyl alcohol 80ml, soaked overnight, adds hot reflux 3 hours, lets cool, be transferred in 100ml volumetric flask, be incorporated in volumetric flask with a small amount of washed with methanol apparatus,Soxhlet's, add methanol constant volume to scale, shake up, filter, get subsequent filtrate, to obtain final product.
Determination method: accurate absorption reference substance solution and each 10 μ l of need testing solution respectively, injection liquid chromatography, measures, to obtain final product.
In eight taste filler sheets, the high-efficient liquid phase chromatogram determining content of the psoralen and isopsorapen of Psoralea corylifolia is with psoralen (C
11h
6o
3) and Isopsoralen (C
11h
6o
3) calculation of total, be not less than 0.30mg.
Eight tastes stop the every sheet of white tiles containing Psoralea corylifolia, with psoralen (C
11h
6o
3) and Isopsoralen (C
11h
6o
3) calculation of total gets eight tastes and stop white tiles sample products (lot number: x110401, x110402, x110403) and stop white tiles assay according to Psoralea corylifolia quality standard draft to eight tastes, the results are shown in Table 8.
Table 1. three batch sample assay result
X110401, X110402, X110403 tri-batches of products, are produced by Shanghai Baisuixing Pharmaceutical Co., Ltd.
Embodiment 2
(1) get eight tastes and stop each 10 of white tiles X110401, X110402, X110403, porphyrize, takes 2g respectively, all adds ethyl acetate 20mL, ultrasonic process 15min, and filter, filtrate evaporate to dryness, residue adds ethyl acetate 1mL makes dissolving, as need testing solution.Separately get medicine biological standardization institute of Psoralea corylifolia single control medicinal material China) 0.5g, be made in the same way of positive control solution.And blank sample is made in the same way of negative placebo solution (will to remove stir-baked SEMEN PSORALEAE with salt solution medicinal material).Test according to thin-layered chromatography (Chinese Pharmacopoeia version in 2010 VI B), draw above-mentioned positive control medicinal material solution 5 μ l, test sample and each 10 μ l of negative control solution, put respectively on same silica gel g thin-layer plate, with normal hexane-ethyl acetate (4:1V/V) for developping agent, ascending development, exhibition is apart from about 8cm, take out, dry, spray with the potassium hydroxide methanol solution of 10% (or replacing with 10% sodium hydrate methanol solution), inspect under putting ultraviolet lamp (365nm).In test sample chromatogram, with on control medicinal material solution relevant position, show identical blue and white fluorescence spot, and negative control solution occurs without same blob.See accompanying drawing 1.
(2) get eight tastes and stop each 10 of white tiles X110401, X110402, X110403, porphyrize, takes 2g respectively, all add water 50ml, place in water-bath and reflux 30 minutes, let cool, centrifugal, get supernatant, extract 2 times with ether jolting, each 50ml, discards ether solution, use water saturated normal butyl alcohol again, jolting extracts 2 times, each 50ml, merges n-butanol extracting liquid, wash with the ammonia solution of equivalent, use the water washing 2 times that the normal butyl alcohol of equivalent is saturated again, normal butyl alcohol evaporate to dryness, residue adds methyl alcohol 1mL makes dissolving, centrifugal, get supernatant as need testing solution.Separately get Astragaloside IV control medicinal material 1g (Chinese medicine biological standardization institute), be made in the same way of single medicinal material and control medicinal material solution.And blank sample method makes negative placebo solution (will to remove Milkvetch Root), gets Astragaloside IV reference substance, add methyl alcohol and make the solution of every 1ml containing 0.5mg, product solution in contrast.Test according to thin-layered chromatography (Chinese Pharmacopoeia version in 2010 VI B), draw each 5 μ l of above-mentioned 5 kinds of solution, put respectively on same silica gel g thin-layer plate, with ethyl acetate, alcohol and water (8:2:1V/V) for developping agent, developing tank presaturation 5 minutes (20 DEG C).Ascending development, exhibition is apart from about 8cm, and take out, dry, spray the ethanol solution of sulfuric acid with 10%, 105 DEG C are heated to clear spot.In test sample chromatogram, with on reference substance and control medicinal material solution relevant position, show identical colored speckles, and negative control solution occurs without same blob.See accompanying drawing 2.
(3) get eight tastes and stop each 10 of white tiles X110401, X110402, X110403, porphyrize, takes 2g respectively, all add 20mL water, ultrasonic process 30min dissolves, and adds 0.1mol/l hydrochloric acid solution 10ml, mixing, centrifugal 10min, gets supernatant, add equivalent extracted by ether 2 times, merge extract, centrifugal layering (abolishing emulsification), draw ether extracted liquid, volatilize, make dissolving, as need testing solution with absolute ethyl alcohol 1mL.Separately get red sage root control medicinal material (Chinese medicine biological standardization institute) 0.5g and be made in the same way of control medicinal material solution.And blank sample is made in the same way of negative placebo solution (will to remove red rooted salvia).Test according to thin-layered chromatography (Chinese Pharmacopoeia version in 2010 VI B), draw above-mentioned 3 kinds of solution 5 μ l, put respectively on same silica gel g thin-layer plate, with ethyl acetate-chloroform-benzene-formic acid (6:5:3:1V/V) for developping agent, developing tank presaturation 5min.Ascending development, exhibition is apart from about 8cm, and take out, dry, spray the ferric trichloride ethanolic solution with 10%, 105 DEG C are heated to clear spot.In test sample chromatogram, with on control medicinal material solution relevant position, show identical colored speckles, and negative control solution occurs seeing accompanying drawing 3 without same blob.
Psoralea corylifolia-psoralen in embodiment 3 eight taste filler sheet.The research of the high-performance liquid chromatogram determination method of Isopsoralen
Measure according to high performance liquid chromatography (Chinese Pharmacopoeia version in 2010 annex VI D).
System forms: detecting device photodiode array detector SPD-M20A
Data workstation LCSolution
Pillar Kromasil 100-5C18 250*4.6mm
The 30 DEG C of inspections of testing conditions INSTRUMENT MODEL LC-20AT high performance liquid chromatograph temperature
Survey wavelength 246nm
Mobile phase methanol-water (50:50V/V) sample size 10 μ L
Reagent, standard items
Methyl alcohol specification: chromatographically pure
Psoralen specification: reference substance lot number: 110739-200814
Isopsoralen specification: reference substance lot number: 110738-201012
The inventive method is undertaken selecting and verifying by following experiment:
This experiment take octadecylsilane chemically bonded silica as filling agent; Selection determined wavelength is 246nm, and mobile phase is methanol-water (55:45V/V), and flow velocity is 1.0ml/min, column temperature 30 DEG C.Theoretical cam curve, in psoralen, should be not less than 3000.
Prepared by reference substance solution: be taken at drying under reduced pressure (vacuum tightness-0.1Mpa, temperature 60 C) the psoralen and isopsorapen reference substance of 12 hours appropriate, accurately weighed, adds methyl alcohol and makes every 1ml respectively containing the solution of 20 μ g, to obtain final product.
The experiment of different extraction time:
Get this product 20, fine ground, get about 2.5g, accurately weighed, put in apparatus,Soxhlet's, add methyl alcohol 80ml, adopt within 2,4 hours, add hot reflux respectively, spend the night after soaking and add hot reflux in 2,3,4 hours, let cool, be transferred in 100ml volumetric flask, add methanol constant volume to scale, shake up, filter, get subsequent filtrate, to obtain final product.Concrete data see the following form 1.
Table 2. different extraction time extracts situation
This method all can realize.
The experiment of different sample size:
The preparation of need testing solution: get this product 20, fine ground, get about 2.5g, 3.5g, 4.5g respectively, accurately weighed, put respectively in apparatus,Soxhlet's, add methyl alcohol 80ml, add hot reflux 4 hours, let cool, be transferred in 100ml volumetric flask, add methanol constant volume to scale, shake up, filter, get subsequent filtrate, to obtain final product.
The different sampling amount of table 3. measures situation
Said method all can be implemented.
Method specificity:
By above-mentioned high-efficient liquid phase chromatogram condition, enter blank sample respectively, reference substance solution, each 10 μ l of test sample eight taste filler sheet solution, determine retention time, and the degree of separation of related substances.
Table 4 blank sample, reference substance solution, eight taste filler sheet test sample chromatograms
Psoralea corylifolia blank sample, by preparation method's process of need testing solution, by above-mentioned chromatographic condition sample introduction 10 μ l respectively, going out peak place at psoralen and isopsorapen reference substance does not have absorption peak.
Precision test:
Be equipped with reference substance solution on request, get psoralen and isopsorapen reference substance appropriate, accurately weighed, add methyl alcohol and make every 1ml respectively containing the solution of 20 μ g, sample introduction 10 μ l, continuous sample introduction 5 pin:
Table 5 Precision test result
Average content X
aVG=2232808, RSD=0.09%, n=5, meet content assaying method regulation.Conclusion: Precision test result shows that this assay method precision is good
Sample solution stability:
Preparing solution by preparing need testing solution method, obtaining final product.Get preparation rear 0,2,4,8,12,24 hours, by above-mentioned liquid phase chromatogram condition sample introduction 10 μ L, record peak area: RSD=0.25%
The stability test result of table 6 sample solution
Linear relationship:
Prepare the reference substance solution of variable concentrations respectively, concentration is respectively 6.537,13.074,26.148,39.222,65.37 μ g/ml.Under selected chromatographic condition, sample introduction 10 μ l, records each chromatographic peak area, sees the following form respectively
Table 7 psoralen and Isopsoralen standard curve determination result
With peak area A (mAU*s) for Y, with the concentration C (μ g/ml) of psoralen and Isopsoralen summation for X, try to achieve Psoralea corylifolia content measuring standard curve, the range of linearity is regression equation and the related coefficient of 6.537-65.37 μ g/ml, regression equation is A=86671.24C-52446.8, r=0.9997, and the range of linearity is 6.537-65.37 μ g/ml, good relationship, meets assay requirement.
Reappearance is tested:
Get 6 parts, the sample of same lot number X110401, press the process of surname extraction sample extraction method respectively, psoralen and isopsorapen relative peak area and calculating data see the following form.
The reproducible test results of table 8 sample solution
Psoralen and isopsorapen average content X
aVG=2.325mg, RSD=1.23%, n=6, meet content assaying method regulation.
Application of sample recovery test:
Take 9 parts, the sample of lot number X110401 respectively, every part takes about 1.25g, accurately weighed, adds a certain amount of psoralen and Isopsoralen reference substance, then presses preparation method's process of sample solution, measures its relative peak area, calculates.The results are shown in following table.
Table 9 psoralen and Isopsoralen assay average recovery test findings
Result: the recovery is respectively 95.33%, 96.36%, 95.79%, 94.90%, 95.79%, 95.36%, 96.99%, 96.60%, 97.15%, average recovery rate is 96.03%, RSD=0.82%, and this method measures content, the average recovery of three concentration is good
Conclusion: method determination of recovery rates result shows the recovery 96.03%, RSD=0.82%, n=9, meets the requirements.
Claims (6)
1. the detection method of Chinese medicine eight taste filler sheet, it is characterized in that, the method comprises the following steps:
(1) discrimination method of Psoralea corylifolia, the Radix Astragali and the red sage root in Chinese medicine eight taste filler sheet:
(1) the Psoralea corylifolia microscopical characters in Chinese medicine eight taste filler sheet;
(2) TLC distinguish of the Radix Astragali in Chinese medicine eight taste filler sheet;
(3) TLC distinguish of the red sage root in Chinese medicine eight taste filler sheet;
(2) in Chinese medicine eight taste filler sheet, survey picked up by the high performance liquid chromatography of the psoralen and isopsorapen of Psoralea corylifolia, and content, with psoralen and isopsorapen calculation of total, is not less than 0.30mg.
2. the detection method of Chinese medicine eight taste filler sheet according to claim 1, is characterized in that, the Psoralea corylifolia microscopical characters method in step (one) (1) Chinese medicine eight taste filler sheet is:
Get eight taste filler sheets, porphyrize, get 2g and add ethyl acetate or methyl alcohol, ultrasonic or hot reflux, filter, filtrate evaporate to dryness, residue adds ethyl acetate or methyl alcohol makes dissolving, as need testing solution; Separately get Psoralea corylifolia control medicinal material, be made in the same way of control medicinal material solution; Test according to thin-layered chromatography, draw above-mentioned control medicinal material solution, test sample, put respectively on same silica gel g thin-layer plate, with n-hexane-ethyl acetate 4:1V/V or petroleum ether-ethyl acetate 4:1V/V for developping agent, launch, take out, dry, under putting ultraviolet lamp 365nm or spray with 10% sodium hydrate methanol solution put ultraviolet lamp 365nm under inspect, in test sample chromatogram, with on control medicinal material solution relevant position, show same color spot.
3. the detection method of Chinese medicine eight taste filler sheet according to claim 1, is characterized in that, in step (one) (2) Chinese medicine eight taste filler sheet, the thin-layer identification method of the Radix Astragali is:
Get eight taste filler sheets, porphyrize, get 2g and add water or appropriate amount of ethanol, put in water-bath and add hot reflux or ultrasonic, let cool, get supernatant, use ether jolting, washing, discards ether solution, carry for several times with water saturated normal butyl alcohol jolting again, merge n-butanol extracting liquid, wash with the ammonia solution of equivalent, the water washing several using the normal butyl alcohol of equivalent saturated again, normal butyl alcohol liquid evaporate to dryness, residue adds methyl alcohol 1ml makes dissolving, centrifugal, get supernatant as need testing solution; Separately get Radix Astragali control medicinal material appropriate, be made in the same way of control medicinal material solution; Get Astragaloside IV reference substance, add methyl alcohol and make in right amount, in contrast product solution; Test according to thin-layered chromatography, draw above-mentioned three kinds of solution appropriate, put respectively on same silica gel g thin-layer plate, with ethyl acetate, alcohol and water 8:2:1V/V or methenyl choloride-methanol-water 12:5:3V/V for developping agent, launch, take out, dry, direct heating or the ethanol solution of sulfuric acid sprayed with 10%, 105 DEG C are heated to clear spot.In test sample chromatogram, on the position corresponding to control medicinal material chromatogram, the principal spot of aobvious same color and fluorescence principal spot.
4. the detection method of Chinese medicine eight taste filler sheet according to claim 1, is characterized in that, in step (one) (3) Chinese medicine eight taste filler sheet, the thin-layer identification method of the red sage root is:
Get eight taste filler sheet porphyrizes, 2g water or ethanol alcohol in right amount, are put in water-bath and are added hot reflux or ultrasonic, dissolve, add hydrochloric acid solution appropriate, mixing, centrifugal, get supernatant, add equivalent extracted by ether 2 times, merge extract, centrifugal layering, draws ether extracted liquid, volatilizes, dissolving is made in right amount, as need testing solution with absolute ethyl alcohol; Separately get red sage root control medicinal material appropriate, be made in the same way of control medicinal material solution; Test according to thin-layered chromatography, draw above-mentioned two kinds of solution appropriate, put respectively on same silica gel g thin-layer plate, with ethyl acetate-methenyl choloride-benzene-formic acid 6:5:3:1V/V or methenyl choloride-acetone-formic acid 8:2:1V/V for developping agent, launch, take out, dry, spray the ferric trichloride ethanolic solution with 10%, 105 DEG C are heated to clear spot, in test sample chromatogram, with on control medicinal material solution relevant position, show identical colored speckles.
5. the detection method of Chinese medicine eight taste filler sheet according to claim 1, is characterized in that, in step (two) Chinese medicine eight taste filler sheet, the high-performance liquid chromatogram determination method of the psoralen and isopsorapen of Psoralea corylifolia comprises the following steps:
Chromatographic condition and system suitability: take octadecylsilane chemically bonded silica as filling agent; With methanol-water 50:50V/V or 55:45V/V for mobile phase; Determined wavelength is 246nm, and theoretical cam curve calculates should be not less than 3000 by Psoralea corylifolia peak;
The preparation of reference substance solution: be taken at drying under reduced pressure (vacuum tightness-0.1Mpa, temperature 60 C) the psoralen and isopsorapen reference substance of 12-24 hour appropriate, accurately weighed, add methyl alcohol and make every 1ml respectively containing the solution of 15-20 μ g, to obtain final product;
The preparation of need testing solution: get eight taste filler sheet 20, fine ground, accurately weighed 2.0g, puts in apparatus,Soxhlet's or round-bottomed flask, adds methyl alcohol or ethanol 80-120ml, adds hot reflux 2-5 hour; Or soaked overnight, then add hot reflux 2-4 hour, let cool, be transferred in 100ml volumetric flask, add methanol constant volume to scale, shake up, filter, get subsequent filtrate, obtain test sample;
Measure: accurate absorption reference substance solution and need testing solution each 10-20 μ l respectively, injection liquid chromatography, picks up survey and measures.
6. the detection method of Chinese medicine eight taste filler sheet according to claim 5, it is characterized in that, in step (two) Chinese medicine eight taste filler sheet, survey picked up by the high performance liquid chromatography of the psoralen and isopsorapen of Psoralea corylifolia, content, with psoralen and isopsorapen calculation of total, is not less than 0.30mg.
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