CN104849397A - Diabetes treating traditional Chinese medicine preparation identification method - Google Patents
Diabetes treating traditional Chinese medicine preparation identification method Download PDFInfo
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- CN104849397A CN104849397A CN201410052626.7A CN201410052626A CN104849397A CN 104849397 A CN104849397 A CN 104849397A CN 201410052626 A CN201410052626 A CN 201410052626A CN 104849397 A CN104849397 A CN 104849397A
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Abstract
The present invention discloses a diabetes treating traditional Chinese medicine preparation identification method. According to the present invention, the traditional Chinese medicine preparation is prepared from Keyaoluoqu, plantago depressa willd, agrimonia pilosa ledeb, and lonicera confusa dc; the identification is carrying out thin-layer chromatography on Keyaoluoqu, plantago depressa willd, agrimonia pilosa ledeb, and lonicera confusa dc, wherein the Keyaoluoqu identification method is the thin-layer chromatography method adopting a luteoloside reference substance as a control and adopting a mixed solution comprising ethyl acetate, acetone, formic acid and water according to a ratio of 7:3:1:1.2 as a developing solvent, the plantago depressa willd identification method is the thin-layer chromatography method adopting a plantamajoside reference substance as a control and adopting a mixed solution comprising ethyl acetate, acetone, formic acid and water according to a ratio of 7:3:1:1.2 as a developing solvent, the lonicera confusa dc identification method is the thin-layer chromatography method adopting an isochlorogenic acid A reference substance as a control and adopting a mixed solution comprising ethyl acetate, acetone, formic acid and water according to a ratio of 7:3:1:1.2 as a developing solvent; and the identification method of the present invention has characteristics of high sensitivity, good reproducibility, convenience, feasibility, and no negative control interference.
Description
Technical field
The present invention relates to a kind of discrimination method for the treatment of the Chinese medicine preparation of diabetes, belong to technical field of traditional Chinese medicines.
Background technology
Diabetes are common frdquently encountered diseases of serious harm human health, and the chaff urine patient of current China increases year by year, and exceeded more than 2,000 ten thousand people, global diabetic has exceeded 100,000,000 people, and already, diabetes have become a worldwide problem.Distribution of diabetes is only second to malignant tumour and angiocardiopathy, is the third-largest disease jeopardizing human life.
Application number is 200910312208.6, a kind of discrimination method for the treatment of Asiatic plantain in the Chinese medicine preparation of urinary infection is disclosed in the patented claim that name is called " method of quality control of the Chinese medicine preparation for the treatment of urinary infection ", but how it also unexposedly differentiate the Asiatic plantain in the Chinese medicine preparation for the treatment of diabetes, in addition, also openly how the native honeysuckle in the Chinese medicine preparation for the treatment of diabetes is differentiated in prior art, thus a set of strict quality inspection standard reliably can not be formed, if there is no strict quality standard, the product obtained can not guarantee its quality, result will affect the clinical efficacy of this medicine, think the therapeutic action of Chinese medicine preparation improving treatment diabetes, guarantee medication safe, effectively and product quality stable, formulating a strict reliable quality standard becomes the basic demand of ensuring drug quality.
Summary of the invention
The object of the invention is to, a kind of discrimination method for the treatment of the Chinese medicine preparation of diabetes is provided, the method is highly sensitive, reproducible, it is feasible to facilitate, negative control is noiseless, use it in the quality determining method of the Chinese medicine preparation for the treatment of diabetes, effectively can control drug quality, thus guarantee the safe, effective of the stable of product quality and clinical application.
For solving the problems of the technologies described above, the present invention adopts following technical scheme: a kind of discrimination method for the treatment of the Chinese medicine preparation of diabetes, described Chinese medicine preparation by section die young Luo Qu, Asiatic plantain, hairyvein agrimony and soil honeysuckle be prepared from; Wherein, the section Luo Qu that dies young carries out glycolysis by fresh Snakegourd Fruit, fresh reticulate millettia, fresh stringy stonecrop, flour and wheat bran through song system and forms; Described discriminating be to section die young Luo Qu, Asiatic plantain, soil honeysuckle indentification by TLC; The die young discrimination method of Luo Qu of section is with galuteolin reference substance for contrast, with ethyl acetate-acetone-formic acid-water=7: be the thin-layered chromatography of developping agent at 3: 1: 1.2; The discrimination method of Asiatic plantain is with greater plantain glycosides reference substance for contrast, with ethyl acetate-acetone-formic acid-water=7: be the thin-layered chromatography of developping agent at 3: 1: 1.2; The discrimination method of soil honeysuckle is with 3,5-Dicaffeoylquinic acid reference substance for contrast, with ethyl acetate-acetone-formic acid-water=7: be the thin-layered chromatography of developping agent at 3: 1: 1.2.
Concrete, section's sieve Qu Caiyong following methods of dying young is differentiated:
Get this product content 1g, add 10ml methyl alcohol, ultrasonic process 30 minutes, filter, get filtrate 5ml, add the magnesium powder of 0.2g, drip 12 ~ 14 concentrated hydrochloric acids, water bath processing 1 ~ 2min, solution becomes reddish violet.
The section Luo Qu that dies young also can adopt following methods to differentiate:
(1) preparation of need testing solution: get this product content 1.8 ~ 3g, adds 95% ethanol 50ml, refluxing extraction 2 hours, filters, gets filtrate and be concentrated into 5ml, to obtain final product;
(2) preparation of reference substance solution: get galuteolin reference substance, adds methyl alcohol and makes the solution of every 1ml containing 0.4mg, to obtain final product;
(3) discrimination method: according to the annex VI B thin-layered chromatography test of Chinese Pharmacopoeia version in 2010, draw need testing solution 3 ~ 6 μ l, reference substance solution 1 ~ 2 μ l, put respectively on same silica gel g thin-layer plate, with ethyl acetate-acetone-formic acid-water=7: 3: 1: 1.2 for developping agent, launch, take out, dry, spray with 5% aluminium choride ethanolic solution, in 105 DEG C of heating 10 minutes, let cool, inspect under putting 365nm uviol lamp; In test sample chromatogram, on the position corresponding to reference substance chromatogram, the fluorescence spot of display same color.
The concrete discrimination method of aforesaid Asiatic plantain comprises the following steps:
(1) preparation of need testing solution: get this product content 2g, adds 95% ethanol 50ml, refluxing extraction 2 hours, filters, gets filtrate and be concentrated into 10ml, to obtain final product;
(2) preparation of reference substance solution: get greater plantain glycosides reference substance, adds 60% methyl alcohol and makes the solution of every 1ml containing 0.1mg, to obtain final product;
(3) discrimination method: according to the annex VI B thin-layered chromatography test of Chinese Pharmacopoeia version in 2010, draw need testing solution 2 ~ 4 μ l, reference substance solution 1 ~ 2 μ l, put respectively on same polyamide film, with ethyl acetate-acetone-formic acid-water=7: 3: 1: 1.2 for developping agent, launch, exhibition, apart from being 5cm, is taken out, heat after 10 minutes at 60 DEG C, inspect under being placed in 365nm uviol lamp; In test sample chromatogram, on the position corresponding to reference substance chromatogram, the fluorescence spot of display same color.
The concrete discrimination method of aforesaid soil honeysuckle comprises the following steps:
(1) preparation of need testing solution: get this product content 2g, add methyl alcohol 30ml, refluxing extraction 3 hours, filters, gets filtrate and be concentrated into 10ml, to obtain final product;
(2) preparation of reference substance solution: get 3,5-Dicaffeoylquinic acid reference substance, adds methyl alcohol and makes the solution of every 1ml containing 0.4mg, to obtain final product;
(3) discrimination method: according to the annex VI B thin-layered chromatography test of Chinese Pharmacopoeia version in 2010, draw need testing solution 2 ~ 3 μ l, reference substance solution 1 μ l, put respectively on same polyamide film, with ethyl acetate-acetone-formic acid-water=7: 3: 1: 1.2 for developping agent, launch, exhibition, apart from being 5cm, is taken out, heat after 10 minutes at 60 DEG C, inspect under being placed in 365nm uviol lamp; In test sample chromatogram, on the position corresponding to reference substance chromatogram, the fluorescence spot of display same color.
Aforesaid Chinese medicine preparation, calculates by weight, is prepared from by following bulk drug:
Section dies young Luo Qu 400 ~ 550 portions of Asiatic plantains 300 ~ 400 portions of hairyvein agrimonies 300 ~ 400 parts of native honeysuckles 300 ~ 400 parts; Described section dies young Luo Qu, calculate by weight, by the fresh Snakegourd Fruit of medicinal material 2 parts, fresh reticulate millettia 2 parts and fresh stringy stonecrop 1 part of potpourri formed after pressure extracting juice, the dry fine powder of the dregs of a decoction mixes with flour 2 parts, 1 part, wheat bran again, add concoction, carry out glycolysis through song system and form.
Described native honeysuckle is the dry flower of caprifoliaceae plant largeflower-like honeysuckle flower LoniceramacranthoidesHand.-Mazz. or is with the flower just opened, and is a kind in " Chinese Pharmacopoeia " version one in 2010 28 pages " Honeysuckle flower ".
Preferably, described Chinese medicine preparation, calculates by weight, is prepared from by following bulk drug:
Section dies young Luo Qu 480 portions of Asiatic plantains 350 portions of hairyvein agrimonies 335 parts of native honeysuckles 335 parts.
Preferably, the potpourri of the dry fine powder of the described dregs of a decoction and flour and wheat bran, adds concoction and stirs evenly and make softwood, steams thoroughly, maintenance temperature is 18 ~ 26 DEG C, humidity is 45% ~ 65%, adds bent essence fermentation 20 days ~ 30 days, taking-up, dry, pulverize, Ji get section dies young Luo Qu.
Chinese medicine preparation described in the present invention, is prepared from accordance with the following methods: calculate by weight, and the section of taking dies young Luo Qu, Asiatic plantain, hairyvein agrimony and soil honeysuckle, adds 6 ~ 10 times of water gagings, soaks 30 ~ 120 minutes, decoct 1 ~ 2 hour, filtration, and it is for subsequent use to get filtrate; The dregs of a decoction add 6 ~ 10 times amount soak by water 1 ~ 2 hour, filter; Merge twice filtrate, filter, concentrated, dry, pulverize, add auxiliary material, make corresponding Chinese medicine preparation, to obtain final product.
Preferably, described Chinese medicine preparation, is prepared from accordance with the following methods: calculate by weight, and the section of taking dies young Luo Qu, Asiatic plantain, hairyvein agrimony and soil honeysuckle, Asiatic plantain and hairyvein agrimony are cut into 1.0cm section, adds 8 times of water gagings, soak 30 minutes, decoct 2 hours, filter, it is for subsequent use to get filtrate; The dregs of a decoction add 6 times amount soak by water 1 hour, filter, merge twice filtrate, filter, and the relative density being condensed into 60 DEG C is the clear cream of 1.2 ~ 1.3, under the condition of temperature 55 ~ 75 DEG C, vacuum tightness 0.08 ~ 0.1Mpa, clear cream drying under reduced pressure is become dry cream; Pulverize, add auxiliary material, make corresponding Chinese medicine preparation, to obtain final product.
Preferred, described Chinese medicine preparation, is prepared from accordance with the following methods: calculate by weight, and the section of taking dies young Luo Qu, Asiatic plantain, hairyvein agrimony and soil honeysuckle, Asiatic plantain and hairyvein agrimony are cut into 1.0cm section, adds 8 times of water gagings, soak 30 minutes, decoct 2 hours, filter, it is for subsequent use to get filtrate; The dregs of a decoction add 6 times amount soak by water 1 hour, filter, merge twice filtrate, filter, and the relative density being condensed into 60 DEG C is the clear cream of 1.2, under the condition of temperature 65 DEG C, vacuum tightness 0.1Mpa, clear cream drying under reduced pressure is become dry cream; Pulverize, whole grain, adds the silicon dioxide of 0.37% of dry cream gross weight, and mixing, incapsulates, obtain capsule.
In order to ensure effect of the present invention, applicant has carried out corresponding experimental study and demonstration to the discrimination method of hawthorn in preparation, specific as follows:
One, instrument and reagent
1, instrument
Thin layer chromatogram scanner (Switzerland (CAMAG) Ka Ma company);
KQ-250DB type numerical control ultrasonic cleaner (Kunshan Ultrasonic Instruments Co., Ltd.);
GZX-9146MBE digital display air dry oven (Medical Equipment Plant of Shanghai Boxun Industrial Co., Ltd.);
Microsyringe (Town in Shanghai booth microsyringe factory);
Electronic balance (plum Teller-Tuo benefit), 100,000/precision.
2, medicine and reagent
Chinese medicine preparation of the present invention: adopt method of the present invention to be prepared from;
Reagent:
Methyl alcohol, acetone, formic acid (Tianjin Kermel Chemical Reagent Co., Ltd.), be analysis pure; 95% ethanol (Shanghai development chemical industry one factory), analyzes pure; Anhydrous Aluminum chloride (Tianjin recovery fine chemistry industry research institute), analyzes pure; Ethyl acetate (Shanghai Shen Bo Chemical Co., Ltd.), analyzes pure; Silica G plate (subsidiary factory of Haiyang Chemical Plant, Qingdao); Polyamide film (the biochemical plastic molding and processing plant of City of Taizhou road and bridge tetramethyl); Reference substance: greater plantain glycosides, galuteolin, 3,5-Dicaffeoylquinic acid are (Nat'l Pharmaceutical & Biological Products Control Institute's buying).
Two, method and result
1, section dies young the discriminating of Luo Qu
(1) the bulk drug section of this product flavone compound in Luo Qu of dying young is more, and hydrochloric acid-magnesium powder reaction to check in Chinese medicine whether one of most popular method having flavone compound.The present invention adds the jolting of 0.2g magnesium powder in the methanol solution of sample, drip 12 ~ 14 concentrated hydrochloric acids again, namely heating water bath 1 ~ 2min shows color, and this product hydrochloric acid-magnesium powder reaction solution is obvious, therefore under the physics and chemistry that typing the present invention treats the Chinese medicine preparation of diabetes differentiates item.
(2) Study on Extraction Method
The present invention adopts thin-layer identification method, in the other side, section dies young sieve Qu Jinhang discriminating, take galuteolin as index composition, according to the character of this compound being soluble in hot water, hot methanol and ethanol, the extracting method of galuteolin is studied, compare water, 50% methyl alcohol, 70% ethanol, 95% alcohol reflux extraction and ultrasonic extraction, quantity of sample handling is 3g, filter, get filtrate and be concentrated into 5ml, as need testing solution.Galuteolin adds methyl alcohol and makes the solution of every 1ml containing 0.4mg, product solution in contrast.Test with reference to Chinese Pharmacopoeia thin-layer identification method, choose test sample point sample amount 3 ~ 6 μ l, reference substance point sample amount is 1 ~ 2 μ l, puts respectively on same silica gel g thin-layer plate, with ethyl acetate-acetone-formic acid-water=7:3:1:1.2 for developping agent launches, take out, dry, spray with 5% aluminium choride ethanolic solution, in 105 DEG C of heating 10 minutes, inspect under putting ultraviolet lamp (365nm), thin layer experimental result is as shown in table 1:
Table 1 Study on Extraction Method
Through relatively and verify, 95% alcohol reflux extracts point sample best results after 2h, and the preferred point sample amount of test sample is 3 ~ 4 μ l, and the preferred point sample amount of reference substance is 1 μ l; The best point sample amount of test sample is 3.5 μ l, and the best point sample amount of reference substance is 1 μ l.With on reference substance relevant position, the aobvious clear spot of same color, spot rounding, good separating effect, negative control is noiseless.This method specificity is strong, and spot development is clear.
(3) specificity is investigated
The sample of continuous use three batches, tests according to said method, and as shown in Figure 1, wherein, 1,2,3 is 3 batch sample to result, and 4 is negative control, and s is galuteolin contrast; As shown in Figure 1: with on reference substance relevant position, the aobvious clear spot of same color, spot rounding, good separating effect, negative control is noiseless.This method specificity is strong, and spot development is clear.
Through above-mentioned quality standards in Chinese drugs analytical approach checking, show that this thin-layer identification method is feasible, specificity is strong, simple to operate.
(4) durability is investigated
The sample of continuous use three batches, test according to said method:
The investigation result of a, different label silica gel g thin-layer plate, as shown in Figure 2 and Figure 3: in Fig. 2 and Fig. 3,1,2,3 is 3 batch samples, and s is galuteolin contrast.
The investigation of b, different temperatures and relative humidity, as shown in table 2, investigate result as shown in Fig. 4, Fig. 5, Fig. 6: in Fig. 4, Fig. 5 and Fig. 6,1,2,3 is 3 batch samples, s is galuteolin contrast.
The investigation of table 2 different temperatures and relative humidity
Investigation experimental result from above-mentioned different label silica gel g thin-layer plates, different temperatures and relative humidity: in test sample chromatogram, on the position corresponding to reference substance chromatogram, the fluorescence spot of aobvious same color; Spot rounding, good separating effect, Rf value is moderate, and negative control is noiseless; Tiny variation, this discrimination method is still effective.
(5) check
Carry out general inspection by Luo Qu tri-batch sample moisture of dying young to section, acid-insoluble ash, its result meets quality standard draft regulation, as shown in table 3:
Table 3 section dies young the three batch sample inspection result data of Luo Qu
Therefore sieve Qu Shuifen that section died young is decided to be and is no more than 9.0%, acid-insoluble ash is not higher than 1.0%.
2, the discriminating of Asiatic plantain
(1) adopt thin-layer identification method, in the other side, Asiatic plantain is differentiated, with specificity composition greater plantain glycosides for index, choosing sample extraction method is: sample 2g, adds 95% ethanol 50ml, refluxing extraction 2 hours, filter, get filtrate and be concentrated into 10ml, as need testing solution; Separately get greater plantain glycosides reference substance, add methyl alcohol and make the solution of every 1ml containing 0.1mg, product solution in contrast.Choosing developping agent is ethyl acetate-acetone-formic acid-water=7:3:1:1.2, chooses need testing solution 2 ~ 4 μ l, reference substance solution 1 ~ 2 μ l.
(2) single factor exploration is carried out to different thin layer plate, the results are shown in Table 4:
The investigation result of the different thin layer plate separating effect of table 4
As shown in Table 4: the separating effect effect of polyamide film is best, therefore elects thin layer plate as polyamide film.
(3) with ethyl acetate-acetone-formic acid-water=7:3:1:1.2 for developping agent launches, to difference exhibition apart from carrying out single factor exploration, result is as shown in table 5:
The exhibition of table 5 difference is apart from single factor exploration result
As shown in Table 5: exhibition apart from be 5cm time effect best, therefore by exhibition distance be decided to be 5cm; Take out, heat 10 minutes at 60 DEG C, inspect under putting ultraviolet lamp (365nm); Test with reference to Chinese Pharmacopoeia thin-layer identification method, empirical tests, the best point sample amount of test sample is 2 μ l, and the best point sample amount of reference substance is 2 μ l, with on reference substance relevant position, and aobvious blue-fluorescence spot.
(4) specificity is investigated
The sample of continuous use three batches, tests according to said method, and as shown in Figure 7, in Fig. 7,1,2,3 is three batch samples to result, and 4 is negative sample, and S is greater plantain glycosides.
Through above-mentioned quality standards in Chinese drugs analytical approach checking, show that this thin-layer identification method is feasible, specificity is strong, simple to operate.
(5) durability is investigated
The sample of continuous use three batches, test according to said method:
The investigation of a, different label polyamide film plate, as shown in Figure 8, Figure 9, in Fig. 8 and Fig. 9,1,2,3 is 3 batch samples to result, and s is the contrast of greater plantain glycosides.
Investigation (consistent with the table 2) result of b, different temperatures and relative humidity is as shown in Figure 10, Figure 11 and Figure 12: in Figure 10, Figure 11 and Figure 12, and 1,2,3 is 3 batch samples, and s is the contrast of greater plantain glycosides.
Above-mentioned experiment is investigated different label polyam ide TLC plate, different temperatures and relative humidity, and result shows: in test sample chromatogram, on the position corresponding to reference substance chromatogram, the fluorescence spot of aobvious same color; Spot rounding, good separating effect, Rf value is moderate, and negative control is without any interference; Tiny variation, this discrimination method is still effective.
3, the discriminating of soil honeysuckle
(1) adopt thin-layer identification method, in the other side, soil honeysuckle is differentiated.Take 3,5-Dicaffeoylquinic acid as index composition, be very easily dissolved in the character of methyl alcohol according to this compound, extracting method is elected as: get this product 2g, adds methyl alcohol 30ml, refluxing extraction 3 hours, filters, gets filtrate and be concentrated into 10ml, as need testing solution; Separately get 3,5-Dicaffeoylquinic acid reference substance, add methyl alcohol and make the solution of every 1ml containing 0.4mg, product solution in contrast; Test according to thin-layered chromatography (annex VIB), choose need testing solution 2 ~ 3 μ l, reference substance solution 1 μ l, put respectively on same polyamide film, with ethyl acetate-acetone-formic acid-water=7:3:1:1.2 for developping agent launches, exhibition distance is 5cm, take out, heat 10 minutes at 60 DEG C, inspect under putting ultraviolet lamp (365nm).Through experiment, the best point sample amount of test sample is 2 μ l, and the best point sample amount of reference substance is 1 μ l, in test sample chromatogram, with on reference substance chromatogram relevant position, shows blue-fluorescence spot.Empirical tests, result shows that blue spot colour developing is clear, and the method specificity is strong, therefore is incorporated into standard body.
(2) specificity is investigated
The sample of continuous use three batches, soil honeysuckle is lacked according to making not containing the capsule of soil honeysuckle under method for making item again by the prescription ratio of invention formulation, make by the method under need testing solution preparation and lack soil honeysuckle negative controls, test according to said method, result as shown in figure 13, Tu13Zhong, 1,2,3 is 3 batches, sample, 4 is negative sample, and s is 3,5-Dicaffeoylquinic acid reference substance.
Through above-mentioned quality standards in Chinese drugs analytical approach checking, show that this thin-layer identification method is feasible, specificity is strong, simple to operate.
(3) durability is investigated
The sample of continuous use three batches, test according to said method:
The investigation result of a, different label polyamide film plate is as shown in Figure 14, Figure 15, and in Figure 14 and Figure 15,1,2,3 is 3 batch samples, and s is 3,5-Dicaffeoylquinic acid contrast.
Investigation (consistent with the table 2) result of b, different temperatures and relative humidity is as shown in Figure 16, Figure 17, Figure 18, and in Figure 16, Figure 17 and Figure 18,1,2,3 is 3 batch samples, and S is 3,5-Dicaffeoylquinic acid contrast.
Above-mentioned experiment is investigated different label silica gel g thin-layer plates, different temperature and relative humidity, and result shows: in test sample chromatogram, on the position corresponding to reference substance chromatogram, the fluorescence spot of aobvious same color; Spot rounding, good separating effect, Rf value is moderate, and negative control is noiseless; Tiny variation, this discrimination method is still effective.
4, the discriminating of hairyvein agrimony
Hairyvein agrimony is as one of four medicines in prescribed preparation of the present invention, and the phenolic constituent that the relative content wherein in hairyvein agrimony is higher and characteristic chemical constituent are agrimophol B, to intend with characteristic chemical constituent agrimophol B, for index, carrying out indentification by TLC.Through By consulting literatures and in conjunction with the structural property of this compound, but due to the symmetry of the chemical constitution of agrimophol B strong, polarity is relatively little, and the solubleness in water is extremely low.In said preparation, because the Extraction solvent of hairyvein agrimony is water, this solvent polarity is large, so be difficult to agrimophol B to extract, therefore does not include hairyvein agrimony in discriminating item.
5, assay
This product Fang Zhongke die young sieve Qu Weimiao cure peculiar, tool replenishes qi to invigorate the spleen, the effect of quenching one's thirst of promoting the production of body fluid, function of spleen and stomach regulating, benefit liquid fills as the medicine of a warm nature, enters cold warp, owner's medicine, through consulting, research data associated is at present few, finds that its principal ingredient is flavonoid glycoside through lot of experiments, because this medicine is made up of four traditional Chinese medicine material, composition is comparatively complicated, and purification procedures is loaded down with trivial details, by HPLC method separate active ingredients difficulty, serious interference, the reasons such as contrast disappearance, therefore do not list assay in text.TLC distinguish and physics and chemistry are differentiated the important step as quality control, carries out quality control with discriminating 1 and discriminating 2.In side, greater plantain glycosides is very unstable in addition, and storage temperature is strict, and namely changes to impinging upon in 5 hours after configuration, therefore also adopts TLC distinguish to carry out quality control; Hairyvein agrimony principal ingredient fails agrimophol B to propose at leaching process, and role is booster action in side, therefore in the non-typing of assay item.
6, check
By carrying out general inspection to this product three batch sample weight differential, content uniformity, moisture, disintegration time limited, microbial limit, its result meets quality standard regulation.
Three batches of table 6 Chinese medicine preparation of the present invention check data
7, determination of extractives
This product is not containing toxic medicinal material, assay really acquires a certain degree of difficulty, the mensuration of this project is increased according to " Guizhou Province's Preparation in medical units Technical Review main points-Chinese medicine ethnic drug " (trying), assay method is measured according to " Chinese Pharmacopoeia " version in 2010 annex Ⅹ A Extract mensuration hot dipping, test agent extract content in testing 3 batches, the results are shown in following table 7:
The determination of extractives of table 7 the present invention 3 batches of Chinese medicine preparations
therefore this product water-soluble extractives content is not less than 50.0%.
The related content of the Chinese medicine preparation of the treatment diabetes 8, described in the present invention and preparation method thereof is shown in the patent of invention " a kind of Chinese medicine preparation for the treatment of diabetes and preparation method thereof " that this application people and the present invention apply on the same day.
Three, pharmacodynamics test
Chinese medicine preparation of the present invention is to the experimental study of diabetes B rat blood sugar reducing function
1. materials and methods
1.1 material
1.1.1 animal cleaning grade male SD rat, body weight 150 ~ 180g, is purchased from Chongqing and rises prosperous Bill animal used as test sale company limited, animal credit number SCXK (Chongqing), 2012-0006.
1.1.2 medicine and reagent
1.1.2.1 Chinese medicine preparation of the present invention: provided (hereinafter referred to as TNTL) by Braun Guizhou Pharmaceutical Enterprise Group Co, adult's clinical dosage is 0.045g/kg, and rat dose,equivalent and people are scaled 7 times of people's clinical dosages.Face the used time content (brown powder) the taking-up pure water of Chinese medicine preparation is prepared.During test, rat TNTL low dose group is 0.63g/kg(bis-times of clinical dosages), high dose group is 1.26g/kg(tetra-times of clinical dosages).
1.1.2.2 Streptozotocin (Streptozotocin STZ), Beijing is won Alto and is reached Science and Technology Ltd., lot number: 040M1367.
1.1.2.3 rat insulin kit: Manufactured by Mercodia AB, Sylveniusgatan 8A, SE-754 50Uppsala, Sweden.Lot number: 20518, used time by specification operation.
1.1.2.4 rat C peptide reagent box: upper sea blue base Science and Technology Ltd., lot number: 20130617.Used time by specification operation.
1.1.2.5 melbine: be Tian An board Dimethyldiguanide hydrochloride enteric solubility tablet, adult's clinical dosage is 0.03g/kg every day, is scaled rat equivalent 0.21g/kg.
1.1.2.6 normal diet, is purchased from Chongqing and rises prosperous Bill animal used as test sale company limited.
1.1.2.7 high-sugar-fat-diet makes: white granulated sugar, lard, egg are all purchased from supermarket, and egg boils only with yolk, and normal diet is broken into powder.Feed formula: sell company limited containing normal diet 35g(Chongqing Teng Xin Bill animal used as test in every 100g high lipid food), lard 15g, white granulated sugar 25g, egg yolk 25g, feeding animals 20 days.
1.2 animal grouping and medications
1.2.1 give TNTL in advance to test the impact of type II diabetes rat: rat is divided into 3 groups, be respectively normal group, model group, TNTL group.Normal group gives normal diet, model group and TNTL group high-sugar-fat-diet are fed, TNTL group gavage every day gives Chinese medicine preparation of the present invention (0.63g/kg, 1 times/day), after 20 days simultaneously, model group and TNTL group lumbar injection STZ(face the used time and prepare with citrate buffer, pH value 4.2,30mg/kg, completed injection in 30 minutes), manufacture diabetes model, filter out modeling successful rat normal diet and feed 12 days (the omnidistance administration of TNTL group 32 days).
1.2.2TNTL the impact of diabetes B rat is tested: rat is divided into 5 groups, normal group, model group, melbine group, TNTL low dose group, TNTL high dose group.Normal group gives normal diet, and other 4 groups of high-sugar-fat-diets feed modeling (the same) after 20 days, and the successful rat of modeling is used normal diet instead and feeds, and each group starts administration, 2 times/d, each 1 time of upper and lower noon simultaneously.Melbine group ig0.21g/kg, TNTL low dosage ig0.63g/kg, (two times of clinical dosages), TNTL high dose ig1.26g/kg(tetra-times of clinical dosages).Omnidistance administration 27 days.
1.2.3TNTL test with the impact of Western medicine conbined usage on diabetes B rat and normal rat: rat is divided into 4 groups, normal group, model group, melbine+TNTL low dose group, normal+TNTL high dose group.Normal group and normal+TNTL high dose group give normal diet, model group, melbine+TNTL low dose group high lipid food feed modeling (the same) after 20 days, and the successful rat of screening modeling is used normal diet instead and feeds, and each group starts administration simultaneously, 2 times/d, each 1 time of upper and lower noon.Melbine+TNTL low dose group first gives melbine 27 days (ig, 0.21g/kg), then changes TNTL low dosage (ig, 0.63g/kg, two times of clinical dosages) 21 days into.Normally+TNTL high dose group was to TNTL high dose (ig, 1.26g/kg, four times of clinical dosages) 21 days.
1.3 detection methods and instrument
1.3.1 insulin, C peptide all detect by ELISA method, detecting instrument Biotek Synergy2 fluorescence microplate reader, U.S. Biotek Products.
1.3.2 blood-sugar detecting instrument and test paper: U.S.'s Roche Products, ACCU-CHEK Pertorma.
1.4 check pathological section rat limosis are after 16 hours, and sacrificed by decapitation, gets pancreas and be placed in the fixing preservation of neutral formalin liquid, do pathology detection.This detection completes in pathology department of No. 2 Affiliated Hospital of Guiyang College of Traditional Chinese Medicine.Be divided into normal and atrophy according to the atrophy degree after pancreas is damaged, carry out statistics (card side) and check by often organizing atrophy and normal number of elements and process.
1.5 statistical methods: use SPSS17.0 statistical software, measurement data with
express, enumeration data is with percentage expression, and after group, measurement data compares with T inspection, and enumeration data compares with Chi-square Test.
2. result
2.1 give the impact of TNTL on type II diabetes rat in advance
2.1.1 sugar tolerance: rat limosis is after 16 hours, gives glucose solution (gavage, 20g/100mL, 3mL/ are only), measures empty stomach, 1h, 2h, 3h blood glucose value of rat, the results are shown in Table 8, Figure 19:
Table 8 gives Chinese medicine preparation of the present invention on the impact of diabetes B rat sugar tolerance in advance
Note: compare with normal group,
*p<0.05,
*p<0.01; Compare with model group,
#p<0.05,
##p<0.01
Be starkly lower than model group (P<0.05) from the blood glucose value of table 8, Figure 19, TNTL group 3h, other two time points also have reduction.
2.1.2 pancreatic tissue check pathological section
Pathology detection result point out, in advance to TNTL group comparatively model group Pancreas pathology damage obviously alleviate.In table 9, Figure 20-Figure 22:
Table 9 gives Chinese medicine preparation of the present invention on the impact (unit: only) of diabetes B pancreas in rat in advance
Note: compare with normal group,
*p<0.05; Compare with model group,
#p<0.05
2.2TNTL is on the impact of diabetes B rat
2.2.1 body weight
TNTL significantly can improve the body weight that diabetes rat reduces, and the results are shown in Table 10, Figure 23:
Table 10 Chinese medicine preparation of the present invention is on the impact of diabetes B rat body weight
Note: compare with normal group,
*p<0.05,
*p<0.01; Compare with model group,
#p<0.05,
##p<0.01
2.2.2 sugar tolerance
Rat limosis, after 16 hours, gives glucose solution (gavage, 20g/100mL, 3mL/ are only), uses Roche blood glucose meter to measure empty stomach, 1h, 2h, 3h blood glucose value.The results are shown in Table 11, Figure 24:
Table 11 Chinese medicine preparation of the present invention is on the impact of diabetes B rat sugar tolerance
Note: compare with normal group,
*p<0.05,
*p<0.01; Compare with model group, #p<0.05, ##p<0.01
From table 11, Figure 24, the fasting blood-glucose of each group rat does not have significant difference, the blood glucose value of TNTL low dosage and each time point of high dose all lower than model group, difference significance (p<0.01), but still variant with normal group.There was no significant difference between Chinese medicine TNTL low dose group and Chinese medicine TNTL high dose group.
2.2.3 insulin and C peptide
Rat limosis, after 16 hours, gets blood centrifuging and taking supernatant, uses rat insulin ELISA kit (Mercodia), rat C peptide ELISA kit (BluGene), detects the OD value at 450nm wavelength place, the results are shown in Table 12:
Table 12 Chinese medicine preparation of the present invention is on the impact of diabetes B rat limosis insulin, C peptide
Note: compare with normal group,
*p<0.05,
*p<0.01; Compare with model group,
#p<0.05,
##p<0.01
As shown in Table 12, the insulin of model group is significantly lower than normal group, and TNTL high and low dose group insulin comparatively model group has increase trend, but still is starkly lower than normal group.C peptide result shows, and model group decreases, and TNTL high and low dose group comparatively model group has and slightly increases trend.
2.2.4 Pancreas pathology inspection
Gather pancreas and be placed in that neutral formalin liquid is fixing to be preserved, check pathological section, the results are shown in Table 13, Figure 25-Figure 29:
Table 13 Chinese medicine preparation of the present invention is on the impact (unit: only) of diabetes B pancreas
Note: compare with normal group,
*p<0.05; Compare with model group, #p<0.05
2.3TNTL and melbine conbined usage are on the impact of diabetes B rat
2.3.1 sugar tolerance:
Rat limosis, after 16 hours, gives glucose solution (gavage, 20g/100mL, 3mL/ are only), measures empty stomach, 1h, 2h, 3h blood glucose value of rat.The results are shown in Table 14, Figure 30:
Table 14 drug combination is on the impact of diabetes B rat sugar tolerance
Note: compare with normal group,
*p<0.05,
*p<0.01; Compare with model group, #p<0.05, ##p<0.01
From table 14, Figure 30, melbine+TNTL low dose group fasting blood-glucose is significantly lower than model group, and normal+TNTL high dose group and normal group sugar tolerance value do not have difference, illustrate that this TNTL does not affect blood glucose level normal.
2.3.2 Pancreas pathology inspection
Gather pancreas and be placed in that neutral formalin liquid is fixing to be preserved, do pathology detection, the results are shown in Table 15, Figure 31-Figure 34:
Table 15 drug combination is on the impact (unit: only) of pancreas in rat check pathological section
Note: compare with normal group,
*p<0.05
This first 3 groups of test compares, and result melbine+TNTL low dose group has certain protective role to rat Langerhans islet, but does not have statistical significance.Normally+TNTL high dose group compares with normal group, does not have otherness between two groups, and the TNTL of high dose does not affect normal pancreas in rat.
Conclusion:
This experiment male SD rat, feeds with high calorie and adds the classical way of half amount Streptozotocin partial destruction B cell, successfully construct diabetes B rat model.
This research finds, it is no matter the method with the method for administration in advance or conventional therapy administration, no matter be individually dosed method or the method with melbine administering drug combinations, no matter be observe with sugar tolerance or observe with conventional blood sugar, TNTL all obviously can reduce diabetes B rat blood sugar (the equal <0.05 of P value), shows the obvious blood sugar reducing function of TNTL tool.
This research also finds, after TNTL acts on diabetes B rat, while blood sugar obviously reduces, its FPI and C peptide there is no obvious change, point out this blood sugar reducing function mechanism not necessarily B cell is short and secrete effect, may be relevant with improving the factors such as insulin resistance (IR).
This research observes the change of the Neo-Confucianism of the Pancreas Disease after TNTL effect, finds that its atrophy number is starkly lower than the model group (P<0.05) without TNTL effect, shows that TNTL is to the pancreas islet protective effect to a certain degree of diabetes B rat tool.
TNTL is acted on normal SD rats by this research, and it found that the blood sugar of normal SD rats is unchanged before and after TNTL effect, and its pancreas, also without pathological change, shows TNTL to normal rat without blood sugar influence, also has no significant effect its pancreas islet.This result also points out the blood sugar reducing function of TNTL may not realize mainly through promotion B cell excreting insulin further.
Compared with prior art, the discrimination method of the Chinese medicine preparation for the treatment of diabetes provided by the present invention is highly sensitive, reproducible, it is feasible to facilitate, negative control is noiseless, use it in the quality determining method of the Chinese medicine preparation for the treatment of diabetes, effectively can control drug quality, thus guarantee the safe, effective of the stable of product quality and clinical application.
Accompanying drawing explanation
Fig. 1 is that in invention formulation, galuteolin specificity investigates result schematic diagram;
Fig. 2 adopts self-control silica gel g thin-layer plate to the investigation result schematic diagram of galuteolin;
Fig. 3 adopts machine-processed silica gel g thin-layer plate to the investigation result schematic diagram of galuteolin;
Fig. 4 is the investigation result schematic diagram adopting temperature 20 DEG C of humidity 50% pair of galuteolin;
Fig. 5 is the investigation result schematic diagram adopting temperature 25 DEG C of humidity 55% pair of galuteolin;
Fig. 6 is the investigation result schematic diagram adopting temperature 35 DEG C of humidity 70% pair of galuteolin;
Fig. 7 is that greater plantain glycosides specificity investigates result schematic diagram;
Fig. 8 adopts self-control silica gel g thin-layer plate to the investigation result schematic diagram of greater plantain glycosides;
Fig. 9 adopts machine-processed silica gel g thin-layer plate to the investigation result schematic diagram of greater plantain glycosides;
Figure 10 is the investigation result schematic diagram adopting temperature 20 DEG C of humidity 50% pair of greater plantain glycosides;
Figure 11 is the investigation result schematic diagram adopting temperature 25 DEG C of humidity 55% pair of greater plantain glycosides;
Figure 12 is the investigation result schematic diagram adopting temperature 35 DEG C of humidity 70% pair of greater plantain glycosides;
Figure 13 is that result schematic diagram is investigated in the experiment of 3,5-Dicaffeoylquinic acid specificity;
Figure 14 adopts self-control silica gel g thin-layer plate to the investigation result schematic diagram of 3,5-Dicaffeoylquinic acid;
Figure 15 adopts machine-processed silica gel g thin-layer plate to the investigation result schematic diagram of 3,5-Dicaffeoylquinic acid;
Figure 16 is the investigation result schematic diagram adopting temperature 20 DEG C of humidity 50% pair of 3,5-Dicaffeoylquinic acid;
Figure 17 is the investigation result schematic diagram adopting temperature 25 DEG C of humidity 55% pair of 3,5-Dicaffeoylquinic acid;
Figure 18 is the investigation result schematic diagram adopting temperature 35 DEG C of humidity 70% pair of 3,5-Dicaffeoylquinic acid;
Figure 19 is rat limosis after 16 hours, gives glucose solution (gavage, 20g/100mL, 3mL/ are only), measures empty stomach, 1h, 2h, 3h blood glucose value result curve of rat;
Figure 20 is that normal group is to type II diabetes pancreas in rat pathological change schematic diagram;
Figure 21 is that model group is to type II diabetes pancreas in rat pathological change schematic diagram;
Figure 22 is that model group is to type II diabetes pancreas in rat pathological change schematic diagram;
Figure 23 is that Chinese medicine preparation of the present invention affects schematic diagram to type II diabetes rat body weight;
Figure 24 is that Chinese medicine preparation of the present invention affects schematic diagram to diabetes B rat sugar tolerance;
Figure 25 is the section schematic diagram of normal rats pancreas;
Figure 26 is the section schematic diagram of model group diabetic rat pancreas;
Figure 27 is that the section of melbine group on diabetic rat pancreas affects schematic diagram;
Figure 28 is that the section of TNTL low dose group on diabetic rat pancreas affects schematic diagram;
Figure 29 is that the section of TNTL high dose group on diabetic rat pancreas affects schematic diagram;
Figure 30 is that drug combination affects schematic diagram to diabetes B rat sugar tolerance;
Figure 31 is the pancreas schematic diagram of normal rats;
Figure 32 is the pancreas schematic diagram of model group rats;
Figure 33 is that melbine+TNTL low dose group drug combination affects schematic diagram to pancreas in rat;
Figure 34 is that normal+TNTL high dose group affects schematic diagram to pancreas in rat.
Below in conjunction with the drawings and specific embodiments, the present invention is further illustrated.
Embodiment
Embodiment 1:
Section dies young the preparation method of Luo Qu: the potpourri fresh for medicinal material Snakegourd Fruit 2g, fresh reticulate millettia 2g and fresh stringy stonecrop 1g formed is after pressure extracting juice, the dry fine powder of the dregs of a decoction mixes with flour 2g, wheat bran 1g again, add concoction and water to stir evenly and make softwood, thoroughly, maintenance temperature is 22 DEG C, humidity is 50% in steaming, adds bent smart fermentation 25 days, take out, dry, pulverize, to obtain final product.Described fresh Snakegourd Fruit also can be one or more in Chinese Drug Gualouzi, PERICARPIUM TRICHOSANTHIS and root of Chinese trichosanthes.
The preparation method of capsule for the treatment of diabetes: the section of taking dies young Luo Qu 480g, Asiatic plantain 350g, hairyvein agrimony 335g and soil honeysuckle 335g, Asiatic plantain and hairyvein agrimony is cut into 1.0cm section, adds 8 times of water gagings, soak 30 minutes, decoct 2 hours, filter, it is for subsequent use to get filtrate; The dregs of a decoction add 6 times amount soak by water 1 hour, filter, merge twice filtrate, filter, and the relative density being condensed into 60 DEG C is the clear cream of 1.2, under the condition of temperature 65 DEG C, vacuum tightness 0.1Mpa, clear cream drying under reduced pressure is become dry cream; Pulverize, whole grain, adds 1.13g silicon dioxide, and mixing, incapsulates, i.e. obtained capsule.Instructions of taking: in crude drug powder, 3g/ time, daily 3 times, daily crude drug powder meter 9-12g, bfore meals; In capsule: because medicinal material inventory is 1500g, paste-forming rate is 20.3%, thus calculates dry cream and is about 304.5g, auxiliary material silica 1 .13g again, amount to 305.63g, make 1000, every heavy 0.3056g, namely every is equivalent to raw medicinal herbs 1.5g, on average every is heavily 0.30g, therefore designs each serving with 2-3 grain (0.30 dry cream/grain), every day 3 times, with daily 9-12g crude drug powder is suitable, bfore meals.
Proterties: this product is hard shell capsules, content should be brown to tan powder, bitter.
Discrimination method:
Section dies young the discriminating of Luo Qu:
Get this product content 1g, add 10ml methyl alcohol, ultrasonic process 30 minutes, filter, get filtrate 5ml, add the magnesium powder of 0.2g, drip 12 ~ 14 concentrated hydrochloric acids, water bath processing 1 ~ 2min, solution becomes reddish violet.
The discriminating of Asiatic plantain:
(1) preparation of need testing solution: get this product content 2g, adds 95% ethanol 50ml, refluxing extraction 2 hours, filters, gets filtrate and be concentrated into 10ml, to obtain final product;
(2) preparation of reference substance solution: get greater plantain glycosides reference substance, adds 60% methyl alcohol and makes the solution of every 1ml containing 0.1mg, to obtain final product;
(3) discrimination method: according to the annex VI B thin-layered chromatography test of Chinese Pharmacopoeia version in 2010, draw need testing solution 2 ~ 4 μ l, reference substance solution 1 ~ 2 μ l, put respectively on same polyamide film, with ethyl acetate-acetone-formic acid-water=7: 3: 1: 1.2 for developping agent, launch, exhibition, apart from being 5cm, is taken out, heat after 10 minutes at 60 DEG C, inspect under being placed in 365nm uviol lamp; In test sample chromatogram, on the position corresponding to reference substance chromatogram, the fluorescence spot of display same color.
The discriminating of soil honeysuckle:
(1) preparation of need testing solution: get this product content 2g, add methyl alcohol 30ml, refluxing extraction 3 hours, filters, gets filtrate and be concentrated into 10ml, to obtain final product;
(2) preparation of reference substance solution: get 3,5-Dicaffeoylquinic acid reference substance, adds methyl alcohol and makes the solution of every 1ml containing 0.4mg, to obtain final product;
(3) discrimination method: according to the annex VI B thin-layered chromatography test of Chinese Pharmacopoeia version in 2010, draw need testing solution 2 ~ 3 μ l, reference substance solution 1 μ l, put respectively on same polyamide film, with ethyl acetate-acetone-formic acid-water=7: 3: 1: 1.2 for developping agent, launch, exhibition, apart from being 5cm, is taken out, heat after 10 minutes at 60 DEG C, inspect under being placed in 365nm uviol lamp; In test sample chromatogram, on the position corresponding to reference substance chromatogram, the fluorescence spot of display same color.
Embodiment 2:
Section dies young the preparation method of Luo Qu: the potpourri fresh for medicinal material Snakegourd Fruit 2g, fresh reticulate millettia 2g and fresh stringy stonecrop 1g formed is after pressure extracting juice, the dry fine powder of the dregs of a decoction mixes with flour 2g, wheat bran 1g again, add concoction to stir evenly and make softwood, thoroughly, maintenance temperature is 26 DEG C, humidity is 65% in steaming, adds bent smart fermentation 30 days, take out, dry, pulverize, Ji get section dies young Luo Qu.
The preparation method of tablet for the treatment of diabetes: the section of taking dies young Luo Qu 400g, Asiatic plantain 300g, hairyvein agrimony 400g and soil honeysuckle 400g, Asiatic plantain and hairyvein agrimony is cut into 1.0cm section, adds 10 times of water gagings, soak 120 minutes, decoct 2 hours, filter, it is for subsequent use to get filtrate; The dregs of a decoction add 10 times amount soak by water 2 hours, filter, merge twice filtrate, filter, and the relative density being condensed into 60 DEG C is the clear cream of 1.3, under the condition of temperature 75 DEG C, vacuum tightness 0.1Mpa, clear cream drying under reduced pressure is become dry cream; Pulverize, add the corresponding auxiliary material of tablet, obtain 1000, tablet.Instructions of taking: every day 3 times, each 2, every heavy 0.15g, bfore meals.
Discrimination method:
Section dies young the discriminating of Luo Qu:
(1) preparation of need testing solution: get this product content 1.8 ~ 3g, adds 95% ethanol 50ml, refluxing extraction 2 hours, filters, gets filtrate and be concentrated into 5ml, to obtain final product;
(2) preparation of reference substance solution: get galuteolin reference substance, adds methyl alcohol and makes the solution of every 1ml containing 0.4mg, to obtain final product;
(3) discrimination method: according to the annex VI B thin-layered chromatography test of Chinese Pharmacopoeia version in 2010, draw need testing solution 3 ~ 6 μ l, reference substance solution 1 ~ 2 μ l, put respectively on same silica gel g thin-layer plate, with ethyl acetate-acetone-formic acid-water=7: 3: 1: 1.2 for developping agent, launch, take out, dry, spray with 5% aluminium choride ethanolic solution, in 105 DEG C of heating 10 minutes, let cool, inspect under putting 365nm uviol lamp; In test sample chromatogram, on the position corresponding to reference substance chromatogram, the fluorescence spot of display same color.
The discrimination method of Asiatic plantain and soil honeysuckle is with embodiment 1.
Embodiment 3:
Section dies young the preparation method of Luo Qu: the potpourri fresh for medicinal material Snakegourd Fruit 2g, fresh reticulate millettia 2g and fresh stringy stonecrop 1g formed is after pressure extracting juice, the dry fine powder of the dregs of a decoction mixes with flour 2g, wheat bran 1g again, add concoction to stir evenly and make softwood, thoroughly, maintenance temperature is 18 DEG C, humidity is 45% in steaming, adds bent smart fermentation 20 days, take out, dry, pulverize, Ji get section dies young Luo Qu.
The preparation method of granule for the treatment of diabetes: the section of taking dies young Luo Qu 500g, Asiatic plantain 400g, hairyvein agrimony 300g and soil honeysuckle 300g, Asiatic plantain and hairyvein agrimony is cut into 1.0cm section, adds 6 times of water gagings, soak 90 minutes, decoct 1 hour, filter, it is for subsequent use to get filtrate; The dregs of a decoction add 8 times amount soak by water 1 hour, filter, merge twice filtrate, filter, and the relative density being condensed into 60 DEG C is the clear cream of 1.2, under the condition of temperature 55 DEG C, vacuum tightness 0.08Mpa, clear cream drying under reduced pressure is become dry cream; Pulverize, add the corresponding auxiliary material of granule, obtain granule 1000.Instructions of taking: every day 3 times, each 2, every heavy 0.15g, bfore meals.
Luo Qu, Asiatic plantain and the discrimination method of soil honeysuckle are died young with embodiment 1 by section.
Embodiment 4: the preparation method of dripping pill for the treatment of diabetes: the section taking preparation in embodiment 1 dies young Luo Qu 550g, Asiatic plantain 350g, hairyvein agrimony 300g and soil honeysuckle 300g, Asiatic plantain and hairyvein agrimony are cut into 1.0cm section, add 10 times of water gagings, soak 120 minutes, decoct 2 hours, filter, it is for subsequent use to get filtrate; The dregs of a decoction add 10 times amount soak by water 2 hours, filter, merge twice filtrate, filter, and the relative density being condensed into 60 DEG C is the clear cream of 1.3, under the condition of temperature 75 DEG C, vacuum tightness 0.1Mpa, clear cream drying under reduced pressure is become dry cream; Pulverize, add the corresponding auxiliary material of dripping pill, obtain pill.Instructions of taking: every day 3 times, each 2-3 grain, every heavy 0.3g, bfore meals.
Luo Qu, Asiatic plantain and the discrimination method of soil honeysuckle are died young with embodiment 2 by section.
Claims (5)
1. treat a discrimination method for the Chinese medicine preparation of diabetes, described Chinese medicine preparation by section die young Luo Qu, Asiatic plantain, hairyvein agrimony and soil honeysuckle be prepared from; Wherein, the section Luo Qu that dies young carries out glycolysis by fresh Snakegourd Fruit, fresh reticulate millettia, fresh stringy stonecrop, flour and wheat bran through song system and forms; It is characterized in that: described discriminating be to section die young Luo Qu, Asiatic plantain, soil honeysuckle indentification by TLC; The die young discrimination method of Luo Qu of section is with galuteolin reference substance for contrast, with ethyl acetate-acetone-formic acid-water=7: be the thin-layered chromatography of developping agent at 3: 1: 1.2; The discrimination method of Asiatic plantain is with greater plantain glycosides reference substance for contrast, with ethyl acetate-acetone-formic acid-water=7: be the thin-layered chromatography of developping agent at 3: 1: 1.2; The discrimination method of soil honeysuckle is with 3,5-Dicaffeoylquinic acid reference substance for contrast, with ethyl acetate-acetone-formic acid-water=7: be the thin-layered chromatography of developping agent at 3: 1: 1.2.
2. the discrimination method of the Chinese medicine preparation for the treatment of diabetes according to claim 1, it is characterized in that: the die young concrete discrimination method of Luo Qu of section comprises the following steps: get this product content 1g, add 10ml methyl alcohol, ultrasonic process 30 minutes, filters, gets filtrate 5ml, add the magnesium powder of 0.2g, drip 12 ~ 14 concentrated hydrochloric acids, water bath processing 1 ~ 2min, solution becomes reddish violet.
3. the discrimination method of the Chinese medicine preparation for the treatment of diabetes according to claim 1, is characterized in that: the die young concrete discrimination method of Luo Qu of section comprises the following steps:
(1) preparation of need testing solution: get this product content 1.8 ~ 3g, adds 95% ethanol 50ml, refluxing extraction 2 hours, filters, gets filtrate and be concentrated into 5ml, to obtain final product;
(2) preparation of reference substance solution: get galuteolin reference substance, adds methyl alcohol and makes the solution of every 1ml containing 0.4mg, to obtain final product;
(3) discrimination method: according to the annex VI B thin-layered chromatography test of Chinese Pharmacopoeia version in 2010, draw need testing solution 3 ~ 6 μ l, reference substance solution 1 ~ 2 μ l, put respectively on same silica gel g thin-layer plate, with ethyl acetate-acetone-formic acid-water=7: 3: 1: 1.2 for developping agent, launch, take out, dry, spray with 5% aluminium choride ethanolic solution, in 105 DEG C of heating 10 minutes, let cool, inspect under putting 365nm uviol lamp; In test sample chromatogram, on the position corresponding to reference substance chromatogram, the fluorescence spot of display same color.
4., according to the discrimination method of the Chinese medicine preparation of the arbitrary described treatment diabetes of claims 1 to 3, it is characterized in that: the concrete discrimination method of Asiatic plantain comprises the following steps:
(1) preparation of need testing solution: get this product content 2g, adds 95% ethanol 50ml, refluxing extraction 2 hours, filters, gets filtrate and be concentrated into 10ml, to obtain final product;
(2) preparation of reference substance solution: get greater plantain glycosides reference substance, adds 60% methyl alcohol and makes the solution of every 1ml containing 0.1mg, to obtain final product;
(3) discrimination method: according to the annex VI B thin-layered chromatography test of Chinese Pharmacopoeia version in 2010, draw need testing solution 2 ~ 4 μ l, reference substance solution 1 ~ 2 μ l, put respectively on same polyamide film, with ethyl acetate-acetone-formic acid-water=7: 3: 1: 1.2 for developping agent, launch, exhibition, apart from being 5cm, is taken out, heat after 10 minutes at 60 DEG C, inspect under being placed in 365nm uviol lamp; In test sample chromatogram, on the position corresponding to reference substance chromatogram, the fluorescence spot of display same color.
5. the discrimination method of the Chinese medicine preparation for the treatment of diabetes according to claim 4, is characterized in that: the concrete discrimination method of soil honeysuckle comprises the following steps:
(1) preparation of need testing solution: get this product content 2g, add methyl alcohol 30ml, refluxing extraction 3 hours, filters, gets filtrate and be concentrated into 10ml, to obtain final product;
(2) preparation of reference substance solution: get 3,5-Dicaffeoylquinic acid reference substance, adds methyl alcohol and makes the solution of every 1ml containing 0.4mg, to obtain final product;
(3) discrimination method: according to the annex VI B thin-layered chromatography test of Chinese Pharmacopoeia version in 2010, draw need testing solution 2 ~ 3 μ l, reference substance solution 1 μ l, put respectively on same polyamide film, with ethyl acetate-acetone-formic acid-water=7: 3: 1: 1.2 for developping agent, launch, exhibition, apart from being 5cm, is taken out, heat after 10 minutes at 60 DEG C, inspect under being placed in 365nm uviol lamp; In test sample chromatogram, on the position corresponding to reference substance chromatogram, the fluorescence spot of display same color.
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| CN106177580A (en) * | 2016-08-30 | 2016-12-07 | 贵州百灵企业集团制药股份有限公司 | A kind of for granule treating type 2 diabetes mellitus and preparation method thereof |
| CN106177580B (en) * | 2016-08-30 | 2019-11-19 | 贵州百灵企业集团制药股份有限公司 | A kind of granule and preparation method thereof for treating diabetes B |
| CN108614065A (en) * | 2016-12-09 | 2018-10-02 | 内蒙古天奇中蒙制药股份有限公司 | A kind of method of quality control of mountain chrysanthemum Jiangya Granula |
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