CN108760945B

CN108760945B - Detection method of astragalus membranaceus and astragalus membranaceus leucocyte increasing capsule

Info

Publication number: CN108760945B
Application number: CN201810971715.XA
Authority: CN
Inventors: 葛秋平; 张仕林; 尚秘; 吴劲勇; 毛松; 王海洋; 沈开文
Original assignee: Hanfang Pharma Co ltd
Current assignee: Hanfang Pharma Co ltd
Priority date: 2018-08-24
Filing date: 2018-08-24
Publication date: 2021-04-13
Anticipated expiration: 2038-08-24
Also published as: CN108760945A

Abstract

A detection method of a stilbene gel Shengbai Capsule is characterized in that the medicine is prepared from Chinese date, donkey-hide gelatin, blood ginseng, epimedium herb, radix sophorae flavescentis, astragalus and angelica according to the following method: pulverizing radix Angelicae sinensis except colla Corii Asini into fine powder; decocting the rest five materials such as fructus Jujubae in water for three times (2 hr for the first time, 1.5 hr for the second time, and 1 hr for the third time), mixing decoctions, filtering, adding melted colla Corii Asini into the filtrate, stirring, concentrating to soft extract with relative density of 1.30 at 75-85 deg.C, adding the above radix Angelicae sinensis fine powder, mixing, granulating, drying, and making into capsule; the detection method comprises an identification method and a content determination method; the identification method comprises the thin-layer identification of Chinese date, the thin-layer identification of Chinese angelica, the thin-layer identification of lightyellow sophora root, the thin-layer identification of astragalus and/or the thin-layer identification of epimedium; the content determination method comprises the content determination of matrine and/or icariin.

Description

Detection method of astragalus membranaceus and astragalus membranaceus leucocyte increasing capsule

Technical Field

The invention relates to a detection method of a stilbene gel Shengbai capsule, belonging to the technical field of medicines;

background

The astragalus glue leucocyte increasing capsule is a ethnic medicine which is complementary to Miao medicine and traditional Chinese medicine, mainly comprises Chinese date, donkey-hide gelatin, blood ginseng, epimedium herb, lightyellow sophora root, membranous milkvetch root, Chinese angelica and the like, has the effects of enriching blood and tonifying qi, and is clinically used for treating dizziness, short breath, hypodynamia, spontaneous perspiration, night sweat, leukopenia and the like caused by deficiency of qi and blood.

Currently, the executed standard of the astragalus membranaceus and astragalus membranaceus leukogenic capsule is the national food and drug administration standard (number WS-10026 (ZD-0026-. However, the astragalus membranaceus and astragalus membranaceus whitening capsules serving as a traditional Chinese medicine compound preparation consisting of 7 traditional Chinese medicinal materials are complex in formula components, and the quality standard cannot be better controlled according to the current quality standard, so that the inherent quality of the product is improved.

Disclosure of Invention

The invention aims to provide a detection method of a stilbene gel leucocyte increasing capsule, which is accurate and has strong specificity and can be used as a quality control method of the stilbene gel leucocyte increasing capsule. The invention carries out thin-layer identification on Chinese date, Chinese angelica, lightyellow sophora root, membranous milkvetch root and epimedium herb in the astragalus membranaceus and astragalus membranaceus whitening capsule; measuring the content of matrine and/or icariin, and performing thin-layer chromatography identification on Chinese date; the method for determining the amount of matrine in the sophora flavescens medicinal material provides data reference for establishing an effective and reliable quality standard and accurately reflecting the whole quality of the traditional Chinese medicine. Can better control the treatment of the product and ensure the clinical curative effect.

In order to solve the technical problems, the invention adopts the following technical scheme: a detection method of astragalus membranaceus and astragalus membranaceus leukocyte increasing capsule comprises the following steps of preparing 260 parts of Chinese date 240-: pulverizing radix Angelicae sinensis except colla Corii Asini into fine powder; decocting the rest five materials such as fructus Jujubae in water for three times (2 hr for the first time, 1.5 hr for the second time, and 1 hr for the third time), mixing decoctions, filtering, adding melted colla Corii Asini into the filtrate, stirring, concentrating to soft extract with relative density of 1.30 at 75-85 deg.C, adding the above radix Angelicae sinensis fine powder, mixing, granulating, drying, and making into capsule; the detection method comprises an identification method and a content determination method; the identification method comprises the thin-layer identification of Chinese date, the thin-layer identification of Chinese angelica, the thin-layer identification of lightyellow sophora root, the thin-layer identification of astragalus and/or the thin-layer identification of epimedium; the content determination method comprises the content determination of matrine and/or icariin.

In the detection method of the astragalus membranaceus and astragalus membranaceus whitening capsules, the thin layer identification of the Chinese dates is as follows: taking 3g of medicinal granule content, adding 30ml of ethyl acetate, performing ultrasonic treatment for 30 minutes, filtering, evaporating filtrate to dryness, and dissolving residue with 0.5ml of methanol to obtain sample solution; taking 2g of Chinese date reference medicinal material, adding 50ml of water, heating and refluxing for 2 hours, filtering while the solution is hot, volatilizing water in filtrate to prepare thick Chinese date paste, adding 30ml of ethyl acetate into the paste, carrying out ultrasonic treatment for 30 minutes, filtering, evaporating the filtrate to dryness, adding 1ml of methanol to dissolve the filtrate to obtain a reference medicinal material solution, sucking 5 mu l of a test sample solution and 10 mu l of the reference medicinal material solution, respectively dropping the control medicinal material solution and the test sample solution on a same silica gel H thin-layer plate, taking a developing agent of toluene-ethyl acetate-glacial acetic acid (60:6:0.05), developing, taking out, airing, spraying a 10% sulfuric acid ethanol solution, heating at 105 ℃ until spots are clear, placing the spots under an ultraviolet lamp with the wavelength of 365nm, and observing the spots which are fluorescent in the same color in the test sample chromatogram and the positions corresponding to the control product chromatogram.

In the detection method of the astragalus membranaceus and astragalus membranaceus whitening capsules, the thin-layer identification method of the angelica sinensis comprises the following steps: taking 5g of the content of the drug particles, adding 20ml of n-hexane, carrying out ultrasonic treatment for 20 minutes, filtering, and volatilizing the filtrate to 1ml to be used as a test solution; preparing 0.5g of radix Angelicae sinensis control medicinal material, and preparing control medicinal solution by the same method; performing thin-layer chromatography (appendix VI B of 2010 edition of Chinese pharmacopoeia), sucking the two solutions, respectively dropping the two solutions in 5 μ l onto the same silica gel G thin-layer plate, developing with n-hexane-ethyl acetate 9:1 as developing agent, taking out, air drying, inspecting under an ultraviolet lamp with wavelength of 365nm, and performing fluorescence spot with the same color on the corresponding position of the chromatogram of the control medicinal material;

the thin-layer identification method of the sophora flavescens comprises the following steps: taking 1g of the content of the medicine particles, adding 30ml of ethanol, heating and refluxing for 30 minutes, cooling, filtering, adding 20ml of water into the residue after the filtrate is evaporated to dissolve, filtering, placing the filtrate in a separating funnel, adding 0.5ml of concentrated ammonia test solution with the concentration of 25% -28%, shaking and extracting for 2 times by using trichloromethane, 10ml each time, combining the trichloromethane solution, evaporating to dryness, and adding 1ml of absolute ethanol into the residue to dissolve to obtain a sample solution; taking matrine and sophoridine reference substances, respectively adding anhydrous ethanol to obtain solutions containing 1mg per 1ml, using as reference substance solutions, performing thin layer chromatography (appendix VI B of 2010 edition of Chinese pharmacopoeia), sucking 5 μ l of each of the three solutions, respectively dropping on the same silica gel G thin layer plate, placing in an ammonia vapor presaturation tank with cyclohexane-acetone-ethyl acetate-concentrated ammonia solution (2: 3:4: 0.5) as developing agent, presaturating for 30 min, taking out, air drying, and spraying diluted bismuth potassium iodide solution; spots with the same color appear on the chromatogram of the test solution at the corresponding positions of the chromatogram of the reference solution;

the thin-layer identification method of the astragalus comprises the following steps: taking 5g of medicine particle content, adding 30ml of petroleum ether at the temperature of 60-90 ℃, carrying out ultrasonic treatment for 20 minutes, filtering, volatilizing petroleum ether in medicine residue, adding 50ml of methanol, carrying out heating reflux for 30 minutes, filtering, washing filter residue with a small amount of methanol, combining washing liquor and filtrate, drying by distillation, adding 10ml of water into residue, heating for dissolving, cooling, centrifuging for 10 minutes in a centrifuge tube, taking supernate, shaking and extracting for 2 times by using trichloromethane, 15ml each time, discarding trichloromethane liquid, shaking and extracting for 3 times by using water-saturated n-butyl alcohol, adding 10ml for the first time, adding 10ml for the second time, adding 5ml for the third time, combining n-butyl alcohol liquid, washing for 3 times by adding ammonia test solution, discarding 15ml each time, and discarding ammonia solution; washing n-butanol solution with n-butanol saturated water to neutrality, discarding water solution, evaporating n-butanol solution to dryness, and dissolving the residue with 1ml methanol to obtain sample solution; adding methanol into astragaloside IV reference substance to obtain 1mg solution per 1ml as reference substance solution; performing thin-layer chromatography (appendix VI B of the first part of the 2010 edition of Chinese pharmacopoeia), sucking 10 μ l of a test solution and 5 μ l of a reference solution, respectively dropping the solutions on the same silica gel G thin-layer plate, developing the solution with chloroform-methanol-water (13: 7: 2) as a developing agent at a lower layer below 10 ℃, taking out, drying in the air, spraying with 10% sulfuric acid ethanol solution, and heating at 105 ℃ until spots are clearly developed; spots with the same color appear on the chromatogram of the test solution at the positions corresponding to the chromatograms of the reference solution; then placing under an ultraviolet lamp with the wavelength of 365nm for inspection, and displaying fluorescent spots with the same color in the chromatogram of the test sample and at the position corresponding to the chromatogram of the reference sample;

the thin-layer identification method of the epimedium comprises the following steps: taking 5g of medicine particle content, adding 30ml of petroleum ether at the temperature of 60-90 ℃, carrying out ultrasonic treatment for 20 minutes, filtering, volatilizing petroleum ether in medicine residue, adding 50ml of methanol, carrying out heating reflux for 30 minutes, filtering, washing filter residue with 5ml of methanol, combining washing liquor and filtrate, drying by distillation, adding 10ml of water into residue, heating for dissolving, cooling, centrifuging in a centrifuge tube for 10 minutes, taking supernate, shaking and extracting with chloroform for 2 times, 15ml each time, discarding chloroform solution, shaking and extracting with water-saturated n-butyl alcohol for 3 times, adding 10ml for the first time, adding 10ml for the second time, adding 5ml for the third time, combining n-butyl alcohol solution, adding ammonia test solution and washing for 3 times, 15ml each time, discarding ammonia solution; washing n-butanol solution with n-butanol saturated water to neutrality, discarding water solution, evaporating n-butanol solution to dryness, and dissolving the residue with 1ml methanol to obtain sample solution; adding methanol into icariin control to obtain 1mg solution per 1ml as control solution; performing thin-layer chromatography (appendix VI B of the first part of the 2010 edition of the Chinese pharmacopoeia), sucking 2-4 μ l of a test solution and 5 μ l of a reference solution, respectively dropping the two solutions on the same silica gel G thin-layer plate, developing the solution with chloroform-methanol-water of 13:7:2 and a lower layer solution placed below 10 ℃ as a developing agent, taking out the solution, drying the solution in the air, spraying 10% sulfuric acid ethanol solution, and heating the solution at 105 ℃ until spots are clearly developed; spots with the same color appear on the chromatogram of the test solution at the positions corresponding to the chromatograms of the reference solution; and viewing under 365nm ultraviolet lamp to show fluorescent spots of the same color in the chromatogram of the test solution at the position corresponding to the chromatogram of the reference solution.

In the detection method of the stilbene gel leucocyte increasing capsule, the content determination method of the matrine comprises the following steps: the determination is carried out according to a high performance liquid chromatography (the four parts 0512 method of the 2015 version of Chinese pharmacopoeia):

chromatographic conditions and system applicability test: octadecylsilane chemically bonded silica is used as a filling agent; mobile phase: methanol is used as phase A, 0.1% triethylamine is used as phase B, and the elution conditions are as follows: 53% A-47% B; the detection wavelength is 220 nm; the theoretical plate number is not less than 4000 calculated according to matrine peak;

preparation of control solutions: precisely weighing matrine reference substance, adding methanol to obtain solution containing 0.2mg per 1ml, and shaking;

preparation of a test solution: taking 1.0g of the content of the medicine particles, precisely weighing, placing in a conical flask with a plug, adding 2ml of concentrated ammonia test solution with the concentration of 25-28% for wetting, adding 30ml of trichloromethane, heating and refluxing for 1.5 hours, filtering, evaporating filtrate to dryness, dissolving residues in 10ml of methanol, and shaking up to obtain the medicine;

the determination method comprises the following steps: respectively sucking 5 μ l of each of the reference solution and the sample solution, injecting into liquid chromatograph, and measuring.

In the detection method of the astragalus membranaceus-leucocyte increasing capsule, the method for measuring the content of icariin comprises the following steps: according to the high performance liquid chromatography (appendix VI D of the first part of the Chinese pharmacopoeia 2010 edition):

chromatographic conditions and system applicability test: octadecylsilane chemically bonded silica is used as a filling agent; mobile phase: acetonitrile is phase A, water is phase B, and elution conditions are as follows: 25.5% A-74.5% B; the detection wavelength is 270 nm; the theoretical plate number is not less than 3000 calculated according to icariin peak;

preparation of control solutions: weighing icariin reference substance, precisely weighing, and adding methanol to obtain solution containing 25 μ g of icariin per 1 ml;

preparation of a test solution: taking the medicine content, grinding, taking 1.5g, precisely weighing, placing in a conical flask with a plug, precisely adding 25ml of 70% ethanol, sealing the plug, weighing, carrying out ultrasonic treatment for 1 hour, carrying out ultrasonic power of 250W and ultrasonic frequency of 35kHz, taking out, cooling, weighing again, complementing the weight loss by 70% ethanol, shaking up, filtering, and taking the subsequent filtrate to obtain the medicine;

the determination method comprises the following steps: precisely sucking 10 μ l of each of the reference solution and the sample solution, injecting into liquid chromatograph, and measuring.

In the detection method of the astragalus membranaceus and leucocyte increasing capsule, the medicine is detected as follows:

(1) the characteristics are as follows: the hard capsule content is brown granule and powder; is bitter in taste;

(2) and (3) identification: comprises the thin-layer identification of Chinese date, the thin-layer identification of Chinese angelica, the thin-layer identification of lightyellow sophora root, the thin-layer identification of astragalus and/or the thin-layer identification of epimedium;

the thin layer identification of the Chinese dates is as follows: taking 3g of medicinal granule content, adding 30ml of ethyl acetate, performing ultrasonic treatment for 30 minutes, filtering, evaporating filtrate to dryness, and dissolving residue with 0.5ml of methanol to obtain sample solution; taking 2g of a Chinese date reference medicinal material, adding 50ml of water, heating and refluxing for 2 hours, filtering while the Chinese date reference medicinal material is hot, volatilizing water in filtrate to prepare a Chinese date thick paste, adding 30ml of ethyl acetate into the paste, carrying out ultrasonic treatment for 30 minutes, filtering, evaporating the filtrate to dryness, adding 1ml of methanol to dissolve the filtrate to obtain a reference medicinal material solution, sucking 5 mu l of a test sample solution and 10 mu l of the reference medicinal material solution, respectively dropping the control sample solution and the reference medicinal material solution on a same silica gel H thin-layer plate, taking a developing agent of toluene-ethyl acetate-glacial acetic acid which is 60:6:0.05 as a developing agent, taking out, airing, spraying a 10% sulfuric acid ethanol solution, heating at 105 ℃ until spots are clear in color, placing the spots under an ultraviolet lamp with the wavelength of 365nm, and viewing fluorescent spots with the same color in a test sample chromatogram at positions corresponding;

the thin-layer identification method of the angelica comprises the following steps: taking 5g of the content of the drug particles, adding 20ml of n-hexane, carrying out ultrasonic treatment for 20 minutes, filtering, and volatilizing the filtrate to 1ml to be used as a test solution; preparing 0.5g of radix Angelicae sinensis control medicinal material, and preparing control medicinal solution by the same method; performing thin-layer chromatography (appendix VI B of 2010 edition of Chinese pharmacopoeia), sucking the two solutions, respectively dropping the two solutions in 5 μ l onto the same silica gel G thin-layer plate, developing with n-hexane-ethyl acetate 9:1 as developing agent, taking out, air drying, inspecting under an ultraviolet lamp with wavelength of 365nm, and performing fluorescence spot with the same color on the corresponding position of the chromatogram of the control medicinal material;

the thin-layer identification method of the epimedium comprises the following steps: taking 5g of medicine particle content, adding 30ml of petroleum ether at the temperature of 60-90 ℃, carrying out ultrasonic treatment for 20 minutes, filtering, volatilizing petroleum ether in medicine residue, adding 50ml of methanol, carrying out heating reflux for 30 minutes, filtering, washing filter residue with 5ml of methanol, combining washing liquor and filtrate, drying by distillation, adding 10ml of water into residue, heating for dissolving, cooling, centrifuging in a centrifuge tube for 10 minutes, taking supernate, shaking and extracting with chloroform for 2 times, 15ml each time, discarding chloroform solution, shaking and extracting with water-saturated n-butyl alcohol for 3 times, adding 10ml for the first time, adding 10ml for the second time, adding 5ml for the third time, combining n-butyl alcohol solution, adding ammonia test solution and washing for 3 times, 15ml each time, discarding ammonia solution; washing n-butanol solution with n-butanol saturated water to neutrality, discarding water solution, evaporating n-butanol solution to dryness, and dissolving the residue with 1ml methanol to obtain sample solution; adding methanol into icariin control to obtain 1mg solution per 1ml as control solution; performing thin-layer chromatography (appendix VI B of the first part of the 2010 edition of the Chinese pharmacopoeia), sucking 2-4 μ l of a test solution and 5 μ l of a reference solution, respectively dropping the two solutions on the same silica gel G thin-layer plate, developing the solution with chloroform-methanol-water of 13:7:2 and a lower layer solution placed below 10 ℃ as a developing agent, taking out the solution, drying the solution in the air, spraying 10% sulfuric acid ethanol solution, and heating the solution at 105 ℃ until spots are clearly developed; spots with the same color appear on the chromatogram of the test solution at the positions corresponding to the chromatograms of the reference solution; viewing under 365nm ultraviolet lamp to display fluorescent spots of the same color in the chromatogram of the test solution at the position corresponding to the chromatogram of the reference solution;

(3) content determination: comprises measuring the content of matrine and/or icariin;

the method for measuring the content of the matrine comprises the following steps: the determination is carried out according to a high performance liquid chromatography (the four parts 0512 method of the 2015 version of Chinese pharmacopoeia):

the determination method comprises the following steps: respectively sucking 5 μ l of reference solution and sample solution, injecting into liquid chromatograph, and measuring;

the method for measuring the content of the icariin comprises the following steps: according to the high performance liquid chromatography (appendix VI D of the first part of the Chinese pharmacopoeia 2010 edition):

preparation of a test solution: taking the medicine contents with different filling amounts, grinding, taking 1.5g, precisely weighing, placing in a conical flask with a plug, precisely adding 25ml of 70% ethanol, sealing, weighing, ultrasonically treating for 1 hour, carrying out ultrasonic power of 250W and ultrasonic frequency of 35kHz, taking out, cooling, weighing again, complementing the lost weight with 70% ethanol, shaking up, filtering, and taking the subsequent filtrate to obtain the medicine;

The inventors have conducted a number of experiments and the following is a study of the detection method of the present invention

Experimental example 1: thin layer identification of Chinese date

1.1 instruments and reagents

ZF-20D ultraviolet analyzer; silica gel G plate (100X 100mm, lot # 20100321; 100X 200 mm; lot # 20100317, manufacturer: Qingdao oceanic plant), silica gel H plate (100X 100mm, lot # 20100503; 100X 200 mm; lot # 20100417, manufacturer: Qingdao oceanic plant); silica gel H thin layer plate (home made).

Absolute ethanol (batch No. 20100705; manufacturer: national drug group chemical Co., Ltd.), ethyl acetate (batch No. 1100101; manufacturer: Shanghai Bo chemical Co., Ltd.), methanol (batch No. 20100901; manufacturer: national drug group chemical Co., Ltd.), sulfuric acid (batch No. 20090201; manufacturer: Chongqing Chundong chemical Co., Ltd.), chloroform (batch No. 0907142; manufacturer: West Longsco chemical Co., Ltd.), ether (batch No. 20101125; manufacturer: national drug group chemical Co., Ltd.), toluene (batch No. 20101225; manufacturer: national drug group chemical Co., Ltd.), glacial acetic acid batch No.: S189K040), ethyl acetate (batch No.: 20090418, respectively; kyropoulos chemical company).

Self-making a thin-layer plate: taking 1 part of silica gel H, uniformly mixing with 3 parts of 1% CMC-Na aqueous solution, paving a plate, airing, drying at 105 ℃ for 30 minutes, taking out, and putting into a dryer for later use.

Astragalus membranaceus and astragalus membranaceus-containing Shengbai capsules (batch numbers: 2527025, 2527026 and 2528001), a Chinese date negative sample (batch number: 20171218) and a Chinese date control medicinal material (batch number: 121040-201408). Purchasing a reference medicinal material to a Chinese medicine biological product identification institute; n-hexane, chloroform, methanol, diethyl ether, ethyl acetate, glacial acetic acid, toluene, ethanol and the like are all analytically pure.

1.2 examination of thin-layer identification Experimental conditions

1.2.1 examination of preparation conditions of test solutions

Condition 1: collecting 3g of the content of the product, adding 30ml of ethyl acetate, performing ultrasonic treatment for 30 minutes, filtering, evaporating the filtrate to dryness, and dissolving the residue in 0.5ml of methanol to obtain the product.

Condition 2: taking 3g of the content of the product, adding 30ml of diethyl ether, performing ultrasonic treatment for 30 minutes, filtering, evaporating the filtrate to dryness, and adding 0.5ml of methanol into residues for dissolving to obtain the product.

Condition 3: taking 3g of the content of the product, adding 30ml of methanol, performing ultrasonic treatment for 30 minutes, filtering, evaporating the filtrate to dryness, and adding 0.5ml of methanol into residues for dissolving to obtain the product.

1.2.2 preparation of reference drug solution

Condition 1: taking 2g of Chinese date as a reference medicinal material, adding 30ml of ethyl acetate, carrying out ultrasonic treatment for 30 minutes, filtering, evaporating filtrate to dryness, and dissolving residues in 1ml of methanol to obtain the Chinese date extract.

Condition 2: collecting fructus Jujubae control medicinal material 2g, adding water 50ml, heating and refluxing for 2 hr, filtering while hot, volatilizing water from filtrate to obtain fructus Jujubae soft extract, adding ethyl acetate 50ml, ultrasonic treating for 30 min, filtering, evaporating filtrate to dryness, and dissolving with methanol 1ml to obtain the final product.

Condition 3: collecting fructus Jujubae control medicinal material 2g, adding water 50ml, heating and refluxing for 2 hr, filtering while hot, volatilizing water from filtrate to obtain fructus Jujubae soft extract, adding diethyl ether 50ml, ultrasonic treating for 30 min, filtering, evaporating filtrate to dryness, and dissolving with methanol 1ml to obtain the final product.

Condition 4: collecting fructus Jujubae control medicinal material 2g, adding water 50ml, heating and refluxing for 2 hr, filtering while hot, volatilizing water from filtrate to obtain fructus Jujubae soft extract, adding methanol 50ml, ultrasonic treating for 30 min, filtering, evaporating filtrate to dryness, adding methanol 1ml for dissolving to obtain the final product

1.2.3 preparation of negative test solution: taking 3g of the negative sample lacking the Chinese date, and preparing a negative control solution according to the preparation method of the test solution.

1.2.4 unfolding and inspection Condition inspection

Condition 1: performing thin layer chromatography (the method of 0502 in four parts of 2015 th edition of Chinese pharmacopoeia)^[3]) The test comprises respectively dispensing 10 μ l of control medicinal material solution and 5 μ l of test solution on the same silica gel H thin layer plate, spreading with toluene-ethyl acetate-glacial acetic acid (60:6:0.05) and n-hexane-ethyl acetate (3:1) as developing agent, taking out, air drying, spraying 10% sulphuric acid ethanol developer, heating at 105 deg.C until the spots are clear, and viewing under ultraviolet lamp (365 nm).

Condition 2: performing thin layer chromatography (the four parts 0502 method of the year 2015 in pharmacopoeia of China), respectively dropping 10 μ l of control solution and 5 μ l of test solution on the same silica gel G thin layer plate, spreading with toluene-ethyl acetate-glacial acetic acid (60:6:0.05) as developing agent, taking out, air drying, spraying 10% sulphuric acid ethanol color developing agent, heating at 105 deg.C until the spots are clear, and inspecting under ultraviolet lamp (365 nm).

Condition 3: according to the test of thin-layer chromatography (the four parts 0502 method of the year 2015 edition in China pharmacopoeia), the sample application amount is inspected: spreading 10 μ l of control medicinal material solution, 3 μ l of test solution, 5 μ l of test solution, 10 μ l of test solution, and 15 μ l of test solution on the same silica gel H thin layer plate, spreading with toluene-ethyl acetate-glacial acetic acid (60:6:0.05) as developing agent, taking out, air drying, spraying 10% sulphuric acid ethanol developer, heating at 105 deg.C until spots are clear, and viewing under ultraviolet lamp (365 nm).

The result shows that the content of the product of 3g is taken, ethyl acetate, ether and methanol are added to extract the jujube components, and the extraction effect of the ether and the methanol on the jujube is weaker than that of the ethyl acetate as shown in a chromatogram, as shown in figure 1:

comparing chromatograms of chromatographic conditions, the H thin layer plate and toluene-ethyl acetate-glacial acetic acid (60:6:0.05) have good separation degree and clear color development, as shown in figure 2, figure 3 and figure 4: the map is examined by taking the sample amount of 5 mul as the optimal condition as shown in figure 5:

combining the above experiment, collecting 3g of the content of the product, adding 30ml of ethyl acetate, performing ultrasonic treatment for 30 minutes, filtering, evaporating the filtrate, and dissolving the residue with 0.5ml of methanol to obtain a sample. Taking 2g of fructus Jujubae as reference material, adding 50ml of water, heating and refluxing for 2 hr, filtering while hot, volatilizing water from filtrate to obtain fructus Jujubae soft extract, adding 50ml of ethyl acetate, ultrasonic treating for 30 min, filtering, evaporating filtrate to dryness, dissolving with 1ml of methanol to obtain reference material solution, sucking 5 μ l of sample solution and 10 μ l of reference material solution, respectively dropping on the same silica gel H thin layer plate, developing with toluene-ethyl acetate-glacial acetic acid (60:6:0.05) as developer, taking out, air drying, spraying with 10% ethanol sulfate solution, heating at 105 deg.C until the spots are clearly developed, viewing under ultraviolet lamp (365nm), subjecting to sample chromatography, fluorescent spots with the same color are displayed at the positions corresponding to the chromatogram of the reference substance, the separation degree of the spots is high, the color development is clear, and the negative control is not interfered (as shown in figure 6), so the method is selected and listed in the text of quality standard.

1.3 durability

1.3.1 comparison of different humidities

At a temperature of 25 ℃, development is carried out under the conditions of 35% humidity and 75% humidity respectively. The results are shown (FIGS. 7 and 8).

1.3.2 comparison of different temperatures

At a humidity of 75%, development was carried out at a temperature of 4 ℃, 10 ℃ and 40 ℃ respectively. The results are shown (FIGS. 9, 10, 11).

1.3.3 different brands of veneer sheets (results see FIGS. 12-14)

Experimental example 2 matrine content measurement

2.1 instruments and reagents

Shimadzu LC-30AD high performance liquid chromatograph; agilent 1260 high performance liquid chromatograph; a KQ-500V DB type double-frequency numerical control ultrasonic cleaner (ultrasonic instruments, Inc., Kunshan city); the astragalus glue Shengbai capsule is provided by Guizhou Han prescription pharmaceutical industry Co., Ltd; matrine reference substance (China institute for testing and testing food and drug, batch No. 110805-200508 for content determination); reagent: methanol (chromatographically pure, analytically pure), ethanol (analytically pure), water (WP-UP-LH-40 physiochemical analytical ultrapure water machine), triethylamine (analytically pure), chloroform (analytically pure), n-hexane (analytically pure), diethyl ether (analytically pure), phosphoric acid (analytically pure).

2.2 methods of measurement

1.2.1 chromatographic condition selection:

(1) a chromatographic column: rishuan wet tissue and Rishuan lotion of reference company^[5]And in the determination of the matrine content, a column taking octadecylsilane chemically bonded silica as a filler is selected for investigation. The result shows that the column separation effect is better by adopting octadecylsilane chemically bonded silica as the filler.

(2) Mobile phase: reference 2015 edition pharmacopoeia^[4]And company product standard, and through experiment, the matrine and other impurities to be detected are well separated by adopting methanol-0.1% triethylamine water solution (53:47) as a mobile phase.

(3) Detection wavelength: referring to the content determination of matrine in the sophora flavescens medicinal material in the pharmacopoeia of 2015 edition, the detection wavelength is selected to be 220 nm.

(4) Theoretical plate number: the theoretical plate number is not less than 4000 according to the determination of matrine content in the product Rishuan wet tissue and Rishuan lotion of the company.

1.2.2 preparation of test solutions:

taking 1.0g of the content of the product, precisely weighing, placing in a conical flask with a plug, adding 2ml of concentrated ammonia test solution for wetting, adding 30ml of trichloromethane, heating and refluxing for 1.5 hours, filtering, evaporating filtrate to dryness, dissolving residues in 10ml of methanol, and shaking uniformly to obtain the product.

1.2.3 preparation of control solutions:

accurately weighing appropriate amount of matrine reference substance, adding methanol to obtain solution containing 0.2mg per 1ml, and shaking.

1.3 method verification

1.3.1 accuracy test

Taking about 1.0g of the stilbene gel Shengbai capsule (batch number: 2527025, average content: 1.7499mg/g) with the determined content, precisely weighing, placing in a conical flask with a plug, precisely adding 7ml of matrine reference substance solution (0.22100mg/ml), preparing a sample solution according to the preparation method of the test sample solution, determining the content of matrine according to the chromatographic conditions, and calculating the recovery rate, wherein the results are shown in table 1 and the sample amount of an accuracy chromatogram (figure 15) is 5 mul, which indicates that the recovery rate is better.

Table 1 recovery test data (n ═ 6)

1.3.2 repeatability tests

Taking the same batch of QIJIAOSHENGBAI capsules (2527025), preparing 6 parts according to the preparation method of the test solution, determining matrine content according to the above chromatographic conditions, and finding the results in Table 2 and the repeatability chromatogram (FIG. 16) to show that the repeatability is good.

Table 2 repeatability tests (n ═ 6)

1.3.3 precision test

Precisely measuring 5 μ l of the same stilbene gel Shengbai capsule sample solution, continuously introducing sample for 6 times, and recording chromatogram, wherein the results are shown in Table 3 and the precision chromatogram (see figure 17), which indicates that the precision is better.

TABLE 3 precision test (n ═ 6)

1.3.4 specificity

Preparing a sample solution and a negative sample solution from the stilbene gel Shengbai capsule (2527025) and the negative sample according to the preparation method of the test solution, respectively injecting 5 mu l of the reference solution, the sample solution and the negative sample solution into a liquid chromatograph according to the chromatographic conditions, wherein the chromatogram of the sample solution is at the corresponding chromatographic peak of the chromatogram of the reference solution, and the negative sample solution has no interference, and the chromatogram is a special chromatogram (see figure 18).

1.3.5 Linear relationship investigation

Precisely taking 3 μ l, 5 μ l, 7 μ l, 9 μ l, 11 μ l and 13 μ l of matrine control (0.22100mg/ml) and sequentially injecting into liquid chromatograph under the above chromatographic conditions, and measuring peak area. Taking the sample amount X of the reference substance as a horizontal coordinate and the peak area Y as a vertical coordinate, performing linear regression calculation, drawing a working curve (figure 19), and obtaining a linear regression equation as follows: y is 48.811X +17.542, R2 is 0.999. The matrine sample amount and peak area of the reference substance have good linear relationship in the range of 0.663 mu g-2.873 mu g. See linear chromatograms (fig. 20).

TABLE 4 matrine control Linear relationship

1.3.6 range

The precision of the measurement at high and low sampling was examined by sampling 80% and 120% of the normal measurement samples. The QIJIAOSHENGBAI Capsule (2527025) is prepared by sampling 6 parts of the above materials, preparing test solution, and determining matrine content according to the above chromatographic conditions, and the results are shown in Table 5.

TABLE 5 results of measurement of the ranges

1.3.7 durability

1.3.7.1 selection of sample extraction solvent

Taking a sample of the astragalus membranaceus Shengbai capsule (2527025), grinding, uniformly mixing, taking 3 parts, each part being about 1.0g, precisely weighing, respectively placing into a conical flask with a plug, respectively adding 2ml of concentrated ammonia test solution for wetting, then precisely adding 30ml of diethyl ether, trichloromethane and methanol, respectively, heating and refluxing for 1.5 hours, taking out, filtering, drying the filtrate to dryness, dissolving the residue in 10ml of methanol to obtain a test solution, respectively taking 5 mul of the 4 parts of solution, respectively injecting into a liquid chromatograph, and calculating, wherein the result is shown in table 6, which indicates that the content of the product is more completely extracted by adopting trichloromethane as an extraction solvent.

TABLE 6 selection of extraction solvents

1.3.7.2 selection of sample extraction method

Taking a sample of the astragalus membranaceus and astragalus membranaceus whiting capsules (2527025), grinding, uniformly mixing, taking 2 parts of each part of the astragalus membranaceus and astragalus membranaceus whiting capsules (1.0 g), precisely weighing, respectively placing into conical bottles with stoppers, respectively adding 2ml of concentrated ammonia test solution for wetting, precisely adding 30ml of trichloromethane, ultrasonically treating one part of the mixture for 1 hour, heating and refluxing the other part of the mixture for extraction for 1 hour, taking out, filtering, and evaporating filtrate to dryness. Dissolving the residue with 10ml methanol to obtain sample solution, respectively adding 5 μ l each of the 2 parts solution into liquid chromatograph, calculating to obtain results shown in Table 7, and performing ultrasonic treatment to obtain more complete reflux extraction content, so heating reflux extraction is selected.

TABLE 7 selection of extraction methods

1.3.7.3 sample extraction time Effect

Taking a astragalus membranaceus and astragalus membranaceus Shengbai capsule (2527025) sample, grinding, uniformly mixing, taking 3 parts, weighing 1.0g of each part precisely, placing the 3 parts in conical flasks with plugs respectively, wetting with 2ml of concentrated ammonia test solution respectively, adding 30ml of trichloromethane precisely, heating and refluxing for 1.0, 1.5 and 2.0 hours respectively, taking out, filtering, evaporating filtrate, dissolving residues with 10ml of methanol to obtain test solution, respectively taking 5 mul of each of the 3 parts of solution, injecting into a liquid chromatograph, calculating, the result is shown in table 8, comprehensively considering, and selecting the heating and refluxing for 1.5 hours as the sample extraction time.

TABLE 8 extraction time impact

1.3.7.4 Effect of different brands of posts

The content of matrine in the stilbene gel whitening capsule (2527025) is respectively measured by adopting columns with different brands of octadecylsilane chemically bonded silica as filling agents, and the influence of chromatographic columns with different brands is inspected. The chromatogram is shown in a durability chromatogram, and the content measurement result is shown in Table 9. The result shows that chromatographic columns using octadecylsilane chemically bonded silica of different brands as filling agents have no influence on content measurement.

TABLE 9 measurement results of columns of different brands

1.3.7.5 influence of the proportion of the mobile phase

Under the condition of not changing the components of the mobile phase, the proportion of the mobile phase is changed, the influence of the mobile phase with different proportions is examined, and the result that the separation degree of the sample is better under the selected proportion of the mobile phase is shown in the table 10 and the figure 21.

TABLE 10 measurement results under different flow phase ratio conditions

1.3.7.6 sample stability test

The stilbene gel Shengbai Capsule (2527025) is prepared by the method for preparing test solution, and the content of matrine is determined according to the above chromatographic conditions, and the result is shown in Table 11 and FIG. 22.

TABLE 11 sample stability test

1.4 sample determination

15 batches of samples were tested under the conditions described above and the results are shown in Table 12.

TABLE 12 results of sample measurement

The product is prepared by extracting radix Sophorae Flavescentis, so every 1g of radix Sophorae Flavescentis contains matrine (C)₁₅H₂₄N₂O), the content limit should be: in the prescription, the content of radix sophorae flavescentis is limited to radix sophorae flavescentis (total content of matrine and oxymatrine)/the number of prepared capsules is 0.7-250 g, 1.2%/1000 capsules, 0.7-1000 mg and 2.10mg, and each capsule is 0.5g, so that the content of radix sophorae flavescentis is 4.2mg/g calculated by every 1 g.

Because the matrine is partially lost in the process of water boiling in the production process and is mixed with the ointment powder, the matrine is seriously lost in the drying process at 75-95 ℃, the transfer rate of the water boiling matrine in the two processes is up to 58 percent, the matrine can only reach 41.5 percent in the total mixing and drying process, the total transfer rate can only reach about 40 percent and can not reach 70 percent, and the results of the matrine transfer test and the chromatographic chart of the matrine transfer test are simulated in a table 13 (figure 23).

Therefore, it is tentatively determined that every 1g of it contains matrine (C)₁₅H2₄N₂O) is not less than 1.3 mg/g.

TABLE 13 simulation of matrine transfer test results

The invention has the beneficial effects that: the invention provides a detection method of a stilbene gel Shengbai capsule, which comprises the steps of taking matrine and icariin as effective components as indexes, and determining the content of the matrine and the icariin by adopting a high performance liquid chromatography; and thin-layer chromatography (TLC) identification method of fructus Jujubae, radix Angelicae sinensis, radix Sophorae Flavescentis, radix astragali, and herba Epimedii in the medicine; the detection method is accurate, high in sensitivity, good in repeatability and stable in detection result, and the thin-layer chromatography identification of the Chinese dates is increased; the method for determining the amount of matrine in the sophora flavescens medicinal material provides data reference for establishing an effective and reliable quality standard and accurately reflecting the whole quality of the traditional Chinese medicine. The quality of the stilbene gel Shengbai capsule can be effectively controlled, which is more beneficial to monitoring the product quality by manufacturers and supervision and management departments and provides better guarantee for the treatment of medical departments and patients.

Drawings

FIG. 1 is a view of the examination results of the development and inspection conditions (1, 2, 3; 4, 1, 5, 2, 6, 3);

fig. 2 is H-plate toluene: ethyl acetate: from chromatogram of chromatographic conditions when glacial acetic acid is 60:6: 0.05; (1, Chinese date reference medicinal material solution; 2, 2527025; 3, 2527026; 4, 2528001);

FIG. 3 is H-plate n-hexane: from the chromatogram of the chromatographic conditions in ethyl acetate (3: 1); (1, Chinese date reference medicinal material solution; 2, 2527025; 3, 2527026; 4, 2528001);

fig. 4 is G thin layer plate, toluene: ethyl acetate: from the chromatogram of the chromatographic conditions in glacial acetic acid (60:6: 0.05); (1, Chinese date reference medicinal material solution; 2, 2527025; 3, 2527026; 4, 2528001);

FIG. 5 is a different point sample size spectrum (1, 10. mu.l of the Chinese date reference medicinal material solution, 2, 3. mu.l of the sample, 3, 5. mu.l of the sample, 4, 10. mu.l of the sample, 5, 15. mu.l of the sample);

FIG. 6 is a specific map (1, jujube control solution 2, 25270253, 25270264, 25280015, lack jujube negative sample);

FIG. 7 is a graph showing durability results at 25 ℃ and 35% humidity (1, jujube control solution; 2, 2527025; 3, 2527026; 4, 2528001);

FIG. 8 is a graph showing durability results at 25 ℃ and 75% humidity (1, jujube control solution; 2, 2527025; 3, 2527026; 4, 2528001);

FIG. 9 is a graph showing durability results at a humidity of 75% and a temperature of 4 ℃ (1, jujube control solution; 2, 2527025; 3, 2527026; 4, 2528001);

FIG. 10 is a graph showing durability results at humidity of 75% and temperature of 10 ℃ (1, jujube control solution; 2, 2527025; 3, 2527026; 4, 2528001);

FIG. 11 is a graph showing durability results at a humidity of 75% and a temperature of 40 ℃ (1, jujube control solution; 2, 2527025; 3, 2527026; 4, 2528001);

FIG. 12 is a graph showing the durability results of Qingdao ocean chemical plant brand thin-layer plates at humidity of 75% and temperature of 25 deg.C (1, jujube control solution; 2, 2527025; 3, 2527026; 4, 2528001);

FIG. 13 is a graph of the durability results of Qingdao ocean wave chemical plant brand thin-layer plates at 75% humidity and 25 deg.C; (1, Chinese date reference medicinal material solution 2, 25270253, 25270264, 2528001)

FIG. 14 is a graph showing the durability results of a home-made H-sheet laminate at a humidity of 75% and a temperature of 25 ℃ (1, jujube control solutions 2, 2527025; 25270264, 2528001)

Figure 15 is matrine accuracy chromatogram; (a-matrine control 1, test times 1; b-matrine control 2, test times 2; c-matrine control 3, test times 3; d-matrine control 4, test times 4; e-matrine control 5, test times 5; f-matrine control 6, test times 6);

FIG. 16 is a matrine reproducibility chromatogram (a-matrine control 1, test times 1; b-matrine control 2, test times 2; c-matrine control 3, test times 3; d-matrine control 4, test times 4; e-matrine control 5, test times 5; f-matrine control 6, test times 6);

FIG. 17 shows a precision chromatogram of matrine (a-matrine control 1, test number 1; b-matrine control 2, test number 2; c-matrine control 3, test number 3; d-matrine control 4, test number 4; e-matrine control 5, test number 5; f-matrine control 6, test number 6);

FIG. 18 is a matrine specificity chromatogram (a-matrine control 1, test times 1; b-matrine control 2, test times 2; c-matrine control 3);

FIG. 19 is a linear regression graph showing the measurement of matrine content;

FIG. 20 is a linear chromatogram of matrine content measurement (a-matrine control 1, test times 1; b-matrine control 2, test times 2; c-matrine control 3, test times 3; d-matrine control 4, test times 4; e-matrine control 5, test times 5; f-matrine control 6, test times 6);

FIG. 21 is a graph showing the effect of mobile phase ratio on matrine content measurement; (a-methanol: 0.1% aqueous triethylamine solution 53: 47; b-methanol: 0.1% aqueous triethylamine solution 50: 45; c-methanol: 0.1% aqueous triethylamine solution 50: 50);

FIG. 22 is a stability chromatogram when measuring the matrine content;

FIG. 23 is a process simulated chromatogram of matrine when matrine content is measured; wherein a is a process mimic sequence 1 (matrine control); b is a process simulation sequence 2(25278001 total extract before mixing); c is process simulation sequence 3(25278001 after total mixing); d is a process simulation sequence 4(25278002 total pre-mixing extract); e is process simulation sequence 5(25278002 after total mixing); f is a process simulation sequence 6 (matrine and oxymatrine mixed contrast); g is a process simulation sequence 7 (lightyellow sophora root medicinal material ZY 17112001); h is a process simulation sequence 8 (radix sophorae flavescentis medicinal material ZY 17112002).

The invention will be further illustrated by the following examples

Detailed Description

Example 1: the detection method of the astragalus membranaceus and astragalus membranaceus whitening capsules comprises the following steps

The formula is as follows: 250g of Chinese date, 250g of donkey-hide gelatin, 375g of blood ginseng, 375g of epimedium herb, 250g of radix sophorae flavescentis, 750g of astragalus mongholicus and 250g of angelica sinensis.

The process comprises the following steps: pulverizing radix Angelicae sinensis except colla Corii Asini into fine powder; decocting the rest five materials such as fructus Jujubae in water for three times (2 hr for the first time, 1.5 hr for the second time, and 1 hr for the third time), mixing decoctions, filtering, adding melted colla Corii Asini into the filtrate, stirring, concentrating to obtain soft extract with relative density of 1.30 at 75-85 deg.C, adding the above radix Angelicae sinensis fine powder, mixing, granulating, drying, and making into capsule.

The detection method comprises the following steps:

Claims

1. A detection method of a stilbene gel Shengbai capsule is characterized in that: the astragalus membranaceus and astragalus membranaceus leukocyte increasing capsule is prepared from 240 portions of Chinese date, 240 portions of donkey-hide gelatin, 250 portions of blood ginseng, 365 portions of blood, 385 portions of epimedium, 365 portions of epimedium, 260 portions of radix sophorae flavescentis, 740 portions of astragalus membranaceus and 760 portions of angelica sinensis and 260 portions of angelica sinensis by the following method: pulverizing radix Angelicae sinensis except colla Corii Asini into fine powder; decocting the rest five materials such as fructus Jujubae in water for three times (2 hr for the first time, 1.5 hr for the second time, and 1 hr for the third time), mixing decoctions, filtering, adding melted colla Corii Asini into the filtrate, stirring, concentrating to soft extract with relative density of 1.30 at 75-85 deg.C, adding the above radix Angelicae sinensis fine powder, mixing, granulating, drying, and making into capsule; the detection method comprises the thin-layer identification of Chinese date, the thin-layer identification of Chinese angelica, the thin-layer identification of lightyellow sophora root, the thin-layer identification of astragalus root and the thin-layer identification of epimedium herb, and also comprises the content determination of matrine and icariin;

the thin-layer identification method of the angelica comprises the following steps: taking 5g of the content of the drug particles, adding 20ml of n-hexane, carrying out ultrasonic treatment for 20 minutes, filtering, and volatilizing the filtrate to 1ml to be used as a test solution; preparing 0.5g of radix Angelicae sinensis control medicinal material, and preparing control medicinal solution by the same method; according to a thin-layer chromatography Chinese pharmacopoeia 2010 edition I appendix VI B test, absorbing 5 mul of each of the two solutions, respectively dropping the two solutions on the same silica gel G thin-layer plate, developing by using n-hexane-ethyl acetate 9:1 as a developing agent, taking out, airing, placing under an ultraviolet lamp with the wavelength of 365nm for inspection, and displaying fluorescent spots with the same color on the positions corresponding to the color spectrum of a reference medicinal material in the color spectrum of a test product;

the thin-layer identification method of the sophora flavescens comprises the following steps: taking 1g of the content of the medicine particles, adding 30ml of ethanol, heating and refluxing for 30 minutes, cooling, filtering, adding 20ml of water into the residue after the filtrate is evaporated to dissolve, filtering, placing the filtrate in a separating funnel, adding 0.5ml of concentrated ammonia test solution with the concentration of 25% -28%, shaking and extracting for 2 times by using trichloromethane, 10ml each time, combining the trichloromethane solution, evaporating to dryness, and adding 1ml of absolute ethanol into the residue to dissolve to obtain a sample solution; taking matrine and sophoridine reference substances, respectively adding anhydrous ethanol to prepare a solution containing 1mg per 1ml, taking the solution as a reference substance solution, performing a test according to an appendix VI B of a 2010 version of Chinese pharmacopoeia of thin-layer chromatography, sucking 5 mu l of each of the three solutions, respectively dropping the three solutions on a same silica gel G thin-layer plate, placing the solution in a stretching cylinder presaturated with ammonia vapor by taking cyclohexane-acetone-ethyl acetate-concentrated ammonia solution as a developing agent, presaturating for 30 minutes, stretching, taking out, drying in the air, and spraying a diluted bismuth potassium iodide test solution; spots with the same color appear on the chromatogram of the test solution at the positions corresponding to the chromatograms of the reference solution;

the thin-layer identification method of the astragalus comprises the following steps: taking 5g of medicine particle content, adding 30ml of petroleum ether at the temperature of 60-90 ℃, carrying out ultrasonic treatment for 20 minutes, filtering, volatilizing petroleum ether in medicine residue, adding 50ml of methanol, carrying out heating reflux for 30 minutes, filtering, washing filter residue with a small amount of methanol, combining washing liquor and filtrate, drying by distillation, adding 10ml of water into residue, heating for dissolving, cooling, centrifuging for 10 minutes in a centrifuge tube, taking supernate, shaking and extracting for 2 times by using trichloromethane, 15ml each time, discarding trichloromethane liquid, shaking and extracting for 3 times by using water-saturated n-butyl alcohol, adding 10ml for the first time, adding 10ml for the second time, adding 5ml for the third time, combining n-butyl alcohol liquid, washing for 3 times by adding ammonia test solution, discarding 15ml each time, and discarding ammonia solution; washing n-butanol solution with n-butanol saturated water to neutrality, discarding water solution, evaporating n-butanol solution to dryness, and dissolving the residue with 1ml methanol to obtain sample solution; adding methanol into astragaloside IV reference substance to obtain 1mg solution per 1ml as reference substance solution; according to a test of an appendix VI B of the 2010 version of the Chinese pharmacopoeia of the thin-layer chromatography, 10 mu l of a test solution and 5 mu l of a reference solution are sucked and respectively spotted on the same silica gel G thin-layer plate, a lower layer solution which is placed at the temperature of below 10 ℃ and is chloroform-methanol-water of 13:7:2 is used as a developing agent, the developing agent is developed, taken out and dried, a 10% sulfuric acid ethanol solution is sprayed, and the heating is carried out at the temperature of 105 ℃ until spots are clearly developed; spots with the same color appear on the chromatogram of the test solution at the positions corresponding to the chromatograms of the reference solution; then placing under an ultraviolet lamp with the wavelength of 365nm for inspection, and displaying fluorescent spots with the same color in the chromatogram of the test sample and at the position corresponding to the chromatogram of the reference sample;

the thin-layer identification method of the epimedium comprises the following steps: taking 5g of medicine particle content, adding 30ml of petroleum ether at the temperature of 60-90 ℃, carrying out ultrasonic treatment for 20 minutes, filtering, volatilizing petroleum ether in medicine residue, adding 50ml of methanol, carrying out heating reflux for 30 minutes, filtering, washing filter residue with 5ml of methanol, combining washing liquor and filtrate, drying by distillation, adding 10ml of water into residue, heating for dissolving, cooling, centrifuging in a centrifuge tube for 10 minutes, taking supernate, shaking and extracting with chloroform for 2 times, 15ml each time, discarding chloroform solution, shaking and extracting with water-saturated n-butyl alcohol for 3 times, adding 10ml for the first time, adding 10ml for the second time, adding 5ml for the third time, combining n-butyl alcohol solution, adding ammonia test solution and washing for 3 times, 15ml each time, discarding ammonia solution; washing n-butanol solution with n-butanol saturated water to neutrality, discarding water solution, evaporating n-butanol solution to dryness, and dissolving the residue with 1ml methanol to obtain sample solution; adding methanol into icariin control to obtain 1mg solution per 1ml as control solution; according to a test of an appendix VI B of the 2010 version of the Chinese pharmacopoeia of thin-layer chromatography, sucking 2-4 mu l of a test solution and 5 mu l of a reference solution, respectively dropping the test solution and the reference solution on the same silica gel G thin-layer plate, developing the test solution and the reference solution by taking chloroform-methanol-water as a developing agent, taking out the developed solution, drying the developed solution in the air, spraying a 10% sulfuric acid ethanol solution, and heating the developed solution at 105 ℃ until spots are clearly developed; spots with the same color appear on the chromatogram of the test solution at the positions corresponding to the chromatograms of the reference solution; viewing under 365nm ultraviolet lamp to display fluorescent spots of the same color in the chromatogram of the test solution at the position corresponding to the chromatogram of the reference solution;

the method for measuring the content of the matrine comprises the following steps: the determination is carried out according to a method 0512 of four parts of Chinese pharmacopoeia 2015 year edition by high performance liquid chromatography:

the method for measuring the content of the icariin comprises the following steps: the determination is carried out according to the appendix VI D of the first part of the Chinese pharmacopoeia 2010 edition of high performance liquid chromatography:

2. The method for detecting the stilbene gel capsules for increasing the white blood cells as claimed in claim 1, which comprises the following steps: the characteristics of the astragalus membranaceus and astragalus membranaceus whitening capsules are as follows: the hard capsule content is brown granule and powder; is bitter in taste.