Quality control method of warm-face powder

Abstract

The invention relates to a surface warming powder quality control method, and belongs to the technical field of traditional Chinese medicine quality detection. According to the method, the microscopic characteristics of scorpion, stiff silkworm and rhizoma typhonii in the powder for warming the face are taken as identification indexes for the first time, an angelica dahurica reference medicinal material is taken as an index for thin-layer chromatography identification, and cinnamaldehyde in cassia twig and imperatorin in angelica dahurica are taken as indexes for high performance liquid chromatography content determination; the method can be used for accurately measuring the five medicinal materials and the index components thereof in the Wenfengsan, and is provided for quality control of the Wenfengsan. The detection method disclosed by the invention is simple and convenient to operate, good in repeatability, high in accuracy, good in detected index stability and suitable for quality control application of the Wenfengsan.

Description

Technical Field

[0001] The invention relates to the technical field of quality detection of traditional Chinese medicines, in particular to a quality control method of Wenmiansan. Background Art

[0002] According to statistics, among the common Bell's palsy patients in clinic, about 1 / 5 suffer from refractory facial palsy, of which 33.3% suffer from partial paralysis and 66.7% suffer from complete paralysis. Patients will still suffer from various sequelae after receiving existing treatments. At present, we have not yet sorted out the pathogenesis of refractory facial palsy. The deterioration of refractory facial palsy is mostly due to the delay in medical treatment of peripheral facial palsy, improper treatment methods or drug application in the early stage of the disease, and the patient's physical endowment and mental factors, which lead to the prolongation of the disease.

[0003] The "mouth habit", "hanging line wind", "mouth abnormality" and "mouth crookedness" often mentioned in traditional Chinese medicine include difficult-to-treat and stubborn facial paralysis. From the perspective of traditional Chinese medicine, refractory facial paralysis is caused by insufficient positive energy and empty meridians, which allows external evil energy to take the opportunity to invade, and then causes wind phlegm to block the meridians and meridians. As time goes by, people are prone to fall into a complex relationship between positive deficiency and evil love, showing the complex characteristics of the interweaving of virtuality and reality. These symptoms are interrelated and cause and effect. The longer the illness lasts, the longer the evil energy enters the body, and the positive energy in the human body will gradually be depleted, which is what is often said as "long-term illness will lead to deficiency". In addition, the deficiency of positive energy in the human body cannot promote the circulation of qi, blood and body fluids in the body. Quantitative changes cause qualitative changes. Over time, it will turn from virtual to real, and produce pathological products such as phlegm, fluid and blood stasis, thereby blocking the meridians and affecting the function of facial muscles.

[0004] Wenmiansan, a clinical prescription of Hai'an Traditional Chinese Medicine Hospital, is a prescription developed for intractable facial paralysis. Wenmiansan is composed of 5 Chinese herbal medicines, including white aconite, silkworm, scorpion, angelica dahurica and cinnamon twig. Modern pharmacological studies have shown that cinnamon twig in the prescription of Wenmiansan contains multiple ingredients such as cinnamaldehyde, and angelica dahurica contains multiple ingredients such as imperatorin, which have anti-inflammatory and skin vascular dilation effects; white aconite contains β-sitosterol, which makes it have strong antioxidant capacity and the function of inhibiting oxygen free radicals; substances such as scorpion toxins contained in silkworm and scorpion can optimize microcirculation and play a positive role in promoting nerve recovery. In the formula of Wenmiansan, aromatic borneol and transdermal absorbents are often added to help it be better absorbed by the body. Therefore, Wenmiansan can optimize the blood circulation of the facial nerve and help the nerve recover quickly.

[0005] At present, the preparation method of Wenmiansan is to grind white aconite root, bombyx batryticatus, scorpion, cinnamon twig and angelica dahurica into extremely fine powder and mix them evenly. However, there are no relevant literature and reports on the quality testing of Wenmiansan. It is urgent to formulate a truly feasible quality standard method to ensure the product quality and clinical efficacy of Wenmiansan. Summary of the invention

[0006] Therefore, the technical problem to be solved by the present invention is to overcome the problem that there is a lack of a quality control method for temperature surface dispersion in the prior art.

[0007] In order to solve the above technical problems, the present invention provides a quality control method for Wenmian San. After extensive exploration and research, the present invention proposes for the first time to use scorpion, silkworm and white aconite in Wenmian San as indicators for microscopic identification, Angelica dahurica as an indicator for thin layer chromatography identification, cinnamaldehyde in cassia twig and imperatorin in Angelica dahurica as indicators for high performance liquid chromatography content determination, which can achieve effective detection of characteristic medicinal materials and indicator components of Wenmian San.

[0008] The first object of the present invention is to provide a quality control method for temperature-controlled dispersion, wherein the quality control method is selected from one or more of the following methods:

[0009] (1) using microscopic identification to identify that the Wenmian powder contains scorpion, silkworm and white aconite;

[0010] (2) using thin layer chromatography to identify the presence of Angelica dahurica in the Wenmian Powder, wherein the Wenmian Powder is used as the sample to be tested, Angelica dahurica is used as the positive control, and a silica gel G thin layer plate is used for identification, and a sample lacking Angelica dahurica is used as a negative control, wherein the sample lacking Angelica dahurica is composed of Radix Aconiti Lateralis, Bombyx Batryticatus, Scorpio and Cinnamomum cassiae;

[0011] (3) High performance liquid chromatography was used to quantitatively determine cinnamaldehyde and imperatorin in the Wenmiansan.

[0012] Furthermore, the microscopic identification method is to prepare the Wenmiansan into chloral hydrate tablets or water-mounted tablets and observe under a microscope:

[0013] The calcium oxalate needle crystals in the white aconite are scattered or bundled in the mucous cells, and the needle crystals are about 97-136 μm long; the diameters of the spiral ducts and the annular ducts are 9-45 μm;

[0014] The mycelium of Bombyx batryticatus is nearly colorless, slender, curled and tangled in the body wall; the tracheal wall fragments are slightly curved or arc-shaped, with brown or dark brown spiral filaments; the surface of the epidermal tissue has a grid-like wrinkled texture and small spikes formed by the texture protrusions, with round hair pits and yellow edges; the bristles are yellow or yellow-brown, with a smooth surface and slightly thick walls; the undigested mulberry leaf tissue mostly contains clusters or prismatic crystals of calcium oxalate;

[0015] The outer epidermis of the scorpion body wall fragment has a polygonal grid-like texture on the surface, and is densely covered with fine particles, with visible hair pits, tiny round holes and light brown or nearly colorless tumor-like protrusions; the inner epidermis is colorless with transverse stripes, and the inner and outer epidermis are traversed by many microscopic pores of varying lengths; the striated muscle fibers are mostly broken, the light band is wider than the dark band, there is a dark line in the light band, and the dark band has dense short longitudinal textures.

[0016] Further, the thin layer chromatography method comprises the following steps:

[0017] S1, the Wenmian powder is dissolved in an organic solvent as a sample to be tested; the Angelica dahurica is ground into powder and dissolved in an organic solvent, centrifuged and the supernatant is taken, and the filtrate is filtered after adding an organic solvent to dissolve as a positive control; white aconite, bombyx batryticatus, scorpion and cinnamon twig are mixed and ground into powder, dissolved in an organic solvent, centrifuged and the supernatant is taken, and the filtrate is filtered after adding an organic solvent to dissolve as a negative control;

[0018] S2. The sample to be tested, the positive control and the negative control are respectively aspirated, and are spotted on a silica gel G thin layer plate, which is developed in a developing agent, taken out, air-dried, and examined under an ultraviolet lamp; if the chromatogram of the sample to be tested shows a spot of the same color at the position corresponding to the chromatogram of the positive control, then the Wenmiansan contains Angelica dahurica; the chromatogram of the negative control does not show a spot of the same color at the position corresponding to the positive control.

[0019] Furthermore, the organic solvent includes methanol.

[0020] Furthermore, the developing agent comprises a mixed solution of petroleum ether and ethyl ether, wherein the volume ratio of petroleum ether to ethanol is 3:2.

[0021] Furthermore, the high performance liquid chromatography method for cinnamaldehyde is as follows: the extract obtained by ultrasonic extraction of the Wenmian powder with an organic solvent is used as a sample to be tested, cinnamaldehyde is dissolved in an organic solvent as a standard, cinnamon twig powder is dissolved in an organic solvent as a positive control, white aconite powder, scorpion powder, bombyx batryticatus powder and angelica dahurica powder are mixed and dissolved in an organic solvent to obtain a negative control;

[0022] The test samples, standard products, positive controls and negative controls were tested using a high performance liquid chromatograph. The liquid chromatograms of the test samples and the positive control showed chromatographic peaks with the same retention time. The content was determined using the external standard method based on the peak area.

[0023] Furthermore, the detection conditions of the high performance liquid chromatograph are as follows: chromatographic column 5C18-MS-Ⅱ (4.6ID×250mm); isocratic elution process of mobile phase including water and acetonitrile (48:52); flow rate of 0.9-1.1mL per minute; column temperature of 25-35°C; wavelength for detection of 280-300nm; sample input volume of 10-20μL.

[0024] Furthermore, the high performance liquid chromatography method for imperatorin is as follows: the extract obtained by ultrasonic extraction of the Wenmian powder with an organic solvent is used as a sample to be tested, imperatorin is dissolved in an organic solvent as a standard, Angelica dahurica powder is dissolved in an organic solvent as a positive control, and Rhizoma Aconiti Lateralis Preparata powder, Bombyx Batryticatus powder, Scorpio powder and Cinnamomum cassia powder are mixed and dissolved in an organic solvent to obtain a negative control;

[0025] The test samples, standard products, positive controls and negative controls were tested using a high performance liquid chromatograph. The liquid chromatograms of the test samples and the positive control showed chromatographic peaks with the same retention time. The content was determined using the external standard method based on the peak area.

[0026] Furthermore, the detection conditions of the high performance liquid chromatograph are as follows: chromatographic column 5C18-MS-Ⅱ (4.6ID×250mm); isocratic elution process of mobile phase including water and acetonitrile (48:52); flow rate of 0.9-1.1mL per minute; column temperature of 25-35°C; wavelength for detection of 290-300nm; sample input volume of 10-20μL.

[0027] Furthermore, the organic solvent includes methanol.

[0028] Beneficial effects of the present invention:

[0029] The present invention uses scorpion, silkworm and white aconite in Wenmian San as indicators for microscopic identification, uses Angelica dahurica as an indicator for thin-layer chromatography identification, and uses cinnamaldehyde in cassia twig and imperatorin in Angelica dahurica as indicators for high-performance liquid chromatography identification, so as to accurately identify the effective ingredients in Wenmian San and provide a method for quality control of Wenmian San. Meanwhile, the detection method of the present invention is simple to operate, has good repeatability, high accuracy, and good stability of indicator substances, and is suitable for quality control application of Wenmian San. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to make the content of the present invention more clearly understood, the present invention is further described in detail below according to specific embodiments of the present invention in conjunction with the accompanying drawings, wherein

[0031] Figure 1 This is a microscopic feature image of Bombyx batryticatus in Wenmian powder (×400), where: 1 is mycelium, 2 is hyphae, 3 is epidermal cells, 4 is bristles, 5 is tracheal wall fragments, 6 is undigested mulberry leaf tissue, 7 and 8 are quasi-crystals;

[0032] Figure 2 This is a microscopic feature image of Radix Aconiti Lateralis Preparata in Wenmian Powder (×400), where: 1 is a thin-walled cell fragment, 2 is a calcium oxalate needle crystal, 3 is a vessel, and 4 is a gelatinized starch granule after heating and permeabilization with chloral hydrate;

[0033] Figure 3These are the powder characteristics of the whole scorpion in Wenmiansan (×400), where 1 is bristle, 2 is body wall fragment, 3 is striated muscle fiber, and 4 is fat oil droplet;

[0034] Figure 4 This is the TLC identification diagram of Angelica dahurica in Wenmian San, where: 1 is the positive control of Angelica dahurica, 2 is the Wenmian San sample to be tested, and 3 is the negative control lacking Angelica dahurica;

[0035] Figure 5 This is the TLC identification diagram of cinnamon twig in Wenmiansan, where: 1 is the positive control of cinnamon twig, 2 is the sample to be tested in Wenmiansan, and 3 is the negative control without cinnamon twig;

[0036] Figure 6 This is the TLC identification chart of whole scorpion in Wenmian San, where: 1 is the whole scorpion positive control, 2 is the Wenmian San sample to be tested, and 3 is the negative control lacking whole scorpion;

[0037] Figure 7 It is the TLC identification diagram of other medicinal materials in Wenmian Powder, where: A is the TLC identification diagram of Bombyx Batryticatus; B is the TLC identification diagram of Radix Aconiti Lateralis Preparata; 1 is the positive control of medicinal materials, 2 is the sample to be tested in Wenmian Powder, and 3 is the negative control;

[0038] Figure 8 It is a specific HPLC chromatogram for the determination of cinnamaldehyde content in Wenmiansan compound, wherein: A is the cinnamaldehyde reference solution, B is the Wenmiansan compound solution, C is the cinnamaldehyde-deficient negative sample solution, and D is the standard curve for the determination of cinnamaldehyde content in Wenmiansan;

[0039] Fig. 9 It is a specific HPLC chromatogram for the determination of imperatorin content in Wenmiansan compound, wherein: A is the imperatorin reference solution, B is the Wenmiansan compound solution, C is the Angelica dahurica-deficient negative sample solution, and D is the standard curve for the determination of imperatorin content in Wenmiansan compound. DETAILED DESCRIPTION

[0040] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0041] Example 1: Microscopic Identification

[0042] Preparation of Wenmiansan: Take 200g of white aconite root, bombyx batryticatus, scorpion, cinnamon twig and angelica dahurica, grind them into a very fine powder, seal them in a brown can and set aside.

[0043] Following the standard operating procedures specified in the Microscopic Identification Method (General Chapter 2001) of Part 4 of the 2020 edition of the Chinese Pharmacopoeia, the Wenmiansan compound powder samples were prepared into tablets, chloral hydrate tablets and water-mounted tablets, and the microscopic characteristics of scorpion, silkworm and white aconite were observed under a microscope.

[0044] (1) Microscopic identification of Bombyx batryticatus in Wenmiansan

[0045] The Wenmiansan compound powder sample was sliced ​​and the microscopic features of Bombyx batryticatus were observed under a microscope. The results are as follows: Figure 1 As shown, the mycelium is nearly colorless, slender and curled, intertwined and interwoven, and there are also scattered hyphae. The epidermal cells are grayish yellow with extremely wrinkled grid-like textures. The bristles are yellow-brown, with slightly thick walls, clear medullary cavities, and often brittle. The tracheal wall fragments are slightly curved, with spiral filaments arranged in a palisade shape. Undigested mulberry leaf tissue is yellow-green, and often has prismatic or clustered calcium oxalate crystals arranged irregularly in it.

[0046] (2) Microscopic identification of white aconite root in Wenmiansan

[0047] The Wenmiansan compound powder sample was sliced ​​and the microscopic characteristics of Radix Aconiti Lateralis Preparata were observed under a microscope. The results were as follows: Figure 2 As shown in Figure 1, calcium oxalate needles in Rhizoma Aconiti Lateralis Preparata are scattered or bundled in mucous cells, with a length of about 97-136 μm; the diameters of the spiral and annular vessels are 9-45 μm.

[0048] (3) Microscopic identification of scorpions in Wenmiansan

[0049] The Wenmiansan compound powder sample was sliced ​​and the microscopic features of Bombyx batryticatus were observed under a microscope. The results are as follows: Figure 3 As shown. The number of fat droplets is relatively large, usually colorless or light yellow; the body wall fragments are polygonal, and its surface is covered with many fine particles, and hair pits, tiny round holes and almost colorless or light brown tumor-like protrusions can be seen. The fibers of striated muscle are mostly broken, the bandwidth of the light band is relatively wider than the dark band, and there is a dark line in the light band, and the dark band has dense short longitudinal textures. The color of the bristles is reddish brown, easy to break, sharp at the end, and with longitudinal textures, and its medullary cavity is very narrow.

[0050] Example 2: Thin layer identification

[0051] 1. Thin layer identification of Angelica dahurica

[0052] (1) Preparation of the Wenmiansan sample to be tested: 1.0 g of Wenmiansan compound powder was taken, 10 mL of methanol was added, and ultrasonic treatment was performed at 30°C for 30 minutes. After the ultrasonic treatment, the mixture was centrifuged at 3000 r / min for 15 minutes. The supernatant was taken and filtered using a 0.22 μm organic microporous filter membrane. The filtrate was placed in a 60°C water bath and rotary evaporated to near dryness. 2 mL of methanol was added again and ultrasonic treatment was performed at 30°C to dissolve the residue, which was then taken out, filtered, and set aside.

[0053] (2) Preparation of positive control of Angelica dahurica: Take 0.50 g of Angelica dahurica control medicinal material, add 5 mL of methanol, and dissolve it under ultrasonication for 30 min. Then put it into a centrifuge tube for centrifugation, and then centrifuge it at a speed of 3000 r / min for 15 min. Then, take out the supernatant, filter it, and evaporate it to dryness at 50°C. Then add 400 μL of methanol to dissolve it, take it out, filter it, and set it aside.

[0054] (3) Preparation of negative control lacking Angelica dahurica: Take about 0.2 g of powder of each of the four medicinal materials, namely, Radix Aconiti Lateralis, Bombyx Batryticatus, Scorpio, and Cinnamomum cassia, mix them evenly, add 8 mL of methanol, and dissolve them by ultrasonication for 30 min. Then put them into a centrifuge tube for centrifugation, and then centrifuge them at a speed of 3000 r / min for 15 min. After that, take out the supernatant, filter it, and evaporate it to dryness at 50°C. Then add 400 μL of methanol to dissolve it, take it out, filter it and set it aside for use.

[0055] (4) Follow the standard operating procedure of thin layer chromatography, accurately pipette 10 μL of the three solutions, and spot the Wenmiansan sample, Angelica dahurica positive control, and Angelica dahurica negative control on the same silica gel G thin layer plate, and place it in a pre-saturated chromatography tank. The developing agent is a solvent mixed with petroleum ether (30-60℃) and ether in a ratio of 3:2, develop at 25℃, take out, and place the dried thin layer plate under ultraviolet light (365nm) for inspection.

[0056] Depend on Figure 4 The TLC graph of Angelica dahurica in Wenmiansan compound shows that the thin layer chromatograms of Wenmiansan compound sample and Angelica dahurica reference medicinal material show the same blue and yellow fluorescent spots at the corresponding positions, and the Angelica dahurica negative reference does not show spots of the same color at the corresponding positions, which does not interfere with the detection of the target components. The spots are clearly separated and the spots are clear and distinct. The results meet the quality standards and can be listed as an inspection item under the quality standards of Wenmiansan.

[0057] 2. Thin layer identification of cinnamon twig

[0058] (1) Positive control: Take about 0.2 g of cinnamon twig powder, add about 2 mL of methanol, and dissolve it under ultrasonication for 30 min. Then put it into a centrifuge tube for centrifugation, and then centrifuge it at 3000 r / min for 15 minutes. Then take out the supernatant on the upper layer, filter it, and evaporate it to dryness at 50°C. Then add 400 μL of methanol to dissolve it, take it out, filter it, and set it aside.

[0059] (2) Negative control lacking cinnamon twig: Take about 0.2 g of powder of each of the four medicinal materials, namely, white aconite root, bombyx batryticatus, scorpion, and angelica dahurica, mix them evenly, add about 8 mL of methanol, and dissolve them under ultrasonication for 30 min. Then put them into a centrifuge tube for centrifugation, and then centrifuge them at a speed of 3000 r / min for 15 min. After that, take out the supernatant, filter it, and evaporate it to dryness at 50°C. Then add 400 μL of methanol to dissolve it, take it out, filter it and set it aside for use.

[0060] (3) Sample to be tested: Take about 1 g of warm noodle powder, add 10 mL of methanol, dissolve it under ultrasonication for 30 min, put it into a centrifuge tube for centrifugation, and then centrifuge it at 3000 r / min for 15 min. Then take out the supernatant, filter it, evaporate it to dryness at 50°C, add 400 μL of methanol to dissolve it, take it out, filter it and set it aside.

[0061] (4) According to the provisions of thin layer chromatography (General Rules 0502 of Part IV of the Chinese Pharmacopoeia), 15 μL of the filtrate of the sample to be tested, the positive control and the negative control lacking cinnamon twigs were accurately taken and spotted on the same silica gel G plate, placed in a pre-saturated chromatography cylinder, and placed in a developing solvent of petroleum ether-ethyl acetate (17:3). After that, it was developed and taken out to dry, and finally sprayed with vanillin sulfuric acid test solution. The spots were clearly colored and observed under sunlight.

[0062] The thin layer chromatography identification results of cinnamon twigs showed that the negative control sample without cinnamon twigs had interference and could not be identified. Later, liquid chromatography was used for identification and content determination.

[0063] 3. Thin layer identification of whole scorpion

[0064] (1) Positive control: Take about 0.2 g of scorpion powder, add about 2 mL of methanol, dissolve it in ultrasound for 30 min, put it into a centrifuge tube for centrifugation, and then centrifuge it at 3000 r / min for 15 min. Then take out the supernatant, filter it, evaporate it to dryness at 50°C, add 400 μL of methanol to dissolve it, take it out, filter it, and set it aside.

[0065] (2) Negative control without whole scorpion: Take about 0.2 g of powder of each of the four medicinal materials, namely, Rhizoma Aconiti Lateralis, Bombyx Batryticatus, Angelica Dahurica, and Cinnamomum cassia, mix them evenly, add about 8 mL of methanol, and dissolve them by ultrasonication for 30 min. Then put them into a centrifuge tube for centrifugation, and then centrifuge them at a speed of 3000 r / min for 15 min. After that, take out the supernatant, filter it, evaporate it to dryness at 50°C, add 400 μL of methanol to dissolve it, take it out, filter it, and set it aside.

[0066] (3) Samples to be tested: Take about 1 g of warm noodle powder, add about 10 mL of methanol, and dissolve it under ultrasonication for 30 min. Centrifuge it in a centrifuge tube at 3000 r / min for 15 min, take out the supernatant, filter it, evaporate it to dryness at 50°C, add 400 μL of methanol to dissolve it, take it out, filter it, and set it aside.

[0067] (4) According to the provisions of thin layer chromatography (General Rules 0502 of Part IV of the Chinese Pharmacopoeia), accurately take 10 μL of the above-prepared filtrate and spot it on the same silica gel G plate. In the development tank, add n-butanol, glacial acetic acid and water (8:3:1) as the developing agent, and then develop the silica gel G plate. When it reaches the corresponding liquid level, take it out and dry it. Next, spray it with 0.5% ninhydrin ethanol solution and heat it until the color of the spot becomes clearly visible.

[0068] The results of TLC identification of whole scorpion showed that the negative control sample without whole scorpion had interference and could not be identified. Microscopic identification was used.

[0069] 4. Thin layer identification of silkworm

[0070] (1) Samples to be tested: 1.0 g of Wenmiansan compound powder was accurately weighed, and 10 mL of methanol was added. The mixture was ultrasonically treated at 30°C for 30 min to dissolve the mixture. The mixture was centrifuged at 3000 r / min for 15 min, and the supernatant was collected and filtered using a 0.22 μm organic microporous filter membrane. The filtrate was rotary evaporated in a 60°C water bath until it was nearly dry. 2 mL of methanol was added, and the mixture was ultrasonically treated at 30°C to dissolve the mixture. The mixture was collected, filtered, and set aside.

[0071] (2) Positive control: Take about 0.2043 g of Bombyx batryticatus powder, add about 2 mL of methanol, and dissolve it under ultrasonication for 30 minutes. Then put it into a centrifuge tube for centrifugation, and then centrifuge it at 3000 r / min for 15 minutes. Then take out the supernatant on the upper layer, filter it, and evaporate it to dryness at 50°C. Then add 400 μL of methanol to dissolve it, take it out, filter it, and set it aside.

[0072] (3) Negative control lacking Bombyx Batryticatus: Take about 0.2 g of powder of each of the four medicinal materials, namely, Radix Aconiti Lateralis, Radix Angelicae Dahuricae, Scorpio, and Cinnamomum cassiae, mix them evenly, add about 8 mL of methanol, and dissolve them by ultrasonication for 30 min. Then, put them into a centrifuge tube for centrifugation, and then centrifuge them at a speed of 3000 r / min for 15 min. After that, take out the supernatant, filter it, and evaporate it to dryness at 50°C. Then, add 400 μL of methanol to dissolve it, take it out, filter it and set it aside for use.

[0073] (4) According to the standard operating procedure of thin layer chromatography, 5 μL of each of the three solutions was accurately pipetted, and the positive control, the sample to be tested, and the negative control lacking Bombyx batryticatus were spotted on the same silica gel G thin layer plate, and placed in a pre-saturated chromatography tank. A solvent mixed with n-butanol, water, and glacial acetic acid in a ratio of 4:1:1 was used as the developing agent. After the development was completed, the plate was taken out and evenly sprayed with 2% ninhydrin ethanol solution as a color developer on the dried thin layer plate, heated for 2 minutes, and then placed under visible light and ultraviolet light (365nm) for inspection.

[0074] The results of TLC identification of Bombyx Batryticatus showed that the negative control sample of Bombyx Batryticatus was not identified due to interference. Microscopic identification was used.

[0075] 5. Thin layer identification of white aconite

[0076] (1) Sample to be tested: Accurately weigh 0.98 g of Wenmiansan compound powder, add 10 ml of methanol, ultrasonicate at 30°C for 30 min, centrifuge at 3000 r / min for 15 min, take the supernatant, filter, and place the filtrate in a 60°C water bath and rotary evaporate to near dryness, then add 2 ml of methanol, ultrasonicate at 30°C to dissolve, take out, filter, and set aside.

[0077] (2) Positive control of Radix Aconiti Lateralis Preparata: Accurately weigh 0.2020 g of Radix Aconiti Lateralis Preparata control medicinal material, add about 2 mL of methanol, and dissolve it under ultrasonication for 30 min. Then, put it into a centrifuge tube for centrifugation, and then centrifuge it at a speed of 3000 r / min for 15 min. Then, remove the supernatant from the upper layer, filter it, and evaporate it to dryness at 50°C. Then, add 400 μL of methanol to dissolve it, remove it, filter it, and set it aside.

[0078] (3) Negative control sample lacking white aconite root: Take about 0.2 g of each of the four medicinal materials powder of Angelica dahurica, Bombyx batryticatus, Scorpio, and Cinnamomum cassia, mix them evenly, add about 8 mL of methanol, and dissolve them by ultrasonication for 30 min. Then put them into a centrifuge tube for centrifugation, and then centrifuge them at a speed of 3000 r / min for 15 min. After that, take out the supernatant, filter it, and evaporate it to dryness at 50°C. Then add 400 μL of methanol to dissolve it, take it out, filter it and set it aside for use.

[0079] (4) According to the standard operating procedure of thin layer chromatography, 5 μL of each of the three solutions was accurately pipetted, and the positive control, the sample to be tested, and the negative control solution lacking white aconite were spotted on the same silica gel G thin layer plate, placed in a pre-saturated chromatography cylinder, and a solvent mixed with chloroform and acetone in a ratio of 25:1 was used as the developing agent. After the development was completed, the plate was taken out and 10% sulfuric acid ethanol solution was evenly sprayed on the dried thin layer plate as a color developer, heated until the spots were clearly colored, and placed under ultraviolet light (365nm) for inspection.

[0080] The thin layer chromatography identification results of Radix Aconiti Lateralis Radix showed that the negative control sample without Radix Aconiti Lateralis Radix had interference and could not be identified. Microscopic identification was used.

[0081] Example 4: Identification of cinnamaldehyde and imperatorin content in Wenmiansan by HPLC

[0082] 1. Determination of cinnamaldehyde content

[0083] (1) HPLC conditions

[0084] Chromatographic column: COSMOSIL 5C18-MS-Ⅱ chromatographic column (4.6×250mm); the mobile phase includes an isocratic elution process of water (A) and acetonitrile (B) (48:52); the flow rate is 1 mL per minute; the column temperature is 30°C; the wavelength used for detection is 290 nm; the sample input volume is 10 μL.

[0085] (2) Solution preparation

[0086] A. Preparation of standard solution

[0087] First, accurately weigh 0.1517g of cinnamaldehyde and place it in a 20mL volumetric flask. Next, add an appropriate amount of methanol to dissolve, adjust the volume, and shake well to make a stock solution. The mass concentration of cinnamaldehyde is 758.5μg / mL. After that, extract 1.4mL from this stock solution with a pipette and place it in a 100mL volumetric flask. Finally, add an appropriate amount of methanol to dissolve, adjust the volume, and shake well to make a stock solution 2.

[0088] B. Preparation of Control Solution

[0089] Positive control: Weigh 0.4005g of cinnamon powder accurately and put it into a 50mL bottle. Next, add 45mL of methanol and treat it in ultrasound for 30 minutes. After that, take it out and cool it, then add methanol to the specified scale, shake well and filter. Finally, take 1mL of the filtrate and put it into a 10mL bottle, add methanol to the scale and shake well, so that the required positive control solution is obtained.

[0090] Sample to be tested: Place 2.001g of precisely weighed Wenmiansan powder into a 50mL bottle, then add 45mL of methanol and perform ultrasonic treatment for 30 minutes. Take it out and cool it, add methanol to the scale, shake it well and filter it. Then, take 1mL of the filtrate and place it in a 10mL volumetric bottle, add methanol to the scale and shake it well, so that the required Wenmiansan solution is obtained.

[0091] Negative control without cinnamon twig: Weigh about 0.40g of each of white aconite, scorpion, silkworm and angelica dahurica accurately and put them into a 50mL bottle. Then, add 45mL of methanol and treat it in ultrasound for 30 minutes. Then take it out and cool it, add methanol to the specified scale, shake it well and filter it. Then, take 1mL of the filtrate, put it into a 10mL volumetric flask, add methanol to the scale, shake it well to prepare the negative control solution.

[0092] (3) Methodological investigation

[0093] A. Specificity test

[0094] Take 10 μL of each of the "standard", "positive control", "test sample" and "negative control lacking cinnamon twig" for HPLC measurement. For specific results, see Figure 8 . After comparing the liquid chromatography peaks with those of the standard samples, it was found that at the same detection wavelength, the liquid chromatography of Guizhi control medicinal materials and Wenmiansan showed chromatographic peaks with the same retention time, while in the negative control sample of Guizhi, there was a similar chromatographic peak with a relatively small peak area, and the interference peak was 1.37% calculated by peak area. The results showed that the interference of the negative was very small, and the specificity of this method was excellent.

[0095] B. Linear relationship investigation

[0096] Accurately pipette 0.2, 0.5, 1.0, 1.5, 2.0, and 3.0 mL of the standard respectively, and put them into 10 mL volumetric flasks, fix the volume, and perform high performance liquid chromatography analysis. Under the same experimental conditions, the relative percentage content of samples from different sources was determined, and the linear relationship and standard curve between the components were drawn. Among them, the horizontal axis (x) represents the mass concentration of the standard (μg / mL), and the vertical axis (y) is the integral value of the peak area, which is used to calculate the linear regression equation and its applicable range. The obtained data are plotted into a standard curve and a linear relationship curve diagram, and the difference between the absorbance and sample amount during the determination process of the two methods is compared, and the precision is investigated. The linear result of the determination of cinnamaldehyde is Y=110120X-58370(R 2 =0.9992).

[0097] In addition, the results of methodological tests showed that the RSD in the precision experiment of this method was 1.26%, the RSD in the repeatability experiment was 0.62%, the RSD in the 24h stability experiment was 1.36%, and the average recovery rate in the sample recovery rate experiment was 99.3% and the RSD was 0.61%, indicating that the methodology is feasible.

[0098] C. Sample content determination

[0099] Take 3 portions of the same batch of Wenmiansan and prepare the solution of the sample to be tested by the same method as (2). After the preparation is completed, accurately draw 1 mL of the liquid and put it into a 10 mL volumetric flask and shake it well. Then, extract the filtrate through a 0.22 μm organic microporous filter membrane and accurately draw 10 μL. Then, perform high performance liquid chromatography on the sample to calculate the cinnamaldehyde content in the Wenmiansan. The average content of cinnamaldehyde in the above Wenmiansan was determined to be 150.8 mg / g.

[0100] 2. Determination of imperatorin content

[0101] (1) HPLC conditions

[0102] Chromatographic column: COSMOSIL 5C18-MS-Ⅱ chromatographic column (4.6×250mm); the mobile phase includes an isocratic elution process of water (A) and acetonitrile (B) (48:52); the flow rate is 1 mL per minute; the column temperature is 30°C; the wavelength used for detection is 290 nm; the sample input volume is 10 μL.

[0103] (2) Solution preparation

[0104] A. Preparation of Standard Solution

[0105] Accurately weigh 2.5401 mg of imperatorin, place in a 25 mL volumetric flask, add appropriate amount of methanol to dissolve, dilute to volume, and shake well. Pipette 1 mL of the above imperatorin stock solution, transfer to a 10 mL volumetric flask, add methanol to dissolve, dilute to volume, and shake well to obtain.

[0106] B. Preparation of Control Solution

[0107] Sample to be tested: accurately weigh 1.997 g of the compound powder of Dingwen Miansan, transfer it to a 50 mL volumetric flask, add about 45 mL of methanol, ultrasonicate at 30°C for 30 min, take it out, let it stand, add methanol to the scale, shake it well, filter it through a 0.22 μm organic microporous filter membrane, and take the filtrate to obtain the sample.

[0108] Preparation of negative control solution lacking Angelica dahurica: Accurately weigh 1.5956 g of Angelica dahurica negative control powder (composed of 0.40 g each of scorpion, cinnamon twig, white aconite root, and bombyx batryticatus), transfer it to a 50 mL volumetric flask, add about 45 mL of methanol, ultrasonically treat at 30°C for 30 min, add methanol to make up to volume, shake well, take an appropriate amount of solution, filter through a 0.22 μm organic microporous filter membrane, and take the filtrate to obtain.

[0109] (3) Methodological investigation

[0110] A. Specificity Experiment

[0111] Accurately pipette 20 μL of each standard solution, sample solution to be tested, and negative control solution, and perform HPLC detection to verify the specificity of the method. Fig. 9 The results showed that at the same detection wavelength, the chromatogram of the Wenmiansan compound test solution and the chromatogram of the imperatorin reference solution showed consistent imperatorin chromatographic peaks at the same retention time, and the negative sample solution lacking Angelica dahurica had no corresponding chromatographic peak at the corresponding time. This shows that the negative sample lacking Angelica dahurica has no interference with the detection of the target component, and the above method has good specificity.

[0112] B. Linear relationship experiment

[0113] Accurately pipette 0.2, 0.5, 1, 2, and 3 mL of imperatorin standard respectively, place in a 10 mL volumetric flask, add appropriate amount of methanol to dissolve, fix to volume, shake well, filter, take the filtrate, and prepare a series of reference solutions with imperatorin mass concentrations of 2.012, 5.029, 10.06, 20.12, and 30.18 μg / mL respectively. Accurately pipette 20 μL of each of the above solutions, perform HPLC analysis respectively, calculate the linear regression equation and range, and draw the standard curve. The linear regression equation for the determination of imperatorin is Y=15.709×10 7 X-13501(R 2 =0.9983), and imperatorin showed a good linear relationship with the peak area in the range of 2.012-30.17 μg / mL.

[0114] In addition, the results of methodological tests showed that the RSD of the precision experiment of this method was 1.29%, the RSD of the repeatability experiment was 1.62%, the RSD of the 24h stability experiment was 1.33%, and the average recovery rate of the sample recovery experiment was 101.3% and the RSD was 1.47%, indicating that the methodology is feasible.

[0115] The results of the determination of the content of imperatorin in the Wenmiansan compound showed that the content of imperatorin in the Wenmiansan compound was 15.3 mg / g.

[0116] Obviously, the above embodiments are merely examples for clear explanation and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived from these are still within the protection scope of the invention.

Claims

1. A quality control method for warm noodles, characterized in that: The quality control method is selected from one or more of the following methods: (1) using microscopic identification to identify that the Wenmian powder contains scorpion, silkworm and white aconite; (2) using thin layer chromatography to identify the presence of Angelica dahurica in the Wenmian Powder, wherein the Wenmian Powder is used as a sample to be tested, Angelica dahurica is used as a positive control, and a silica gel G thin layer plate is used for identification; (3) High performance liquid chromatography was used to quantitatively determine the presence of cinnamaldehyde and imperatorin in the Wenmiansan.

2. The quality control method according to claim 1, characterized in that: The microscopic identification method of white aconite is to prepare the warm surface powder into chloral hydrate tablets or water-mounted tablets, and observe under a microscope: the calcium oxalate needle crystals in the white aconite are scattered or bundled in the mucous cells, and the needle crystals are about 97-136μm long; the diameters of the spiral ducts and annular ducts are 9-45μm.

3. The quality control method according to claim 1, characterized in that: The microscopic identification method for Bombyx batryticatus is to prepare the Wenmiansan into chloral hydrate tablets or water-mounted tablets, and observe them under a microscope: the Bombyx batryticatus mycelium is nearly colorless, slender, curled and entangled in the body wall; the tracheal wall fragments are slightly curved or arc-shaped, with brown or dark brown spiral threads; the surface of the epidermal tissue has a grid-like wrinkled texture and small spikes formed by the texture protrusions, with round hair pits and yellow edges; the bristles are yellow or yellow-brown, with a smooth surface and slightly thick walls; the undigested mulberry leaf tissue mostly contains clusters or square crystals of calcium oxalate.

4. The quality control method according to claim 1, characterized in that: The microscopic identification method for the whole scorpion is to prepare the warm surface powder into chloral hydrate tablets or water-mounted tablets, and observe them under a microscope: the surface of the outer epidermis of the whole scorpion body wall fragments presents a polygonal grid-like texture, and the surface is densely covered with fine particles, and hair pits, fine round holes and light brown or nearly colorless tumor-like protrusions can be seen; the inner epidermis is colorless, with horizontal stripes, and the inner and outer epidermis are vertically traversed by many microscopic channels of varying lengths; the striated muscle fibers are mostly broken, the light band is wider than the dark band, there is a dark line in the light band, and the dark band has dense short longitudinal textures.

5. The quality control method according to claim 1, characterized in that: The thin layer chromatography method comprises the following steps: S1, using the extract obtained by ultrasonic extraction of the Wenmian powder with an organic solvent as a test sample; grinding Angelica dahurica into powder and ultrasonically extracting it with an organic solvent as a positive control; S2. The sample to be tested and the positive control are respectively aspirated, and are spotted on a silica gel G thin layer plate, developed in a developing agent, taken out, air-dried, and examined under an ultraviolet lamp; if the chromatogram of the sample to be tested shows spots of the same color at the corresponding positions of the chromatogram of the positive control, then the Wenmiansan contains Angelica dahurica.

6. The quality control method according to claim 5, characterized in that: The developing agent comprises a mixed solution of petroleum ether and ether.

7. The quality control method according to claim 1, characterized in that: The high performance liquid chromatography method for cinnamaldehyde is as follows: the extract obtained by ultrasonic extraction of the Wenmiansan with an organic solvent is used as a sample to be tested, and cinnamaldehyde is dissolved in an organic solvent as a standard; a high performance liquid chromatograph is used to detect the sample to be tested and the standard respectively, and the content of cinnamaldehyde in the sample to be tested is determined.

8. The quality control method according to claim 7, characterized in that: The detection conditions of the high performance liquid chromatograph are: carbon 18 reverse phase silica gel chromatographic column; isocratic elution process of mobile phase including water and acetonitrile; flow rate is 1.0 mL per minute; column temperature is 25-35°C; wavelength for detection is 290-300 nm; sample input volume is 10-20 μL.

9. The quality control method according to claim 1, characterized in that: The high performance liquid chromatography method for imperatorin is as follows: the extract obtained by ultrasonic extraction of Wenmiansan with an organic solvent is used as the sample to be tested, and imperatorin is dissolved in the organic solvent as the standard; the sample to be tested and the standard are tested using a high performance liquid chromatograph to determine the content of imperatorin in the sample to be tested.

10. The quality control method according to claim 9, characterized in that: The detection conditions of the high performance liquid chromatograph are: carbon 18 reverse phase silica gel chromatographic column; isocratic elution process of mobile phase including water and acetonitrile; flow rate is 1.0 mL per minute; column temperature is 25-35°C; wavelength for detection is 290-300 nm; sample input volume is 10-20 μL.

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