Detection method of gastrodia elata naolingling capsule

Abstract

The invention belongs to the technical field of medicine detection, and particularly relates to a detection method of a gastrodia elata Taifengling capsule, the gastrodia elata Taifengling capsule is prepared from gastrodia elata, achyranthes bidentata, radix scrophulariae, rehmannia, angelica sinensis, eucommia ulmoides, ligusticum wallichii, mistletoe, wild chrysanthemum flower and uncaria, and the detection method comprises the following steps: carrying out microscopic identification on the gastrodia elata by adopting a microscopic identification method; carrying out thin-layer chromatography identification on radix scrophulariae, ligusticum wallichii, angelica sinensis and oleanolic acid by adopting a thin-layer chromatography method; and determining the content of gastrodin in the gastrodia elata medicinal material and the content of linarin in the wild chrysanthemum medicinal material by adopting a high performance liquid chromatography. According to the detection method disclosed by the invention, a quality control system of the gastrodia elata naolingling capsule is established, the adopted detection method is scientific and reasonable, high in accuracy and good in reproducibility, the quality of the gastrodia elata naolingling capsule can be comprehensively and effectively controlled, and the clinical curative effect of the product is ensured.

Description

Technical Field

[0001] The present invention belongs to the technical field of drug detection, and particularly relates to a detection method for Tianma Toufengling Capsules. Background Art

[0002] Tianma Toufengling Capsule is a traditional Chinese medicine preparation made from Gastrodia elata, Achyranthes bidentata, Scrophularia ningpoensis, Rehmannia glutinosa, Angelica sinensis, Eucommia ulmoides, Ligusticum chuanxiong, Viscum coloratum, Chrysanthemum indicum, and Uncaria rhynchophylla. In the formula, Gastrodia elata suppresses liver yang and dispels wind and dredges collaterals, and is good at treating headache and dizziness caused by hyperactivity of liver yang; Achyranthes bidentata supplements the liver and kidneys and strengthens the tendons and bones, Scrophularia ningpoensis nourishes yin and clears heat, Ligusticum chuanxiong promotes blood circulation and qi movement, dispels wind and relieves pain, Uncaria rhynchophylla suppresses liver wind and clears heat and relieves convulsion, Chrysanthemum indicum clears the liver and improves eyesight, and clears heat and detoxifies; Rehmannia glutinosa clears heat and cools blood, nourishes yin and promotes fluid production, Angelica sinensis nourishes blood and promotes blood circulation, and Viscum coloratum and Eucommia ulmoides supplement the liver and kidneys and strengthen the tendons and bones. Multiple herbs are used together to nourish yin and suppress yang, dispel wind, and strengthen the tendons and bones, and are used for intractable headache, long-term numbness of hands and feet, and chronic low back and leg pain.

[0003] At present, the quality standard of Tianma Toufengling Capsule is included in the 16th volume of the Drug Standards of Traditional Chinese Medicine Formulas Prepared by the Ministry of Health. In the standard, there are only thin-layer identifications of oleanolic acid and Angelica sinensis; with the development of society, the requirements for the quality control of drugs are becoming stricter and stricter. In order to better control the product quality, the applicant has conducted in-depth research on the detection method of Tianma Toufengling Capsule. Summary of the Invention

[0004] In view of the imperfect detection method in the prior art for Tianma Toufengling Capsule, the present invention provides a detection method for Tianma Toufengling Capsule.

[0005] The detection method for Tianma Toufengling Capsule is to identify Scrophularia ningpoensis and any one or more of Ligusticum chuanxiong, Angelica sinensis, and oleanolic acid by thin-layer chromatography. The identification method for Scrophularia ningpoensis is as follows:

[0006] Take 2 - 3 g of the content of Tianma Toufengling Capsule, grind it finely, add 20 - 30 ml of water, ultrasonically treat for 20 - 30 minutes, filter, extract the filtrate with 30 - 40 ml of ethyl acetate by shaking twice, combine the ethyl acetate extracts, evaporate to dryness, dissolve the residue in 1 ml of methanol to obtain the test solution; take another 0.5 g of the control medicinal material of Scrophularia ningpoensis and prepare the control medicinal material solution in the same way. Then take the harpagoside reference substance and make a solution containing 1 mg per 1 ml with methanol as the reference substance solution. According to the thin-layer chromatography test, absorb 3 - 5 μl of the test solution, the control medicinal material solution, and the reference substance solution respectively, and spot them on the same silica gel G thin-layer plate. Use ethyl acetate: formic acid: water = 8:1:1 as the developing agent, develop, take out, dry in the air, spray with 5% vanillin sulfuric acid solution, heat at 105 °C until the spots are clearly visible, and view under daylight.

[0007] The identification method of Ligusticum chuanxiong is as follows: Take 2 - 3 g of the content of Tianma Toufengling Capsule, grind it finely, add 20 - 30 ml of ether, tightly stopper, ultrasonically treat in an ice - water bath for 30 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 1 ml of chloroform to obtain the test solution; additionally, take 0.2 g of the reference medicinal material of Ligusticum chuanxiong and prepare the reference medicinal material solution in the same method. According to the thin - layer chromatography test, suck 1 - 5 μL of each of the above two solutions and respectively spot them on the same silica gel G thin - layer plate. Use cyclohexane:ethyl acetate = 3:1 as the developing agent, develop, take out, dry in air, and examine under a 365 nm ultraviolet lamp.

[0008] The identification method of Angelica sinensis is as follows: Take 5 g of the content of Tianma Toufengling Capsule, grind it finely, add 30 ml of the mixed solution of chloroform:methanol = 2:1, shake well, soak for 1 hour and then ultrasonically treat for 30 minutes, filter, evaporate the filtrate to dryness, add 1 ml of chloroform to the residue, stir well, and take the supernatant as the test solution; additionally, take 1 g of the reference medicinal material of Angelica sinensis and prepare the reference medicinal material solution in the same method; according to the thin - layer chromatography test, suck 10 μl of the test solution and 1 μl of the reference medicinal material solution, respectively spot them on the same silica gel G thin - layer plate. Use n - hexane:ethyl acetate = 9:1 as the developing agent, develop, take out, dry in air, and examine under a 365 nm ultraviolet lamp;

[0009] The identification method of oleanolic acid is as follows: Take 2 g of the content of Tianma Toufengling Capsule, grind it finely, add 20 ml of ethanol, heat under reflux for 40 minutes, and let it stand; take 10 ml of the supernatant, add 1 ml of hydrochloric acid, heat under reflux for 1 hour and then concentrate to about 5 ml, add 10 ml of water, extract with 20 ml of petroleum ether, evaporate the extract to dryness, dissolve the residue in 1 ml of ethanol to obtain the test solution; additionally, take oleanolic acid reference substance and prepare a solution containing 1 mg per 1 ml with ethanol as the reference substance solution; according to the thin - layer chromatography test, suck 2 μl of each of the above two solutions and respectively spot them on the same silica gel G thin - layer plate. Use chloroform:methanol = 40:1 as the developing agent, develop, take out, dry in air, spray with 5% ethanolic solution of phosphomolybdic acid, bake at 110 °C for about 10 minutes, and examine under daylight.

[0010] Furthermore, the detection method of Tianma Toufengling Capsule also includes the microscopic identification of Gastrodia elata Blume medicinal material. Specifically: Take the content of Tianma Toufengling Capsule and observe it under a microscope: Calcium oxalate raphides occur in bundles or scattered, with a length of 25 - 77 μm, and the diameters of spiral vessels, reticulate vessels, and annular vessels are 8 - 41 μm

[0011] Preferably, the microscopic identification method is as follows: Take a little of the content of Tianma Toufengling Capsule and place it on a glass slide, add distilled water, chloral hydrate test solution, or dilute glycerol test solution, etc. (when necessary, heat for clearing), cover with a cover glass, and observe, measure, and photograph under a simple polarizing microscope in normal light field and polarized - light dark field.

[0012] Furthermore, the detection method of the Tianma Toufengling Capsule also includes a method for determining the content of gastrodin and / or limonene.

[0013] Among them, the method for determining the content of gastrodin is: using octadecylsilane bonded silica gel as a filler; using methanol as mobile phase A and 0.1% phosphoric acid water as mobile phase B, and performing gradient elution; the detection wavelength is 220nm; the column temperature is 30°C; the flow rate is 0.8ml / min; the gradient elution is: 0-15min, mobile phase A1%→7%; 15-16min, mobile phase A7%→90%.

[0014] Preparation of reference solution: Take an appropriate amount of gastrodin reference substance, weigh accurately, and add 70% methanol to make a solution containing 30 μg of gastrodin per 1 ml;

[0015] Preparation of test solution: Take the contents of Tianma Toufengling Capsule, grind it into powder, take about 1g, weigh it accurately, put it in a stoppered conical bottle, add 25ml of 70% methanol accurately, stopper it tightly, weigh it, treat it with ultrasound for 30 minutes, let it cool, weigh it again, make up the lost weight with 70% methanol, shake it well, filter it, and take the filtrate to obtain the solution;

[0016] Determination method: Accurately pipette 5 μl of reference solution and test solution respectively, inject into liquid chromatograph for determination.

[0017] In some embodiments, the method for determining the content of montanol is: using octadecylsilane bonded silica gel as a filler; using acetonitrile as mobile phase A, and 0.1% phosphoric acid water as mobile phase B, for gradient elution; the detection wavelength is 334nm; the column temperature is 30°C, and the flow rate is 1.0ml / min; the gradient elution is: 0-5min, mobile phase A 25%; 5-15min, mobile phase A 25%→27%; 15-20min, mobile phase A 27%→30%.

[0018] Preparation of reference solution: Take an appropriate amount of lignoside reference substance, weigh accurately, and add 70% methanol to make a solution containing 30 μg of lignoside per 1 ml;

[0019] Preparation of test solution: Take the contents of Tianma Toufengling Capsule, grind it into powder, take about 1g, weigh it accurately, put it in a stoppered conical bottle, add 25ml of 70% methanol accurately, stopper it tightly, weigh it, treat it with ultrasound for 30 minutes, let it cool, weigh it again, make up the lost weight with 70% methanol, shake it well, filter it, and take the filtrate to obtain the solution;

[0020] Determination method: Accurately pipette 10 μl of reference solution and test solution respectively, inject into liquid chromatograph and determine.

[0021] In some embodiments, the Tianma Toufengling Capsule is prepared from the following raw materials in parts by weight: 50 parts of Gastrodia elata, 50 parts of Achyranthes bidentata, 50 parts of Scrophularia ningpoensis, 50 parts of Rehmannia glutinosa, 215 parts of Angelica sinensis, 43 parts of Eucommia ulmoides, 50 parts of Ligusticum chuanxiong, 215 parts of Mistletoe, 50 parts of Chrysanthemum indici, and 50 parts of Uncaria rhynchophylla; its preparation method is: among the above ten ingredients, Gastrodia elata is crushed into fine powder, and the remaining nine ingredients including Scrophularia ningpoensis are decocted in water three times, each time for 1.5 to 2 hours, the decoctions are combined, filtered, the filtrate is concentrated under reduced pressure to a thick paste, Gastrodia elata powder is added to mix well, dried, crushed into fine powder or made into granules, and dried to obtain the obtained product.

[0022] Beneficial effects:

[0023] 1. The present invention adds microscopic identification of Gastrodia elata medicinal materials on the basis of the original quality standards, thereby strengthening the quality control of the main drug Gastrodia elata.

[0024] 2. The present invention also adds a thin layer chromatography identification method for Scrophularia ningpoensis and Chuanxiong, which is simple to operate, has strong specificity, good reproducibility, and is beneficial to product quality control.

[0025] 3. The present invention also adds an HPLC method for determining the contents of gastrodin and montmorillonine. This method has high accuracy, strong specificity, good reproducibility, and convenient operation. It can effectively control the quality of Tianma Toufengling Capsules and ensure the clinical efficacy of the product. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Microscopic features of Tianma Tou Feng Ling Capsule (×40) 1. Calcium oxalate needle crystals (a. Features under an optical microscope, b. Features under a polarizing microscope); 2. Catheter

[0027] Figure 2 Figure 1. Investigation of the preparation method of Scrophularia ningpoensis thin layer identification test samples, including 1. Scrophularia ningpoensis negative; 2. Batch 01 sample; 3. Batch 02 sample; 4. Batch 03 sample; 5. Scrophularia ningpoensis reference medicinal material; 6. Harpaoside reference substance

[0028] Figure 3 Figure 1. Investigation of the preparation method of the sample for identification of Chuanxiong TLC, including 1. Negative sample without Chuanxiong; 2. Sample of batch 01; 3. Sample of batch 02; 4. Sample of batch 03; 5. Chuanxiong control medicinal material

[0029] Figure 4 The image of the development agent for the identification of Chuanxiong TLC, where 1. Negative without Chuanxiong; 2. Batch 01 sample; 3. Batch 02 sample; 4. Batch 03 sample; 5. Chuanxiong control medicinal material

[0030] Figure 5 HPLC chromatogram of the suitability experiment of the gastrodin content determination system

[0031] Figure 6 HPLC spectra of linaloside detection at different wavelengths

[0032] Figure 7 System suitability test HPLC chromatogram for determination of buddleoside content Detailed implementation manners

[0033] To make the technical solutions of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are described clearly and completely. The following described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Combining the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts fall within the protection scope of the present invention.

[0034] In a specific embodiment of the present application, a detection method for Tianma Toufengling Capsules is provided, and the identification items include the following:

[0035] (1) Take this product and observe it under a microscope: Calcium oxalate prisms occur in bundles or scattered, 25-77 μm in length, and the diameters of spiral ducts, reticulate ducts and annular ducts are 8-41 μm.

[0036] (2) Take the contents of 10 capsules of this product, grind them finely, add 20 ml of ethanol, heat under reflux for 40 minutes, and let it stand. Take 10 ml of the supernatant, add 1 ml of hydrochloric acid, heat under reflux for 1 hour, then concentrate to about 5 ml, add 10 ml of water, extract with 20 ml of petroleum ether (60-90 °C), evaporate the extract to dryness, dissolve the residue in 1 ml of ethanol to make a test solution. Separately take oleanolic acid reference substance, and make a solution containing 1 mg per 1 ml with ethanol as the reference solution. According to the thin-layer chromatography method (Appendix Ⅵ B in Part I of Chinese Pharmacopoeia 2000 Edition), take 2 μl of each of the above two solutions, spot them on the same silica gel G thin-layer plate respectively, use chloroform-methanol (40:1) as the developing agent, develop, take out, dry in air, spray with 5% ethanolic solution of phosphomolybdic acid, and bake at 110 °C for about 10 minutes. In the chromatogram of the test solution, at the position corresponding to the chromatogram of the reference solution, there are spots showing the same color.

[0037] (3) Take the contents of 25 capsules of this product, grind them finely, add 30 ml of a mixed solution of chloroform and methanol (2:1), shake well, soak for 1 hour, then ultrasonically treat for 30 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 1 ml of chloroform, stir well, and take the supernatant as the test solution. Separately take 1 g of Angelica sinensis reference medicinal material and make a reference medicinal material solution in the same way. According to the thin-layer chromatography method (Appendix Ⅵ B in Part I of Chinese Pharmacopoeia 2000 Edition), take 10 μl of the test solution and 1 μl of the reference medicinal material solution, spot them on the same silica gel G thin-layer plate respectively, use n-hexane-ethyl acetate (9:1) as the developing agent, develop, take out, dry in air, and examine under an ultraviolet lamp (365 nm). In the chromatogram of the test solution, at the position corresponding to the chromatogram of the reference medicinal material, there are two fluorescent spots showing the same color.

[0038] (4) Take 2.5 g of the contents of this product, grind it finely, add 20 ml of water, tightly stopper, ultrasonically treat for 30 minutes, filter, extract the filtrate twice with 40 ml of ethyl acetate by shaking, combine the ethyl acetate extracts, evaporate to dryness, dissolve the residue in 1 ml of methanol to obtain the test solution. Separately take 0.5 g of the control crude drug of Scrophulariae Radix, and prepare the control crude drug solution in the same method. Then take harpagide reference substance, add methanol to make a solution containing 1 mg per 1 ml, which is used as the reference substance solution. According to the thin-layer chromatography method (General Principles 0502) for testing, absorb 5 μl of the test solution and the control crude drug solution respectively, and 3 μl of the reference substance solution, spot them separately on the same silica gel G thin-layer plate to form strips, use ethyl acetate - formic acid - water (8:1:1) as the developing solvent, pre-equilibrate for 20 minutes, develop, take out, air dry, spray with 5% vanillin sulfuric acid solution, heat at 105 °C until the spots are clearly developed, and examine under daylight. In the chromatogram of the test solution, at the positions corresponding to the chromatograms of the control crude drug and the reference substance, there are strip spots of the same color.

[0039] (5) Take 2.5 g of the contents of this product, grind it finely, add 20 ml of ether, tightly stopper, ultrasonically treat in an ice-water bath for 30 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 1 ml of chloroform to obtain the test solution. Separately take 0.2 g of the control crude drug of Chuanxiong Rhizoma, and prepare the control crude drug solution in the same method. According to the thin-layer chromatography method (General Principles 0502) for testing, absorb 1 μL of each of the above two solutions, spot them separately on the same silica gel G thin-layer plate, use cyclohexane - ethyl acetate (3:1) as the developing solvent, pre-equilibrate for 20 minutes, develop, take out, air dry, and examine under an ultraviolet lamp (365 nm). In the chromatogram of the test solution, at the position corresponding to the chromatogram of the control crude drug, there are spots of the same color.

[0040] In addition to the identification method, in some embodiments, there is also a content determination item. Among them, the content determination method of gastrodin is as follows:

[0041] Determine according to the high performance liquid chromatography method (General Principles 0512).

[0042] Chromatographic conditions and system suitability test Use octadecylsilane chemically bonded silica gel as the filler; use methanol as mobile phase A and 0.1% phosphoric acid solution as mobile phase B, and perform gradient elution according to the regulations in the following table; the detection wavelength is 220 nm; the column temperature is 30 °C. The number of theoretical plates calculated based on the gastrodin peak is not less than 6000.

[0043]

[0044] Preparation of the reference substance solution Take an appropriate amount of gastrodin reference substance, accurately weigh it, add 70% methanol to make a solution containing 30 μg of gastrodin per 1 ml, and that is it.

[0045] Preparation of the test solution: Take the content of this product under the difference in filling quantity, grind it finely, take about 1 g, weigh it accurately, place it in a stoppered conical flask, accurately add 25 ml of 70% methanol, stopper it tightly, weigh it, ultrasonically treat it (power 250 W, frequency 40 kHz) for 30 minutes, let it cool, weigh it again, make up the lost weight with 70% methanol, shake well, filter, and take the subsequent filtrate, that is obtained.

[0046] Assay method: Accurately pipette 5 μl each of the reference solution and the test solution, inject them into the liquid chromatograph, and determine, that is obtained.

[0047] In some embodiments, it also includes the method for determining the content of luteoloside, and the specific method is as follows:

[0048] Determine according to the high performance liquid chromatography method (General Principles 0512).

[0049] Chromatographic conditions and system suitability test: Use octadecylsilane chemically bonded silica as the filler; use acetonitrile as mobile phase A and 0.1% phosphoric acid solution as mobile phase B, and carry out gradient elution according to the regulations in the following table; the detection wavelength is 334 nm; the column temperature is 30 °C. The number of theoretical plates calculated based on the luteoloside peak is not less than 10000.

[0050]

[0051] Preparation of the reference solution: Take an appropriate amount of luteoloside reference substance, weigh it accurately, and make a solution containing 30 μg of luteoloside per 1 ml with 70% methanol, that is obtained.

[0052] Preparation of the test solution: Take the content of this product under the difference in filling quantity, grind it finely, take about 1 g, weigh it accurately, place it in a stoppered conical flask, accurately add 25 ml of 70% methanol, stopper it tightly, weigh it, ultrasonically treat it (power 250 W, frequency 40 kHz) for 30 minutes, let it cool, weigh it again, make up the lost weight with 70% methanol, shake well, filter, and take the subsequent filtrate, that is obtained.

[0053] Assay method: Accurately pipette 10 μl each of the reference solution and the test solution, inject them into the liquid chromatograph, and determine, that is obtained.

[0054] The technical solutions provided by the present invention will be described in detail below in conjunction with specific embodiments.

[0055] In the examples, all the original reagent materials can be obtained commercially. The experimental methods without specific conditions are the conventional methods and conventional conditions well known in the field, or the conditions recommended by the instrument manufacturer.

[0056] Apparatus:

[0057]

[0058] Drugs and reagents: chromatographic grade methanol, chromatographic grade acetonitrile (Merck Millipore, Germany); Ligusticum chuanxiong (120918 - 201813), Scrophularia ningpoensis (121008 - 202110), harpagide (111730 - 202110) were all purchased from the National Institutes for Food and Drug Control, China.

[0059] Example 1 Preparation method of Tianma Toufengling Capsules

[0060] Weigh 50 g of Gastrodia elata and crush it into fine powder. Then weigh 50 g of Achyranthes bidentata, 50 g of Scrophularia ningpoensis, 50 g of Rehmannia glutinosa, 215 g of Angelica sinensis, 43 g of Eucommia ulmoides, 50 g of Ligusticum chuanxiong, 215 g of Taxillus chinensis, 50 g of Chrysanthemum indicum, and 50 g of Uncaria rhynchophylla. Decoct them with water three times, each time for 2 hours. Combine the decoctions, filter, concentrate the filtrate under reduced pressure to a thick paste, add the Gastrodia elata powder and mix well, dry, crush into fine powder, and fill into capsules to obtain the product.

[0061] Example 2 Detection method of Tianma Toufengling Capsules

[0062]

Appearance

[0063]

Identification

[0064] (2) Take 2 g of the content of this product, grind it finely, add 20 ml of ethanol, heat under reflux for 40 minutes, and let it stand. Take 10 ml of the supernatant, add 1 ml of hydrochloric acid, heat under reflux for 1 hour, then concentrate to about 5 ml, add 10 ml of water, extract with 20 ml of petroleum ether (60 - 90 °C), evaporate the extract to dryness, dissolve the residue in 1 ml of ethanol to obtain the test solution. Take oleanolic acid reference substance, dissolve it in ethanol to make a solution containing 1 mg per 1 ml as the reference solution. According to the thin-layer chromatography method (Appendix Ⅵ B, Volume I, Chinese Pharmacopoeia 2000 Edition), absorb 2 μl of each of the above two solutions and spot them on the same silica gel G thin-layer plate. Use chloroform - methanol (40:1) as the developing agent, develop, take out, dry, spray with 5% ethanolic solution of phosphomolybdic acid, and bake at 110 °C for about 10 minutes. In the chromatogram of the test solution, at the position corresponding to the chromatogram of the reference solution, there are spots of the same color.

[0065] (3) Take 5 g of the content of this product, grind it finely, add 30 ml of a mixed solution of chloroform and methanol (2:1), shake well, soak for 1 hour, then ultrasonically treat for 30 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 1 ml of chloroform, stir well, and take the supernatant as the test solution. Separately take 1 g of the control crude drug Angelica sinensis, and prepare the control crude drug solution in the same method. According to the thin-layer chromatography method (Appendix Ⅵ B in Volume I of the Chinese Pharmacopoeia 2000 Edition), take 10 μl of the test solution and 1 μl of the control crude drug solution, and respectively spot them on the same silica gel G thin-layer plate. Use hexane-ethyl acetate (9:1) as the developing solvent, develop, take out, dry in air, and examine under an ultraviolet lamp (365 nm). In the chromatogram of the test solution, at the corresponding positions to the chromatogram of the control crude drug, two fluorescent spots of the same color appear.

[0066] (4) Take 2.5 g of the content of this product, grind it finely, add 20 ml of water, tightly stopper, ultrasonically treat for 30 minutes, filter, extract the filtrate with 40 ml of ethyl acetate in two portions by shaking, combine the ethyl acetate extracts, evaporate to dryness, dissolve the residue in 1 ml of methanol to obtain the test solution. Separately take 0.5 g of the control crude drug Scrophulariae Radix, and prepare the control crude drug solution in the same method. Then take harpagide reference substance, dissolve it in methanol to prepare a solution containing 1 mg per 1 ml as the reference substance solution. According to the thin-layer chromatography method (General Rule 0502), take 5 μl each of the test solution and the control crude drug solution, and 3 μl of the reference substance solution, and respectively spot them on the same silica gel G thin-layer plate to form strips. Use ethyl acetate-formic acid-water (8:1:1) as the developing solvent, pre-equilibrate for 20 minutes, develop, take out, dry in air, spray with 5% vanillin sulfuric acid solution, heat at 105 °C until the spots are clearly developed, and examine under daylight. In the chromatogram of the test solution, at the corresponding positions to the chromatograms of the control crude drug and the reference substance, strip spots of the same color appear.

[0067] (5) Take 2.5 g of the content of this product, grind it finely, add 20 ml of ether, tightly stopper, ultrasonically treat in an ice-water bath for 30 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 1 ml of chloroform to obtain the test solution. Separately take 0.2 g of the control crude drug Rhizoma Chuanxiong, and prepare the control crude drug solution in the same method. According to the thin-layer chromatography method (General Rule 0502), take 1 μL each of the above two solutions, and respectively spot them on the same silica gel G thin-layer plate. Use cyclohexane-ethyl acetate (3:1) as the developing solvent, pre-equilibrate for 20 minutes, develop, take out, dry in air, and examine under an ultraviolet lamp (365 nm). In the chromatogram of the test solution, at the corresponding positions to the chromatogram of the control crude drug, spots of the same color appear.

[0068]

Examination

[0070]

Content Determination

[0071] Chromatographic conditions and system suitability test: Octadecylsilane bonded silica gel was used as filler; methanol was used as mobile phase A, 0.1% phosphoric acid water was used as mobile phase B, gradient elution was performed according to the provisions in the following table; the detection wavelength was 220nm; the column temperature was 30°C. The theoretical plate number calculated based on the gastrodin peak was not less than 6000.

[0072]

[0073] Preparation of reference solution: Take an appropriate amount of gastrodin reference solution, weigh it accurately, add 70% methanol to make a solution containing 30 μg of gastrodin per 1 ml.

[0074] Preparation of test solution: Take the contents of the product under the item of filling amount difference, grind it into powder, take out about 1 g, weigh it accurately, put it in a stoppered conical flask, accurately add 25 ml of 70% methanol, stopper it tightly, weigh it, treat it with ultrasound (power 250 W, frequency 40 kHz) for 30 minutes, cool it, weigh it again, make up the lost weight with 70% methanol, shake it well, filter it, and take the filtrate to obtain the solution.

[0075] Determination method: Accurately pipette 5μl of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0076] [Content Determination] Menghuaside was determined according to high performance liquid chromatography (General Rule 0512).

[0077] Chromatographic conditions and system suitability test: Octadecylsilane bonded silica gel was used as filler; acetonitrile was used as mobile phase A, 0.1% phosphoric acid water was used as mobile phase B, gradient elution was performed according to the provisions in the following table; the detection wavelength was 334nm; the column temperature was 30°C. The theoretical plate number calculated based on the limonene peak was not less than 10,000.

[0078]

[0079] Preparation of reference solution: Take an appropriate amount of limonene glycoside reference substance, weigh it accurately, and add 70% methanol to prepare a solution containing 30 μg of limonene glycoside per 1 ml.

[0080] Preparation of test solution: Take the contents of the product under the item of filling amount difference, grind it into powder, take out about 1 g, weigh it accurately, put it in a stoppered conical flask, accurately add 25 ml of 70% methanol, stopper it tightly, weigh it, treat it with ultrasound (power 250 W, frequency 40 kHz) for 30 minutes, cool it, weigh it again, make up the lost weight with 70% methanol, shake it well, filter it, and take the filtrate to obtain the solution.

[0081] Determination method: Accurately pipette 10μl of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0082] Example 3 Investigation of Microscopic Identification Method of Gastrodia elata in Tianma Tou Fengling Capsules

[0083] 3.1 Method: Take a small amount of the contents of 9 batches of Tianma Toufengling Capsules and place them on a glass slide. Add distilled water, chloral hydrate test solution or dilute glycerol test solution (heat to permeabilize if necessary), cover with a cover slip, and observe, measure and photograph the normal light field and polarized dark field under a simple polarizing microscope.

[0084] 3.2 Results: The microscopic characteristics of the 9 batches of Tianma Toufengling Capsules were as follows: calcium oxalate needles were bundled or scattered, 21 to 77 μm long, and the diameter of the tubes was 8 to 41 μm ( Figure 1 ).

[0085] Example 4 Study on the Thin-Layer Identification Method of Scrophularia ningpoensis in Tianma Tou Feng Ling Capsule

[0086] 4.1 Investigation of test sample preparation methods

[0087] Method 1: Take 2.5g of the contents of Tianma Toufengling Capsule, grind it into powder, put it in a stoppered conical flask, add 20mL of water, seal it, shake it, ultrasonically treat it in an ice bath for 30 minutes, filter it, extract the filtrate with petroleum ether (60-90℃) (20mL×2), separate the water layer, extract it with chloroform (20mL×2), take the chloroform layer, evaporate it to dryness, add 1mL of methanol to dissolve the residue, and use it as the test solution. Take 0.5g of Scrophularia control medicinal material and 2.5g of Scrophularia negative sample, and prepare Scrophularia control medicinal material solution and Scrophularia negative sample solution in the same way.

[0088] Method 2: Take 2.5g of the contents of Tianma Toufengling Capsule, grind it into powder, put it in a stoppered conical bottle, add 20mL of water, seal it tightly, shake it, ultrasonically treat it in an ice bath for 30 minutes, filter it, extract the filtrate with petroleum ether (60-90℃) (20mL×2), separate the water layer, separate the water layer, evaporate it in a water bath, and dissolve the residue in 1mL of methanol as the test solution. Take 0.5g of Scrophularia control medicinal material and 2.5g of Scrophularia negative sample, and prepare Scrophularia control medicinal material solution and Scrophularia negative sample solution in the same way.

[0089] Method 3: Take 2.5g of the contents of Tianma Toufengling Capsule, grind it into powder, put it in a stoppered conical bottle, add 20mL of water, seal it tightly, shake it, ultrasonically treat it in an ice bath for 30 minutes, filter it, extract the filtrate with ethyl acetate (20mL×2), take the ethyl acetate layer, evaporate it in a water bath, and dissolve the residue in 1mL of methanol as the test solution. Take 0.5g of Scrophularia control medicinal material and 2.5g of Scrophularia negative sample, and prepare Scrophularia control medicinal material solution and Scrophularia negative sample solution in the same way.

[0090] Take the harpaoside reference substance and add methanol to prepare a solution containing 1 mg per 1 mL as the reference substance solution.

[0091] Three batches of Tianma Toufengling Capsules were prepared into test solutions by methods 1, 2, and 3 respectively. According to the thin-layer chromatography method (General Principles 0502), 5 μL of the test solution, the negative solution without Scrophulariae Radix, the solution of Scrophulariae Radix reference medicinal material, and 3 μL of the harpagoside reference substance solution were respectively spotted on the same silica gel G thin-layer plate. Ethyl acetate - formic acid - water (8﹕1﹕1) was used as the developing solvent. The plate was developed in a developing tank pre-equilibrated with the developing solvent for 20 minutes, taken out, dried, sprayed with 5% vanillin sulfuric acid solution, heated at 105 °C until the spots developed clearly, and examined under daylight.

[0092] By comparing different preparation methods of the test samples, it was found that the developing effect of method 3 was better. The main spots of each batch of samples were clear, the separation effect was good, and the Rf value was appropriate. Therefore, method 3 was selected as the preparation method for the test samples. Figure 2 )

[0093] 4.2 Investigation of the developing solvent

[0094] Three batches of Tianma Toufengling Capsules were prepared into test solutions according to method 3 under item 4.1. According to the thin-layer chromatography method (General Principles 0502), 5 μL of the test solution, the negative solution without Scrophulariae Radix, the solution of Scrophulariae Radix reference medicinal material, and 3 μL of the harpagoside reference substance solution were respectively spotted on the same silica gel G thin-layer plate. Petroleum ether - ethyl acetate - water (4﹕1﹕0.5), dichloromethane - formic acid - water (3﹕1﹕0.5), and ethyl acetate - formic acid - water (8﹕1﹕1) were used as the developing solvents. The plate was developed in a developing tank pre-equilibrated with the developing solvent for 20 minutes, taken out, dried, sprayed with 5% vanillin sulfuric acid solution, heated at 105 °C until the spots developed clearly, and examined under daylight.

[0095] The results showed that when petroleum ether - ethyl acetate - water (4﹕1﹕0.5), dichloromethane - formic acid - water (3﹕1﹕0.5), and ethyl acetate - formic acid - water (8﹕1﹕1) were used as the developing solvents, among the three developing conditions, only when the developing solvent was ethyl acetate - formic acid - water (8﹕1﹕1), the spots were clear, the Rf value was relatively appropriate, and the separation effect was good. Therefore, ethyl acetate - formic acid - water (8﹕1﹕1) was selected as the developing solvent for the subsequent experiments.

[0096] 4.3 Investigation of different color development methods

[0097] Taking the test solution under item 4.2, according to the thin-layer chromatography method (General Principles 0502), 5 μL of the test solution, the negative solution without Scrophulariae Radix, the solution of Scrophulariae Radix reference medicinal material, and 3 μL of the harpagoside reference substance solution were respectively spotted on the same silica gel G thin-layer plate. Ethyl acetate - formic acid - water (8﹕1﹕1) was used as the developing solvent. The plate was developed in a developing tank pre-equilibrated with the developing solvent for 20 minutes, taken out, dried, sprayed with 5% vanillin sulfuric acid solution, heated at 105 °C until the spots developed clearly; examined under an ultraviolet lamp (365 nm).

[0098] The results showed that when sprayed with 5% vanillin sulfuric acid solution, the test sample and the control medicinal material solution showed spots of the same color at the corresponding positions, but when placed under ultraviolet light at 365nm, no obvious characteristic spots appeared at the corresponding positions of the test sample. Therefore, it was selected to spray with 5% vanillin sulfuric acid solution, heat at 105℃ until the spots were clearly colored, and then inspected under sunlight.

[0099] 4.4 Temperature and humidity inspection

[0100] Take the test solution under Item 4.2 and test it according to the thin layer chromatography method (General Rule 0502). Pipette 5μL of the test solution, the negative solution lacking Scrophularia ningpoensis, the control medicinal material solution of Scrophularia ningpoensis, and 3μL of the harpaoside reference solution, and spot them on the same silica gel G thin layer plate respectively. Use ethyl acetate-formic acid-water (8:1:1) as the developing solvent. After pre-balancing with the developing solvent for 20 minutes, develop at a humidity of 40% and 80% and a temperature of 10℃ and 25℃, respectively. Take out, dry, spray with 5% vanillin sulfuric acid solution, heat at 105℃ until the spots are clearly colored, and inspect under sunlight.

[0101] The results showed that the temperature and humidity had no significant effect on the separation degree and the clarity of the main spots of the Scrophularia ningpoensis sample. Therefore, the humidity R = 40% and the temperature of 25°C were selected as the development conditions.

[0102] 4.5 Inspection of thin-layer boards from different manufacturers

[0103] Take the test solution under Item 4.2 and test it according to the thin layer chromatography method (General Rule 0502). Pipette 5μL of the test solution, the negative solution lacking Scrophularia ningpoensis, the control medicinal material solution of Scrophularia ningpoensis, and 3μL of the harpaoside reference solution, and spot them on silica gel G thin layer plates from different manufacturers (Yantai Chemical Silica G Plate, Qingdao Ocean Silica G Plate, Qingdao Dingkang Silica G Plate), use ethyl acetate-formic acid-water (8:1:1) as the developing agent, develop, take out, dry, spray with 5% vanillin sulfuric acid solution, heat at 105°C until the spots are clearly colored, and inspect under sunlight.

[0104] The results showed that there was no significant difference in the silicone G thin layer sheets between different manufacturers.

[0105] Finally, take 10 batches of Tianmatoufengling Capsule samples, prepare the test solution, Scrophulariae Radix control medicinal material solution, and Scrophulariae Radix-free negative solution according to the test solution preparation method of the optimized Method 3. Pipette 5 μL of the test solution, Scrophulariae Radix-free negative solution, and Scrophulariae Radix control medicinal material solution, and 3 μL of the harpagoside reference substance solution. At 25 °C and R = 40%, develop with ethyl acetate - formic acid - water (8:1:1) as the developing solvent. Take out, dry in air, spray with 5% vanillin sulfuric acid solution, heat at 105 °C until the spots are clearly developed, and examine under daylight. The results show that this method is stable and reliable, with good chromatographic separation, clear main spots, and no interference from the negative control.

[0106] Study on the TLC Identification Method of Ligustici Chuanxiong Rhizoma in Tianmatoufengling Capsule - Example 5

[0107] 5.1 Investigation on the Test Solution Preparation Method

[0108] Method 1: Take 2.5 g of the content of Tianmatoufengling Capsule sample, grind it finely, place it in a stoppered conical flask, add 20 mL of petroleum ether, stopper tightly, shake well, ultrasonically treat in an ice bath for 30 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 1 mL of chloroform to obtain the test solution. Separately take 0.2 g of Ligustici Chuanxiong Rhizoma control medicinal material and 2.5 g of Ligustici Chuanxiong Rhizoma-free negative sample, and prepare the Ligustici Chuanxiong Rhizoma control medicinal material solution and Ligustici Chuanxiong Rhizoma-negative sample solution in the same way.

[0109] Method 2: Take 2.5 g of the content of Tianmatoufengling Capsule sample, grind it finely, place it in a stoppered conical flask, add 20 mL of ethyl acetate, stopper tightly, shake well, ultrasonically treat in an ice bath for 30 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 1 mL of chloroform to obtain the test solution. Separately take 0.2 g of Ligustici Chuanxiong Rhizoma control medicinal material and 2.5 g of Ligustici Chuanxiong Rhizoma-free negative sample, and prepare the Ligustici Chuanxiong Rhizoma control medicinal material solution and Ligustici Chuanxiong Rhizoma-negative sample solution in the same way.

[0110] Method 3: Take 2.5 g of the content of Tianmatoufengling Capsule sample, grind it finely, place it in a stoppered conical flask, add 20 mL of ether, stopper tightly, shake well, ultrasonically treat in an ice bath for 30 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 1 mL of chloroform to obtain the test solution. Separately take 0.2 g of Ligustici Chuanxiong Rhizoma control medicinal material and 2.5 g of Ligustici Chuanxiong Rhizoma-free negative sample, and prepare the Ligustici Chuanxiong Rhizoma control medicinal material solution and Ligustici Chuanxiong Rhizoma-negative sample solution in the same way.

[0111] Take 3 batches of Tianmatoufengling Capsule and prepare the test solutions respectively using Method 1, 2, and 3. According to the thin-layer chromatography method (General Rule 0502), pipette 1 μL of the test solution, Ligustici Chuanxiong Rhizoma control medicinal material solution, and Ligustici Chuanxiong Rhizoma-free negative solution, and spot them on the same silica gel G thin-layer plate respectively. Use cyclohexane - ethyl acetate (3:1) as the developing solvent, develop in a developing tank pre-equilibrated with the developing solvent for 20 minutes, take out, dry in air, and examine under an ultraviolet lamp (365 nm).

[0112] As a result, by comparing different sample preparation methods, it was found that Method 3 had a better development effect ( Figure 3 ), the main spots were clear, the separation effect was good, and the Rf value was appropriate. Therefore, Method 3 was selected as the sample preparation method for the test samples.

[0113] 5.2 Investigation of the developing solvent

[0114] Three batches of Tianmatoufengling capsules were prepared into test solution according to Method 3 under item 5.1. The test was carried out according to the thin-layer chromatography method (General Principles 0502). 1 μL of each of the above solutions was respectively spotted on the same silica gel G thin-layer plate. Using A: petroleum ether - ethyl acetate (3:1), B: n-hexane - ethyl acetate (3:1), C: cyclohexane - ethyl acetate (3:1) as the developing solvents, the development was carried out in a developing tank pre-equilibrated with the developing solvent for 20 minutes. After taking out, it was dried, and inspected under an ultraviolet lamp (365 nm).

[0115] The results showed that by comparing the three developing conditions of petroleum ether - ethyl acetate (3:1), n-hexane - ethyl acetate (3:1), and cyclohexane - ethyl acetate (3:1), characteristic spots appeared under conditions B and C, and the Rf value under condition C was more appropriate, and the separation effect was good ( Figure 4 ). Therefore, cyclohexane - ethyl acetate (3:1) was selected as the developing solvent.

[0116] 5.3 Investigation of different color development methods

[0117] The test solution under item 5.2 was taken, and the test was carried out according to the thin-layer chromatography method (General Principles 0502). 1 μL of each of the above solutions was respectively spotted on the same silica gel G thin-layer plate. Using cyclohexane - ethyl acetate (3:1) as the developing solvent, the development was carried out in a developing tank pre-equilibrated with the developing solvent for 20 minutes. After taking out, it was dried, sprayed with 5% vanillin sulfuric acid solution, and heated at 105 °C until the spots developed clearly; inspected under an ultraviolet lamp (365 nm).

[0118] The results showed that when sprayed with 5% vanillin sulfuric acid solution, the test samples and the solution of the control crude drug did not show the same color spots at the corresponding positions, while when inspected under an ultraviolet lamp at 365 nm, obvious characteristic spots appeared at the positions corresponding to the test samples. Therefore, inspection under an ultraviolet lamp at 365 nm was selected.

[0119] 5.4 Investigation of temperature and humidity

[0120] The test solution under item 5.2 was taken, and the test was carried out according to the thin-layer chromatography method (General Principles 0502). 1 μL of each of the above solutions was respectively spotted on the same silica gel G thin-layer plate. Using cyclohexane - ethyl acetate (3:1) as the developing solvent, after pre-equilibrating with the developing solvent for 20 minutes, the development was carried out at a humidity of 40% and 80% and a temperature of 10 °C and 25 °C respectively. After taking out, it was dried and inspected under an ultraviolet lamp (365 nm).

[0121] The results showed that temperature and humidity had no obvious effect on the resolution and clarity of the main spots of the Chuanxiong samples. Therefore, a humidity of R = 40% and a temperature of 25 °C were selected as the developing conditions.

[0122] 5.5 Investigation of TLC plates from different manufacturers

[0123] Take the test solution under item 5.2, and conduct the test according to the thin-layer chromatography method (General Rule 0502). Pipette 1 μL of each of the above solutions and spot them on silica gel G TLC plates from different manufacturers (Yantai Chemical Industry Silica Gel G Plate, Qingdao Ocean Silica Gel G Plate, Qingdao Dingkang Silica Gel G Plate) respectively. Use cyclohexane-ethyl acetate (3:1) as the developing solvent, develop, take out, air dry, and examine under an ultraviolet lamp (365 nm).

[0124] The results showed that there were no significant differences among the silica gel G TLC plates from different manufacturers.

[0125] Finally, take 10 batches of Tianmatoufengling capsules samples, prepare the test solution, the reference medicinal material solution of Chuanxiong, and the negative solution without Chuanxiong according to the test solution preparation method of the optimized Method 3. Pipette 1 μL of each of the above solutions, and under the conditions of 25 °C and R = 40%, use cyclohexane-ethyl acetate (3:1) as the developing solvent, develop, take out, air dry, and examine under an ultraviolet lamp (365 nm). The results showed that this method was stable and reliable, with good chromatographic separation, clear main spots, and no interference from the negative control.

[0126] Example 6 Investigation of the content determination method of gastrodin in Tianmatoufengling capsules

[0127] Gastrodia elata is the main medicinal material in Tianmatoufengling capsules, and gastrodin is the main active ingredient in Gastrodia elata. It belongs to the monobenzyl class of compounds and has various pharmacological effects such as sedation, analgesia, and blood pressure lowering. Previous studies found that the content of gastrodin was relatively high, with good peak shape and resolution, and strong specificity. Therefore, gastrodin was selected as the index component for evaluating the content determination of Tianmatoufengling capsules.

[0128] 6.1 Chromatographic conditions

[0129] Use a Waters Symmetry C18 chromatographic column (4.6 * 150 mm, 5 μm); mobile phase: methanol (A) - 0.1% phosphoric acid water (B); column temperature: 30 °C; injection volume: 5 μL; detection wavelength: 220 nm. The elution gradient is shown in Table 1.

[0130] Table 1 Elution gradient program

[0131]

[0132] 6.2 System suitability test

[0133] 5 μL of each of the gastrodin reference solution, the Tianma Tou Feng Ling test solution, and the negative control solution lacking the gastrodia elata medicinal material were injected into the liquid chromatograph and the chromatogram was recorded. Figure 5 ). As can be seen from the figure, the retention time of gastrodin is 12.6 minutes. Under the experimental conditions, the separation degree between gastrodin and other component peaks is greater than 1.5. The chromatogram of the test sample has the same chromatographic peak at the corresponding retention time with the chromatogram of the reference sample, and there is no interference with the negative control, indicating that this method has good specificity.

[0134] 6.3 Investigation of the preparation method of the test solution

[0135] 6.3.1 Investigation of extraction solvent

[0136] Take about 1g of Tianma Tou Fengling Capsule powder, weigh it accurately, put it in a stoppered conical bottle, and accurately add 25mL of 30% methanol, 50% methanol, 70% methanol and methanol respectively, stopper it tightly, weigh it, treat it with ultrasound (power 250W, frequency 40kHz) for 30 minutes, let it cool, weigh it again, make up the lost weight with the corresponding solvent, shake it well, filter it, and take the filtrate as the test solution. Accurately take 5μL of each test solution, inject it into the liquid chromatograph, and determine it according to the chromatographic conditions under "6.1" to calculate the content of gastrodin. The results are shown in Table 2.

[0137] Table 2 Comparison of different extraction solvents

[0138]

[0139] The results showed that when 70% methanol was used as the extraction solvent, the content of gastrodin was higher, so 70% methanol was considered to be selected as the extraction solvent.

[0140] 6.3.2 Investigation of different extraction methods

[0141] Take about 1g of Tianma Tou Fengling Capsule powder, weigh it accurately, put it in a stoppered conical bottle, add 25mL of 70% methanol accurately, stopper it, weigh it, ultrasonically treat it (power 250W, frequency 40kHz), heat it under reflux, shake it for 30min, let it cool, weigh it again, make up the lost weight with 70% methanol, shake it well, filter it, take the filtrate, and use it as the test solution. Accurately take 5μL of each test solution, inject it into the liquid chromatograph, and determine it according to the chromatographic conditions under "6.1" to calculate the content of gastrodin. The results are shown in Table 3.

[0142] Table 3 Comparison of different extraction methods

[0143]

[0144] The results showed that compared with shaking extraction and reflux extraction, the content of gastrodin was higher during ultrasonic extraction, and ultrasonic extraction was selected as the extraction method for Tianma Toufengling Capsules.

[0145] 6.3.3 Investigation of different extraction times

[0146] Take about 1g of Tianma Tou Fengling Capsule powder, weigh it accurately, put it in a stoppered conical bottle, add 25mL of 70% methanol accurately, weigh it, extract it by ultrasonic for 0.25h, 0.5h, 1h, 2h, take it out, cool it, weigh it again, make up the lost weight with 70% methanol, shake it well, filter it, take the filtrate, and use it as the test solution. Accurately take 5μL of each test solution, inject it into the liquid chromatograph, and determine it according to the chromatographic conditions under "6.1" to calculate the content of gastrodin. The results are shown in Table 4.

[0147] Table 4 Comparison of different extraction times

[0148]

[0149] The results showed that after the extraction time was 0.5 h, the content of gastrodin did not change much, indicating that the extraction was complete in 0.5 h. Therefore, the extraction time was considered to be 0.5 h.

[0150] 6.4 Preparation of test solution

[0151] Take about 1 g of the powder of this product, weigh it accurately, put it in a stoppered conical flask, add 25 mL of 70% methanol accurately, weigh it, treat it with ultrasound (power 250 W, frequency 40 kHz) for 30 minutes, let it cool, weigh it again, make up the lost weight with 70% methanol, shake it well, filter it, and take the filtrate as the test solution.

[0152] Example 7 Methodological Verification of the Determination of Gastrodin in Tianma Tou Feng Ling Capsules

[0153] 7.1 Linear Investigation

[0154] Accurately weigh the gastrodin reference substance, add an appropriate amount of 70% methanol to dissolve and dilute to make a solution containing 4.97μg, 9.94μg, 24.86μg, 49.72μg, 99.44μg, and 124.30μg of gastrodin per 1ml; accurately draw 5μL of the reference solution of each concentration, inject it into the liquid chromatograph, and record the chromatographic peak area. Draw a standard curve with the peak area as the ordinate (y) and the injection concentration as the abscissa (x).

[0155] The results showed that the linear regression equation of gastrodin was: y = 2038x + 0.4196, R 2 =0.9998. This indicates that the concentration of gastrodin in the range of 4.97 to 124.30 μg / mL has a good linear relationship with the peak area. The results of substituting the peak area of ​​the measured sample into the standard curve and using the external standard one-point method for calculation are not much different, so the external standard one-point method is used to calculate the content in the main text.

[0156] 7.2 Repeatability Test

[0157] Take 50%, 100%, and 150% (0.5 g, 1.0 g, 1.5 g) of the amounts taken in the preparation method of the test solution under item "6.4" respectively, weigh 3 portions for each weighing, accurately weigh the test substance, prepare the test solution according to the preparation method of the test solution under item "6.4", inject 5 μL, determine, and calculate the content and RSD. The results show that the method has good repeatability within the range of 0.5 - 1.5 g of the test solution taken. See Table 5 for details.

[0158] Table 5 Results of Repeatability Test for Determination of Gastrodin Content in Tianma Toufengling Capsules

[0159]

[0160] 7.3 Intermediate Precision Test

[0161] Prepare 3 portions of the test solution respectively by different experimenters at different times according to the method in "7.2" and determine. The results show that the intermediate precision of the method is good. See Table 6.

[0162] Table 6 Results of Intermediate Precision Test for Determination of Gastrodin Content in Tianma Toufengling Capsules

[0163]

[0164] 7.4 Accuracy

[0165] The accuracy of the method was investigated by the recovery test. Accurately weigh the prepared preparation (gastrodin content: 0.8544 mg / g), accurately add the reference substance according to 50%, 100%, and 150% of the mass number of the reference substance based on the amount of the sample taken, prepare the test solution according to the preparation method of the test solution, determine, and the results show that the average recovery rate is 98.5% - 100.8%, and the RSD is 0.2% - 2.2%. See Table 7.

[0166] Table 7 Results of Recovery Test for Determination of Gastrodin Content in Tianma Toufengling Capsules

[0167]

[0168] 7.5 Durability Investigation

[0169] To investigate the durability of the established analytical method, the test solution was determined respectively with different brands of chromatographic columns, different column temperatures, different flow rates, and different brands of instruments. At the same time, the stability of the test solution placed at room temperature for 24 hours was also investigated.

[0170] 7.5.1 Stability investigation: Take Tianma Toufengling Capsules, prepare the test solution according to the preparation method of the test solution, and inject and analyze at 0, 2, 4, 8, 12, and 24 hours respectively; the average value of the gastrodin peak area is 7.4041, and the RSD is 3.0%, indicating that the test solution is stable within 24 hours.

[0171] 7.5.2 Chromatographic column investigation: Take Tianma Toufengling Capsules, prepare the test solution according to the preparation method of the test solution, and determine the content of gastrodin using three chromatographic columns: 1: ACE Excel 5C18-PFP (ACE, 4.6*150mm, 5μm); 2: Hypesil BDS C18 (Elite, 4.6*250mm, 5μm); 3: Symmetry C18 (Waters, 4.6*150mm, 5μm); the results show that different brands of chromatographic columns have good resolution and durability for gastrodin.

[0172] 7.5.3 Flow rate investigation: Take Tianma Toufengling Capsules, prepare the test solution according to the preparation method of the test solution, and set three flow rates of 0.6 mL / min, 0.8 mL / min, and 1.0 mL / min to determine the content of gastrodin; the results show that when the flow rate is in the range of 0.6 mL / min to 1.0 mL / min, the determination results of gastrodin are not affected much, but the resolution is better at a flow rate of 0.8 mL / min. Therefore, a flow rate of 0.8 mL / min is selected for subsequent experiments.

[0173] 7.5.4 Column temperature investigation: By investigating three temperatures of 25°C, 30°C, and 35°C, the results show that the resolution of each peak is good at 25°C to 35°C, and the durability is good within the range of column temperature change from 25°C to 35°C. According to the peak elution situation of the sample, 30°C is selected as the detection temperature.

[0174] 7.5.5 Investigation of different models of instruments: By investigating the determination using two instruments, Thermo ULmate3000 and Shimadzu LC-2040C 3D, the results show that different brands of instruments have good resolution and durability for gastrodin. Investigation of the determination method of acacetin in Tianma Toufengling Capsules in Example 8

[0175] 8.1 Chromatographic conditions and system suitability test

[0176] 8.1.1 Selection of detection wavelength

[0177] Perform a full wavelength scan on the acacetin reference substance. The results show that acacetin has the maximum absorption near 269 nm and 334 nm; therefore, take a sample and scan at these two wavelengths of 269 nm and 334 nm respectively. The results are as follows: there is no interference at 334 nm. Considering comprehensively, the wavelength of 334 nm is selected as the wavelength for the determination of acacetin in Tianma Toufengling Capsules (Figure 6 )

[0178] 8.1.2 Chromatographic Conditions

[0179] Use an ACE Excel 5C18 - PFP chromatographic column (4.6 * 250 mm, 5 μm); Mobile phase: acetonitrile (A) - 0.1% phosphoric acid in water (B); Column temperature: 30 °C; Injection volume: 10 μL; Detection wavelength: 334 nm. The elution gradient is shown in Table 8.

[0180] Table 8 Elution Gradient Program

[0181]

[0182] 8.1.3 System Suitability Test

[0183] Precisely pipette 10 μL each of the luteoloside reference substance solution, the test solution of Tianma Toufengling Capsule, and the negative control solution without Flos Chrysanthemi Indici into the liquid chromatograph, and record the chromatogram ( Figure 7 ). It can be seen from the figure that the retention time of luteoloside is 15.6 minutes. Under the test conditions, the resolution between luteoloside and other component peaks is greater than 1.5. The test solution chromatogram has the same chromatographic peaks at the retention time corresponding to the reference substance chromatogram, and the negative control shows no interference, indicating that this method has good specificity.

[0184] 8.2 Investigation on the Preparation Method of Test Solution

[0185] 8.2.1 Investigation on Extraction Solvents

[0186] Take about 1 g of the powder of Tianma Toufengling Capsule, accurately weigh it, place it in a stoppered conical flask, accurately add 25 mL each of water, 30% methanol, 50% methanol, 70% methanol, and methanol respectively, stopper tightly, weigh, ultrasonically treat (power 250 W, frequency 40 kHz) for 30 minutes, let it cool, weigh again, make up the lost weight with the corresponding solvent, shake well, filter, and take the subsequent filtrate as the test solution. Precisely pipette 10 μL each of the test solutions, inject them into the liquid chromatograph, and determine the content of luteoloside according to the chromatographic conditions under "8.1.2". The results are shown in Table 9.

[0187] Table 9 Comparison of Different Extraction Solvents

[0188]

[0189] The results show that when 70% methanol is used as the extraction solvent, the content of luteoloside is relatively high. Therefore, 70% methanol is considered as the extraction solvent.

[0190] 8.2.2 Investigation on Different Extraction Methods

[0191] Take about 1 g of Gastrodia Rhizome and Headache and Vertigo Capsule powder, accurately weigh it, place it in a stoppered conical flask, accurately add 25 mL of 70% methanol respectively, stopper it, weigh it, ultrasonically treat (power 250 W, frequency 40 kHz), heat under reflux, and shake and extract for 30 minutes respectively, cool it, weigh it again, make up the lost weight with 70% methanol, shake well, filter, take the subsequent filtrate as the test solution. Accurately absorb 10 μL of each test solution respectively, inject it into the liquid chromatograph, and determine according to the chromatographic conditions under item "8.1.2", and calculate the content of luteolin. The results are shown in Table 10.

[0192] Table 10 Comparison of different extraction methods

[0193]

[0194] The results show that compared with shaking extraction and reflux extraction, the content of luteolin is higher during ultrasonic extraction, so ultrasonic extraction is selected as the extraction method for Gastrodia Rhizome and Headache and Vertigo Capsule.

[0195] 8.2.3 Investigation of different extraction times

[0196] Take about 1 g of Gastrodia Rhizome and Headache and Vertigo Capsule powder, accurately weigh it, place it in a stoppered conical flask, accurately add 25 mL of 70% methanol, weigh it, ultrasonically extract for 0.25 h, 0.5 h, 1 h, and 2 h respectively, take it out, cool it, weigh it again, make up the lost weight with 70% methanol, shake well, filter, take the subsequent filtrate as the test solution. Accurately absorb 10 μL of each test solution respectively, inject it into the liquid chromatograph, and determine according to the chromatographic conditions under item "8.1.2", and calculate the content of luteolin. The results are shown in Table 11.

[0197] Table 11 Comparison of different extraction times

[0198]

[0199] The results show that when the extraction time is 0.5 h, the change in the content of luteolin is not significant, indicating that the extraction is complete in 0.5 h. Therefore, the extraction time of 0.5 h is considered for selection.

[0200] 8.2.4 Preparation of test solution

[0201] Take about 1 g of the powder of this product, accurately weigh it, place it in a stoppered conical flask, accurately add 25 mL of 70% methanol, weigh it, ultrasonically treat (power 250 W, frequency 40 kHz) for 30 minutes, cool it, weigh it again, make up the lost weight with 70% methanol, shake well, filter, take the subsequent filtrate as the test solution.

[0202] Example 9 Methodology verification for the determination of luteolin content in Gastrodia Rhizome and Headache and Vertigo Capsule

[0203] 9.1 Linear investigation

[0204] Accurately weigh the control substance of luteolin, dissolve it with an appropriate amount of 70% methanol and dilute to prepare solutions containing 2.5 μg, 5.0 μg, 12.5 μg, 25.0 μg, 50.0 μg, and 62.5 μg of luteolin per 1 mL; accurately pipette 10 μL of the control substance solutions at each concentration and inject them into the liquid chromatograph, and record the chromatographic peak areas. Taking the peak area as the ordinate (y) and the injection concentration as the abscissa (x), plot the standard curve.

[0205] The results showed that the linear regression equation of luteolin was: y = 0.206x - 0.1465, R2 = 0.9989. It indicated that there was a good linear relationship between the luteolin concentration in the range of 2.5 - 62.5 μg / mL and the peak area. Substituting the measured peak area of the sample into the standard curve for calculation and using the external standard single-point method for calculation, the results were not significantly different. Therefore, the external standard single-point method was used to calculate the content in the main text.

[0206] 9.2 Repeatability test

[0207] Respectively take 50%, 100%, and 150% (0.5 g, 1.0 g, 1.5 g) of the amounts according to the preparation method of the test solution in "8.2.4", weigh 3 portions for each weighing, accurately weigh the test substance, prepare the test solution according to the preparation method of the test solution in "8.2.4", inject 10 μL, determine, calculate the content and RSD. The results showed that the method had good repeatability within the range of 0.5 - 1.5 g of the test solution taken, as shown in Table 12.

[0208] Table 12 Results of the repeatability test for the determination of luteolin content in Tianma Toufengling Capsules

[0209]

[0210] 9.3 Intermediate precision

[0211] Prepare 3 portions of the test solution respectively by different experimenters at different times according to the above method and determine. The results showed that the intermediate precision of the method was good, as shown in Table 13.

[0212] Table 13 Results of the intermediate precision test for the determination of luteolin content in Tianma Toufengling Capsules

[0213]

[0214] 9.4 Accuracy

[0215] The accuracy of the method was investigated by the recovery test. Gastrodia Headache and Wind-Clearing Capsules with determined content (scopolin content: 0.6478 mg / g) were accurately weighed. The reference substance was precisely added in amounts of 50%, 100%, and 150% of the quality of the reference substance corresponding to the amount of the sample taken. The test solution was prepared according to the method for preparing the test solution of the sample and determined. The results showed that the average recovery rate was 98.6% - 99.9%, and the RSD was 0.7% - 2.1% (Table 14).

[0216] Table 14 Results of the recovery test for the determination of scopolin content in Gastrodia Headache and Wind-Clearing Capsules

[0217]

[0218] 9.5 Investigation of durability

[0219] To investigate the durability of the established analytical method, the test solution was determined using chromatographic columns of different brands, different column temperatures, different detection wavelengths, different flow rates, and instruments of different brands. At the same time, the stability of the test solution after being placed at room temperature for 24 hours was also investigated. The results are shown in the following tests.

[0220] 9.5.1 Investigation of the stability of the test solution

[0221] For the same test sample, the test solution was prepared according to the method for preparing the test solution. Samples were injected at 0, 2, 4, 8, 12, and 24 hours. The average value of the scopolin peak area was 5.8002, and the RSD was 2.7%, indicating that the test solution was stable within 24 hours.

[0222] 9.5.2 Investigation of the chromatographic column

[0223] Approximately 1 g of Gastrodia Headache and Wind-Clearing Capsules powder was taken, and the test solution was prepared according to the method for preparing the test solution under item "8.2.4". Three chromatographic columns were used for the analysis of Gastrodia Headache and Wind-Clearing Capsules samples: 1: ACE Excel 5C18-PFP (ACE, 4.6 * 250 mm, 5 μm); 2: Hypesil BDS C18 (Thermo Scientific, 4.6 * 250 mm, 5 μm); 3: XB-C18 (Welch, 4.6 * 250 mm, 5 μm). The results showed that the resolution of scopolin by chromatographic columns of different brands was good, and the durability was good. ACE Excel 5C18-PFP (4.6 * 250 mm, 5 μm) was used for subsequent determination.

[0224] 9.5.3 Investigation of the flow rate

[0225] Take about 1 g of Gastrodia Rhizome and Headache-Relieving Capsule powder, and prepare the test solution according to the preparation method of the test solution under item "8.2.4". Examine three flow rates: 0.8 mL / min, 1.0 mL / min, and 1.2 mL / min. The results show that when the flow rate is in the range of 0.8 mL / min to 1.2 mL / min, the determination result of buddleoside is less affected, the resolution is good, and the durability is good. The subsequent method uses 1.0 mL / min.

[0226] 9.5.4 Column Temperature Investigation

[0227] Take about 1 g of Gastrodia Rhizome and Headache-Relieving Capsule powder, and prepare the test solution according to the preparation method of the test solution under item "8.2.4". Examine three temperatures: 25 °C, 30 °C, and 35 °C. The results show that the resolution of each peak is better at 25 °C to 35 °C, and the durability is good within the range of column temperature change from 25 °C to 35 °C. According to the peak elution situation of the sample, 30 °C is selected as the detection temperature.

[0228] 9.5.5 Comparison of Analysis Results at Different Detection Wavelengths

[0229] Take about 1 g of Gastrodia Rhizome and Headache-Relieving Capsule powder, and prepare the test solution according to the preparation method of the test solution under item "8.2.4". Examine three wavelengths: 329 nm, 334 nm, and 339 nm. The results show that the resolution of each peak is better at 329 nm to 339 nm, and the durability is good within the range of wavelength change from 329 nm to 339 nm. A small change in wavelength has basically no effect on the determination result.

[0230] 9.5.6 Comparison of Analysis Results of Different Instrument Models

[0231] Take about 1 g of Gastrodia Rhizome and Headache-Relieving Capsule powder, and prepare the test solution according to the preparation method of the test solution under item "8.2.4". Examine two instruments: Thermo ULmate3000 and Shimadzu LC-2040C 3D. The results show that the resolution of buddleoside by instruments of different brands is good, and the durability is good. Thermo ULmate3000 is used for determination in subsequent research.

[0232] Example 10 Determination of the Contents of Gastrodin and Buddleoside in Gastrodia Rhizome and Headache-Relieving Capsule

[0233] Take 10 batches of Gastrodia Rhizome and Headache-Relieving Capsule, and prepare the test solution and reference solution according to the determination method of the contents of gastrodin and buddleoside in Example 2, and inject them for analysis respectively. The results are shown in Table 15. As a result, the content of gastrodin is 0.09 - 0.20 mg / capsule, and the content of buddleoside is 0.04 - 0.20 mg / capsule.

[0234] Table 15 Determination Results of the Contents of 10 Batches of Gastrodia Rhizome and Headache-Relieving Capsule (mg / capsule)

[0235]

[0236] The above embodiments are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. A detection method for Tianma Toufengling capsules, wherein the Tianma Toufengling capsules are made from Gastrodia elata, Achyranthes bidentata, Scrophularia ningpoensis, Rehmannia glutinosa, Angelica sinensis, Eucommia ulmoides, Ligusticum chuanxiong, Viscum coloratum, Chrysanthemum indicum, and Uncaria rhynchophylla, and are characterized in that, The detection method is to identify Scrophulariae Radix and any one or more of Ligustici Chuanxiong Rhizoma, Angelicae Sinensis Radix, and oleanolic acid by thin-layer chromatography. The method for identifying Scrophulariae Radix is as follows: Take 2 - 3 g of the content of Tianmatoufengling Capsules, grind it finely, add 20 - 30 ml of water, ultrasonically treat for 20 - 30 minutes, filter, extract the filtrate with 30 - 40 ml of ethyl acetate by shaking twice, combine the ethyl acetate extracts, evaporate to dryness, dissolve the residue in 1 ml of methanol to obtain the test solution. Additionally, take 0.5 g of the control medicinal material of Scrophulariae Radix and prepare the control medicinal material solution in the same way. Further, take the harpagoside reference substance, add methanol to make a solution containing 1 mg per 1 ml as the reference substance solution. According to the thin-layer chromatography test, absorb 3 - 5 μl of each of the test solution, the control medicinal material solution, and the reference substance solution, respectively spot them on the same silica gel G thin-layer plate, use ethyl acetate: formic acid: water = 8:1:1 as the developing agent, develop, take out, dry in air, spray with 5% vanillin sulfuric acid solution, heat at 105 °C until the spots are clearly visible, and examine under daylight.

2. The detection method according to claim 1, characterized in that: The method for identifying Ligustici Chuanxiong Rhizoma is as follows: Take 2 - 3 g of the content of Tianmatoufengling Capsules, grind it finely, add 20 - 30 ml of ether, tightly stopper, ultrasonically treat in an ice-water bath for 30 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 1 ml of chloroform to obtain the test solution. Additionally, take 0.2 g of the control medicinal material of Ligustici Chuanxiong Rhizoma and prepare the control medicinal material solution in the same way. According to the thin-layer chromatography test, absorb 1 - 5 μL of each of the test solution and the control medicinal material solution, respectively spot them on the same silica gel G thin-layer plate, use cyclohexane: ethyl acetate = 3:1 as the developing agent, develop, take out, dry in air, and examine under a 365 nm ultraviolet lamp.

3. The detection method according to claim 1, wherein: The method for identifying Angelicae Sinensis Radix is as follows: Take 5 g of the content of Tianmatoufengling Capsules, grind it finely, add 30 ml of a mixed solution of chloroform: methanol = 2:1, shake, soak for 1 hour, then ultrasonically treat for 30 minutes, filter, evaporate the filtrate to dryness, add 1 ml of chloroform to the residue, stir well, take the supernatant as the test solution. Additionally, take 1 g of the control medicinal material of Angelicae Sinensis Radix and prepare the control medicinal material solution in the same way. According to the thin-layer chromatography test, absorb 10 µl of the test solution and 1 µl of the control medicinal material solution, respectively spot them on the same silica gel G thin-layer plate, use n-hexane: ethyl acetate = 9:1 as the developing agent, develop, take out, dry in air, and examine under a 365 nm ultraviolet lamp.

4. The detection method according to claim 1, characterized in that: The method for identifying oleanolic acid is as follows: Take 2 g of the content of Tianmatoufengling Capsules, grind it finely, add 20 ml of ethanol, heat under reflux for 40 minutes, and let it stand. Take 10 ml of the supernatant, add 1 ml of hydrochloric acid, heat under reflux for 1 hour, then concentrate to about 5 ml, add 10 ml of water, extract with 20 ml of petroleum ether, evaporate the extract to dryness, dissolve the residue in 1 ml of ethanol to obtain the test solution. Additionally, take the oleanolic acid reference substance, add ethanol to make a solution containing 1 mg per 1 ml as the reference substance solution. According to the thin-layer chromatography test, absorb 2 µl of each of the above two solutions, respectively spot them on the same silica gel G thin-layer plate, use chloroform: methanol = 40:1 as the developing agent, develop, take out, dry in air, spray with 5% ethanolic phosphomolybdic acid solution, heat at 110 °C until the spots are clearly visible, and examine under daylight.

5. The detection method according to claim 1, characterized in that: The detection method also includes microscopic identification. For microscopic identification, the content of Tianma Toufengling Capsule is placed under a microscope for observation: calcium oxalate needle crystals are in bundles or scattered, 25-77 μm in length, and the diameters of spiral ducts, reticulate ducts and annular ducts are 8-41 μm.

6. The detection method according to any one of claims 1 or 2, characterized in that: The detection method also includes the method for determining the content of gastrodin and / or buddleoside.

7. The detection method according to claim 6, wherein The method for determining the content of gastrodin is as follows: octadecylsilane chemically bonded silica gel is used as the filler; methanol is used as mobile phase A, and 0.1% phosphoric acid water is used as mobile phase B for gradient elution; the detection wavelength is 220 nm; the column temperature is 30 °C; the flow rate is 0.8 ml / min; the gradient elution is as follows: 0-15 min, mobile phase A 1% → 7%; 15-16 min, mobile phase A 7% → 90%.

8. The detection method according to claim 7, wherein In the detection method: Preparation of reference solution: An appropriate amount of gastrodin reference substance is accurately weighed and dissolved in 70% methanol to prepare a solution containing 30 μg of gastrodin per 1 ml, and that is the reference solution; Preparation of test solution: The content of Tianma Toufengling Capsule is ground finely, 1 g is taken, accurately weighed, placed in a stoppered conical flask, 25 ml of 70% methanol is accurately added, stoppered, weighed, ultrasonically treated for 30 minutes, cooled, weighed again, and the lost weight is made up with 70% methanol, shaken well, filtered, and the subsequent filtrate is taken, and that is the test solution; Determination method: 5 μl of the reference solution and the test solution are respectively accurately pipetted and injected into the liquid chromatograph for determination.

9. The detection method according to claim 6, wherein The method for determining the content of buddleoside is as follows: octadecylsilane chemically bonded silica gel is used as the filler; acetonitrile is used as mobile phase A, and 0.1% phosphoric acid water is used as mobile phase B for gradient elution; the detection wavelength is 334 nm; the column temperature is 30 °C, and the flow rate is 1.0 ml / min; the gradient elution is as follows: 0-5 min, mobile phase A 25%; 5-15 min, mobile phase A 25% → 27%; 15-20 min, mobile phase A 27% → 30%; Preparation of reference solution: An appropriate amount of buddleoside reference substance is accurately weighed and dissolved in 70% methanol to prepare a solution containing 30 μg of buddleoside per 1 ml, and that is the reference solution; Preparation of test solution: The content of Tianma Toufengling Capsule is ground finely, 1 g is taken, accurately weighed, placed in a stoppered conical flask, 25 ml of 70% methanol is accurately added, stoppered, weighed, ultrasonically treated for 30 minutes, cooled, weighed again, and the lost weight is made up with 70% methanol, shaken well, filtered, and the subsequent filtrate is taken, and that is the test solution; Determination method: 10 μl of the reference solution and the test solution are respectively accurately pipetted and injected into the liquid chromatograph for determination.

10. The detection method according to any one of claims 1-9, characterized in that, The Tianma Toufengling Capsule is prepared from the following raw materials in parts by weight: 50 parts of Gastrodia elata, 50 parts of Achyranthes bidentata, 50 parts of Scrophularia ningpoensis, 50 parts of Rehmannia glutinosa, 215 parts of Angelica sinensis, 43 parts of Eucommia ulmoides, 50 parts of Ligusticum chuanxiong, 215 parts of Taxillus chinensis, 50 parts of Chrysanthemum indicum, 50 parts of Uncaria rhynchophylla; for the above ten herbs, Gastrodia elata is pulverized into fine powder, and the other nine herbs including Scrophularia ningpoensis are decocted with water three times, each time for 1.5-2 hours. The decoction liquids are combined, filtered, and the filtrate is concentrated under reduced pressure to a thick paste, mixed with the Gastrodia elata powder, dried, pulverized into fine powder or made into granules, and dried to obtain the product.

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