Quality control method of traditional Chinese medicine composition preparation

Abstract

The invention provides a quality control method of a traditional Chinese medicine composition preparation. A traditional Chinese medicine composition is composed of astragalus membranaceus, atractylodes macrocephala koidz and other components. The quality control method of the traditional Chinese medicine composition comprises an identification method and a content determination method. Through combination of thin layer chromatography identification and high performance liquid chromatography content determination, the quality of the traditional Chinese medicine composition preparation is rapidly and effectively controlled.

Description

Technical Field

[0001] The present invention belongs to the technical field of medicine, and particularly relates to a quality control method for a traditional Chinese medicine composition preparation. Background Art

[0002] Xinqitongbi Tablets (formerly known as Yuping Tongqiao Tablets) are used to replenish qi and strengthen the superficial resistance, invigorate the spleen and open the orifices. It is a traditional Chinese medicine for persistent allergic rhinitis with deficiency of both the lung and spleen. Its effective active ingredients include Astragalus membranaceus, Atractylodes macrocephala, Saposhnikovia divaricata, Magnolia officinalis, Angelica dahurica, Alpinia officinarum, Notopterygium incisum, Paeonia suffruticosa, Cryptotympana pustulata, Prunus mume, and Glycyrrhiza uralensis. Based on the classic prescription Yupingfeng San, this medicine adds pungent-warm herbs to replenish qi and strengthen the superficial resistance, expel wind and dispel cold. At the same time, it is combined with Magnolia officinalis and Angelica dahurica to dispel wind and cold, dredge the nasal orifices, Alpinia officinarum to tonify the lung qi and benefit the spleen and stomach, Notopterygium incisum to expel wind and cold, and Cryptotympana pustulata and Paeonia suffruticosa, which are sweet and cool herbs, to prevent the side effects of consuming qi and injuring body fluids. Prunus mume is sour and astringent to astringe yin and prevent excessive dryness caused by pungent-warm herbs from secretly consuming body fluids. It is suitable for the syndrome of deficiency of both the lung and spleen and invasion of exogenous pathogens. As a pure traditional Chinese medicine preparation, it avoids the side effects caused by western medicine due to long-term medication and ensures the safety of medication.

[0003] The applicant of the present invention applied for a patent for invention named "A Pharmaceutical Composition for Treating Allergic Rhinitis and Its Preparation Method" on August 23, 2021, and requested to protect a traditional Chinese medicine composition with a weight part composition of "10 - 20 parts of Astragalus membranaceus, 5 - 15 parts of Atractylodes macrocephala, 5 - 15 parts of Saposhnikovia divaricata, 5 - 15 parts of Magnolia officinalis, 5 - 15 parts of Angelica dahurica, 5 - 15 parts of Alpinia officinarum, 5 - 15 parts of Notopterygium incisum, 5 - 15 parts of Paeonia suffruticosa, 5 - 15 parts of Cryptotympana pustulata, 5 - 15 parts of Prunus mume, and 4 - 8 parts of Glycyrrhiza uralensis". This patent provides the composition and preparation method of the traditional Chinese medicine composition, but does not provide a quality control method for the traditional Chinese medicine composition preparation. Summary of the Invention

[0004] The purpose of the present invention is to provide a quality control method for the above-mentioned traditional Chinese medicine composition.

[0005] In order to achieve the above purpose, the inventor provides the following technical solutions.

[0006] A quality control method for a traditional Chinese medicine composition preparation, the raw material composition of the traditional Chinese medicine composition by weight includes: 10 - 20 parts of Astragalus membranaceus, 5 - 15 parts of Atractylodes macrocephala, 5 - 15 parts of Saposhnikovia divaricata, 5 - 15 parts of Magnolia officinalis, 5 - 15 parts of Angelica dahurica, 5 - 15 parts of Alpinia officinarum, 5 - 15 parts of Notopterygium incisum, 5 - 15 parts of Paeonia suffruticosa, 5 - 15 parts of Cryptotympana pustulata, 5 - 15 parts of Prunus mume, and 4 - 8 parts of Glycyrrhiza uralensis;

[0007] The quality control method for the traditional Chinese medicine composition includes an identification method and a content determination method.

[0008] The identification method includes any one of the following identification methods (1) to (5):

[0009] (1) A thin-layer chromatography method using magnolia flower as the reference crude drug and cyclohexane-ethyl acetate with a volume ratio of 1:1 as the developing solvent;

[0010] (2) A thin-layer chromatography method using angelica root as the reference crude drug and cyclohexane-ethyl acetate-formic acid with a volume ratio of 1:2:0.1 as the developing solvent;

[0011] (3) A thin-layer chromatography method using cineole as the reference substance and cyclohexane-ethyl acetate with a volume ratio of 12:1 as the developing solvent;

[0012] (4) A thin-layer chromatography method using paeonol as the reference substance and cyclohexane-ethyl acetate with a volume ratio of 3:1 as the developing solvent;

[0013] (5) A thin-layer chromatography method using liquorice root as the reference crude drug and the lower layer solution of chloroform-methanol-water (kept below 10 °C) with a volume ratio of 13:6:2 as the developing solvent;

[0014] The content determination method includes the content determination methods of astragalus root and ledebouriella root, and the contents of astragaloside IV and 5-O-methylvisammioside are determined by high performance liquid chromatography.

[0015] For the quality control method of the above traditional Chinese medicine composition, the identification method in item (1) includes the following steps:

[0016] Take the content of the traditional Chinese medicine composition preparation, dissolve it with methanol, filter, and use the filtrate as the test solution; take another magnolia flower reference crude drug, extract it with methanol, filter, and use the filtrate as the reference crude drug solution; perform the test according to the thin-layer chromatography method. Pipette the test solution and the reference crude drug solution, and spot them on the same silica gel G thin-layer plate respectively. Use cyclohexane-ethyl acetate with a volume ratio of 1:1 as the developing solvent, develop, take out, dry, spray with 10% sulfuric acid ethanol solution, heat until the spots are clearly visible. In the test solution chromatogram, at the position corresponding to the magnolia flower reference crude drug chromatogram, there are spots showing the same color.

[0017] The identification method in item (2) of the above identification method includes the following operating steps:

[0018] Take the content of the traditional Chinese medicine composition preparation, dissolve it with methanol, filter, and use the filtrate as the test solution; take another angelica root reference crude drug, extract it with methanol, filter, and use the filtrate as the reference crude drug solution; perform the test according to the thin-layer chromatography method. Pipette the test solution and the reference crude drug solution, and spot them on the same silica gel G thin-layer plate respectively. Use cyclohexane-ethyl acetate-formic acid with a volume ratio of 1:2:0.1 as the developing solvent, develop, take out, dry, spray with 10% sulfuric acid ethanol solution, heat until the spots are visible, and examine under an ultraviolet lamp (365 nm); in the test solution chromatogram, at the position corresponding to the reference crude drug chromatogram, there are main fluorescent spots showing the same color.

[0019] The identification method in item (3) includes the following operation steps:

[0020] Take the content of the traditional Chinese medicine composition preparation, dissolve it with methanol, filter, and use the filtrate as the test solution; separately take cineole reference substance, dissolve it with methanol to prepare a solution, and use it as the reference solution; according to the thin-layer chromatography test, absorb the test solution and the reference solution, and spot them on the same silica gel G thin-layer plate respectively. Use cyclohexane-ethyl acetate at a ratio of 12:1 as the developing agent, develop, take out, dry in the air, spray with 1% vanillin sulfuric acid solution, and heat until the spots develop color; in the test solution chromatogram, at the position corresponding to the reference solution chromatogram, there are spots showing the same color.

[0021] The identification method in item (4) includes the following operation steps:

[0022] Take the content of the traditional Chinese medicine composition preparation, dissolve it with methanol, filter, and use the filtrate as the test solution; separately take paeonol reference substance, dissolve it with methanol to prepare a solution, and use it as the reference solution; according to the thin-layer chromatography test, absorb the test solution and the reference solution, and spot them on the same silica gel G thin-layer plate respectively. Use cyclohexane-ethyl acetate at a ratio of 3:1 as the developing agent, develop, take out, dry in the air, spray with 5% ferric chloride ethanol solution in hydrochloric acid acidity, and heat until the spots develop color; in the test solution chromatogram, at the position corresponding to the reference solution chromatogram, there are spots showing the same color.

[0023] The identification method in item (5) includes the following operation steps:

[0024] Take the content of the traditional Chinese medicine composition preparation, dissolve it with methanol, filter, and use the filtrate as the test solution; separately take the control crude drug of licorice, extract it with methanol, filter, and use the filtrate as the control crude drug solution; according to the thin-layer chromatography test, absorb the test solution and the control crude drug solution, and spot them on the same silica gel G thin-layer plate respectively. Use the lower layer solution of chloroform-methanol-water at a ratio of 13:6:2 placed below 10°C as the developing agent, develop, take out, dry in the air, spray with 10% sulfuric acid ethanol solution, and heat until the spots develop color; in the test solution chromatogram, at the position corresponding to the control crude drug chromatogram, there are spots showing the same color.

[0025] Preferably, the above identification method includes the following steps:

[0026] The identification method in item (1) includes the following operation steps:

[0027] Take the content of the traditional Chinese medicine composition preparation, dissolve it in methanol, filter, and use the filtrate as the test solution; separately take the control crude drug of Magnolia flower, extract it by ultrasonic treatment with methanol, filter, and use the filtrate as the control crude drug solution; according to the thin layer chromatography test, absorb the test solution and the control crude drug solution, and spot them on the same silica gel G thin layer plate respectively. Use cyclohexane - ethyl acetate (1:1) as the developing agent, develop, take out, dry in air, spray with 10% sulfuric acid ethanol solution, heat at 105 °C until the spots are clearly developed. In the test solution chromatogram, at the position corresponding to the control crude drug chromatogram of Magnolia flower, there is 1 main spot of the same color;

[0028] The identification method in item (2) of the said identification method includes the following operation steps:

[0029] Take the content of the traditional Chinese medicine composition preparation, dissolve it in methanol, filter, and use the filtrate as the test solution; separately take the control crude drug of Angelica dahurica, extract it by ultrasonic treatment with methanol, filter, and use the filtrate as the control crude drug solution; according to the thin layer chromatography test, absorb the test solution and the control crude drug solution, and spot them on the same silica gel G thin layer plate respectively. Use cyclohexane - ethyl acetate - formic acid (1:2:0.1) as the developing agent, develop, take out, dry in air, spray with 10% sulfuric acid ethanol solution, heat at 105 °C until the spots are clearly developed, and examine under an ultraviolet lamp (365 nm); in the test solution chromatogram, at the position corresponding to the control crude drug chromatogram, there is a main fluorescent spot of the same color;

[0030] The identification method in item (3) of the said identification method includes the following operation steps:

[0031] Take the content of the traditional Chinese medicine composition preparation, dissolve it in methanol, filter, and use the filtrate as the test solution; separately take the reference substance of cineole, dissolve it in methanol to prepare a solution, and use it as the reference substance solution; according to the thin layer chromatography test, absorb the test solution and the reference substance solution, and spot them on the same silica gel G thin layer plate respectively. Use cyclohexane - ethyl acetate (12:1) as the developing agent, develop, take out, dry in air, spray with 1% vanillin sulfuric acid solution, heat at 105 °C until the spots are clearly developed; in the test solution chromatogram, at the position corresponding to the reference substance chromatogram, there are spots of the same color;

[0032] The identification method in item (4) of the said identification method includes the following operation steps:

[0033] Take the content of the traditional Chinese medicine composition preparation, dissolve it in methanol, filter, and use the filtrate as the test solution; separately take the reference substance of paeonol, dissolve it in methanol to prepare a solution, and use it as the reference substance solution; according to the thin layer chromatography test, absorb the test solution and the reference substance solution, and spot them on the same silica gel G thin layer plate respectively. Use cyclohexane - ethyl acetate (3:1) as the developing agent, develop, take out, dry in air, spray with 5% ferric chloride ethanol solution with hydrochloric acid acidity, heat until the spots are clearly developed; in the test solution chromatogram, at the position corresponding to the reference substance chromatogram, there are spots of the same color;

[0034] The identification method in item (5) of the above identification method includes the following operation steps:

[0035] Take the content of the traditional Chinese medicine composition preparation, dissolve it with methanol, filter, and use the filtrate as the test solution; take the control medicinal material of licorice, extract it ultrasonically with methanol, filter, and use the filtrate as the control medicinal material solution; according to the thin-layer chromatography test, absorb the test solution and the control medicinal material solution, and spot them on the same silica gel G thin-layer plate respectively. Use the lower layer solution of chloroform-methanol-water (13:6:2) placed below 10 °C as the developing agent, develop, take out, dry in the air, spray with 10% sulfuric acid ethanol solution, and heat at 105 °C until the spots are clearly developed; in the test solution chromatogram, at the position corresponding to the control medicinal material chromatogram, there are spots showing the same color.

[0036] In the above quality control method, the content determination method of astragalus membranaceus includes the following operation steps:

[0037] Chromatographic conditions: The chromatographic column is packed with octadecylsilane-bonded silica gel; the mobile phase is methanol-water (75:25); the flow rate is 1.0 ml / min; evaporative light scattering detector; the column temperature is 30 °C;

[0038] Preparation of internal standard stock solution: Take an appropriate amount of ginsenoside Rb3, weigh it accurately, dissolve it with methanol to make a solution, and you will get it;

[0039] Preparation of astragaloside IV stock solution: Take an appropriate amount of astragaloside IV, weigh it accurately, dissolve it with methanol to make a solution, and you will get it;

[0040] Preparation of standard curve: Accurately measure appropriate amounts of astragaloside IV stock solution and internal standard stock solution respectively, and make 70% methanol solutions containing 38 μg of astragaloside IV and 70 μg of ginsenoside Rb3 per 1 ml and 70% methanol solutions containing 128 μg of astragaloside IV and 70 μg of ginsenoside Rb3 per 1 ml. Accurately absorb them respectively, inject them into the liquid chromatograph, and measure. Use as the ordinate and as the abscissa to draw the standard curve;

[0041] Preparation of test solution: Take the content of the traditional Chinese medicine composition preparation, weigh it accurately, accurately add methanol, tightly stopper, weigh, ultrasonically treat, cool, weigh again, make up the lost weight with methanol, shake well, filter, accurately measure the continuous filtrate, evaporate to dryness in a water bath, add water to the residue, dissolve it in portions into a separatory funnel, extract with water-saturated n-butanol to obtain the n-butanol solution. First wash it with 1% NaOH, then wash it with water saturated with n-butanol. Accurately add the internal standard stock solution to the obtained n-butanol solution, evaporate to dryness, dissolve the residue with 70% methanol, shake well, filter, and you will get it;

[0042] Determination method: Accurately absorb the test solution, inject it into the liquid chromatograph, measure, and calculate by the standard curve method to get it.

[0043] In the above quality control method, the content determination method of Fangfeng includes the following operating steps:

[0044] Chromatographic conditions: The chromatographic column is packed with octadecylsilane chemically bonded silica; the mobile phase is acetonitrile - water (16:84); the flow rate is 1.0 ml / min; the detection wavelength is 254 nm; the column temperature is 25°C;

[0045] Preparation of reference substance solution: Weigh an appropriate amount of 5-O-methylvisammioside reference substance accurately, dissolve it in 60% methanol to make a solution, and that's it;

[0046] Preparation of test solution: Take the content of the traditional Chinese medicine composition preparation, weigh it accurately, add methanol accurately, tightly stopper, weigh, ultrasonically treat, let it cool, weigh again, make up the lost weight with methanol, shake well, filter, accurately measure the subsequent filtrate, load it onto a neutral alumina column, elute with water, collect the eluate, evaporate to dryness, dissolve the residue in water, shake well, filter, and that's it;

[0047] Determination method: Accurately pipette the reference substance solution and the test solution, inject them into the liquid chromatograph, and determine, and that's it.

[0048] In the quality control method provided by the present invention, the preparation dosage form of the traditional Chinese medicine composition is capsule, tablet, pill, oral liquid, granule, injection or powder.

[0049] The operating steps of the preparation process of the tablets include:

[0050] A. Coarsely crush Atractylodes macrocephala and Notopterygium incisum in the formula, and coarsely crush and break the shell of Magnolia liliflora; Weigh Atractylodes macrocephala, Fangfeng, Alpinia officinarum, Notopterygium incisum, and Magnolia liliflora according to the prescription amount, add 6 - 12 times the amount of water, extract the volatile oil by steam distillation for 6 - 10 hours, collect the volatile oil for later use; Filter the aqueous solution after distillation, concentrate it under reduced pressure for later use;

[0051] B. Weigh Astragalus membranaceus, Cryptotympana pustulata, and Glycyrrhiza uralensis according to the prescription amount, decoct with 9 - 13 times the amount of water for three times, each time for 1 - 3 hours, filter, concentrate the extract under reduced pressure, combine it with the concentrated volatile oil solution and concentrate under reduced pressure to a relative density of 1.15 - 1.20 for later use;

[0052] C. Weigh Angelica dahurica and Prunus mume according to the prescription amount, extract with 5 - 8 times the amount of 50 - 80% ethanol twice, the first time for 1 - 3 hours, the second time for 1 - 3 hours, filter, recover the ethanol from the extract under reduced pressure, and concentrate under reduced pressure to a relative density of 1.15 - 1.20 for later use;

[0053] D. Sterilize Cortex Moutan and crush it into fine powder for later use;

[0054] E. Using the fine powder of Cortex Moutan as the base material, granulate, size the granules, and press tablets according to the conventional process to obtain the tablets.

[0055] Preferably, the operation steps of the preparation process of the tablets include:

[0056] A. Coarsely crush Atractylodes macrocephala and Notopterygium incisum in the formula, and coarsely crush and break the shell of Magnolia liliflora; Weigh Atractylodes macrocephala, Saposhnikovia divaricata, Alpinia officinarum, Notopterygium incisum, and Magnolia liliflora according to the prescription amount, add 8-10 times the amount of water, and extract the volatile oil by steam distillation for 8 hours. Collect the volatile oil and reserve it; Filter the aqueous solution after distillation, concentrate it under reduced pressure, and reserve it;

[0057] B. Weigh Astragalus membranaceus, Cryptotympana pustulata, and Glycyrrhiza uralensis according to the prescription amount, decoct with 10-12 times the amount of water for three times, each time for 1 hour, filter, concentrate the extract under reduced pressure, and combine it with the volatile oil extract and concentrate it under reduced pressure to a relative density of 1.15-1.20, and reserve it;

[0058] C. Weigh Angelica dahurica and Prunus mume according to the prescription amount, extract with 6-7 times the amount of 60-70% ethanol twice, the first time for 2 hours and the second time for 1.5 hours, filter, recover the ethanol from the extract under reduced pressure, and concentrate it under reduced pressure to a relative density of 1.15-1.20, and reserve it;

[0059] D. Sterilize Paeonia suffruticosa and pulverize it into fine powder for reserve;

[0060] E. Using the fine powder of Paeonia suffruticosa as the base material, granulate, size the granules, and press tablets according to the conventional process to obtain the tablets.

[0061] In the quality control method provided by the present invention, the raw material composition of the traditional Chinese medicine composition is preferably: 10 parts of Astragalus membranaceus, 15 parts of Atractylodes macrocephala, 5 parts of Saposhnikovia divaricata, 15 parts of Magnolia liliflora, 5 parts of Angelica dahurica, 15 parts of Alpinia officinarum, 5 parts of Notopterygium incisum, 15 parts of Paeonia suffruticosa, 5 parts of Cryptotympana pustulata, 15 parts of Prunus mume, and 4 parts of Glycyrrhiza uralensis.

[0062] The raw material composition of the traditional Chinese medicine composition can also be preferably: 20 parts of Astragalus membranaceus, 5 parts of Atractylodes macrocephala, 15 parts of Saposhnikovia divaricata, 5 parts of Magnolia liliflora, 15 parts of Angelica dahurica, 5 parts of Alpinia officinarum, 15 parts of Notopterygium incisum, 5 parts of Paeonia suffruticosa, 15 parts of Cryptotympana pustulata, 5 parts of Prunus mume, and 8 parts of Glycyrrhiza uralensis.

[0063] The raw material composition of the traditional Chinese medicine composition can also be preferably: 15 parts of Astragalus membranaceus, 10 parts of Atractylodes macrocephala, 10 parts of Saposhnikovia divaricata, 10 parts of Magnolia liliflora, 10 parts of Angelica dahurica, 10 parts of Alpinia officinarum, 10 parts of Notopterygium incisum, 10 parts of Paeonia suffruticosa, 10 parts of Cryptotympana pustulata, 10 parts of Prunus mume, and 6 parts of Glycyrrhiza uralensis.

[0064] The raw material composition of the traditional Chinese medicine composition can also be preferably: 16 parts of Astragalus membranaceus, 8 parts of Atractylodes macrocephala, 8 parts of Saposhnikovia divaricata, 8 parts of Magnolia liliflora, 8 parts of Angelica dahurica, 8 parts of Alpinia officinarum, 8 parts of Notopterygium incisum, 8 parts of Paeonia suffruticosa, 8 parts of Cryptotympana pustulata, 8 parts of Prunus mume, and 5 parts of Glycyrrhiza uralensis.

[0065] The quality control method provided by the present invention realizes the quality control of the traditional Chinese medicine composition of the present invention through thin-layer chromatography identification and high-performance liquid chromatography for content determination. The identification method uses Magnolia flower, Dahurian angelica root, eucalyptol, paeonol, and licorice as reference medicinal materials / reference substances respectively, and in combination with specific developing agents and chromatography conditions, realizes the identification of Magnolia flower, Dahurian angelica root, galangal, moutan bark, and licorice in the traditional Chinese medicine composition of the present invention by thin-layer chromatography. The spots are clear, and the resolution and reproducibility are good. At the same time, the present invention also establishes a high-performance liquid chromatography method for the content determination of astragalus root and divaricate saposhnikovia root, with good specificity, good repeatability, good stability, good durability, high precision and accuracy. The advantages of the quality control method provided by the present invention are also reflected in multiple identification methods. The preparation of all test samples is very simple. Just dissolving with methanol can achieve the purpose of identification; the preparation of all reference substances is also very simple, and it can be completed just by adding methanol for dissolution or extraction. The simple and easy operation method brings great convenience to daily inspection, not only shortening the processing time, but also reducing the operation error, thus realizing the combined use of thin-layer chromatography identification and high-performance liquid chromatography content determination, and finally realizing the rapid and effective control of the quality of the traditional Chinese medicine composition preparation of the present invention. Description of the Drawings

[0066] Figure 1 It is the thin-layer identification chromatogram of Magnolia flower in Example 6. In the figure, the samples from left to right are Magnolia flower reference medicinal material, test sample A2101001, test sample A2101002, test sample A2101003, and negative sample.

[0067] Figure 2 It is the thin-layer identification chromatogram of Dahurian angelica root in Example 6. In the figure, the samples from left to right are Dahurian angelica root reference medicinal material, test sample A2101001, test sample A2101002, test sample A2101003, and negative sample.

[0068] Figure 3 It is the thin-layer identification chromatogram of eucalyptol in Example 6. In the figure, the samples from left to right are eucalyptol reference substance, test sample A2101001, test sample A2101002, test sample A2101003, galangal, and Magnolia flower negative sample.

[0069] Figure 4 It is the thin-layer identification chromatogram of moutan bark in Example 6. In the figure, the samples from left to right are paeonol reference substance, test sample A2101001, test sample A2101002, test sample A2101003, and moutan bark negative sample.

[0070] Figure 5It is the thin-layer identification chromatogram of liquorice in Example 6. In the figure, the samples from left to right are the control crude drug of liquorice, test sample A2101001, test sample A2101002, test sample A2101003, and the negative sample of liquorice.

[0071] Figure 6 It is the HPLC chromatogram of the specificity of astragaloside IV in Example 6.

[0072] Figure 7 It is the standard curve of astragaloside IV in Example 6.

[0073] Figure 8 It is the HPLC chromatogram of the specificity of ledebouriella root in Example 6.

[0074] Figure 9 It is the standard curve of prim-O-glucosylcimifugin in Example 6.

[0075] Figure 10 It is the standard curve of 5-O-methylvisammioside in Example 6. Detailed implementation mode

[0076] The following further elaborates on the content of the present invention in combination with specific embodiments.

[0077] Example 1 Quality inspection of the traditional Chinese medicine composition tablets of the present invention

[0078] Raw material formula:

[0079] Astragalus membranaceus 206g, Atractylodes macrocephala 137g, Ledebouriella seseloides 137g, Magnolia liliflora Desr. 137g, Angelica dahurica (Fisch.) Benth. et Hook. f. ex Franch. et Sav. 137g, Alpinia officinarum Hance 137g, Notopterygium incisum Ting ex H. T. Chang 137g, Paeonia suffruticosa Andr. 137g, Cryptotympana pustulata Fabricius 137g, Prunus mume Sieb. et Zucc. 137g, Glycyrrhiza uralensis Fisch. 82g.

[0080] Preparation of tablets:

[0081] A. Coarsely crush Atractylodes macrocephala and Notopterygium incisum in the formula, and coarsely crush Magnolia liliflora Desr. to break the shell; weigh Atractylodes macrocephala, Ledebouriella seseloides, Alpinia officinarum Hance, Notopterygium incisum, and Magnolia liliflora Desr. according to the prescription amount, add 8 times the amount of water, and extract the volatile oil by steam distillation for 8 hours. Collect the volatile oil and reserve it; filter the aqueous solution after distillation, concentrate it under reduced pressure, and reserve it;

[0082] B. Weigh Astragalus membranaceus, Cryptotympana pustulata Fabricius, and Glycyrrhiza uralensis Fisch. according to the prescription amount, decoct with 11 times the amount of water for three times, each time for 1 hour, filter, concentrate the extract under reduced pressure, and combine it with the concentrated volatile oil solution and concentrate it under reduced pressure to a relative density of 1.15 - 1.20, and reserve it;

[0083] C. Weigh Angelica dahurica (Fisch.) Benth. et Hook. f. ex Franch. et Sav. and Prunus mume Sieb. et Zucc. according to the prescription amount, add 6 times the amount of 60% ethanol, extract twice, each time for 1.5 hours, filter, recover the ethanol from the extract under reduced pressure, and concentrate it under reduced pressure to a relative density of 1.15 - 1.20, and reserve it;

[0084] D. Pulverize the cortex moutan into fine powder, sterilize it, and reserve for use;

[0085] E. Using the fine powder of cortex moutan as the base material, granulate it by conventional process, size the granules, press into 1000 tablets, and coat with film coating to obtain the product.

[0086] Identification:

[0087] (1) Take 10 tablets of this product, remove the coating, grind them finely, add 8 ml of methanol, ultrasonically treat for 30 minutes, filter, and use the filtrate as the test solution. Separately, take 1 g of the control crude drug of magnolia flower (Magnolia biondii Pamp.), add 8 ml of methanol, ultrasonically treat for 30 minutes, filter, and use the filtrate as the control crude drug solution. According to the thin-layer chromatography method (General Principles 0502 of the Chinese Pharmacopoeia 2020 Edition) for testing, absorb 5 - 10 μl of the test solution and 5 μl of the control crude drug solution, respectively spot them on the same silica gel G thin-layer plate, use cyclohexane - ethyl acetate (1:1) as the developing agent, develop, take out, dry, spray with 10% sulfuric acid ethanol solution, and heat at 105 °C until the spots are clearly visible. In the chromatogram of the test solution, at the position corresponding to the chromatogram of the control crude drug of magnolia flower (Magnolia biondii Pamp.), there is 1 main spot of the same color.

[0088] (2) Take the test solution under Identification (1) as the test solution. Separately, take 0.5 g of the control crude drug of angelica dahurica, add 10 ml of methanol, ultrasonically treat for 30 minutes, filter, and use the filtrate as the control crude drug solution. According to the thin-layer chromatography method (General Principles 0502 of the Chinese Pharmacopoeia 2020 Edition) for testing, absorb 5 - 10 μl of the test solution and the control crude drug solution respectively, spot them on the same silica gel G thin-layer plate, use cyclohexane - ethyl acetate - formic acid (1:2:0.1) as the developing agent, develop, take out, dry, spray with 10% sulfuric acid ethanol solution, heat at 105 °C until the spots are clearly visible, and examine under ultraviolet light (365 nm). In the chromatogram of the test solution, at the position corresponding to the chromatogram of the control crude drug, there is a main fluorescent spot of the same color.

[0089] (3) Take the test solution under Identification (1) as the test solution. Separately, take eucalyptol reference substance, add methanol to make a solution containing 2 mg per 1 ml as the reference substance solution. According to the thin-layer chromatography method (General Principles 0502 of the Chinese Pharmacopoeia 2020 Edition) for testing, absorb 5 - 10 μl of the test solution and 3 μl of the reference substance solution respectively, spot them on the same silica gel G thin-layer plate, use cyclohexane - ethyl acetate (12:1) as the developing agent, develop, take out, dry, spray with 1% vanillin sulfuric acid solution, and heat at 105 °C until the spots are clearly visible. In the chromatogram of the test solution, at the position corresponding to the chromatogram of the reference substance, there are spots of the same color.

[0090] (4) Take the test solution under Identification (1) as the test solution. Separately, take paeonol reference substance, dissolve it in methanol to prepare a solution containing 2 mg per 1 ml as the reference solution. Perform the test according to the thin-layer chromatography method (General Principles 0502 of the Chinese Pharmacopoeia 2020 Edition). Pipette 5 - 10 μl of the test solution and 5 μl of the reference solution respectively, and spot them on the same silica gel G thin-layer plate. Use cyclohexane - ethyl acetate (3:1) as the developing solvent, develop, take out, air dry, spray with 5% ferric chloride ethanol solution in hydrochloric acid, and heat until the spots are clearly visible. In the chromatogram of the test solution, at the position corresponding to the chromatogram of the reference substance, spots of the same color appear.

[0091] (5) Take the test solution under Identification (1) as the test solution. Separately, take 1 g of the reference crude drug of Glycyrrhizae Radix et Rhizoma, add 8 ml of methanol, ultrasonically treat for 30 minutes, filter, and use the filtrate as the reference crude drug solution. Perform the test according to the thin-layer chromatography method (General Principles 0502 of the Chinese Pharmacopoeia 2020 Edition). Pipette 5 - 10 μl of the test solution and 5 μl of the reference crude drug solution respectively, and spot them on the same silica gel G thin-layer plate. Use the lower layer solution of chloroform - methanol - water (13:6:2) placed at a temperature below 10℃ as the developing solvent, develop, take out, air dry, spray with 10% sulfuric acid ethanol solution, and heat at 105℃ until the spots are clearly visible. In the chromatogram of the test solution, at the position corresponding to the chromatogram of the reference crude drug, spots of the same color appear.

[0092] Content determination:

[0093] (1) Content determination of Astragali Radix

[0094] Chromatographic conditions and system suitability test: Use octadecylsilane chemically bonded silica gel as the filler; methanol - water (75:25) as the mobile phase; flow rate 1.0 ml / min; evaporative light scattering detector; column temperature: 30℃; the number of theoretical plates calculated based on astragaloside IV should be not less than 4000.

[0095] Preparation of internal standard stock solution: Take an appropriate amount of ginsenoside Rb3 and place it in a volumetric flask, dissolve it in methanol to prepare a methanol solution containing 0.35 mg per 1 ml, that is, obtain the ginsenoside Rb3 internal standard stock solution.

[0096] Preparation of astragaloside IV stock solution: Take an appropriate amount of astragaloside IV and place it in a volumetric flask, dissolve it in methanol to prepare a methanol solution containing 0.32 mg per 1 ml, that is, obtain the astragaloside IV stock solution.

[0097] Preparation of standard curve: Accurately pipette appropriate amounts of astragaloside IV stock solution and internal standard stock solution respectively, prepare 70% methanol solutions containing 38 μg of astragaloside IV and 70 μg of ginsenoside Rb3 per 1 ml and 70% methanol solutions containing 128 μg of astragaloside IV and 70 μg of ginsenoside Rb3 per 1 ml. Accurately pipette 10 μl respectively, inject into the liquid chromatograph, determine, with as the ordinate, with Using it as the abscissa, plot the standard curve.

[0098] Preparation of the test solution: Take this product, remove the film coating, grind it finely, take 2.5 g, weigh accurately, place it in a stoppered conical flask, accurately add 50 ml of methanol, stopper tightly, weigh, ultrasonically treat for 30 minutes (power 250 W, frequency 40 kHz), cool, weigh again, make up the lost weight with methanol, shake well, filter, accurately measure 25 ml of the subsequent filtrate (reserve the remaining filtrate), evaporate to dryness in a water bath, dissolve the residue in 25 ml of water in portions and transfer it to a separating funnel, extract with water-saturated n-butanol 3 times, 25 ml each time, combine the n-butanol layers, first wash with 1% NaOH 3 times, 20 ml each time, then wash with water saturated with n-butanol 3 times, 20 ml each time, accurately add 1 ml of the internal standard stock solution to the n-butanol layer, evaporate to dryness, dissolve the residue in 70% methanol and transfer it to a 5-ml volumetric flask, add 70% methanol to the scale, shake well, filter, and you will get the solution.

[0099] Assay method: Accurately pipette 10 μl of the test solution, inject it into the liquid chromatograph for determination, and calculate by the standard curve method to obtain the result.

[0100] (2) Content determination of Saposhnikovia divaricata

[0101] Chromatographic conditions and system suitability test: Use octadecylsilane chemically bonded silica gel as the filler; acetonitrile-water (16:84) as the mobile phase; flow rate 1.0 ml / min; detection wavelength 254 nm; column temperature: 25 °C; the number of theoretical plates calculated by 5-O-methylvisammioside should be not less than 5000.

[0102] Preparation of the reference solution: Accurately weigh an appropriate amount of 5-O-methylvisammioside reference substance, add 60% methanol to make a solution containing 60 μg per 1 ml, and you will get the solution.

[0103] Preparation of the test solution: Accurately measure 10 ml of the subsequent filtrate reserved in the content determination of Astragalus membranaceus, load it onto a neutral alumina column (100 - 200 mesh, 3 g, inner diameter 1.0 cm), elute with 15 ml of water, collect the eluate, evaporate to dryness, dissolve the residue in water and transfer it to a 5-ml volumetric flask, add water to the scale, shake well, filter, and you will get the solution.

[0104] Assay method: Accurately pipette 10 μl each of the reference solution and the test solution, inject them into the liquid chromatograph for determination, and you will get the result.

[0105] Example 2 Quality inspection of the traditional Chinese medicine composition capsule of the present invention

[0106] Raw material formula:

[0107] Astragalus membranaceus 240 g, Atractylodes macrocephala 120 g, Saposhnikovia divaricata 120 g, Magnolia liliflora 120 g, Angelica dahurica 120 g, Alpinia officinarum 120 g, Notopterygium incisum 120 g, Paeonia suffruticosa 120 g, Cryptotympana pustulata 120 g, Prunus mume 120 g, Glycyrrhiza uralensis 75 g.

[0108] Preparation of capsules:

[0109] A. Coarsely crush Atractylodes macrocephala and Notopterygium incisum in the formula, and coarsely crush and break the shell of Magnolia liliflora; Weigh Atractylodes macrocephala, Saposhnikovia divaricata, Alpinia officinarum, Notopterygium incisum, and Magnolia liliflora according to the prescription amount, add 10 times the amount of water, and extract the volatile oil by steam distillation for 8 hours. Collect the volatile oil for later use; Filter the aqueous solution after distillation, concentrate it under reduced pressure for later use;

[0110] B. Weigh Astragalus membranaceus, Cryptotympana pustulata, and Glycyrrhiza uralensis according to the prescription amount, decoct with 12 times the amount of water for three times, each time for 1 hour, filter, concentrate the extract under reduced pressure, and combine it with the concentrated volatile oil extract and concentrate under reduced pressure to a relative density of 1.15 - 1.20 for later use;

[0111] C. Weigh Angelica dahurica and Prunus mume according to the prescription amount, extract with 7 times the amount of 70% ethanol twice, the first time for 2 hours and the second time for 1.5 hours, filter, recover the ethanol from the extract under reduced pressure, and concentrate it under reduced pressure to a relative density of 1.15 - 1.20 for later use;

[0112] D. Powder Paeonia suffruticosa into fine powder, sterilize it for later use;

[0113] E. Using the fine powder of Paeonia suffruticosa as the base material, granulate and size the granules according to the conventional process, and fill 1000 capsules to obtain the product.

[0114] Identification:

[0115] Same as Example 1.

[0116] Content determination:

[0117] Same as Example 1.

[0118] Example 3 Quality inspection of the traditional Chinese medicine composition granule of the present invention

[0119] Raw material formula:

[0120] Astragalus membranaceus 137 g, Atractylodes macrocephala 205 g, Saposhnikovia divaricata 68 g, Magnolia liliflora 205 g, Angelica dahurica 68 g, Alpinia officinarum 205 g, Notopterygium incisum 68 g, Paeonia suffruticosa 205 g, Cryptotympana pustulata 68 g, Prunus mume 205 g, Glycyrrhiza uralensis 55 g.

[0121] Preparation of granules:

[0122] A. Coarsely crush Atractylodes macrocephala and Notopterygium incisum in the formula, and coarsely crush and break the shell of Magnolia liliflora; Weigh Atractylodes macrocephala, Saposhnikovia divaricata, Alpinia officinarum, Notopterygium incisum, and Magnolia liliflora according to the prescription amount, add 9 times the amount of water, and extract the volatile oil by steam distillation for 8 hours. Collect the volatile oil for later use; Filter the aqueous solution after distillation, concentrate it under reduced pressure for later use;

[0123] B. Weigh the astragalus root, cicada slough, and liquorice according to the prescription amount, add 11 times the amount of water, decoct three times for 1 hour each time, filter, concentrate the extract under reduced pressure, combine it with the concentrated volatile oil solution, and concentrate under reduced pressure to a relative density of 1.15 - 1.20 for standby.

[0124] C. Weigh the dahurian angelica root and smoked plum according to the prescription amount, add 6 times the amount of 70% ethanol, extract twice, 2 hours for the first time and 1.5 hours for the second time, filter, recover the ethanol from the extract under reduced pressure, and concentrate under reduced pressure to a relative density of 1.15 - 1.20 for standby.

[0125] D. Pulverize the cortex moutan into fine powder, sterilize it for standby.

[0126] E. Using the fine powder of cortex moutan as the base material, granulate and size the granules according to the conventional process to make 1000 bags of granules.

[0127] Identification:

[0128] Same as Example 1.

[0129] Content determination:

[0130] Same as Example 1.

[0131] Example 4 Preparation of the traditional Chinese medicine composition pill of the present invention

[0132] Raw material formula:

[0133] Astragalus root 300g, atractylodes macrocephala 75g, ledebouriella divaricata 225g, magnolia flower bud 75g, dahurian angelica root 225g, galangal 75g, notopterygium root 225g, cortex moutan 75g, cicada slough 225g, smoked plum 75g, liquorice 120g.

[0134] Preparation of pills:

[0135] A. Coarsely crush the atractylodes macrocephala and notopterygium root in the formula, and coarsely crush the magnolia flower bud to break the shell; weigh the atractylodes macrocephala, ledebouriella divaricata, galangal, notopterygium root, and magnolia flower bud according to the prescription amount, add 10 times the amount of water, and extract the volatile oil by steam distillation for 8 hours, collect the volatile oil for standby; filter the aqueous solution after distillation, concentrate it under reduced pressure for standby.

[0136] B. Weigh the astragalus root, cicada slough, and liquorice according to the prescription amount, add 10 times the amount of water, decoct three times for 1 hour each time, filter, concentrate the extract under reduced pressure, combine it with the concentrated volatile oil solution, and concentrate under reduced pressure to a relative density of 1.15 - 1.20 for standby.

[0137] C. Weigh the dahurian angelica root and smoked plum according to the prescription amount, add 6 times the amount of 60% ethanol, extract twice, 2 hours for the first time and 1.5 hours for the second time, filter, recover the ethanol from the extract under reduced pressure, and concentrate under reduced pressure to a relative density of 1.15 - 1.20 for standby.

[0138] D. Pulverize the cortex moutan into fine powder, sterilize it, and reserve for later use;

[0139] E. Using the fine powder of cortex moutan as the base material, prepare 1000 pills according to the conventional process.

[0140] Identification:

[0141] Same as Example 1.

[0142] Content determination:

[0143] Same as Example 1.

[0144] Example 5 Quality inspection of the powder of the traditional Chinese medicine composition of the present invention

[0145] Raw material formula:

[0146] Astragalus membranaceus 180g, Atractylodes macrocephala 90g, Saposhnikovia divaricata 90g, Magnolia liliflora 90g, Angelica dahurica 90g, Alpinia officinarum 90g, Notopterygium incisum 90g, Cortex moutan 90g, Cryptotympana pustulata 90g, Fructus mume 90g, Licorice 60g.

[0147] Preparation of the powder:

[0148] A. Coarsely crush the Atractylodes macrocephala and Notopterygium incisum in the formula, and coarsely crush the Magnolia liliflora to break the shell; Weigh the Atractylodes macrocephala, Saposhnikovia divaricata, Alpinia officinarum, Notopterygium incisum, and Magnolia liliflora according to the prescription amount, add 10 times the amount of water, and extract the volatile oil by steam distillation for 8 hours. Collect the volatile oil and reserve for later use; Filter the aqueous solution after distillation, concentrate it under reduced pressure, and reserve for later use;

[0149] B. Weigh the Astragalus membranaceus, Cryptotympana pustulata, and Licorice according to the prescription amount, decoct with 10 times the amount of water three times, each time for 1 hour, filter, concentrate the extract under reduced pressure, and combine it with the concentrated volatile oil extract and concentrate under reduced pressure to a relative density of 1.15 - 1.20, and reserve for later use;

[0150] C. Weigh the Angelica dahurica and Fructus mume according to the prescription amount, extract with 7 times the amount of 70% ethanol twice, the first time for 2 hours and the second time for 1.5 hours, filter, recover the ethanol from the extract under reduced pressure, and concentrate it under reduced pressure to a relative density of 1.15 - 1.20, and reserve for later use;

[0151] D. Pulverize the cortex moutan into fine powder, sterilize it, and reserve for later use;

[0152] E. Using the fine powder of cortex moutan as the base material, prepare the powder according to the conventional process.

[0153] Identification:

[0154] Same as Example 1.

[0155] Content determination:

[0156] Same as Example 1.

[0157] Example 6

[0158] During the research process, the inventor investigated the quality control method of the traditional Chinese medicine composition preparation of the present invention, which is as follows.

[0159] (I) Identification

[0160] 1. Materials, Instruments and Reagents

[0161] The test samples were the traditional Chinese medicine composition tablets prepared according to Example 1, with batch numbers 080401, 081101, 081102, 081103, A2101001, A2101002, A2101003.

[0162] KQ-250 type ultrasonic cleaner

[0163] AT201 METTLER TOLEDO 1 / 100,000 analytical balance

[0164] 2. Identification Tests

[0165] (1) Thin-layer chromatography (TLC) identification of Magnoliae Flos. The sample extracted by ultrasonic extraction with methanol was used as the test sample, and chloroform-ether (5:1) was used as the developing agent. After development, the spots at the corresponding positions of the test sample and the control crude drug were not very clear. Then, cyclohexane-ethyl acetate (3:1) was used as the developing agent for development, and 1% vanillin sulfuric acid solution was used as the color-developing agent. As a result, the spots were clearly colored, but the Rf value was on the low side. The developing agent was adjusted to cyclohexane-ethyl acetate (1:1), and the Rf value was moderate, and the spots were clear, but there was interference from the negative control. Then, 10% sulfuric acid ethanol solution was used for color development. The spots were clearly colored, and the Rf value was moderate, and there was no interference from the negative control. After testing three batches of samples, the repeatability was good, so it was included in the control method. (The TLC chromatogram is shown in the appendix Figure 1 ).

[0166] (2) TLC identification of Angelicae Dahuricae Radix. The research was carried out with reference to the identification method under Angelicae Dahuricae Radix in Chinese Pharmacopoeia (2005 Edition). The sample extracted by ultrasonic extraction with methanol was used as the test sample, and petroleum ether (30-60°C)-ether (3:2) was used as the developing agent. Development was carried out below 25°C, taken out, dried, and examined under an ultraviolet lamp (365 nm). As a result, the spots on the test sample chromatogram at the corresponding positions of the reference substance chromatogram were not clearly colored.

[0167] Subsequently, a re-study on the identification of Angelica dahurica was carried out. The sample extracted by ultrasonic extraction with methanol was used as the test sample, and petroleum ether (30 - 60°C) - ether (3:2) was used as the developing agent, and it was inspected under an ultraviolet lamp (365 nm). As a result, the spots on the test sample chromatogram at the corresponding positions of the reference crude drug chromatogram were not clearly colored, and there was interference from the negative control; later, using the reference crude drug of Angelica dahurica, imperatorin, and isoimperatorin as references, cyclohexane - ethyl acetate (8:2) and cyclohexane - ethyl acetate (7:4) were used as the developing agents. After development, it was directly inspected under an ultraviolet lamp (365 nm or 254 nm). As a result, in the test sample chromatogram, two spots of the same color appeared at the corresponding positions of the reference crude drug chromatogram, which were isoimperatorin and imperatorin respectively. Among them, there was interference from the negative control for isoimperatorin, and the spot of imperatorin was not clear. Later, the proportion of the developing agent and the coloring method were adjusted. Cyclohexane - ethyl acetate (4:7) and cyclohexane - ethyl acetate (1:3) were tried as the developing agents. After development, it was directly inspected under an ultraviolet lamp (254 nm) or sprayed with 10% sulfuric acid ethanol and heated at 105°C until the spots were clear, and then inspected under an ultraviolet lamp (365 nm). As a result, after spraying with 10% sulfuric acid ethanol and coloring, when inspected under an ultraviolet lamp (365 nm), the same spots appeared, and there was no interference from the negative control, but the Rf value was small. Later, the proportion of the developing agent was adjusted to cyclohexane - ethyl acetate - formic acid (1:2:0.1). Using the reference crude drug of Angelica dahurica as the reference, after development and coloring, in the test sample chromatogram, at the corresponding positions of the reference crude drug chromatogram, fluorescent spots of the same color appeared, the spots were clear, the resolution was good, and there was no interference from the negative control. After testing three batches of samples, the repeatability was good, so it was included in the quality standard. (The TLC chromatogram is shown in the appendix Figure 2 )

[0168] (3) Thin layer identification of cineole. Cineole is a volatile component in Alpinia officinarum. The sample extracted by ultrasonic extraction with methanol was used as the test sample, and cyclohexane - ethyl acetate (5:1) was used as the developing agent. After development, it was taken out, dried, sprayed with 1% vanillin sulfuric acid solution, and heated at 105°C until the spots were clearly colored. As a result, the Rf value was too large. Later, the developing agent was adjusted to cyclohexane - ethyl acetate (12:1). As a result, the Rf value was appropriate, the spots were clearly colored, and there was no interference from the negative control. After testing three batches of samples, the repeatability was good, and it was included in the quality standard.

[0169] Subsequently, during the research on the determination method of volatile oil content by gas chromatography, it was found that there were many overlapping chromatographic peaks between Alpinia officinarum Hance and Magnolia liliflora Desr., and both had chromatographic peaks of cineole. Therefore, the specificity of cineole identification was studied. The results showed that when the volatile oil added to the negative sample of Alpinia officinarum Hance was equivalent to that in the finished product, there was no interference in the negative sample of Alpinia officinarum Hance and the specificity was good. When the amount of volatile oil added to the negative sample of Alpinia officinarum Hance was about 2.5 times that in the finished product, there was interference in the negative sample of Alpinia officinarum Hance; the negative sample of Magnolia liliflora Desr. also had interference with cineole; the double negative sample without Alpinia officinarum Hance and Magnolia liliflora Desr. had good specificity and no negative interference, so it was included in the quality standard. (The TLC chromatogram is shown in the appendix Figure 3 ).

[0170] (4) Thin-layer identification of Cortex Moutan. The sample extracted by ultrasonic extraction with methanol was used as the test sample, cyclohexane-ethyl acetate (3:1) was used as the developing agent, developed, taken out, dried, sprayed with 5% ferric trichloride ethanol solution in hydrochloric acid, and heated until the spots were clearly colored. This method is the identification method of Cortex Moutan medicinal materials under the Chinese Pharmacopoeia 2005 edition. The spots at the corresponding positions of the test sample and the reference substance were clearly colored, and there was no negative interference. After testing three batches of samples, the repeatability was good and it was included in the quality standard. (The TLC chromatogram is shown in the appendix Figure 4 ).

[0171] (5) Thin-layer identification of Glycyrrhiza uralensis Fisch. The sample extracted by ultrasonic extraction with methanol was used as the test sample. Chloroform-methanol (5:1) and chloroform-methanol-formic acid (5:1:0.1) were used as the developing agents, developed, first examined under an ultraviolet lamp (254 nm), then sprayed with 10% sulfuric acid ethanol solution, heated at 105 °C until the spots were clearly colored, and then examined under an ultraviolet lamp (365 nm). Saposhnikovia divaricata (Turcz.) Schischk., Glycyrrhiza uralensis Fisch. and Notopterygium incisum Ting ex H. T. Chang were identified simultaneously, but there was interference in the negative sample of Notopterygium incisum Ting ex H. T. Chang. Later, chloroform-methanol (5:1) and chloroform-methanol-formic acid (5:1:0.2) were used as the two developing agents to identify Saposhnikovia divaricata (Turcz.) Schischk. and Glycyrrhiza uralensis Fisch. simultaneously. The results showed that the chloroform-methanol-formic acid (5:1:0.2) developing system had better separation, clear spot color development, moderate Rf value, and no negative interference. After testing three batches of samples, the repeatability was good and it was included in the quality standard.

[0172] During the implementation of the standard, it was found that the main spot of Saposhnikovia divaricata (Turcz.) Schischk. was not clear and the method reproducibility was poor. Considering that there was already a determination item for the content of Saposhnikovia divaricata (Turcz.) Schischk. in the standard, the thin-layer identification method of Saposhnikovia divaricata (Turcz.) Schischk. was deleted. After deleting the identification of Saposhnikovia divaricata (Turcz.) Schischk., there was no need to consider the chromatographic spot situation of Saposhnikovia divaricata (Turcz.) Schischk., so the thin-layer identification method of Glycyrrhiza uralensis Fisch. was optimized.

[0173] The sample extracted by ultrasonic with methanol was used as the test sample, and the control crude drug of licorice was used as the control. Attempts were made to use the lower layer solution of ethyl acetate - formic acid - glacial acetic acid - water (8:1:1:2) and chloroform - methanol - water (13:6:2) placed below 10 °C as the developing agent. After development, it was sprayed with 10% sulfuric acid ethanol solution and heated at 105 °C until the spots were clear for inspection. Under the conditions of the two developing agents, in the test sample chromatogram, main spots of the same color appeared at the corresponding positions of the control crude drug chromatogram. According to the separation effect and the clarity of the main spots, the lower layer solution of chloroform - methanol - water (13:6:2) placed below 10 °C was selected as the developing agent. The specificity test showed that there was no interference from the negative sample. The three batches of samples were detected according to the selected method, and the repeatability was good, so it was included in the quality standard. (The TLC chromatogram is shown in the appendix Figure 5 ).

[0174] (6) TLC identification study was carried out on Saposhnikovia divaricata in the traditional Chinese medicine composition formula. A TLC identification method for simultaneously identifying Saposhnikovia divaricata and licorice on the same TLC plate was established. For details, see the research description of the licorice identification method. However, during the implementation of the method, it was found that the main spot of Saposhnikovia divaricata was not clear and the method reproducibility was poor. Considering that there was already a determination item for the content of Saposhnikovia divaricata in quality control, the TLC identification method for Saposhnikovia divaricata was deleted and not included in the quality standard.

[0175] (7) TLC identification study was carried out on Atractylodes macrocephala in the traditional Chinese medicine composition formula. The identification method under Atractylodes macrocephala in the Chinese Pharmacopoeia 2005 edition was referred to for the study. The sample extracted by ultrasonic with methanol was used as the test sample, and petroleum ether (60 - 90 °C) - ethyl acetate (50:1), petroleum ether (60 - 90 °C) - ethyl acetate (10:1) were used as the developing agents. After development, it was taken out, dried, sprayed with 5% vanillin sulfuric acid solution and heated until the spots were clearly colored. No corresponding spots were found in both cases; using chloroform - ether (5:1) as the developing agent, spraying with 10% sulfuric acid ethanol solution and heating at 105 °C until the spots were clearly colored, no corresponding spots were found either; so it was not included in the quality standard.

[0176] (8) TLC identification study was carried out on Notopterygium incisum in the traditional Chinese medicine composition formula. The sample extracted by ultrasonic with methanol was used as the test sample. Chloroform - methanol (5:1) and chloroform - methanol - formic acid (5:1:0.1) were used as the developing agents. After development, it was sprayed with 10% sulfuric acid ethanol solution and heated at 105 °C until the spots were clearly colored, and then inspected under an ultraviolet lamp (365 nm). The results showed that there was interference from the negative samples in both cases. Later, the developing system and the coloring system were changed to find other characteristic spots. Using chloroform - ether (5:1) as the developing agent, spraying with 10% sulfuric acid ethanol solution and heating at 105 °C until the spots were clearly colored, no characteristic spots were found; using cyclohexane - ethyl acetate (3:1) as the developing agent and 1% vanillin sulfuric acid for coloring, the spots interfered with those of Saposhnikovia divaricata. Therefore, no TLC identification method was established.

[0177] Subsequently, a further study on the identification of Notopterygium incisum was carried out. The sample extracted by ultrasonic treatment with methanol was used as the test sample. Using chloroform - methanol (4:1), ethyl acetate - methanol - water (8:1:1), chloroform - methanol - ammonia water 7:2:0.5 as the developing agents, nodakenin as the control, they were spotted on silica gel G or silica gel G thin - layer plates prepared with 3% sodium acetate solution respectively, and inspected under an ultraviolet lamp (365 nm). The results showed that there were interferences of varying degrees in the negative samples.

[0178] The developing agents and coloring methods were studied. Using nodakenin reference substance as the control, with chloroform - methanol - ammonia water (7:2:0.5), the upper layer solution of ethyl acetate - methanol - water - ammonia water (10:2:3:0.5), ethyl formate - methanol - ammonia water (10:4:0.5) as the developing agents, after development, directly inspected under an ultraviolet lamp (365 nm) or sprayed with 2% sulfuric acid - ethanol solution, heated for coloring and then inspected under an ultraviolet lamp (254 nm or 365 nm). The results showed that in the test sample chromatogram, there were fluorescent spots of the same color at the corresponding positions of the reference substance chromatogram, but there were interferences in the negative samples.

[0179] Subsequently, using the Notopterygium incisum medicinal material as the control, with the upper layer solution of ethyl acetate - methanol - water - ammonia water (10:2:3:0.5) as the developing agent, after development, inspected under an ultraviolet lamp (365 nm) or sprayed with 10% sulfuric acid - ethanol solution, heated for coloring and then inspected under an ultraviolet lamp (365 nm). The results showed that in the test sample chromatogram, no specific and clear spots were found; also, using ethyl formate - methanol - ammonia water (8:2:0.5), ethyl formate - methanol - ammonia water (10:4:0.5) as the developing agents, after spraying with aluminum trichloride solution and inspected under an ultraviolet lamp at 365 nm, or after coloring with 2% sulfuric acid - ethanol and 10% sulfuric acid - ethanol and inspected under an ultraviolet lamp (254 nm, 365 nm), the results showed that there were spots of the same color at the corresponding positions of the chromatogram of the Notopterygium incisum medicinal material, but there were interferences in the negative samples.

[0180] Since the identification method could not be successfully established for the sample extracted by ultrasonic treatment with methanol, the preparation method of the test sample was studied. The test sample solution was prepared by the method of reflux extraction with ammonia - ether mixed solution respectively, or by ultrasonic extraction with ethyl acetate and n - butanol respectively. Using petroleum ether (60 - 90 °C) - ether - triethylamine (3:2:0.2) or the upper layer solution of ethyl acetate - methanol - water - ammonia water (10:2:3:0.5) as the developing agent, and coloring with 10% sulfuric acid - ethanol, no characteristic spots were found in the results, so it was not included in the quality standard.

[0181] (9) TLC identification study was carried out on Fructus Mume in the traditional Chinese medicine composition. The identification method under Fructus Mume in Chinese Pharmacopoeia (2005 Edition) was referred to for the study. The sample extracted by ultrasonic with methanol was used as the test sample, and cyclohexane - chloroform - ethyl acetate - formic acid (20:5:8:0.1) was used as the developing agent. After development, when the spotting volume of the test sample solution increased to 15 μl, the spots at the corresponding positions of the chromatogram of the reference substance and the reference crude drug were still not clear, and the resolution was poor. Using the upper layer solution of butyl acetate - formic acid - water (4:2:2) as the developing agent and 0.1% bromocresol green alcohol solution as the color developing agent, there was a spot in the ethanol solution of the test sample at the position corresponding to the reference substance of citric acid, but the tailing was serious, so it was not included in the quality standard.

[0182] Subsequently, further research on the identification of Fructus Mume was carried out: The sample extracted by ultrasonic with methanol was used as the test sample, and cyclohexane - chloroform - ethyl acetate - formic acid (20:5:8:0.1), cyclohexane - ethyl acetate - formic acid (20:8:0.3), toluene - acetone 5:1 were used as the developing agents for development. Using the reference crude drug of Fructus Mume and ursolic acid as the references, sprayed with 10% sulfuric acid ethanol solution, heated at 105 °C until the spots were clearly developed, and examined under daylight and ultraviolet lamp (365 nm). The results showed that the negatives had interference to varying degrees; developed with toluene - ethyl acetate - glacial acetic acid (12:4:0.5) as the developing agent, sprayed with 10% sulfuric acid ethanol solution, heated at 105 °C until the spots were clearly developed, and examined under daylight. The spots in the chromatogram of the test sample were not clear. Examined under ultraviolet lamp (365 nm), the results showed that the resolution of the spots was poor and the negatives had interference. So it was not included in the quality standard.

[0183] (10) TLC identification study of volatile components. Essential oils were extracted from Saposhnikoviae Radix, Notopterygii Rhizoma, Atractylodis Macrocephalae Rhizoma, Magnoliae Flos, and Alpiniae Officinarum Rhizoma in the traditional Chinese medicine composition. Therefore, the identification method of volatile components was studied using the reference crude drugs of Saposhnikoviae Radix, Notopterygii Rhizoma, Atractylodis Macrocephalae Rhizoma, Magnoliae Flos, and Alpiniae Officinarum Rhizoma as references. The research contents are as follows:

[0184] "Take this product, remove the coating, grind it finely, weigh 5 g, place it in a round-bottomed flask, add 200 ml of water, connect the volatile oil detector, add water from the upper end of the detector to fill the graduated part and overflow into the flask, add 2 ml of n-hexane, connect the reflux condenser, heat to gentle boiling and maintain for 2 hours, cool, and take the n-hexane solution" as the test solution; take the control medicinal materials of Saposhnikovia divaricata, Notopterygium incisum, Atractylodes macrocephala, Magnolia liliflora, and Alpinia officinarum, and prepare the control medicinal material solution in the same way. Spot it on a silica gel GF254 thin-layer plate, and use "cyclohexane-isopropanol (20:1), petroleum ether (60-90°C)-ethyl acetate (5:2), upper layer solution of petroleum ether (60-90°C)-ethyl acetate-formic acid (15:5:2), petroleum ether (60-90°C)-ethyl acetate (10:1), petroleum ether (60-90°C)-ethyl acetate (15:1), chloroform-ethyl acetate (9:1), chloroform-ethyl acetate (15:1), n-hexane-ethyl acetate (7:2), petroleum ether (60-90°C)-ethyl acetate-triethylamine (10:1:0.2)" as the developing agent, develop, spray with 10% sulfuric acid ethanol solution or spray with 5% vanillin sulfuric acid ethanol solution, heat at 105°C until the spots are clearly visible, and examine under daylight or ultraviolet light. No good thin-layer effect was obtained in all cases.

[0185] Adjust the developing agent to "cyclohexane-ethyl acetate-dichloromethane-formic acid (10:1:1:0.2), cyclohexane-ethyl acetate-dichloromethane-formic acid (15:2:2:0.2)", spray with 10% sulfuric acid ethanol solution, heat at 105°C until the spots are clearly visible, or spray with 2% p-dimethylaminobenzaldehyde 40% sulfuric acid ethanol solution, and heat with a hot air blower until the spots are clearly visible. Examine under daylight and ultraviolet light. As a result, the separation degree of the test solution chromatographic spots is poor, and no good thin-layer effect is obtained.

[0186] Subsequently, attempts were made to adjust the developing agent, change the test solution treatment method, replace the silica gel plate, etc. for research, but no ideal thin-layer effect was obtained.

[0187] In the quality control method, there is already a thin-layer identification of cineole in the volatile oil (as described above). Therefore, the thin-layer identification method of cineole is retained in the quality control method, and no other identification methods for volatile components are added.

[0188] (11) For Astragalus membranaceus in the formula of the traditional Chinese medicine composition of the present invention, a corresponding content determination quality standard has been established, so no thin-layer identification method is established.

[0189] (12) Cicada slough mainly contains amino acids. The thin-layer identification method has poor specificity and there is no legal control medicinal material, so it is not included in the quality control method.

[0190] (II) Content determination

[0191] 1 Content determination of Astragalus membranaceus

[0192] The content of astragaloside IV, an active ingredient of Astragalus membranaceus in the prescription, was determined by HPLC-ELSD internal standard method. Since astragaloside IV has end absorption and a low response value, the interference of ultraviolet detector is relatively large. ELSD is a general quality detector that can detect semi-volatile or non-volatile compounds. Its working principle is that under the action of auxiliary gas, the mobile phase is atomized to form a liquid mist (mist droplets), which is evaporated by heating. At this time, the sample that is not easily volatilized and dissolved in the mobile phase forms particulate matter, and these particulate matters are pushed by the auxiliary gas into the light beam channel, causing light beam scattering. By measuring the intensity of the scattered light, the number of sample particles can be predicted, and thus the sample concentration can be determined. From the working principle of ELSD, it can be seen that ELSD has many influencing factors, so its stability is poor, and sometimes the intraday precision cannot meet the analysis requirements. In this experiment, if the peak area of astragaloside IV is calculated separately in the precision test, the RSD of the reference substance is 3.31% and that of the sample is 2.18%, while the RSD of the reference substance and the sample in the precision test using internal standard are 0.33% and 0.22% respectively; if the peak area of astragaloside IV in the 24-hour stability test is calculated separately, the RSD of the reference substance is 8.63% and that of the sample reaches 11.3%, while the RSD using internal standard are 0.74% and 0.35% respectively; and the content of astragaloside IV in the sample is very low. Considering the above factors, the internal standard method is used to detect astragaloside IV in the prescription.

[0193] 1.1 Materials, Instruments and Reagents

[0194] The test samples were the traditional Chinese medicine composition tablets prepared according to Example 1, with batch numbers 080401, 081101, 081102, 081103, A2101001, A2101002, A2101003

[0195] Agilent 1100 liquid chromatography system; evaporative light scattering detector (Aotai 2000ES); chromatographic column (Shiseido MG C18 5μm 4.6*250mm)

[0196] KQ-250 type ultrasonic cleaner

[0197] AT201 METTLER TOLEDO 1 / 100,000 analytical balance

[0198] Methanol: Fisher, chromatographically pure

[0199] Water: ultrapure water, Milli-Q Advantage A10 ultrapure water system

[0200] 1.2 Selection of Mobile Phase for Astragaloside IV

[0201] After the sample was extracted with water-saturated n-butanol, methanol-water (75:25) was used as the mobile phase at a flow rate of 1.0 ml / min. The retention time was appropriate, the peak shape was symmetrical, and baseline separation was achieved, obtaining a very good separation effect, so it was included in the main text.

[0202] 1.3 Chromatographic conditions

[0203] Chromatographic column: Packed with octadecylsilane-bonded silica gel

[0204] Column length: 250 mm

[0205] Mobile phase: Methanol-water (75:25)

[0206] Flow rate: 1.0 ml / min

[0207] Evaporative light scattering detector: Drift tube temperature 80 °C; Gas flow rate 3.0 L / min

[0208] Column temperature: 30 °C

[0209] Injection volume: 10 μl

[0210] Theoretical plate number: Calculated by astragaloside IV, it should not be less than 4000

[0211] 1.4 Selection of internal standard

[0212] A series of compounds with similar chemical properties and stable properties to astragaloside IV were selected. Under these chromatographic conditions, only ginsenoside Rb2 and ginsenoside Rb3 had good resolution from astragaloside IV and appropriate retention times. However, since the price of ginsenoside Rb2 is twice that of ginsenoside Rb3 and the amount of internal standard used is large, considering the cost, ginsenoside Rb3 was selected as the internal standard.

[0213] 1.5 Preparation of reference substance solution

[0214] Preparation of ginsenoside Rb3 internal standard stock solution: Take an appropriate amount of ginsenoside Rb3 and dissolve it in methanol to make a methanol solution containing 0.35 mg per 1 ml, that is, the ginsenoside Rb3 internal standard stock solution is obtained.

[0215] Preparation of astragaloside IV stock solution: Take an appropriate amount of astragaloside IV and dissolve it in methanol to make a methanol solution containing 0.32 mg per 1 ml, that is, the astragaloside IV stock solution is obtained.

[0216] Preparation of low-concentration reference substance solution: Precisely measure 3 ml of astragaloside IV stock solution and 5 ml of ginsenoside Rb3 internal standard stock solution, place them in a 25-ml volumetric flask, and make a 70% methanol solution, that is, it is obtained.

[0217] Preparation of high-concentration reference solution: Accurately measure 10 ml of astragaloside stock solution and 5 ml of ginsenoside Rb3 internal standard stock solution, place them in a 25-ml volumetric flask, and prepare a 70% methanol solution, which is ready.

[0218] 1.6 Preparation of test solution

[0219] 1.6.1 Investigation of test solution preparation method

[0220] Since the test sample is a compound preparation and there are many components in the sample, to reduce the interference of other components in the determination on astragaloside and reduce the contamination of the chromatographic column by the test sample, the preparation method of the test sample was investigated.

[0221] The method is as follows: Take 2.5 g of the test sample, add 50 ml of methanol, ultrasonically treat for 20 minutes, filter, accurately measure 25 ml of the continuous filtrate, evaporate to dryness in a water bath, dissolve the residue in 25 ml of water, extract with chloroform 4 times, 25 ml each time, collect the chloroform layers separately, evaporate to dryness, dissolve the residue in 70% methanol and transfer to a 5-ml volumetric flask as test samples 1, 2, 3, and 4; extract the aqueous layer with water-saturated n-butanol 5 times, 25 ml each time, combine the first 3 times, evaporate the 4th and 5th times to dryness separately, dissolve the residue in 70% methanol and transfer to a 5-ml volumetric flask as test samples 5 and 6; wash the first 3 times of n-butanol solution with 1% sodium hydroxide solution 3 times, 20 ml each time, combine the 1% NaOH washing solutions, extract with water-saturated n-butanol 2 times, 20 ml each time, combine the back-extracted n-butanol solution, evaporate to dryness, dissolve the residue in 70% methanol and transfer to a 5-ml volumetric flask as test sample 7; wash the first 3 times of n-butanol solution with water-saturated with n-butanol 3 times, 20 ml each time, add 1 ml of internal standard stock solution to the n-butanol solution, evaporate to dryness in a water bath, dissolve the residue in 70% methanol and transfer to a 5-ml volumetric flask as test sample 8; extract the washing solution with water-saturated n-butanol 2 times, 20 ml each time, combine the n-butanol, evaporate to dryness, dissolve the residue in 70% methanol and transfer to a 5-ml volumetric flask as test sample 9.

[0222] The test results are shown in Table 1.

[0223] Table 1 Test results of investigation of test solution preparation method

[0224]

[0225] *Since astragaloside was not detected in test sample 5, test sample 6 was not detected.

[0226] It can be seen from the test results that astragaloside was not detected in the alkaline water washing solution or the n-butanol saturated water washing solution, so back extraction was no longer carried out; astragaloside was not detected in the chloroform layer either, and there was no absorption peak at the corresponding position of ginsenoside Rb3. It can be seen that chloroform extraction did not significantly improve the chromatographic behavior of astragaloside and ginsenoside Rb3, and chloroform has high toxicity. Considering production costs and environmental protection, chloroform was not used for extraction.

[0227] 1.6.2 Investigation on the dosage of extraction solvent

[0228] The dosage of the extraction solvent has a great influence on the dissolution of astragaloside, so the dosage of the extraction solvent was investigated. The method is as follows: The sample was ground fine and extracted with different amounts of methanol. The test results are shown in Table 2.

[0229] Table 2 Influence of the dosage of extraction solvent on the content of astragaloside

[0230]

[0231] It can be seen from the test results that 50 ml of methanol can completely extract astragaloside from the sample, so 50 ml of methanol was used for extraction.

[0232] 1.6.3 Investigation on ultrasonic time

[0233] A certain time is required for the dissolution of astragaloside, so the extraction time was investigated. The method was: The sample was ground fine and ultrasonically extracted with methanol for different times. The results are shown in Table 3.

[0234] Table 3 Influence of ultrasonic time on the content of astragaloside

[0235]

[0236] It can be seen from the results that astragaloside can be completely extracted by ultrasonic treatment for 30 minutes, so the ultrasonic time was set at 30 minutes.

[0237] 1.6.4 Summary of the test on the preparation method of the test solution

[0238] Through the above tests, various factors affecting the determination of the content of astragaloside were investigated, and the preparation method of the test solution was determined.

[0239] Preparation method of test solution: Take 10 tablets of the test sample, remove the film coating, grind them finely, take 2.5 g, weigh accurately, place in a stoppered conical flask, accurately add 50 ml of methanol, stopper tightly, weigh, ultrasonically treat for 30 minutes (power 250 W, frequency 40 kHz), cool, weigh again, make up the lost weight with methanol, shake well, filter, accurately measure 25 ml of the subsequent filtrate, evaporate to dryness in a water bath, dissolve the residue in 25 ml of water in portions and transfer to a separating funnel, extract with water-saturated n-butanol 3 times, 25 ml each time, combine the n-butanol layers, first wash with 1% NaOH 3 times, 20 ml each time, then wash with water saturated with n-butanol 3 times, 20 ml each time, accurately add 1 ml of the internal standard stock solution to the n-butanol layer, evaporate to dryness, dissolve the residue in 70% methanol and dilute to a volume of 5 ml in a volumetric flask, shake well, filter, and obtain the solution.

[0240] 1.7 Specificity test

[0241] Weigh about 1 / 10 of the prescription amount of the Chinese herbal medicines in the prescription, remove Astragali Radix, prepare the negative sample according to the preparation process, and treat it according to the treatment method of the test solution to obtain the negative sample solution. Pipette the negative sample solution, reference solution and sample solution, and perform detection according to the selected determination conditions. The results show that the negative sample has no interference on astragaloside IV and ginsenoside Rb3 (see the attached Figure 6 ).

[0242] 1.8 Investigation of linear relationship

[0243] Accurately pipette 2 ml, 3 ml, 5 ml, 10 ml, 15 ml of the astragaloside IV reference solution with a concentration of 0.3184 mg / ml into 25-ml volumetric flasks, respectively add 5 ml of the accurately pipetted ginsenoside Rb3 reference solution with a concentration of 0.3422 mg / ml, dilute to the mark, accurately pipette 10 μl, inject into the liquid chromatograph, and measure the peak areas. Use as the ordinate and as the abscissa to plot the standard curve. The measurement results are shown in Table 4;

[0244] Table 4 Standard curve of astragaloside IV

[0245]

[0246] Note: The concentration unit is μg / ml.

[0247] The results show that there is a good linear relationship between the logarithmic ratio of the concentrations of astragaloside IV and ginsenoside Rb3 in the range of 0.7661 - 1.2429. The regression equation is: y = 1.1374x - 0.1356, r = 0.9994 (see the attached standard curve Figure 7 ).

[0248] 1.9 Precision test

[0249] Absorb the mixed solution of astragaloside IV and ginsenoside Rb3 reference substances and the same sample solution, inject each for 5 times, and measure the logarithmic ratio of the peak areas of astragaloside IV and ginsenoside Rb3. The test results are shown in Table 5.

[0250] Table 5 Results of precision test

[0251]

[0252] The results show that the RSD value of the logarithmic ratio of the reference substance peak areas is 0.33%, and the RSD value of the logarithmic ratio of the peak areas in the sample is 0.22%, indicating that the precision of the instrument is good.

[0253] 1.10 Stability test

[0254] Absorb the mixed solution of astragaloside IV and ginsenoside Rb3 reference substances and the corresponding sample solution, inject at regular intervals, and measure the logarithmic ratio of the peak areas of astragaloside IV and ginsenoside Rb3. The test results are shown in Table 6.

[0255] Table 6 Results of astragaloside IV stability test

[0256]

[0257]

[0258] The results show that within 24 hours, the logarithmic ratio of the reference substance peak areas and the logarithmic ratio of the peak areas are stable, and the RSD values are 0.74% and 0.35% respectively, indicating that the sample is stable within 24 hours.

[0259] 1.11 Repeatability test

[0260] Take the same batch of samples, take three sample amounts of high, medium and low respectively, with 3 portions for each sample amount, extract, prepare and measure according to the preparation method of the sample solution. The measurement results are shown in Table 7.

[0261] Table 7 Results of repeatability test for astragaloside IV content

[0262]

[0263] The results show that the average content of astragaloside IV is 0.3390 mg / g, and the RSD is 1.61%, indicating that the reproducibility of the method is very good.

[0264] 1.12 Recovery test

[0265] Adopt the addition recovery test. Take 9 portions of the same batch of samples, divide them into three groups, add 50 ml of methanol solutions of astragaloside IV reference substances with high, medium and low concentrations respectively, extract by ultrasonic wave, measure, and calculate the recovery rate. The results are shown in Table 8.

[0266] Table 8 Determination Results of Astragaloside Recovery Rate

[0267]

[0268] The results showed that the average recovery rate was 97.92%, and the RSD was 1.40%, indicating that the accuracy of the method was good.

[0269] 1.13 Durability Test

[0270] 1.13.1 Effect of Evaporative Light Scattering Gas Flow Rate on Astragaloside Content

[0271] Take the same test sample, change the gas flow rate, and investigate the effect of different gas flow rates on the astragaloside content. The test results are shown in Table 9:

[0272] Table 9 Effect of Evaporative Light Scattering Gas Flow Rate on Astragaloside Content

[0273]

[0274] It can be seen from the test results that the gas flow rate (2.7 - 3.3 L / min) does not affect the determination result of astragaloside content, and its RSD is 1.60%.

[0275] 1.13.2 Effect of Column Temperature on Astragaloside Content

[0276] Take the same test sample, adjust the column temperature, and investigate the effect of column temperature on the astragaloside content. The test results are shown in Table 10.

[0277] Table 10 Effect of Column Temperature on Astragaloside Content

[0278]

[0279] It can be seen from the above table that when the column temperature is in the range of 25 - 35 °C, the column temperature does not affect the determination result of astragaloside content, and its RSD is 1.02%.

[0280] 1.13.3 Effect of Drift Tube Temperature on Astragaloside Content

[0281] Take the same test sample, adjust the drift tube temperature, and investigate the effect of drift tube temperature on the astragaloside content. The test results are shown in Table 11.

[0282] Table 11 Effect of Drift Tube Temperature on Astragaloside Content

[0283]

[0284] It can be seen from the above table that when the drift tube temperature is in the range of 75 - 85 °C, the drift tube temperature does not affect the determination result of astragaloside content, and its RSD is 1.92%.

[0285] 1.13.4 Influence of Chromatographic Column and Mobile Phase Ratio on the Content of Astragaloside IV

[0286] Take the same test sample and replace different chromatographic columns to investigate the influence of different column packings on the content of astragaloside IV. The test results are shown in Table 12:

[0287] Table 12 Influence of Chromatographic Column on the Content of Astragaloside IV

[0288]

[0289] Note: Chromatographic Column 1: Shiseido MG C18 4.6*250mm 5μm

[0290] Chromatographic Column 2: Phenomenex Gemini C18 4.6*150mm 5μm

[0291] Chromatographic Column 3: Waters Sunfire C18 4.6*150mm 5μm

[0292] It can be seen from the test results that slight changes in the three different brand chromatographic columns and the mobile phase ratio have no significant influence on the content of astragaloside IV.

[0293] 1.14 Content Determination

[0294] 1.14.1 Content Determination Results of Pilot-Scale Samples

[0295] Using the established content determination method, the 3 batches of pilot-scale samples were determined, and the transfer rate was calculated. The test results are shown in Table 13:

[0296] Table 13 Content Determination Results of Astragaloside IV

[0297]

[0298] 1.14.2 Content Determination of Large-Scale Production Samples

[0299] Using the established content determination method, the content of astragaloside IV in 6 batches of large-scale production samples was determined, and the transfer rate was calculated. The test results are shown in Table 14.

[0300] Table 14 Content Determination Results of Large-Scale Production Samples

[0301]

[0302] 1.14.3 Optimization of Content Calculation Method

[0303] (1) The method for determining the content of Astragalus membranaceus: The test solution is prepared from the plain tablets after removing the coating, and the content determination result is calculated based on the average tablet weight of the plain tablets. Since the tablet core is easily damaged when removing the coating, if the tablet core is damaged, the average tablet weight of the plain tablets will be lower than the actual value, affecting the accuracy of the content determination result. Therefore, caution must be exercised during operation, resulting in low efficiency.

[0304] In the content determination items of Zhitong Huazheng Tablets and Juanxiao Tablets in the 2020 edition of the Chinese Pharmacopoeia, the test solution is prepared from the plain tablets after removing the coating, and the content determination result is calculated based on the average tablet weight of the coated tablets under the weight variation item. This method can simplify the test operation and improve the accuracy of the content determination result.

[0305] When the Chinese medicinal composition tablets of the present invention are film-coated tablets, the technological requirement is that the coating weight gain is 3%. The difference in tablet weight between the plain tablets and the coated tablets is small. Therefore, the inspection data of large production batches of Xinqi Tongbi Tablets were reviewed. The average tablet weight of the coated tablets of 6 batches of samples is about 3% higher than the average tablet weight of the plain tablets after removing the coating, which is consistent with the coating weight gain of 3% in the process. Therefore, if the test solution for content determination is prepared from the plain tablets after removing the coating and the content determination result is calculated based on the average tablet weight of the coated tablets under the weight variation item, theoretically it is about 3% higher than the actual measured value, and the average content determination value of 6 batches of samples also differs by about 3%. This difference can be controlled by revising the content limit.

[0306] Therefore, referring to the content determination methods of Zhitong Huazheng Tablets and Juanxiao Tablets in the 2020 edition of the Chinese Pharmacopoeia, the method of preparing the test solution for content determination from the plain tablets after removing the coating remains unchanged, and it is revised to calculate the content determination result based on the average tablet weight of the coated tablets under the weight variation item. This revision is only a change in the calculation method and does not affect the quality control of the product. However, since the test operation is simplified and the test error is reduced, the accuracy of the content determination result is improved.

[0307] The average tablet weight under the weight variation item of the coated tablets was used to recalculate the 6 batches of inspection data, and the results are shown in Table 15.

[0308] Table 15 Summary of the results of the determination of astragaloside IV content

[0309]

[0310] Note: Batch A2101001 is a Phase II clinical sample, and batch A2101002 is a Phase III clinical sample

[0311] (2) The optimization of the method for determining the content of Astragalus membranaceus does not change the method for preparing the test solution and the chromatographic conditions, but only changes the calculation method of the finished product content. The optimized method for determining the content is as follows:

[0312] Chromatographic conditions and system suitability test: Use octadecylsilane chemically bonded silica gel as the filler; methanol-water (75:25) as the mobile phase; flow rate 1.0 ml / min; evaporative light scattering detector; column temperature: 30 °C; the number of theoretical plates calculated with astragaloside IV should be not less than 4000.

[0313] Preparation of internal standard stock solution: Take an appropriate amount of ginsenoside Rb3 and place it in a volumetric flask, add methanol to make a methanol solution containing 0.35 mg per 1 ml, namely the ginsenoside Rb3 internal standard stock solution.

[0314] Preparation of astragaloside IV stock solution: Take an appropriate amount of astragaloside IV and place it in a volumetric flask, add methanol to make a methanol solution containing 0.32 mg per 1 ml, namely the astragaloside IV stock solution.

[0315] Preparation of standard curve: Accurately measure appropriate amounts of astragaloside IV stock solution and internal standard stock solution respectively, and make 70% methanol solutions containing 38 μg of astragaloside IV and 70 μg of ginsenoside Rb3 per 1 ml and 70% methanol solutions containing 128 μg of astragaloside IV and 70 μg of ginsenoside Rb3 per 1 ml. Accurately pipette 10 μl respectively, inject into the liquid chromatograph for determination. Using as the ordinate and as the abscissa, plot the standard curve.

[0316] Preparation of test solution: Take the sample under weight variation, remove the film coating, grind it finely, take 2.5 g, accurately weigh it, place it in a stoppered conical flask, accurately add 50 ml of methanol, stopper it, weigh it, ultrasonically treat it for 30 minutes (power 250 W, frequency 40 kHz), let it cool, weigh it again, make up the lost weight with methanol, shake well, filter, accurately pipette 25 ml of the subsequent filtrate (reserve the remaining filtrate), evaporate to dryness in a water bath, add 25 ml of water to the residue, dissolve it in portions and transfer it to a separating funnel, extract with water-saturated n-butanol 3 times, 25 ml each time, combine the n-butanol layers, first wash with 1% NaOH 3 times, 20 ml each time, then wash with n-butanol-saturated water 3 times, 20 ml each time. Accurately add 1 ml of internal standard stock solution to the n-butanol layer, evaporate to dryness, dissolve the residue with 70% methanol and transfer it to a 5 ml volumetric flask, add 70% methanol to the scale, shake well, filter, namely obtain.

[0317] Determination method: Accurately pipette 10 μl of the test solution, inject into the liquid chromatograph for determination, and calculate by the standard curve method, namely obtain.

[0318] 2 Determination of the content of Saposhnikovia divaricata (Turcz.) Schischk.

[0319] 2.1 Materials, instruments and reagents

[0320] The test samples were the Chinese medicine composition tablets prepared according to Example 1, with batch numbers 080401, 081101, 081102, 081103, A2101001, A2101002, and A2101003.

[0321] Agilent 1100 liquid chromatography system; chromatographic column (Shiseido MG C18 5μm 4.6*250mm)

[0322] KQ-250 type ultrasonic cleaner

[0323] AT201 METTLER TOLEDO 1 / 100,000 analytical balance

[0324] Methanol: Fisher, chromatographically pure

[0325] Water: ultrapure water, Milli-Q Advantage A10 ultrapure water system

[0326] 2.2 Selection of mobile phase for prim-O-glucosylcimifugin and 5-O-methylvisammioside

[0327] Referring to the method for the determination of the content of Saposhnikoviae Radix in the first part of the Chinese Pharmacopoeia 2005 edition, methanol-water (40:60) was used as the mobile phase, but the resolution was not good and a good baseline separation could not be achieved. Subsequently, acetonitrile-water (16:84) was used as the mobile phase, with a symmetric peak shape and baseline separation achieved, obtaining a good separation effect, so it was included in the quality standard.

[0328] 2.3 Chromatographic conditions

[0329] Chromatographic column: packed with octadecylsilane chemically bonded silica gel

[0330] Column length: 250mm

[0331] Mobile phase: acetonitrile-water (16:84)

[0332] Flow rate: 1.0 ml / min

[0333] Detection wavelength: 254nm

[0334] Column temperature: 25°C

[0335] Injection volume: 10 μl each for the reference substance and the sample

[0336] Theoretical plate number: calculated by the prim-O-glucosylcimifugin peak should be not less than 2000

[0337] 2.4 Preparation of the reference substance solution

[0338] Weigh accurately an appropriate amount of cimifugin reference substance and 5-O-methylvisammioside reference substance, add 60% methanol to make a mixed solution containing 120 μg and 60 μg per 1 ml respectively, and you will get it.

[0339] 2.5 Preparation of test solution

[0340] 2.5.1 Investigation of purification method

[0341] Since the test sample is a compound preparation and there are many components in the sample, in order to reduce the interference of other components in the determination on cimifugin and 5-O-methylvisammioside, and reduce the pollution of the test sample to the chromatographic column, the preparation method of the test sample was investigated. The method is as follows: Take the test sample, grind it finely, accurately add 20 ml of methanol, ultrasonically treat for 30 minutes, filter, accurately measure 5 ml of the subsequent filtrate, evaporate to dryness in a water bath, dissolve the residue with 7 ml of water, load it onto a polyamide column (100 - 200 mesh, 2.0 g, inner diameter 1.0 cm), elute with 5 ml and 5 ml of water respectively, collect the eluates respectively, evaporate to dryness in a water bath, dissolve the residue with water and make the volume constant to 5 ml in a volumetric flask as test samples 1 and 2; then elute with 30 ml of methanol, collect the eluate, evaporate to dryness in a water bath, dissolve the residue with methanol and make the volume constant to 5 ml in a volumetric flask as test sample 3.

[0342] Accurately measure 5 ml of the subsequent filtrate, load it onto a neutral alumina column (100 - 200 mesh, 3.0 g, inner diameter 1.0 cm), first elute with 10 ml of methanol, then elute with 30 ml of water, collect the eluate, evaporate to dryness in a water bath, dissolve the residue with methanol and make the volume constant to 5 ml in a volumetric flask as test samples 4 and 5; The test results are shown in Table 16:

[0343] Table 16 Test results of purification methods

[0344]

[0345] From the test results, cimifugin and 5-O-methylvisammioside can be eluted with water on both the polyamide column and the neutral alumina column. Therefore, using water as the elution solvent, the purification methods of the two columns were compared. The method is as follows:

[0346] Take the test sample and grind it finely, accurately add 20 ml of methanol, ultrasonically treat for 30 minutes, and filter. Accurately measure 5 ml of the subsequent filtrate, load it onto a neutral alumina column (100 - 200 mesh, 3.0 g, inner diameter 1.0 cm), elute with 30 ml of water, collect the eluate, evaporate to dryness in a water bath, dissolve the residue with water and make the volume constant to 5 ml in a volumetric flask as test sample 1; Accurately measure 5 ml of the subsequent filtrate, evaporate to dryness in a water bath, dissolve the residue with 10 ml of water, load it onto a polyamide column (100 - 200 mesh, 2.0 g, inner diameter 1.0 cm), elute with 20 ml of water, collect the eluate, evaporate to dryness in a water bath, dissolve the residue with water and make the volume constant to 5 ml in a volumetric flask as test sample 2. The test results are shown in Table 17:

[0347] Table 17 Test Results of Purification Methods

[0348]

[0349] Results showed that there was no difference in the content between the two methods. However, preparing the test sample using a neutral alumina column was simpler than using a polyamide column, and the sample on the neutral alumina column had a better return to the baseline. Therefore, the neutral alumina column was used to prepare the test sample.

[0350] 2.5.2 Investigation of the Elution Solvent Dosage

[0351] To ensure that cimifugin and 5-O-methylvisammioside in the sample could be fully eluted without introducing too many impurities that would affect the peak separation effect, the elution solvent dosage was investigated. The method was as follows: Remove the coating from the test sample, grind it finely, take 2.5 g, accurately add 50 ml of methanol, ultrasonically treat for 30 minutes, filter, accurately measure 10 ml of the subsequent filtrate, load it onto a neutral alumina column (100 - 200 mesh, 3.0 g, inner diameter 1.0 cm), elute with 15 ml, 5 ml, 5 ml, and 5 ml of water respectively, collect the eluates separately, evaporate to dryness in a water bath, dissolve the residue in water and make up the volume to 5 ml in a volumetric flask to obtain test sample solutions 1, 2, 3, and 4. The test results are shown in Table 18:

[0352] Table 18 Investigation Results of the Elution Solvent Dosage

[0353]

[0354] Results showed that 15 ml of water was sufficient for complete elution. Therefore, the elution solvent dosage was set at 15 ml of water for elution.

[0355] 2.5.3 Investigation of the Ultrasonic Time

[0356] It takes a certain amount of time for the dissolution of cimifugin and 5-O-methylvisammioside, so the ultrasonic time was investigated. The method was as follows: Grind the sample finely, ultrasonically extract with methanol for different times respectively. The results are shown in Table 19:

[0357] Table 19 Investigation Results of the Ultrasonic Time

[0358]

[0359] Results showed that the ultrasonic time had no significant effect on the content determination of the sample. To ensure sufficient extraction of the sample and save time, the ultrasonic time was set at 30 minutes.

[0360] 2.5.4 Investigation of the Extraction Solvent Dosage

[0361] The amount of extraction solvent used has an impact on the dissolution of prim-O-glucosylcimifugin and 5-O-methylvisammioside. Therefore, the amount of extraction solvent used was investigated as follows: The sample was ground finely and extracted with different amounts of methanol. The test results are shown in Table 20:

[0362] Table 20 Results of the investigation on the amount of extraction solvent used

[0363]

[0364] As can be seen from the results, within the range of 25 ml to 100 ml of the solvent amount, there is no impact on the content determination. Considering that it can be combined with the test sample of astragaloside IV, the amount of extraction solvent used was set at 50 ml.

[0365] 2.5.5 Investigation on the amount of neutral alumina used

[0366] The amount of neutral alumina used has a great impact on the separation and purification effect of prim-O-glucosylcimifugin and 5-O-methylvisammioside. Therefore, the amount of neutral alumina used was investigated as follows: The sample was ground finely, extracted with methanol by ultrasonic treatment, 10 ml of the subsequent filtrate was precisely measured, and adsorbed onto neutral alumina columns (with amounts of 2.0 g, 3.0 g, 4.0 g, 5.0 g, and an inner diameter of 1.0 cm) respectively, and then desorbed. The test results are shown in Table 21:

[0367] Table 21 Results of the investigation on the amount of neutral alumina used

[0368]

[0369] As can be seen from the results, the measured contents of each sample have little difference. To ensure sufficient impurity removal without affecting the measured content, the amount of neutral alumina used was set at 3.0 g.

[0370] 2.5.6 Summary of the test on the preparation method of the test solution

[0371] Through the above tests, various factors affecting the content determination of prim-O-glucosylcimifugin and 5-O-methylvisammioside were investigated, and the preparation method of the test solution was determined.

[0372] Preparation method of the test solution: Take 10 tablets of this product, remove the film coating, weigh accurately, grind finely, take 2.5 g, weigh accurately, place in a stoppered conical flask, accurately add 50 ml of methanol, stopper tightly, weigh, ultrasonically treat for 30 minutes (power 250 W, frequency 40 kHz), cool, weigh again, make up the lost weight with methanol, shake well, filter, accurately measure 10 ml of the subsequent filtrate, load onto a neutral alumina column (100 - 200 mesh, 3.0 g, inner diameter 1.0 cm), elute with 15 ml of water, collect the eluate, evaporate to dryness in a water bath, dissolve the residue in water, dilute to a volume of 5 ml in a volumetric flask, shake well, filter through a 0.22 μm filter membrane, and you will get it.

[0373] 2.6 Specificity Test

[0374] Weigh about 1 / 10 of the prescription amount of the traditional Chinese medicinal materials, remove Saposhnikovia divaricata, and prepare a negative sample according to the preparation process. Treat it according to the treatment method of the test solution to obtain a negative sample solution. Pipette the reference solution, negative sample solution and sample solution, and perform detection according to the selected determination conditions. The results show that the negative sample has no interference. (See the attached Figure 8 )

[0375] 2.7 Linear Relationship

[0376] Precisely pipette 1 μl, 2 μl, 5 μl, 10 μl, 15 μl, 20 μl, 30 μl, 40 μl of the reference solutions of prim-O-glucosylcimifugin with a concentration of 0.1512 mg / ml and 5-O-methylvisammioside with a concentration of 0.060 mg / ml respectively, inject them into the high performance liquid chromatograph, and determine the peak areas. Take the integrated peak area values as the ordinate and the injection volume as the abscissa to plot the standard curve. The determination results are shown in Table 22.

[0377] Table 22 Standard Curves of Prim-O-glucosylcimifugin and 5-O-methylvisammioside

[0378]

[0379] The results show that: there is a good linear relationship between the injection volume of prim-O-glucosylcimifugin in the range of 0.1512 μg - 6.0480 μg, and the regression equation is: y = 1476.6995x + 29.5334, r = 0.99999; there is a good linear relationship between the injection volume of 5-O-methylvisammioside in the range of 0.0600 μg - 2.4000 μg, and the regression equation is: y = 1900.9052x + 5.2463, r = 1.00000. (See the attached Figure 9 , attached Figure 10 )

[0380] 2.8 Precision Test

[0381] Pipette the reference solutions of prim-O-glucosylcimifugin and 5-O-methylvisammioside and the same sample solution, inject each for 6 times, and determine the peak areas. The test results are shown in Table 23:

[0382] Table 23 Results of Precision Test

[0383]

[0384] As can be seen from the above results, the RSD value of prim-O-glucosylcimifugin reference substance was 0.19%, and that of 5-O-methylvisammioside reference substance was 0.15%. The RSD value of prim-O-glucosylcimifugin in the sample was 0.37%, and that of 5-O-methylvisammioside was 0.50%, indicating good instrument precision.

[0385] 2.9 Stability test

[0386] Absorb the solutions of prim-O-glucosylcimifugin and 5-O-methylvisammioside reference substances and the corresponding test solutions, inject samples at regular intervals, measure the peak areas, and the test results are shown in Table 24:

[0387] Table 24 Results of stability test

[0388]

[0389] As can be seen from the above results, within 20 hours, the peak areas of prim-O-glucosylcimifugin, 5-O-methylvisammioside reference substances and the sample were all stable, and the RSD values were 0.97%, 0.18%, 0.74%, 0.40% respectively.

[0390] 2.10 Repeatability test

[0391] Take the same batch of samples, take high, medium and low sample amounts respectively, with 3 portions for each sample amount. According to the preparation method of the test solution, prepare each test solution according to law and conduct the determination. The determination results are shown in Table 25:

[0392] Table 25 Results of repeatability test

[0393]

[0394] As can be seen from the determination results, the RSD of prim-O-glucosylcimifugin was 1.30%, the RSD of 5-O-methylvisammioside was 1.11%, and the RSD of the total content was 0.75%, indicating good repeatability of the method.

[0395] 2.11 Recovery test

[0396] Adopt the spiked recovery test. Take 9 portions of the same batch of samples, divide them into three groups, and add 50 ml of methanol solutions of prim-O-glucosylcimifugin and 5-O-methylvisammioside reference substances with high, medium and low concentrations respectively. Conduct ultrasonic extraction, determination, and calculate the recovery rate. The results are shown in Table 26:

[0397] Table 26 Results of recovery rate determination

[0398]

[0399]

[0400] The results showed that the average recovery rate of prim-O-glucosylcimifugin was 101.79% and the RSD was 1.44%; the average recovery rate of 5-O-methylvisammioside was 101.32% and the RSD was 1.40%, indicating that the accuracy of the method was good.

[0401] 2.12 Durability Test

[0402] 2.12.1 Effect of Detection Wavelength on the Contents of Prim-O-glucosylcimifugin and 5-O-methylvisammioside

[0403] Take the same test sample, adjust the range of detection wavelength, and investigate the effect of detection wavelength on the contents of prim-O-glucosylcimifugin and 5-O-methylvisammioside. The test results are shown in Table 27:

[0404] Table 27 Effect of Detection Wavelength on the Contents of Prim-O-glucosylcimifugin and 5-O-methylvisammioside

[0405]

[0406] It can be seen from the test results that within the range of 249 - 259 nm of the detection wavelength, the detection wavelength will not affect the determination of the contents of prim-O-glucosylcimifugin and 5-O-methylvisammioside.

[0407] 2.12.2 Effect of Column Temperature on the Contents of Prim-O-glucosylcimifugin and 5-O-methylvisammioside

[0408] Take the same test sample, adjust the column temperature, and investigate the effect of column temperature on the contents of prim-O-glucosylcimifugin and 5-O-methylvisammioside. The test results are shown in Table 28:

[0409] Table 28 Effect of Column Temperature on the Contents of Prim-O-glucosylcimifugin and 5-O-methylvisammioside

[0410]

[0411]

[0412] It can be seen from the test results that when the column temperature is within the range of 20 - 30 °C, the column temperature does not affect the determination results of the contents of prim-O-glucosylcimifugin and 5-O-methylvisammioside, so the column temperature is set at 25 °C.

[0413] 2.12.3 Effect of Neutral Aluminum Oxide on the Contents of Prim-O-glucosylcimifugin and 5-O-methylvisammioside

[0414] Use neutral aluminum oxide from different manufacturers to prepare test samples according to the test sample preparation method, and investigate the effect of neutral aluminum oxide on the contents of prim-O-glucosylcimifugin and 5-O-methylvisammioside. The test results are shown in Table 29:

[0415] Table 29 Influence of Neutral Aluminum Oxide Manufacturers on the Contents of Cimifugin and 5-O-Methylvisammioside

[0416]

[0417] Note: Manufacturer 1: Sinopharm Chemical Reagent Co., Ltd., Batch No.: F20040304.

[0418] Manufacturer 2: Shanghai Wusi Chemical Reagent Co., Ltd., Batch No.: 040804.

[0419] Manufacturer 3: Shanghai Nahui Drying Reagent Factory, Batch No.: 20080308.

[0420] It can be seen from the test results that neutral aluminum oxides from different manufacturers and batches will not affect the content determination of cimifugin and 5-O-methylvisammioside.

[0421] 2.12.4 Influence of Chromatographic Column on the Contents of Cimifugin and 5-O-Methylvisammioside

[0422] Take the same test sample, replace different chromatographic columns, and investigate the influence of different column packings on the contents of cimifugin and 5-O-methylvisammioside. The test results are shown in Table 30:

[0423] Table 30 Influence of Chromatographic Column on the Contents of Cimifugin and 5-O-Methylvisammioside

[0424]

[0425]

[0426] Note: Chromatographic Column 1: Shiseido MG C 18 5μm 4.6×250mm

[0427] Chromatographic Column 2: Phenomenex Gemini C 18 5μm 4.6×150mm

[0428] Chromatographic Column 3: Waters Symmetry C 18 5μm 4.6×250mm

[0429] It can be seen from the test results that the three different brand chromatographic columns have no significant influence on the content determination of cimifugin and 5-O-methylvisammioside.

[0430] 2.13 Content Determination

[0431] 2.13.1 Content Determination Results of Pilot Batch Samples

[0432] The 3 batches of pilot-scale samples were determined using the established content determination method, and the transfer rate was calculated. The results are shown in Table 31:

[0433] Table 31 Results of the determination of the total content of cimifugin and 5-O-methylvisammioside

[0434]

[0435] 2.13.2 Content determination of large-scale production samples

[0436] Using the established content determination method, the contents of cimifugin and 5-O-methylvisammioside in 6 batches of large-scale production samples were determined, and the transfer rate was calculated. The test results are shown in Table 32.

[0437] Table 32 Results of the content determination of large-scale production samples

[0438]

[0439]

[0440] 2.13.3 Optimization of the content determination method

[0441] (1) For the content determination method of Saposhnikovia divaricata, the test solution was prepared with the plain tablets after removing the coating, and the content determination result was calculated based on the average tablet weight of the plain tablets. Since the tablet core is easily damaged when removing the coating, if the tablet core is damaged, the average tablet weight of the plain tablets will be lower than the actual value, affecting the accuracy of the content determination result. Therefore, caution must be exercised during operation, resulting in low efficiency.

[0442] In the content determination items of Zhitong Huazheng Tablets and Juanxiao Tablets in the Chinese Pharmacopoeia (2020 Edition), the test solution was prepared with the plain tablets after removing the coating, and the content determination result was calculated based on the average tablet weight of the coated tablets under the weight variation item. This method can simplify the test operation and improve the accuracy of the content determination result.

[0443] The test sample is a film-coated tablet, and the process requires a 3% coating weight gain. The weight difference between the plain tablets and the coated tablets is small. Therefore, the inspection data of the large-scale production batches of Xinqi Tongbi Tablets were reviewed. The average tablet weight of the coated tablets of 6 batches of samples was about 3% higher than the average tablet weight of the plain tablets after removing the coating, which is consistent with the 3% coating weight gain in the process. Therefore, if the test sample for content determination is prepared with the plain tablets after removing the coating and the content determination result is calculated based on the average tablet weight of the coated tablets under the weight variation item, theoretically it will be about 3% higher than the actual measured value, and the average content determination value of the 6 batches of samples also differs by about 3%. This difference can be controlled by revising the content limit.

[0444] Therefore, referring to the content determination methods of Zhitong Huazheng Tablets and Juanxiao Tablets in the Chinese Pharmacopoeia 2020 Edition, the method for preparing the test samples for content determination by using the plain tablets after removing the coating remains unchanged, and it is revised to calculate the content determination results based on the average tablet weight of the coated tablets under the weight variation test. This revision only changes the calculation method and does not affect the product quality control. However, since the test operation is simplified and the test error is reduced, the accuracy of the content determination results is improved.

[0445] The average tablet weights under the weight variation test of the coated tablets were used to recalculate the 6 batches of inspection data, and the results are shown in Table 33.

[0446] Table 33 Summary of the Results of the Content Determination of Saposhnikoviae Radix (Calculated Based on the Average Tablet Weight of the Coated Tablets)

[0447]

[0448]

[0449] Note: Batch A2101001 is a Phase II clinical sample, and batch A2101002 is a Phase III clinical sample.

[0450] (2) For the content determination of Saposhnikoviae Radix, the contents of prim-O-glucosylcimifugin and 5-O-methylvisammioside were detected simultaneously by the same method. During the implementation of the quality standard, it was found that there were many impurity peaks before and after prim-O-glucosylcimifugin and it could not return to the baseline, while there were fewer impurity peaks before and after 5-O-methylvisammioside and it could achieve baseline separation.

[0451] Starting from the Phase II clinical samples, 6 batches of large-scale production samples were successively prepared, and the content data of prim-O-glucosylcimifugin and 5-O-methylvisammioside in the 6 batches of large-scale production samples were summarized, and the transfer rates were calculated. The test results are shown in Tables 34 and 35.

[0452] Table 34 Results of the Content Determination of 5-O-methylvisammioside (Calculated Based on the Average Tablet Weight of the Coated Tablets)

[0453]

[0454] Table 35 Results of the Content Determination of prim-O-glucosylcimifugin (Calculated Based on the Average Tablet Weight of the Coated Tablets)

[0455]

[0456] It can be seen from the test data that the transfer rates of the two components, prim-O-glucosylcimifugin and 5-O-methylvisammioside, are basically the same, and both are components in Saposhnikoviae Radix. If the content determination item of prim-O-glucosylcimifugin is deleted and only the content determination method of 5-O-methylvisammioside is retained, the product quality control requirements will not be reduced. Therefore, the content determination method of the prim-O-glucosylcimifugin component was deleted.

[0457] The optimized method for the determination of the windproof content is as follows:

[0458] Chromatographic conditions and system suitability test: Use octadecylsilane chemically bonded silica gel as the filler; acetonitrile - water (16:84) as the mobile phase; flow rate 1.0 ml / min; detection wavelength 254 nm; column temperature: 25 °C; the number of theoretical plates calculated by 5-O-methylvisamminol glycoside should be not less than 5000.

[0459] Preparation of the reference substance solution: Weigh accurately an appropriate amount of 5-O-methylvisamminol glycoside reference substance, and make a solution containing 60 μg per 1 ml with 60% methanol, that is obtained.

[0460] Preparation of the test solution: Precisely measure 10 ml of the reserved successive filtrate under the determination of the astragalus content, load it onto a neutral alumina column (100 - 200 mesh, 3 g, inner diameter 1.0 cm), elute with 15 ml of water, collect the eluate, evaporate to dryness, dissolve the residue with water, transfer it to a 5-ml volumetric flask, add water to the scale, shake well, filter, that is obtained.

[0461] Determination method: Precisely pipette 10 μl each of the reference substance solution and the test solution, inject them into the liquid chromatograph, and determine, that is obtained.

Claims

1. A quality control method for a traditional Chinese medicine composition preparation, characterized in that, The raw materials of the Chinese medicine composition by weight include: 10-20 parts of astragalus, 5-15 parts of atractylodes, 5-15 parts of siler, 5-15 parts of magnolia, 5-15 parts of angelica, 5-15 parts of galangal, 5-15 parts of notopterygium, 5-15 parts of peony bark, 5-15 parts of cicada shell, 5-15 parts of ebony, and 4-8 parts of liquorice; The quality control method of the traditional Chinese medicine composition includes an identification method and a content determination method. The identification method includes at least one of the following identification methods (1) to (5): (1) Thin layer chromatography using magnolia as the reference herb and cyclohexane-ethyl acetate in a volume ratio of 1:1 as the developing solvent; (2) Thin layer chromatography using Angelica dahurica as the reference herb and cyclohexane-ethyl acetate-formic acid in a volume ratio of 1:2:0.1 as the developing solvent; (3) Thin layer chromatography using eucalyptus oil as the reference substance and cyclohexane-ethyl acetate with a volume ratio of 12:1 as the developing solvent; (4) Thin layer chromatography using paeonol as the reference substance and cyclohexane-ethyl acetate with a volume ratio of 3:1 as the developing solvent; (5) TLC method using licorice as the reference herb and a lower layer solution of chloroform-methanol-water at a volume ratio of 13:6:2 placed below 10°C as the developing solvent; The content determination method includes a content determination method of Astragalus and a content determination method of Saposhnikovia divaricata, and adopts high performance liquid chromatography to determine the content of Astragaloside IV and 5-O-methylvisaside.

2. The quality control method according to claim 1, characterized in that, The identification method of item (1) in the identification method comprises the following steps: Take the contents of the Chinese medicine composition preparation, add methanol to dissolve, filter, and use the filtrate as the test solution; take another magnolia control medicinal material, add methanol to extract, filter, and use the filtrate as the control medicinal material solution; according to the thin layer chromatography test, take the test solution and the control medicinal material solution, respectively spot them on the same silica gel G thin layer plate, use 1:1 cyclohexane-ethyl acetate as a developing solvent, develop, take out, dry, spray with 10% sulfuric acid ethanol solution, heat until the spots are clearly colored, and in the test sample chromatogram, a spot of the same color appears at the corresponding position of the magnolia control medicinal material chromatogram; The identification method of item (2) in the identification method comprises the following steps: Take the contents of the Chinese medicine composition preparation, add methanol to dissolve, filter, and use the filtrate as the test solution; take another reference medicinal material of Angelica dahurica, add methanol to extract, filter, and use the filtrate as the reference medicinal material solution; according to the thin layer chromatography test, take the test solution and the reference medicinal material solution, respectively spot them on the same silica gel G thin layer plate, use 1:2:0.1 cyclohexane-ethyl acetate-formic acid as the developing agent, develop, take out, dry, spray with 10% sulfuric acid ethanol solution, heat until the spots are colored, and place under ultraviolet light (365nm) for inspection; in the chromatogram of the test sample, at the corresponding position of the chromatogram of the reference medicinal material, a main fluorescent spot of the same color is displayed; The identification method of item (3) in the identification method comprises the following steps: Take the content of the traditional Chinese medicine composition preparation, dissolve it in methanol, filter, and use the filtrate as the test solution. Separately, take the reference substance of eucalyptol, dissolve it in methanol to prepare a solution as the reference solution. According to the thin-layer chromatography test, absorb the test solution and the reference solution, and spot them respectively on the same silica gel G thin-layer plate. Use cyclohexane-ethyl acetate at a ratio of 12:1 as the developing agent, develop, take out, dry in the air, spray with 1% vanillin sulfuric acid solution, and heat until the spots are colored. In the chromatogram of the test solution, at the position corresponding to the chromatogram of the reference substance, there are spots of the same color. The identification method in item (4) of the said identification method includes the following operation steps: Take the content of the traditional Chinese medicine composition preparation, dissolve it in methanol, filter, and use the filtrate as the test solution. Separately, take the reference substance of paeonol, dissolve it in methanol to prepare a solution as the reference solution. According to the thin-layer chromatography test, absorb the test solution and the reference solution, and spot them respectively on the same silica gel G thin-layer plate. Use cyclohexane-ethyl acetate at a ratio of 3:1 as the developing agent, develop, take out, dry in the air, spray with 5% ferric trichloride ethanol solution with hydrochloric acid acidity, and heat until the spots are colored. In the chromatogram of the test solution, at the position corresponding to the chromatogram of the reference substance, there are spots of the same color. The identification method in item (5) of the said identification method includes the following operation steps: Take the content of the traditional Chinese medicine composition preparation, dissolve it in methanol, filter, and use the filtrate as the test solution. Separately, take the control crude drug of liquorice, extract it with methanol, filter, and use the filtrate as the control crude drug solution. According to the thin-layer chromatography test, absorb the test solution and the control crude drug solution, and spot them respectively on the same silica gel G thin-layer plate. Use the lower layer solution of chloroform-methanol-water at a ratio of 13:6:2 placed below 10 °C as the developing agent, develop, take out, dry in the air, spray with 10% sulfuric acid ethanol solution, and heat until the spots are colored. 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.

3. The quality control method according to claim 2, characterized in that, The identification method in item (1) of the said identification method includes the following operation steps: Take the content of the traditional Chinese medicine composition preparation, dissolve it in methanol, filter, and use the filtrate as the test solution. Separately, take the control crude drug of magnolia flower, extract it by ultrasonic extraction with methanol, filter, and use the filtrate as the control crude drug solution. According to the thin-layer chromatography test, absorb the test solution and the control crude drug solution, and spot them respectively on the same silica gel G thin-layer plate. Use cyclohexane-ethyl acetate at a ratio of 1:1 as the developing agent, develop, take out, dry in the air, spray with 10% sulfuric acid ethanol solution, and heat at 105 °C until the spots are clearly colored. In the chromatogram of the test solution, at the position corresponding to the chromatogram of the magnolia flower control crude drug, there is 1 main spot of the same color. The identification method in item (2) of the said identification method includes the following operation steps: Take the content of the traditional Chinese medicine composition preparation, dissolve it in methanol, filter, and use the filtrate as the test solution; separately take the control crude drug of Angelica dahurica, extract it by ultrasonic treatment with methanol, filter, and use the filtrate as the control crude drug solution; according to the thin-layer chromatography test, absorb the test solution and the control crude drug solution, and spot them respectively on the same silica gel G thin-layer plate. Use cyclohexane-ethyl acetate-formic acid (1:2:0.1) as the developing agent, develop, take out, dry in the air, spray with 10% sulfuric acid ethanol solution, heat at 105°C until the spots are clearly developed, and examine under an ultraviolet lamp (365 nm); in the chromatogram of the test sample, at the position corresponding to the chromatogram of the control crude drug, there are main fluorescent spots of the same color; The identification method in item (3) of the said identification method includes the following operating steps: Take the content of the traditional Chinese medicine composition preparation, dissolve it in methanol, filter, and use the filtrate as the test solution; separately take the reference substance of cineole, dissolve it in methanol to prepare a solution, and use it as the reference substance solution; according to the thin-layer chromatography test, absorb the test solution and the reference substance solution, and spot them respectively on the same silica gel G thin-layer plate. Use cyclohexane-ethyl acetate (12:1) as the developing agent, develop, take out, dry in the air, spray with 1% vanillin sulfuric acid solution, and heat at 105°C until the spots are clearly developed; in the chromatogram of the test sample, at the position corresponding to the chromatogram of the reference substance, there are spots of the same color; The identification method in item (4) of the said identification method includes the following operating steps: Take the content of the traditional Chinese medicine composition preparation, dissolve it in methanol, filter, and use the filtrate as the test solution; separately take the reference substance of paeonol, dissolve it in methanol to prepare a solution, and use it as the reference substance solution; according to the thin-layer chromatography test, absorb the test solution and the reference substance solution, and spot them respectively on the same silica gel G thin-layer plate. Use cyclohexane-ethyl acetate (3:1) as the developing agent, develop, take out, dry in the air, spray with 5% ferric trichloride ethanol solution with hydrochloric acid acidity, and heat until the spots are clearly developed; in the chromatogram of the test sample, at the position corresponding to the chromatogram of the reference substance, there are spots of the same color; The identification method in item (5) of the said identification method includes the following operating steps: Take the content of the traditional Chinese medicine composition preparation, dissolve it in methanol, filter, and use the filtrate as the test solution; separately take the control crude drug of liquorice, extract it by ultrasonic treatment with methanol, filter, and use the filtrate as the control crude drug solution; according to the thin-layer chromatography test, absorb the test solution and the control crude drug solution, and spot them respectively on the same silica gel G thin-layer plate. Use the lower layer solution of chloroform-methanol-water (13:6:2) placed at a temperature below 10°C as the developing agent, develop, take out, dry in the air, spray with 10% sulfuric acid ethanol solution, and heat at 105°C until the spots are clearly developed; in the chromatogram of the test sample, at the position corresponding to the chromatogram of the control crude drug, there are spots of the same color.

4. The quality control method according to claim 1, characterized in that, The content determination method of the said Astragalus membranaceus includes the following operating steps: Chromatographic conditions: The chromatographic column is packed with octadecylsilane chemically bonded silica gel; the mobile phase is methanol-water (75:25); the flow rate is 1.0 ml / min; evaporative light scattering detector; the column temperature is 30°C; Preparation of the internal standard stock solution: Take an appropriate amount of ginsenoside Rb3, accurately weigh it, dissolve it in methanol to prepare a solution, and obtain it; Preparation of the astragaloside IV stock solution: Take an appropriate amount of astragaloside IV, accurately weigh it, dissolve it in methanol to prepare a solution, and obtain it; Preparation of standard curve: Accurately pipette appropriate amounts of astragaloside IV stock solution and internal standard stock solution respectively to prepare 70% methanol solutions containing 38 μg of astragaloside IV and 70 μg of ginsenoside Rb3 per 1 ml, and 70% methanol solutions containing 128 μg of astragaloside IV and 70 μg of ginsenoside Rb3 per 1 ml. Accurately pipette and inject them into the liquid chromatograph for determination. Using as the ordinate and as the abscissa, plot the standard curve; Preparation of the test solution: Take the content of the traditional Chinese medicine composition preparation, weigh it precisely, add methanol precisely, stopper tightly, weigh again, perform ultrasonic treatment, let it cool, weigh again, make up the lost weight with methanol, shake well, filter, precisely measure the successive filtrate, evaporate to dryness in a water bath, add water to the residue, dissolve it in portions into a separating funnel, extract with water-saturated n-butanol to obtain the n-butanol solution, first wash it with 1% NaOH, then wash it with water saturated with n-butanol, precisely add the internal standard stock solution to the obtained n-butanol solution, evaporate to dryness, dissolve the residue with 70% methanol, shake well, filter, and you will get it; Determination method: Precisely absorb the test solution, inject it into the liquid chromatograph for determination, and calculate by the standard curve method to obtain it.

5. The quality control method according to claim 1, wherein, The content determination method of the Saposhnikovia divaricata Turcz. includes the following operating steps: Chromatographic conditions: The chromatographic column uses octadecylsilane chemically bonded silica as the filler; the mobile phase is acetonitrile-water at 16:84; the flow rate is 1.0 ml / min; the detection wavelength is 254 nm; the column temperature is 25 °C; Preparation of the reference solution: Precisely weigh an appropriate amount of the 5-O-methylvisammioside reference substance, weigh it precisely, and make a solution with 60% methanol to obtain it; Preparation of the test solution: Take the content of the traditional Chinese medicine composition preparation, weigh it precisely, add methanol precisely, stopper tightly, weigh again, perform ultrasonic treatment, let it cool, weigh again, make up the lost weight with methanol, shake well, filter, precisely measure the successive filtrate, load it onto a neutral alumina column, elute with water, collect the eluate, evaporate to dryness, dissolve the residue with water, shake well, filter, and you will get it; Determination method: Precisely absorb the reference solution and the test solution, inject them into the liquid chromatograph for determination, and you will get it.

6. The quality control method according to claim 1, wherein, The preparation dosage form of the traditional Chinese medicine composition is capsule, tablet, pill, granule or powder.

7. The quality control method according to claim 6, wherein, The operating steps of the preparation process of the tablet include: A. Coarsely crush Atractylodes macrocephala Koidz. and Notopterygium incisum Ting ex Hsiao & K. C. Hsia in the prescription, and coarsely crush the shell of Magnolia liliflora Desr.; Weigh Atractylodes macrocephala Koidz., Saposhnikovia divaricata Turcz., Alpinia officinarum Hance, Notopterygium incisum Ting ex Hsiao & K. C. Hsia, and Magnolia liliflora Desr. according to the prescription amount, add 6 - 12 times the amount of water, extract the volatile oil by steam distillation for 6 - 10 hours, collect the volatile oil for standby; Filter the aqueous solution after distillation, concentrate it under reduced pressure for standby; B. Weigh Astragalus membranaceus (Fisch.) Bunge, Cryptotympana pustulata Fabricius, and Glycyrrhiza uralensis Fisch. according to the prescription amount, decoct with 9 - 13 times the amount of water for three times, each time for 1 - 3 hours, filter, concentrate the extract under reduced pressure, and combine it with the volatile oil extract and concentrate it under reduced pressure to a relative density of 1.15 - 1.20 for standby; C. Weigh Angelica dahurica (Fisch.) Benth. et Hook. f. ex Franch. et Sav. and Prunus mume Sieb. et Zucc. according to the prescription amount, add 5 - 8 times the amount of 50 - 80% ethanol, extract twice, the first time for 1 - 3 hours, the second time for 1 - 3 hours, filter, recover the ethanol from the extract under reduced pressure, and concentrate it under reduced pressure to a relative density of 1.15 - 1.20 for standby; D. Sterilize and pulverize Paeonia suffruticosa Andr. into fine powder for standby; E. Use the fine powder of Paeonia suffruticosa Andr. as the base material, granulate by the conventional process, size the granules, and press into tablets to obtain them.

8. The quality control method according to claim 6, wherein, The operating steps of the preparation process of the tablet include: A. Coarsely crush Atractylodes macrocephala Koidz. and Notopterygium incisum Ting ex Hsiao & K. C. Hsia in the prescription, and coarsely crush the shell of Magnolia liliflora Desr.; Weigh Atractylodes macrocephala Koidz., Saposhnikovia divaricata Turcz., Alpinia officinarum Hance, Notopterygium incisum Ting ex Hsiao & K. C. Hsia, and Magnolia liliflora Desr. according to the prescription amount, add 8 - 10 times the amount of water, extract the volatile oil by steam distillation for 8 hours, collect the volatile oil for standby; Filter the aqueous solution after distillation, concentrate it under reduced pressure for standby; B. Weigh the astragalus root, cicada slough, and liquorice root according to the prescription amount, add 10 - 12 times the amount of water, decoct for three times, 1 hour each time, filter, concentrate the extract under reduced pressure, combine it with the volatile oil extract, and concentrate under reduced pressure to a relative density of 1.15 - 1.20 for standby. C. Weigh the dahurian angelica root and smoked plum according to the prescription amount, add 6 - 7 times the amount of 60 - 70% ethanol, extract twice, 2 hours for the first time and 1.5 hours for the second time, filter, recover the ethanol from the extract under reduced pressure, and concentrate under reduced pressure to a relative density of 1.15 - 1.20 for standby. D. Sterilize the cortex moutan and pulverize it into fine powder for standby. E. Using the fine powder of cortex moutan as the base material, granulate, screen the granules, and press tablets according to the conventional process to obtain the product.

9. The quality control method according to claim 1, wherein, The raw material composition of the traditional Chinese medicine composition by weight parts is as follows: 10 parts of astragalus root, 15 parts of atractylodes macrocephala, 5 parts of ledebouriella root, 15 parts of magnolia flower bud, 5 parts of dahurian angelica root, 15 parts of galangal, 5 parts of incised notopterygium root, 15 parts of cortex moutan, 5 parts of cicada slough, 15 parts of smoked plum, and 4 parts of liquorice root.

10. The quality control method according to claim 1, wherein, The raw material composition of the traditional Chinese medicine composition by weight parts is as follows: 20 parts of astragalus root, 5 parts of atractylodes macrocephala, 15 parts of ledebouriella root, 5 parts of magnolia flower bud, 15 parts of dahurian angelica root, 5 parts of galangal, 15 parts of incised notopterygium root, 5 parts of cortex moutan, 15 parts of cicada slough, 5 parts of smoked plum, and 8 parts of liquorice root.

11. The quality control method according to claim 1, wherein, The raw material composition of the traditional Chinese medicine composition by weight parts is as follows: 15 parts of astragalus root, 10 parts of atractylodes macrocephala, 10 parts of ledebouriella root, 10 parts of magnolia flower bud, 10 parts of dahurian angelica root, 10 parts of galangal, 10 parts of incised notopterygium root, 10 parts of cortex moutan, 10 parts of cicada slough, 10 parts of smoked plum, and 6 parts of liquorice root.

12. The quality control method according to claim 1, wherein, The raw material composition of the traditional Chinese medicine composition by weight parts is as follows: 16 parts of astragalus root, 8 parts of atractylodes macrocephala, 8 parts of ledebouriella root, 8 parts of magnolia flower bud, 8 parts of dahurian angelica root, 8 parts of galangal, 8 parts of incised notopterygium root, 8 parts of cortex moutan, 8 parts of cicada slough, 8 parts of smoked plum, and 5 parts of liquorice root.

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