A quality detection method for a traditional Chinese medicine composition for treating gynecological blood diseases

Abstract

The present invention belongs to the technical field of analytical chemistry, and specifically relates to a quality detection method for a traditional Chinese medicine composition for treating gynecological blood diseases. The quality detection method includes a thin layer chromatography qualitative detection method and a content detection method. The thin layer chromatography qualitative detection method uses isochlorogenic acid A and isochlorogenic acid B as reference substances, and is used in conjunction with Yunnan Guiyina Balsamifera reference medicinal materials to identify the sample. The method is used to analyze the sample, and the obtained thin layer chromatography separation effect is good, the spots are round and clear, and the development rate is fast. The result is easy to judge, and the sample can be quickly and accurately identified. The content detection method of the present invention has a relatively appropriate peak time and good separation. After methodological investigation, it was found that the specificity, stability, repeatability, intermediate precision, accuracy, etc. of the two methods all meet the requirements. The quality detection method provided by the present invention can be used to comprehensively control the quality of the traditional Chinese medicine composition, and further ensure the stability and controllability of the efficacy of the traditional Chinese medicine composition.

Description

Technical Field

[0001] The invention belongs to the technical field of analytical chemistry, and particularly relates to a quality detection method for a traditional Chinese medicine composition for treating gynecological blood diseases. Background Art

[0002] Yixue'an Granules are a traditional Chinese medicine for treating gynecological blood disorders. Its main ingredients include Artemisia argyi, Motherwort, Corydalis yanhusuo (vinegar-processed), and licorice. They have the effects of promoting blood circulation, stopping bleeding, promoting qi circulation, and relieving pain. Clinically, they are used for persistent lochia after childbirth, post-abortion uterine bleeding, and those diagnosed with blood stasis according to Traditional Chinese Medicine.

[0003] In the National Food and Drug Administration standard YBZ00292008, the content determination method of Yixuean Granules uses a constant-speed mobile phase to only determine the content of protocatechuic acid, and the test solution is prepared by extraction with a water-saturated n-butanol solution; this method has a large negative interference on the main active ingredient protocatechuic acid, the determination is inaccurate, and cannot effectively control the content of protocatechuic acid in the finished product.

[0004] Artemisia annua is one of the main medicinal materials in Yixuean Granules that plays a role in promoting blood circulation and stopping hemostasis. Modern research data show that phenolic acid compounds are one of the main active ingredients of Artemisia annua; Artemisia annua has the effects of promoting blood circulation, stopping hemostasis and promoting diuresis, and the ethyl acetate part of Artemisia annua (mainly containing phenolic acid compounds) has good hemostatic activity, indicating that isochlorogenic acid A and isochlorogenic acid B are closely related to the hemostatic effect of Artemisia annua.

[0005] Therefore, using isochlorogenic acid A and isochlorogenic acid B as the indicator components of Artemisia argyi in Yixuean granules will improve the quality standards of Yixuean granules and provide a scientific basis for better monitoring and evaluation of the quality of Yixuean granules. Summary of the Invention

[0006] The present invention aims to solve the above technical problems and provide a quality detection method for a traditional Chinese medicine composition for treating gynecological blood diseases, which can be used to comprehensively control the quality of the traditional Chinese medicine composition of the present invention and further ensure the stability and controllability of the therapeutic effect of the traditional Chinese medicine composition.

[0007] The technical solution of the present invention is:

[0008] A method for detecting the quality of a traditional Chinese medicine composition for treating gynecological blood disorders, wherein the traditional Chinese medicine composition comprises Artemisia argyi, Leonurus japonicus, Corydalis yanhusuo and Licorice, and the method for detecting the quality comprises a thin layer chromatography qualitative detection;

[0009] The thin layer chromatography qualitative detection comprises the following steps:

[0010] 1) Preparation of test solution and control medicinal material solution: Take the traditional Chinese medicine composition, dissolve it in water, adjust the pH to 10, sonicate, filter, extract the filtrate with chloroform, discard the chloroform layer, adjust the pH of the aqueous layer to 2, filter, extract the filtrate with ethyl acetate, combine the ethyl acetate layers, evaporate to dryness, and dissolve the residue in methanol to prepare the test solution; take Artemisia balsamifera as a control medicinal material and prepare a control medicinal material solution in the same manner;

[0011] 2) Preparation of reference solution: Take isochlorogenic acid A reference substance and isochlorogenic acid B reference substance, add methanol to make a solution as the reference solution;

[0012] 3) Thin layer chromatography identification: Spot the test sample solution, control medicinal material solution, and reference substance solution on the same thin layer plate, use ethyl acetate-formic acid-water upper solution as the developing agent, develop, take out, dry, and examine under ultraviolet light. In the test sample chromatogram, spots of the same color appear at the corresponding positions in the control medicinal material and reference substance chromatograms.

[0013] Furthermore, step 1) is: taking the traditional Chinese medicine composition, grinding it, adding 50 ml of water to dissolve it, adjusting the pH to 10 with sodium hydroxide, ultrasonically treating it for 30 minutes, filtering, extracting the filtrate with chloroform twice, 20 ml each time, discarding the chloroform layer, adjusting the pH of the aqueous layer to 2 with dilute hydrochloric acid, filtering, extracting the filtrate with ethyl acetate twice, 20 ml each time, combining the ethyl acetate layers, evaporating them to dryness, and adding an appropriate amount of methanol to the residue to dissolve it as the test solution; taking another 2 g of Yunnan gui Artemisia balsamifera control medicinal material, and preparing a control medicinal material solution in the same way.

[0014] Furthermore, step 2) is: taking isochlorogenic acid A reference substance and isochlorogenic acid B reference substance, adding methanol to prepare a solution containing 1 mg per 1 ml as a reference solution.

[0015] Furthermore, step 3) is: aspirate 5-10 μl of the test sample solution, 5-10 μl of the control medicinal material solution, and 3-5 μl of the reference substance solution, and spot them respectively on the same high-efficiency silica gel GF254 thin layer plate, use the upper layer solution of ethyl acetate-formic acid-water as the developing agent, develop, take out, dry, and place under an ultraviolet lamp with a wavelength of 254 nm for inspection. In the chromatogram of the test sample, spots of the same color appear at the corresponding positions of the chromatograms of the control medicinal material and the reference substance.

[0016] Furthermore, in the ethyl acetate-formic acid-water, the volume ratio of ethyl acetate, formic acid and water is 12:0.5:0.1.

[0017] Furthermore, the quality detection method also includes a method for detecting the content of isochlorogenic acid A and isochlorogenic acid B.

[0018] Furthermore, the method for detecting the content of isochlorogenic acid A and isochlorogenic acid B comprises the following steps:

[0019] Step S1, preparation of reference solution: taking isochlorogenic acid A and isochlorogenic acid B reference substances, adding methanol solution to prepare a mixed reference solution, that is, obtaining a reference solution;

[0020] Step S2, preparation of a test solution: taking the traditional Chinese medicine composition, dissolving it in water, adjusting the pH to 10, ultrasonically treating, filtering, extracting the filtrate with chloroform, discarding the chloroform layer, adjusting the pH of the aqueous layer to 2, filtering, extracting the filtrate with ethyl acetate, combining the ethyl acetate layers, evaporating to dryness, and dissolving the residue with methanol to obtain a test solution;

[0021] Step S3, content determination: Take the reference solution in step S1 and the test solution in step S2 respectively and detect them according to the following chromatographic conditions: octadecylsilane bonded silica gel as the filler; acetonitrile-0.05% phosphoric acid solution as the mobile phase; and the detection wavelength is 330 nm.

[0022] Furthermore, step S1 is: taking an appropriate amount of isochlorogenic acid A and isochlorogenic acid B reference substances, accurately weighing them, and adding methanol solution to prepare a mixed reference solution containing 20 μg of isochlorogenic acid A and isochlorogenic acid B per 1 ml, thereby obtaining a reference solution.

[0023] Furthermore, step S2 is: taking the traditional Chinese medicine composition, accurately weighing it, placing it in a stoppered conical flask, accurately adding 50 ml of water to dissolve it, adjusting the pH to 10 with sodium hydroxide, ultrasonically treating it for 30 minutes, filtering it, and extracting the filtrate with chloroform twice, 20 ml each time, discarding the chloroform layer, adjusting the water layer to pH 2 with dilute hydrochloric acid, filtering it, and extracting the filtrate with ethyl acetate twice, 20 ml each time, combining the ethyl acetate layers, evaporating them to dryness, dissolving the residue with methanol, and making the volume to 25 ml, shaking it well, and filtering it through a microporous filter membrane to obtain the test solution.

[0024] Furthermore, in the acetonitrile-0.05% phosphoric acid solution of step S3, the volume ratio of acetonitrile to 0.05% phosphoric acid is 25:75.

[0025] Due to the adoption of the above technical solution, the beneficial effects of the present invention are:

[0026] The quality detection method described in the present invention includes a thin-layer chromatography qualitative detection method and a content detection method. The thin-layer chromatography qualitative detection method uses isochlorogenic acid A and isochlorogenic acid B as reference substances, and is used in combination with the Yunnan cinnamon Artemisia balsamifera reference medicinal material to identify the sample. When the method is used to analyze the sample, the obtained thin-layer chromatography separation effect is good, the spots are round and clear, the development rate is fast, the results are easy to judge, and the sample can be quickly and accurately identified.

[0027] The content detection method described in the present invention has a relatively appropriate peak time and good separation. Methodological investigations have found that the specificity, stability, repeatability, intermediate precision, accuracy, etc. of the two methods all meet the requirements.

[0028] The quality detection method provided by the present invention can be used to comprehensively control the quality of the traditional Chinese medicine composition, and further ensure the stability and controllability of the therapeutic effect of the traditional Chinese medicine composition. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 The thin layer chromatogram of Artemisia balsamifera in Yixue'an granules of the present invention is shown in the figure. In the figure, the characteristic spots are: A: isochlorogenic acid A, Rf is about 0.78; B: isochlorogenic acid B, Rf is about 0.43; the order of spotting is: 1-10 test solution (batch number: 210807, 210808, 210902, 220101, 220103, 220202, 220401, 220402, 220403, 220901); 11. Artemisia balsamifera control medicinal material; 12. isochlorogenic acid A reference substance; 13. isochlorogenic acid B reference substance; 14. Artemisia balsamifera negative sample lacking Artemisia balsamifera;

[0030] Figure 2 The thin layer chromatogram of Artemisia balsamifera in Yixue'an granules of the present invention (sugar-free) is shown in the figure. In the figure, the characteristic spots are: A: isochlorogenic acid A, Rf is about 0.81; B: isochlorogenic acid B, Rf is about 0.49; the order of spotting is: 1. Negative sample lacking Artemisia balsamifera; 2-12 test solution (batch number: 200301, 220102, 220501, 220801, 220802, 220803, 220804, 220805, 220806, 220807, 220902); 13. Artemisia balsamifera reference medicinal material; 14. Isochogenic acid A reference substance; 15. Isochogenic acid B reference substance;

[0031] Figure 3 This is a thin layer chromatogram of Artemisia argyi in Yixuean granules for identification (1). In the figure, 1. test solution (batch number: 210807); 2. isochlorogenic acid A reference substance; 3. test solution (batch number: 220102); 4. isochlorogenic acid B reference substance; 5. Artemisia argyi reference medicinal material solution;

[0032] Figure 4 This is the thin layer chromatogram of Artemisia argyi in Yixuean granules for identification (2). In the figure, 1. test solution (batch number: 210807); 2. isochlorogenic acid A reference substance; 3. test solution (batch number: 220102); 4. isochlorogenic acid B reference substance; 5. Artemisia argyi reference medicinal material solution;

[0033] Figure 5This is a thin layer chromatogram of Artemisia argyi in another Yixuean granule for identification (2). In the figure, 1. test solution (batch number: 210807); 2. isochlorogenic acid A reference substance; 3. test solution (batch number: 220102); 4. isochlorogenic acid B reference substance; 5. Artemisia argyi reference medicinal material solution;

[0034] Figure 6 This is a thin layer chromatogram of Artemisia argyi in Yixue'an granules for [Identification] (3), in which: 1. Test solution (batch number: 210807); 2. Test solution (batch number: 220102); 3. Isochlorogenic acid A reference substance and Isochlorogenic acid B reference substance.

[0035] Figure 7 This is a thin layer chromatogram of Artemisia argyi in Yixue'an granules for [Identification] (4). In the figure, 1. test sample solution (batch number: 210807); 2. test sample solution (batch number: 220102); 3. test sample solution (batch number: 220901); 4. Artemisia argyi reference material; 5. isochlorogenic acid A reference substance; 6. isochlorogenic acid B reference substance; 7. negative control sample solution lacking Artemisia argyi.

[0036] Figure 8 This is a thin layer chromatogram of Artemisia argyi in Yixue'an granules for [Identification] (5), in which: 1. Test sample solution (batch number: 220102); 2. Isochlorogenic acid A reference substance; 3. Isochlorogenic acid B reference substance.

[0037] Figure 9 This is a thin layer chromatogram of Artemisia argyi in Yixue'an granules for [Identification] (6), in which: 1. Test solution (batch number: 210807); 2. Test solution (batch number: 220102); 3. Isochlorogenic acid A reference substance and Isochlorogenic acid B reference substance.

[0038] Figure 10 This is a chromatogram of the content determination of isochlorogenic acid A and isochlorogenic acid B in Yixue'an granules. In the figure, 1. isochlorogenic acid A; 2. isochlorogenic acid B;

[0039] Figure 11 This is a chromatogram of the content determination of the mixed reference substance. In the figure, 1. isochlorogenic acid A; 2. isochlorogenic acid B;

[0040] Figure 12 This is a chromatogram of the content determination of Yixuean granules sample (with sugar). In the figure, 1. isochlorogenic acid A; 2. isochlorogenic acid B;

[0041] Figure 13 This is a chromatogram of the content determination of Yixuean granules sample (sugar-free), in which: 1. isochlorogenic acid A; 2. isochlorogenic acid B;

[0042] Figure 14This is a chromatogram of the content determination of the medicinal material sample of Artemisia argyi. In the figure, 1. isochlorogenic acid A; 2. isochlorogenic acid B;

[0043] Figure 15 This is the chromatogram of the content determination of the negative sample (containing sugar) lacking Artemisia yunnanensis;

[0044] Figure 16 This is the chromatogram of the content determination of the negative sample lacking Artemisia argyi (no sugar);

[0045] Figure 17 is the standard curve of isochlorogenic acid A;

[0046] Figure 18 is the standard curve of isochlorogenic acid B;

[0047] Figure 19 This is the lower limit of quantification and noise chromatogram of Yixuean granules;

[0048] Figure 20 This is a diagram of the stability test of isochlorogenic acid A and isochlorogenic acid B in Yixuean granules (containing sugar);

[0049] Figure 21 This is a diagram of the stability test of isochlorogenic acid A and isochlorogenic acid B in Yixuean granules (sugar-free);

[0050] Figure 22 This is a repeatability test diagram of isochlorogenic acid A and isochlorogenic acid B in Yixuean granules (containing sugar);

[0051] Figure 23 This is the repeatability test diagram of isochlorogenic acid A and isochlorogenic acid B in Yixuean granules (sugar-free); DETAILED DESCRIPTION

[0052] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0053] It should be noted that the traditional Chinese medicine composition (Yixuean granules) of the present invention can be prepared by the following preparation method:

[0054]

Prescription

[0055] Corydalis yanhusuo (vinegar-processed) 133.3g, licorice 66.7g

[0056] [Preparation method] The above four ingredients, Corydalis yanhusuo, are crushed into coarse powder, and the percolation method under the extract and extract item (Chinese Pharmacopoeia 2020 Edition Part IV General Rules 0189) is used, using 70% ethanol as the solvent. After soaking for 24 hours, slowly percolate at a rate of 3 ml per minute, collect about 1000 ml of the percolate, recover the ethanol, and concentrate it to a clear paste with a relative density of 1.10-1.20 (60°C); the other three ingredients, including Yunnan guimei linalool, are decocted three times with water (12, 10, and 8 times), and each The decoction is then combined for 1.5 hours, filtered, and the filtrate is concentrated to a relative density of 1.12-1.17 (60°C). The mixture is allowed to stand for more than 8 hours, filtered, and the filtrate is concentrated to a clear paste with a relative density of 1.20-1.28 (60°C). The above two clear pastes are added with appropriate amounts of sucrose powder and soluble starch, made into granules, dried, and made into 1000g [specification (1)]; or added with appropriate amounts of soluble starch, made into granules, dried, and made into 533g [specification (2)].

[0057] Example 1 Quality research and literature data of the present invention

[0058] 1. Source and batch number of the test sample

[0059] (1) Yixuean Granules [Specification (1)] (Contains sugar) (Guangxi Wanshoutang Pharmaceutical Co., Ltd.) Batch numbers: 210807, 210808, 210902, 220101, 220103, 220202, 220401, 220402, 220403, 220901.

[0060] (2) Yixuean Granules [Specification (2)] (Sugar-free) (Guangxi Wanshoutang Pharmaceutical Co., Ltd.) Batch No.: 200301, 220102, 220501, 220801, 220802, 220803, 220804, 220805, 220806, 220807, 220902.

[0061] (3) Negative control sample: The three medicinal materials except Artemisia argyi were prepared in-house according to the prescription ratio and preparation method in the main text.

[0062] 2. Source, batch number, and content of the reference substances and reference medicinal materials used in the test

[0063] Isochlorogenic acid A (purity > 98%, RFS-Y06801911011, for content determination, Chengdu Ruifensi Biotechnology Co., Ltd.), isochlorogenic acid B (purity ≥ 98%, DST210625-037, for content determination, Lemeitian Pharmaceutical / Desit Biotechnology).

[0064] The reference medicinal material of Artemisia argyi was provided by the China National Institute for the Control of Pharmaceutical and Biological Products, batch number: batch number 121647-201602, for identification purposes.

[0065] 3. Identification

[0066] 3.1 Thin layer chromatography identification of Artemisia balsamifera

[0067] (1) Take 30g [Specification (1)] (containing sugar) or 16g [Specification (2)] (without sucrose) of this product, grind it into powder, add 50ml of water to dissolve it, adjust the pH to 10 with sodium hydroxide, ultrasonicate it for 30 minutes, filter it, extract the filtrate with chloroform twice, 20ml each time, discard the chloroform layer, adjust the pH of the water layer to 2 with dilute hydrochloric acid, filter it, extract the filtrate with ethyl acetate twice, 20ml each time, combine the ethyl acetate layers, evaporate to dryness, and dissolve the residue in methanol to prepare the test solution. Take 2g of the control medicinal material of Artemisia argyi and prepare the control medicinal material solution in the same way. Take the reference substance of isochlorogenic acid A and the reference substance of isochlorogenic acid B, add methanol to prepare a solution containing 1mg per 1mL, and prepare the reference solution. According to the thin layer chromatography method (General Rules 0502 of Part IV of the 2020 edition of the Chinese Pharmacopoeia), 5-10 μl of the test solution, 5-10 μl of the reference medicinal material solution, and 3-5 μl of the reference solution were respectively applied to the same high-efficiency silica gel GF 254 For thin layer plates, use the upper layer solution of ethyl acetate-formic acid-water (12:0.5:0.1) as the developing solvent, develop, remove, dry, and examine under ultraviolet light (254nm). In the chromatogram of the test sample, spots of the same color appear at the corresponding positions in the chromatograms of the reference medicinal material and the reference sample. Figure 1 and Figure 2 .

[0068] Negative control: According to the prescribed amount, take Leonurus japonicus, Corydalis yanhusuo (vinegar-processed), and Licorice root herbs, and make granules lacking Artemisia argyi and Artemisia argyi according to the provisions under the above [Preparation Method]. Weigh an appropriate amount of granules and make a negative sample lacking Artemisia argyi and Artemisia argyi according to the same method as the test sample. The negative control test was carried out. The results showed that the other three herbs had no interference with this identification. Figure 1 and Figure 2 .

[0069] After the identification test of multiple batches of this product, the test sample chromatogram showed spots of the same color at the corresponding positions of the reference sample and reference medicinal material chromatograms. Figure 1 and Figure 2 The experimental results show that this thin layer chromatography identification has good reproducibility and strong specificity.

[0070] In addition to the thin layer chromatography identification mentioned above, this application also conducted the following comparative tests:

[0071]

Identification

[0072] [Identification] (2): Take the test solution under [Identification] (1) as the test solution. Take the reference solution under [Identification] (1) as the reference solution. According to the thin layer chromatography method (General Rules 0502 of Part Four of the 2020 edition of the Chinese Pharmacopoeia), 5-10 μL of the test solution and 3-5 μL of the reference solution were taken and spotted on the same polyamide film respectively. 36% glacial acetic acid solution was used as the developing agent. The film was developed, taken out, and dried. ① It was placed under ultraviolet light (365 nm) for inspection; ② It was sprayed with 2% aluminum chloride ethanol solution, and hot air was blown until the spots were clearly colored, and then placed under ultraviolet light (365 nm) for inspection. The results showed that Figure 4 and Figure 5 The chromatogram of the test sample showed fluorescent spots of the same color at the corresponding positions of the chromatogram of the reference sample, but the spot separation was poor. Even after adjusting the concentrations of glacial acetic acid solutions, effective separation was not possible.

[0073]

Identification

Identification

Identification

[0074]

Identification

Identification

[0075]

Identification

Identification

[0076]

Identification

[0077] 4. Content determination

[0078] 4.1 Solution Preparation

[0079] (1) Preparation of reference solution: Take appropriate amounts of isochlorogenic acid A and isochlorogenic acid B reference substances, place them in the same 10 mL volumetric flask, and add methanol to make a mixed reference solution containing 20 μg of each of isochlorogenic acid A and isochlorogenic acid B per 1 mL.

[0080] (2) Preparation of test solution Take Yixuean granules, mix them, take an appropriate amount, grind them into powder, take Yixuean granules (sugar 1g / sugar-free 0.5g), weigh them accurately, place them in a stoppered conical flask, accurately add 50mL of water to dissolve them, adjust the pH to 10 with sodium hydroxide, ultrasonically treat (power 320W, frequency 80kHz) for 30 minutes, filter, extract the filtrate with chloroform twice, 20mL each time, discard the chloroform layer, adjust the water layer to pH 2 with dilute hydrochloric acid, filter, extract the filtrate with ethyl acetate twice, 20mL each time, combine the ethyl acetate layers, evaporate to dryness, dissolve the residue with methanol, and make up to 25mL, shake well, filter through a microporous filter membrane, and obtain the test solution.

[0081] (3) Preparation of control medicinal material solution: Extract the control medicinal material sample solution according to the preparation method of the test solution.

[0082] (4) Preparation of negative sample solution: According to the prescribed amount, take the medicinal materials such as Leonurus japonicus, Corydalis yanhusuo (vinegar-processed), and Licorice root, and prepare granules lacking Artemisia argyi according to the provisions under the above-mentioned [Preparation Method]. Accurately weigh an appropriate amount of granules and extract them according to the preparation method of the test solution to prepare a negative sample solution.

[0083] 4.2 Chromatographic conditions

[0084] Column: Waters Xbridge-C18 Column (5 μm, 4.6×250 mm), column temperature: 25°C; mobile phase: acetonitrile-0.05% phosphoric acid aqueous solution (25:75); injection volume: 10 μL; wavelength: 330 nm; theoretical plate number calculated based on the isochlorogenic acid A peak is not less than 3000.

[0085] 4.3 System suitability test

[0086] 10 μL of each of the reference solution, test solution, control medicinal material sample solution, and negative sample solution were injected. Under the above chromatographic conditions, the reference, medicinal material, test, and negative samples were analyzed. The retention time of isochlorogenic acid A was approximately 18 minutes, and the retention time of isochlorogenic acid B was approximately 17 minutes. The test sample was completely separated, and there was basically no interference with the negative sample at 16 and 18 minutes. Therefore, it can be used for quantitative analysis ( Figure 10 ).

[0087] 4.4 Specificity test

[0088] Yixuean granules ([Specification (1)] Batch No.: 220101 (with sugar); [Specification (2)] 220102 (without sugar)) were prepared according to the method under "Preparation of test solution", the reference substance was prepared according to the method under "Preparation of reference solution", and the negative control was prepared according to "Preparation of negative control solution". The chromatographic conditions were used for analysis. The results showed that the content determination of isochlorogenic acid A and isochlorogenic acid B was not interfered by factors such as solvents and other medicinal materials, and had good specificity. Figures 11-16 .

[0089] 4.5 Linear Relationship and Range

[0090] Take 19.15 mg of isochlorogenic acid A reference substance, accurately weighed, placed in a 25 mL volumetric flask, added methanol to dissolve and dilute to the scale; take 21.05 mg of isochlorogenic acid B reference substance, accurately weighed, placed in a 10 mL volumetric flask, added methanol to dissolve and dilute to the scale; as the reference substance stock solution.

[0091] The stock solutions of isochlorogenic acid A (30 μL, 60 μL, 120 μL, 240 μL, 480 μL, 960 μL) and isochlorogenic acid B (30 μL, 60 μL, 120 μL, 240 μL, 480 μL, 960 μL) were accurately measured and numbered 1-6, and placed in 10 mL volumetric flasks numbered 1-6. Methanol was added to dilute to the scale, shaken, and a series of concentrations of mixed reference solutions were prepared; 10 μL of each of the 6 mixed reference solutions with different concentrations was drawn, injected, and measured. The injection volume of each component was used as the abscissa (X) and the peak area was used as the ordinate (Y). The standard curve was drawn to obtain the regression equation and correlation coefficient. The results showed that the standard curves had good linearity within the linear range (the determination results are shown in Tables 1 and 2, the linear range is shown in Table 3, and the standard curve is shown in Table 3). Figure 17 、 Figure 18 ).

[0092] Table 1 Isochlorogenic acid A determination results

[0093]

[0094] Table 2 Isochlorogenic acid B determination results

[0095]

[0096] Table 3 Regression equations and linear ranges of the two components

[0097]

[0098]

[0099] 4.6 Lower limit of quantification

[0100] Take an appropriate amount of isochlorogenic acid A (concentration: 0.0569 μg / mL) and isochlorogenic acid B reference solution (concentration: 0.0617 μg / mL), inject 10 μL, and determine according to the method. When the signal-to-noise ratio is greater than 10, the corresponding isochlorogenic acid A and isochlorogenic acid B are 0.0057 and 0.0062 μg, respectively. After 5 consecutive injections, the peak area RSD values ​​were 1.49% and 2.33% (Table 4). Therefore, the quantitative limits of isochlorogenic acid A and isochlorogenic acid B of this content determination method are 0.0057 and 0.0062 μg, respectively ( Figure 19 ).

[0101] Table 4 Quantitative limits of isochlorogenic acid A and isochlorogenic acid B

[0102]

[0103] 4.7 Precision test

[0104] Prepare isochlorogenic acid A and isochlorogenic acid B reference substances according to the method under "Preparation of reference solution", and prepare the test solution according to the method under "Preparation of test solution". According to the above chromatographic conditions, inject the samples continuously 6 times on the high performance liquid chromatograph, record the peak areas of isochlorogenic acid A and isochlorogenic acid B, and calculate the RSD values.

[0105] Results The RSD of the peak area of ​​isochlorogenic acid A in the reference sample was 1.55%, and the RSD of the peak area of ​​isochlorogenic acid B was 1.27%. The RSD of the peak area of ​​isochlorogenic acid A in the sugar-containing sample was 1.43%, and the RSD of the peak area of ​​isochlorogenic acid B in the sugar-containing sample was 0.99%. The RSD of the peak area of ​​isochlorogenic acid A in the sugar-free sample was 1.66%, and the RSD of the peak area of ​​isochlorogenic acid B in the sugar-free sample was 1.24%, indicating that the method had good precision (Table 5).

[0106] Table 5 Precision test results

[0107]

[0108] 4.8 Intermediate Precision

[0109] The same batch of Yixuean granules ([Specification (1)] sugar-containing batch number: 220101 and [Specification (2)] sugar-free batch number: 220102) were taken and processed according to the method under the "Preparation of Test Solution" under different time, different analysts, different equipment and other variable factors. The content was determined according to the above chromatographic conditions, and the relative average deviation (RAD) of the content of isochlorogenic acid A and isochlorogenic acid B was calculated. The results showed that the relative average deviation values ​​of the content of isochlorogenic acid A and isochlorogenic acid B in Yixuean granules under different analysts, different dates and different equipment conditions were within a reasonable range, indicating that the intermediate precision of this method was good. The results are shown in Tables 6 to 9.

[0110] Table 6 Intermediate precision test for determination of isochlorogenic acid A content in Yixuean granules (containing sugar)

[0111]

[0112]

[0113] Table 7 Intermediate precision test for determination of isochlorogenic acid B content in Yixuean granules (containing sugar)

[0114]

[0115] Table 8 Intermediate precision test for determination of isochlorogenic acid A content in Yixuean granules (sugar-free)

[0116]

[0117]

[0118] Table 9 Intermediate precision test for determination of isochlorogenic acid B content in Yixuean granules (sugar-free)

[0119]

[0120] 4.9 Stability test

[0121] A batch of Yixuean granules test solution ([Specification (1)] sugar-containing batch number: 220101 and [Specification (2)] sugar-free batch number: 220102) was prepared according to the method under "Preparation of test solution". The isochlorogenic acid A reference substance and isochlorogenic acid B reference substance were prepared according to the method under "Preparation of reference solution". The content was determined according to the above chromatographic conditions. The samples were injected at 0h, 2h, 4h, 6h, 8h, and 10h. The peak areas of isochlorogenic acid A and isochlorogenic acid B were measured and the RSD values ​​were calculated. The RSD of the peak area of ​​the sugar-containing test sample (isochlorogenic acid A) was 1.59%, and the RSD of the peak area of ​​the sugar-containing test sample (isochlorogenic acid B) was 2.95%; the RSD of the peak area of ​​the sugar-free test sample (isochlorogenic acid A) was 2.26%, and the RSD of the peak area of ​​the test sample (isochlorogenic acid B) was 0.94%. The results showed that both sugar-containing and sugar-free test solutions had good stability after 10 hours. Figure 20 and Figure 21 , Table 10 and Table 11.

[0122] Table 10 Stability test results (sugar content)

[0123]

[0124]

[0125] Table 11 Stability test results (sugar-free)

[0126]

[0127] 4.10. Repeatability test 1 [Specification (1)] (Containing sugar)

[0128] Take an appropriate amount of the same batch of this product (batch number: 220101), grind it into powder, take 6 samples respectively, each weighing about 1g, accurately weigh, extract and determine according to the method under "Preparation of test solution", and calculate the average content and RSD% of isochlorogenic acid A and isochlorogenic acid B in the test solution. The average content of isochlorogenic acid A in 6 sugar-containing test samples was 0.5142mg / g, and the RSD% value was 2.30%. The average content of sugar-containing isochlorogenic acid B was 0.9177mg / g, and the RSD% value was 1.19%. The results show that this method has good repeatability, as shown in the results. Figure 22 , Table 12, Table 13.

[0129] Table 12 Repeatability test results of test sample (isochlorogenic acid A) (n=6) (containing sugar)

[0130]

[0131] Table 13 Repeatability test results of test sample (isochlorogenic acid B) (n=6) (containing sugar)

[0132]

[0133]

[0134] Reproducibility test 2 [Specification (2)] (no sucrose) Take an appropriate amount of the same batch of this product (batch number: 220102), grind it into powder, take 6 samples respectively, each weighing about 0.5g, accurately weigh, extract and determine according to the method under "Preparation of test solution", and calculate the average content and RSD% of isochlorogenic acid A and isochlorogenic acid B in the test solution. The average content of isochlorogenic acid A in 6 sugar-free test samples was 1.0410mg / g, and the RSD% value was 2.47%; the average content of isochlorogenic acid B was 1.8102mg / g, and the RSD% value was 0.92%. The results show that the method has good reproducibility, as shown in the results. Figure 23 , Table 14, Table 15.

[0135] Table 14 Repeatability test results of test sample (isochlorogenic acid A) (n=6) (sugar-free)

[0136]

[0137] Table 15 Repeatability test results of test sample (isochlorogenic acid B) (n=6) (sugar-free)

[0138]

[0139] 4.11. Sample recovery test 1 [Specification (1)] (containing sucrose)

[0140] Take 0.5 g of each sample with known content (batch number 220101, isochlorogenic acid A content of 0.5142 mg / g, isochlorogenic acid B content of 0.9177 mg / g), a total of 6 portions, take 0.34 mL of isochlorogenic acid A (concentration of 0.766 mg / mL) and 0.56 mL of isochlorogenic acid B (concentration of 0.842 mg / mL), add to the sample, mix well, extract according to the above test solution preparation method, and measure the peak area according to the above chromatographic conditions. Calculate the content and recovery rate of isochlorogenic acid A and isochlorogenic acid B. The results show that the average recovery rate of isochlorogenic acid A is 101.30%, RSD = 2.21%; the average recovery rate of isochlorogenic acid B is 101.88%, RSD = 2.37%. The test results are shown in Tables 16 and 17.

[0141] Table 16 Sample recovery test results (sugar-containing)

[0142]

[0143] Table 17 Sample recovery test results (sugar-containing)

[0144]

[0145] Sample recovery test 2 [Specification (2)] (sugar-free) Take 0.25 g of each sample with known content (batch number 220102, isochlorogenic acid A content of 1.0410 mg / g, isochlorogenic acid B content of 1.7988 mg / g), a total of 6 parts, take 0.34 mL of isochlorogenic acid A (concentration of 0.766 mg / mL) and 0.54 mL of isochlorogenic acid B (concentration of 0.842 mg / mL), add to the sample, mix well, extract according to the above test solution preparation method and determine the peak area according to the above chromatographic conditions and calculate the content and recovery of isochlorogenic acid A and isochlorogenic acid B. Results The average recovery rate of isochlorogenic acid A was 100.99%, RSD = 1.82%; the average recovery rate of isochlorogenic acid B was 101.24%, RSD = 1.31%. The test results are shown in Tables 18 and 19.

[0146] Table 18 Sample recovery test results (sugar-free)

[0147]

[0148] Table 19 Sample recovery test results (sugar-free)

[0149]

[0150] 4.12, Durability 1 [Specification (1)] (Sugar-containing)

[0151] The same batch of Yixuean granules (batch number: 220101) were taken and treated according to the method under "Preparation of test solution". The content was determined according to the above chromatographic conditions. Waters Xbridge-C 18 Column (5 μm, 4.6 × 250 mm), ECOSILODS-C 18 Column (5 μm, 4.6 × 250 mm), Dalian Yilite-C 18 The contents of isochlorogenic acid A and isochlorogenic acid B were determined using a 5 μm column (4.6 × 250 mm). The relative standard deviations (RSDs) for the contents of isochlorogenic acid A and isochlorogenic acid B were calculated. The RSD values ​​for isochlorogenic acid A were 2.03%, and for isochlorogenic acid B were 0.39%. The results demonstrate the robustness of this method. The results are shown in Tables 20 and 21.

[0152] Table 20 Determination of the content of isochlorogenic acid A in Yixuean granules Durability test (containing sugar)

[0153]

[0154]

[0155] Table 21 Durability test for determination of isochlorogenic acid B content in Yixuean granules (sugar-containing)

[0156]

[0157] Durability 2 [Specification (2)] (Sugar-free)

[0158] The same batch of Yixuean granules (batch number: 220102) were taken and treated according to the method under "Preparation of test solution". The content was determined according to the above chromatographic conditions. Waters Xbridge-C 18 Column (5 μm, 4.6 × 250 mm), ECOSILODS-C 18 Column (5 μm, 4.6 × 250 mm), Dalian Yilite-C 18 The contents of isochlorogenic acid A and isochlorogenic acid B were determined using a 5 μm column (4.6 × 250 mm). The relative standard deviations (RSDs) for the contents of isochlorogenic acid A and isochlorogenic acid B were calculated. The RSD values ​​for isochlorogenic acid A were 2.28%, and for isochlorogenic acid B were 2.02%. The results demonstrate the robustness of this method. The results are shown in Tables 22 and 23.

[0159] Table 22 Determination of the content of isochlorogenic acid A in Yixuean granules Durability test (sugar-free)

[0160]

[0161] Table 23 Determination of the content of isochlorogenic acid B in Yixuean granules Durability test (sugar-free)

[0162]

[0163] 4.13. Content Determination of Yixuean Granules

[0164] (1) Determination of Yixuean Granules Content [Specification (1)] (Contains sucrose)

[0165] Ten batches of Yixuean granules produced by Guangxi Wanshoutang Pharmaceutical Co., Ltd. were prepared according to the method under "Preparation of test sample solution", and the reference sample was prepared according to the method under "Preparation of reference sample solution". The content was determined using the above-mentioned chromatographic conditions, and the contents of isochlorogenic acid A and isochlorogenic acid B in the ten batches of Yixuean granules were calculated.

[0166] Each 15g bag of this product contained 0.2955-0.6354 mg / g of isochlorogenic acid A in 10 batches of Yixue'an Granules, or 4.4325-9.5310 mg / bag. The isochlorogenic acid B content ranged from 0.4669-1.0559 mg / g, or 7.0035-15.8388 mg / bag. The results are shown in Tables 36 and 37. The results showed that the contents of isochlorogenic acid A and B varied significantly between batches of finished Yixue'an Granules. Preliminary analysis revealed that the differences in the contents of isochlorogenic acid A and B in different batches of the medicinal materials resulted in significant variations in the contents of these acids across batches of finished Yixue'an Granules. The results are shown in Tables 24 and 25.

[0167] Table 24 Determination of isochlorogenic acid A content in samples (n=2) (containing sugar)

[0168]

[0169]

[0170] Table 25 Determination of isochlorogenic acid B content in samples (n=2) (containing sugar)

[0171]

[0172]

[0173] (2) Determination of the content of Yixuean granules [Specification (2)] (sugar-free)

[0174] Eleven batches of Yixuean granules produced by Guangxi Wanshoutang Pharmaceutical Co., Ltd. were prepared according to the method under "Preparation of test sample solution", and the reference sample was prepared according to the method under "Preparation of reference sample solution". The content was determined using the above-mentioned chromatographic conditions, and the contents of isochlorogenic acid A and isochlorogenic acid B in the 11 batches of Yixuean granules were calculated.

[0175] Each bag of this product is 8g. The isochlorogenic acid A content in 11 batches of Yixue'an Granules ranged from 0.5561 to 1.2270 mg / g, or 4.4489 to 9.8157 mg / bag. The isochlorogenic acid B content ranged from 0.8927 to 2.0849 mg / g, or 7.1416 to 16.6794 mg / bag. The results are shown in Tables 38 and 39. The results showed that the contents of isochlorogenic acid A and B varied significantly among different batches of finished Yixue'an Granules. Preliminary analysis revealed that the differences in the contents of isochlorogenic acid A and B in different batches of the medicinal materials resulted in significant variations in the contents of these acids among different batches of finished Yixue'an Granules. The results are shown in Tables 26 and 27.

[0176] Table 26 Determination of isochlorogenic acid A content in samples (n=2) (sugar-free)

[0177]

[0178]

[0179] Table 27 Determination of isochlorogenic acid B content in samples (n=2) (sugar-free)

[0180]

[0181] The formulation of the content standard value is based on comprehensive consideration of the following factors:

[0182] Ten and eleven batches of Yixuean granules of two specifications were tested respectively. The content of isochlorogenic acid A in the finished product containing sucrose [Specification (1)] was the highest at 9.5310 mg / bag, the lowest at 4.4325 mg / bag, and the average was 6.9114 mg / bag; the content of isochlorogenic acid B was the highest at 15.8388 mg / bag, the lowest at 7.0035 mg / bag, and the average was 11.4289 mg / bag; the content of isochlorogenic acid A in the finished product without sugar [Specification (2)] was the highest at 9.8157 mg / bag, the lowest at 4.4236 mg / bag, and the average was 6.6620 mg / bag; the content of isochlorogenic acid B was the highest at 16.6794 mg / bag, the lowest at 7.1416 mg / bag, with an average of 11.3923 mg / bag; based on the experimental measurement results, and taking into account the different contents of isochlorogenic acid A and isochlorogenic acid B from different medicinal materials and the losses in industrial production, the uniformity of different batches and the stability test of retained samples, the limits of the contents of isochlorogenic acid A and isochlorogenic acid B in the finished products are appropriately lowered. Therefore, it is temporarily stipulated that the limits of the contents of isochlorogenic acid A and isochlorogenic acid B in sugar-containing finished products shall not be less than 3.9 mg and 6.1 mg per bag respectively, and the total content of the two items shall not be less than 10.0 mg; the limits of the contents of isochlorogenic acid A and isochlorogenic acid B in sugar-free finished products shall not be less than 3.8 mg and 6.2 mg per bag respectively, and the total content of the two items shall not be less than 10.0 mg.

[0183] The above description is a detailed description of the preferred embodiments of the present invention, but the embodiments are not intended to limit the scope of the patent application of the present invention. Any equivalent changes or modifications completed under the technical spirit suggested by the present invention should fall within the patent scope covered by the present invention.

Claims

1. A quality detection method for a traditional Chinese medicine composition for treating gynecological blood diseases, characterized in that: The traditional Chinese medicine composition comprises Artemisia argyi, Leonurus japonicus, Corydalis yanhusuo and Licorice, and the quality detection method comprises a thin layer chromatography qualitative detection; The thin layer chromatography qualitative detection comprises the following steps: 1) Preparation of test solution and control medicinal material solution: Take the traditional Chinese medicine composition, dissolve it in water, adjust the pH to 10, sonicate, filter, extract the filtrate with chloroform, discard the chloroform layer, adjust the pH of the aqueous layer to 2, filter, extract the filtrate with ethyl acetate, combine the ethyl acetate layers, evaporate to dryness, and dissolve the residue in methanol to prepare the test solution; take Artemisia balsamifera as a control medicinal material and prepare a control medicinal material solution in the same manner; 2) Preparation of reference solution: Take isochlorogenic acid A reference substance and isochlorogenic acid B reference substance, add methanol to make a solution as the reference solution; 3) Thin layer chromatography identification: The test sample solution, control medicinal material solution, and reference substance solution are spotted on the same thin layer plate, and the upper layer solution of ethyl acetate-formic acid-water is used as the developing solvent. The plate is developed, removed, air-dried, and examined under ultraviolet light. In the chromatogram of the test sample, spots of the same color appear at the corresponding positions in the chromatograms of the control medicinal material and reference substance. The volume ratio of ethyl acetate, formic acid, and water in the ethyl acetate-formic acid-water solution is 12:0.5:0.

1. The quality detection method also includes a method for detecting the content of isochlorogenic acid A and isochlorogenic acid B; The method for detecting the content of isochlorogenic acid A and isochlorogenic acid B comprises the following steps: Step S1, preparation of reference solution: taking isochlorogenic acid A and isochlorogenic acid B reference substances, adding methanol solution to prepare a mixed reference solution, that is, obtaining a reference solution; Step S2, preparation of a test solution: taking the traditional Chinese medicine composition, dissolving it in water, adjusting the pH to 10, ultrasonically treating, filtering, extracting the filtrate with chloroform, discarding the chloroform layer, adjusting the pH of the aqueous layer to 2, filtering, extracting the filtrate with ethyl acetate, combining the ethyl acetate layers, evaporating to dryness, and dissolving the residue with methanol to obtain a test solution; Step S3, content determination: The reference solution in step S1 and the test solution in step S2 were respectively taken and detected according to the following chromatographic conditions: octadecylsilane bonded silica gel as a filler; acetonitrile-0.05% phosphoric acid solution as a mobile phase; the detection wavelength was 330 nm, wherein the volume ratio of acetonitrile and 0.05% phosphoric acid in the acetonitrile-0.05% phosphoric acid solution was 25:

75.

2. The quality detection method of the Chinese medicine composition for treating gynecological blood diseases according to claim 1, wherein: Step 1) is: take the traditional Chinese medicine composition, grind it, add 50 ml of water to dissolve it, adjust the pH to 10 with sodium hydroxide, ultrasonicate for 30 minutes, filter, extract the filtrate with chloroform twice, 20 ml each time, discard the chloroform layer, adjust the pH of the aqueous layer to 2 with dilute hydrochloric acid, filter, extract the filtrate with ethyl acetate twice, 20 ml each time, combine the ethyl acetate layers, evaporate to dryness, and add an appropriate amount of methanol to dissolve the residue as the test solution; take 2 g of Yunnan gui Artemisia balsamifera control medicinal material and prepare a control medicinal material solution in the same way.

3. The quality detection method of the Chinese medicine composition for treating gynecological blood disease according to claim 1, wherein: Step 2) is: take isochlorogenic acid A reference substance and isochlorogenic acid B reference substance, add methanol to make a solution containing 1 mg per 1 ml, and use it as the reference solution.

4. The quality detection method of the Chinese medicine composition for treating gynecological blood diseases according to claim 1, wherein: Step 3) is as follows: 5-10 μl of the test solution, 5-10 μl of the control medicinal material solution, and 3-5 μl of the reference substance solution are respectively spotted on the same high-efficiency silica gel GF254 thin layer plate, and the upper layer solution of ethyl acetate-formic acid-water is used as the developing agent. The plate is developed, removed, dried, and examined under an ultraviolet lamp with a wavelength of 254 nm. In the chromatogram of the test sample, spots of the same color appear at the corresponding positions of the chromatograms of the control medicinal material and the reference substance.

5. The quality detection method of the Chinese medicine composition for treating gynecological blood diseases according to claim 1, wherein: Step S1 is: taking an appropriate amount of isochlorogenic acid A and isochlorogenic acid B reference substances, accurately weighing them, and adding methanol solution to prepare a mixed reference solution containing 20 μg of isochlorogenic acid A and isochlorogenic acid B per 1 ml, thereby obtaining a reference solution.

6. The quality testing method of the Chinese medicine composition for treating gynecological blood diseases according to claim 1, wherein: Step S2 is: taking the traditional Chinese medicine composition, accurately weighing it, placing it in a stoppered conical flask, accurately adding 50 ml of water to dissolve it, adjusting the pH to 10 with sodium hydroxide, ultrasonically treating it for 30 minutes, filtering it, and extracting the filtrate with chloroform twice, 20 ml each time, discarding the chloroform layer, and adjusting the pH of the aqueous layer to 2 with dilute hydrochloric acid. Filter, extract the filtrate with ethyl acetate twice, 20 ml each time, combine the ethyl acetate layers, evaporate to dryness, dissolve the residue with methanol, and adjust the volume to 25 ml, shake well, and filter through a microporous filter membrane to obtain the test solution.

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