Quality detection method of traditional Chinese medicine preparation for treating hypertension

Abstract

The invention belongs to the field of quality detection of traditional Chinese medicines, and discloses a quality detection method of a traditional Chinese medicine preparation for treating hypertension, which comprises a thin-layer chromatography identification method of moutan bark, sunflower leaf, selfheal, uncaria and kudzu vine root, and a high performance liquid chromatography content determination method of paeoniflorin and puerarin. The quality detection method covers all the medicines in the preparation prescription, is simple and convenient to operate, high in specificity and good in reproducibility, particularly, the same test solution is creatively adopted, and the moutan bark and the sunflower leaves are identified on the same thin-layer plate, so that the detection time is shortened, the detection cost is reduced, and the detection efficiency is improved. A new thought is provided for thin-layer identification of the traditional Chinese medicine compound preparation, and the quality standard of traditional Chinese medicine is improved. In the aspect of content determination, a multi-component high performance liquid phase content determination method is established, the specificity is high, the accuracy is high, the precision is good, the quality of the traditional Chinese medicine preparation can be effectively controlled, the stability of a product production process is monitored, and the stability, uniformity and controllability of the product quality are ensured.

Description

Technical Field

[0001] The invention belongs to the field of drug preparation detection, and in particular relates to a quality detection method for a traditional Chinese medicine preparation for treating hypertension. Background Art

[0002] Hypertension is a common and frequently occurring disease in clinical practice. It can lead to damage to target organs such as the heart, brain, and kidneys, and further cause stroke, coronary heart disease, and heart and kidney failure. It has a high disability and mortality rate, and seriously threatens human health and quality of life. It is one of the most common diseases that endanger human health at this stage, a major risk factor for death, and has become one of the serious global public health challenges.

[0003] To this end, the inventor disclosed a Chinese medicine preparation for treating hypertension in an invention patent (patent number: ZL200310105398.7). The prescription of the Chinese medicine preparation is: 10-38% of Uncaria rhynchophylla, 10-35% of Paeonia suffruticosa, 5-32% of Prunella vulgaris, 5-28% of sunflower leaves, and 4-30% of Pueraria lobata. The preparation method is: extract Uncaria rhynchophylla and Pueraria lobata with 60-80% ethanol twice, the first time for 2 hours and the second time for 1 hour, combine the extracts, filter, recover ethanol from the filtrate and concentrate it into a concentrated solution with a relative density of 1.16-1.25; then take Paeonia suffruticosa moutan to distill paeonol; decoct the residue and Prunella vulgaris with water twice, each time 1 hour, combine the extracts, filter, and concentrate the filtrate; take another sunflower leaf and add water to decoct twice, each time for 1 hour, combine the extracts, filter, and concentrate the filtrate; take the above two kinds of concentrated solutions of peony bark and sunflower leaf, combine them, add ethanol to make the alcohol content reach 50-70%, precipitate with alcohol, refrigerate for 24 hours, filter, recycle the ethanol in the filtrate and concentrate it into a concentrated solution with a relative density of 1.16-1.25; combine the above two kinds of concentrated solutions, continue to concentrate into a thick paste, which can be dried by different methods, crushed, add paeonol, mix, sieve, and obtain the extracted dry powder of the present invention; add one or more auxiliary materials to the powder to make granules, capsules, and tablets. The whole Chinese medicine preparation has the effects of calming the liver and suppressing yang, clearing away heat and activating blood circulation, and is used to treat hypertension. The syndrome differentiation of traditional Chinese medicine is liver yang hyperactivity syndrome, and the symptoms are: dizziness, headache, irritability, palpitations, insomnia, tinnitus, red face and eyes, red tongue, and stringy pulse.

[0004] The results of pharmacological and efficacy studies showed that the acute hypotensive effect of the Chinese medicine preparation of the present invention on spontaneously hypertensive rats occurred 4 hours after administration, but there was still an obvious hypotensive effect 10 hours after administration; the blood pressure of spontaneously hypertensive rats had dropped significantly on the 3rd day after continuous administration, and the blood pressure reduction amplitude reached the maximum after 7-11 days of continuous administration, and there was a certain maintenance hypotensive effect after drug withdrawal; the blood pressure of renal hypertensive rats showed obvious hypotension 2 hours after administration, and the blood pressure reduction amplitude reached the maximum 4-7 hours after administration, and there was still an obvious hypotensive effect 10 hours after administration; the blood pressure reduction amplitude of awake RHR basically reached the maximum after 8-10 days of continuous administration, and there was a maintenance hypotensive effect after drug withdrawal, and the blood pressure of the high and medium dose groups was still significantly different from that of the control group on the 4th day after drug withdrawal (P<0.05 or P<0.01). In addition, after continuous administration of the Chinese medicine preparation of the present invention for 15 days, the concentration of the pressor active substances in the blood of RHR: angiotensin II and cortisol can be significantly reduced (P<0.05 or P<0.01, compared with the control group), thereby producing a hypotensive effect; the high-dose group can significantly reduce the systolic blood pressure, diastolic blood pressure, mean arterial pressure, left ventricular intraventricular pressure, left ventricular end-diastolic pressure, ejection time, total oxygen consumption index and total peripheral vascular resistance of anesthetized dogs, and increase coronary flow (compared with the saline group, P<0.05). The improvement of some hemodynamic indicators after administration is conducive to the exertion of the antihypertensive effect, and may also be conducive to reducing complications caused by hypertension (such as ventricular hypertrophy, etc.).

[0005] The results of 220 phase II clinical trials with placebo as control showed that the total effective rate of the Chinese medicine preparation of the present invention for type II hypertension (systolic blood pressure 160-179 mmHg, diastolic blood pressure 100-109 mmHg) was 78.33%, and that of the control group was 41.38%; the blood pressure reaching standard rate was 40.00% in the test group and 13.79% in the control group, and the difference between the two groups was statistically significant (P < 0.05); the TCM syndrome efficacy control rate was 40.00% in the test group and 3.45% in the control group, and the difference between the two groups was statistically significant (P < 0.05); the improvement of single TCM symptoms, dizziness, and headache between the two groups had statistically significant differences (P < 0.05), the test group was better than the control group, and there was no statistically significant difference in the improvement of other single TCM symptoms between the groups (P > 0.05).

[0006] The results of the Phase III clinical trial of 480 cases with positive drugs as controls showed that the total effective rate of blood pressure efficacy of the Chinese medicine preparation of the present invention was 82.16% in the test group and 62.03% in the control group, and the blood pressure compliance rate was 48.41% in the test group and 21.52% in the control group, with a statistically significant difference between the two groups (P < 0.05). The control rate of TCM syndrome efficacy was 47.13% in the test group and 27.85% in the control group, with a statistically significant difference between the two groups (P < 0.05). As for the changes in the scores of individual TCM symptoms, there were significant differences between the two groups in the improvement of the scores of individual TCM symptoms such as dizziness, headache, irritability, tinnitus, and flushed face and eyes (P<0.05). The total effective rate of TCM liver yang hyperactivity syndrome control was 92.99%, indicating that it has good clinical efficacy for type Ⅱ hypertension. In addition, the results of the clinical trial of the antihypertensive effect of the present invention combined with Western medicine showed that the Chinese medicine preparation tablets of the present invention can increase the antihypertensive effect of Western medicine, with a total effective rate of 87.10%, a blood pressure compliance rate of 43.87%, and a total effective rate of clinical control of TCM liver yang hyperactivity syndrome of 89.67%.

[0007] Based on the results of the pharmacological test and the results of the Phase II and Phase III clinical trials, it can be seen that the Chinese medicine preparation of the present invention has a significant antihypertensive effect and a definite antihypertensive mechanism. It is superior to the control placebo and brain-clearing antihypertensive tablets in the treatment of hypertension (liver yang hyperactivity syndrome) when used alone or in combination with Western medicine, and has a good therapeutic effect. However, the quality inspection method of the Chinese medicine preparation for treating hypertension is not disclosed in the invention patent (patent number: ZL200310105398.7). In order to further follow the development law of traditional Chinese medicine, adhere to the combination of inheritance and innovation, and embody the concept of drug quality life cycle management, the inventor has established a scientific, reasonable and feasible quality inspection method for the Chinese medicine preparation based on in-depth research and using modern science and technology, which effectively ensures the controllable product quality, thereby further ensuring the clinical efficacy of the Chinese medicine preparation. Summary of the invention

[0008] The purpose of the present invention is to provide a quality detection method for a Chinese medicine preparation for treating hypertension, including a thin layer identification method for all medicinal flavors in the prescription of the preparation, and a method for determining the content of paeoniflorin and puerarin, the effective components of the main medicinal flavors of Paeonia suffruticosa and Pueraria root. The thin layer identification method in the technical solution of the present invention has strong specificity, good reproducibility, rapidity and convenient operation; through systematic methodological research and analytical method verification of content determination indicators, it is shown that the high-performance liquid phase content determination method of paeoniflorin and puerarin determined by the present invention is reasonable, feasible, high in accuracy, good in precision, and meets the requirements for stability. It can be used as a quality detection method for Chinese medicine preparations of the present invention to further ensure the stability and efficacy of the quality of the drug product.

[0009] The traditional Chinese medicine preparation of the invention is composed of 10-38% of Uncaria rhynchophylla, 10-35% of Paeonia suffruticosa, 5-32% of Prunella vulgaris, 5-28% of sunflower leaf and 4-30% of Pueraria root.

[0010] A more preferred formulation is 10-30% Uncaria rhynchophylla, 10-32% Paeonia suffruticosa, 5-26% Prunella vulgaris, 5-25% Helianthus annuus leaves, and 4-22% Pueraria root.

[0011] The Chinese medicine preparation of the present invention is prepared by the following process: extracting Uncaria rhynchophylla and Pueraria root with 60-80% ethanol twice, combining the extracts, filtering, recovering ethanol from the filtrate and concentrating it into a concentrated solution; then distilling paeonol from peony bark; boiling the residues and Prunella vulgaris with water twice, combining the extracts, filtering, and concentrating the filtrate; separately boiling sunflower leaves with water twice, combining the extracts, filtering, and concentrating the filtrate, combining the peony bark and sunflower leaf concentrates, adding ethanol to make the alcohol content reach 50-70%, precipitating with alcohol, refrigerating for 24 hours, filtering, recovering ethanol from the filtrate and concentrating it into a concentrated solution; combining the two concentrates, continuing to concentrate into a thick paste, drying, crushing, adding paeonol, mixing, sieving, adding one or more auxiliary materials, and then preparing granules, capsules, and tablets.

[0012] The process after further optimizing the process parameters is as follows: the Chinese medicine preparation is composed of 10-30% of Uncaria rhynchophylla, 10-32% of Paeonia suffruticosa, 5-26% of Prunella vulgaris, 5-25% of sunflower leaves, and 4-22% of Pueraria lobata, and is prepared by the following process: extracting Uncaria rhynchophylla and Pueraria lobata with 70-80% ethanol twice, the first time for 2-3 hours and the second time for 1-2 hours, combining the extracts, filtering, recovering ethanol from the filtrate and concentrating it into a concentrated solution with a relative density of 1.20-1.25; then distilling paeonol from Paeonia suffruticosa; decocting the residues and Prunella vulgaris with water twice, each time for 1-2 hours, Combine the extracts, filter, and concentrate the filtrate; take another sunflower leaf and add water to boil twice, each time for 1 to 2 hours, combine the extracts, filter, and concentrate the filtrate, combine the two concentrated solutions of peony bark and sunflower leaves, add ethanol to make the alcohol content reach 55 to 70%, precipitate with alcohol, refrigerate for 24 hours, filter, recover ethanol from the filtrate and concentrate it into a concentrated solution with a relative density of 1.18 to 1.25; combine the above two concentrated solutions, continue to concentrate into a thick paste, dry, crush, add paeonol, mix well, sieve, add one or more auxiliary materials, and then it can be made into granules, capsules, and tablets.

[0013] The technical solution of the patent application of this invention is:

[0014] 1. The thin layer identification method of peony bark and sunflower leaf of the present invention comprises the following steps:

[0015] (1) Preparation of solution: Take the Chinese medicine preparation, grind it, add hot water to dissolve it, extract it with water-saturated ether for 3 times, combine the ether solutions, wash it with water-saturated ether for 2 times, discard the water solution, evaporate the ether solution, add acetone to dissolve the residue, and use it as the test solution; take the paeonol reference substance, add acetone to prepare a solution, and use it as the reference substance solution; take the sunflower leaf reference medicinal material, add water to reflux extraction, filter, extract the filtrate with water-saturated ether for 3 times, combine the ether solutions, evaporate, and add acetone to dissolve the residue, and use it as the reference medicinal material solution;

[0016] (2) According to the thin layer chromatography test, the above three solutions were respectively spotted on the same silica gel G thin layer plate, and cyclohexane-ethyl acetate with a volume ratio of 2.7:1 was used as the developing solvent. The plate was developed, taken out, dried, and examined under a UV lamp at 365 nm; in the chromatogram of the test sample, fluorescent spots of the same color appeared at the corresponding positions of the chromatogram of the sunflower leaf reference medicinal material; then the plate was sprayed with a 5% ferric chloride ethanol solution in hydrochloric acid, and heated at 105°C until the spots were clearly colored; in the chromatogram of the test sample, the same blue-brown spots appeared at the corresponding positions of the chromatogram of the paeonol reference material.

[0017] Furthermore, in the method for identifying peony bark and sunflower leaves, the thin layer development temperature is below 10°C.

[0018] 2. The thin layer identification method of Uncaria rhynchophylla, Prunella vulgaris and Pueraria lobata of the present invention comprises the following steps:

[0019] (1) Identification method of Uncaria rhynchophylla: Take the above-mentioned Chinese medicine preparation, grind it into powder, add hot water to dissolve it, add concentrated ammonia test solution to adjust the pH value to 9-10, extract it with chloroform three times, combine the chloroform solutions, extract it with 3% sulfuric acid solution three times, combine the acid water solutions, add concentrated ammonia test solution to adjust the pH value to 9-10, extract it with chloroform three times, combine the chloroform solutions, evaporate to dryness, add methanol to dissolve the residue, and use it as the test solution; take Uncaria rhynchophylla control medicinal material, add methanol to ultrasonically treat it, filter it, evaporate the filtrate to dryness, add hot water to dissolve the residue, add concentrated ammonia test solution to adjust the pH value to 9-10, and use chloroform to Extract three times, combine the chloroform solution, evaporate to dryness, add methanol to the residue to dissolve it, and use it as the control medicinal material solution; according to the thin layer chromatography test, absorb the above two solutions and spot them on the same silica gel G thin layer plate, use the upper layer solution of toluene-acetone-ethanol-concentrated ammonia test solution with a volume ratio of 8:6:1:4 as the developing agent, develop, take out, dry, spray with a mixed solution of dilute potassium bismuth iodide test solution-1% ferric chloride anhydrous ethanol solution with a volume ratio of 2:1, and blow with cold wind until the spots are clearly colored; in the chromatogram of the test sample, the same orange-red main spot appears at the corresponding position of the chromatogram of the control medicinal material;

[0020] (2) Identification method of Prunella vulgaris: take the Chinese medicine preparation, grind it, add water to dissolve it, filter it, extract the filtrate with ethyl acetate three times, combine the ethyl acetate solution, wash it with water saturated with ethyl acetate twice, discard the water solution, evaporate the ethyl acetate solution to dryness, add methanol to dissolve the residue, and use it as the test solution; take another Prunella vulgaris control medicinal material, add methanol to ultrasonic treatment, filter it, evaporate the filtrate to dryness, add methanol to dissolve the residue, and use it as the control medicinal material solution; according to the thin layer chromatography test, absorb the above two solutions and spot them on the same silica gel G thin layer plate, use a mixed solution of chloroform-methanol-water with a volume ratio of 10:3:1 and formic acid with a volume ratio of 7:0.3 as the developing agent, develop it, take it out, dry it, spray it with hydrochloric acid acid 5% ferric chloride ethanol solution, and heat it at 105°C until the spots are clearly colored; in the chromatogram of the test sample, spots of the same color appear at the corresponding positions of the chromatogram of the control medicinal material;

[0021] (3) Identification method of Pueraria root: Take the above-mentioned Chinese medicine preparation, grind it into powder, add methanol for ultrasonic treatment, filter it, and use the filtrate as the test solution; take another puerarin reference substance, add methanol to make a solution, and use it as the reference solution; according to the thin layer chromatography test, take the above two solutions and spot them on the same silica gel G thin layer plate, use chloroform-methanol-water with a volume ratio of 6-9:2.5:0.25 as the developing solvent, develop, take out, dry, and examine under ultraviolet light at 365nm; in the chromatogram of the test substance, at the corresponding position of the chromatogram of the reference substance, a fluorescent spot of the same color appears.

[0022] Furthermore, in the thin-layer identification method of Uncaria rhynchophylla, the color developer diluted potassium bismuth iodide test solution is freshly prepared. In the thin-layer identification method of Pueraria lobata, the ratio of the developing agent is preferably chloroform-methanol-water in a volume ratio of 7:2.5:0.25.

[0023] 3. The method for determining the content of paeoniflorin described in the present invention comprises the following steps:

[0024] (1) Chromatographic conditions: The mobile phase was a mixture of methanol, 0.05 mol / L potassium dihydrogen phosphate solution (pH 3.0), and water in a volume ratio of 30:12:58; the detection wavelength was 230 nm;

[0025] (2) Preparation of reference solution: Take an appropriate amount of paeoniflorin reference substance, weigh accurately, and add methanol to prepare a solution containing 15-25 μg per 1 ml.

[0026] (3) Preparation of test solution: Take the Chinese medicine preparation, grind it into powder, weigh it accurately, add water for ultrasonic treatment, shake well, centrifuge, and take the supernatant;

[0027] (4) Determination method: Accurately pipette the reference solution and the test solution separately, inject them into the liquid chromatograph, and determine the result.

[0028] Furthermore, the pH value of the mobile phase in the liquid chromatography conditions for determining the content of paeoniflorin is adjusted by using glacial acetic acid.

[0029] 4. The method for determining the content of puerarin of the present invention comprises the following steps:

[0030] (1) Chromatographic conditions: methanol as mobile phase A, water as mobile phase B, gradient elution; detection wavelength at 250 nm;

[0031] (2) Preparation of reference solution: Take an appropriate amount of puerarin reference substance, weigh it accurately, and add methanol to make a solution containing 50-70 μg per 1 ml.

[0032] (3) Preparation of test solution: Take the Chinese medicine preparation, grind it into powder, weigh it accurately, add 30% ethanol for ultrasonic treatment, shake well, centrifuge, and take the supernatant.

[0033] (4) Determination method: Accurately pipette the reference solution and the test solution separately, inject them into the liquid chromatograph, and determine the result.

[0034] Furthermore, the gradient elution method in the liquid chromatography conditions for the determination of puerarin content is: 0-35 minutes, mobile phase A: 17%, mobile phase B: 83%; 35-40 minutes, mobile phase A: 100%, mobile phase B: 0%; 40-50 minutes, mobile phase A: 17%, mobile phase B: 83%.

[0035] The beneficial effects of the technical solution of the present invention are as follows:

[0036] 1. The quality detection method of the traditional Chinese medicine preparation provided by the present invention has established a thin layer identification method for all medicinal flavors in the preparation prescription, which can comprehensively reflect the quality of the traditional Chinese medicine preparation and meet the most stringent requirements of the state for the quality standards of traditional Chinese medicine preparations.

[0037] 2. The thin layer identification method in the traditional Chinese medicine preparation provided by the present invention, through repeated comparison of the test solution preparation method and screening research experiments with different compositions and proportions of the developing agent, finally determined the appropriate identification conditions, the negative control solution has no interference, has the advantages of strong specificity and high sensitivity, and the preparation process is simple, with the advantages of good reproducibility, rapidity and convenient operation.

[0038] 3. The thin layer identification method of the traditional Chinese medicine preparation provided by the present invention has the following outstanding features: the same test sample solution is creatively used for the peony bark and sunflower leaf in the preparation prescription, and the identification is carried out on the same thin layer plate, that is, two medicinal flavors can be identified at the same time in one test, which greatly facilitates the actual operation of quality inspection, shortens the inspection time, reduces the inspection cost, provides a new idea for thin layer identification in traditional Chinese medicine compound preparations, and is conducive to improving the quality inspection standards of traditional Chinese medicine.

[0039] 4. The quality detection method of the Chinese medicine preparation provided by the present invention fully considers the correlation between the determination index and the process and stability in terms of content determination, and gives priority to the chemical components associated with the safety and effectiveness of the drug. After in-depth liquid phase condition exploration and comparison test, sample preparation methodology research and analytical method verification, a high-efficiency liquid phase content determination method of multiple components and multiple indicators (peonidin and puerarin) is established. The results of the peonidin content determination research test show that its precision test RSD≤0.88%, repeatability test RSD≤0.66%, stability test RSD≤0.13%, and sample recovery test RSD≤0.71%; the results of the puerarin content determination research test show that its precision test RSD≤0.20%, repeatability test RSD≤1.09%, stability test RSD≤0.92%, and sample recovery test RSD≤1.14%; the above test data show that the content determination method established by the present invention is reasonable, feasible, has strong specificity, high accuracy, good precision, and stability meets the requirements.

[0040] 5. Since the 2020 edition of the Chinese Pharmacopoeia does not specify the method for determining the content of paeoniflorin under the item of Paeonia suffruticosa, the method for determining the content of paeoniflorin in the traditional Chinese medicine preparation provided by the present invention can provide a new idea for the quality inspection of Paeonia suffruticosa medicinal materials contained in traditional Chinese medicine compound preparations, which is conducive to improving the quality inspection standards of traditional Chinese medicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 -Thin layer identification chromatogram of sunflower leaves, wherein 1-negative control solution of peony bark, 2-paeonol reference solution, 3-test solution batch number-01, 4-test solution batch number-02, 5-test solution batch number-03, 6-sunflower leaf control medicinal material solution, 7-sunflower leaf negative control solution;

[0042] Figure 2 -Thin layer identification chromatogram of peony bark, wherein 1-negative control solution of peony bark, 2-paeonol reference solution, 3-batch number of test solution-01, 4-batch number of test solution-02, 5-batch number of test solution-03, 6-sunflower leaf control medicinal material solution, 7-sunflower leaf negative control solution;

[0043] Figure 3 -Thin layer identification chromatogram of Uncaria rhynchophylla, wherein 1-Uncaria rhynchophylla control medicinal material solution, 2-test solution batch number-01, 3-test solution batch number-02, 4-test solution batch number-03, 5-negative control solution;

[0044] Figure 4- Prunella vulgaris thin layer identification chromatogram, wherein 1-Prunella vulgaris control medicinal material solution, 2-test solution batch number-01, 3-test solution batch number-02, 4-test solution batch number-03, 5-negative control solution;

[0045] Figure 5 - Pueraria thin layer identification chromatogram, where 1-puerarin reference solution, 2-test solution batch number-01, 3-test solution batch number-02, 4-test solution batch number-03, 5-negative control solution;

[0046] Figure 6 - Peony glycoside content determination chart, wherein 1-peony glycoside reference solution, 2-test solution, 3-negative control solution;

[0047] Figure 7 -Purarin content determination chart, wherein 1-Purarin reference solution, 2-test solution, 3-negative control solution. DETAILED DESCRIPTION

[0048] Unless otherwise defined, the technical terms or scientific terms used in the patent application specification and claims of the present invention shall have the common meanings understood by persons having ordinary skills in the field to which the present invention belongs.

[0049] Example 1: Thin layer identification method of peony bark and sunflower leaves

[0050] Before establishing the thin layer identification method of the Chinese medicine preparation Moutan Cortex of the present invention, first refer to the thin layer identification method commonly used in the pharmacopoeia. It was found in the experiment that there was a purple-red spot above the paeonol spot in the thin layer spectrum of the test solution. If it was developed with the cyclohexane-ethyl acetate (3:1) development system commonly used in the pharmacopoeia, the purple-red spot partially overlapped with the paeonol spot. Therefore, the ratio of cyclohexane-ethyl acetate was explored, and the results are shown in Table 1.

[0051] Table 1 Effect of different developing agent ratios on identification of paeonol

[0052] test Developing agent ratio Thin layer results 1 3:1 There is a purple spot above the paeonol spot 2 2.8:1 The purple spots intersect with paeonol, which is interfering 3 2.5:1 Paeonol spot Rf value is too low 4 2.7:1 Paeonol and purple spots can be separated well

[0053] The results showed that after adjusting the developing solvent to cyclohexane-ethyl acetate (2.7:1), the separation of paeonol and purple-red spots was improved, but it was still not satisfactory. The development temperature was continued to be below 10°C. The results showed that the spots of the two were well separated and the thin layer spectrum was satisfactory. Under this condition, the spots in the test solution corresponding to the sunflower leaf control medicinal material were also well separated. Therefore, the optimal developing solvent system for Paeonia suffruticosa and sunflower leaves was finally determined.

[0054] In summary, the identification methods of peony bark and sunflower leaves are as follows:

[0055] Take 3 tablets of the Chinese medicine preparation of the present invention, grind them, add 25ml of hot water to dissolve them, cool them, extract them 3 times with ether saturated with water, 15ml each time, combine the ether solution, wash them 2 times with water saturated with ether, 20ml each time, discard the water solution, evaporate the ether solution, add 2ml of acetone to the residue to dissolve it, and use it as the test solution. Take another 4g of sunflower leaf control medicinal material, add 40ml of water, reflux extraction for 30 minutes, filter, extract the filtrate with ether saturated with water saturated with water 3 times, 15ml each time, combine the ether solution, evaporate, and add 1ml of acetone to the residue to dissolve it, and use it as the control medicinal material solution. Take the paeonol reference substance again, add acetone to make a solution containing 1mg per 1ml, and use it as the reference substance solution. According to the thin layer chromatography method (Chinese Pharmacopoeia 2020 Edition Part IV General Rules 0502), 8μl of the test solution, 8μl of the control medicinal material solution, and 3μl of the reference substance solution were taken and spotted on the same silica gel G thin layer plate, respectively, with cyclohexane-ethyl acetate (2.7:1) as the developing agent, developed below 10°C, taken out, dried, and inspected under ultraviolet light (365nm). In the chromatogram of the test sample, fluorescent spots of the same color appear at the corresponding positions of the chromatogram of the control medicinal material. Then spray with 5% ferric chloride ethanol solution in hydrochloric acid and heat at 105°C until the spots are clearly colored. In the chromatogram of the test sample, the same blue-brown spots appear at the corresponding positions of the chromatogram of the reference substance.

[0056] Embodiment 2: Uncaria rhynchophylla thin layer identification method

[0057] Uncaria rhynchophylla contains a variety of alkaloid components. In the preparation of the present invention, an alcohol extraction process is used. Therefore, when exploring the preparation method of the test solution, the following different methods are compared. The results are shown in Table 2.

[0058] Table 2 Effects of different test solution preparation methods on identification of Uncaria rhynchophylla

[0059]

[0060] The results showed that the thin layer development effect was the best when the test solution was prepared by repeated acid-base extraction. Therefore, the preparation method of the thin layer identification test solution of Uncaria rhynchophylla was determined. In addition, the type and composition ratio of the developing agent were further explored and studied with the separation degree of the thin layer spots as an indicator, and the best developing agent system for Uncaria rhynchophylla was determined. The results are shown in Table 3.

[0061] Table 3 Effects of different types and proportions of developing agents on identification of Uncaria rhynchophylla

[0062]

[0063] In summary, the identification methods of Uncaria rhynchophylla are as follows:

[0064] Take 5 tablets of the Chinese medicine preparation of the present invention, grind them into powder, add 25ml of hot water to dissolve them, cool them, add concentrated ammonia test solution to adjust the pH value to 9-10, extract them with chloroform 3 times, 15ml each time, combine the chloroform solution, extract them with 3% sulfuric acid solution 3 times, 15ml each time, combine the acid water solution, add concentrated ammonia test solution to adjust the pH value to 9-10, extract them with chloroform 3 times, 15ml each time, combine the chloroform solution, evaporate to dryness, add 1ml of methanol to the residue to dissolve it as the test solution. Take 3g of Uncaria rhynchophylla control medicinal material, add 30ml of methanol, ultrasonically treat for 30 minutes, filter, evaporate the filtrate to dryness, add 25ml of hot water to dissolve the residue, cool them, add concentrated ammonia test solution to adjust the pH value to 9-10, extract them with chloroform 3 times, 15ml each time, combine the chloroform solution, evaporate to dryness, add 1ml of methanol to the residue to dissolve it as the control medicinal material solution. According to the thin layer chromatography method (Chinese Pharmacopoeia 2020 Edition Part IV General Rules 0502), 8 μl of each of the above two solutions were taken and spotted on the same silica gel G thin layer plate, and the upper layer solution of toluene-acetone-ethanol-concentrated ammonia test solution (8:6:1:4) was used as the developing agent. After development, the plate was taken out, dried, and sprayed with a freshly prepared mixed solution of dilute bismuth potassium iodide test solution-1% ferric chloride anhydrous ethanol solution (2:1), and the spots were blown with cold wind until the color was clear. In the chromatogram of the test sample, the same orange-red main spot appeared at the corresponding position of the chromatogram of the control medicinal material.

[0065] Embodiment 3: Prunella vulgaris thin layer identification method

[0066] The main component of Prunella vulgaris is saponin. A water extraction process is used in the preparation of the present invention. Therefore, when exploring the preparation method of the test solution, the following different methods were compared. The results are shown in Table 4.

[0067] Table 4 Effects of different test solution preparation methods on identification of Prunella vulgaris

[0068]

[0069] The results showed that the sample was dissolved in hot water and extracted with ethyl acetate, and impurities were removed by filtration and water washing. The thin layer separation effect was good and the negative control solution had no interference.

[0070] In summary, the identification method of Prunella vulgaris is as follows:

[0071] Take 5 tablets of the Chinese medicine preparation of the present invention, grind them into powder, add 20 ml of hot water to dissolve them, filter them, extract the filtrate with ethyl acetate 3 times, 15 ml each time, combine the ethyl acetate solution, wash it twice with water saturated with ethyl acetate, 20 ml each time, discard the water solution, evaporate the ethyl acetate solution to dryness, add 2 ml of methanol to dissolve the residue, and use it as the test solution. Take 2 g of Prunella vulgaris control medicinal material, add 30 ml of methanol, ultrasonically treat for 30 minutes, filter, evaporate the filtrate to dryness, add 1 ml of methanol to dissolve the residue, and use it as the control medicinal material solution. According to the thin layer chromatography method (Chinese Pharmacopoeia 2020 Edition Part IV General Rules 0502), 8 μl of the test solution and 5 μl of the control medicinal material solution were taken and spotted on the same silica gel G thin layer plate, respectively, and a mixed solution of [chloroform-methanol-water (10:3:1) lower layer liquid]-formic acid (7:0.3) was used as the developing agent. After development, the plate was taken out, dried, sprayed with hydrochloric acid acid 5% ferric chloride ethanol solution, and heated at 105°C until the spots were clearly colored. In the chromatogram of the test sample, spots of the same color appeared at the corresponding position of the chromatogram of the control medicinal material.

[0072] Example 4: Pueraria lobata thin layer identification method

[0073] Take 2 tablets of the Chinese medicine preparation of the present invention, grind them, add 10 ml of methanol, ultrasonically treat for 15 minutes, filter, and use the filtrate as the test solution. Take another puerarin reference substance, add methanol to make a solution containing 0.5 mg per 1 ml as the reference solution. According to the thin layer chromatography method (Chinese Pharmacopoeia 2020 Edition Four General Rules 0502), take 2 μl of each of the above two solutions, and spot them on the same silica gel G thin layer plate, respectively, with chloroform-methanol-water (7:2.5:0.25) as the developing agent, develop, take out, dry, and inspect under ultraviolet light (365nm). In the test sample chromatogram, fluorescent spots of the same color appear at the corresponding position of the reference chromatogram.

[0074] Example 5: Method for determining the content of paeoniflorin

[0075] 1. Instruments and test drugs

[0076] HP1100 high performance liquid chromatograph, quaternary pump, DAD detector

[0077] HP Chemstation data processing software, 7725i manual injection valve.

[0078] Methanol and glacial acetic acid were of chromatographic grade, and potassium dihydrogen phosphate and other reagents were of analytical grade.

[0079] Paeoniflorin reference substance (provided by the National Institute for the Control of Pharmaceutical and Biological Products, batch number 736-200217).

[0080] 2. Study on the preparation method of test solution

[0081] 2.1 Selection of extraction solvent

[0082] Take an appropriate amount of the Chinese medicine preparation tablets of the present invention with batch number 20020702, grind them into powder, take 0.4 g of fine powder, take 2 portions in total, put them into 25 ml measuring bottles respectively, use water and dilute ethanol as solvents respectively, ultrasonically treat for 30 minutes, cool, add water to the scale, shake well, centrifuge (3000 rpm) for 5 minutes, accurately measure 1 ml of the supernatant, put it into a 2 ml measuring bottle, add water to the scale, shake well, and obtain.

[0083] Take 5 mg of paeoniflorin reference substance, accurately weigh it, place it in a 25 ml volumetric flask, add methanol to the mark, and shake well; accurately measure 1 ml, place it in a 10 ml volumetric flask, add water to the mark, shake well, and the result is (i.e., each 1 ml contains 20 μg of paeoniflorin).

[0084] Octadecylsilane bonded silica gel was used as filler; methanol-0.05 mol / L potassium dihydrogen phosphate solution (adjusted to pH 3.0 with glacial acetic acid)-water (30:12:58) was used as mobile phase; the detection wavelength was 230 nm. The theoretical plate number calculated based on the paeoniflorin peak should be no less than 2500.

[0085] Accurately pipette 20 μl of reference solution and test solution respectively, inject into liquid chromatograph, and measure. And measure the content, the results are shown in Table 5.

[0086] Table 5 Comparison of paeoniflorin content in different extraction solvents

[0087]

[0088] The results showed that the content of paeoniflorin was high and the extraction was more complete when water was used as the extraction solvent, so water was determined to be used as the extraction solvent.

[0089] 2.2 Selection of extraction method

[0090] Take an appropriate amount of the Chinese medicine preparation tablets of batch number 20020702 of the present invention, grind them into powder, and extract them according to the following methods.

[0091] Ultrasonic extraction method: Take 0.4g of fine powder, weigh it accurately, put it in a 25ml volumetric flask, add water and ultrasonically treat it for 30 minutes, let it cool, add water to the scale, shake well, centrifuge (3000 rpm) for 5 minutes, accurately measure 1ml of supernatant, put it in a 2ml volumetric flask, add water to the scale, shake well, and you have it.

[0092] Reflux extraction method: Take 0.4 g of fine powder, weigh accurately, place in a 50 ml round-bottom flask, add 25 ml of water accurately, weigh accurately, heat and reflux for 30 minutes, cool, add water to the original weight, shake well, centrifuge (3000 rpm) for 5 minutes, and prepare the reflux extraction test solution in the same way.

[0093] Take the above test solution and determine the content according to the above method. The results are shown in Table 6.

[0094] Table 6 Comparison of paeoniflorin content by different extraction methods

[0095]

[0096] The results showed that there was no significant difference in the content determination results of the two methods, and ultrasonic extraction was simple to operate, so ultrasonic extraction was determined to be used.

[0097] 2.3 Selection of extraction time

[0098] Take an appropriate amount of the Chinese medicine tablets of the present invention with batch number 20020702, grind them into powder, take 0.4 g, take 3 portions in total, accurately weigh them, put them in 25 ml volumetric bottles, and ultrasonically treat them for 15 minutes, 30 minutes, and 45 minutes respectively according to the above method to prepare the test solution. The content was determined according to the above method. The results are shown in Table 7.

[0099] Table 7 Effect of different extraction time on paeoniflorin content

[0100]

[0101] The results showed that different ultrasonic times had no significant effect on the content of paeoniflorin, and 30 minutes of extraction was more complete, so the ultrasonic extraction time was determined to be 30 minutes.

[0102] 3. Selection of HPLC conditions

[0103] Preparation of reference solution: Take 5 mg of paeoniflorin reference substance, accurately weigh it, place it in a 25 ml volumetric flask, add methanol to the mark, shake well, accurately measure 1 ml, place it in a 10 ml volumetric flask, add water to the mark, shake well, and obtain (each 1 ml contains 20 μg of paeoniflorin).

[0104] Preparation of test solution: Take 20 tablets of the Chinese medicine preparation of the present invention, accurately weigh, grind, take 0.4g, accurately weigh, put in a 25ml volumetric flask, first add 10ml of water, shake well, continue to add water to about 23ml, ultrasonically treat for 30 minutes, cool, add water to the scale, shake well, centrifuge (3000 rpm) for 5 minutes, accurately measure 1ml of the supernatant, put in a 2ml volumetric flask, add water to the scale, shake well, and obtain.

[0105] Preparation of negative control solution: Remove the ingredients except the peony bark from the prescription and prepare a negative sample according to the prescription ratio and preparation process; then prepare a negative control solution according to the above-mentioned test sample solution preparation method and set aside.

[0106] HPLC conditions: After multiple tests, various mobile phases with different proportions were selected to eliminate the interference of the negative control solution. The results are shown in Table 8.

[0107] Table 8 Effect of different mobile phases on the separation of paeoniflorin

[0108]

[0109] The results showed that the mobile phase of methanol-0.05 mol / L potassium dihydrogen phosphate solution (pH adjusted to 3.0 with glacial acetic acid)-water (30:12:58) had high separation of paeoniflorin and good peak shape, and there was no interference from the negative control solution.

[0110] 3.1 Chromatographic conditions and quantitative methods

[0111] Chromatographic column: HP1100, Nova-Pak C 18 , 4μm, 4.6×250mm stainless steel column

[0112] Column temperature: 35℃ Flow rate: 1.0ml / min Detection wavelength: 230nm

[0113] Mobile phase: methanol-0.05 mol / L potassium dihydrogen phosphate solution (adjust pH to 3.0 with glacial acetic acid)-water (30:12:58)

[0114] 3.2 Investigation of linear relationship

[0115] Take 5 mg of paeoniflorin reference substance, accurately weigh it, put it in a 25 ml volumetric flask, add methanol to the scale, shake well, then accurately measure 0.25 ml, 0.5 ml, 1 ml, 2 ml, 2.5 ml, and 4 ml respectively, put it in a 10 ml volumetric flask, add water to the scale, shake well, as the reference substance solution; accurately pipette 20 μl of each of the above reference substance solutions, inject them into the liquid chromatograph, record the chromatogram and peak area, the results are shown in Table 9.

[0116] Table 9 Linear range investigation results

[0117] Concentration (μg / ml) 4.9 9.8 19.6 39.2 49.0 78.4 Peak area 97.940 199.111 395.394 822.346 1036.306 1692.175

[0118] The regression analysis of the data obtained in the above table yielded a regression equation of Y = 21.697X-19.26, with a correlation coefficient of r = 1.000. It can be seen that the concentration of the paeoniflorin reference substance showed a good linear relationship in the range of 4.9 μg / ml to 78.4 μg / ml.

[0119] 3.3 Stability test

[0120] Accurately pipette 20 μl of the same reference solution and test solution respectively, and inject once at regular intervals. The peak area values ​​are shown in Table 10.

[0121] Table 10 Stability test results of test solution and reference solution

[0122]

[0123] The results showed that the test solution and the reference solution were stable within 48 hours.

[0124] 3.4 Precision test

[0125] Accurately pipette 20 μl of the same reference solution and inject it 6 times continuously. The peak area values ​​are shown in Table 11.

[0126] Table 11 Precision test results

[0127] serial number 1 2 3 4 5 6 RSD(%) Peak area of ​​reference substance 406.384 404.231 409.401 400.684 409.889 403.378 0.88

[0128] The results showed that this method had good precision.

[0129] 3.5 Repeatability test

[0130] Take the Chinese medicine preparation tablets of the present invention with batch number 20020702, prepare 6 test sample solutions according to the above method, and determine the content according to the above method. The results are shown in Table 12.

[0131] Table 12 Repeatability test results

[0132]

[0133] The results showed that this method had good reproducibility.

[0134] 3.6 Recovery test

[0135] Take the tablets of the Chinese medicine preparation of the present invention with batch number 20020702, grind them into powder, take 5 portions of fine powder (content 2.836 mg / tablet, average tablet weight 0.5955 g / tablet), 0.2 g per portion, accurately weigh, accurately add 1 ml of paeoniflorin reference solution (0.926 mg / ml), respectively, and measure according to the method. The results are shown in Table 13.

[0136] Table 13 Recovery test results

[0137]

[0138] From the results in the above table, it can be seen that this method has a good recovery rate.

[0139] 4. Sample determination

[0140] Several batches of the Chinese medicine preparation tablets of the present invention were tested, and the results are shown in Table 14.

[0141] Table 14 Determination results of paeoniflorin content in several batches of samples

[0142]

[0143] 5. Determination of the content limit of paeoniflorin

[0144] According to national requirements, the content limit should generally be 80% of the average value of the test data of multiple batches of samples. In view of the large difference in the content of paeoniflorin in medicinal materials from different sources, and based on the results of content determination of several batches of samples, it is tentatively determined that each tablet of the Chinese medicine preparation of the present invention contains paeoniflorin (C 28 H 23 O 11 ) shall not be less than 1.1 mg.

[0145] Example 6: Puerarin content determination method

[0146] 1. Instruments and test drugs

[0147] Waters 2998 Diode Array Detector (PDA)

[0148] Waters SymmetryShield RP18 5μm 4.6×150mm stainless steel column

[0149] Puerarin reference substance (provided by the National Institute for the Control of Pharmaceutical and Biological Products, batch number 110752-200511)

[0150] The mobile phase used methanol of chromatographic grade, water of ultrapure water, and the rest of the water were of analytical grade.

[0151] 2. Study on the preparation method of test solution

[0152] 2.1 Selection of extraction method

[0153] Take 20 tablets of the Chinese medicine preparation of the present invention with batch number 20121101, accurately weigh, grind, and extract according to the following methods:

[0154] Ultrasonic extraction method: Take 0.1g of fine powder, weigh accurately, put it in a 25ml volumetric flask, add about 20ml of 30% ethanol, ultrasonically treat for 30 minutes, cool, add 30% ethanol to the scale, shake well, filter, and take the filtrate as the test solution.

[0155] Reflux extraction method: Take 0.1g of fine powder, weigh it accurately, put it in a 100ml round-bottom flask, add 25ml of 30% ethanol accurately, weigh it, heat and reflux for 30 minutes, cool, weigh it again, make up the lost weight with 30% ethanol, shake well, filter, and take the filtrate as the test solution.

[0156] Take 5 mg of puerarin reference substance, accurately weigh it, place it in a 10 ml volumetric flask, add 30% ethanol to the scale, and shake well; accurately measure 1.2 ml, place it in a 10 ml volumetric flask, add 30% ethanol to the scale, shake well, and the result is (i.e., each 1 ml contains 60 μg of puerarin).

[0157] Octadecylsilane bonded silica gel was used as filler; methanol was used as mobile phase A and water was used as mobile phase B for gradient elution; the detection wavelength was 250 nm. 10 μl of reference solution and test solution were accurately drawn respectively, injected into liquid chromatograph, and measured. The results were shown in Table 15.

[0158] Table 15 Comparison of puerarin content in different extraction methods

[0159]

[0160] The results showed that there was no significant difference in the content of puerarin between ultrasonic extraction and reflux extraction. Since reflux operation was complicated and ultrasonic operation was simple, ultrasonic extraction was chosen.

[0161] 2.2 Selection of different extraction solvents

[0162] Take the tablets of the Chinese medicine preparation of the present invention with batch number 20121101, accurately weigh, grind, take 0.1g of fine powder respectively, accurately weigh, put in a 25ml volumetric bottle, add about 20ml of methanol, ethanol, 30% ethanol, and 80% ethanol respectively, ultrasonically treat for 30 minutes, cool, add corresponding solvent to scale, shake well, filter, and take the filtrate as the test solution. Determine the content according to the above method, and the results are shown in Table 16.

[0163] Table 16 Comparison of puerarin content in different extraction solvents

[0164]

[0165] The results showed that the content of puerarin extracted by ethanol was low, and there was no significant difference in the content of puerarin extracted by methanol, 30% ethanol and 80% ethanol. Considering factors such as the extraction method of Pueraria lobata and liquid chromatogram, 30% ethanol was selected as the extraction solvent.

[0166] 2.3 Selection of ultrasound time

[0167] Take the tablets of the Chinese medicine preparation of the present invention with batch number 20121101, accurately weigh, grind, take 0.1g of fine powder, take three portions in total, accurately weigh, put in a 25ml volumetric flask, add about 20ml of 30% ethanol, ultrasonicate for 15 minutes, 30 minutes, and 45 minutes respectively, cool, add 30% ethanol to the scale respectively, shake well, filter, and take the filtrate as the test solution. Determine the content according to the above method. The results are shown in Table 17.

[0168] Table 17 Effect of different ultrasound time on puerarin content

[0169]

[0170] The test results show that there is no significant difference in puerarin content when ultrasonicated for 15 minutes, 30 minutes and 45 minutes. In order to ensure complete extraction of puerarin, the ultrasonication time is selected as 30 minutes.

[0171] 3. Selection of HPLC Conditions

[0172] Preparation of reference solution: Accurately weigh an appropriate amount of puerarin reference substance and add 30% ethanol to make a solution containing about 60 μg per 1 ml as the reference solution.

[0173] Preparation of test solution: Take the Chinese medicine preparation tablets of the present invention with batch number 20121101, accurately weigh, grind, take 0.1g, accurately weigh, put into a 25ml volumetric flask, add about 20ml of 30% ethanol, ultrasonically treat for 30 minutes, cool, add 30% ethanol to the scale, shake well, filter, and take the filtrate.

[0174] Preparation of negative control solution: Remove Pueraria root from the prescription, prepare a negative sample according to the prescription ratio and preparation process, and prepare a negative control solution according to the preparation method of the test solution.

[0175] 3.1 Chromatographic determination conditions and quantitative methods

[0176] Waters e2695 HPLC Waters 2998 diode array detector (PDA)

[0177] Chromatographic column: Waters SymmetryShield RP18 5μm 4.6×150mm stainless steel column Column temperature: 35℃ Flow rate: 1.0ml / min Injection volume: 10μl Detection wavelength: 250nm

[0178] Use methanol as mobile phase A and water as mobile phase B, and perform gradient elution as specified in Table 18.

[0179] Table 18 Gradient elution table

[0180] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0～35 17 83 35～40 100 0 40～50 17 83

[0181] 3.2 Investigation of linear relationship

[0182] Accurately weigh 5.94 mg of puerarin reference substance, place it in a 10 ml volumetric flask, add 30% ethanol to dissolve and dilute to the mark, shake well, and use it as the puerarin reference substance stock solution; accurately measure 2.0, 1.0, 0.50, 0.40, 0.20, and 0.10 ml of the above stock solution, place it in 6 5 ml volumetric flasks, add 30% ethanol to dilute to the mark, shake well, and use it as the linear 1 # ~6 #Solution. 10 μl of each of the six solutions of different concentrations was accurately drawn and injected into the liquid chromatograph, and the chromatogram and peak area were recorded. The results are shown in Table 15. With the peak area A as the abscissa and the reference substance concentration C (μg / ml) as the ordinate, linear regression was performed, and the regression equation was: C=0.00002A-0.17227, and the correlation coefficient r=0.99995. The results are shown in Table 19. The results showed that the concentration of puerarin showed a good linear relationship in the range of 237.60-11.88 μg / ml.

[0183] Table 19 Linear range investigation results

[0184] serial number 1# 2# 3# 4# 5# 6# Concentration (μg / ml) 237.60 118.80 59.40 47.52 23.76 11.88 Peak area 9687557 4767443 2424965 1957512 974375 503248

[0185] 3.3 Stability test

[0186] Accurately pipette 10 μl of the same reference solution and test solution respectively, and inject once at regular intervals. The peak area values ​​are shown in Table 20.

[0187] Table 20 Stability test results of reference solution and test solution

[0188]

[0189] The results showed that both the reference solution and the test solution were stable within 24 hours.

[0190] 3.4 Precision test

[0191] Accurately pipette 10 μl of puerarin reference solution (concentration of 60.28 μg / ml) and inject it 6 times continuously. The peak area values ​​are shown in Table 21.

[0192] Table 21 Precision test results

[0193] serial number 1 2 3 4 5 6 average RSD(%) Peak area 2427364 2421861 2415664 2427353 2428393 2426760 2424566 0.20

[0194] The results showed that this method had good precision.

[0195] 3.5 Repeatability test

[0196] Take the tablets of the Chinese medicine preparation of the present invention with batch number 20121101, grind them into powder, take 0.1 g of the fine powder, accurately weigh it, take 6 portions in total, prepare the test solution according to the above method and determine the content. The determination results are shown in Table 22.

[0197] Table 22 Repeatability test results

[0198] sample Puerarin content (mg / tablet) Average (mg / tablet) RSD(%) 1 8.32 2 8.29 3 8.27 8.36 1.09 4 8.49 5 8.46 6 8.33

[0199] The results showed that this determination method had good repeatability.

[0200] 3.6 Recovery test

[0201] Take a known content (puerarin content 8.36 mg / tablet), take about 0.05 g of the fine powder of the Chinese medicine preparation tablets of the present invention with batch number 20121101, take 6 portions in total, accurately weigh, put into a 25 ml volumetric flask, accurately add 2.0 ml of puerarin reference substance (0.346 mg / ml) solution, and determine according to the above method. The results are shown in Table 23.

[0202] Table 23 Puerarin recovery test results

[0203]

[0204] The results showed that puerarin had a good recovery rate.

[0205] 4. Sample determination

[0206] Several batches of samples were measured according to the above method, and the results are shown in Table 24.

[0207] Table 24 Puerarin content determination results in several batches of samples

[0208]

[0209] 5. Determination of Puerarin Content Limit

[0210] According to national requirements, the content limit should generally be 80% of the average value of the test data of multiple batches of samples. In view of the large difference in the content of puerarin in medicinal materials from different sources, and based on the results of content determination of several batches of samples, it is tentatively determined that each tablet of the Chinese medicine preparation of the present invention contains puerarin (C 21 H 20 O9), not less than 7mg.

Claims

1. A quality inspection method for a traditional Chinese medicine preparation for treating hypertension, wherein the traditional Chinese medicine preparation is composed of 10-38% Uncaria rhynchophylla, 10-35% Paeonia suffruticosa, 5-32% Prunella vulgaris, 5-28% Helianthus annuus leaves, and 4-30% Pueraria lobata, and is prepared by the following process: extracting Uncaria rhynchophylla and Pueraria lobata twice with 60-80% ethanol, combining the extracts, filtering, recovering ethanol from the filtrate and concentrating it into a concentrated solution; then distilling paeonol from Paeonia suffruticosa; decocting the residues and Prunella vulgaris with water twice, combining the extracts, filtering, , the filtrate is concentrated; another sunflower leaf is taken and boiled twice with water, the extracts are combined, filtered, the filtrate is concentrated, the two concentrated solutions of peony bark and sunflower leaf are combined, ethanol is added to make the alcohol content reach 50-70%, alcohol precipitation, refrigerated for 24 hours, filtered, the filtrate recovers ethanol and is concentrated into a concentrated solution; the two concentrated solutions are combined, and further concentrated into a thick paste, dried, crushed, paeonol is added, mixed, sieved, and one or more auxiliary materials are added to make granules, capsules, and tablets; characterized in that, The quality inspection method of the traditional Chinese medicine preparation includes a thin layer identification method of peony bark, sunflower leaf, selfheal, Uncaria rhynchophylla, and kudzu root, and a method for determining the content of paeoniflorin and puerarin; Wherein, the thin layer identification method of peony bark and sunflower leaf comprises the following steps: (1) Preparation of solution: Take the Chinese medicine preparation, grind it into powder, add hot water to dissolve it, extract it with water-saturated ether for 3 times, combine the ether solutions, wash it with water-saturated ether for 2 times, discard the water solution, evaporate the ether solution, add acetone to dissolve the residue, and use it as the test solution; take the paeonol reference substance, add acetone to prepare a solution, and use it as the reference substance solution; take the sunflower leaf reference medicinal material, add water to reflux extraction, filter, extract the filtrate with water-saturated ether for 3 times, combine the ether solutions, evaporate, and add acetone to dissolve the residue, and use it as the reference medicinal material solution; (2) According to the thin layer chromatography test, the above three solutions were respectively spotted on the same silica gel G thin layer plate, and cyclohexane-ethyl acetate with a volume ratio of 2.7:1 was used as the developing solvent. The plate was developed, taken out, dried, and examined under a UV lamp at 365nm; in the chromatogram of the test sample, fluorescent spots of the same color appeared at the corresponding positions of the chromatogram of the sunflower leaf reference medicinal material; then the plate was sprayed with a 5% ferric chloride ethanol solution in hydrochloric acid, and heated at 105℃ until the spots were clearly colored; in the chromatogram of the test sample, the same blue-brown spots appeared at the corresponding positions of the chromatogram of the paeonol reference material.

2. The method for quality testing of a traditional Chinese medicine preparation for treating hypertension according to claim 1, characterized in that: The Chinese medicine preparation is composed of 10-30% Uncaria rhynchophylla, 10-32% Paeonia suffruticosa, 5-26% Prunella vulgaris, 5-25% Helianthus annuus leaves and 4-22% Pueraria lobata, and is prepared by the following process: extracting Uncaria rhynchophylla and Pueraria lobata with 70-80% ethanol for two times, the first time for 2-3 hours and the second time for 1-2 hours, combining the extracts, filtering, recovering ethanol from the filtrate and concentrating it into a concentrated solution with a relative density of 1.20-1.25; distilling paeonol from Paeonia suffruticosa; decocting the residue and Prunella vulgaris with water for two times, each time for 1-2 hours, combining the extracts, filtering The filtrate is concentrated; sunflower leaves are separately boiled in water for two times, each time for 1 to 2 hours, the extracts are combined, filtered, the filtrate is concentrated, the two concentrated solutions of peony bark and sunflower leaves are combined, ethanol is added to make the alcohol content reach 55 to 70%, alcohol precipitation is performed, refrigerated for 24 hours, filtered, the filtrate is recovered with ethanol and concentrated into a concentrated solution with a relative density of 1.18 to 1.25; the two concentrated solutions are combined, and further concentrated into a thick paste, dried, crushed, paeonol is added, mixed, sieved, and one or more auxiliary materials are added to make granules, capsules, and tablets; Wherein, in the method for identifying peony bark and sunflower leaves, the thin layer development temperature is below 10°C.

3. The quality detection method of a traditional Chinese medicine preparation for treating hypertension according to claim 1, characterized in that: The thin layer identification method of Uncaria rhynchophylla, Prunella vulgaris and Pueraria lobata in the quality detection method comprises the following steps: (1) Identification method of Uncaria rhynchophylla: Take the above-mentioned Chinese medicine preparation, grind it into powder, add hot water to dissolve it, add concentrated ammonia test solution to adjust the pH value to 9-10, extract it with chloroform three times, combine the chloroform solutions, extract it with 3% sulfuric acid solution three times, combine the acid water solutions, add concentrated ammonia test solution to adjust the pH value to 9-10, extract it with chloroform three times, combine the chloroform solutions, evaporate to dryness, add methanol to dissolve the residue, and use it as the test solution; take Uncaria rhynchophylla control medicinal material, add methanol to ultrasonic treatment, filter, evaporate the filtrate to dryness, add hot water to dissolve the residue, add concentrated ammonia test solution to adjust the pH value to 9-10, and use chloroform to Extract three times, combine the chloroform solution, evaporate to dryness, add methanol to the residue to dissolve it, and use it as the control medicinal material solution; according to the thin layer chromatography test, absorb the above two solutions and spot them on the same silica gel G thin layer plate, use the upper layer solution of toluene-acetone-ethanol-concentrated ammonia test solution with a volume ratio of 8:6:1:4 as the developing agent, develop, take out, dry, spray with a mixed solution of dilute potassium bismuth iodide test solution-1% ferric chloride anhydrous ethanol solution with a volume ratio of 2:1, and blow with cold wind until the spots are clearly colored; in the chromatogram of the test sample, the same orange-red main spot appears at the corresponding position of the chromatogram of the control medicinal material; (2) Identification method of Prunella vulgaris: Take the Chinese medicine preparation, grind it, add water to dissolve it, filter it, extract the filtrate with ethyl acetate three times, combine the ethyl acetate solution, wash it with water saturated with ethyl acetate twice, discard the water solution, evaporate the ethyl acetate solution to dryness, add methanol to dissolve the residue, and use it as the test solution; take another Prunella vulgaris control medicinal material, add methanol to ultrasonic treatment, filter it, evaporate the filtrate to dryness, add methanol to dissolve the residue, and use it as the control medicinal material solution; according to the thin layer chromatography test, absorb the above two solutions and spot them on the same silica gel G thin layer plate, use the lower layer liquid of chloroform-methanol-water with a volume ratio of 10:3:1 and the mixed solution of formic acid with a volume ratio of 7:0.3 as the developing agent, develop it, take it out, dry it, spray it with hydrochloric acid acid 5% ferric chloride ethanol solution, and heat it at 105°C until the spots are clearly colored; in the chromatogram of the test sample, spots of the same color appear at the corresponding positions of the chromatogram of the control medicinal material; (3) Identification method of Pueraria root: Take the above-mentioned Chinese medicine preparation, grind it into powder, add methanol for ultrasonic treatment, filter it, and use the filtrate as the test solution; take another puerarin reference substance, add methanol to make a solution, and use it as the reference solution; according to the thin layer chromatography test, take the above two solutions and spot them on the same silica gel G thin layer plate, use chloroform-methanol-water with a volume ratio of 6-9:2.5:0.25 as the developing solvent, develop, take out, dry, and examine under ultraviolet light at 365nm; in the chromatogram of the test substance, at the corresponding position of the chromatogram of the reference substance, a fluorescent spot of the same color appears.

4. The quality detection method of a traditional Chinese medicine preparation for treating hypertension as claimed in claim 3, characterized in that: In the thin-layer identification method of Uncaria rhynchophylla, the color developing agent dilute potassium bismuth iodide test solution is freshly prepared.

5. The quality detection method of a traditional Chinese medicine preparation for treating hypertension as claimed in claim 3, characterized in that: In the thin-layer identification method for Pueraria root, the ratio of the developing agent is preferably chloroform-methanol-water in a volume ratio of 7:2.5:0.

25.

6. The quality inspection method of a traditional Chinese medicine preparation for treating hypertension according to claim 1, characterized in that: The method for determining the content of paeoniflorin in the quality detection method comprises the following steps: (1) Chromatographic conditions: The mobile phase was a mixture of methanol, 0.05 mol / L potassium dihydrogen phosphate solution (pH 3.0), and water in a volume ratio of 30:12:58; the detection wavelength was 230 nm; (2) Preparation of reference solution: Take an appropriate amount of paeoniflorin reference substance, weigh accurately, and add methanol to make a solution containing 15-25 μg per 1 ml. (3) Preparation of test solution: Take the Chinese medicine preparation, grind it into powder, weigh it accurately, add water for ultrasonic treatment, shake well, centrifuge, and take the supernatant; (4) Determination method: Accurately pipette the reference solution and the test solution separately, inject them into the liquid chromatograph, and determine the result.

7. The quality detection method of a traditional Chinese medicine preparation for treating hypertension according to claim 6, characterized in that: In the method for determining the content of paeoniflorin, the pH value of the mobile phase is adjusted by using glacial acetic acid.

8. The quality detection method of a traditional Chinese medicine preparation for treating hypertension according to claim 1, characterized in that: The method for determining the content of puerarin comprises the following steps: (1) Chromatographic conditions: methanol as mobile phase A, water as mobile phase B, gradient elution; detection wavelength is 250 nm; (2) Preparation of reference solution: Take an appropriate amount of puerarin reference substance, weigh accurately, and add methanol to make a solution containing 50-70 μg per 1 ml. (3) Preparation of test solution: Take the Chinese medicine preparation, grind it into powder, weigh it accurately, add 30% ethanol for ultrasonic treatment, shake well, centrifuge, and take the supernatant; (4) Determination method: Accurately pipette the reference solution and the test solution separately, inject them into the liquid chromatograph, and determine the result.

9. The quality detection method of a traditional Chinese medicine preparation for treating hypertension according to claim 8, characterized in that: In the puerarin content determination method, the gradient elution mode is: 0-35 minutes, mobile phase A: 17%, mobile phase B: 83%; 35-40 minutes, mobile phase A: 100%, mobile phase B: 0%; 40-50 minutes, mobile phase A: 17%, mobile phase B: 83%.

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