Method for simultaneously determining contents of 11 chemical components in Shuangdan prescription, Shuangdan ligusticum wallichii prescription, Shuangdan radix cyathulae prescription and single-line traditional Chinese medicine extract

Abstract

The invention belongs to the technical field of traditional Chinese medicines, and particularly relates to a method for simultaneously determining the contents of 11 chemical components in a Shuandang prescription, a Shuandang ligusticum wallichii prescription, a Shuandang radix cyathulae prescription and a single-row traditional Chinese medicine extract, which comprises the following steps: S1, water-extracting a single-row prescription and a Shuandang modified prescription; s2, preparing a test solution; s3, preparing a standard solution; s4, performing chromatographic detection on the mixed standard substance solution, and fitting to obtain a linear regression equation; and S5, performing chromatographic detection on the test solution, and calculating the average content of each chemical component in each test solution according to the fitted linear regression equation. According to the invention, through a stable and reliable high performance liquid chromatography analysis method, 11 chemical components in a Shuangdang prescription, a Shuangdang ligusticum wallichii prescription, a Shuangdang radix cyathulae prescription and a single-line traditional Chinese medicine extract can be subjected to component analysis and content determination at the same time, and the attribution and content of the chemical components in different formulas are determined; and data support is provided for subsequent preliminary pharmacokinetics and tissue distribution research of different components, so that a new thought and a new method can be provided for developing new drugs.

Description

Technical Field

[0001] The invention belongs to the technical field of traditional Chinese medicines, and particularly relates to a method for simultaneously determining the contents of 11 chemical components in Shuangdan prescription, Shuangdan Chuanxiong prescription, Shuangdan Chuanxiong prescription and single-line traditional Chinese medicine extracts. Background Art

[0002] Shuangdan Recipe comes from Shi Jinmo's Collection of Clinical Experiences on Drugs. It is one of the classic prescriptions for promoting blood circulation and removing blood stasis. It is composed of Danshen and Moutan Cortex based on the mutual use of the seven emotions in traditional Chinese medicine. Danshen in the recipe is bitter and slightly cold in nature. It can move blood and remove blood stasis, relieve pain, and remove blood stasis and generate new blood, cool blood, relieve restlessness and calm the mind; Moutan Cortex is bitter and slightly cold in nature. It specifically enters the blood, cools blood and promotes blood circulation, can clear the hidden fire in the blood, and disperse blood stasis. Danshen is good at promoting blood circulation and removing blood stasis, while Moutan Cortex is good at cooling blood and dispersing blood stasis, clearing the hidden heat in the yin, and the two medicines are compatible, cooling blood and promoting blood circulation, removing blood stasis and generating new blood, and increasing the power of clearing away evil heat. This recipe has the effects of promoting blood circulation and removing blood stasis, unblocking meridians and relieving pain. It is mainly used clinically to treat symptoms such as chest pain and heart pain caused by blood stasis and obstruction, and is often used to treat coronary heart disease and angina pectoris. At present, the common preparations of this prescription include Shuangdan granules, Shuangdan capsules, Shuangdan oral liquid, Shuangdan dripping pills, Shuangdan tablets and Shuangdan dispersible tablets, etc. Among them, Shuangdan oral liquid is included in the 2020 edition of the "Chinese Pharmacopoeia", and its effects are to promote blood circulation, remove blood stasis, dredge meridians and relieve pain.

[0003] Meridian-guiding drugs are also called meridian-guiding messenger drugs. They can guide other drugs to the site of the disease or a certain meridian, so as to better play a therapeutic role. Modern research has found that Chinese medicine meridian-guiding drugs can promote the absorption or distribution of other drugs in target organ tissues, and their targeting may be related to opening the physiological barrier of the target site. The mechanism of action of meridian-guiding drugs also includes changing the cell membrane structure or permeability, changing the intracellular pH value, playing a role in enhancing the targeting of Chinese medicine, inhibiting the activity of P-glycoprotein or other membrane transport proteins, inhibiting their expression, and thus promoting the absorption of Chinese medicine ingredients. The meridian-guiding mechanism may also be closely related to its regulation of the levels of biochemical substances (such as NO, cAMP, cGMP, neurotransmitters, etc.) in various tissues of the body; or by directly affecting the level of neurotransmitters and regulating the levels of two or more biochemical substances to play a meridian-guiding role; meridian-guiding drugs may also increase the solubility of drugs and promote the dissolution of active ingredients. Guiding the meridians and returning to the meridians are closely related. The theory of guiding the meridians is further developed based on the theory of returning to the meridians. At the same time, it is combined with the theory of compatibility, becoming a major feature of Chinese medicine. In the compound prescription, by combining the guiding meridian drugs, it is possible to coordinate the various drugs to move toward the diseased area and play a directional role, thereby improving the efficacy.

[0004] "Jiaoyao" refers to a method of using three Chinese medicines in combination, guided by the basic theories of traditional Chinese medicine, based on syndrome differentiation and treatment, and based on the principles of compatibility based on the smell, properties, and seven emotions of Chinese medicine. Jiaoyao is between Chinese medicine and prescriptions, and is an extension of medicine, but not a simple increase in the number of medicines. Jiaoyao plays a major or auxiliary role in prescriptions, or is an independent prescription. In clinical applications, it can reduce toxicity, increase efficacy, and expand the scope of drug use, providing new ideas and methods for the development of new drugs.

[0005] The Shennong's Herbal Classic records that Chuanxiong is "spicy and warm in nature, and is used to treat strokes that enter the brain". The Ming Dynasty's Materia Medica Collection also says that Chuanxiong "goes up to the head and eyes, regulates menstrual fluid, and relieves depression in the middle, and is a blood qi medicine". Chuanxiong promotes the qi in the blood, and is also a wind medicine, which is good at ascending and dispersing, and can reach the head and eyes, promote qi and activate blood circulation. The Qing Dynasty's Depei Materia Medica states: "Headache is ineffective without a medicine to guide it. Chuanxiong is effective for Jueyin headaches, and Chuanxiong is a medicine to guide the meridians for Jueyin headaches". In the treatment method for swelling recorded in the Ming Dynasty's Shoushi Baoyuan, if the forehead does not swell after taking Baoyuan Decoction, six parts of Chuanxiong can be added as a guide, indicating that Chuanxiong can be used as a guide to guide the medicine to the forehead. In modern research, Wang et al. found that after adding Chuanxiong or borneol, the content of jujube saponin A, the main active ingredient in the compound Shuyu Jiannao prescription, in brain tissue increased. Liu Lirong et al. found that after Angelica dahurica and Chuanxiong were combined, the blood concentration and Cmax of four coumarin compounds in Angelica dahurica could be increased to varying degrees, promoting the absorption of active ingredients in the body, while slowing down their clearance rate in the body and improving bioavailability. Zhao Qian et al. found that Chuanxiong, as a meridian-guiding drug, did not promote brain metastasis of melanoma while opening the blood-brain barrier, and combined with temozolomide, it could enhance the therapeutic effect of temozolomide on brain metastasis melanoma B16F10. When Chuanxiong is used as a meridian-guiding drug in combination with other drugs, it can achieve the effect of guiding the drug to the head by increasing the concentration of the drug in the brain tissue, increasing the blood concentration, and enhancing the drug effect.

[0006] "Chinese Materia Medica" records that both Sichuan Achyranthes and Huai Achyranthes can promote blood circulation and menstruation, nourish the liver and kidney, strengthen tendons and bones, promote diuresis and relieve stranguria, and guide fire (blood) downward, but Sichuan Achyranthes is better at promoting blood circulation and menstruation, while Huai Achyranthes is better at nourishing the liver and kidney and strengthening tendons and bones. As a medicine for guiding the meridians downward, Sichuan Achyranthes is often used in combination with other medicines to guide the meridians to the part to exert therapeutic effects. Jiawei Shenqi Pills, which warms and nourishes kidney yang, promotes diuresis and reduces swelling, uses Sichuan Achyranthes (soaked in wine) in the recipe. "Yi Fang Jijie" records that adding Sichuan Achyranthes can benefit the liver and kidney and guide downward. Modern studies have shown that Chuanxiong, Ermiao Pills, and Sanmiao Pills can reduce the inflammatory response of the ankle joints of rats with acute gouty arthritis by downregulating the expression of NF-κB p65 protein, inhibiting the levels of TNF-α, IL-6, and IL-8, and reducing joint swelling. The efficacy of Sanmiao Pills is particularly significant. Sanmiao Pills is composed of Ermiao Pills and Chuanxiong, which may be related to the fact that Chuanxiong has the effects of removing blood stasis, dredging collaterals, and nourishing the liver and kidneys, as well as the effect of "leading the medicine downward". Shen Liang et al.'s research showed that Sijunzi Decoction and Sijunzi plus Chuanxiong can accelerate fibroblasts from G0 / G1 phase to S phase, promote fibroblast proliferation, and promote wound healing. The proportion of fibroblasts in the S phase of the Sijunzi plus Chuanxiong group was significantly higher than that of the Sijunzi Decoction group, suggesting that the mechanism of Chuanxiong's meridian-guiding effect may be a synergistic effect of increasing the proportion of fibroblasts in the S phase and promoting fibroblast proliferation.

[0007] Shuangdan Jiawei Fang is based on the theory of guiding the meridians and the method of combining corner medicines. It uses the classic Shuangdan Fang with Chuanxiong or Chuanxiong to form corner medicines, namely, Salvia miltiorrhiza-Paeonia suffruticosa-Liuxiong and Salvia miltiorrhiza-Paeonia suffruticosa-Chuanxiong. It explores the guiding effect of Chuanxiong / Chuanxiong, that is, whether it can change the ownership and content of the main active ingredients of Shuangdan Fang in different prescriptions, which can provide new ideas and methods for developing new drugs.

[0008] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention and should not be regarded as an acknowledgment or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art. Summary of the invention

[0009] The purpose of the present invention is to provide a method for simultaneously determining the contents of 11 chemical components in Shuangdan Recipe, Shuangdan Chuanxiong Recipe, Shuangdan Chuanxiong Recipe and single-line Chinese medicine extracts, which can provide new ideas and methods for developing new drugs.

[0010] To achieve the above object, the present invention provides the following technical solutions:

[0011] A method for simultaneously determining the contents of 11 chemical components in Shuangdan Recipe, Shuangdan Chuanxiong Recipe, Shuangdan Chuanxiong Recipe and single-line Chinese medicine extracts, comprising the following steps:

[0012] S1. Water Extract Single Prescription and Shuangdan Jiawei Prescription

[0013] The prescription was extracted with water twice, the extracts were combined, filtered, concentrated under reduced pressure, and spray-dried to obtain dry powder;

[0014] S2. Prepare the test solution

[0015] Prepare the test solution from the prescription dry powder;

[0016] S3. Preparation of standard solution

[0017] Accurately weigh gallic acid, danshensu, paeoniflorin, ferulic acid, cyasterone, ligusticolide I, rosmarinic acid, lithospermic acid, salvianolic acid B, salvianolic acid A and paeonol standards respectively to prepare a mixed standard solution;

[0018] S4. Chromatographic detection

[0019] Mixed standard solutions were taken according to the volume gradient, fixed to volume with 50% methanol, and detected by chromatographic column. The concentration of the mixed standard solution was used as the horizontal axis, and the peak area of ​​each component was used as the vertical axis. The linear regression equation was obtained by fitting.

[0020] S5. Test the test solution

[0021] The test solution prepared by S2 was detected by chromatographic column, and the average content of each chemical component in each test solution was calculated according to the fitted linear regression equation.

[0022] Further, in step S1, the single-line prescription includes Danshen single-line prescription, Moutan bark single-line prescription, Chuanxiong single-line prescription and Chuanxiong single-line prescription; the double-dan added prescription includes Shuangdan prescription, Shuangdan plus Chuanxiong prescription and Shuangdan plus Chuanxiong prescription.

[0023] Furthermore, in step S1, the specific operation of water extraction is: add 8 times the amount of water for soaking for 30 minutes for the first time, and reflux extraction for 1.5 hours; add 6 times the amount of water for the second time, reflux extraction for 1 hour, combine the two extracts, filter, and concentrate under reduced pressure to a relative density of 1:1.15-1.18 at 80°C.

[0024] Furthermore, in step S2, the test sample solution is prepared by mixing the water extract sample with 50% methanol in a mass volume ratio of 1 g:50 mL, ultrasonicating for 30 min, cooling, supplementing the weight loss with 50% methanol, filtering through a 0.45 μm filter membrane, and taking the filtrate to obtain the test sample solution.

[0025] Further, in step S3, the concentrations of gallic acid, danshensu, paeoniflorin, ferulic acid, cyperasterone, olibanolide I, rosmarinic acid, lithospermic acid, salvianolic acid B, salvianolic acid A and paeonol in the mixed standard solution are 105.64 μg / mL, 521.36 μg / mL, 247.35 μg / mL, 100.94 μg / mL, 107.80 μg / mL, 102.51 μg / mL, 108.19 μg / mL, 99.57 μg / mL, 707.56 μg / mL, 532.14 μg / mL and 102.20 μg / mL, respectively.

[0026] Furthermore, in steps S4 and S5, the chromatographic column is Eclipse XDB-C 18 (4.6mm×250mm, 5μm) column, the mobile phase was acetonitrile A-0.1% phosphoric acid aqueous solution B, gradient elution, flow rate was 1.0mL / min, detection wavelength was 265nm, column temperature was 25℃, injection volume was 20μL.

[0027] Furthermore, the elution conditions are:

[0028]

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] The present invention uses an established stable and reliable high performance liquid chromatography analysis method to simultaneously perform component analysis and content determination on 11 chemical components in Shuangdan Recipe, Shuangdan Chuanxiong Recipe, Shuangdan Chuanxiong Recipe and single-line Chinese medicine extracts, determine the ownership and content of the chemical components in different prescriptions, provide data support for subsequent preliminary pharmacokinetic and tissue distribution studies of different prescriptions, and further provide new ideas and methods for the development of new drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 is the chromatogram of mixed standard;

[0032] Figure 2 This is the HPLC chromatogram of the water extract of Salvia miltiorrhiza;

[0033] Figure 3 This is the HPLC chromatogram of Paeonia suffruticosa water extract;

[0034] Figure 4 This is the HPLC chromatogram of the water extract of Ligusticum chuanxiong;

[0035] Figure 5 This is the HPLC chromatogram of Achyranthes bidentata water extract;

[0036] Figure 6 This is the HPLC chromatogram of the water extract of Shuangdan Recipe;

[0037] Figure 7 This is the HPLC chromatogram of the water extract of Shuangdan Chuanxiong prescription;

[0038] Figure 8 This is the HPLC chromatogram of the water extract of Shuangdanchuan Achyranthes bidentata recipe.

[0039] Description of main reference numerals:

[0040] Figure 1-8 Among them, 1-gallic acid; 2-danshensu; 3-peonidin; 4-ferulic acid; 5-calixasterone; 6-oligospermum officinale I; 7-rosmarinic acid; 8-lithospermic acid; 9-salvianolic acid B; 10-salvianolic acid A; 11-paeonol. DETAILED DESCRIPTION

[0041] The following is a clear and complete description of the technical solution of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by technicians in this field without creative work are within the scope of protection of the present invention.

[0042] 1. Analysis and content determination of the main chemical components of Shuangdan Jiawei Fang

[0043] The double-dan prescription (Danshen + Paeonia suffruticosa) was combined with the guiding herbs Chuanxiong and Rhizoma Cyathulae to form horn medicine, and extracted by traditional decoction method. The extract powder was obtained by spray drying and used as test drug for subsequent experiments. A high-performance liquid chromatography (HPLC) analysis method was established to analyze the chemical components and determine the content of extracts from different prescriptions, laying a foundation for subsequent in vivo drug research.

[0044] 1.1 Experimental instruments and reagents

[0045] 1.1.1 Experimental instruments

[0046] The main experimental instruments and manufacturers are shown in Table 1.1.

[0047] Table 1.1 Experimental instruments and manufacturers

[0048]

[0049] 1.1.2 Drugs and reagents

[0050] The main experimental reagents and standards are shown in Table 1.2.

[0051] Table 1.2 Reagents / standard products and manufacturers

[0052]

[0053] 1.3 Experimental methods and results

[0054] 1.3.1 Preparation of Shuangdan Jiawei Formula Water Extract

[0055] The four medicinal materials of Salvia miltiorrhiza, Paeonia suffruticosa, Ligusticum chuanxiong and Cyathula were weighed according to the following dosages and extracted separately.

[0056] Danshen single prescription: Danshen 1.2kg

[0057] Mudan Cortex single prescription: Mudan Cortex 1.2kg

[0058] Chuanxiong single prescription: Chuanxiong 1.2kg

[0059] Sichuan Achyranthes bidentata single prescription: Sichuan Achyranthes bidentata 1.2kg

[0060] Double Dan Recipe: Danshen 1.2kg + Paeonia suffruticosa 0.6kg

[0061] Shuangdan plus Chuanxiong prescription: Danshen 1.2kg + Paeonia suffruticosa 0.6kg + Chuanxiong 0.6kg

[0062] Double Dan plus Sichuan Cyathula formula: Danshen 1.2kg + Paeonia suffruticosa 0.6kg + Sichuan Cyathula 0.6kg

[0063] Extraction method: The above prescription medicinal materials are extracted twice with water. The first time, add 8 times the amount of water to soak for 30 minutes, and reflux extraction for 1.5 hours; the second time, add 6 times the amount of water to reflux extraction for 1 hour; combine the two extracts, filter, and reduce the pressure to concentrate to a relative density of 1:1.15-1.18 (80℃), spray dry, and obtain dry powder, which is packaged in aluminum foil bags of 100g each for standby use. See Table 1.3 for details.

[0064] Table 1.3 Preparation information of Shuangdan Jiawei Fang water extract

[0065]

[0066] 1.3.2 Preparation of test solution

[0067] Take about 1.0g of Shuangdan Chuanxiong Fang / Shuangdan Chuanxiong Fang water extract sample, weigh accurately, place in a 150mL conical flask, add about 50mL of 50% methanol, ultrasonicate for 30min, cool, add 50% methanol to make up the weight loss, shake well, filter through a 0.45μm microporous filter membrane, take the filtrate, and get the test solution. The remaining parties are prepared in the same way.

[0068] 1.3.3 Preparation of standard solution

[0069] Accurately weigh appropriate amounts of gallic acid, danshensu, paeoniflorin, ferulic acid, cyasterone, ligusticolide I, rosmarinic acid, lithospermic acid, salvianolic acid B, salvianolic acid A, and paeonol standards into a 10 mL volumetric flask, dissolve them in 50% methanol and dilute to the mark to prepare a mixed reference solution. The concentrations of each standard in the mixed standard solution were as follows: gallic acid was 105.64 μg / mL, danshensu was 521.36 μg / mL, paeoniflorin was 247.35 μg / mL, ferulic acid was 100.94 μg / mL, cyasterone was 107.80 μg / mL, ligusticolide I was 102.51 μg / mL, rosmarinic acid was 108.19 μg / mL, lithospermic acid was 99.57 μg / mL, salvianolic acid B was 707.56 μg / mL, salvianolic acid A was 532.14 μg / mL, and paeonol was 102.20 μg / mL. The mixed standard solution was stored in a 4°C refrigerator for later use.

[0070] 1.3.4 Chromatographic conditions

[0071] The chromatographic column used is Eclipse XDB-C 18 (4.6mm×250mm, 5μm) column, the mobile phase was acetonitrile (A)-0.1% phosphoric acid aqueous solution (B), gradient elution, the chromatographic conditions were as shown in Table 1.4, the flow rate was 1.0mL / min, the detection wavelength was 265nm, the column temperature was 25℃, and the injection volume was 20μL.

[0072] Table 1.4 HPLC elution conditions

[0073]

[0074] 1.3.5 System adaptability experiment

[0075] The above mixed reference solution and test solution are tested according to the chromatographic conditions shown under "1.3.4". See the chromatogram for details. Figure 1-8 .

[0076] like Figure 1-8 As shown, a total of 11 components were detected, namely gallic acid, danshensu, paeoniflorin, ferulic acid, cyperasterone, olibanolide I, rosmarinic acid, lithospermic acid, salvianolic acid B, salvianolic acid A, and paeonol.

[0077] 1.3.6 Linear relationship investigation

[0078] Take 10μL, 20μL, 50μL, 100μL, 250μL, 500μL, 750μL, 1000μL of the mixed standard solution under "1.3.3" respectively, place each in a 1mL volumetric flask, dilute with 50% methanol, and obtain a series of mixed reference solutions respectively, and detect according to the chromatographic conditions under "2.3.4". With the concentration of the reference solution as the horizontal axis (X) and the peak area of ​​each component as the vertical axis (Y), the linear equation is fitted to obtain the linear regression equation. The results are shown in Table 1.5, indicating that the linear relationship is good within the corresponding concentration range.

[0079] Table 1.5 Linear relationship investigation results (n=6)

[0080]

[0081] 1.3.7 Precision experiment

[0082] Take the mixed standard solution under "1.3.3" and repeat the injection 6 times according to the chromatographic conditions shown in "1.3.4". Calculate the RSD of the peak area of ​​each component. The results are shown in Table 1.6.

[0083] Table 1.6 Precision investigation results (n=6)

[0084]

[0085]

[0086] As can be seen from Table 1.6, the RSDs of gallic acid, danshensu, paeoniflorin, ferulic acid, cyperasterone, olibanolide I, rosmarinic acid, lithospermic acid, salvianolic acid B, salvianolic acid A, and paeonol are 0.05%, 0.05%, 0.27%, 0.08%, 0.09%, 0.07%, 0.10%, 0.73%, 0.62%, 0.44%, and 0.19%, respectively. The RSD values ​​are all less than 3%, indicating that the precision of the instrument is good.

[0087] 1.3.8 Stability test

[0088] Accurately weigh the water extract of Shuangdan Chuanxiong Fang / Shuangdan Chuanxiong Fang, prepare the test solution according to the preparation method under "1.3.2", inject the test solution and the standard solution in "1.3.3" at 0, 2, 4, 6, 8, 12, 24, and 48 hours after preparation according to the chromatographic conditions shown in "1.3.4", record the peak area of ​​each component in the chromatogram of the standard solution and the test solution, and the results are shown in Tables 1.7, 1.8 and 1.9.

[0089] Table 1.7 Standard stability test results (n = 8)

[0090]

[0091] Table 1.8 Shuangdan Chuanxiong prescription stability test results (n = 8)

[0092]

[0093] Table 1.9 Shuangdanchuan Niuxi prescription stability test results (n = 8)

[0094]

[0095] Combined with Tables 1.7-1.9, it can be seen that in the standard solution, the RSDs of gallic acid, danshensu, paeoniflorin, ferulic acid, cyperasterone, olibanolide I, rosmarinic acid, lithospermic acid, salvianolic acid B, salvianolic acid A, and paeonol were 0.08%, 0.50%, 0.40%, 0.41%, 0.34%, 0.48%, 0.48%, 0.83%, 0.85%, 2.44%, and 0.58%, respectively. The RSD values ​​were all less than 3%, indicating that the standard solution had good stability within 48 hours. In the test solution of Shuangdan Chuanxiong recipe, the RSDs of gallic acid, danshensu, paeoniflorin, ferulic acid, ligusticolide I, rosmarinic acid, lithospermic acid, salvianolic acid B, salvianolic acid A and paeonol were 0.75%, 1.54%, 1.43%, 0.84%, 0.77%, 0.39%, 0.89%, 0.26%, 4.99% and 0.89%, respectively. Except for salvianolic acid A, the RSD values ​​were all less than 3%. The RSD of salvianolic acid A within 12 hours was 0.84%, which was less than 3%, indicating that the test solution of Shuangdan Chuanxiong recipe had good stability within 12 hours. In the test solution of Shuangdan Niuxi prescription, the RSDs of gallic acid, danshensu, paeoniflorin, cyasterone, rosmarinic acid, lithospermic acid, salvianolic acid B, salvianolic acid A and paeonol were 0.66%, 0.56%, 0.91%, 1.85%, 0.69%, 0.76%, 0.76%, 7.70% and 0.67%, respectively, and the RSD values ​​were all less than 3%, except for salvianolic acid A, the RSD values ​​of which were all less than 3%. The RSD of salvianolic acid A was 1.54% within 12 hours, which was less than 3%, indicating that the test solution of Shuangdan Niuxi prescription had good stability within 12 hours.

[0096] 1.3.9 Repeatability Experiment

[0097] Accurately weigh the water extract of Shuangdan Chuanxiong Fang / Shuangdan Chuanxiong Fang, prepare 6 test solutions in parallel according to the preparation method under "1.3.2", and inject and measure them according to the chromatographic conditions shown in "1.3.4" respectively, and calculate the RSD% of each component. The results are shown in Tables 1.10 and 1.11.

[0098] Table 1.10 Repeatability test results of Shuangdan Chuanxiong prescription (n=6)

[0099]

[0100] Table 1.11 Repeatability test results of Shuangdanchuan Niuxi prescription (n=6)

[0101]

[0102] As shown in Tables 1.10 and 1.11, the RSDs of gallic acid, danshensu, paeoniflorin, ferulic acid, ligusticolide I, rosmarinic acid, lithospermic acid, salvianolic acid B, salvianolic acid A, and paeonol in the test solution of Shuangdan Chuanxiong prescription were 0.82%, 0.71%, 0.76%, 0.95%, 0.88%, 0.65%, 1.08%, 1.31%, 1.57%, and 0.90%, respectively. 4%. In the test solution of Shuangdan Niuxi prescription, the RSDs of gallic acid, danshensu, paeoniflorin, cyperasterone, rosmarinic acid, lithospermic acid, salvianolic acid B, salvianolic acid A and paeonol were 0.33%, 0.40%, 0.70%, 1.57%, 0.53%, 0.60%, 0.61%, 1.13% and 0.37%, respectively. The RSD values ​​were all less than 3%, indicating that the method had good repeatability.

[0103] 1.3.10 Sample recovery experiment

[0104] Accurately weigh 6 portions of 0.5g Shuangdan Chuanxiong Fang / Shuangdan Chuanxiong Fang water extract and place them in 150mL flasks respectively. Accurately add a certain amount of gallic acid, danshensu, paeoniflorin, ferulic acid, cyperasterone, ligusticolide I, rosmarinic acid, lithospermic acid, salvianolic acid B, salvianolic acid A, and paeonol standards. According to the method under "1.3.2", prepare 6 test solutions. According to the chromatographic conditions shown in "1.3.4", calculate the RSD value of the recovery rate of each component in the 6 test solutions. The results are shown in Tables 1.12 and 1.13.

[0105] Table 2.12 Determination results of the recovery of 10 chemical components in the water extract of Shuangdan Chuanxiong prescription (n=6)

[0106]

[0107]

[0108]

[0109] Table 1.13 Determination results of the recovery rates of 9 chemical components in the water extract of Shuangdanchuan Achyranthes bidentata prescription (n=6)

[0110]

[0111]

[0112] It can be seen from Tables 1.12 and 1.13 that the average recovery rate of each component is greater than 96%, and the RSD value is less than 3%, indicating that the sample recovery rate of this method is stable and good.

[0113] 1.3.11 Content determination

[0114] Prepare the test solutions of seven kinds of water extract samples according to the method in "1.3.2", inject and measure according to the chromatographic conditions shown in "1.3.4", calculate the average content of each component in each test solution, and the results are shown in Table 1.14.

[0115] Table 1.14 Contents of 11 chemical components in seven water extracts of Salvia miltiorrhiza, Paeonia suffruticosa, Ligusticum chuanxiong, and Cyathula officinalis (mg / g)

[0116]

[0117] 1.3.12 Determination of content of compound mixture

[0118] The four water extracts of Salvia miltiorrhiza, Paeonia suffruticosa, Ligusticum chuanxiong and Cyathula were compounded, and the test solutions of the four compound mixtures were prepared according to the preparation method under "2.3.2". The samples were injected and measured according to the chromatographic conditions shown in "1.3.4", and the average content of each chemical component in each test solution was calculated. The results are shown in Table 1.15.

[0119] Table 1.15 Content of chemical components in three compound mixtures (mg / g)

[0120]

[0121]

[0122] Since Shuangdan Jiawei Fang contains many ingredients with different properties, isocratic elution is difficult to meet the analytical requirements. Therefore, the gradient elution method was used to select different chromatographic columns such as Eclipse XDB-C 18 (4.6mm×250mm, 5μm), Stamsil C 18 (4.6mm×250mm, 5μm), Shim-pack GIST C 18 -AQ (4.6mm×250mm, 5μm), different mobile phases (acetonitrile-0.1% phosphoric acid aqueous solution, acetonitrile-0.1% formic acid aqueous solution), different detection wavelengths (265nm, 280nm), different flow rates (0.8mL / min, 1.0mL / min, 1.2mL / min), different column temperatures (20℃, 25℃, 30℃), and different injection volumes (5μL, 10μL, 20μL) were investigated to observe and compare the separation. The final detection chromatographic conditions used were Eclipse XDB-C 18(4.6mm×250mm, 5μm) column, mobile phase system acetonitrile-0.1% phosphoric acid aqueous solution, flow rate of 1.0mL / min, detection wavelength of 265nm, column temperature of 25℃, injection volume of 20μL. In the method of preparing the test solution, different solvents (methanol, water, 50% methanol) and different ultrasonic times (15min, 30min, 60min) were investigated. Taking all the above factors into consideration, 50% methanol and ultrasonic treatment for 30min were selected as the ideal test solution preparation conditions. The quantitative analysis method of 11 chemical components in Shuangdan Jiawei Fang was established by high performance liquid chromatography. The method performed well in system adaptability, precision, stability, repeatability and recovery tests.

[0123] By analyzing the components of water extracts of different prescriptions, five chemical components were detected in the water extract of Salvia miltiorrhiza, namely, danshensu, rosmarinic acid, lithospermic acid, salvianolic acid B, and salvianolic acid A; three components were detected in the water extract of Paeonia suffruticosa, namely, gallic acid, paeoniflorin, and paeonol; two components were detected in the water extract of Ligusticum chuanxiong, namely, ferulic acid and ligusticolide I; and one component was detected in the water extract of Cyathula officinalis, namely, cyperasterone. After the four herbs were extracted in the formula (Salvia miltiorrhiza + Paeonia suffruticosa, Salvia miltiorrhiza + Paeonia suffruticosa + Ligusticum chuanxiong, Salvia miltiorrhiza + Paeonia suffruticosa + Cyathula officinalis), the chemical components were not lost. The results of the determination of the chemical component contents in the seven water extracts of different formulas were analyzed, and it was found that the contents of most chemical components in the water extracts of the three combinations were lower than those in the water extracts of the corresponding single medicinal flavors. The single water extracts were recombined and the contents of the same chemical components were tested. The results showed that the contents of each chemical component in the three compounds were generally higher than those in the water extracts of the three combinations. After the Shuangdan formula was extracted with Chuanxiong or Chuanxiong, the contents of gallic acid, danshensu, salvianolic acid A and paeonol decreased, which may be related to the interaction between the Chinese medicine compound during mixed extraction.

[0124] Through research, a stable and reliable high performance liquid chromatography (HPLC) analysis method was established, and 11 chemical components were analyzed and determined at the same time to determine the identity and content of the chemical components in different formulations, providing data support for subsequent preliminary pharmacokinetic and tissue distribution studies of different formulations in rats.

[0125] The foregoing description of specific exemplary embodiments of the present invention is for the purpose of illustration and demonstration. These descriptions are not intended to limit the present invention to the precise form disclosed, and it is clear that many changes and variations can be made based on the above teachings. The purpose of selecting and describing the exemplary embodiments is to explain the specific principles of the present invention and its practical application, so that those skilled in the art can realize and utilize various different exemplary embodiments of the present invention and various different selections and changes. The scope of the present invention is intended to be limited by the claims and their equivalents.

Claims

1. A method for simultaneously determining the contents of 11 chemical components in Shuangdan Recipe, Shuangdan Chuanxiong Recipe, Shuangdan Chuanxiong Recipe and single-line Chinese medicine extracts, characterized in that: The following steps are involved: S1. Water Extract Single Prescription and Shuangdan Jiawei Prescription The prescription was extracted with water twice, the extracts were combined, filtered, concentrated under reduced pressure, and spray-dried to obtain dry powder; S2. Prepare the test solution Prepare the test solution from the prescription dry powder; S3. Preparation of standard solution Accurately weigh gallic acid, danshensu, paeoniflorin, ferulic acid, cyasterone, ligusticolide I, rosmarinic acid, lithospermic acid, salvianolic acid B, salvianolic acid A and paeonol standards respectively to prepare a mixed standard solution; S4. Chromatographic detection Mixed standard solutions were taken according to the volume gradient, fixed to volume with 50% methanol, and detected by chromatographic column. The concentration of the mixed standard solution was used as the horizontal axis, and the peak area of ​​each component was used as the vertical axis. The linear regression equation was obtained by fitting. S5. Test the test solution The test solution prepared by S2 was detected by chromatographic column, and the average content of each chemical component in each test solution was calculated according to the fitted linear regression equation.

2. The method for simultaneously determining the contents of 11 chemical components in Shuangdan Recipe, Shuangdan Chuanxiong Recipe, Shuangdan Chuanxiong Recipe and single-line Chinese medicine extracts according to claim 1, characterized in that: In step S1, the single-line prescription includes Danshen single-line prescription, Moutan bark single-line prescription, Chuanxiong single-line prescription and Chuanxiong single-line prescription; the double-dan added-flavor prescription includes Shuangdan prescription, Shuangdan plus Chuanxiong prescription and Shuangdan plus Chuanxiong prescription.

3. The method for simultaneously determining the contents of 11 chemical components in Shuangdan Recipe, Shuangdan Chuanxiong Recipe, Shuangdan Chuanxiong Recipe and single-line Chinese medicine extracts according to claim 1, characterized in that: In step S1, the specific operation of water extraction is: add 8 times the amount of water for the first time, soak for 30 minutes, and reflux extraction for 1.5 hours; add 6 times the amount of water for the second time, reflux extraction for 1 hour, combine the two extracts, filter, and concentrate under reduced pressure to a relative density of 1:1.15-1.18 at 80°C.

4. The method for simultaneously determining the contents of 11 chemical components in Shuangdan Recipe, Shuangdan Chuanxiong Recipe, Shuangdan Chuanxiong Recipe and single-line Chinese medicine extracts according to claim 1, characterized in that: In step S2, the test sample solution is prepared by mixing the water extract sample with 50% methanol in a mass volume ratio of 1 g:50 mL, ultrasonicating for 30 min, cooling, supplementing the weight loss with 50% methanol, filtering through a 0.45 μm filter membrane, and taking the filtrate to obtain the test sample solution.

5. The method for simultaneously determining the contents of 11 chemical components in Shuangdan Recipe, Shuangdan Chuanxiong Recipe, Shuangdan Chuanxiong Recipe and single-line Chinese medicine extracts according to claim 1, characterized in that: In step S3, the concentrations of gallic acid, danshensu, paeoniflorin, ferulic acid, cyperasterone, olibanolide I, rosmarinic acid, lithospermic acid, salvianolic acid B, salvianolic acid A and paeonol in the mixed standard solution are 105.64 μg / mL, 521.36 μg / mL, 247.35 μg / mL, 100.94 μg / mL, 107.80 μg / mL, 102.51 μg / mL, 108.19 μg / mL, 99.57 μg / mL, 707.56 μg / mL, 532.14 μg / mL and 102.20 μg / mL, respectively.

6. The method for simultaneously determining the contents of 11 chemical components in Shuangdan Recipe, Shuangdan Chuanxiong Recipe, Shuangdan Chuanxiong Recipe and single-line Chinese medicine extracts according to claim 1, characterized in that: In steps S4 and S5, the chromatographic column is Eclipse XDB-C 18 (4.6mm×250mm, 5μm) column, the mobile phase was acetonitrile A-0.1% phosphoric acid aqueous solution B, gradient elution, flow rate was 1.0mL / min, detection wavelength was 265nm, column temperature was 25℃, injection volume was 20μL.

7. The method for simultaneously determining the contents of 11 chemical components in Shuangdan Recipe, Shuangdan Chuanxiong Recipe, Shuangdan Chuanxiong Recipe and single-line Chinese medicine extracts according to claim 6, characterized in that: The elution conditions are:

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