A method for simultaneously determining the contents of 11 chemical components in Shuangdan Decoction, Shuangdan Chuanqiong Decoction, Shuangdan Chuanxiong Decoction, Shuangdan Chuanniaoxi Decoction and single traditional Chinese medicine extract

Abstract

The application belongs to the technical field of traditional Chinese medicine, and particularly relates to a method for simultaneously determining the contents of 11 chemical components in Shuangdan Decoction, Shuangdan Chuanqiong Decoction, Shuangdan Chuanniuxi Decoction and single traditional Chinese medicine extracts, which comprises the following steps: S1. water extraction of single decoction and Shuangdan Decoction; S2. preparation of test sample solution; S3. preparation of standard sample solution; S4. chromatographic detection of mixed standard sample solution, and fitting to obtain a linear regression equation; and S5. chromatographic detection of test sample solution, and calculation of the average content of each chemical component in each test sample solution according to the fitted linear regression equation. The application can simultaneously analyze and determine the contents of 11 chemical components in Shuangdan Decoction, Shuangdan Chuanqiong Decoction, Shuangdan Chuanniuxi Decoction and single traditional Chinese medicine extracts by using a stable and reliable high-performance liquid chromatography analysis method, so as to determine the attribution and content of the chemical components in different prescriptions, provide data support for subsequent preliminary pharmacokinetic and tissue distribution research of different prescriptions, and further provide new ideas and methods for the development of new drugs.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine technology, specifically relating to a method for simultaneously determining the content of 11 chemical components in Shuangdanfang, Shuangdan Chuanxiongfang, Shuangdan Chuanniuxifang, and single-herb extracts. Background Technology

[0002] The Shuangdan formula, originating from *Shi Jinmo's Collection of Clinical Experiences in Paired Herbs*, is a classic and effective formula for promoting blood circulation and removing blood stasis. It consists of Danshen (Salvia miltiorrhiza) and Mudanpi (Paeonia suffruticosa), combined based on the principle of synergistic effects among the seven emotions in traditional Chinese medicine. Danshen, bitter and slightly cold in nature, enters the blood level, promoting blood circulation, removing blood stasis, relieving pain, promoting blood regeneration, cooling the blood, relieving irritability, and calming the mind. Mudanpi, bitter and slightly cold in nature, specifically enters the blood level, both cooling and promoting blood circulation, clearing latent heat in the blood, and dispersing blood stasis. Danshen excels at promoting blood circulation and removing blood stasis, while Mudanpi excels at cooling the blood, dispersing blood stasis, and clearing latent heat in the yin level. The combination of these two herbs cools and promotes blood circulation, removes blood stasis, promotes blood regeneration, and enhances the ability to clear pathogenic heat. This formula has the effects of promoting blood circulation, removing blood stasis, unblocking the meridians, and relieving pain. Clinically, it is mainly used to treat symptoms such as chest pain and angina caused by blood stasis, and is often used in the treatment of coronary heart disease and angina pectoris. Currently, common preparations of this formula include Shuangdan granules, Shuangdan capsules, Shuangdan oral liquid, Shuangdan dripping pills, Shuangdan tablets, and Shuangdan dispersible tablets. Among them, Shuangdan oral liquid is included in the 2020 edition of the Chinese Pharmacopoeia, and its efficacy is to promote blood circulation, remove blood stasis, unblock meridians, and relieve pain.

[0003] Guiding drugs, also known as meridian-guiding agents, are drugs that direct other medications to the site of the disease or a specific meridian, thereby enhancing their therapeutic effect. Modern research has found that guiding drugs in traditional Chinese medicine can promote the absorption or distribution of other drugs in target organs and tissues. Their targeted action may be related to opening the physiological barriers at the target site. The mechanisms of action of guiding drugs also include altering cell membrane structure or permeability, changing intracellular pH to enhance the targeting effect of traditional Chinese medicine, inhibiting the activity and expression of P-glycoproteins or other membrane transport proteins, and thus promoting the absorption of traditional Chinese medicine components. The 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 it may exert its guiding effect by directly affecting neurotransmitter levels and regulating the levels of two or more biochemical substances; guiding drugs may also increase drug solubility and promote the dissolution of active ingredients. The concepts of guiding drugs to the meridian and returning drugs to the meridian are closely related. The theory of guiding drugs to the meridian is further developed based on the theory of returning drugs to the meridian, and at the same time, it is combined with the theory of drug compatibility, becoming a major feature of TCM drug use. In compound prescriptions, by combining drugs that guide drugs to the meridian, the drugs can be coordinated to move towards the disease site and exert a directional effect, thereby improving the efficacy.

[0004] "Cornered medicine" refers to a method of medication that combines three Chinese herbs, guided by the basic theories of Traditional Chinese Medicine (TCM), based on syndrome differentiation and treatment, and adhering to the principles of herbal compatibility based on their aroma, properties, and the seven emotions. Cornered medicine lies between a single herb and a prescription; it is an expansion of the medicine, but not simply an increase in the number of herbs. Cornered medicine plays a primary or auxiliary role in a prescription, or it can function independently. In clinical application, it can reduce toxicity and enhance efficacy, expand the scope of drug use, and provide new ideas and methods for developing new drugs.

[0005] The *Shennong Bencao Jing* records that Chuanxiong (Ligusticum striatum) is "pungent and warm in nature, and treats stroke affecting the head." The Ming Dynasty's *Bencao Huiyan* also states that Chuanxiong "ascends to the head and eyes, regulates menstruation, and relieves stagnation; it is a qi-regulating herb in the blood." Chuanxiong moves qi in the blood and is also a wind-dispelling herb, readily dispersing and penetrating, reaching the head and eyes to promote qi and blood circulation. The Qing Dynasty's *Depei Bencao* states, "Headache is ineffective without a guiding herb; Chuanxiong is effective for Jueyin headaches, serving as a guiding herb." The Ming Dynasty's *Shoushi Baoyuan* records a treatment for bloating where, after taking Baoyuan Tang, the bloating in the head and forehead does not subside; six fen of Chuanxiong can be added as a guiding herb, indicating that Chuanxiong can be used as a guiding herb to reach the head and forehead. In modern research, Wang et al., when studying the effects of borneol and chuanxiong on the blood-brain barrier penetration of the compound Shuyu Jiannao formula, found that the addition of chuanxiong or borneol increased the content of jujube seed saponin A, the main active ingredient in the formula, in brain tissue. Liu Lirong et al. found that the combination of angelica dahurica and chuanxiong could increase the blood concentration and Cmax of four coumarin compounds in angelica dahurica to varying degrees, promoting the absorption of active ingredients in the body while slowing their clearance rate and improving bioavailability. Zhao Qian et al. found that chuanxiong, as a guiding herb, opened the blood-brain barrier without promoting melanoma brain metastasis, and that its combination with temozolomide enhanced the therapeutic effect of temozolomide on brain metastatic melanoma B16F10. When chuanxiong is used as a guiding herb in combination with other drugs, it can achieve the effect of guiding drugs upwards to the head by increasing drug concentration in brain tissue, increasing blood drug concentration, and enhancing drug effects.

[0006] According to traditional Chinese medicine, both Achyranthes bidentata (Chuan Niu Xi) and Achyranthes bidentata (Huai Niu Xi) can invigorate blood circulation, regulate menstruation, tonify the liver and kidneys, strengthen tendons and bones, promote urination, and guide fire (blood) downwards. However, Achyranthes bidentata excels in invigorating blood circulation and regulating menstruation, while Achyranthes bidentata excels in tonifying the liver and kidneys and strengthening tendons and bones. As a guiding herb, Achyranthes bidentata is often combined with other herbs to exert its therapeutic effects by directing the medicine to specific areas along the meridian. The modified Shenqi Wan formula, which warms and tonifies kidney yang and promotes diuresis and reduces swelling, uses Achyranthes bidentata (soaked in wine). The *Yifang Jijie* (Collection of Medical Prescriptions) records that adding Achyranthes bidentata can benefit the liver and kidneys, thereby promoting downward movement. Modern research shows that Achyranthes bidentata, Ermiao Wan, and Sanmiao Wan can reduce the inflammatory response of the ankle joint in rats with acute gouty arthritis by downregulating NF-κB p65 protein expression, inhibiting the levels of TNF-α, IL-6, and IL-8, and reducing joint swelling. Sanmiao Wan is particularly effective, as it is composed of Ermiao Wan combined with Achyranthes bidentata. This may be related to the fact that Achyranthes bidentata, in addition to its effects of promoting blood circulation, clearing the meridians, and tonifying the liver and kidneys, also has a "guiding the medicine downwards" effect. Studies by Shen Liang et al. have shown that Sijunzi Decoction and Sijunzi Decoction plus Achyranthes bidentata can accelerate the entry of fibroblasts from the G0 / G1 phase into the S phase, promote fibroblast proliferation, and promote wound healing. Furthermore, the proportion of fibroblasts in the S phase in the Sijunzi Decoction group was significantly higher than that in the Sijunzi Decoction group, suggesting that the mechanism of Achyranthes bidentata's guiding effect may be a synergistic effect by increasing the proportion of fibroblasts in the S phase and promoting fibroblast proliferation.

[0007] Based on the theory of guiding herbs and the compatibility of complementary herbs, the modified Shuangdan formula adds Chuanxiong or Achyranthes bidentata to the classic Shuangdan formula to form complementary herbs, namely Danshen-Mudanpi-Chuanxiong or Danshen-Mudanpi-Chuanniuxi. The study explores the guiding effect of Chuanxiong / Achyranthes bidentata, that is, whether it can change the attribution and content of the main effective components of Shuangdan formula in different formulations, which can provide new ideas and methods for the development of new drugs.

[0008] The information disclosed in this background section is intended only to enhance the understanding of the overall background of the invention and should not be construed as an admission or in any way implying that the information constitutes prior art known to those skilled in the art. Summary of the Invention

[0009] The purpose of this invention is to provide a method for simultaneously determining the content of 11 chemical components in Shuangdanfang, Shuangdan Chuanxiongfang, Shuangdan Chuanniuxifang, and single-herb extracts, which can provide new ideas and methods for developing new drugs.

[0010] To achieve the above objectives, the present invention provides the following technical solution:

[0011] A method for simultaneously determining the content of 11 chemical components in Shuangdanfang, Shuangdanchuanxiongfang, Shuangdanchuanniuxifang, and single-herb extracts includes the following steps:

[0012] S1. Water-extracted single-ingredient formula and double-ingredient modified formula

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

[0014] S2. Preparation of the test solution

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

[0016] S3. Preparation of standard solutions

[0017] Accurately weigh gallic acid, tanshinone, paeoniflorin, ferulic acid, cylindricalanone, ligustrol I, rosmarinic acid, lithospermic acid, salvianolic acid B, salvianolic acid A and paeonol standards respectively, and prepare a mixed standard solution.

[0018] S4. Chromatographic detection

[0019] According to the volume gradient, the mixed standard solution was taken separately, diluted to volume with 50% methanol, and detected by chromatographic column. The concentration of the mixed standard solution was used as the abscissa and the peak area of ​​each component was used as the ordinate to obtain the linear regression equation.

[0020] S5. Detection of the test solution

[0021] The test solutions prepared by S2 were detected using a chromatographic column. Based on the fitted linear regression equation, the average content of each chemical component in each test solution was calculated.

[0022] Furthermore, in step S1, the single-ingredient formulas include the Danshen single-ingredient formula, the Mudanpi single-ingredient formula, the Chuanxiong single-ingredient formula, and the Chuanniuxi single-ingredient formula; the double-Dans-added formulas include the double-Dans formula, the double-Dans plus Chuanxiong formula, and the double-Dans plus Chuanniuxi formula.

[0023] Furthermore, in step S1, the specific operation of water extraction is as follows: first, add 8 times the amount of water to soak for 30 minutes, and then reflux for 1.5 hours; second, add 6 times the amount of water to reflux 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℃.

[0024] Furthermore, in step S2, the preparation method of the test sample solution is as follows: the water extract sample is mixed with 50% methanol at a mass-volume ratio of 1g:50mL, sonicated for 30min, cooled, and the weight loss is replenished with 50% methanol. The mixture is then filtered through a 0.45μm filter membrane, and the filtrate is collected to obtain the test sample solution.

[0025] Furthermore, in step S3, the concentrations of gallic acid, tanshinone, paeoniflorin, ferulic acid, cylindrica, ligustrol I, rosmarinic acid, lithospermic acid, salvianolic acid B, salvianolic acid A, and paeonol in the mixed standard solution are, respectively: 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.

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

[0027] Furthermore, the elution conditions are as follows:

[0028]

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

[0030] This invention utilizes a stable and reliable high-performance liquid chromatography (HPLC) method to simultaneously analyze and determine the content of 11 chemical components in Shuangdan Formula, Shuangdan Chuanxiong Formula, Shuangdan Chuanniuxi Formula, and single-herb extracts. This method identifies the chemical components and their content in different formulations, providing data support for subsequent preliminary pharmacokinetic and tissue distribution studies of different formulations. Ultimately, it can offer new ideas and methods for developing new drugs. Attached Figure Description

[0031] Figure 1 Chromatogram of mixed standards;

[0032] Figure 2 This is the HPLC chromatogram of the aqueous extract of Salvia miltiorrhiza.

[0033] Figure 3 Here is the HPLC chromatogram of the aqueous extract of Paeonia suffruticosa root bark;

[0034] Figure 4 Here is the HPLC chromatogram of the aqueous extract of Ligusticum chuanxiong.

[0035] Figure 5 Here is the HPLC chromatogram of the aqueous extract of Achyranthes bidentata;

[0036] Figure 6 This is the HPLC chromatogram of the aqueous extract of Shuangdanfang.

[0037] Figure 7 The HPLC chromatogram of the aqueous extract of Shuangdan Chuanxiong Decoction;

[0038] Figure 8 This is the HPLC chromatogram of the aqueous extract of Shuangdanchuan Niuxi Fang.

[0039] Explanation of key figure labels:

[0040] Figure 1-8 In the formula, 1-gallic acid; 2-tanshinone; 3-paeonoside; 4-ferulic acid; 5-crissanosterone; 6-limonene lactone I; 7-rosmarinic acid; 8-lithocholic acid; 9-tanshinone B; 10-tanshinone A; 11-paeonol. Detailed Implementation

[0041] The technical solution of this invention patent will be clearly and completely described below. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without inventive effort are within the scope of protection of this invention.

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

[0043] The two-component formula (Danshen + Mudanpi) was combined with the guiding herbs Chuanxiong and Chuanniuxi to form a compound. The extract was obtained by traditional decoction method and spray-dried to obtain dry powder, which was used as a reagent for subsequent experiments. A high-performance liquid chromatography (HPLC) method was established to analyze the chemical composition and determine the content of the extracts of different formulas, laying the foundation for subsequent in vivo drug research.

[0044] 1.1 Experimental Apparatus and Reagents

[0045] 1.1.1 Experimental Apparatus

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

[0047] Table 1.1 Experimental Instruments and Manufacturers

[0048]

[0049] 1.1.2 Medicines and Reagents

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

[0051] Table 1.2 Reagents / Standards and Manufacturers

[0052]

[0053] 1.3 Experimental Methods and Results

[0054] 1.3.1 Preparation of water extract of Shuangdan Jiawei Fang

[0055] The four medicinal materials, namely Salvia miltiorrhiza, Paeonia suffruticosa, Ligusticum chuanxiong, and Achyranthes bidentata, were weighed according to the following dosage and extracted separately.

[0056] Danshen single-ingredient formula: Danshen 1.2kg

[0057] Peony bark single-ingredient formula: 1.2 kg of peony bark

[0058] Chuanxiong (Ligusticum striatum) alone: ​​1.2 kg of Chuanxiong

[0059] A single-ingredient formula for Achyranthes bidentata: 1.2 kg of Achyranthes bidentata

[0060] Double Dan Formula: 1.2kg of Danshen (Salvia miltiorrhiza) + 0.6kg of Mudanpi (Paeonia suffruticosa).

[0061] Double Danshen plus Chuanxiong formula: Danshen 1.2kg + Mudanpi 0.6kg + Chuanxiong 0.6kg

[0062] Double Danshen plus Achyranthes bidentata formula: Danshen 1.2kg + Mudanpi 0.6kg + Chuanniuxi 0.6kg

[0063] Extraction method: The above-mentioned prescription herbs were extracted twice with water. For the first extraction, the herbs were soaked in 8 times the amount of water for 30 minutes and then refluxed for 1.5 hours. For the second extraction, the herbs were refluxed for 1 hour with 6 times the amount of water. The two extracts were combined, filtered, concentrated under reduced pressure to a relative density of 1:1.15-1.18 (80℃), and spray-dried to obtain a dry powder. Each 100g packet was packaged in an aluminum foil bag for later use. See Table 1.3 for details.

[0064] Table 1.3 Information on the preparation of water extracts from Shuangdan Jiawei Formula

[0065]

[0066] 1.3.2 Preparation of the test solution

[0067] Accurately weigh approximately 1.0 g of the aqueous extract sample of Shuangdan Chuanxiong Formula / Shuangdan Chuanniuxi Formula and place it in a 150 mL Erlenmeyer flask. Add approximately 50 mL of 50% methanol, sonicate for 30 min, cool, and replenish the lost weight with 50% methanol. Shake well and filter through a 0.45 μm microporous membrane. Collect the filtrate to obtain the test solution. The other formulas are prepared using the same method.

[0068] 1.3.3 Preparation of Standard Solutions

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

[0070] 1.3.4 Chromatographic conditions

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

[0072] Table 1.4 HPLC elution conditions

[0073]

[0074] 1.3.5 System Adaptability Experiment

[0075] The above-mentioned mixed reference solution and test solution were analyzed under the chromatographic conditions shown in section "1.3.4". The chromatogram is shown below. Figure 1-8 .

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

[0077] 1.3.6 Examination of Linear Relationships

[0078] Take 10 μL, 20 μL, 50 μL, 100 μL, 250 μL, 500 μL, 750 μL, and 1000 μL of the mixed standard solution from section "1.3.3", and place each into a 1 mL volumetric flask. Dilute to volume with 50% methanol to obtain a series of mixed reference solutions. Detect these solutions according to the chromatographic conditions described in section "2.3.4". Plot the concentration of the reference solution as the abscissa (X) and the peak area of ​​each component as the ordinate (Y). Fit a linear equation to obtain the linear regression equation. The results are shown in Table 1.5, indicating a good linear relationship within the corresponding concentration range.

[0079] Table 1.5 Results of the linear relationship examination (n=6)

[0080]

[0081] 1.3.7 Precision Experiment

[0082] Take the mixed standard solution under section “1.3.3” and inject it repeatedly 6 times under the chromatographic conditions shown in “1.3.4”. Calculate the peak area RSD of each component. The results are shown in Table 1.6.

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

[0084]

[0085]

[0086] As shown in Table 1.6, the RSDs of gallic acid, tanshinone, paeoniflorin, ferulic acid, cylindrica, ligustrol 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. All RSD values ​​are less than 3%, indicating that the instrument has good precision.

[0087] 1.3.8 Stability Test

[0088] Accurately weigh the aqueous extracts of Shuangdan Chuanxiong Fang / Shuangdan Chuanniuxi Fang, and prepare the test solution according to the preparation method in section "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 h after preparation, respectively, according to the chromatographic conditions shown in "1.3.4". Record the peak areas of each component in the chromatograms of the standard solution and the test solution. The results are shown in Tables 1.7, 1.8, and 1.9.

[0089] Table 1.7 Results of stability tests on standard samples (n=8)

[0090]

[0091] Table 1.8 Stability test results of Shuangdan Chuanxiong Formula (n=8)

[0092]

[0093] Table 1.9 Stability test results of Shuangdanchuan Niuxi Formula (n=8)

[0094]

[0095] As shown in Tables 1.7-1.9, the RSDs of gallic acid, tanshinone, paeoniflorin, ferulic acid, cylindrica, ligustrol I, rosmarinic acid, lithospermic acid, salvianolic acid B, salvianolic acid A, and paeonol in the standard solution are 0.08%, 0.50%, 0.40%, 0.41%, 0.34%, 0.48%, 0.48%, 0.83%, 0.85%, 2.44%, and 0.58%, respectively. All RSD values ​​are less than 3%, indicating that the standard solution has good stability within 48 hours. In the test solution of Shuangdan Chuanxiong Formula, the RSDs of gallic acid, tanshinone, paeoniflorin, ferulic acid, ligustilide 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 is less than 3%, indicating that the test solution of Shuangdan Chuanxiong Formula has good stability within 12 hours. In the test solution of Shuangdan Niuxi Fang, the RSDs of gallic acid, tanshinone, paeoniflorin, cylindrica sterol, 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, all less than 3%. Except for salvianolic acid A, the RSDs of all other compounds were less than 3%. The RSD of salvianolic acid A within 12 hours was 1.54%, less than 3%, indicating that the test solution of Shuangdan Niuxi Fang had good stability within 12 hours.

[0096] 1.3.9 Repeatability Experiment

[0097] Accurately weigh the aqueous extracts of Shuangdan Chuanxiong Fang / Shuangdan Chuanniu Xi Fang, and prepare 6 test solutions in parallel according to the preparation method in section "1.3.2". Inject and determine the contents of each component according to the chromatographic conditions shown in "1.3.4". Calculate the RSD% of each component. The results are shown in Tables 1.10 and 1.11.

[0098] Table 1.10 Results of repeatability experiments of Shuangdan Chuanxiong formula (n=6)

[0099]

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

[0101]

[0102] As shown in Tables 1.10 and 1.11, the RSDs of gallic acid, tanshinone, paeoniflorin, ferulic acid, ligustilide I, rosmarinic acid, lithospermic acid, salvianolic acid B, salvianolic acid A, and paeonol in the Shuangdan Chuanxiong formula test solution were 0.82%, 0.71%, 0.76%, 0.95%, 0.88%, 0.65%, 1.08%, 1.31%, 1.57%, and 0.9%, respectively. In the test solution of Shuangdan Niuxi Fang, the RSDs of gallic acid, tanshinone, paeoniflorin, cylindrica sterol, 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. All RSDs were less than 3%, indicating good repeatability of the method.

[0103] 1.3.10 Spiking and Recovery Experiment

[0104] Accurately weigh 0.5g of the aqueous extracts of Shuangdan Chuanxiong Fang / Shuangdan Chuanniuxi Fang into 6 portions, and place them into 150mL flasks respectively. Accurately add a certain amount of gallic acid, tanshinone, paeoniflorin, ferulic acid, cylindrica sterol, chuanxiong lactone I, rosmarinic acid, lithospermic acid, salvianolic acid B, salvianolic acid A, and paeonol standards. Prepare 6 test solutions according to the method in section "1.3.2". Inject and determine the components according to the chromatographic conditions shown in "1.3.4". Calculate the RSD values ​​of the recoveries of each component in the 6 test solutions. The results are shown in Tables 1.12 and 1.13.

[0105] Table 2.12 Results of recovery rate determination of 10 chemical components in the aqueous extract of Shuangdan Chuanxiong formula (n=6)

[0106]

[0107]

[0108]

[0109] Table 1.13 Results of recovery rate determination of 9 chemical components in the aqueous extract of Shuangdanchuan Niuxi Fang (n=6)

[0110]

[0111]

[0112] As shown in Tables 1.12 and 1.13, the average recovery rate of each component is greater than 96%, and the RSD value is less than 3%, indicating that the recovery rate of this method is stable and good.

[0113] 1.3.11 Content Determination

[0114] Seven water extract samples were prepared according to the method in "1.3.2" and injected for determination according to the chromatographic conditions shown in "1.3.4". The average content of each component in each test sample solution was calculated and the results are shown in Table 1.14.

[0115] Table 1.14 Content (mg / g) of 11 chemical components in seven water extracts of Salvia miltiorrhiza, Paeonia suffruticosa, Ligusticum chuanxiong, and Achyranthes bidentata.

[0116]

[0117] 1.3.12 Determination of content in compound mixtures

[0118] Four water extracts of Salvia miltiorrhiza, Paeonia suffruticosa, Ligusticum chuanxiong, and Achyranthes bidentata were compounded and prepared into test solutions according to the preparation method in section "2.3.2". The solutions were injected and determined according to the chromatographic conditions shown in "1.3.4". The average content of each chemical component in each test solution was calculated and the results are shown in Table 1.15.

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

[0120]

[0121]

[0122] Because Shuangdan Jiawei Formula contains numerous components with varying properties, isocratic elution is insufficient for analytical requirements. Therefore, a gradient elution method is employed, using 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 The chromatographic conditions were investigated using an AQ column (4.6 mm × 250 mm, 5 μm) with different mobile phases (acetonitrile-0.1% phosphoric acid aqueous solution, acetonitrile-0.1% formic acid aqueous solution), different detection wavelengths (265 nm, 280 nm), different flow rates (0.8 mL / min, 1.0 mL / min, 1.2 mL / min), different column temperatures (20℃, 25℃, 30℃), and different injection volumes (5 μL, 10 μL, 20 μL). The resolution was observed and compared. The final detection chromatographic conditions used were those of an Eclipse XDB-C column. 18A 4.6 mm × 250 mm, 5 μm column was used. The mobile phase consisted of acetonitrile-0.1% phosphoric acid aqueous solution at a flow rate of 1.0 mL / min, a detection wavelength of 265 nm, a column temperature of 25 °C, and an injection volume of 20 μL. Different solvents (methanol, water, 50% methanol) and different ultrasonic treatment times (15 min, 30 min, 60 min) were investigated for the preparation of the test solution. Considering all factors, 50% methanol and ultrasonic treatment for 30 min were selected as the ideal conditions for preparing the test solution. A quantitative analysis method for 11 chemical components in Shuangdan Jiawei Formula was established using high-performance liquid chromatography (HPLC). This method demonstrated good performance in system suitability, precision, stability, repeatability, and recovery tests.

[0123] Component analysis of water extracts from different formulations revealed five chemical components in the Salvia miltiorrhiza water extract: tanshinone, rosmarinic acid, lithospermic acid, salvianolic acid B, and salvianolic acid A; three components in the Paeonia suffruticosa water extract: gallic acid, paeoniflorin, and paeonol; two components in the Ligusticum chuanxiong water extract: ferulic acid and ligustrol I; and one component in the Achyranthes bidentata water extract: cylindrica sterol. No chemical components were lost when the four herbs were combined into formulations (Salvia miltiorrhiza + Paeonia suffruticosa, Salvia miltiorrhiza + Paeonia suffruticosa + Ligusticum chuanxiong, Salvia miltiorrhiza + Paeonia suffruticosa + Achyranthes bidentata). Analysis of the chemical component content in the seven water extracts from different formulations showed that the content of most chemical components in the water extracts of the three combinations was lower than that in the water extracts of the corresponding single herbs. The single-herb aqueous 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 compound extracts were generally higher than those in the aqueous extracts of the three combinations. After extracting Shuangdanfang with the addition of Chuanxiong or Chuanniuxi, the contents of gallic acid, tanshinone, salvianolic acid A, and paeonol decreased, which may be related to the interaction between the traditional Chinese medicine compound extracts during the mixed extraction process.

[0124] This study established a stable and reliable high-performance liquid chromatography (HPLC) method, and simultaneously analyzed and determined the content of 11 chemical components. The study identified the chemical components and their contents 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 invention is for illustrative and explanatory purposes. These descriptions are not intended to limit the invention to the precise forms disclosed, and it will be apparent that many changes and variations can be made in accordance with the foregoing teachings. The exemplary embodiments were chosen and described in order to explain the specific principles of the invention and its practical application, thereby enabling those skilled in the art to implement and utilize various different exemplary embodiments of the invention, as well as various different choices and variations. The scope of the invention is intended to be defined by the claims and their equivalents.

Claims

1. A method for simultaneously determining the content of 11 chemical components in Shuangdan Fang, Shuangdan Chuanxiong Fang, Shuangdan Chuanniuxi Fang, and single-herb extracts, characterized in that, Includes the following steps: S1. Water-extracted single-ingredient formula and double-ingredient modified formula The prescription was extracted twice with water, the extracts were combined, filtered, concentrated under reduced pressure, and spray-dried to obtain a dry powder. S2. Preparation of the test solution Prepare a test solution from the dry powder of the prescription; S3. Preparation of standard solutions Accurately weigh gallic acid, tanshinone, paeoniflorin, ferulic acid, cylindricalanone, ligustrol I, rosmarinic acid, lithospermic acid, salvianolic acid B, salvianolic acid A and paeonol standards respectively, and prepare a mixed standard solution. S4. Chromatographic detection According to the volume gradient, the mixed standard solution was taken separately, diluted to volume with 50% methanol, and detected by chromatographic column. The concentration of the mixed standard solution was used as the abscissa and the peak area of ​​each component was used as the ordinate to obtain the linear regression equation. S5. Detection of the test solution The test solutions prepared in S2 were analyzed using a chromatographic column. Based on the fitted linear regression equation, the average content of each chemical component in each test solution was calculated; among which, In steps S4 and S5, the chromatographic column is an Eclipse XDB-C. 18 5 μm, 4.6 mm × 250 mm; mobile phase: acetonitrile A - 0.1% phosphoric acid aqueous solution B, gradient elution, flow rate: 1.0 mL / min, detection wavelength: 265 nm, column temperature: 25 ℃, injection volume: 20 μL; elution conditions: 。 2. The method for simultaneously determining the content of 11 chemical components in Shuangdanfang, Shuangdan Chuanxiongfang, Shuangdan Chuanniuxifang, and single-herb extracts according to claim 1, characterized in that, In step S1, the single-ingredient formulas include the Danshen single-ingredient formula, the Mudanpi single-ingredient formula, the Chuanxiong single-ingredient formula, and the Chuanniuxi single-ingredient formula; the double-Dans-modified formulas include the double-Dans formula, the double-Dans-plus-Chuanxiong formula, and the double-Dans-plus-Chuanniuxi formula.

3. The method for simultaneously determining the content of 11 chemical components in Shuangdanfang, Shuangdan Chuanxiongfang, Shuangdan Chuanniuxifang, and single-herb extracts according to claim 1, characterized in that, In step S1, the specific operation of water extraction is as follows: first, add 8 times the amount of water and soak for 30 minutes, then reflux for 1.5 hours; second, add 6 times the amount of water and reflux 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℃.

4. The method for simultaneously determining the content of 11 chemical components in Shuangdanfang, Shuangdan Chuanxiongfang, Shuangdan Chuanniuxifang, and single-herb extracts according to claim 1, characterized in that, In step S2, the preparation method of the test sample solution is as follows: the water extract sample and 50% methanol are mixed at a mass-volume ratio of 1g:50mL, sonicated for 30min, cooled, and the weight loss is replenished with 50% methanol. The mixture is then filtered through a 0.45μm filter membrane, and the filtrate is collected to obtain the test sample solution.

5. The method for simultaneously determining the content of 11 chemical components in Shuangdanfang, Shuangdan Chuanxiongfang, Shuangdan Chuanniuxifang, and single-herb extracts according to claim 1, characterized in that, In step S3, the concentrations of gallic acid, tanshinone, paeoniflorin, ferulic acid, cylindrica, ligustrol I, rosmarinic acid, lithospermic acid, salvianolic acid B, salvianolic acid A, and paeonol in the mixed standard solution were, respectively: 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.

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