Screening and detection method of traditional Chinese medicine quality markers for yiqi tongluo capsule against myocardial ischemia and application thereof

Abstract

The application discloses a screening and detection method of traditional Chinese medicine quality markers of Yiqi Tongluo Capsules (YTC) against myocardial ischemia and application of the method. The application screens quality markers (Q-markers) of Yiqi Tongluo Capsules for treating myocardial ischemia, optimizes an extraction process of YTC and establishes a fingerprint spectrum on the basis of finding Q-marker components, so as to be beneficial to optimization of the extraction process of YTC, quality control and development of modern preparations.

Description

Technical Field

[0001] This invention specifically relates to the screening and detection method of quality markers of Yiqi Tongluo capsules for anti-myocardial ischemia and their application. Background Technology

[0002] In recent years, cardiovascular diseases have become increasingly prevalent, becoming the number one killer of human health. In particular, with the increasing prevalence of obesity, hyperlipidemia, and aging, the number of people suffering from ischemic heart disease has also increased significantly. Myocardial ischemia refers to a pathophysiological state in which reduced blood perfusion and oxygen supply to the heart from the coronary arteries leads to abnormal energy metabolism and an inability to maintain normal cardiac function. In traditional Chinese medicine, it falls under the category of "chest pain and heart pain," clinically manifesting as dull pain behind the sternum or in the precordial region, sometimes radiating to the back, causing shortness of breath and inability to lie down, and even threatening life.

[0003] Yiqi-Tongluo capsule (YTC) has the effects of invigorating qi, promoting blood circulation, and clearing the meridians. It is included in the "List of Traditional Chinese Medicine Preparations for Dispensing in Medical Institutions of Sichuan Province (First Batch)" and is a traditional Chinese medicine commonly used in clinical practice to treat myocardial ischemia and coronary heart disease. It has the characteristics of definite efficacy and few side effects. YTC is composed of 12 herbs including Astragalus membranaceus, Angelica sinensis, Pheretima aspergillum, Paeonia lactiflora, Ligusticum chuanxiong, and Carthamus tinctorius. Astragalus membranaceus is sweet and slightly warm in nature. As a representative of Qi-tonifying herbs, it has the effects of nourishing blood and promoting body fluid production, and relieving stagnation and pain. Angelica sinensis is warm in nature and sweet, pungent, and bitter in taste. It enters the heart meridian and is an essential medicine for blood diseases. It has the effects of tonifying blood and unblocking collaterals without harming blood. Pheretima aspergillum is salty and cold in nature. It unblocks collaterals and promotes the circulation of the medicine throughout the body. It is mainly used to treat Qi deficiency and blood stasis syndrome in stroke. Paeonia lactiflora is bitter and cold. It has the effects of clearing heat and cooling blood, dispersing blood stasis and relieving pain. Ligusticum chuanxiong is pungent, dispersing, warm and unblocking. It can both invigorate blood and remove blood stasis and promote the flow of Qi in the blood. It is a Qi-tonifying herb in the blood. All the herbs are combined to achieve the effects of "tonifying Qi and invigorating blood, relieving stagnation and pain". However, YTC lacks scientific and reasonable quality control methods, and its material basis for treating myocardial ischemia and reasonable extraction methods have not been systematically studied, which seriously limits the clinical application and promotion of this traditional Chinese medicine compound and its modern formulation development. Summary of the Invention

[0004] To address the above problems, this invention provides a method for screening quality markers of traditional Chinese medicine (TCM) for the anti-myocardial ischemia effect of Yiqi Tongluo capsules, which includes the following steps:

[0005] (1) Take blank serum and drug-containing serum from animals respectively, and detect them by UPLC-Q-TOF-MS / MS. Compare the total ion chromatograms obtained by detection, and compare the chromatographic information of peaks with differences with literature to determine the blood-entering components.

[0006] (2) Take the blood-entering components obtained in step (1), use the oxidative stress injury cardiomyocyte model to evaluate the cardiomyocyte protective activity of the blood-entering components, and screen for components that can increase cardiomyocyte cell vitality, which are the quality markers of traditional Chinese medicine for Yiqi Tongluo capsules to resist myocardial ischemia.

[0007] Further, the chromatographic conditions of the UPLC-Q-TOF-MS / MS in step (1) are as follows: mobile phase acetonitrile-0.1% formic acid aqueous solution, gradient elution program: 0-5 min, 5% acetonitrile; 5-15 min, 5%-15% acetonitrile; 15-25 min, 15%-30% acetonitrile; 25-30 min, 30%-55% acetonitrile; 30-40 min, 55%-75% acetonitrile; 40-45 min, 75%-85% acetonitrile; 45-50 min, 85% acetonitrile; flow rate 0.2 mL / min, column temperature 35℃, injection volume 10 μL.

[0008] Further, the mass spectrometry conditions of the UPLC-Q-TOF-MS / MS in step (1) are as follows: electrospray ion source, negative ion scanning, capillary voltage 3500V, nebulizing gas pressure 45Pa, drying gas flow rate 10L / min, heating capillary temperature 350℃, source pyrolysis voltage 145V, and mass number scanning range m / z 100~1200.

[0009] This invention also provides a method for preparing Qi-boosting and meridian-clearing capsules, characterized by comprising the following steps:

[0010] Take the raw materials for Yiqi Tongluo Capsules, soak them in 8 times the amount of water for 1 hour, and then decoct and extract them 3 times, each time for 1.5 hours; the raw materials are composed of 304 parts Astragalus membranaceus, 304 parts Codonopsis pilosula, 114 parts Dalbergia odorifera, 190 parts Angelica sinensis, 114 parts Pheretima aspergillum, 152 parts Paeonia lactiflora, 190 parts Ligusticum chuanxiong, 76 parts Carthamus tinctorius, 114 parts Prunus persica, 190 parts Allium macrostemon, 190 parts Trichosanthes kirilowii peel, and 76 parts Mentha haplocalyx.

[0011] This invention also provides a quality marker of traditional Chinese medicine for the anti-myocardial ischemia effect of Yiqi Tongluo capsules, which is obtained by screening according to the aforementioned method. It is composed of paeoniflorin, ferulic acid, isoflavone, ligustilide A, n-butylphthalide, ligustilide, and angelicin A. The peak position of the quality marker in the control fingerprint spectrum of Yiqi Tongluo capsules is as follows: Figure 5 As shown.

[0012] Furthermore, the liquid chromatography conditions for obtaining the reference fingerprint were as follows: Column: Hypersil GOLD TMLiquid chromatography column (4.6 × 250 mm, 5 μm); mobile phase: acetonitrile-0.1% formic acid aqueous solution; flow rate: 1 mL / min; detection wavelength: 280 nm; column temperature: 35 ℃; injection volume: 10 μL; gradient elution program: 0–5 min, 5% acetonitrile; 5–15 min, 5%–20% acetonitrile; 15–23 min, 20% acetonitrile; 23–25 min, 20%–25% acetonitrile; 25–30 min, 25% acetonitrile; 30–35 min, 25%–50% acetonitrile; 35–40 min, 50%–62% acetonitrile; 40–55 min, 62% acetonitrile; 55–58 min, 62%–5% acetonitrile.

[0013] Finally, this invention provides a method for detecting the quality markers of Yiqi Tongluo capsules for anti-myocardial ischemia in traditional Chinese medicine, which includes the following steps:

[0014] a. Preparation of reference solution: Take the reference standard of the quality marker of traditional Chinese medicine, dissolve it in methanol to obtain the solution;

[0015] b. Preparation of test solution: Take Yiqi Tongluo capsules, extract with methanol, and filter the extract to obtain the solution;

[0016] c. Inject the reference solution and the test solution separately into the high-performance liquid chromatograph under the following chromatographic conditions:

[0017] Column: Octadecylsilane-bonded silica gel as packing material; Mobile phase: Acetonitrile-0.1% formic acid solution; Gradient elution program: 0–5 min, 5% acetonitrile; 5–15 min, 5%–20% acetonitrile; 15–23 min, 20% acetonitrile; 23–25 min, 20%–25% acetonitrile; 25–30 min, 25% acetonitrile; 30–35 min, 25%–50% acetonitrile; 35–40 min, 50%–62% acetonitrile; 40–55 min, 62% acetonitrile; 55–58 min, 62%–5% acetonitrile.

[0018] Further, the reference solution in step a contains 1-160 μg / mL of traditional Chinese medicine quality markers per 1 mL; the traditional Chinese medicine quality markers are paeoniflorin, ferulic acid, isoflavone, ligustilide A, n-butylphthalide, ligustilide and / or angelicalide A.

[0019] Further, in step b, the mass-to-volume ratio of the contents of the Yiqi Tongluo capsule to methanol is 1g: 2-100mL.

[0020] Further, in the chromatographic conditions described in step c, the chromatographic column is: Hypersil GOLD. TM 4.6×250mm 5μm; Flow rate: 1mL / min; Detection wavelength: 280nm; Column temperature: 35℃; Injection volume: 10μL.

[0021] This invention explores the quality marker (Q-marker) of Yiqi Tongluo Capsules (YTC) in the treatment of myocardial ischemia. Based on the discovery of Q-marker components, the extraction process is optimized and a YTC fingerprint spectrum is established, which will benefit the optimization of the extraction process, quality control, and the development of modern formulations. Furthermore, the detection method of YTC as a quality marker for anti-myocardial ischemia in this invention has high specificity, good precision, good repeatability, and high accuracy, enabling quality control of YTC, ensuring its efficacy, and possessing practical application value.

[0022] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions, or alterations can be made without departing from the basic technical concept of the present invention.

[0023] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0024] Figure 1 Total ion chromatogram of Yiqi Tongluo Capsules (A: YTC negative ion mode; B: YTC positive ion mode; C: Blood negative ion mode; D: Blood positive ion mode)

[0025] Figure 2 Compound structural formula;

[0026] Figure 3 Effects of different control substances on isoproterenol (ISO)-induced survival of cardiomyocytes H9C2 (compared with the model group; **P<0.01, *P<0.05; A: effect of ISO on H9C2 cells; BK: represent the protective effects of paeoniflorin, ferulic acid, verrucoside, astragaloside A, ligustilide A, n-butylphthalide, ligustilide, angelicin A, amygdalin, and arachidonic acid on ISO-induced cardiomyocytes, respectively).

[0027] Figure 4 Chromatogram of the mixed reference solution;

[0028] Figure 5 Overlay chromatograms and reference chromatograms of 15 batches of YTCHPLC;

[0029] Figure 6YTC HPLC chromatograms (A: HPLC chromatogram of the test solution; B: HPLC chromatogram of the negative control solution lacking paeoniflorin; C: HPLC chromatogram of the negative control solution lacking ferulic acid, ligustilide A, n-butylphthalide, ligustilide, and angelica lactone A; D: HPLC chromatogram of the negative control solution lacking isoflavone; E: HPLC chromatogram of the reference solution, containing 1-paeoniflorin, 2-ferulic acid, 3-isoflavone, 4-ligustilide A, 5-n-butylphthalide, 6-ligustilide, and 7-angelica lactone A, respectively).

[0030] Figure 7 Standard curves (A: paeoniflorin standard curve; B: ferulic acid standard curve; C: verbascoside standard curve; D: ligustilide A standard curve; E: n-butylphthalide standard curve; F: ligustilide standard curve; G: angelica lactone A standard curve);

[0031] Figure 8 The effect of different soaking times on the comprehensive score of 7 components in YTC;

[0032] Figure 9 The effect of different water addition amounts on the comprehensive score of 7 components in YTC;

[0033] Figure 10 The effect of different extraction times on the comprehensive score of seven components in YTC;

[0034] Figure 11 The effect of different extraction times on the comprehensive score of 7 components in YTC. Detailed Implementation

[0035] The reagents, reagents, and equipment used in the specific embodiments of this invention are all known products and were obtained by purchasing commercially available products.

[0036] Example 1: Study on the quality markers of Yiqi Tongluo capsules for anti-myocardial ischemia in traditional Chinese medicine. I. Blood component analysis of Yiqi Tongluo capsules.

[0037] 1. Blood sample preparation

[0038] Six mice were randomly divided into two groups (n=3 per group): a control group and a Yiqi Tongluo capsule group. The Yiqi Tongluo capsule was administered by gavage at a concentration of 30 mg / mL, while the control group was administered the same volume of physiological saline by gavage. Sixty minutes after administration, blood was collected by enucleation. Whole blood was allowed to stand at 4°C for 30 minutes, then centrifuged (3000 rpm, 4°C, 15 min). 0.5 mL of each sample was collected, and three times the volume of acetonitrile was added. The mixture was immediately vortexed for 3 min and then centrifuged (10000 rpm, 4°C, 10 min). The supernatant was collected and subjected to nitrogen blowing at 40°C. After drying, 200 μL of methanol was added to reconstitute the supernatant, followed by centrifugation (1000 rpm, 4°C, 5 min). The mixture was filtered to obtain serum samples, which were then filtered through a 0.22 μm microporous membrane. The initial filtrate was discarded and used as the test solution for later use.

[0039] 2. UPLC-MS Analysis

[0040] Chromatographic conditions: mobile phase acetonitrile (A)-0.1% formic acid aqueous solution (B), gradient elution: 0–5 min, 5% A; 5–15 min, 5%–15% A; 15–25 min, 15%–30% A; 25–30 min, 30%–55% A; 30–40 min, 55%–75% A; 40–45 min, 75%–85% A; 45–50 min, 85% A; flow rate 0.2 mL / min, column temperature 35℃, injection volume 10 μL.

[0041] Mass spectrometry conditions: Electrospray ionization source, negative ion scanning, capillary voltage 3500V, nebulizing gas pressure 45Pa, 10L / min, capillary temperature 350℃, source fragmentation voltage 145V, mass number scanning range m / z 100~1200.

[0042] Based on relevant research literature, octadecylsilane-bonded silica gel was selected as the packing material, and the mobile phase was acetonitrile (A)-0.1% formic acid aqueous solution (B). Gradient elution was adopted (Table 1), with a flow rate of 0.2 mL / min; column temperature of 35℃; injection volume of 10 μL; and sampling time of 50 min.

[0043] Table 1 Gradient elution settings

[0044]

[0045]

[0046] UPLC-Q-TOF-MS / MS analysis was performed, with two injections, one in positive ion mode and the other in negative ion mode, to obtain the total ion chromatogram of the YTC-infused blood sample solution. Figure 1 As shown.

[0047] The molecular weight and theoretical values ​​of primary fragment ions of the compounds were calculated using the Molecular Weight Calculator tool in MassLynx V4.2 software. Based on the retention time, peak intensity, primary and secondary fragment ion information, and mass spectrometry fragmentation patterns corresponding to the chromatographic peaks, and after consulting relevant Chinese and English literature, with an allowable error of less than 5 ppm, a total of 10 YTC components entering the bloodstream were identified, namely: ferulic acid, astragaloside A, Z-ligustilide, verbascoside, ligustilide A, angelicalide A, n-butylphthalide, paeoniflorin, etc. (Tables 2-3). The chemical structural formulas are shown in Table 2. Figure 2 .

[0048] A YTC database was established using UPLC-Q-TOF-MS / MS technology, and the effective components among the blood-entering components were screened. The selected components were chosen as the main active ingredients of YTC for anti-MI.

[0049] Compound 1: In negative ion mode, the retention time is 12.78 min, showing a quasi-molecular ion [MH]-m / z of 193.0523. Based on the precise mass number of the quasi-molecular ion peak, this compound is identified as C. 10 H 10 O4, a quasi-molecular ion, loses one molecule of CH3 to produce a characteristic fragment ion with m / z 178.9796. Further loss of CO2 forms a fragment ion with m / z 134.9881, which is identified as ferulic acid through literature comparison.

[0050] Compound 2: In negative ion mode, the retention time is 18.29 min, showing a quasi-molecular ion peak [MH]. - m / z 783.4490. Based on the precise mass number of the quasi-molecular ion peak, the molecular formula of this compound is determined to be C. 41 H 68 O 14 The ion formed by losing a neutral fragment of glucose (162 Da) at m / z 621.3502, and then losing a neutral fragment of xylopyranose (132 Da) at m / z 489.3199, was identified as astragaloside A by literature comparison.

[0051] Compound 3: In positive ion mode, the retention time is 19.41 min, showing a quasi-molecular ion peak at m / z 191.1088. Based on the precise mass number of the quasi-molecular ion peak, the molecular formula of this compound is determined to be C. 12 H 14 O2, m / z 173.0961 is the quasi-molecular ion peak [M+H]. +Fragment ions generated by the removal of H2O at m / z 191.1088, fragment ions generated by the removal of CO from the quasi-molecular ion peak at m / z 145.1067, and fragment ions generated by the further removal of CO from the quasi-molecular ion peak after the removal of H2O, followed by the neutral loss of C2H4 to form fragment ions at m / z 117.0772 and m / z 163.1115, respectively, and fragment ions generated by the removal of C2H4 from the quasi-molecular ion peak at m / z 149.0235, respectively. These fragment ions were identified as Z-ligustilide by literature comparison.

[0052] Compound 4: At a retention time of 20.84 min, the quasi-molecular ion peak is [M+H]. + 285.0741. Based on the precise mass number of the quasi-molecular ion peak, the molecular formula of this compound is determined to be C. 16 H 12 O5, m / z 239.1228 is an ion fragment generated by the loss of one H2O (18 Da) and one CO (28 Da) from the quasi-molecular ion peak, and m / z 225.1757 is a fragment ion generated by the further loss of CH2 from this peak. Based on literature comparison, it was identified as verbascoside isoflavone.

[0053] Compound 5: In positive ion mode, the retention time is 30.48 min, showing a quasi-molecular ion peak [M+H]. + m / z 193.1228, based on the precise mass number of the quasi-molecular ion peak, the molecular formula of this compound is determined to be C. 12 H 16 O2. m / z 175.0876 is the fragment ion produced by the removal of H2O from the quasi-molecular ion peak, and m / z 147.1183 is the fragment ion produced by the further removal of CO from the quasi-molecular ion peak after the removal of H2O. Through literature comparison, it was identified as ligustrolactone A.

[0054] Compound 6: In negative ion mode, the retention time is 30.86 min, showing a quasi-molecular ion peak [MH]-m / z 456.1498. Based on the precise mass number of the quasi-molecular ion peak, the molecular formula of this compound is determined to be C. 20 H 27 NO 11 The ion with m / z 323.1000 is a fragment ion generated by the loss of C7H4NO2. It was identified as amygdalin through literature comparison.

[0055] Compound 7: In positive ion mode, the quasi-molecular ion peak is 381.2061 at a retention time of 35.65 min. Based on the precise mass number of the quasi-molecular ion peak, the molecular formula of this compound is determined to be C. 24 H 28O4. m / z 191.1136 is a fragment ion produced after the cleavage of phthalide dimer into phthalide monomer; m / z 173.0961 is produced after the cleavage of monomer loses one molecule of H2O; further removal of CO produces fragment ions m / z 145.9556 and m / z 149.0278, which are fragment ions produced after the cleavage of monomer loses one C3H6 free radical. Based on literature comparison, these fragment ions are identified as angelica lactone A.

[0056] Compound 8: In negative ion mode, the retention time is 40.20 min, showing a quasi-molecular ion peak [MH]-m / z 303.2327. Based on the precise mass number of the quasi-molecular ion peak, the molecular formula of this compound is determined to be C. 20 H 32 O2, m / z 285.2189 is a fragment ion generated by the loss of H2O from the quasi-molecular ion peak, and m / z 259.2439 is a fragment ion generated by the loss of CO2 from the quasi-molecular ion peak. They were identified as arachidonic acid through literature comparison.

[0057] Compound 9: In positive ion mode, the retention time is 44.77 min, showing a quasi-molecular ion peak [M+H]. + m / z 191.1088. Based on the precise mass number of the quasi-molecular ion peak, the molecular formula of this compound is determined to be C. 12 H 14 O2, m / z 173.1053 is the fragment ion generated by the removal of H2O from the quasi-molecular ion peak, and m / z 145.1025 is the fragment ion generated by the further removal of CO. Through literature comparison, it was identified as n-butylphthalide.

[0058] Compound 10: In negative ion mode, the retention time is 62.21 min, showing a quasi-molecular ion peak at m / z 479.1581. Based on the precise mass number of the quasi-molecular ion peak, the molecular formula of this compound is determined to be C. 23 H 28 O 11 The peak at m / z 449.1407 represents the quasi-molecular ion peak, formed by the rearrangement of a single CH₂O molecule after the loss of a benzoic acid molecule. Further loss of a benzoic acid molecule results in the peak at m / z 327.2318. The peak at m / z 165.0413 represents fragments of the pinyl basic framework, and the peak at m / z 121.0274 represents fragment ions formed from the loss of the single benzoic acid molecule. Based on literature comparison, it was identified as paeoniflorin.

[0059]

[0060]

[0061] II. Screening of YTC cardiomyocyte protective components

[0062] 1. CCK-8 method to explore ISO modeling concentration

[0063] H9C2 cells were seeded in 96-well plates and treated with ISO at concentrations of 0.16 mol / L, 0.24 mol / L, 0.32 mol / L, and 0.40 mol / L, respectively. Absorbance was measured at 450 nm using the CCK-8 assay. Cell viability = (A... 450 Sample-A 450 Blank) / (A 450 Normal-A 450 (blank), see Figure 3 The ISO concentration shown in A is 0.32 mmol / L, which is the most suitable.

[0064] 2. CCK-8 assay for screening components with protective effects against H9C2 cells.

[0065] H9C2 cells in logarithmic growth phase were divided into 5 groups: normal group (cultured normally in DEME complete medium), model group (ISO concentration of 0.32 mmol / L in complete medium), low-dose group (ISO + low-dose drug), medium-dose group (ISO + medium-dose drug), and high-dose group (ISO + high-dose drug). After digestion, the cells were cultured in an incubator for 12 h, followed by administration of the corresponding drug solution. After 12 h of drug administration, the cells were treated with the same dose of ISO (0.32 mol / L) for 24 h.

[0066] The concentrations of paeoniflorin used were: 0.03, 0.07, and 0.13 mmol / L; the concentrations of ferulic acid used were: 0.08, 0.16, and 0.33 mmol / L; the concentrations of verbascoside used were: 0.21, 0.28, and 0.35 mmol / L; the concentrations of ligustrazine A used were: 0.05, 0.10, and 0.21 mmol / L; the concentrations of n-butylphthalide used were: 0.21, 0.32, and 0.42 mmol / L; and the concentrations of ligusticum used were: The concentrations of lactones used were 0.05, 0.11, and 0.21 mmol / L; the concentrations of angelicin A used were 0.007, 0.013, and 0.026 mmol / L; the concentrations of astragaloside A used were 0.05, 0.08, and 0.10 mmol / L; the concentrations of amygdalin used were 0.01, 0.02, and 0.03 mmol / L; and the concentrations of arachidonic acid used were 0.01, 0.02, and 0.03 mmol / L.

[0067] 3. Experimental Results

[0068] After treating H9C2 cells with 0.32 mmol / L ISO for 24 h, the survival rate of H9C2 cells decreased by nearly 60% compared with the normal group (P<0.01). Figure 3As shown in the figure. Compared with the model group, after pretreatment with paeoniflorin, ferulic acid, verbascoside, ligustilide A, n-butylphthalide, ligustilide, angelica lactone A, and astragaloside A for 12 hours, cell viability increased in a concentration-dependent manner after stimulation with 0.32 mmol / L ISO. However, cell viability did not increase after stimulation with 0.32 mmol / L ISO for amygdalin and arachidonic acid. Because the detection conditions for astragaloside A require an evaporative light scattering detector, in order to simplify the detection conditions, avoid adding additional detection instruments, and reduce the number of components, we selected seven components—paeoniflorin, ferulic acid, verbascoside, ligustilide A, n-butylphthalide, ligustilide, and angelica lactone A—as indicator components in the next step of fingerprinting research.

[0069] III. Qualitative Analysis of YTC Fingerprint Spectra

[0070] 1. Drug preparation

[0071] Formula: Astragalus membranaceus 304g, Codonopsis pilosula 304g, Dalbergia odorifera 114g, Angelica sinensis 190g, Pheretima aspergillum 114g, Paeonia lactiflora 152g, Ligusticum chuanxiong 190g, Carthamus tinctorius 76g, Prunus persica 114g, Allium macrostemon 190g, Trichosanthes kirilowii peel 190g, Mentha haplocalyx 76g.

[0072] Weigh out the medicinal materials according to the prescription dosage. Grind Angelica sinensis, earthworm, Codonopsis pilosula, safflower, and peppermint into fine powder, sieve, mix well, and set aside. Add water to the coarse powder and the remaining seven herbs, including Astragalus membranaceus, and decoct three times, one hour each time. Combine the decoctions, filter, and concentrate the filtrate to a thick paste with a relative density of 1.32-1.35 (60℃). Mix the paste with the fine powder, freeze-dry at low temperature, and pulverize to obtain YTC powder.

[0073] 2. Preparation of the test solution

[0074] Preparation of the test solution: Weigh 1g of YTC powder accurately, place it in a 10mL volumetric flask, add 10mL of methanol, sonicate (power 240W, frequency 45kHz) for 30min, cool, shake well, filter through a 0.22μm microporous membrane, and collect the filtrate to obtain the test solution.

[0075] 3. Preparation of mixed reference solution

[0076] Preparation of mixed reference solutions: Accurately weigh appropriate amounts of paeoniflorin, ferulic acid, verbascoside, ligustilide A, n-butylphthalide, ligustilide, and angelicalide A reference standards, dissolve and dilute with methanol to obtain mixed reference solutions with mass concentrations of 159.76 μg / mL, 63.52 μg / mL, 10 μg / mL, 11.12 μg / mL, 1.68 μg / mL, 155.68 μg / mL, and 1.44 μg / mL, respectively, and store at 4℃ for later use.

[0077] 4. Chromatographic conditions

[0078] Column: Hypersil GOLD TM The liquid chromatography column was 4.6 × 250 mm × 5 μm; the mobile phase was acetonitrile-0.1% formic acid aqueous solution; the flow rate was 1 mL / min; the detection wavelength was 280 nm; the column temperature was 35 ℃; and the injection volume was 10 μL. The elution gradient is shown in Table 4.

[0079] Table 4 Elution gradient

[0080]

[0081] 5. Methodological Investigation

[0082] 5.1 Precision Test

[0083] Take the same compound YTC test solution (sample S1), and inject it six times according to the chromatographic conditions under "III. Qualitative Analysis of YTC Fingerprint" section 4, and record the chromatograms. Using peak 6 (the chromatographic peak of ligustilide, which has a good peak shape, the same below) as the reference peak, the RSDs of the relative retention times and relative peak areas of the seven common peaks were 0.02%–0.05% and 0.41%–1.25% (n=6), respectively, indicating that the method has good precision.

[0084] 5.2 Stability Test

[0085] Take the same compound YTC test solution (sample S1) and inject it according to the chromatographic conditions under "III. Qualitative Analysis of YTC Fingerprint" (section 4) at room temperature for 0, 2, 4, 6, 12, and 24 hours. Record the chromatograms. Using peak 6 as the reference peak, the RSDs of the relative retention times and relative peak areas of the seven common peaks were 0.01%–0.06% and 0.35%–1.84% (n=6), respectively, indicating that the test solution has good stability within 24 hours at room temperature.

[0086] 5.3 Repeatability Test

[0087] Six portions of the same batch of compound YTC (sample S1) were taken and injected according to the chromatographic conditions under "III. Qualitative Analysis of YTC Fingerprint" section 4, and the chromatograms were recorded. Using peak 6 as the reference peak, the RSDs of the relative retention times and relative peak areas of the seven common peaks were 0.01%–0.10% and 0.42%–1.33% (n=6), respectively, indicating that the method has good repeatability.

[0088] 6. Fingerprint pattern establishment and similarity analysis

[0089] 6.1 Establishment of fingerprint map

[0090] Fingerprints for 15 batches of YTC were established according to the preparation method of the test solution under "III. Qualitative Analysis of YTC Fingerprints, Section 2" and the chromatographic conditions under "III. Qualitative Analysis of YTC Fingerprints, Section 4". The HPLC chromatograms of the 15 batches of YTC were analyzed using the "Software for Similarity Evaluation of Chromatographic Fingerprints of Traditional Chinese Medicine (2012 Edition)". The median method was used to generate the reference fingerprint. The results showed that 7 common peaks were identified in the 15 batches of YTC samples. Comparison with the chromatogram of the mixed reference solution identified peak 1 as paeoniflorin, peak 2 as ferulic acid, peak 3 as isoflavone, peak 4 as ligustilide A, peak 5 as n-butylphthalide, peak 6 as ligustilide, and peak 7 as angelica lactone A. The chromatogram of the mixed reference solution is shown in the figure. Figure 4 The HPLC fingerprints of 15 batches of YTC samples are shown below. Figure 5 .

[0091] 6.2 Similarity Evaluation

[0092] Data analysis was performed on the chromatograms of 15 batches of YTC samples using the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 Edition)" software. A retention time window width of 0.1 min was set, and automatic matching was performed after multi-point correction. The similarity was calculated using the mean method. Table 5 shows that the similarity comparison of all 15 batches of samples was greater than 0.996, and the similarity comparison between all 15 batches of samples and the control fingerprint chromatogram was greater than 0.998. This indicates that the chemical composition of these 15 batches of YTC samples is basically consistent, the preparation process is relatively stable, and the similarity to the control fingerprint chromatogram is high.

[0093]

[0094] 7 Summary

[0095] Based on the Q-Marker measurability principle, a fingerprint spectrum of YTC was established using HPLC. The study showed that paeoniflorin, ferulic acid, verbascoside, ligustilide A, n-butylphthalide, ligustilide, and angelicin A were common peaks in the fingerprint spectrum and were characteristic components of YTC, which can be used as Q-Markers for YTC.

[0096] Example 2: Optimization of YTC Extraction Process Based on Q-marker

[0097] 1. Preparation of the test solution

[0098] The preparation method is the same as in Example 1, Section 3, "Preparation of the test solution in the qualitative analysis of YTC fingerprint spectrum".

[0099] 2. Preparation of mixed reference solution

[0100] Preparation of mixed reference solution: Accurately weigh appropriate amounts of paeoniflorin, ferulic acid, verbascoside, ligustilide A, n-butylphthalide, ligustilide, and angelicalide A reference standards, place them in the same 25 mL volumetric flask, add an appropriate amount of methanol, and sonicate to completely dissolve them, obtaining a mixed reference solution with mass concentrations of 0.7988 mg / mL, 0.3176 mg / mL, 0.05 mg / mL, 0.0556 mg / mL, 0.0084 mg / mL, 0.7784 mg / mL, and 0.0072 mg / mL, respectively, and store at 4℃ for later use.

[0101] 3. Preparation of negative sample solution

[0102] After removing the remaining herbs from the YTC prescription (Ligusticum striatum, Angelica sinensis, Paeonia lactiflora, Astragalus membranaceus, and Codonopsis pilosula), negative control standards lacking paeoniflorin, ferulic acid, isoflavone, ligustilide A, n-butylphthalide, ligustilide, and angelica lactone A were prepared sequentially according to the YTC preparation method. The negative control solution was then prepared according to the test solution preparation method.

[0103] 4Liquid phase conditions

[0104] The chromatographic conditions are the same as those in Example 1, "III. Qualitative Analysis of YTC Fingerprint" under chromatographic conditions 4.

[0105] 5 System Suitability Test

[0106] Accurately inject 10 μL each of the reference solution, test solution, and negative control solution into the liquid chromatograph under the chromatographic conditions described in section "2.4" and record the chromatograms. The chromatographic peaks of each component showed good resolution with their adjacent peaks, and there was no interference from the negative sample. (See section "2.4"). Figure 6 .

[0107] 6. Examination of linear relationships

[0108] Accurately pipette 0.50, 1.00, 2.00, 4.00, and 8.00 mL of the mixed reference solution from section "2.2" into separate 10 mL volumetric flasks. Dilute to volume with methanol, mix well, and inject for analysis according to the chromatographic conditions described in section "2.4". Perform linear regression with reference solution mass concentration as the abscissa (X) and peak area integral as the ordinate (Y). The results are shown in Table 6. Figure 7 As shown.

[0109]

[0110] 7 Precision Test

[0111] The same compound YTC test solution was injected and determined six times consecutively under the chromatographic conditions described in section “2.4”. The RSDs of the peak areas of paeoniflorin, ferulic acid, verbascoside, ligustilide A, n-butylphthalide, ligustilide, and angelicalide A were 0.51%, 0.91%, 1.25%, 0.95%, 1.24%, 0.41%, and 0.90% (n=6), respectively, indicating that the method has good precision.

[0112] 8. Stability Test

[0113] The same compound YTC test solution was placed at room temperature for 0, 2, 4, 6, 12, and 24 hours, and then injected for determination under the chromatographic conditions in section “2.4”. The RSDs of the peak areas of paeoniflorin, ferulic acid, verbascoside, ligustilide A, n-butylphthalide, ligustilide, and angelica lactone A were 0.35%, 0.75%, 1.49%, 1.84%, 1.66%, 0.72%, and 0.66% (n=6), respectively, indicating that the test solution has good stability within 24 hours at room temperature.

[0114] 9 Repeatability Tests

[0115] Six portions of the same batch of compound YTC were taken, ground into a fine powder, and test solutions were prepared according to the method in section "2.1". The solutions were then injected and determined under the chromatographic conditions in section "2.4". The RSDs of the peak areas of paeoniflorin, ferulic acid, verbascoside, ligustilide A, n-butylphthalide, ligustilide, and angelica lactone A were 0.64%, 0.42%, 1.33%, 0.49%, 0.93%, 0.45%, and 0.75% (n=6), respectively, indicating that the method has good repeatability.

[0116] 10. Recovery test

[0117] Take 0.5g of YTC with known content of each component, 6 portions, and place them in 10mL volumetric flasks. Accurately measure 1mL of reference solutions with mass concentrations of 797.26μg / mL, 177.92μg / mL, 26.45μg / mL, 72.67μg / mL, 12.18μg / mL, 881.87μg / mL, and 6.31μg / mL, respectively. Prepare test solutions according to the method in section “1”, and inject and determine according to the liquid chromatography conditions in section “4”. Record the peak area and calculate the recovery rate and RSD value. The average recoveries of paeoniflorin, ferulic acid, verbascoside, ligustilide A, n-butylphthalide, ligustilide, and angelicalide A were calculated to be 102.51%, 98.38%, 102.08%, 100.43%, 99.78%, 101.86%, and 101.82%, respectively; the RSD values ​​were 0.64%, 1.93%, 1.51%, 1.53%, 1.64%, 1.60%, and 1.92%, respectively. The results are shown in Table 7-13.

[0118] Table 7 Results of the paeoniflorin recovery test (n=6)

[0119]

[0120] Table 8 Results of ferulic acid recovery test (n=6)

[0121]

[0122] Table 9. Results of the recovery test of verbascoside isoflavones (n=6)

[0123]

[0124] Table 10 Results of the recovery test of Ligusticum striatum lactone A (n=6)

[0125]

[0126]

[0127] Table 11 Results of the n-Butylphthalide recovery test (n=6)

[0128]

[0129] Table 12 Results of the recovery test of ligustilide (n=6)

[0130]

[0131] Table 13 Results of the recovery test of angelica lactone A (n=6)

[0132]

[0133] 11 Content Determination

[0134] Accurately weigh 1g of the drug, prepare the test solution according to the method in section "1", and inject the sample under the liquid phase conditions in section "4" to determine the content of paeoniflorin, ferulic acid, verbascoside, ligustilide A, n-butylphthalide, ligustilide, and angelica lactone A in the drug solution.

[0135] 12. Single-factor investigation of decoction conditions

[0136] Formula: Astragalus membranaceus 304g, Codonopsis pilosula 304g, Dalbergia odorifera 114g, Angelica sinensis 190g, Pheretima aspergillum 114g, Paeonia lactiflora 152g, Ligusticum chuanxiong 190g, Carthamus tinctorius 76g, Prunus persica 114g, Allium macrostemon 190g, Trichosanthes kirilowii peel 190g, Mentha haplocalyx 76g.

[0137] Weigh out each medicinal herb according to the prescription dosage. Grind Angelica sinensis, earthworm, Codonopsis pilosula, safflower, and peppermint into fine powder, sieve, mix well, and set aside. Add water to the coarse powder and the remaining seven herbs, including Astragalus membranaceus, decoct, filter, and concentrate the filtrate to a thick paste with a relative density of 1.32-1.35 (60℃). Mix the paste with the fine powder, dry at low temperature, and pulverize to obtain the final product.

[0138] 12.1 Single-factor analysis of soaking time

[0139] Under fixed decoction conditions, the comprehensive scores of seven index components in the combined decoction after soaking for 0, 1, 2, and 3 hours were examined. The results are as follows: Figure 8 As shown. From Figure 8 The effect of different soaking times on the comprehensive score of the seven YTC components showed that the comprehensive score gradually decreased and tended to balance with increasing soaking time. The comprehensive score was highest when no soaking was performed. However, soaking time had a significant impact on the comprehensive score, so soaking times of 0, 1, and 2 hours were selected as the three levels for the orthogonal experiment.

[0140] The weighted average score is calculated as follows: Overall score (%) = (content of compound 1 / highest content of compound 1 in 9 experiments + ... + content of compound 7 / highest content of compound 7 in 9 experiments) × 1 / 7 × 100%.

[0141] 12.2 Single-factor investigation of water addition

[0142] Under fixed decoction conditions, the comprehensive scores of seven index components in the combined decoction were examined when the water volume was 6, 8, 10, and 12 times. The results are as follows: Figure 9 As shown. From Figure 9The effect of different water addition amounts on the comprehensive score of the seven YTC components was examined. The comprehensive score gradually increased with increasing water addition. Three levels of water addition (8, 10, and 12 times the normal amount) were selected for the orthogonal experiment. Results are shown below. Figure 9 .

[0143] 12.3 Examination of extraction time

[0144] Under fixed decoction conditions, the comprehensive scores of seven index components in the combined decoction were examined at extraction times of 0.5, 1, 1.5, and 2 hours. The results are as follows: Figure 10 As shown. From Figure 10 Regarding the impact of different extraction times on the comprehensive score of the seven YTC components, the comprehensive score peaks as the extraction time increases. Therefore, extraction times of 1.0, 1.5, and 2.0 hours should be selected as the three levels for the orthogonal experiment.

[0145] 12.4 Single-factor analysis of extraction frequency

[0146] Under fixed decoction conditions, the comprehensive scores of seven index components in the combined decoctions were examined when the extraction was performed 1, 2, 3, and 4 times. The results are as follows: Figure 11 As shown. From Figure 11 The effect of different extraction times on the comprehensive score of the seven YTC index components was examined. As the number of extraction times increased, the comprehensive score reached a peak. However, when the extraction times were 4, the comprehensive score decreased significantly. Therefore, extraction times of 1, 2, and 3 were selected as the three levels for the orthogonal experiment.

[0147] 13 Orthogonal Experiments on Traditional Decoction Techniques

[0148] 13.1 Orthogonal Experimental Design

[0149] Based on the results of four single-factor studies (decoction time, number of decoctions, soaking time, and amount of water added), a four-factor, three-level orthogonal experiment was conducted, using the comprehensive score of seven components in the decoction (paeoniflorin, ferulic acid, isoflavone, ligustilide A, n-butylphthalide, ligustilide, and angelicalide A) as the evaluation index. Decoction time, number of decoctions, soaking time, and amount of water added were selected as the factors to be studied. The arrangement of factors and levels is shown in Table 14.

[0150] Table 14 Factors and Levels

[0151]

[0152] 13.2 Orthogonal Experiment and its Results

[0153] The contents of paeoniflorin, ferulic acid, verbascoside, ligustilide A, n-butylphthalide, ligustilide, and angelicalide A were determined according to the orthogonal factor level table. The weighted average score was used to calculate the comprehensive score, and the optimal conditions for the extraction process, including the number of extractions, water addition, extraction time, and soaking time, were determined. The results of the orthogonal experiment are shown in Table 15. One-way ANOVA was performed using SPSS 17.0 software, and the results are shown in Table 16.

[0154] Table 16 directly shows that the order of the range R is: D>A>C>B, so D is the most important factor, and A, C, and B are secondary factors; D2>D3>D1, so D2 is the optimal level; A3>A1>A2, so A3 is the optimal level; C2>C1>C3, so C2 is the optimal level; B1>B3>B2, so B1 is the optimal level. Data analysis shows that the optimal extraction process is A3B1C2D2. Further analysis of variance shows that soaking time has a significant impact on the overall score (P<0.05), while other factors have no significant impact on the overall score. Finally, the water addition ratio was determined to be 8 times, extraction was performed 3 times, each time for 1.5 hours, and the soaking time was 1 hour.

[0155]

[0156] Table 16. Analysis of Variance Table (*P<0.05)

[0157]

[0158] 14 Summary

[0159] This study optimized the YTC extraction process based on the comprehensive score of the Q-marker. Therefore, this experiment adopted a combination of single-factor investigation and orthogonal experiment, using the contents of seven extracts—paeoniflorin, ferulic acid, verbascoside, ligustilide A, n-butylphthalide, ligustilide, and angelicin A—as indicators for comprehensive evaluation. The final extraction process was determined as follows: the water addition ratio was determined to be 8 times, extraction was performed 3 times, each time for 1.5 hours, and the soaking time was 1 hour.

[0160] In summary, based on the discovery of the Q-marker component of Yiqi Tongluo Capsules, this invention optimizes the extraction process and establishes the YTC fingerprint spectrum, which will be beneficial for the optimization of the extraction process, quality control, and the development of modern formulations.

Claims

1. A method for detecting quality markers of traditional Chinese medicine in Yiqi Tongluo capsules, characterized in that: It includes the following steps: a. Preparation of reference solution: Take the reference standard of the quality marker of traditional Chinese medicine, dissolve it in methanol to obtain the solution; the quality marker of traditional Chinese medicine is paeoniflorin, ferulic acid, verbascoside, ligusticin A, n-butylphthalide, ligustilide and angelicain A; b. Preparation of test solution: Take Yiqi Tongluo capsules, extract with methanol, and filter the extract to obtain the solution; The preparation method of the Qi-boosting and meridian-clearing capsules includes the following steps: The raw materials for Yiqi Tongluo Capsules are as follows: 304 parts Astragalus membranaceus, 304 parts Codonopsis pilosula, 114 parts Dalbergia odorifera, 190 parts Angelica sinensis, 114 parts Pheretima aspergillum, 152 parts Paeonia lactiflora, 190 parts Ligusticum chuanxiong, 76 parts Carthamus tinctorius, 114 parts Prunus persica, 190 parts Allium macrostemon, 190 parts Trichosanthes kirilowii peel, and 76 parts Mentha haplocalyx. Angelica sinensis, Pheretima aspergillum, Codonopsis pilosula, Carthamus tinctorius, and Mentha haplocalyx are pulverized into fine powder, sieved, and mixed evenly. The coarse powder is soaked in 8 times the amount of water with Astragalus membranaceus, Dalbergia odorifera, Paeonia lactiflora, Ligusticum chuanxiong, Prunus persica, Allium macrostemon, and Trichosanthes kirilowii peel for 1 hour, then decocted and extracted 3 times, 1.5 hours each time. The decoctions are combined, filtered, and the filtrate is concentrated to a thick paste with a relative density of 1.32-1.35 at 60℃. This paste is then mixed with the fine powder, freeze-dried at low temperature, and pulverized to obtain the final product. c. Inject the reference solution and the test solution separately into the high-performance liquid chromatograph under the following chromatographic conditions: Column: Hypersil GOLD TM 4.6 × 250 mm 5 μm; Mobile phase: acetonitrile-0.1% formic acid solution; Gradient elution program: 0–5 min, 5% acetonitrile; 5–15 min, 5%–20% acetonitrile; 15–23 min, 20% acetonitrile; 23–25 min, 20%–25% acetonitrile; 25–30 min, 25% acetonitrile; 30–35 min, 25%–50% acetonitrile; 35–40 min, 50%–62% acetonitrile; 40–55 min, 62% acetonitrile; 55–58 min, 62%–5% acetonitrile; Flow rate: 1 mL / min; detection wavelength: 280 nm.

2. The method according to claim 1, characterized in that: The reference solution described in step a contains 1~160 μg / mL of traditional Chinese medicine quality markers per 1 mL.

3. The method according to claim 1, characterized in that: The mass-to-volume ratio of the contents of the Yiqi Tongluo capsules to methanol in step b is 1g: 2~100mL.

4. The method according to claim 1, characterized in that: The chromatographic conditions described in step c are as follows: column temperature: 35℃; injection volume: 10 μL.

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