Detection method of traditional Chinese medicine composition for treating chronic heart failure

Abstract

The invention provides a detection method of a traditional Chinese medicine composition for treating chronic heart failure. The detection method comprises thin-layer identification of eight medicinal material components, namely salvia miltiorrhiza, radix paeoniae rubra, fructus amomi, cassia twig, semen plantaginis, ginseng, rhizoma alismatis and herba leonuri. The identification method has the advantages of high detection speed and relatively comprehensive relative components of qualitative detection indexes. The invention further establishes a method for detecting the content of six traditional Chinese medicine effective substance components including ginsenosides Rg1, Re and Rb1, salvianolic acid B, paeoniflorin and cinnamaldehyde, and the method has the advantages of accuracy, reliability, simplicity, convenience, feasibility and good stability. The method can be applied to the quality detection standard of the ginseng-monkshood heart-benefiting traditional Chinese medicine composition, so that the internal quality of the composition is guaranteed.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of Chinese medicine chemical component detection, and particularly relates to a detection method of a Chinese medicine composition for treating chronic heart failure. BACKGROUND

[0002] The Chinese medicine composition for treating chronic heart failure is Shenfu Yixin Granules. The medicine is prepared from ginseng, white aconite root tablet, cassia twig, salvia miltiorrhiza, red peony root, motherwort, alisma, polyporus umbellatus, plantago seed, euphorbia fischeriana steud, amomum villosum, pericarpium ziziphi, jujube. The medicine has the functions of warming yang and benefiting qi, promoting blood circulation to remove blood stasis, and removing water and swelling. The medicine is used for treating chronic heart failure (qi deficiency, yang deficiency, blood stasis and water retention), and is used for treating palpitation, shortness of breath, cold limbs, abdominal fullness, lower extremity edema, short urine, dark complexion, purple lips, exposed blue veins on the neck, mass under the rib, pale or dark tongue, white or greasy fur, rapid and thin pulse, and pulse contraction or pulse.

[0003] The present applicant has previously laid out two patent applications for the product, and the technical subject matter relates to "formula, preparation method, detection method" and the like. CN1572312A discloses a Chinese medicine for treating heart failure and a preparation method thereof. The formula is recorded as follows: ginseng 167 parts, white aconite root tablet 278 parts, cassia twig 333 parts, salvia miltiorrhiza 833 parts, red peony root 417 parts, motherwort 833 parts, alisma 417 parts, polyporus umbellatus 417 parts, plantago seed 417 parts, jujube 333 parts, euphorbia fischeriana steud 417 parts, amomum villosum 278 parts, pericarpium ziziphi 417 parts, and appropriate amount of auxiliary materials. The preparation method is as follows: ginseng is extracted by refluxing with ethanol, and the recovered ethanol is concentrated for use; amomum villosum and cassia twig are extracted by distillation with water to obtain volatile oil, and the volatile oil is packaged with β-cyclodextrin. The residue of amomum villosum and cassia twig and the residue of ginseng and the remaining raw materials except ginseng, amomum villosum and cassia twig are decocted with water, filtered, and the filtrate is concentrated, filtered, and concentrated after recovering ethanol. The filtrate is diluted with water, filtered, and the filtrate is concentrated. The ginseng liquid is mixed, dried, crushed, and mixed with the volatile oil package and appropriate amount of lactose, granulated, and packaged to obtain granules.

[0004] CN101199812A discloses a detection method for traditional Chinese medicine granules used to treat heart failure. The detection method includes thin-layer chromatography of ingredients such as Salvia miltiorrhiza, Paeonia lactiflora, Leonurus japonicus, Lepidium apetalum, Ziziphus jujuba, Amomum villosum, Aconitum carmichaelii, and Cinnamomum cassia, as well as the determination of the content of ginsenosides in ginseng. Weigh the granules, dissolve them in water, add 200 ml of anhydrous ethanol, shake gently to mix, filter, discard the initial filtrate, accurately measure the subsequent filtrate, recover the ethanol, dissolve in 50 ml of water, extract three times with 25 ml of ether each time, discard the ether, extract the aqueous layer six times with water-saturated n-butanol solution, combine the n-butanol extracts, wash three times with 2% sodium hydroxide solution, discard the sodium hydroxide solution, wash three times with water, evaporate the n-butanol layer to dryness on a water bath, dissolve the residue in an appropriate amount of methanol, and make up to volume to obtain the test solution; separately, prepare a solution containing 1 mg of ginsenoside Rg1 reference standard in methanol, as the test solution. According to the thin-layer chromatography method in the Chinese Pharmacopoeia, the test solution and the reference solution are spotted separately onto a silica gel G thin-layer plate. The plate is placed in a chromatography tank saturated with ammonia vapor. The upper layer of n-butanol-ethyl acetate-water = 4:1:5 is used as the developing solvent. The plate is developed at a temperature below 10°C. The plate is then removed, dried, sprayed with 10% sulfuric acid ethanol solution, and heated at 100°C until the spots are clearly visible. The plate is then removed and scanned using thin-layer scanning at wavelengths of λS 520nm and λR 690nm. The absorbance integral values ​​of the test sample and the reference sample are measured. The content of ginsenoside Rgl in the granules shall not be less than 0.028%.

[0005] However, because Shenfu Yixin Granules contain as many as thirteen kinds of Chinese medicinal herbs, current internal control standards have the drawback of thin-layer chromatography (TLC) identification, which, while identifying many types, lacks strong specificity. Furthermore, content determination using only TLC scanning to measure the content of one ginsenoside, Rg1, cannot comprehensively and accurately control the overall intrinsic quality of the herbal compound of this invention. Therefore, developing a highly specific quality testing method with multi-index control is particularly important. Summary of the Invention

[0006] This invention provides a detection method for a traditional Chinese medicine composition for treating chronic heart failure. The method includes thin-layer chromatography (TLC) identification of eight medicinal materials: Salvia miltiorrhiza, Paeonia lactiflora, Amomum villosum, Cinnamomum cassia, Plantago asiatica, Panax ginseng, Alisma plantago-aquatica, and Leonurus japonicus. This identification method features rapid detection speed and relatively comprehensive qualitative indicators. This invention also establishes a method for detecting the content of six active ingredients from traditional Chinese medicine: ginsenosides Rg1, Re, Rb1, salvianolic acid B, paeoniflorin, and cinnamaldehyde. This method is accurate, reliable, simple, and stable. It can be used in the quality testing standards of the Shenfu Yixin traditional Chinese medicine composition of this invention to ensure its intrinsic quality.

[0007] The technical solution of this invention patent application is as follows:

[0008] A method for detecting a traditional Chinese medicine composition for treating chronic heart failure, wherein the content detection method includes the following steps for detecting ginsenoside content:

[0009] (1) Preparation of the test solution: Take the Chinese herbal composition of the present invention, grind it into a fine powder, weigh it, take methanol, weigh it, reflux for 1 hour, cool it, weigh it again, make up the weight loss with methanol, shake it well, filter it, take the filtrate, evaporate it to dryness, dissolve the residue in 20% acetonitrile, dilute it, shake it well, filter it, take the filtrate, and the test solution is obtained.

[0010] (2) Preparation of reference solution: Weigh ginsenoside Rg1 reference standard, ginsenoside Re reference standard, and ginsenoside Rb1 reference standard, add 20% acetonitrile to prepare a mixed solution containing 0.10 mg of ginsenoside Rg1, 0.10 mg of ginsenoside Re, and 0.10 mg of ginsenoside Rb1 per ml.

[0011] (3) Chromatographic conditions and system suitability test: Octadecylsilane-bonded silica gel was used as the stationary phase, acetonitrile as mobile phase A, and water as mobile phase B. The gradient elution program was as follows: 0–8 min, 17% A, 83% B; 8–23 min, 17%–19% A, 83%–81% B; 23–30 min, 19%–24% A, 81%–76% B; 30–40 min, 24%–29% A, 76%–71% B; 40–48 min… In 48–68 min, 29% A, 71% B; 48–68 min, 29%–80% A, 71%–20% B; 68–69 min, 80%–17% A, 20%–83% B; 69–80 min, 17% A, 83% B; column temperature 25–35 °C, flow rate 0.8–1.2 mL / min, evaporative light detector; nebulizer with nitrogen or compressed air, nebulization temperature 62–68 °C, drift tube temperature 82–87 °C;

[0012] (4) Content detection: Take the test solution from step (1) and the reference solution from step (2), inject them into the liquid chromatograph, and determine the content of ginsenoside Rg1, ginsenoside Re and ginsenoside Rb1 in the test solution of the traditional Chinese medicine composition according to the chromatographic conditions in step (3).

[0013] Preferably, the chromatographic column model and specifications are: CAPCELL CORE C18, 4.6mm×150mm, 2.7μm.

[0014] Preferably, the column temperature is 30℃, the flow rate is 1mL / min, the evaporative light detector is used, the nebulization temperature is 65℃, and the drift tube temperature is 85℃.

[0015] The method for detecting the content of the traditional Chinese medicine composition also includes the following steps for determining the content of salvianolic acid B:

[0016] (1) Chromatographic conditions: Octadecylsilane-bonded silica gel was used as the stationary phase, acetonitrile-0.2% phosphoric acid water was used as the mobile phase (23:77), the detection wavelength was 286 nm, the flow rate was 0.8–1.2 mL / min, and the column temperature was 23–27 °C.

[0017] (2) Preparation of the test solution: Weigh the Chinese herbal composition of the present invention, add 70-80% methanol, sonicate for 20-40 minutes, cool, dilute to the mark with 70-80% methanol solution, shake well, filter, and take the filtrate to obtain the test solution.

[0018] (3) Preparation of reference solution: Accurately weigh salvianolic acid B reference standard, add 70-80% methanol to prepare a solution containing 0.05 mg per ml;

[0019] (4) Determination method: Accurately pipette the reference solution and the test solution separately, inject them into the liquid chromatograph, and determine the result.

[0020] Preferably, the method for detecting the content of the traditional Chinese medicine composition further includes the following steps for determining the paeoniflorin content:

[0021] (1) Chromatographic conditions and system suitability test: Octadecylsilane-bonded silica gel was used as the stationary phase; acetonitrile-0.05% formic acid solution (14:86) was used as the mobile phase; the detection wavelength was 230 nm; the flow rate was 0.8–1.2 ml / min; and the column temperature was 23–27 °C.

[0022] (2) Preparation of test solution: Take the Chinese herbal medicine composition, grind it into a fine powder, weigh it, add 70-80% methanol, sonicate for 30 minutes, let it cool, dilute it to the mark with 75% methanol solution, shake it well, filter it, and take the filtrate to obtain the test solution.

[0023] (3) Preparation of reference solution: Weigh an appropriate amount of paeoniflorin reference standard, add 70-80% methanol to prepare a solution containing 0.03 mg per ml;

[0024] (4) Determination method: Pipette the reference solution and the test solution into the liquid chromatograph and determine the result.

[0025] Preferably, the chromatographic column model and specifications are: Aglient 5TC-C18, 4.6mm×250mm.

[0026] Preferably, in step (1), the flow rate is 1 mL / min and the column temperature is 25 °C.

[0027] Preferably, in step (2), the concentration of methanol in the preparation of the test solution is 75%, and the ultrasonic time is 30 minutes.

[0028] Preferably, the method for detecting the content of the traditional Chinese medicine composition further includes the following steps for determining the content of cinnamaldehyde:

[0029] (1) Chromatographic conditions and system suitability test: Octadecylsilane-bonded silica gel was used as the stationary phase, acetonitrile-water 30:70 was used as the mobile phase, the detection wavelength was 290 nm, the flow rate was 0.8 to 1.2 ml / min, and the column temperature was 23 to 27 °C.

[0030] (2) Preparation of test solution: Take the Chinese herbal medicine composition, grind it into a fine powder, add acetonitrile, seal it, sonicate for 30 minutes, take it out and let it cool to room temperature, dilute it with acetonitrile, shake it well, filter it, and the test solution is obtained.

[0031] (3) Preparation of reference solution: Weigh cinnamaldehyde reference standard and add acetonitrile to prepare a solution containing 0.05 mg per ml.

[0032] (4) Determination method: Pipette 10 μl of the reference solution and the test solution into the liquid chromatograph and determine the result.

[0033] Preferably, the chromatographic column model and specifications are: Agilent ZORBAX Eclipse Plus C18, 4.6mm*250mm, 5um.

[0034] Preferably, the thin-layer chromatography identification items of the traditional Chinese medicine composition include: one or a combination of two of the thin-layer chromatography identification items of tanshinone or paeoniflorin;

[0035] The thin-layer chromatography identification of Salvia miltiorrhiza is as follows:

[0036] Preparation of test solution: Take the traditional Chinese medicine composition, grind it into a fine powder, add methanol, sonicate, filter, evaporate the filtrate to dryness, dissolve the residue in water, extract with ether twice by shaking, discard the ether solution, add hydrochloric acid to adjust the pH of the aqueous solution to 2-3, extract with ether, discard the ether solution, extract the aqueous solution with ethyl acetate twice by shaking, combine the ethyl acetate solutions, wash, evaporate the ethyl acetate solution to dryness, dissolve the residue in methanol to obtain the test solution;

[0037] Preparation of reference herb solution: Take another Salvia miltiorrhiza reference herb, add hydrochloric acid solution, heat to reflux, filter, extract the filtrate twice with ethyl acetate, combine the ethyl acetate solutions, evaporate to dryness, dissolve the residue in methanol to obtain the reference herb solution. Development and color development conditions: Perform thin-layer chromatography, take the test solution and the reference herb solution, spot them separately on the same silica gel G thin-layer plate, use toluene-ethyl acetate-formic acid 8:5:2 as the developing solvent, develop, remove, air dry, place in ammonia vapor, fumigate for 15 minutes, and examine under ultraviolet light at 365 nm. Standard: In the chromatogram of the test sample, fluorescent spots of the same color appear at the same positions as in the chromatogram of the reference herb.

[0038] The thin-layer chromatography identification of paeoniflorin is as follows:

[0039] Preparation of test solution: Take the traditional Chinese medicine composition, extract it by reflux with anhydrous ethanol, filter it, evaporate the filtrate to dryness in a water bath, dissolve the residue in n-butanol, filter it, evaporate the filtrate to dryness in a water bath, dissolve the residue in methanol to obtain the test solution;

[0040] Preparation of reference solution: Paeoniflorin reference standard was dissolved in methanol to prepare a solution, which was then used as the reference solution.

[0041] Development and color development conditions: Perform the thin-layer chromatography test, take the above two solutions and spot them separately on the same silica gel G thin-layer plate with sodium carboxymethyl cellulose as binder, use chloroform-ethyl acetate-methanol-formic acid 40:5:10:0.2 as the developing solvent, develop, remove, air dry, spray with 5% vanillin-sulfuric acid solution, bake at 105℃ for 10 minutes for color development, standard: in the chromatogram of the test sample, spots of the same color appear at the corresponding positions as in the chromatogram of the reference standard.

[0042] Borneol acetate or cinnamaldehyde thin-layer identification test: one or a combination of two of them;

[0043] Identification of Borneol Acetate:

[0044] Preparation of test solution: Take the traditional Chinese medicine composition, add water to dissolve it, add petroleum ether, heat in a water bath under reflux, separate the petroleum ether layer, evaporate to obtain the test solution;

[0045] Preparation of reference solution: Weigh bornyl acetate reference standard, add ethanol to prepare a solution, and use it as the reference solution;

[0046] Development and color development conditions: Perform thin-layer chromatography test, take the above two solutions and spot them separately on the same silica gel G thin-layer plate, use cyclohexane-ethyl acetate 22:1 as the developing solvent, develop, remove, air dry, and spray with 5% vanillin sulfuric acid solution; Standard: In the chromatogram of the test sample, spots of the same color appear at the corresponding positions as in the chromatogram of the reference sample.

[0047] The identification of cinnamaldehyde:

[0048] Preparation of test solution: Take the traditional Chinese medicine composition, add cyclohexane according to the method for determining volatile oil, heat and extract, separate the cyclohexane layer as the test solution;

[0049] Preparation of reference solution: Weigh cinnamaldehyde reference standard, add cyclohexane to prepare a solution, and use it as the reference solution;

[0050] Development and color development conditions: Perform the thin-layer chromatography test. Take the test solution and the reference solution and spot them separately on the same silica gel G thin-layer plate with sodium carboxymethyl cellulose. Use petroleum ether-ethyl acetate 17:3 as the developing solvent. Develop, remove, and air dry. Spray with dinitrophenylhydrazine ethanol test solution. Standard: In the chromatogram of the test sample, spots of the same color appear at the corresponding positions as in the chromatogram of the reference sample.

[0051] The detection method also includes: thin-layer identification of psyllium husk.

[0052] Preparation of test solution: Take the Chinese herbal medicine composition, add methanol, sonicate, filter, evaporate the filtrate to dryness, add water to dissolve, extract with ethyl acetate three times, discard the ethyl acetate solution, extract the aqueous layer twice with water-saturated n-butanol, combine the n-butanol layers, wash the n-butanol layer twice with water-saturated n-butanol, evaporate the n-butanol layer to dryness, dissolve the residue in methanol to obtain the test solution.

[0053] Preparation of reference solution: Weigh genipin glycoside reference standard and verbascoside reference standard, and add methanol to prepare a 1 mg / ml solution;

[0054] Reference herb solution: Take Plantago seed reference herb, add methanol, sonicate, filter, evaporate the filtrate to dryness, dissolve in water, extract three times with ethyl acetate, 20 mL each time, discard the ether solution, extract the aqueous layer twice with water-saturated n-butanol, 20 mL each time, combine the n-butanol layers, wash the n-butanol layer twice with water-saturated n-butanol, 20 mL each time, evaporate the n-butanol layer to dryness, dissolve the residue in methanol, and use as the reference herb solution;

[0055] Development, color development conditions, and inspection: Perform the thin-layer chromatography test. Apply the test solution, reference solution, and reference medicinal material solution separately to the same silica gel high-performance G thin-layer plate containing sodium carboxymethyl cellulose. Use ethyl acetate-ethanol-formic acid-water 12:3:1:0.8 as the developing solvent. Develop, remove, and air-dry. Spray with 2% vanillin-sulfuric acid solution and develop at 105℃. Examine under 365nm and sunlight. Standard: In the chromatogram of the test sample, spots of the same color should appear at the corresponding positions as in the chromatograms of the reference sample and the reference medicinal material.

[0056] Preferably, the detection method further includes: ginseng thin-layer chromatography identification item:

[0057] Preparation of test solution: Grind the traditional Chinese medicine composition into a fine powder, add methanol, sonicate, filter, evaporate the filtrate to dryness, dissolve in water, extract three times with ethyl acetate, discard the ethyl acetate solution, extract the aqueous layer twice with water-saturated n-butanol, 20 mL each time, combine the n-butanol layers, wash the n-butanol layer twice with water-saturated n-butanol, 20 mL each time, evaporate the n-butanol layer to dryness, dissolve the residue in methanol to obtain the test solution;

[0058] Preparation of reference solution: Weigh ginsenoside Rb1, ginsenoside Re, and ginsenoside Rg1, add methanol to prepare a solution; this is the reference solution.

[0059] Reference herb solution: Take ginseng reference herb, add methanol, sonicate, filter, evaporate the filtrate to dryness, dissolve in water, extract three times with ethyl acetate, discard the ether solution, extract the aqueous layer twice with water-saturated n-butanol, combine the n-butanol layers, wash the n-butanol layer twice with water-saturated n-butanol, evaporate the n-butanol layer to dryness, dissolve the residue in methanol, and use as the reference herb solution;

[0060] Development, color development conditions, and inspection: Perform the thin-layer chromatography test. Apply the test solution, reference solution, and reference medicinal material solution separately to the same silica gel high-performance G thin-layer plate containing sodium carboxymethyl cellulose. Use the upper layer of n-butanol-ethyl acetate-water (4:1:5) as the developing solvent. Develop, remove, and air-dry. Spray with 10% sulfuric acid ethanol solution. Develop the color at 105℃ and inspect at 365 nm and under sunlight. Standard: In the chromatogram of the test sample, spots of the same color should appear at the corresponding positions as in the chromatograms of the reference sample and the reference medicinal material.

[0061] Preferably, the detection method further includes: thin-layer chromatography identification of Alisma plantago-aquatica:

[0062] Preparation of test solution: Weigh the Chinese herbal medicine composition, grind it into a fine powder, add ethyl acetate, sonicate, filter, add activated carbon powder to the filtrate, filter, evaporate to dryness, dissolve the residue in ethyl acetate to obtain the test solution;

[0063] Reference herb solution: Weigh Alisma plantago-aquatica reference herb, add ethyl acetate, sonicate, filter, evaporate to dryness, dissolve the residue in ethyl acetate to obtain the reference herb solution;

[0064] Development, color development conditions, and inspection: Perform the thin-layer chromatography test. Take the test solution and the reference medicinal material solution and spot them separately on the same silica gel high-performance G thin-layer plate with sodium carboxymethyl cellulose. Use cyclohexane-ethyl acetate 1:1 as the developing solvent. Develop, remove, and air dry. Spray with 2,4-dinitrophenylhydrazine hydrochloride ethanol solution. Develop the color at 105℃ and inspect at 365nm and under sunlight. Standard: In the chromatogram of the test sample, spots of the same color appear at the corresponding positions as in the chromatogram of the reference medicinal material.

[0065] Preferably, the detection method further includes: thin-layer chromatography identification of stachydrine hydrochloride.

[0066] Preparation of test solution: Take the Chinese herbal medicine composition, grind it into a fine powder, add anhydrous ethanol, heat under reflux, filter, evaporate the filtrate to dryness, add water to dissolve the residue, pass it through a cation exchange resin column, let it stand, wash with water until the eluent is nearly colorless, discard the aqueous solution, then elute with ammonia solution, collect the eluent, evaporate to dryness, add ethanol to dissolve the residue, and use it as the test solution.

[0067] Preparation of reference solution: Take stachydrine hydrochloride reference standard and add ethanol to prepare a solution containing 2 mg in 1 ml as the reference solution;

[0068] Development, color development conditions, and inspection: Perform the thin-layer chromatography test. Take the test solution and the reference solution and spot them separately on the same silica gel G thin-layer plate. Use n-butanol-ethyl acetate-hydrochloric acid 4:0.5:1.5 as the developing solvent. Add the developing solvent, pre-saturate, develop, remove, air dry, and spray with dilute bismuth potassium iodide test solution. Standard: In the chromatogram of the test sample, spots of the same color appear at the corresponding positions as in the chromatogram of the reference sample.

[0069] To further demonstrate the innovativeness of the technical solution of this invention, we have summarized the process of optimizing some parameters of the technical solution of this invention as follows:

[0070] (1) Optimization of test sample preparation conditions

[0071] This experiment investigated both ultraviolet (UV) and evaporative light (EL) detectors. Through comparative experiments, it was found that using the EL detector resulted in fewer impurity peaks, higher peak values ​​for the target compound, and better recovery in the chromatograms of the sample solution. Under the premise of using the EL detector, the extraction methods (shaking, sonication, reflux), different solid-liquid ratios (6g→50ml, 6g→100ml, 6g→150ml, 3g→100ml), and different reflux extraction times (0.5 hours, 1 hour, 1.5 hours) were examined to compare the determination results of ginsenoside Rg1, ginsenoside Re, and ginsenoside Rb1 in the sample solution under various conditions. The results obtained using the conditions of "reflux, solid-liquid ratio of 3g→100ml, and reflux time of 1 hour" were accurate, stable, and showed good peak separation. Therefore, the EL detector was selected, and the sample preparation conditions of "reflux, solid-liquid ratio of 3g→100ml, and reflux time of 1 hour" were chosen.

[0072] (2) Selection of mobile phase and optimization of gradient ratio

[0073] This invention uses an acetonitrile (A)-water (B) gradient elution with a C18 (4.6*250mm, 5µm) column and an evaporative light detector. The separation of ginsenosides Rg1 and Re was unsatisfactory, and the peak shape was poor. After screening different brands and models of columns, the CAPCELL CORE C18 (4.6mm×150mm, 2.7µm) was finally determined to meet the peak shape requirements and exhibit good symmetry. During the 0-35 minute period, the initial proportion of mobile phase A was adjusted from 19% to 17%, achieving baseline separation of ginsenosides Rg1 and Re. Due to the late elution of the chromatographic peaks and the long acquisition time, the isocratic ratio from 8-35 minutes was adjusted to a gradient: 8-23 minutes, 17%-19% A and 83%-81% B; 23-30 minutes, 19%-24% A and 81%-76% B; 30-40 minutes, 24%-29%... Ginsenoside Rg1 eluted earlier than usual (around 32 minutes) to approximately 23 minutes, achieving baseline separation with Re and meeting the required resolution. At 40-48 minutes, the elution rates were 29% A and 71% B; at 48-68 minutes, 29%-80% A and 71%-20% B; at 68-69 minutes, 80%-17% A and 20%-83% B; and at 69-80 minutes, 17% A and 83% B. By increasing the gradient elution time from 0-30 minutes, the required separation of ginsenoside Rg1 and Re was achieved, while ginsenoside Rb1 did not achieve baseline separation from adjacent peaks. The detection time was reduced from 100 minutes to 80 minutes, significantly improving detection efficiency. Finally, after extensive experimentation and ratio adjustments, the optimal flow ratio was obtained.

[0074] (3) Selection of characteristic indicator components (e.g., ginsenosides (Rg1, Re, Rb1), peony root extract, and salvianolic acid B)

[0075] To comprehensively and conveniently control the quality of the formulation of this invention, ginsenosides Rg1, Re, and Rb1 from ginseng (the principal ingredient) were selected. These ginsenosides can dilate blood vessels, reduce blood viscosity, lower blood pressure, promote blood circulation, and have protective effects against arrhythmia, myocardial hypertrophy, myocardial ischemia, and vascular endothelial cells. The water-soluble component of salvianolic acid B from danshen (Salvia miltiorrhiza) was selected, which acts on key targets of blood stasis such as thrombin, exerting its blood-activating and stasis-removing effects. Cinnamaldehyde from cinnamon twig (an assistant ingredient) and paeoniflorin from red peony root (an adjuvant ingredient) were used as content assay indicators. These six compounds were ultimately determined as content assay indicators. This allows for quantitative control of the formulation's quality, providing an accurate, simple, and reliable comprehensive analytical method for the quality control of Shenfu Yixin Granules.

[0076] The beneficial effects of the technical solution of this invention are as follows:

[0077] (1) This invention establishes a thin-layer chromatography (TLC) identification method for eight medicinal materials: tanshinone, paeoniflorin, bornyl acetate, cinnamaldehyde, plantain seed, ginseng, alisma, and stachydrine hydrochloride. This identification method has the advantages of fast detection speed and relatively comprehensive qualitative detection indicators. The above-mentioned TLC identification method also has the advantages of relatively simple operation and low cost.

[0078] (2) This invention establishes a method for detecting the content of ginsenosides Rg1, Re, and Rb1. Methodological validation revealed the following: ① Repeatability test: The RSD of the three components was 1.28%–1.56%, indicating good repeatability of the detection method. ② Intermediate precision RSD was 1.53%–2.01%, indicating good precision of the method for detecting the three components. ③ Stability test results: The Rg1 content (mg / g) in the test solution was 0.4114, 0.4162, and 0.4149, with an average Rg1 content of 0.4142. The RSD% was 0.6. The Re content (mg / g) in the test solution was 0.4655, 0.4720, and 0.4756, with an average Re content of 0.4710 mg / g. The RSD% was 1.1. The Rb1 content (mg / g) in the test solution was 0.5857, 0.6016, and 0.6093, with an average Rb1 content of 0.5989 and an RSD% of 0.21. ④ The recovery rate test results showed that the recovery rate of Rg1 was 99.09%–105.87%, while the recovery rates of Re and Rb1 were 99.3–110% and 99.66–104.66%, respectively. This indicates that the content detection method established in this invention has good recovery rates.

[0079] (3) This invention establishes a method for detecting the content of salvianolic acid B. Methodological validation revealed the following: ① Repeatability test: The RSD of salvianolic acid B content was 0.4%. ② Intermediate precision: The RSD of salvianolic acid B content was 0.7%. ③ Stability test results: The RSD was 2.0%, indicating good stability of the detection method. ④ Spiking recovery test results: The spiking recovery rate of salvianolic acid B was between 98.06% and 104.21%, indicating that the content detection method established in this invention has good recovery rate.

[0080] (4) This invention establishes a method for detecting the content of paeoniflorin. Methodological validation revealed the following: ① Repeatability test: The RSD of paeoniflorin content was 1.1%. ② Intermediate precision RSD was 0.6. ③ Stability test results: The paeoniflorin content in the test solution was 0.9%. ④ Recovery test results: The recovery rate of paeoniflorin was between 100.40% and 104.60%, with an RSD of 1.5%. This indicates that the content detection method established in this invention has good recovery rate.

[0081] (5) The present invention establishes a method for detecting the content of cinnamaldehyde. Methodological verification shows: ① Repeatability test: the RSD of cinnamaldehyde content is 0.4%. ② Intermediate precision RSD of cinnamaldehyde is 0.5%. ③ Stability test results: the RSD of cinnamaldehyde content is 0.4%. ④ Spiking recovery test results: the spiking recovery rate of cinnamaldehyde is between 97.6% and 103.2%, with an RSD of 2.1%. This indicates that the content detection method established in this invention has a good spiking recovery rate.

[0082] Therefore, the detection method for the content of ginsenosides Rg1, Re, Rb1, salvianolic acid B, paeoniflorin and cinnamaldehyde in Shenfu Yixin Granules established in this invention is accurate, reliable, simple and feasible, and has good stability. It can be used in the quality detection standard of the Shenfu Yixin Traditional Chinese Medicine Composition of this invention to ensure its intrinsic quality. Attached Figure Description

[0083] Figure 1 - Thin-layer chromatography identification of Salvia miltiorrhiza, from left to right: Salvia miltiorrhiza negative sample solution, Salvia miltiorrhiza control material solution, Shenfu Yixin granules test solution, Shenfu Yixin granules test solution;

[0084] Figure 2 - Thin-layer chromatography identification of paeoniflorin, from left to right: Paeonia lactiflora negative sample solution, paeoniflorin reference solution, Ginseng and Aconite Heart-Nourishing Granules test solution, Ginseng and Aconite Heart-Nourishing Granules test solution;

[0085] Figure 3 - Thin-layer chromatography identification of borneol acetate, Amomum villosum negative sample solution, borneol acetate reference solution, Ginseng and Aconite Heart-Nourishing Granules test solution, Ginseng and Aconite Heart-Nourishing Granules test solution;

[0086] Figure 4 - Cinnamaldehyde thin-layer chromatography identification, from left to right: cinnamon twig negative sample solution, cinnamaldehyde reference solution, ginseng and aconite-invigorating granules test solution, ginseng and aconite-invigorating granules test solution.

[0087] Figure 5 - Thin-layer chromatography identification of Plantago asiatica, 365 UV lamp. Note: From left to right: Ginseng and Aconite-Benefiting Granules test solution, Ginseng and Aconite-Benefiting Granules test solution, Ginseng and Aconite-Benefiting Granules test solution, Plantago asiatica negative sample solution, Plantago asiatica reference material solution, Genipin glycoside reference solution, Verbascoside reference solution.

[0088] Figure 6 - Ginseng thin-layer chromatography identification, UV lamp 365 Note: From left to right: mixed reference solution, ginseng negative sample solution, Shenfu Yixin granules test solution, Shenfu Yixin granules test solution, ginsenoside Re reference solution, ginsenoside Rg1 reference solution, ginsenoside Rb1 reference solution.

[0089] Figure 7 -Identification of Alisma plantago-aquatica using thin-layer chromatography. Note: From left to right: Shenfu Yixin Granules test solution, Alisma plantago-aquatica negative sample solution, and Alisma plantago-aquatica reference medicinal material solution;

[0090] Figure 8 - Identification of stachydrine hydrochloride, from left to right: Motherwort negative sample solution, stachydrine hydrochloride reference solution, Ginseng and Aconite Heart-Nourishing Granules test solution, Ginseng and Aconite Heart-Nourishing Granules test solution.

[0091] Figure 9 -Chromatogram of ginsenoside mixed reference solution, the elution order is ginsenoside Rg1, ginsenoside Re, ginsenoside Rb1;

[0092] Figure 10 -Chromatogram of the test sample of Shenfu Yixin Granules. Detailed Implementation

[0093] Unless otherwise defined, the technical or scientific terms used in the specification and claims of this patent application shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0094] Example 1: Thin-layer chromatography identification of various medicinal materials according to the present invention

[0095] 1.1 Thin-layer chromatography identification of Salvia miltiorrhiza in this invention

[0096] 1.1 Test method:

[0097] Preparation of test solution: Take this product, grind it into a fine powder, weigh 18g, add 40ml of methanol, sonicate for 30 minutes, filter, evaporate the filtrate to dryness, add 20ml of water to dissolve the residue, extract twice with 25ml of ether each time, discard the ether solution, add hydrochloric acid to adjust the pH of the aqueous solution to 2-3, extract with 25ml of ether, discard the ether solution, extract the aqueous solution twice with 25ml of ethyl acetate each time, combine the ethyl acetate solutions, wash with 30ml of water, evaporate the ethyl acetate solution to dryness, add 1ml of methanol to dissolve the residue, and use this as the test solution.

[0098] Preparation of the reference herb solution: Take 2g of Salvia miltiorrhiza reference herb, add 25ml of hydrochloric acid solution (1→50), heat under reflux for 1 hour, filter, and extract the filtrate twice with ethyl acetate, 25ml each time. Combine the ethyl acetate extracts, evaporate to dryness, and dissolve the residue in 1ml of methanol to obtain the reference herb solution. Development and colorimetric conditions: Perform the thin-layer chromatography method (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 0502). Apply 5μl each of the test solution and the reference herb solution to the same silica gel G thin-layer plate. Develop using toluene-ethyl acetate-formic acid (8:5:2) as the developing solvent. Remove and air dry. Examine under ammonia vapor for 15 minutes and then under ultraviolet light (365nm). Standard: The test sample chromatogram should show fluorescent spots of the same color at the same positions as the reference herb chromatogram.

[0099] 1.2 Verification Results:

[0100] Specificity: In the chromatogram of the negative sample of *Salvia miltiorrhiza*, no fluorescent spots of the same color were observed at the positions as those in the chromatogram of the control herb, indicating that the specificity of this method meets the requirements. Robustness: Different humidity levels (32%, 58%, 72% relative humidity), different temperatures (30–35℃, 20–25℃, 2–8℃), and different thin-layer chromatography manufacturers (Merck, Qingdao Ocean, Yantai Huayang) have no effect on the test results.

[0101] 1.2 Thin-layer chromatography identification of paeoniflorin

[0102] 1.2.1 Test method:

[0103] Preparation of test solution: Take 2g of this product, reflux with 10ml of anhydrous ethanol for 1 hour, filter, evaporate the filtrate to dryness in a water bath, dissolve the residue in 10ml of n-butanol, filter, evaporate the filtrate to dryness in a water bath, dissolve the residue in 1ml of methanol to obtain the test solution.

[0104] Preparation of reference solution: Paeoniflorin reference standard was prepared by adding methanol to a solution containing 1 mg per ml, which served as the reference solution. Development and colorimetric conditions: Thin-layer chromatography (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 0502) was performed. 10 μl of each of the two solutions were spotted separately onto the same silica gel G thin-layer plate using sodium carboxymethyl cellulose as a binder. The plate was developed using chloroform-ethyl acetate-methanol-formic acid (40:5:10:0.2) as the developing solvent. The plate was then removed, air-dried, sprayed with 5% vanillin-sulfuric acid solution, and dried at 105℃ for 10 minutes for color development. Standard: In the chromatogram of the test sample, spots of the same color appear at the corresponding positions as in the chromatogram of the reference standard.

[0105] 1.2.2 Verification Results

[0106] Specificity: In the chromatogram of the negative sample of Paeonia lactiflora, no spots of the same color as those in the chromatogram of the control herb were observed at the same positions, indicating that the specificity of this method meets the requirements. Robustness: Different humidity levels (32%, 58%, 72% relative humidity), different temperatures (30–35℃, 20–25℃, 2–8℃), and different thin-layer chromatography manufacturers (Merck, Qingdao Ocean, Yantai Huayang) have no effect on the test results.

[0107] 1.3 Identification of Borneol Acetate

[0108] 1.3.1 Test Method

[0109] Preparation of test solution: Take 15g of this product, add 30mL of water to dissolve it, add 40mL of petroleum ether (60~90℃), heat in a water bath under reflux for 1h, separate the petroleum ether layer, evaporate to 1mL, and use it as the test solution.

[0110] Preparation of reference solution: Accurately weigh bornyl acetate reference standard, add ethanol to prepare a solution containing 10 μl per 1 ml, as the reference solution. Development and colorimetric conditions: Perform thin-layer chromatography (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 0502). Apply 1 μl of each of the above two solutions to the same silica gel G thin-layer plate. Develop with cyclohexane-ethyl acetate (22:1) as the developing solvent. Remove, air dry, and spray with 5% vanillin-sulfuric acid solution. Standard: In the chromatogram of the test sample, spots of the same color appear at the corresponding positions as in the chromatogram of the reference standard.

[0111] 1.3.2 Verification Results:

[0112] Specificity: In the chromatogram of the cardamom negative sample, no spots of the same color as those in the chromatogram of the reference herb were observed at the same positions, indicating that the specificity of this method meets the requirements. Robustness: Different humidity levels (32%, 58%, 72% relative humidity), different temperatures (30–35℃, 20–25℃, 2–8℃), and different thin-layer chromatography manufacturers (Merck, Qingdao Ocean, Yantai Huayang) have no effect on the test results.

[0113] 1.4 Identification of Cinnamaldehyde

[0114] 1.4.1 Test method:

[0115] Preparation of test solution: Take 10g of this product and extract it according to the method for determination of volatile oil (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 2204, Method A). Add 2-4ml of cyclohexane to the graduated tube of the extractor, heat and extract for 5 hours, and separate the cyclohexane layer as the test solution.

[0116] Preparation of reference solution: Accurately weigh cinnamaldehyde reference standard and add cyclohexane to prepare a solution containing 0.1 ml per ml, which is used as the reference solution. Development and colorimetric conditions: Perform thin-layer chromatography (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 0502). Apply 10 μl of the test solution and 2 μl of the reference solution separately to the same silica gel G thin-layer plate coated with sodium carboxymethyl cellulose. Develop with petroleum ether (60-90℃)-ethyl acetate (17:3) as the developing solvent. Remove the plate, air dry, and spray with dinitrophenylhydrazine ethanol reagent. Standard: In the chromatogram of the test sample, spots of the same color appear at the corresponding positions as in the chromatogram of the reference standard.

[0117] 1.4.2 Verification Results:

[0118] Specificity: In the chromatogram of the negative cinnamon twig sample, no spots of the same color as those in the chromatogram of the reference medicinal material were observed at the same positions, indicating that the specificity of this method meets the requirements. Robustness: Different humidity levels (32%, 58%, 72% relative humidity), different temperatures (30–35℃, 20–25℃, 2–8℃), and different thin-layer chromatography manufacturers (Merck, Qingdao Ocean, Yantai Huayang) have no effect on the test results.

[0119] 1.5 Identification of Plantago asiatica

[0120] 1.5.1 Test method:

[0121] Preparation of test solution: Grind the product into a fine powder, weigh 5g of the powder, place it in a 100mL Erlenmeyer flask, add 40mL of methanol, sonicate (250W power, 40kHz frequency) for 30 minutes, filter, evaporate the filtrate to dryness, dissolve in 20mL of water, transfer to a separatory funnel, and extract three times with 20mL of ethyl acetate each time, discarding the ethyl acetate solution. Extract the aqueous layer twice with water-saturated n-butanol, 20mL each time, combine the n-butanol layers, and wash the n-butanol layer twice with water-saturated n-butanol, 20mL each time. Evaporate the n-butanol layer to dryness, dissolve the residue in 1mL of methanol, and use this as the test solution.

[0122] Preparation of reference solution: Weigh 2 mg of genipin glycoside reference standard and 2 mg of verbascoside reference standard, and add methanol to prepare a 1 mg / mL solution; this is the reference solution. Reference herb solution: Take 1 g of Plantago asiatica powder, place it in a 100 mL Erlenmeyer flask, add 40 mL of methanol, sonicate (250 W, 40 kHz) for 30 minutes, filter, evaporate the filtrate to dryness, dissolve in 20 mL of water, transfer to a separatory funnel, and extract three times with 20 mL of ethyl acetate each time, discarding the ether extract. Extract the aqueous layer twice with 20 mL of water-saturated n-butanol each time, combining the n-butanol layers, and then wash the n-butanol layer twice with 20 mL of water-saturated n-butanol each time. Evaporate the n-butanol layer to dryness, dissolve the residue in 1 mL of methanol, and this is the reference herb solution. Development and colorimetric conditions: Perform thin-layer chromatography (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 0502). Apply 5 μl each of the test solution, reference solution, and reference medicinal material solution to the same silica gel high-performance G thin-layer plate coated with sodium carboxymethyl cellulose. Develop using ethyl acetate-ethanol-formic acid-water (12:3:1:0.8) as the developing solvent. Remove and air-dry. Spray with 2% vanillin-sulfuric acid solution and develop at 105℃. Examine under 365 nm and sunlight. Standard: The chromatogram of the test sample should show spots of the same color at the corresponding positions as the chromatograms of the reference sample and the reference medicinal material.

[0123] 1.5.2 Verification Results:

[0124] Specificity: In the chromatogram of the negative sample of Plantago asiatica, no spots of the same color as those in the chromatogram of the control herb were observed at the same positions, indicating that the specificity of this method meets the requirements. Robustness: Different humidity levels (32%, 58%, 72% relative humidity), different temperatures (30–35℃, 20–25℃, 2–8℃), and different thin-layer chromatography manufacturers (Merck, Qingdao Ocean, Yantai Huayang) have no effect on the test results.

[0125] 1.6 Ginseng Identification

[0126] 1.6.1 Test method:

[0127] Preparation of the test solution: Grind the sample into a fine powder, weigh 6g, grind it finely, place it in a 100mL Erlenmeyer flask, add 30mL of methanol, sonicate (250W power, 40kHz frequency) for 30 minutes, filter, evaporate the filtrate to dryness, dissolve it in 20mL of water, transfer it to a separatory funnel, and extract it three times with ethyl acetate, 20mL each time, discarding the ethyl acetate solution. Extract the aqueous layer twice with water-saturated n-butanol, 20mL each time, combine the n-butanol layers, and wash the n-butanol layer twice with water-saturated n-butanol, 20mL each time. Evaporate the n-butanol layer to dryness, dissolve the residue in 1mL of methanol, and use it as the test solution.

[0128] Preparation of reference solution: Weigh 1 mg of ginsenoside Rb1 reference standard, 1 mg of ginsenoside Re reference standard, and 1 mg of ginsenoside Rg1 reference standard, and add 1 ml of methanol to prepare a 1 mg / ml solution; this is the reference solution. Reference herb solution: Take 1 g of ginseng reference herb, place it in a 100 mL Erlenmeyer flask, add 40 mL of methanol, sonicate (power 250 W, frequency 40 kHz) for 30 minutes, filter, evaporate the filtrate to dryness, dissolve in 20 mL of water, transfer to a separatory funnel, and extract three times with ethyl acetate, 20 mL each time, discarding the ether solution. Extract the aqueous layer twice with water-saturated n-butanol, 20 mL each time, combine the n-butanol layers, and wash the n-butanol layer twice with water-saturated n-butanol, 20 mL each time. Evaporate the n-butanol layer to dryness, dissolve the residue in 1 mL of methanol, and this is the reference herb solution. Development and color development conditions: Perform thin-layer chromatography (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 0502). Apply 5 μl each of the test solution, reference solution, and reference medicinal material solution to the same silica gel high-performance G thin-layer plate coated with sodium carboxymethyl cellulose. Develop using the upper layer of n-butanol-ethyl acetate-water (4:1:5) as the developing solvent. Remove the plate, air dry, spray with 10% sulfuric acid ethanol solution, and develop the color at 105℃. Examine under 365 nm and sunlight. Standard: In the chromatogram of the test sample, spots of the same color should appear at the corresponding positions as in the chromatograms of the reference solution and the reference medicinal material.

[0129] 1.6.2 Verification Results:

[0130] Specificity: In the chromatogram of the ginseng negative sample, no spots of the same color as those in the chromatogram of the control herb were observed at the same positions, indicating that the specificity of this method meets the requirements. Robustness: Different humidity levels (32%, 58%, 72% relative humidity), different temperatures (30–35℃, 20–25℃, 2–8℃), and different thin-layer chromatography manufacturers (Merck, Qingdao Ocean, Yantai Huayang) have no effect on the test results.

[0131] 1.7 Identification of Alisma plantago-aquatica:

[0132] 1.7.1 Test method:

[0133] Preparation of test solution: Weigh 15g of the product granules, grind them finely, add 50ml of ethyl acetate, sonicate for 30min, filter, add 1.5g of activated carbon powder to the filtrate, filter, evaporate to dryness, add 0.5ml of ethyl acetate to the residue to dissolve it, and use it as the test solution.

[0134] Reference herb solution: Weigh 2g of Alisma plantago-aquatica reference herb, add 30ml of ethyl acetate, sonicate for 30min, filter, evaporate to dryness, dissolve the residue in 0.5ml of ethyl acetate to obtain the reference herb solution. Development and color development conditions: Perform thin-layer chromatography (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 0502). Apply 5μl each of the test solution and the reference herb solution to the same silica gel high-performance G thin-layer plate with sodium carboxymethyl cellulose. Develop with cyclohexane-ethyl acetate (1:1) as the developing solvent. Remove, air dry, spray with 2,4-dinitrophenylhydrazine hydrochloride-ethanol solution, and develop at 105℃. Examine under 365nm and sunlight. Standard: In the chromatogram of the test sample, spots of the same color appear at the corresponding positions as in the chromatogram of the reference herb.

[0135] 1.7.2 Verification Results:

[0136] Specificity: In the chromatogram of the negative sample of Alisma plantago-aquatica, no spots of the same color as those in the chromatogram of the control herb were observed at the same positions, indicating that the specificity of this method meets the requirements. Robustness: Different humidity levels (32%, 58%, 72% relative humidity), different temperatures (30–35℃, 20–25℃, 2–8℃), and different thin-layer chromatography manufacturers (Merck, Qingdao Ocean, Yantai Huayang) have no effect on the test results.

[0137] 1.8 Identification of stachydrine hydrochloride

[0138] 1.8.1 Test method:

[0139] Preparation of test solution: Take 10g of this product, grind it into a fine powder, add 50ml of anhydrous ethanol, heat under reflux for 30 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 5ml of water, and pass it through a 732Na-type strong acid cation exchange resin column (inner diameter 0.9cm, column height 12cm, pretreatment required). When the liquid level is level with the column packing, close the plunger, let it stand for 30 minutes, wash with water until the eluent is nearly colorless, discard the water, then elute with 50ml of ammonia test solution, collect the eluent, evaporate to dryness, dissolve the residue in 1ml of ethanol, and use it as the test solution.

[0140] Preparation of reference solution: Prepare a reference solution by dissolving stachydrine hydrochloride reference standard in ethanol to a concentration of 2 mg / ml. Development and colorimetric conditions: Perform thin-layer chromatography (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 0502). Apply 10 μl of both the test solution and the reference solution separately to the same silica gel G thin-layer plate. Use n-butanol-ethyl acetate-hydrochloric acid (4:0.5:1.5) as the developing solvent. Develop the plate in a developing chamber pre-saturated with the developing solvent for 20 minutes. Remove the plate, air dry, and spray with dilute bismuth potassium iodide reagent. Standard: The test sample chromatogram should show spots of the same color at the corresponding positions as the reference sample chromatogram.

[0141] 1.8.2 Verification Results:

[0142] Specificity: In the chromatogram of the Leonurus japonicus negative sample, no spots of the same color as those in the chromatogram of the reference standard were observed at the same positions, indicating that the specificity of this method meets the requirements. Robustness: Different humidity levels (32%, 58%, 72% relative humidity), different temperatures (30–35℃, 20–25℃, 2–8℃), and different thin-layer chromatography manufacturers (Merck, Qingdao Ocean, Yantai Huayang) have no effect on the test results.

[0143] Example 2: Determination of Ginsenoside Content

[0144] 2.2.1 Test Method

[0145] Chromatographic conditions and system suitability test: Octadecylsilane-bonded silica gel was used as the stationary phase (CAPCELL COREC 184.6mm × 150mm 2.7μm); acetonitrile was used as mobile phase A, and water as mobile phase B, with gradient elution performed according to the table below; evaporative light detector; flow rate 1.0 ml / min; column temperature 30℃. The nebulizer was nitrogen or compressed air, flow rate 1.6 ml / min, nebulization temperature 65℃, drift tube temperature 85℃, and gain factor 6. The theoretical plate number, calculated based on the ginsenoside Rg1 peak, should not be less than 10,000, and the resolution between the chromatographic peaks of ginsenoside Rg1 and ginsenoside Re should be greater than 1.5.

[0146]

[0147] Preparation of the test solution: Take an appropriate amount of this product, grind it into a fine powder, take 3g, accurately weigh it, place it in an Erlenmeyer flask, accurately measure 100ml of methanol, weigh it, reflux for 1 hour, cool it, weigh it again, make up the weight loss with methanol, shake well, filter, accurately measure 50ml of the filtrate, place it in an evaporating dish, evaporate it to dryness, dissolve the residue in 20% acetonitrile, transfer it to a 10ml volumetric flask, dilute it to the mark with 20% acetonitrile, shake well, filter, and take the filtrate to obtain the test solution.

[0148] Preparation of reference solution: Take appropriate amounts of ginsenoside Rg1 reference standard, ginsenoside Re reference standard, and ginsenoside Rb1 reference standard, respectively, accurately weigh them, and add 20% acetonitrile to prepare a mixed solution containing 0.10 mg of ginsenoside Rg1, 0.10 mg of ginsenoside Re, and 0.10 mg of ginsenoside Rb1 per ml.

[0149] Assay: Accurately pipette 10 μl, 15 μl, and 20 μl of the reference solution and 20 μl of the test solution, respectively, and inject them into the liquid chromatograph for determination. Calculate the regression equation between the logarithmic value of the reference substance mass and the logarithmic value of the corresponding main peak area; the correlation coefficient (r) should not be less than 0.99. Use the regression equation to calculate the content of ginsenoside Rg1, ginsenoside Re, and ginsenoside Rb1 in the test sample.

[0150] 2.2.2 Specificity

[0151] Test results: Neither the blank solvent nor the negative solution interfered with the detection of cinnamaldehyde.

[0152] 2.2.3 System Applicability

[0153] Five consecutive injections of the control solution were performed. The chromatographic peak detection results of ginsenosides showed that the RSD (%) of the Rg1 peak was 0.17, the RSD (%) of the Re peak was 0.25, and the RSD of the Rb1 peak was 0.13. The RSD (%) of the logarithm of the ginsenoside peak area was ≤0.3%.

[0154] 2.2.4 Linear Range

[0155] Table 1 Results of linear relationship investigation for each detected component

[0156]

[0157] 2.2.5 Repeatability Test

[0158] An appropriate amount of the traditional Chinese medicine composition of this invention was taken, and six test solutions were prepared by Inspector 1 according to the method under section "2.2.1". The peak areas of ginsenosides Rg1, Re, and Rb1 were measured respectively, and the experimental results are as follows: The content determination results of Rg1 were 0.4114 mg / g, 0.4023 mg / g, 0.4096 mg / g, 0.3987 mg / g, 0.4163 mg / g, and 0.4066 mg / g, with an RSD (%) of 1.56. The content determination results of Re were 0.4655 mg / g, 0.4683 mg / g, 0.4682 mg / g, 0.4590 mg / g, 0.4768 mg / g, and 0.4760 mg / g, with an RSD (%) of 1.42. The results of the Rb1 content determination were 0.5857 mg / g, 0.5914 mg / g, 0.5965 mg / g, 0.5941 mg / g, 0.6081 mg / g, and 0.5907 mg / g, and the RSD (%) of Rb1 was 1.28.

[0159] 2.2.6 Intermediate Precision

[0160] Take an appropriate amount of this product, and according to the method under section "2.2.1", Inspector 2 prepares 6 test solutions, and repeats the injection analysis 6 times consecutively under the chromatographic conditions under section "2.2.1". The detection results are as follows. For a total of 12 test solution samples from Inspectors 1 and 2, the results of the ginsenoside Rg1, Re, and Rb1 content determination are as follows: The intermediate precision content determination results of Rg1 measured by Inspector 2 are 0.3969 mg / g, 0.3912 mg / g, 0.3939 mg / g, 0.4042 mg / g, 0.3913 mg / g, and 0.4032 mg / g, and the RSD (%) of Rg1 is 1.45. The contents of ginsenoside Rg1 were determined by two inspectors, Personnel 1 and 2, to be: 0.4114 mg / g, 0.4023 mg / g, 0.4096 mg / g, 0.3987 mg / g, 0.4163 mg / g, 0.4066 mg / g, 0.3969 mg / g, 0.3912 mg / g, 0.3939 mg / g, 0.4042 mg / g, 0.3913 mg / g, and 0.4032 mg / g. The RSD (%) of Rg1 was 2.01.

[0161] The intermediate precision results of Re content determination, measured by Inspector 2, were 0.4591 mg / g, 0.4499 mg / g, 0.4539 mg / g, 0.4651 mg / g, 0.4532 mg / g, and 0.4674 mg / g, with an RSD (%) of 1.53. Inspectors 1 and 2 determined the ginsenoside Re content to be 0.4655 mg / g, 0.4683 mg / g, and...

[0162] 0.4682mg / g, 0.4590mg / g, 0.4768mg / g, 0.4760mg / g, 0.4591mg / g, 0.4499mg / g,

[0163] The RSD (%) of Re for 0.4539 mg / g, 0.4651 mg / g, 0.4532 mg / g, and 0.4674 mg / g was 1.87.

[0164] The intermediate precision content determination results of Rb1 measured by inspector 2 were: 0.6080 mg / g, 0.5928 mg / g, 0.5906 mg / g, 0.6174 mg / g, 0.5957 mg / g, and 0.6079 mg / g, and the RSD (%) of the Rb1 content determination results was 1.76. The contents of ginsenoside Rb1 were determined by two inspectors, Personnel 1 and 2, and were as follows: 0.4114 mg / g, 0.4023 mg / g, 0.5857 mg / g, 0.5914 mg / g, 0.5965 mg / g, 0.5941 mg / g, 0.6081 mg / g, 0.5907 mg / g, 0.6080 mg / g, 0.5928 mg / g, 0.5906 mg / g, 0.6174 mg / g, 0.5957 mg / g, and 0.6079 mg / g. The RSD (%) of the Rb1 content determination results was 1.62.

[0165] 2.2.7 Recovery rate

[0166] 2.2.7.1 Recovery rate of Rg1

[0167] Six portions of 1.5g of the Shenfu Yixin Granules of this invention were accurately weighed and added to a certain amount of the corresponding reference solution. Sample solutions were prepared according to the method in section "2.2.1" and analyzed under the chromatographic conditions in section "2.1.1". The peak area of ​​Rg1 was recorded, and the recovery rates of Rg1 were calculated to be 104.27%, 103.75%, 99.09%, 105.87%, 100.98%, and 102.80%, respectively. The average recovery rate was 102.80%, and the RSD was 2.37%, indicating that the Rg1 content detection method of this invention has good accuracy.

[0168] 2.2.7.2 Re recovery rate

[0169] Accurately weigh 1.5g of the Shenfu Yixin Granules of this invention, making 6 portions. Add a certain amount of the corresponding reference solution to each portion, prepare sample solutions according to the method in section "2.2.1", and analyze them under the chromatographic conditions in section "2.1.1". Record the Re chromatographic peak area and calculate the Rg1 recovery rates as 100.84%, 103.56%, and 95.76%, respectively.

[0170] The recovery rates were 105.78%, 99.30%, and 102.61%, with an average recovery rate of 101.3% and an RSD of 3.47%, indicating that the Re content detection method of this invention has good accuracy.

[0171] 2.2.7.3 Recovery rate of Rb1

[0172] Six portions of 1.5g of the Shenfu Yixin Granules of this invention were accurately weighed and added to a certain amount of the corresponding reference solution. Sample solutions were prepared according to the method in section "2.2.1" and analyzed under the chromatographic conditions in section "2.1.1". The Re chromatographic peak area was recorded, and the Rb1 recovery rates were calculated to be 100.78%, 104.66%, 103.20%, 104.39%, 101.16%, and 99.66%, respectively. The average recovery rate was 102.3%, and the RSD was 2.02%, indicating that the Rb1 content detection method of this invention has good accuracy.

[0173] 2.2.8 Stability of reference standard and test solution

[0174] Specific concentrations of Rg1, Re, and Rb1 reference solutions were prepared and measured under the chromatographic conditions described in section "2.1.1" at room temperature for 0 h, 16 h, and 26 h, respectively. The results were as follows: Rg1 Log peak areas were 3.30432, 3.31760, and 3.31557, with an average Log peak area of ​​3.3125 and an RSD% of 0.3. Re Log peak areas were 3.34163, 3.34696, and 3.34941, with an average Log peak area of ​​3.346 and an RSD% of 0.2. Rb1 Log peak areas were 3.28652, 3.29321, and 3.29017, with an average Log peak area of ​​3.29 and an RSD% of 0.2. These results indicate that the ginsenoside reference solutions exhibit good stability at room temperature for up to 26 h.

[0175] A test solution of Shenfu Yixin Granules at a certain concentration was prepared and determined according to the chromatographic conditions under section "2.1.1". The chromatographic results were as follows: Rg1 content (mg / g) in the test solution was 0.4114, 0.4162, and 0.4149, with an average Rg1 content of 0.4142. The RSD% was 0.6. Re content (mg / g) in the test solution was 0.4655, 0.4720, and 0.4756, with an average Re content of 0.4710 mg / g. The RSD% was 1.1. Rb1 content (mg / g) in the test solution was 0.5857, 0.6016, and 0.6093, with an average Rb1 content of 0.5989. The RSD% was 0.21. The above test results show that the ginsenosides in the test solution of Shenfu Yixin Granules have good stability within 26 hours at room temperature.

[0176] Example 3: Determination of Danshenol B Content

[0177] 3.1 Test Method

[0178] Chromatographic conditions and system suitability test: Octadecylsilane-bonded silica gel was used as the stationary phase (Aglient 5TC-C184.6mm×250mm); acetonitrile-0.2% phosphoric acid water (23:77) was used as the mobile phase; the detection wavelength was 286nm; the flow rate was 1.0ml / min; and the column temperature was 25℃.

[0179] Preparation of the test solution: Take an appropriate amount of this product, grind it into a fine powder, take 0.4g, weigh it accurately, place it in a 50ml volumetric flask, add about 30ml of 75% methanol, sonicate for 30 minutes, let it cool, dilute it to the mark with 75% methanol solution, shake well, filter, and take the filtrate to obtain the test solution.

[0180] Preparation of reference solution: Accurately weigh an appropriate amount of salvianolic acid B reference standard, add 75% methanol to prepare a solution containing 0.05 mg per ml.

[0181] Determination method: Accurately pipette 10 μl of the reference solution and the test solution into the liquid chromatograph and determine the result.

[0182] 3.2 System Applicability

[0183] Five consecutive injections of the control solution were performed to determine the peak area of ​​salvianolic acid B in the solution of the Shenfu Yixin granules of this invention. The peak area measurements were 1223949, 1232185, 1235859, 1241280, and 1265663, with an RSD of 0.6%.

[0184] 3.3 Linear Range

[0185] Tanshinone B: showed good linearity in the concentration range of 0.01238 mg / ml to 0.19808 mg / ml, with the linear equation being y = 12863251.2832x - 7988.5 and R = 1.0000. The Y-intercept was compared with the Y value at 100% concentration, and the result was 1.3%.

[0186] 3.4 Repeatability

[0187] Six test solutions were prepared, and the content of salvianolic acid B in the Shenfu Yixin Granules solution of this invention was determined. The salvianolic acid B content (mg / g) was 7.35, 7.41, 7.38, 7.37, 7.39, and 7.34, with an RSD (%) of 0.4.

[0188] 3.5 Intermediate Precision

[0189] Inspector 2 prepared six test solutions and determined the content of salvianolic acid B in the Shenfu Yixin Granules solution of this invention. The experimental results showed that the content of salvianolic acid B (mg / g) in the test solutions was 7.54, 7.44, 7.51, 7.58, 7.56, and 7.55, with an RSD (%) of 0.7.

[0190] Two inspectors, 1 and 2, tested 12 sample solutions to determine the content of salvianolic acid B in the Shenfu Yixin Granules solution of this invention. The experimental results showed that the salvianolic acid B content (mg / g) in the sample solutions was 7.35, 7.41, 7.38, 7.37, 7.39, 7.34, 7.54, 7.44, 7.51, 7.58, 7.56, and 7.55. The RSD (%) was 1.2.

[0191] 3.6 Recovery rate

[0192] Accurately weigh 1.5g of the Shenfu Yixin Granules of this invention, making 6 portions. Add a certain amount of the corresponding reference solution to each portion, prepare sample solutions according to the method in section "3.1", and analyze them under the chromatographic conditions in section "3.1". Record the peak area of ​​salvianolic acid B, and calculate the recovery rates of salvianolic acid B as 101.40%, 101.09%, and 101.09%, respectively.

[0193] The recoveries were 104.21%, 101.64%, 101.46%, and 98.06%, with an average recovery rate of 101.3% and an RSD of 2.0%. The results indicate that the method for detecting the content of salvianolic acid B in this invention has good accuracy.

[0194] 3.7 Stability of the test solution

[0195] The content of salvianolic acid B in the Shenfu Yixin Granules solution of this invention was determined by placing the test solution for 0 h, 6 h, 20 h, 48 h, and 72 h. Experimental results: The content of salvianolic acid B (mg / g) in the test solution was 7.64, 7.55, 7.54, 7.60, and 7.45, with RSDs of 2.0%. These data indicate that salvianolic acid B in the Shenfu Yixin Granules test solution exhibits good stability within 72 h at room temperature.

[0196] Example 4: Determination of paeoniflorin content

[0197] 4.1 Test Method

[0198] Chromatographic conditions and system suitability test: Octadecylsilane-bonded silica gel was used as the stationary phase (Aglient 5TC-C18 4.6mm×250mm); acetonitrile-0.05% formic acid solution (14:86) was used as the mobile phase; the detection wavelength was 230nm; the flow rate was 1.0ml / min; and the column temperature was 25℃.

[0199] Preparation of the test solution: Take an appropriate amount of this product, grind it into a fine powder, take 0.4g of Shenfu Yixin granules, weigh it accurately, place it in a 50ml volumetric flask, add about 30ml of 75% methanol, sonicate for 30 minutes, let it cool, dilute it to the mark with 75% methanol solution, shake well, filter, and take the filtrate to obtain the test solution.

[0200] Preparation of reference solution: Accurately weigh an appropriate amount of paeoniflorin reference standard, add 75% methanol to prepare a solution containing 0.03 mg per ml.

[0201] Determination method: Accurately pipette 10 μl of the reference solution and the test solution into the liquid chromatograph and determine the result.

[0202] 4.2 System Applicability

[0203] The paeoniflorin content in the Shenfu Yixin Granules solution of this invention was determined by five consecutive injections of the control solution. The experimental results showed that the chromatographic peak areas of paeoniflorin in the test solution were 384.211, 397.283, 388.310, and 385.690.

[0204] 386.574, with an RSD (%) of 0.9.

[0205] 4.3 Linear Range

[0206] Experimental results: Paeoniflorin showed good linearity in the concentration range of 12.0450 μg / ml to 60.2248 μg / ml, with a linear equation of y = 13.339x - 2.8 and R = 0.9999. The Y-intercept was 0.8% when compared with the Y value at 100% concentration.

[0207] 4.4 Repeatability

[0208] Inspector 1 used 6 test solutions to determine the paeoniflorin content (mg / g) in the Shenfu Yixin Granules solution of this invention. The results were 3.2281, 3.2191, 3.3044, 3.2643, 3.2390, and 3.2242, with an RSD (%) of 1.1.

[0209] 4.5 Intermediate Precision

[0210] Inspector 2 prepared six test solutions and determined the paeoniflorin content in the Shenfu Yixin Granules solution of this invention. The experimental results showed that the paeoniflorin content (mg / g) in the test solutions was 3.3169, 3.3456, 3.3531, 3.3271, 3.3475, and 3.3706, with an RSD (%) of 0.6.

[0211] Two inspectors, 1 and 2, tested 12 sample solutions to determine the paeoniflorin content in the Shenfu Yixin Granules solution of this invention. The experimental results showed that the paeoniflorin content (mg / g) in the sample solutions was 3.2281, 3.2191, and...

[0212] 3.3044, 3.2643, 3.2390, 3.2242, 3.3169, 3.3456, 3.3531, 3.3271, 3.3475, 3.3706. The RSD (%) is 1.8.

[0213] 4.6 Recovery rate

[0214] Six portions of 0.2g of the Shenfu Yixin Granules of this invention were accurately weighed and added to corresponding reference solutions. Sample solutions were prepared according to the method described in section "4.1" and analyzed under the chromatographic conditions described in section "4.1". The peak area of ​​paeoniflorin was recorded. The recoveries of paeoniflorin were calculated to be 103.80%, 100.40%, 102.00%, 104.60%, 101.80%, and 103.00%, respectively, with an average recovery rate of 102.6% and an RSD of [value missing]. The results indicate that the method for detecting paeoniflorin content in this invention has good accuracy.

[0215] 4.7 Stability of the test solution

[0216] The paeoniflorin content in the Shenfu Yixin Granules solution of this invention was determined by placing the test solution for 0 h, 6 h, and 24 h. Experimental results: The paeoniflorin content (mg / g) in the test solution was 3.3835, 3.3706, and 3.42857.45, with RSDs of 0.90%. These data indicate that paeoniflorin in the Shenfu Yixin Granules test solution exhibits good stability within 24 h at room temperature.

[0217] Example 5: Determination of Cinnamaldehyde Content

[0218] 5.1 Test Method

[0219] Chromatographic conditions and system suitability test: Octadecylsilane-bonded silica gel was used as the stationary phase (Agilent ZORBAX Eclipse Plus C18 4.6mm*250mm, 5um); acetonitrile-water (30:70) was used as the mobile phase; the detection wavelength was 290nm; the flow rate was 1.0ml / min; and the column temperature was 25℃.

[0220] Preparation of the test solution: Take an appropriate amount of this product, grind it into a fine powder, accurately weigh 0.3g, place it in a 50ml volumetric flask, add 2ml of water, seal, let stand for 2 hours, add 35ml of acetonitrile, seal, sonicate for 30 minutes (500W, 40KHz), remove and let it reach room temperature, dilute with acetonitrile to the mark, shake well, filter, and the test solution is obtained.

[0221] Preparation of reference solution: Accurately weigh an appropriate amount of cinnamaldehyde reference standard, add acetonitrile to prepare a solution containing 0.05 mg per ml.

[0222] Determination method: Accurately pipette 10 μl of the reference solution and the test solution into the liquid chromatograph and determine the result.

[0223] 5.2 System Applicability

[0224] Five consecutive injections of the control solution were performed to determine the peak area of ​​cinnamaldehyde in the Shenfu Yixin Granules solution of this invention. The peak area measurements were 630649, 632641, 632146, 634457, and 632335, with an RSD (%) of 0.3.

[0225] 5.3 Linear Range

[0226] Cinnamaldehyde showed good linearity in the concentration range of 0.53 μg / ml to 15.86 μg / ml, with the linear equation being y = 119128x + 6212.1 and R = 0.9999. The Y-intercept was 1.0% when compared with the Y value at 100% concentration.

[0227] 5.4 Repeatability

[0228] Six test solutions were prepared, and the cinnamaldehyde content in the Shenfu Yixin Granules solution of this invention was determined. The cinnamaldehyde content (mg / g) was 0.6643, 0.6664, 0.6699, 0.6655, 0.6694, and 0.6696, with an RSD (%) of 0.4.

[0229] 5.5 Intermediate Precision

[0230] Inspector 2 prepared six test solutions to determine the cinnamaldehyde content in the Shenfu Yixin Granules solution of this invention. The experimental results showed that the cinnamaldehyde content (mg / g) in the test solutions was 0.6652, 0.6697, 0.6725, 0.6713, 0.6726, and 0.6672. The RSD (%) was 0.5.

[0231] Two inspectors, Personnel 1 and 2, tested 12 sample solutions to determine the cinnamaldehyde content in the Shenfu Yixin Granules solution of this invention. The experimental results showed that the cinnamaldehyde content (mg / g) in the sample solutions was 0.6643, 0.6664, 0.6699, 0.6655, 0.6694, 0.6696, 0.6652, 0.6697, 0.6725, 0.6713, 0.6726, and 0.6672. The RSD (%) was 0.5.

[0232] 5.6 Recovery rate

[0233] Six portions of the present invention's Shenfu Yixin Granules (0.15g each) were accurately weighed and added to corresponding reference solutions. Sample solutions were prepared according to the method described in section "5.1" and analyzed under the chromatographic conditions described in section "5.1". The peak areas of cinnamaldehyde were recorded, and the recoveries of cinnamaldehyde were calculated to be 100.7%, 103.2%, 102.5%, 102.3%, 100.5%, and 97.6%, respectively, with an average recovery rate of 101.0% and an RSD of 2.1%. The results indicate that the method for detecting cinnamaldehyde content in this invention has good accuracy.

[0234] 5.7 Stability of the test solution

[0235] The cinnamaldehyde content in the Shenfu Yixin Granules solution of this invention was determined at 0h, 5h, 10h, and 20h. Experimental results: The chromatographic peak areas of cinnamaldehyde in the test solution were 479619, 481652, 482113, and 483648, with an average peak area of ​​481758 and RSDs of 0.4%. These data indicate that the cinnamaldehyde component in the Shenfu Yixin Granules test solution exhibits good stability within 20h at room temperature.

Claims

1. A method for detecting a traditional Chinese medicine composition for treating chronic heart failure, characterized in that, The content detection method includes the following steps for detecting ginsenoside content: (1) Preparation of the test solution: Take the Chinese herbal composition of the present invention, grind it into a fine powder, weigh it, take methanol, weigh it, reflux for 1 hour, cool it, weigh it again, make up the weight loss with methanol, shake it well, filter it, take the filtrate, evaporate it to dryness, dissolve the residue in 20% acetonitrile, dilute it, shake it well, filter it, take the filtrate, and the test solution is obtained. (2) Preparation of reference solution: Weigh ginsenoside Rg1 reference standard, ginsenoside Re reference standard, and ginsenoside Rb1 reference standard, add 20% acetonitrile to prepare a mixed solution containing 0.10 mg of ginsenoside Rg1, 0.10 mg of ginsenoside Re, and 0.10 mg of ginsenoside Rb1 per ml. (3) Chromatographic conditions and system suitability test: Octadecylsilane-bonded silica gel was used as the stationary phase, acetonitrile as mobile phase A, and water as mobile phase B. The gradient elution program was as follows: 0–8 min, 17% A, 83% B; 8–23 min, 17%–19% A, 83%–81% B; 23–30 min, 19%–24% A, 81%–76% B; 30–40 min, 24%–29% A, 76%–71% B; 40–48 min… In 48–68 min, 29% A, 71% B; 48–68 min, 29%–80% A, 71%–20% B; 68–69 min, 80%–17% A, 20%–83% B; 69–80 min, 17% A, 83% B; column temperature 25–35 °C, flow rate 0.8–1.2 mL / min, evaporative light detector; nebulizer with nitrogen or compressed air, nebulization temperature 62–68 °C, drift tube temperature 82–87 °C; (4) Content detection: Take the test solution from step (1) and the reference solution from step (2), inject them into the liquid chromatograph, and determine the content of ginsenoside Rg1, ginsenoside Re and ginsenoside Rb1 in the test solution of the traditional Chinese medicine composition according to the chromatographic conditions in step (3).

2. The method for detecting traditional Chinese medicine compositions as described in claim 1, characterized in that, The content detection method also includes the following steps for determining the content of salvianolic acid B: (1) Chromatographic conditions: Octadecylsilane-bonded silica gel was used as the stationary phase, acetonitrile-0.2% phosphoric acid water was used as the mobile phase (23:77), the detection wavelength was 286 nm, the flow rate was 0.8–1.2 mL / min, and the column temperature was 23–27 °C. (2) Preparation of the test solution: Weigh the Chinese herbal composition of the present invention, add 70-80% methanol, sonicate for 20-40 minutes, cool, dilute to the mark with 70-80% methanol solution, shake well, filter, and take the filtrate to obtain the test solution. (3) Preparation of reference solution: Accurately weigh salvianolic acid B reference standard, add 70-80% methanol to prepare a solution containing 0.05 mg per ml; (4) Determination method: Accurately pipette the reference solution and the test solution separately, inject them into the liquid chromatograph, and determine the result.

3. The method for detecting traditional Chinese medicine compositions as described in claim 1, characterized in that, The content detection method also includes a method for determining paeoniflorin content, with the following steps: (1) Chromatographic conditions and system suitability test: Octadecylsilane-bonded silica gel was used as the stationary phase; acetonitrile-0.05% formic acid solution (14:86) was used as the mobile phase; the detection wavelength was 230 nm; the flow rate was 0.8–1.2 ml / min; and the column temperature was 23–27 °C. (2) Preparation of test solution: Take the Chinese herbal medicine composition, grind it into a fine powder, weigh it, add 70-80% methanol, sonicate for 30 minutes, let it cool, dilute it to the mark with 75% methanol solution, shake it well, filter it, and take the filtrate to obtain the test solution. (3) Preparation of reference solution: Weigh an appropriate amount of paeoniflorin reference standard, add 70-80% methanol to prepare a solution containing 0.03 mg per ml; (4) Determination method: Pipette the reference solution and the test solution into the liquid chromatograph and determine the result.

4. The method for detecting traditional Chinese medicine compositions as described in claim 1, characterized in that, The content detection method also includes the following steps for determining the cinnamaldehyde content: (1) Chromatographic conditions and system suitability test: Octadecylsilane-bonded silica gel was used as the stationary phase, acetonitrile-water 30:70 was used as the mobile phase, the detection wavelength was 290 nm, the flow rate was 0.8 to 1.2 ml / min, and the column temperature was 23 to 27 °C. (2) Preparation of test solution: Take the Chinese herbal medicine composition, grind it into a fine powder, add acetonitrile, seal it, sonicate for 30 minutes, take it out and let it cool to room temperature, dilute it with acetonitrile, shake it well, filter it, and the test solution is obtained. (3) Preparation of reference solution: Weigh cinnamaldehyde reference standard and add acetonitrile to prepare a solution containing 0.05 mg per ml. (4) Determination method: Pipette 10 μl of the reference solution and the test solution into the liquid chromatograph and determine the result.

5. The method for detecting traditional Chinese medicine compositions as described in claim 1, characterized in that, The detection method also includes: one or a combination of two of the thin-layer identification items for tanshinone or paeoniflorin; The thin-layer chromatography identification of Salvia miltiorrhiza is as follows: Preparation of test solution: Take the traditional Chinese medicine composition, grind it into a fine powder, add methanol, sonicate, filter, evaporate the filtrate to dryness, dissolve the residue in water, extract with ether twice by shaking, discard the ether solution, add hydrochloric acid to adjust the pH of the aqueous solution to 2-3, extract with ether, discard the ether solution, extract the aqueous solution with ethyl acetate twice by shaking, combine the ethyl acetate solutions, wash, evaporate the ethyl acetate solution to dryness, dissolve the residue in methanol to obtain the test solution; Preparation of reference herb solution: Take another Salvia miltiorrhiza reference herb, add hydrochloric acid solution, heat to reflux, filter, extract the filtrate twice with ethyl acetate, combine the ethyl acetate solutions, evaporate to dryness, dissolve the residue in methanol to obtain the reference herb solution. Development and color development conditions: Perform thin-layer chromatography, take the test solution and the reference herb solution, spot them separately on the same silica gel G thin-layer plate, use toluene-ethyl acetate-formic acid 8:5:2 as the developing solvent, develop, remove, air dry, place in ammonia vapor, fumigate for 15 minutes, and examine under ultraviolet light at 365 nm. Standard: In the chromatogram of the test sample, fluorescent spots of the same color appear at the same positions as in the chromatogram of the reference herb. The paeoniflorin thin-layer chromatography identification method is as follows: Preparation of test solution: Take the traditional Chinese medicine composition, extract it by reflux with anhydrous ethanol, filter it, evaporate the filtrate to dryness in a water bath, dissolve the residue in n-butanol, filter it, evaporate the filtrate to dryness in a water bath, dissolve the residue in methanol to obtain the test solution; Preparation of reference solution: Paeoniflorin reference standard was dissolved in methanol to prepare a solution, which was then used as the reference solution. Development and color development conditions: Perform the thin-layer chromatography test, take the above two solutions and spot them separately on the same silica gel G thin-layer plate with sodium carboxymethyl cellulose as binder, use chloroform-ethyl acetate-methanol-formic acid 40:5:10:0.2 as the developing solvent, develop, remove, air dry, spray with 5% vanillin-sulfuric acid solution, bake at 105℃ for 10 minutes for color development, standard: in the chromatogram of the test sample, spots of the same color appear at the corresponding positions as in the chromatogram of the reference standard.

6. The method for detecting traditional Chinese medicine compositions as described in claim 1, characterized in that, The detection method also includes one or a combination of two of the thin-layer identification items for borneol acetate or cinnamaldehyde. Identification of Borneol Acetate: Preparation of test solution: Take the traditional Chinese medicine composition, add water to dissolve it, add petroleum ether, heat in a water bath under reflux, separate the petroleum ether layer, evaporate to obtain the test solution; Preparation of reference solution: Weigh bornyl acetate reference standard, add ethanol to prepare a solution, and use it as the reference solution; Development and color development conditions: Perform thin-layer chromatography test, take the above two solutions and spot them separately on the same silica gel G thin-layer plate, use cyclohexane-ethyl acetate 22:1 as the developing solvent, develop, take out, air dry, and spray with 5% vanillin sulfuric acid solution; Standard: In the chromatogram of the test sample, spots of the same color appear at the corresponding positions as in the chromatogram of the reference sample; The identification of cinnamaldehyde: Preparation of test solution: Take the traditional Chinese medicine composition, add cyclohexane according to the method for determining volatile oil, heat and extract, separate the cyclohexane layer as the test solution; Preparation of reference solution: Weigh cinnamaldehyde reference standard, add cyclohexane to prepare a solution, and use it as the reference solution; Development and color development conditions: Perform the thin-layer chromatography test. Take the test solution and the reference solution and spot them separately on the same silica gel G thin-layer plate with sodium carboxymethyl cellulose. Use petroleum ether-ethyl acetate 17:3 as the developing solvent. Develop, remove, and air dry. Spray with dinitrophenylhydrazine ethanol test solution. Standard: In the chromatogram of the test sample, spots of the same color appear at the corresponding positions as in the chromatogram of the reference sample.

7. The method for detecting traditional Chinese medicine compositions as described in claim 1, characterized in that, The detection method also includes: thin-layer identification of psyllium husk. Preparation of test solution: Take the Chinese herbal medicine composition, add methanol, sonicate, filter, evaporate the filtrate to dryness, add water to dissolve, extract three times with ethyl acetate, discard the ether solution, extract the aqueous layer twice with water-saturated n-butanol, combine the n-butanol layers, wash the n-butanol layer twice with water-saturated n-butanol, evaporate the n-butanol layer to dryness, dissolve the residue in methanol to obtain the test solution. Preparation of reference solution: Weigh genipin glycoside reference standard and verbascoside reference standard, and add methanol to prepare a 1 mg / ml solution; Reference herb solution: Take Plantago seed reference herb, add methanol, sonicate, filter, evaporate the filtrate to dryness, dissolve in water, extract three times with ethyl acetate, 20 mL each time, discard the ether solution, extract the aqueous layer twice with water-saturated n-butanol, 20 mL each time, combine the n-butanol layers, wash the n-butanol layer twice with water-saturated n-butanol, 20 mL each time, evaporate the n-butanol layer to dryness, dissolve the residue in methanol, and use as the reference herb solution; Development and color development conditions: Perform the thin-layer chromatography test. Take the test solution, reference solution, and reference medicinal material solution and spot them separately on the same silica gel high-performance G thin-layer plate with sodium carboxymethyl cellulose. Use ethyl acetate-ethanol-formic acid-water 12:3:1:0.8 as the developing solvent. Develop, remove, and air dry. Spray with 2% vanillin-sulfuric acid solution and develop the color at 105℃. Examine under 365nm and sunlight. Standard: In the chromatogram of the test sample, spots of the same color appear at the corresponding positions as in the chromatograms of the reference sample and the reference medicinal material.

8. The method for detecting traditional Chinese medicine compositions as described in claim 1, characterized in that, The detection method also includes: ginseng thin-layer chromatography identification item: Preparation of test solution: Grind the traditional Chinese medicine composition into a fine powder, add methanol, sonicate, filter, evaporate the filtrate to dryness, dissolve in water, extract three times with ethyl acetate, discard the ether solution, extract the aqueous layer twice with water-saturated n-butanol, 20 mL each time, combine the n-butanol layers, wash the n-butanol layer twice with water-saturated n-butanol, 20 mL each time, evaporate the n-butanol layer to dryness, dissolve the residue in methanol to obtain the test solution; Preparation of reference solution: Weigh ginsenoside Rb1, ginsenoside Re, and ginsenoside Rg1, add methanol to prepare a solution; this is the reference solution. Reference herb solution: Take ginseng reference herb, add methanol, sonicate, filter, evaporate the filtrate to dryness, dissolve in water, extract three times with ethyl acetate, discard the ether solution, extract the aqueous layer twice with water-saturated n-butanol, combine the n-butanol layers, wash the n-butanol layer twice with water-saturated n-butanol, evaporate the n-butanol layer to dryness, dissolve the residue in methanol, and use as the reference herb solution; Development and color development conditions: Perform the thin-layer chromatography test, take the test solution, reference solution, and reference medicinal material solution, and spot them separately on the same silica gel high-performance G thin-layer plate with sodium carboxymethyl cellulose. Use the upper layer solution of n-butanol-ethyl acetate-water 4:1:5 as the developing solvent, develop, remove, air dry, spray with 10% sulfuric acid ethanol solution, develop the color at 105℃, and examine at 365nm and under sunlight; Standard: In the chromatogram of the test sample, spots of the same color appear at the corresponding positions as in the chromatograms of the reference standard and the reference medicinal material.

9. The method for detecting traditional Chinese medicine compositions as described in claim 1, characterized in that, The detection method also includes: thin-layer chromatography identification of Alisma plantago-aquatica: Preparation of test solution: Weigh the Chinese herbal medicine composition, grind it into a fine powder, add ethyl acetate, sonicate, filter, add activated carbon powder to the filtrate, filter, evaporate to dryness, dissolve the residue in ethyl acetate to obtain the test solution; Reference herb solution: Weigh Alisma plantago-aquatica reference herb, add ethyl acetate, sonicate, filter, evaporate to dryness, dissolve the residue in ethyl acetate to obtain the reference herb solution; Development and color development conditions: Perform the thin-layer chromatography test. Take the test solution and the reference medicinal material solution and spot them separately on the same silica gel high-performance G thin-layer plate with sodium carboxymethyl cellulose. Use cyclohexane-ethyl acetate 1:1 as the developing solvent. Develop, remove, and air dry. Spray with 2,4-dinitrophenylhydrazine hydrochloride ethanol solution and develop the color at 105℃. Examine under 365nm and sunlight. Standard: In the chromatogram of the test sample, spots of the same color appear at the corresponding positions as in the chromatogram of the reference medicinal material.

10. The method for detecting traditional Chinese medicine compositions as described in claim 1, characterized in that, The detection method also includes: thin-layer chromatography identification of stachydrine hydrochloride. Preparation of test solution: Take the Chinese herbal medicine composition, grind it into a fine powder, add anhydrous ethanol, heat under reflux, filter, evaporate the filtrate to dryness, add water to dissolve the residue, pass it through a cation exchange resin column, let it stand, wash with water until the eluent is nearly colorless, discard the aqueous solution, then elute with ammonia solution, collect the eluent, evaporate to dryness, add ethanol to dissolve the residue, and use it as the test solution. Preparation of reference solution: Take stachydrine hydrochloride reference standard and add ethanol to prepare a solution containing 2 mg in 1 ml as the reference solution; Development and colorimetric conditions: Perform the thin-layer chromatography test. Apply the test solution and reference solution separately to the same silica gel G thin-layer plate, using a 4:1 ratio of n-butanol to ethyl acetate to hydrochloric acid. Use a 0.5:1.5 ratio as the developing solvent. Pre-saturate the sample with the developing solvent, develop, remove, air dry, and spray with dilute potassium bismuth iodide solution. Standard: In the chromatogram of the test sample, spots of the same color appear at the corresponding positions as in the chromatogram of the reference sample.

Images