Detection method of blood-activating and pain-stopping compound preparation and application

Abstract

The application provides a detection method and application of a blood-activating and pain-stopping compound preparation, and belongs to the field of drug analysis. The method adopts high performance liquid chromatography to detect the content of ginsenoside Rg1, ginsenoside Rb1 and notoginsenoside R1, and the content of ferulic acid and 11-carbonyl-beta-acetyl frankincense acid; gas chromatography is adopted to detect the content of borneol and isoborneol; the qualitative identification of earthworm is carried out by adopting thin layer chromatography with earthworm as a control medicinal material; the qualitative identification of vinegar frankincense is carried out by adopting thin layer chromatography with frankincense as a control medicinal material, 11-carbonyl-beta-frankincense acid, 11-carbonyl-beta-acetyl frankincense acid, beta-frankincense acid and 3-acetyl-beta-frankincense acid as control samples. The application has strong specificity and good durability, perfects the quality control standard of the blood-activating and pain-stopping capsule, and comprehensively evaluates the drug quality. The application can well control the quality of the blood-activating and pain-stopping capsule, guarantee the stability, uniformity and controllability of the quality, provide a reference basis, realize the quality monitoring of the preparation, and guarantee the curative effect of the drug.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical analysis, specifically relating to a detection method and application of a compound preparation for promoting blood circulation and relieving pain. Background Technology

[0002] The formula for Huoxue Zhitong Capsules consists of Angelica sinensis, Panax notoginseng, vinegar-processed frankincense, borneol, Eupolyphaga sinensis, and calcined natural copper. It has the effects of promoting blood circulation, removing blood stasis, reducing swelling, and relieving pain. It is used for traumatic injuries, bruises, swelling, and pain, and is mainly used clinically to treat acute and chronic soft tissue injuries. This formula has been used clinically for many years with good efficacy and is one of the classic formulas for traumatic injuries, bruises, swelling, and pain. Comprehensive quality control is essential to ensure the quality and safety of Huoxue Zhitong Capsules.

[0003] The current standard for Huoxue Zhitong Capsules is included in Part I of the 2020 edition of the Chinese Pharmacopoeia. The standard only provides thin-layer chromatography identification for Angelica sinensis, Panax notoginseng, and borneol. It also establishes detection methods for Rg1 reference standard, ginsenoside Rb1 reference standard, and Panax notoginsenoside R1 in Panax notoginseng. The quality standard is simple and cannot reflect the characteristics of the overall efficacy of multiple components of traditional Chinese medicine, which seriously restricts the market expansion, clinical application, and internationalization of this product. Summary of the Invention

[0004] To address the shortcomings of the above methods, the purpose of this invention is to provide a detection method and application for a compound preparation for promoting blood circulation and relieving pain.

[0005] To achieve the above objectives, the technical solution provided by this invention is as follows:

[0006] The first aspect of this invention provides a method for detecting a compound preparation for promoting blood circulation and relieving pain, comprising:

[0007] The contents of ginsenoside Rg1, ginsenoside Rb1, and notoginsenoside R1 in the blood-activating and pain-relieving compound preparation were simultaneously detected by high performance liquid chromatography (HPLC); the contents of ferulic acid and 11-carbonyl-β-acetylbosuccinic acid in the blood-activating and pain-relieving compound preparation were also simultaneously detected by HPLC.

[0008] The contents of borneol and isoborneol in the blood-activating and pain-relieving compound preparation were determined by gas chromatography.

[0009] Thin-layer chromatography was used to qualitatively identify the ground beetle and vinegar-processed frankincense in the blood-activating and pain-relieving compound preparation.

[0010] In the aforementioned compound preparation for promoting blood circulation and relieving pain, the qualitative identification of *Eupolyphaga sinensis* was performed using *Eupolyphaga sinensis* as a reference material; in the aforementioned compound preparation for promoting blood circulation and relieving pain, the qualitative identification of *Boswellia carterii* was performed using *Boswellia carterii* as a reference material, with 11-carbonyl-β-boswellia carterii acid, 11-carbonyl-β-acetylboswellia carterii acid, β-boswellia carterii acid, and 3-acetyl-β-boswellia carterii acid as reference standards.

[0011] Preferably, the liquid chromatography conditions for simultaneous detection of ginsenoside Rg1, ginsenoside Rb1, and notoginsenoside R1 include: a C18 column; mobile phase A is acetonitrile, and mobile phase B is water; the detection wavelength is 203 nm; the injection volume is 10 μL; the flow rate is 1 mL / min; the column temperature is 30 °C; and the mobile phase gradient elution conditions are as follows: 0 min–12 min, mobile phase A is 20%, and mobile phase B is 80%; 12 min–40 min, mobile phase A is 20%–30%, and mobile phase B is 80%–70%; 40 min–55 min, mobile phase A is 30%–36%, and mobile phase B is 70%–64%; 55 min–60 min, mobile phase A is 36%–47%, and mobile phase B is 64%–53%.

[0012] Preferably, the liquid chromatography conditions for simultaneous detection of ferulic acid and 11-carbonyl-β-acetylbosuccinic acid include: a C18 column; mobile phase A is acetonitrile, and mobile phase B is 0.1% phosphoric acid water; the detection wavelength is 316 nm from 0 to 8 min, and the detection wavelength is 250 nm from 8 to 37 min; the injection volume is 10 μL; the flow rate is 1 mL / min; the column temperature is 30 °C; and the mobile phase gradient elution conditions are: 0 min to 8 min, mobile phase A is 27% → 30%, and mobile phase B is 73% → 70%; 8 min to 15 min, mobile phase A is 30% → 80%, and mobile phase B is 70% → 20%; 15 min to 37 min, mobile phase A is 80%, and mobile phase B is 20%.

[0013] Preferably, the gas chromatographic conditions for detecting borneol and isoborneol include: a quartz capillary column; a flame ionization detector (FID); a detector temperature of 250°C; an injection port temperature of 220°C; a programmed column temperature increase, starting at 90°C, increasing to 105°C at a rate of 3°C / min, then increasing to 220°C at a rate of 10°C / min and holding for 5 min; an injection volume of 1 μL; and a split ratio of 20:1.

[0014] Preferably, the specific procedure for qualitative identification of *Eupolyphaga sinensis* in the blood-activating and pain-relieving compound preparation is as follows:

[0015] Preparation of the test solution: The contents of the blood-activating and pain-relieving compound preparation were used as the test sample. Methanol was used as the solvent. The mixture was ultrasonically treated, filtered, and evaporated to dryness to obtain the residue. The residue was extracted with petroleum ether, and the petroleum ether solution was separated, evaporated to dryness, and then dissolved in methanol to prepare the test solution. The temperature of the petroleum ether extraction was 60℃-90℃.

[0016] Preparation of the reference medicinal material solution: Using ground beetle as the reference medicinal material, the preparation steps of the test sample solution were repeated to prepare the reference medicinal material solution;

[0017] Spot the test solution and the reference medicinal material solution separately onto the same silica gel G thin-layer plate, develop with the developing solvent, remove, air dry, and examine under 365 nm ultraviolet light; in the chromatogram of the test sample, a fluorescent main spot of the same color appears at the corresponding position as in the chromatogram of the reference medicinal material; the developing solvent is toluene-dichloromethane-acetone-formic acid, with a volume ratio of toluene:dichloromethane:acetone:formic acid = 5:5:0.5:0.1.

[0018] Preferably, the specific procedure for qualitative identification of frankincense in the blood-activating and pain-relieving compound preparation is as follows:

[0019] Preparation of test solution: The contents of the blood-activating and pain-relieving compound preparation are used as the test sample, and methanol is used as the solvent. After ultrasonic treatment and filtration, the filtrate is the test solution.

[0020] Preparation of reference material solution: Frankincense was used as the reference material, methanol was used as the solvent, and the solution was subjected to ultrasonic treatment and filtration. The filtrate was the test solution.

[0021] Preparation of reference solutions: Mix 11-keto-β-boswellic acid and 11-carbonyl-β-acetylboswellic acid with methanol to prepare mixed reference solution 1; mix β-boswellic acid and 3-acetyl-β-boswellic acid with methanol to prepare mixed reference solution 2;

[0022] Take samples of the test solution, reference medicinal material solution, mixed reference solution 1, and mixed reference solution 2, and spot them separately on the same silica gel GF254 thin-layer plate. Develop with the developing solvent, remove and air dry, examine under 254 nm ultraviolet light, then spray with sulfuric acid ethanol solution, and heat until the spots are clearly visible, then examine under sunlight; in the chromatogram of the test sample, spots or fluorescent spots of the same color appear at the corresponding positions as in the chromatograms of the mixed reference standard and the reference medicinal material; the developing solvent is cyclohexane-ethyl acetate-formic acid, with a volume ratio of cyclohexane:ethyl acetate:formic acid = 12:5:0.5.

[0023] The second aspect of this invention provides the application of the above-mentioned detection method for blood-activating and pain-relieving compound preparations in the field of detection of blood-activating and pain-relieving capsules.

[0024] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0025] 1. The method of this invention supplements the deficiencies of the original pharmacopoeia standards, realizing comprehensive quantitative and qualitative detection. This invention adds a thin-layer chromatography identification method for vinegar-processed frankincense and ground beetle, adds a method for determining the content of ferulic acid, 11-carbonyl-β-acetylbosuccinic acid, borneol, and isoborneol, optimizes the method for determining the content of Panax notoginseng, and shortens the analysis time. This method can comprehensively reflect the characteristics of Huoxue Zhitong Capsules, has good specificity, can realize quality monitoring of Huoxue Zhitong Capsules preparations, effectively control the quality of Huoxue Zhitong Capsules, and ensure the efficacy of the drug.

[0026] 2. This invention improves upon thin-layer chromatography (TLC) identification. The 2020 edition of the Chinese Pharmacopoeia, Part I, does not include the identification of vinegar-processed frankincense and *Eupolyphaga sinensis* in the Huoxue Zhitong Capsule standard. The 2020 edition of the Chinese Pharmacopoeia, Part I, also lacks a TLC identification item in the quality standard for acetic acid medicinal materials. However, the quality standard for *Eupolyphaga sinensis* includes a TLC identification item. Using the identification method for *Eupolyphaga sinensis* in the 2020 edition of the Chinese Pharmacopoeia, Part I, the spots in the chromatogram of the test sample are unclear at the corresponding positions as in the reference material, making it unsuitable for identifying *Eupolyphaga sinensis* in the Huoxue Zhitong Capsule compound preparation. To address these shortcomings, this invention, through extensive creative work, establishes a TLC identification method for *Eupolyphaga sinensis* and vinegar-processed frankincense in Huoxue Zhitong Capsule, enabling direct and visual separation of the compound preparation's components.

[0027] 3. In terms of content determination, this invention has established a new HPLC method for the simultaneous determination of ferulic acid and 11-carbonyl-β-acetylbosuccinic acid in compound preparations, a new GC method for the determination of borneol and isoborneol in compound preparations, and optimized the content determination method of Panax notoginseng, thus shortening the analysis time.

[0028] 4. The detection method for the blood-activating and pain-relieving compound preparation described in this invention has strong specificity and good durability, improving and supplementing the quality control standards for blood-activating and pain-relieving capsules, and providing a more comprehensive evaluation of drug quality. The above detection method can effectively control the quality of blood-activating and pain-relieving capsules, providing a reference for ensuring its stable, uniform, and controllable quality, enabling quality monitoring of the preparation, and guaranteeing the efficacy of the drug. Attached Figure Description

[0029] The accompanying drawings, which constitute a part of this invention, are used to further understand the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings:

[0030] Figure 1 The HPLC chromatograms for the determination of the content of ginsenoside Rg1, ginsenoside Rb1, and notoginsenoside R1 in Example 1 are shown below.

[0031] Figure 2 Chromatograms for the determination of ferulic acid and 11-carbonyl-β-acetylbosuccinic acid in Example 2;

[0032] Figure 3 Chromatograms showing the content determination of borneol and isoborneol in Example 3;

[0033] Figure 4 This is a thin-layer chromatogram of the ground beetle in Example 4;

[0034] Figure 5This is a thin-layer chromatogram for identifying acetic acid frankincense in Example 5;

[0035] Figure 6 The following are thin-layer chromatograms of *Eupolyphaga sinensis* extracted with different solvents in Example 6.

[0036] Figure 7 The following are thin-layer chromatograms of *Eupolyphaga sinensis* prepared using different methods in Example 6.

[0037] Figure 8 This is a thin-layer chromatogram for identifying frankincense in vinegar using different developing solvents in Example 7;

[0038] Figure 9 The following are thin-layer chromatograms of *Eupolyphaga sinensis* at different times in Example 8;

[0039] Figure 10 The following are thin-layer chromatograms of different thin-layer plated Eupolyphaga species in Example 8;

[0040] Figure 11 The following are thin-layer chromatograms of *Eupolyphaga sinensis* at different developing temperatures in Example 8;

[0041] Figure 12 This is a thin-layer chromatogram of *Eupolyphaga sinensis* under different humidity conditions in Example 8;

[0042] Figure 13 The following are thin-layer chromatograms of frankincense obtained from vinegar at different times in Example 9;

[0043] Figure 14 The following are thin-layer chromatograms for identifying frankincense using different silica gel plates in Example 9;

[0044] Figure 15 The following are thin-layer chromatograms of acetic acid frankincense at different developing temperatures in Example 9;

[0045] Figure 16 This is a thin-layer chromatogram for identifying acetic frankincense under different humidity conditions in Example 9;

[0046] Figure 17 The thin-layer chromatograms for identifying Eupolyphaga sinensis in 10 batches of Huoxue Zhitong capsules in Example 13 are shown below.

[0047] Figure 18 The thin-layer chromatograms are for identifying the vinegar and frankincense in 10 batches of Huoxue Zhitong capsules in Example 13. Detailed Implementation

[0048] Specific application examples are only used to further illustrate the claims, but do not constitute a further limitation on the present invention. Any technical solution obtained by making limited modifications to the present invention still falls within the scope of protection of the present invention.

[0049] Example 1

[0050] This embodiment describes the determination of the contents of ginsenoside Rg1, ginsenoside Rb1, and notoginsenoside R1.

[0051] Preparation of the test solution of Huoxue Zhitong Capsules: Weigh approximately 2.0 g of the contents of Huoxue Zhitong Capsules accurately and place them in a 50 mL Erlenmeyer flask. Add 20 mL of 70% methanol, weigh the solution, sonicate for 30 min, cool, replenish the lost weight with 70% methanol, filter through a 0.45 μm microporous membrane, and collect the filtrate.

[0052] Preparation of the test solution for Panax notoginseng: The raw medicinal materials in the prescription of Huoxue Zhitong Capsules were pulverized and sieved. Approximately 0.9g of Angelica sinensis, 0.176g of Boswellia carterii, 0.044g of Borneol, 0.445g of Eupolyphaga sinensis, and 0.27g of Calcite were accurately weighed, mixed evenly, and placed in a 50mL Erlenmeyer flask. 20mL of 70% methanol was added, the weight was measured, and the mixture was sonicated for 30min. After cooling, the weight was added, and the mixture was filtered through a 0.45μm microporous membrane. The filtrate was collected.

[0053] Preparation of reference solutions: Accurately weigh appropriate amounts of ginsenoside Rg1, ginsenoside Rb1, and notoginsenoside R1 reference standards, and place them separately in 25 mL volumetric flasks. Add methanol to dissolve to the mark, shake well, and you will get stock solutions of ginsenoside Rg1 (1.420 mg / mL), ginsenoside Rb1 (2.020 mg / mL), and notoginsenoside R1 (1.031 mg / mL). Accurately pipette 2 mL each of the stock solutions of notoginsenoside R1 and ginsenoside Rb1, and 5 mL of ginsenoside Rg1 into 20 mL volumetric flasks, add methanol to the mark, and prepare a mixed reference solution containing 103.1 μg of notoginsenoside R1, 355 μg of ginsenoside Rg1, and 202 μg of ginsenoside Rb1 per mL.

[0054] Chromatographic conditions: Agilent 5TC-C18(2) column (250×4.6mm, 5μm); mobile phase: A-acetonitrile, B-water; detection wavelength: 203nm; injection volume: 10μL; flow rate: 1mL / min; column temperature: 30℃. The gradient elution conditions were as follows: 0–12 min, mobile phase A 20%, mobile phase B 80%; 12–40 min, mobile phase A 20%–30%, mobile phase B 80%–70%; 40–55 min, mobile phase A 30%–36%, mobile phase B 70%–64%; 55–60 min, mobile phase A 36%–47%, mobile phase B 64%–53%.

[0055] Take the test solution, and if necessary, the Panax notoginseng test solution, the mixed reference solution, and the blank solution (70% methanol). Inject them into the HPLC chromatograph according to the selected chromatographic conditions. The HPLC chromatograms are shown in the figure. Figure 1 , Figure 1 In the table, A represents the blank solvent, B represents the mixed reference solution, C represents the test solution, and D represents the test solution lacking Panax notoginseng. The results show that the blank solvent and the test solution lacking Panax notoginseng do not interfere with the peaks of the indicator compounds, indicating good method specificity.

[0056] Example 2

[0057] This example describes the determination of the contents of ferulic acid and 11-carbonyl-β-acetylbosuccinic acid.

[0058] Preparation of the test solution: Take about 2g of the contents of Huoxue Zhitong Capsules (190503), accurately weigh it, place it in a 50mL Erlenmeyer flask, add 20mL of 70% methanol, weigh it, sonicate for 30min, cool it, add weight, filter it through a 0.45μm microporous membrane to obtain the test solution.

[0059] Preparation of the test solution for Angelica sinensis: The six raw medicinal materials were pulverized and sieved. 0.176g of Panax notoginseng, 0.176g of frankincense, 0.044g of borneol, 0.445g of Eupolyphaga sinensis, and 0.27g of calcined natural copper were mixed evenly and placed in a 50mL Erlenmeyer flask. 20mL of 70% methanol was added, the weight was determined, and the mixture was sonicated for 30min. After cooling, the weight was added, and the mixture was filtered through a 0.45μm microporous membrane. The filtrate was collected.

[0060] Preparation of the frankincense-deficient test solution: The six raw medicinal materials were pulverized and sieved. Approximately 0.9g of Panax notoginseng, approximately 0.176g of Angelica sinensis, 0.044g of borneol, 0.445g of Eupolyphaga sinensis, and 0.27g of calcined natural copper were mixed evenly and placed in a 50mL Erlenmeyer flask. 20mL of 70% methanol was added, the weight was determined, and the mixture was sonicated for 30min. After cooling, the weight was added, and the mixture was filtered through a 0.45μm microporous membrane. The filtrate was collected.

[0061] Preparation of reference solution: Accurately weigh an appropriate amount of ferulic acid reference standard, place it in a 10 mL volumetric flask, add methanol to dissolve and dilute to the mark, shake well to obtain ferulic acid reference standard stock solution (538 μg / mL); Take 2 mL of ferulic acid reference standard stock solution and place it in a 20 mL volumetric flask, add about 5 mg of 11-carbonyl-β-acetylbosuccinic acid reference standard, accurately weigh it, and prepare a mixed reference solution containing ferulic acid (53.8 μg / mL) and 11-carbonyl-β-acetylbosuccinic acid (254.5 μg / mL).

[0062] Chromatographic conditions: Column: Agilent 5TC-C18(2) (4.6×250mm, 5μm); Mobile phase: A-acetonitrile, B-0.1% phosphoric acid water; Detection wavelength: 0–8min: 316nm, 8–37min: 250nm; Injection volume: 10μL; Flow rate: 1mL / min; Column temperature: 30℃. The gradient elution conditions were: 0–8min, mobile phase A 27%–30%, mobile phase B 73%–70%; 8–15min, mobile phase A 30%–80%, mobile phase B 70%–20%; 15–37min, mobile phase A 80%, mobile phase B 20%.

[0063] Take the test solution, the frankincense-deficient and angelica-deficient test solutions, the mixed reference solution, and the blank solution (70% methanol), and inject them into the HPLC chromatograph according to the selected chromatographic conditions. The HPLC chromatograms are shown below. Figure 2 , Figure 2 In the table, A is the blank solvent, B is the mixed reference solution, C is the test solution, D is the test solution lacking Angelica sinensis, and E is the test solution lacking Boswellia carterii. Figure 2 Peak 1 is ferulic acid, and peak 2 is 11-carbonyl-β-acetylbosuccinic acid. The results indicate that the blank solvent, the solution lacking frankincense, and the solution lacking angelica sinensis do not interfere with the peaks of the index compounds, demonstrating good method specificity.

[0064] Example 3

[0065] This example describes the determination of the content of borneol and isoborneol.

[0066] Preparation of the test solution: Weigh approximately 2.0 g of the contents of the blood-activating and pain-relieving capsules accurately, place them in a 50 mL Erlenmeyer flask, add 20 mL of ethyl acetate, weigh the contents, sonicate for 30 min, cool, replenish the lost weight with ethyl acetate, filter through a 0.45 μm microporous membrane, and collect the filtrate.

[0067] Preparation of the borneol-deficient test solution: After pulverizing and sieving the six raw medicinal materials, accurately weigh approximately 0.9g of Angelica sinensis, approximately 0.176g of frankincense, approximately 0.176g of Panax notoginseng, 0.445g of Eupolyphaga sinensis, and 0.27g of calcined natural copper. Mix them evenly and place them in a 50mL conical flask. Add 20mL of ethyl acetate, weigh the mixture, sonicate for 30min, cool, add the weight, filter through a 0.45μm microporous membrane, and collect the filtrate.

[0068] Preparation of reference solutions: Accurately weigh appropriate amounts of isoborneol and borneol reference standards, place them separately in 10mL volumetric flasks, dissolve and dilute to the mark with ethyl acetate, and shake well to obtain isoborneol (1.517mg / mL) and borneol reference standard stock solutions (2.043mg / mL); pipette 2.5mL of each of isoborneol and borneol reference standard stock solutions, place them in 5mL volumetric flasks, dilute to the mark with ethyl acetate, and prepare a mixed reference solution containing isoborneol (0.7585mg / mL) and borneol (1.0215mg / mL).

[0069] Chromatographic conditions: Column: DB-WAXETR quartz capillary column

[0070] (30.00mm×0.250mm×0.25μm); Detector: FID; Detector temperature: 250℃; Inlet temperature: 220℃; Column temperature: programmed temperature ramp, initial temperature 90℃, ramp to 105℃ at a rate of 3℃ / min, ramp to 220℃ at a rate of 10℃ / min, hold for 5 min; Injection volume: 1μL; Split ratio: 20:1.

[0071] Take the test solution, the test solution lacking borneol, the test solution lacking angelica borneol, the mixed reference solution, and the blank solution, and inject them into the GC chromatograph according to the selected chromatographic conditions. The chromatograms are shown in the figure. Figure 2 , Figure 3 In the table, A is the blank solvent, B is the mixed reference solution, C is the test solution, and D is the test solution without borneol. Figure 3 Peak 1 is isoborneol, and peak 2 is borneol. The results indicate that the blank solvent, the solution lacking frankincense, and the test solution lacking angelica sinensis do not interfere with the peaks of the index compounds, demonstrating good method specificity.

[0072] Example 4

[0073] This embodiment describes a thin-layer chromatography method for identifying ground beetles.

[0074] Preparation of the test solution: Take 2.5g of the powder of this product, which is equivalent to 0.5g of ground beetle medicinal material, add 10ml of methanol, sonicate for 15 minutes, filter, evaporate the filtrate to dryness, add 10ml of water to the residue and transfer it to a separatory funnel, add 20ml of petroleum ether (60℃-90℃) and shake to extract, separate the petroleum ether solution, evaporate to dryness, dissolve the residue in 1ml of methanol to obtain the test solution.

[0075] Preparation of the control medicinal material solution: Take another 0.5g of ground beetle control medicinal material and prepare the control medicinal material solution in the same way.

[0076] Preparation of negative test solution: Weigh the powder of Eupolyphaga sinensis according to the prescription ratio, mix them, and prepare the negative solution according to the test item.

[0077] Developing solvent: toluene-dichloromethane-acetone-formic acid (5:5:0.5:0.1);

[0078] Sample volume: 10 μL; bandwidth: 10 m; spread distance: approximately 8 cm;

[0079] Development: Take the reference herb solution, test solution, and negative solution in sequence, and perform thin-layer chromatography. Spot them separately on the same silica gel G thin-layer plate. Use toluene-dichloromethane-acetone-formic acid (5:5:0.5:0.1) as the developing solvent. Develop, remove, air dry, and examine at 365 nm.

[0080] Results: Spots of the same color appeared at the corresponding positions on the chromatogram of the ground beetle reference material. No interference was observed from the negative control solution, indicating that this assay method has good specificity. See the thin-layer chromatogram below. Figure 4 , Figure 4 1 is the control solution of ground beetle, 2 is the test solution, and 3 is the negative test solution.

[0081] Example 5

[0082] This example demonstrates the thin-layer chromatography identification of acetic acid frankincense.

[0083] Preparation of the test solution: Take 1.13g of the powder of this product, which is equivalent to 0.1g of frankincense, place it in a 50mL stoppered conical flask, add 10mL of methanol, sonicate for 15 minutes, filter, and the filtrate is the test solution.

[0084] Preparation of the reference herb solution: Take another 0.1g of frankincense reference herb and prepare the reference herb solution using the same method.

[0085] Preparation of reference solutions: Accurately weigh appropriate amounts of 11-keto-β-boswellic acid and 11-carbonyl-β-acetylboswellic acid, and add methanol to prepare a 1 mg / ml mixed reference solution 1; accurately weigh appropriate amounts of β-boswellic acid and 3-acetyl-β-boswellic acid, and add methanol to prepare a 1 mg / ml mixed reference solution 2.

[0086] Preparation of negative test solution: Weigh the frankincense powder (lacking vinegar) according to the prescription ratio, mix them, and prepare the negative solution according to the test item.

[0087] Developing solvent: cyclohexane-ethyl acetate-formic acid (12:5:0.5);

[0088] Sample volume: 10 μL; bandwidth: 8 m; spread distance: approximately 9 cm;

[0089] Development: Sequentially apply mixed reference solution 1, reference herb solution, test solution, negative solution, and mixed reference solution 2, and perform thin-layer chromatography, spotting each onto the same silica gel GF plate. 254On a thin-layer plate, cyclohexane-ethyl acetate-formic acid (12:5:0.5) was pre-saturated for 10 minutes, then developed, removed, and dried. The plate was then examined at 254 nm. After spraying with 10% sulfuric acid ethanol solution, the plate was heated at 120 °C until the spots were clearly visible and examined under sunlight.

[0090] Results: Spots of the same color appeared at the corresponding positions on the chromatograms of frankincense reference material, 11-keto-β-boswellic acid, 11-carbonyl-β-acetylboswellic acid, β-boswellic acid, and 3-acetyl-β-boswellic acid. The negative control solution showed no interference, indicating that this method has good specificity. The thin-layer chromatogram is shown below. Figure 5 , Figure 5 1 is mixed reference solution 1, 2 is frankincense reference material solution, 3 is test solution, 4 is negative test solution, and 5 is mixed reference solution 2.

[0091] Example 6

[0092] This embodiment examines the identification of the extraction solvent of the test sample from *Eupolyphaga sinensis*. The solvent used to prepare the test sample solution in Example 4 was replaced with one of the following five: petroleum ether, toluene, ethyl acetate, anhydrous ethanol, and methanol. Thin-layer chromatography was performed as in Example 4, and the results are shown below. Figure 6 , Figure 6 In the table, 1 represents the reference material of *Eupolyphaga sinensis* (extracted with methanol), 2 with petroleum ether, 3 with toluene, 4 with ethyl acetate, 5 with anhydrous ethanol, 6 with methanol, and 7 is a negative result (extracted with methanol). The results show that when methanol was used as the solvent, the spots in the chromatogram of the test sample at the corresponding positions as the reference material were not clear. Other solvents could extract the main spots of *Eupolyphaga sinensis*, but the main spots were lighter in color, requiring further purification of the sample. Meanwhile, when petroleum ether, toluene, and ethyl acetate were used as extraction solvents, there was less interference from other spots in the chromatogram, making them suitable solvents for the enrichment of *Eupolyphaga sinensis*.

[0093] The sample solution was prepared by two different methods: silica gel column chromatography and petroleum ether extraction. Thin-layer chromatography was performed according to Example 4, and the results are shown in [Figure 4]. Figure 7 , Figure 7 Table 1 shows the reference material of *Eupolyphaga sinensis*, 2 shows the toluene layer from column chromatography, 3 shows ethyl acetate 1 layer from column chromatography, 4 shows ethyl acetate 2 layer from column chromatography, 5 shows the reference material of *Eupolyphaga sinensis*, 6 shows the petroleum ether extraction, and 7 shows ethyl acetate 1 layer from column chromatography. Note: For ease of comparison, two thin-layer chromatography plates are placed side-by-side on one graph. Figure 7 Samples 1 and 5, and samples 3 and 7 are actually the same samples located on two thin-layer plates.

[0094] Compared with column chromatography using ethyl acetate extraction, the extracted spots were clearer and the operation was simpler. Therefore, the sample treatment method chosen was ultrasonic extraction with methanol followed by petroleum ether extraction.

[0095] Example 7

[0096] This embodiment is used to select the developing solvent of acetic acid frankincense. The specific operating steps are the same as in Example 4 to examine the developing system and its effect on the thin-layer chromatography system.

[0097] Developing solvent 1: cyclohexane-ethyl acetate-formic acid (12:5:0.5);

[0098] Developing solvent 2: n-hexane-diethyl ether-acetone-acetic acid (7:3.5:2:0.1);

[0099] Developing solvent 3: Petroleum ether (60-90℃)-cyclohexane-ethyl acetate-formic acid (2:6:3:0.2)

[0100] Thin-layer chromatography was performed according to Example 4, and the results were examined. (See attached figure.) Figure 8 , Figure 8 In the middle, (a), (b), and (c) are the thin-layer chromatograms corresponding to developing solvent 1, developing solvent 2, and developing solvent 3, respectively; Figure 8 In the diagram, 1 is mixed reference solution 1, 2 is reference medicinal material solution, 3 is test solution, 4 is negative test solution, and 5 is mixed reference solution 2; in (a), (b), and (c), the left side is a chromatogram at 254 nm, and the right side is a chromatogram under sunlight.

[0101] Depend on Figure 8 It is known that when using developing solvent 1, the spots are clear, the Rf value is moderate, and the separation is good; when using developing solvent 2, the spot separation is poor and the Rf value is low; when using developing solvent 3, the spots are clear, the Rf value is slightly lower than that of developing solvent 1, and other differences are not significant. Developing solvent 1 was selected as the thin-layer development system for the experiment.

[0102] Example 8

[0103] This embodiment was used to examine the durability of identification of ground beetles. Following the identification method of Example 4, the identification effects under different times, temperatures, humidity levels, and thin-layer plates were examined. (See attached figures.) Figures 9-12 Among them, such as Figure 9 The image shows the identification effect of ground beetle after the test solution was left for different times.

[0104] Figure 9 1 represents the control material of ground beetle, 2 represents 0 days, 3 represents 1 day, 4 represents 2 days, and 5 represents 3 days; Figure 10 Images showing the identification effects of different thin-layered lamellae on ground beetles. Figure 10 1 is Merck G silicone board from Germany, 2 is Qingdao Silicon Innovation G silicone board, 3 is Qingdao Marine G silicone board, 4 is Qingdao Marine High Efficiency G, and 5 is Merck Gaoxiao G from Germany. Figure 11 Images showing the identification effects of ground beetles at different unfolding temperatures. Figure 11In the diagram, 1 represents 10℃, 2 represents 20℃, and 3 represents 30℃. Figure 12 Images illustrating the identification effects of ground beetles at different humidity levels. Figure 12 In the diagram, 1 has an RH of 33%, 2 has an RH of 60%, and 3 has an RH of 92.5%.

[0105] illustrate: Figure 10 , Figure 11 and Figure 12 In Figures 1, 2, and 3, the left side represents the control solution of Eupolyphaga sinensis, and the right side represents the test solution.

[0106] Experimental results show that the main spot separation effect is good under all conditions, indicating that the identification method is highly operable, has good repeatability, and is durable.

[0107] Example 9

[0108] This embodiment examines the durability of the vinegar-based frankincense identification method. Following the identification method in Example 5, the identification effects under different times, temperatures, humidity levels, and thin-layer plates were investigated. (See attached image.) Figures 13-16 .

[0109] in, Figure 13 Images showing the effects of different storage times on the identification of vinegar-flavored frankincense. Figure 13 In the diagram, (a) represents 0 days of storage, (b) represents 1 day of storage, (c) represents 2 days of storage, and (d) represents 3 days of storage. Figure 13 1 is mixed reference solution 1, 2 is reference medicinal material solution, 3 is test solution, 4 is negative test solution, and 5 is mixed reference solution 2;

[0110] Figure 14 Images showing the effects of different silicone plates in identifying the presence of vinegar and frankincense. Figure 14 (a) is a GF254 silicone board, (b) is a high-efficiency GF254 silicone board, and (c) is a CoreSilicon Valley high-efficiency GF254 silicone board. Figure 14 1 is mixed reference solution 1, 2 is reference medicinal material solution, 3 is test solution, 4 is negative test solution, and 5 is mixed reference solution 2;

[0111] Figure 15 Images showing the identification effects of vinegar-flavored frankincense at different development temperatures. Figure 15 (a) is 10℃, (b) is 20℃, and (c) is 30℃. Figure 15 1 is mixed reference solution 1, 2 is reference medicinal material solution, 3 is test solution, 4 is negative test solution, and 5 is mixed reference solution 2;

[0112] Figure 16 Images illustrating the effects of different humidity levels on the identification of vinegar and frankincense. Figure 16 (a) has an RH of 33%, (b) has an RH of 60%, and (c) has an RH of 92.5%. Figure 16 1 is mixed reference solution 1, 2 is reference medicinal material solution, 3 is test solution, 4 is negative test solution, and 5 is mixed reference solution 2.

[0113] illustrate: Figures 13-16 The left side of (a), (b), and (c) are chromatograms at 254 nm, and the right side are chromatograms under sunlight.

[0114] Depend on Figures 13-16 The experimental results show that the separation effect of the main spot is good under all conditions, indicating that the identification method is highly operable, has good repeatability, and is durable.

[0115] Example 10

[0116] This embodiment is a validation of the methodology for determining the content of ginsenoside Rg1, ginsenoside Rb1, and notoginsenoside R1.

[0117] Linearity and Range: Approximately 12 mg of ginsenoside Rg1 was accurately weighed and placed in a 20 mL volumetric flask. Separately, 4 mL each of the stock solutions of notoginsenoside R1 (1.031 mg / mL) and ginsenoside Rb1 (2.020 mg / mL) were accurately pipetted into 20 mL volumetric flasks, dissolved, and diluted to the mark. The solutions were then serially diluted to prepare five linear solutions with different concentration gradients. The determination was performed under the predetermined chromatographic conditions. A standard curve was plotted with concentration as the x-axis and peak area as the y-axis, and the regression equation was obtained. The results are shown in Table 1.

[0118] Table 1. Examination of the linear relationship among the three components

[0119] <![CDATA[Notoginsenoside R1]]> Y = 3.0538X + 4.061 0.9997 20.6 μg / mL 206.2 μg / mL <![CDATA[Ginsenoside Rg1]]> Y = 3.4466X + 3.9939 0.9996 60.5 μg / mL 605.0 μg / mL <![CDATA[Ginsenoside Rb1]]> Y = 2.3185X + 1.9134 0.9997 40.4 μg / mL 404.4 μg / mL

[0120] The results showed that the concentration C of notoginsenoside R1, ginsenoside Rg1, and ginsenoside Rb1 exhibited good linear relationships with their peak areas A within the concentration ranges of 20.6–206.2 μg / mL, 60.5–605.0 μg / mL, and 40.4–404.0 μg / mL, respectively. Therefore, the content of notoginsenoside R1, ginsenoside Rg1, and ginsenoside Rb1 could be determined and calculated using the external standard one-point method.

[0121] Injection precision: A mixed reference solution of notoginsenoside R1, ginsenoside Rg1, and ginsenoside Rb1 was injected five times consecutively and measured according to the method. The RSD value of the peak area of ​​each reference was less than 2.0%.

[0122] Repeatability: The contents of Huoxue Zhitong Capsules (190501) were taken and the test solution was prepared according to the method in Example 1. Six test solutions were prepared in parallel and continuously measured. The content of each index component and its RSD value are shown in Table 2. The test results show that the repeatability RSD of each peak is less than 2.0%, which meets the requirements of the content determination method validation.

[0123] Table 2 Results of repeatability tests (n=6)

[0124]

[0125] Intermediate precision: Six test solutions were reconstituted by another analyst on different dates according to the method under the repeatability test, and the results were compared with the repeatability test results. The results of 12 samples are shown in Table 3. The results show that the RSD of the 12 measurements in both groups is less than 2.0%, and the method precision is good.

[0126] Table 3 Results of intermediate precision test

[0127]

[0128]

[0129] Stability: The contents of Huoxue Zhitong Capsules (190503) were used to prepare the test solution according to the method described in Example 3. After being placed at room temperature for 0, 2, 4, 8, 12, and 24 hours, the peak areas of Panax notoginseng saponin R1, ginsenoside Rg1, and ginsenoside Rb1 were recorded. The RSD value of the peak area for each peak was calculated to be less than 2.0%. The results are shown in Table 4. The results indicate that the RSD values ​​of the peak areas of Panax notoginseng saponin R1, ginsenoside Rg1, and ginsenoside Rb1 in the chromatogram of the test solution were all less than 2.0%, indicating that the test solution was stable at room temperature for 24 hours.

[0130] Table 4. Stability test results (n=6)

[0131]

[0132] Accuracy: Approximately 1 g of the contents (content already determined) of Huoxue Zhitong Capsules (190503) was accurately weighed and placed in a 50 mL Erlenmeyer flask. Simultaneously, 0.6 mL, 1.3 mL, and 2 mL of the reference stock solutions of Panax notoginseng saponin R1 (1.031 mg / mL), Ginsenoside Rb1 (2.022 mg / mL), and Ginsenoside Rg1 (1.420 mg / mL) were accurately pipetted into the same Erlenmeyer flask, respectively. 16 mL of 70% methanol was added, the mixture was weighed, and the solution was sonicated for 30 min, cooled, and the weight was added back. Six parallel preparations were performed, and each was injected into an HPLC chromatograph. The chromatograms were recorded, and the recoveries were calculated using the following formula (Table 5). The results showed that the recoveries of Panax notoginseng saponin R1, Ginsenoside Rg1, and Ginsenoside Rb1 were all between 90% and 108%, meeting the requirements for method validation. This method has good accuracy.

[0133] Table 5 Results of the sample recovery test

[0134]

[0135]

[0136] Example 11

[0137] This example is a methodological validation for the determination of ferulic acid and 11-carbonyl-β-acetylacrylic acid content.

[0138] Linearity and Range: A mixed reference solution of ferulic acid (53.8 μg / mL) and 11-carbonyl-β-acetylbosuccinic acid (254.5 μg / mL) was prepared by serially diluting the solution to prepare five linear solutions with different concentration gradients. The determination was performed under the planned chromatographic conditions. A standard curve was plotted with concentration as the x-axis and peak area as the y-axis, and the regression equation was obtained. The results are shown in Table 6.

[0139] Table 6. Examination of the linear relationship between the two components

[0140] ferulic acid Y = 48.651X - 27.557 0.9995 5.38 μg / mL~53.80 μg / mL 11-Carbonyl-β-acetylboronic acid Y = 31.523X - 34.601 0.9994 25.45 μg / mL~254.50 μg / mL

[0141] The results showed that the concentration C and peak area A of ferulic acid and 11-carbonyl-β-acetylbosuccinic acid were linearly related at concentrations of 5.38 μg / mL to 53.8 μg / mL and 25.45 μg / mL to 254.5 μg / mL, respectively. Therefore, the content of ferulic acid and 11-carbonyl-β-acetylbosuccinic acid could be determined and calculated using the external standard one-point method.

[0142] Injection precision: Linear solutions of ferulic acid and 11-carbonyl-β-acetylbosuccinic acid with concentrations of 26.90 μg / mL and 127.25 μg / mL, respectively, were injected five times consecutively and determined according to the method. The RSD values ​​of the peak areas of each reference standard were calculated, and the results were all less than 2.0%.

[0143] Repeatability: The contents of Huoxue Zhitong Capsules (190503) were taken and the test solution was prepared according to the method in Example 2. Six parallel solutions were prepared and continuously measured. The content of each index component and its RSD value are shown in Table 7. The test results show that the RSD value of each peak is less than 2.0%, which meets the requirements for content determination method validation.

[0144] Table 7. Precision test results (n=6)

[0145]

[0146] Intermediate precision: Six test solutions were reconstituted by another analyst on different dates according to the method under the repeatability test, and the results were compared with the repeatability test results. The results of 12 samples are shown in Table 8. The results show that the RSD of the 12 measurements in both groups is less than 2.0%, and the method precision is good.

[0147] Table 8 Results of intermediate precision test

[0148]

[0149] Stability: The contents of Huoxue Zhitong Capsules (190503) were used to prepare the test solution according to the method described in Example 2. After being left at room temperature for 0, 2, 4, 8, 12, and 24 hours, the samples were injected, and the peak areas of ferulic acid and 11-carbonyl-β-acetylboswellic acid were recorded. The RSD value of the peak area for each peak was calculated to be less than 2.0%. The results are shown in Table 9. The results indicate that the RSD values ​​of the peak areas of ferulic acid and 11-carbonyl-β-acetylboswellic acid in the chromatogram of the test solution were all less than 2.0%, indicating that the test solution was stable at room temperature for 24 hours.

[0150] Table 9 Stability Test Results

[0151]

[0152] Accuracy: Take about 1g of the contents of Huoxue Zhitong Capsules (190503) (content already determined), place them in a 50mL Erlenmeyer flask, and weigh accurately. Accurately pipette 2mL of ferulic acid reference stock solution (538μg / mL) into a 20mL volumetric flask and dilute to the mark to obtain ferulic acid reference solution (53.8μg / mL). Take about 10mg of 11-carbonyl-β-acetylbosuccinic acid reference standard, weigh accurately, place it in a 5mL volumetric flask, add methanol to dissolve and dilute to the mark to obtain 11-carbonyl-β-acetylbosuccinic acid reference solution (2.036mg / mL). Take 3mL of ferulic acid and 1mL of 11-carbonyl-β-acetylbosuccinic acid reference solution respectively into the above Erlenmeyer flasks, add 16mL of 70% methanol, weigh, sonicate for 30min, cool, and weigh again. Six parallel samples were prepared and injected into an HPLC system. The samples were analyzed according to the prescribed method, and the chromatograms were recorded. The recoveries were calculated using the following formula (Table 10). The results showed that the recoveries of ferulic acid and 11-carbonyl-β-acetylbosuccinic acid were both between 90% and 108%, meeting the requirements for method validation. This method has good accuracy.

[0153] Table 10 Results of the recovery rate test

[0154]

[0155]

[0156] Example 12 Methodological validation for the determination of borneol and isoborneol content

[0157] Linearity and Range: Stock solutions of isoborneol (1.517 mg / mL) and borneol (2.043 mg / mL) were prepared by serial dilution to obtain five linear solutions with different concentration gradients. The determinations were performed under the planned chromatographic conditions. A standard curve was plotted with concentration as the x-axis and peak area as the y-axis, and the regression equation was obtained. The results are shown in Table 11.

[0158] Table 11 Examining the linear relationship between the two components

[0159] Cynomorium Y = 0.6652X - 10.702 0.9997 151.7 μg / mL~1517 μg / mL Borneol Y = 0.6921X - 15.217 0.9997 204.3 μg / mL~2043 μg / mL

[0160] The results showed that the concentration C and peak area A of isoborneol and borneol had good linear relationships at concentrations of 151.7 μg / mL to 1517 μg / mL and 204.3 μg / mL to 2043 μg / mL, respectively, indicating that the content of isoborneol and borneol could be determined and calculated using the external standard one-point method.

[0161] Injection precision: A mixed reference solution of isoborneol (0.7585 mg / mL) and borneol (1.0215 mg / mL) was injected five times consecutively and determined according to the method. The RSD value of the peak area of ​​each reference standard was calculated. The results were all less than 2.0%.

[0162] Repeatability: The contents of the Huoxue Zhitong capsule (190503) were taken, and the test solution was prepared according to the method described in Example 3. Six parallel solutions were prepared and continuously measured. The content of each indicator component and its RSD value are shown in the table. 12 The experimental results showed that the RSD values ​​of each peak were all less than 2.0%, which met the requirements for the validation of the content determination method.

[0163] Table 12 Results of repeatability tests (n=6)

[0164]

[0165] Intermediate precision: Six test solutions were reconstituted by another analyst on different dates according to the method under the repeatability test, and the results were compared with the repeatability test results. The results of 12 samples are shown in Table 13. The results show that the RSD of the 12 measurements in both groups is less than 2.0%, and the method precision is good.

[0166] Table 13 Results of intermediate precision test

[0167]

[0168] Stability: The contents of the Huoxue Zhitong capsules were used to prepare the test solution according to the method described in Example 3. After being left at room temperature for 0, 2, 4, 8, 12, and 24 hours, the samples were injected, and the peak areas of isoborneol and borneol were recorded. The RSD value of the peak area for each peak was calculated to be less than 2.0%. The results are shown in Table 14. The results indicate that the RSD values ​​of the peak areas of isoborneol and borneol in the chromatogram of the test solution are all less than 2.0%, indicating that the test solution is stable at room temperature for 24 hours.

[0169] Table 14 Stability Test Results

[0170]

[0171] Accuracy: Approximately 1 g of the contents of the Huoxue Zhitong capsule (content already determined) was placed in a 50 mL Erlenmeyer flask and accurately weighed. Approximately 8 mg of isoborneol reference standard and approximately 10 mg of borneol reference standard were accurately weighed and placed in a 50 mL Erlenmeyer flask. 20 mL of ethyl acetate was added, and the mixture was weighed. The mixture was sonicated for 30 min, cooled, and the weight was added back. Six parallel preparations were performed and injected into a GC chromatograph. The chromatograms were recorded, and the recovery rates were calculated according to the following formula (Table 15). The results showed that the recoveries of isoborneol and borneol were both between 92% and 105%, meeting the requirements for method validation. This method has good accuracy.

[0172] Table 15 Results of the recovery test (n=6)

[0173]

[0174] Example 13

[0175] This implementation is an application of quality control for Huoxue Zhitong Capsules. The content determination method established in Examples 4-5 was used to test 10 batches of Huoxue Zhitong Capsules. Batch numbers are shown in Table 16, content determination results are shown in Table 4, and the thin-layer chromatography identification results of *Eupolyphaga sinensis* and *Boswellia carterii* in the 10 batches of Huoxue Zhitong Capsules are shown in Table 4. Figures 17-18 .

[0176] exist Figure 17 In the table, 1 is the reference solution of ground beetle, 2-11 are the test solutions from 10 batches (batch numbers are: 221001, 221002, 221003, 221004, 221005, 221006, 221007, 220707, 220805, 220909 respectively), and 12 is the negative test solution.

[0177] exist Figure 18 In the table, 1 is mixed reference solution 1, 2 is frankincense reference medicinal material solution, 3 to 12 are 10 batches of test sample solutions (batch numbers are: 221001, 221002, 221003, 221004, 221005, 221006, 221007, 220707, 220805, 220909 respectively), 13 is negative test sample solution, and 14 is mixed reference solution 2.

[0178] illustrate: Figure 18 The top image shows the chromatogram at 254nm, and the bottom image shows the chromatogram under sunlight.

[0179] Table 16. Content analysis of 10 batches of Huoxue Zhitong Capsules (mg / capsule)

[0180]

[0181] The identification results of *Eupolyphaga sinensis* in 10 batches of Huoxue Zhitong Capsules showed that the established method for identifying *Eupolyphaga sinensis* in Huoxue Zhitong Capsules can effectively identify *Eupolyphaga sinensis* in the capsules; the identification spots of *Eupolyphaga sinensis* in the 10 batches of Huoxue Zhitong Capsules were clear, negative, and interference-free, and the quality was stable. The identification results of frankincense in 10 batches of Huoxue Zhitong Capsules showed that the established method for identifying frankincense in Huoxue Zhitong Capsules can comprehensively evaluate the quality of frankincense in Huoxue Zhitong Capsules; the identification spots of frankincense in the 10 batches of Huoxue Zhitong Capsules were clear, negative, and interference-free, and the quality was stable. The content determination results of 10 batches of Huoxue Zhitong Capsules showed that the determination method has strong specificity and good durability. The above detection methods can effectively control the quality of Huoxue Zhitong Capsules, providing a reference for ensuring its quality stability, uniformity, and controllability, enabling quality monitoring of the preparation, and ensuring the efficacy of the drug.

[0182] The above description is only a preferred embodiment of the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for detecting a compound preparation for promoting blood circulation and relieving pain, characterized in that, The detection method includes: The contents of ginsenoside Rg1, ginsenoside Rb1, and notoginsenoside R1 in the blood-activating and pain-relieving compound preparation were simultaneously determined by high-performance liquid chromatography (HPLC). The HPLC conditions included: a C18 column; mobile phase A of acetonitrile and mobile phase B of water; detection wavelength of 203 nm; injection volume of 10 μL; flow rate of 1 mL / min; column temperature of 30 °C; and a gradient elution program for the mobile phase as follows: 0 min–12 min, mobile phase A 20%, mobile phase B 80%; 12 min–40 min, mobile phase A 20%→30%, mobile phase B 80%→70%; 40 min–55 min, mobile phase A 30%→36%, mobile phase B 70%→64%; 55 min–60 min, mobile phase A 36%→47%, mobile phase B 64%→53%. The contents of ferulic acid and 11-carbonyl-β-acetylbosuccinic acid in the aforementioned blood-activating and pain-relieving compound preparation were simultaneously determined by high-performance liquid chromatography (HPLC). The HPLC conditions included: a C18 column; mobile phase A of acetonitrile and mobile phase B of 0.1% phosphoric acid aqueous solution; detection wavelength of 316 nm from 0 min to 8 min, and 250 nm from 8 min to 37 min; injection volume of 10 μL; flow rate of 1 mL / min; column temperature of 30℃; and a gradient elution program for the mobile phase: 0 min to 8 min, mobile phase A 27%→30%, mobile phase B 73%→70%; 8 min to 15 min, mobile phase A 30%→80%, mobile phase B 70%→20%; 15 min to 37 min, mobile phase A 80%, mobile phase B 20%. The contents of borneol and isoborneol in the blood-activating and pain-relieving compound preparation were determined by gas chromatography. The gas chromatography conditions included: a quartz capillary column; an FID detector; a detector temperature of 250℃; an injection port temperature of 220℃; a programmed column temperature ramp: initial temperature 90℃, ramped to 105℃ at a rate of 3℃ / min, and ramped to 220℃ at a rate of 10℃ / min, held for 5 min; an injection volume of 1 μL; and a split ratio of 20:

1. Thin-layer chromatography (TLC) was used to qualitatively identify *Eupolyphaga sinensis* and *Boswellia carterii* in the blood-activating and pain-relieving compound preparation. The specific procedures were as follows: The contents of the blood-activating and pain-relieving compound preparation were used as the test sample. Methanol was used as the solvent. The mixture was ultrasonically treated, filtered, and evaporated to dryness to obtain the residue. The residue was extracted with petroleum ether, and the petroleum ether solution was separated, evaporated to dryness, and dissolved in methanol to prepare the test sample solution. *Eupolyphaga sinensis* was used as the reference herb. The preparation steps of the test sample solution were repeated to prepare the reference herb solution. The test sample solution and the reference herb solution were spotted separately onto the same silica gel G TLC plate. The plate was developed with toluene-dichloromethane-acetone-formic acid as the developing solvent. The plate was then removed, dried, and examined under 365 nm ultraviolet light. In the chromatogram of the test sample, a fluorescent main spot of the same color appeared at the corresponding position as in the chromatogram of the reference herb. In the aforementioned compound preparation for promoting blood circulation and relieving pain, the qualitative identification of *Eupolyphaga sinensis* was performed using *Eupolyphaga sinensis* as a reference material; in the aforementioned compound preparation for promoting blood circulation and relieving pain, the qualitative identification of *Boswellia carterii* was performed using *Boswellia carterii* as a reference material, with 11-carbonyl-β-boswellia carterii acid, 11-carbonyl-β-acetylboswellia carterii acid, β-boswellia carterii acid, and 3-acetyl-β-boswellia carterii acid as reference standards.

2. The detection method for a blood-activating and pain-relieving compound preparation according to claim 1, characterized in that, The temperature for petroleum ether extraction is 60℃~90℃.

3. The detection method for a blood-activating and pain-relieving compound preparation according to claim 1, characterized in that, The specific procedures for qualitative identification of the vinegar-flavored frankincense in the aforementioned blood-activating and pain-relieving compound preparation are as follows: The contents of the blood-activating and pain-relieving compound preparation were used as the test sample, and methanol was used as the solvent. After ultrasonic treatment and filtration, the filtrate was the test sample solution. Frankincense was used as a reference herb, and methanol was used as a solvent. The mixture was subjected to ultrasonic treatment and filtration, and the filtrate was the reference herb solution. Prepare mixed reference solution 1 by mixing 11-keto-β-boswellic acid and 11-carbonyl-β-acetylboswellic acid with methanol; prepare mixed reference solution 2 by mixing β-boswellic acid and 3-acetyl-β-boswellic acid with methanol. Take the test solution, reference medicinal material solution, mixed reference solution 1, and mixed reference solution 2, and spot them separately on the same silica gel GF254 thin-layer plate. Develop with the developing solvent, remove and air dry, examine under 254 nm ultraviolet light, spray with sulfuric acid ethanol solution, and then heat until the spots are clearly visible. Examine under sunlight. In the chromatogram of the test sample, spots or fluorescent spots of the same color appear at the corresponding positions as in the chromatograms of the mixed reference standard and the reference medicinal material.

4. The detection method for the blood-activating and pain-relieving compound preparation according to claim 3, characterized in that: The developing solvent used in the qualitative identification process of the blood-activating and pain-relieving compound preparation of frankincense is cyclohexane-ethyl acetate-formic acid.

5. The application of the detection method for the blood-activating and pain-relieving compound preparation as described in any one of claims 1 to 4 in the field of detecting blood-activating and pain-relieving capsules.

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