Detection method and application of compound preparation for promoting blood circulation to arrest pain

Abstract

The invention provides a detection method and application of a blood-activating and pain-relieving compound preparation, and belongs to the field of pharmaceutical analysis. According to the method, the contents of ginsenoside Rg1, ginsenoside Rb1 and notoginsenoside R1 as well as the contents of ferulic acid and 11-carbonyl-beta-acetyl masticinic acid are detected by adopting a high performance liquid chromatography; detecting the contents of borneol and isoborneol by adopting a gas chromatographic method; ground beetles are used as reference medicinal materials, and qualitative identification is performed on the ground beetles by adopting a thin-layer chromatography method; according to the method, frankincense is taken as a reference medicinal material, 11-carbonyl-beta-masticinic acid, 11-carbonyl-beta-acetyl masticinic acid, beta-masticinic acid and 3-acetyl-beta-masticinic acid are taken as reference substances, and the vinegar frankincense is qualitatively identified by adopting a thin-layer chromatography. The method is high in specificity and good in durability, the quality control standard of the blood-activating and pain-relieving capsules is perfected, and the medicine quality is comprehensively evaluated. According to the invention, the quality of the blood-activating and pain-relieving capsule can be well controlled, the stable, uniform and controllable quality of the capsule is ensured, a reference basis is provided, the quality monitoring of the preparation can be realized, and the curative effect of the medicine is ensured.

Description

Technical Field

[0001] The invention belongs to the field of drug analysis, and specifically relates to a detection method and application of a blood-activating and analgesic compound preparation. Background Art

[0002] Huoxue Zhitong Capsule is a prescription composed of Chinese Angelica, Panax notoginseng, vinegar frankincense, borneol, earthworm, and calcined natural copper. It has the effects of promoting blood circulation, dispersing blood stasis, reducing swelling and relieving pain. It is used for traumatic injuries, blood stasis and swelling and pain. It is mainly used in the clinic to treat acute and chronic soft tissue injury diseases. This prescription has been used clinically for many years with good efficacy. It is one of the classic prescriptions for traumatic injuries, blood stasis and swelling and pain. Controlling its comprehensive quality information is very necessary to ensure the quality and drug safety of Huoxue Zhitong Capsule.

[0003] The current standard for Huoxue Zhitong Capsules is included in the 2020 edition of the Chinese Pharmacopoeia. The standard only identifies Angelica sinensis, Panax notoginseng, and Borneol thin layer, but does not identify Rg 1 Reference substance, ginsenoside Rb 1 The detection method for reference substances and notoginseng saponin R1 has a simple quality standard and cannot reflect the characteristics of the overall efficacy of multi-component Chinese medicine, which seriously restricts the market expansion, clinical application and internationalization process of this product. Summary of the invention

[0004] In order to solve the shortcomings of the above methods, the purpose of the present invention is to provide a detection method and application of a blood-activating and analgesic compound preparation.

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

[0006] The first aspect of the present invention provides a method for detecting a blood-activating and analgesic compound preparation, comprising:

[0007] High performance liquid chromatography was used to simultaneously detect ginsenoside Rg in the blood-activating and analgesic compound preparation. 1 , Ginsenoside Rb 1 、Notoginsenoside R 1 The content of ferulic acid and 11-carbonyl-β-acetyl boswellic acid in the compound preparation for promoting blood circulation and relieving pain is simultaneously detected by high performance liquid chromatography;

[0008] The content of borneol and isoborneol in the blood-activating and analgesic compound preparation is detected by gas chromatography;

[0009] The TLC method is used to qualitatively identify the Eupolyphaga sinensis and the vinegar frankincense in the blood-activating and analgesic compound preparation;

[0010] Among them, the qualitative identification of the earthworm in the said blood-activating and analgesic compound preparation uses the earthworm as the reference medicinal material; the qualitative identification of the vinegar frankincense in the said blood-activating and analgesic compound preparation uses frankincense as the reference medicinal material, and 11-carbonyl-β-boswellic acid, 11-carbonyl-β-acetylboswellic acid, β-boswellic acid, and 3-acetyl-β-boswellic acid are used as reference substances.

[0011] Preferably, ginsenoside Rg is detected simultaneously 1 , Ginsenoside Rb 1 、Notoginsenoside R 1 The liquid chromatography conditions include: C18 chromatographic column; mobile phase A is acetonitrile, mobile phase B is water; detection wavelength is 203nm; injection volume is 10μL; flow rate is 1mL / min; column temperature is 30℃; mobile phase gradient elution conditions are: 0min~12min, mobile phase A is 20%, mobile phase B is 80%; 12min~40min, mobile phase A is 20%→30%, mobile phase B is 80%→70%; 40min~55min, mobile phase A is 30%→36%, mobile phase B is 70%→64%; 55min~60min, mobile phase A is 36%→47%, mobile phase B is 64%→53%.

[0012] Preferably, the liquid chromatography conditions for the simultaneous detection of ferulic acid and 11-carbonyl-β-acetylboswellic acid include: a C18 chromatographic 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.; 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 chromatography conditions for detecting borneol and isoborneol include: a quartz capillary chromatographic column; a flame ionization detector (FID); a detector temperature of 250°C; an injection port temperature of 220°C; a programmed temperature rise of the column temperature, with an initial temperature of 90°C, rising to 105°C at a rate of 3°C / min, and rising to 220°C at a rate of 10°C / min, and maintaining for 5 min; injection volume: 1 μL; split ratio: 20:1.

[0014] Preferably, the specific operation of qualitative identification of Eupolyphaga sinensis in the blood-activating and analgesic compound preparation is as follows:

[0015] Preparation of the test solution: the contents of the blood-activating and analgesic compound preparation are used as the test sample, methanol is used as the solvent, ultrasonic treatment, filtration, evaporation to obtain the residue, the residue is extracted with petroleum ether, the petroleum ether liquid is separated, evaporated to dryness, and methanol is added to dissolve to prepare the test solution; wherein the temperature of the petroleum ether extraction is 60°C-90°C;

[0016] Preparation of control medicinal material solution: using Eupolyphaga sinensis as the control medicinal material, repeat the preparation steps of the test solution to prepare a control medicinal material solution;

[0017] The test sample solution and the control medicinal material solution are respectively spotted on the same silica gel G thin layer plate, developed with a developing agent, taken out, dried, and examined under 365nm ultraviolet light; in the test sample chromatogram, a main fluorescent spot of the same color appears at the corresponding position of the control medicinal material chromatogram; the developing agent is toluene-dichloromethane-acetone-formic acid, and the volume ratio is toluene: dichloromethane: acetone: formic acid = 5:5:0.5:0.1.

[0018] Preferably, the specific operation of qualitative identification of vinegar frankincense in the compound preparation for promoting blood circulation and relieving pain is as follows:

[0019] Preparation of test solution: The contents of the compound preparation for promoting blood circulation and relieving pain are used as the test sample, methanol is used as the solvent, and the filtrate is the test solution after ultrasonic treatment and filtration;

[0020] Preparation of reference medicinal material solution: Use frankincense as the reference medicinal material and methanol as the solvent. After ultrasonic treatment and filtration, the filtrate is the test solution.

[0021] Preparation of reference solution: separately take 11-keto-β-boswellic acid and 11-carbonyl-β-acetylboswellic acid, mix them and add methanol to prepare mixed reference solution 1; separately take β-boswellic acid and 3-acetyl-β-boswellic acid, mix them and add methanol to prepare mixed reference solution 2;

[0022] Pipette the test solution, control medicinal material solution, mixed reference solution 1 and mixed reference solution 2, spot them on the same silica gel GF254 thin layer plate respectively, develop with a developing agent, take out and dry, examine under 254nm ultraviolet light, spray with sulfuric acid ethanol solution, and then heat until the spots are clearly colored, and examine under sunlight; in the test sample chromatogram, spots of the same color or fluorescent spots appear at the corresponding positions of the mixed reference substance and control medicinal material chromatograms; the developing agent is cyclohexane-ethyl acetate-formic acid, and the volume ratio is cyclohexane: ethyl acetate: formic acid = 12:5:0.5.

[0023] The second aspect of the present invention provides the application of the above-mentioned detection method of the blood-activating and analgesic compound preparation in the field of blood-activating and analgesic capsule detection.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] 1. The method of the present invention supplements the deficiencies of the original pharmacopoeia standards and realizes comprehensive quantitative and qualitative detection. The present invention adds a thin layer identification method for vinegar frankincense and earthworm, adds a content determination method for ferulic acid, 11-carbonyl-β-acetyl boswellic acid, borneol, and isoborneol, optimizes the content determination method of Panax notoginseng, and shortens the analysis time. The method can comprehensively reflect the characteristics of Huoxue Zhitong Capsule, has good specificity, can realize the quality monitoring of Huoxue Zhitong Capsule preparations, effectively controls the quality of Huoxue Zhitong Capsule, and ensures the efficacy of the drug.

[0026] 2. The present invention has made improvements in thin layer identification. There is no identification of vinegar frankincense and earthworm in the standard of Huoxue Zhitong Capsule in the 2020 edition of the Chinese Pharmacopoeia. There is no thin layer chromatography identification item in the quality standard of acetolactic acid medicinal materials included in the 2020 edition of the Chinese Pharmacopoeia. There is a thin layer chromatography identification item in the quality standard of earthworm medicinal materials. The identification method of earthworm medicinal materials included in the 2020 edition of the Chinese Pharmacopoeia is adopted. The spots in the position corresponding to the control medicinal materials in the chromatogram of the test sample are not clear, which is not suitable for the identification of earthworm in the compound preparation of Huoxue Zhitong Capsule. In view of these shortcomings, the present invention has established a thin layer chromatography identification method of earthworm and vinegar frankincense in Huoxue Zhitong Capsule through a lot of creative labor from the preparation of the test sample solution, the selection of the developing agent, and the elimination of the interference of the negative control, so that the components of the compound preparation can be intuitively and visually separated.

[0027] 3. In terms of content determination, the present invention has newly established a HPLC method for simultaneously determining the contents of ferulic acid and 11-carbonyl-β-acetylboswellic acid in the compound preparation, a GC method for determining the contents of borneol and isoborneol in the compound, and optimized the content determination method of Panax notoginseng, thereby shortening the analysis time.

[0028] 4. The detection method of the compound preparation for promoting blood circulation and relieving pain of the present invention has strong specificity and good durability, improves and supplements the quality control standard of the blood circulation and relieving pain capsule, and evaluates the quality of the drug more comprehensively. The above detection method can well control the quality of the blood circulation and relieving pain capsule, ensure the stability, uniformity and controllability of its quality, provide a reference basis, realize the quality monitoring of the preparation, and ensure the efficacy of the drug. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] The accompanying drawings, which constitute part of the present invention, are used to further understand the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention. In the accompanying drawings:

[0030] Figure 1 Ginsenoside Rg in Example 1 1 , Ginsenoside Rb 1 , Panax notoginsenoside R1 HPLC chromatogram of the content determination;

[0031] Figure 2 The chromatogram is a content determination chromatogram of ferulic acid and 11-carbonyl-β-acetylboswellic acid in Example 2;

[0032] Figure 3 It is the content determination chromatogram of borneol and isoborneol in Example 3;

[0033] Figure 4 The thin layer identification chromatogram of the earthworm in Example 4;

[0034] Figure 5 The thin layer identification chromatogram of vinegar frankincense in Example 5;

[0035] Figure 6 The thin layer identification chromatograms of Eupolyphaga sinensis under different extraction solvents in Example 6;

[0036] Figure 7 The thin layer identification chromatograms of Eupolyphaga sinensis prepared by different methods in Example 6 are shown;

[0037] Figure 8 The thin layer identification chromatograms of vinegar frankincense under different developing agents in Example 7;

[0038] Fig. 9 The chromatograms of thin layer identification of Eupolyphaga sinensis at different times in Example 8 are shown;

[0039] Fig.10 The thin layer identification chromatograms of different thin layer plates of earthworm in Example 8;

[0040] Fig.11 The thin layer identification chromatograms of Eupolyphaga sinensis at different development temperatures in Example 8;

[0041] Fig.12 The chromatograms of thin layer identification of earthworms at different humidity in Example 8 are shown;

[0042] Fig.13 The thin layer identification chromatograms of vinegar frankincense at different times in Example 9;

[0043] Fig.14 The thin layer identification chromatograms of vinegar frankincense on different silica gel plates in Example 9;

[0044] Fig.15 The thin layer identification chromatograms of vinegar frankincense at different development temperatures in Example 9;

[0045] Fig.16 The thin layer identification chromatograms of vinegar frankincense at different humidity in Example 9;

[0046] Fig.17This is a thin layer chromatogram of identification of Eupolyphaga sinensis in 10 batches of Huoxue Zhitong Capsules in Example 13;

[0047] Fig.18 This is a thin layer chromatogram for identification of 10 batches of Huoxue Zhitong Capsules with vinegar and frankincense in Example 13. DETAILED DESCRIPTION

[0048] The specific application examples are only used to further illustrate the claims, but do not constitute further limitations to the present invention. Any technical solutions obtained by making limited modifications to the present invention still fall within the scope of protection of the present invention.

[0049] Example 1

[0050] This example is ginsenoside Rg 1 , Ginsenoside Rb 1 , Panax notoginsenoside R 1 The content of the measurement.

[0051] Preparation of the test solution of Huoxue Zhitong Capsule: Take about 2.0g of the content of Huoxue Zhitong Capsule, weigh it accurately, put it in a 50mL conical flask, add 20mL of 70% methanol, weigh it, treat it with ultrasound for 30min, cool it, make up the lost weight with 70% methanol, filter it with 0.45μm microporous membrane, and take the filtrate to obtain the product.

[0052] Preparation of the test solution of Notoginseng: Grind the original medicinal materials in the prescription of Huoxue Zhitong Capsule and sieve them, take about 0.9g of Angelica sinensis, about 0.176g of frankincense, about 0.044g of borneol, about 0.445g of Eupolyphaga sinensis, and about 0.27g of forged natural copper, accurately weigh, mix evenly, put into a 50mL conical flask, add 20mL of 70% methanol, weigh the weight, ultrasonically treat for 30min, cool, make up the weight, filter through 0.45μm microporous membrane, and take the filtrate to obtain.

[0053] Preparation of reference solution: Take ginsenoside Rg 1 , Ginsenoside Rb 1 , Panax notoginsenoside R 1 An appropriate amount of each reference substance was accurately weighed and placed in a 25 mL volumetric flask. Methanol was added to dissolve to the mark and shaken to obtain ginsenoside Rg. 1 (1.420mg / mL), ginsenoside Rb 1 (2.020 mg / mL) and notoginsenoside R 1 (1.031 mg / mL) reference substance stock solution; accurately aspirate notoginsenoside R 1 and ginsenoside Rb 1 2mL of reference stock solution, ginsenoside Rg 1 5 mL was placed in a 20 mL volumetric flask, and methanol was added to the mark to prepare a solution containing notoginseng saponin R per mL.1 103.1μg, Ginsenoside Rg 1 355μg, Ginsenoside Rb 1 202μg of mixed reference solution.

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

[0055] Take the test solution, the test solution lacking Panax notoginseng, 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 chromatogram is shown in Figure 1 , Figure 1 A is blank solvent, B is mixed reference solution, C is test solution, and D is notoginseng test solution. The results show that blank solvent and notoginseng test solution have no interference at the peak of index compound, and the method has good specificity.

[0056] Example 2

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

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

[0059] Preparation of the test solution lacking Angelica sinensis: Grind the six original medicinal materials and sieve them, take 0.176g of Panax notoginseng, 0.176g of frankincense, 0.044g of borneol, 0.445g of Eupolyphaga sinensis, and 0.27g of forged natural copper, mix them evenly, put them in a 50mL conical flask, add 20mL of 70% methanol, weigh the weight, ultrasonically treat for 30min, cool, make up the weight, filter through a 0.45μm microporous membrane, and take the filtrate to obtain the solution.

[0060] Preparation of the test solution lacking frankincense: grind the six original medicinal materials and sieve them, take about 0.9g of Panax notoginseng, about 0.176g of Angelica sinensis, 0.044g of Borneol, 0.445g of Eupolyphaga sinensis, and 0.27g of forged natural copper, mix them evenly, put them in a 50mL conical flask, add 20mL of 70% methanol, weigh the weight, ultrasonically treat for 30min, cool, make up the weight, filter through a 0.45μm microporous membrane, and take the filtrate to obtain the solution.

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

[0062] Chromatographic conditions: Chromatographic 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°C. Mobile phase gradient elution conditions: 0-8min, mobile phase A is 27%-30%, mobile phase B is 73%-70%; 8-15min, mobile phase A is 30%-80%, mobile phase B is 70%-20%; 15-37min, mobile phase A is 80%, mobile phase B is 20%;

[0063] Take the test solution, the test solution lacking frankincense and angelica, 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 chromatogram is shown in Figure 2 , Figure 2 A is blank solvent, B is mixed reference solution, C is test solution, D is test solution lacking Angelica sinensis, and E is test solution lacking Frankincense; Figure 2 Peak 1 is ferulic acid and peak 2 is 11-carbonyl-β-acetyl boswellic acid. The results show that blank solvent, frankincense-deficient and angelica-deficient test solutions have no interference at the peaks of the index compounds, and the method has good specificity.

[0064] Example 3

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

[0066] Preparation of test solution: About 2.0 g of the content of Huoxue Zhitong Capsule is accurately weighed and placed in a 50 mL conical flask. 20 mL of ethyl acetate is added and weighed. Ultrasonic treatment is performed for 30 min. The solution is cooled and the lost weight is supplemented with ethyl acetate. The solution is filtered through a 0.45 μm microporous membrane and the filtrate is taken to obtain the test solution.

[0067] Preparation of the test solution lacking borneol: grind the six original medicinal materials and sieve them, take about 0.9g of Angelica sinensis, about 0.176g of frankincense, about 0.176g of Panax notoginseng, 0.445g of Eupolyphaga sinensis, and 0.27g of forged natural copper, accurately weigh, mix evenly, put into a 50mL conical flask, add 20mL of ethyl acetate, weigh the weight, ultrasonically treat for 30min, cool, make up the weight, filter through a 0.45μm microporous membrane, and take the filtrate to obtain.

[0068] Preparation of reference solution: Take appropriate amount of isoborneol and borneol reference, accurately weigh them, place them in 10mL volumetric flasks, dissolve them with ethyl acetate and dilute to the scale, shake well to obtain isoborneol (1.517mg / mL) and borneol reference stock solutions (2.043mg / mL); take 2.5mL of isoborneol and borneol reference stock solutions, place them in 5mL volumetric flasks, dilute to the scale with ethyl acetate, and prepare a mixed reference solution containing isoborneol (0.7585mg / mL) and borneol (1.0215mg / mL).

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

[0070] (30.00mm×0.250mm×0.25μm); detector: FID; detector temperature: 250℃; injection port temperature: 220℃; column temperature: programmed temperature, initial temperature 90℃, increase to 105℃ at a rate of 3℃ / min, increase to 220℃ at a rate of 10℃ / min, and maintain for 5min; 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 chromatogram is shown in Figure 2 , Figure 3 A is the blank solvent, B is the mixed reference solution, C is the test solution, and D is the test solution lacking borneol; Figure 3 Peak 1 is isoborneol and peak 2 is borneol. The results show that blank solvent, frankincense-deficient and angelica-deficient test solutions have no interference at the peaks of the index compounds, and the method has good specificity.

[0072] Example 4

[0073] The present embodiment is a thin layer identification method of earthworm.

[0074] Preparation of test solution: Take 2.5g of powder of this product, equivalent to 0.5g of Chinese earthworm medicinal material, add 10ml of methanol, ultrasonically treat 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 liquid, evaporate to dryness, add 1ml of methanol to the residue and dissolve it as the test solution.

[0075] Preparation of control medicinal material solution: Take another 0.5 g of Eupolyphaga sinensis 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 medicinal materials according to the prescription ratio, mix them, and prepare the negative solution according to the test sample item.

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

[0078] Spotting volume: 10μL; bandwidth: 10m; extension distance: about 8cm;

[0079] Development: Pipette the control medicinal material solution, test sample solution and negative solution in turn, and spot them on the same silica gel G thin layer plate according to the thin layer chromatography test. Use toluene-dichloromethane-acetone-formic acid (5:5:0.5:0.1) as the developing agent, develop, take out, dry, and examine at 365nm.

[0080] Results: Spots of the same color appeared at the corresponding position of the chromatogram of the control medicinal material of Eupolyphaga sinensis, and the negative control solution had no interference. This determination method has good specificity. Figure 4 , Figure 4 In the middle, 1 is the control medicinal material solution of Eupolyphaga sinensis, 2 is the test solution, and 3 is the negative test solution.

[0081] Example 5

[0082] The present embodiment is the thin layer identification of vinegar frankincense.

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

[0084] Preparation of control medicinal material solution: Take another 0.1 g of frankincense control medicinal material and prepare the control medicinal material solution in the same way.

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

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

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

[0088] Spotting volume: 10μL; bandwidth 8m; extension distance: about 9cm;

[0089] Development: Pipette mixed reference solution 1, reference medicinal material solution, test solution, negative solution, and mixed reference solution 2 in turn, and spot them on the same silica gel GF 254 On the thin layer plate, pre-saturate with 1-cyclohexane-ethyl acetate-formic acid (12:5:0.5) for 10 minutes, then develop, take out, dry, examine at 254nm, spray with 10% sulfuric acid ethanol test solution, heat at 120℃ until the spots are clearly colored, and examine under sunlight.

[0090] Results: Spots of the same color appeared at the corresponding positions of the chromatograms of frankincense reference materials, 11-keto-β-boswellic acid, 11-carbonyl-β-acetylboswellic acid, β-boswellic acid and 3-acetyl-β-boswellic acid, and the negative control solution had no interference. This determination method has good specificity. See the thin layer chromatogram for details. Figure 5 , Figure 5 Among them, 1 is mixed reference solution 1, 2 is frankincense reference medicinal material solution, 3 is test solution, 4 is negative test solution, and 5 is mixed reference solution 2.

[0091] Example 6

[0092] This example investigates the extraction solvent of the test sample for identification of Eupolyphaga sinensis. The solvent for preparing the test sample solution in Example 4 was replaced with the following five solvents: petroleum ether, toluene, ethyl acetate, anhydrous ethanol, and methanol for extraction. Thin layer chromatography was performed according to Example 4. The results are shown in FIG. Figure 6 , Figure 61 is the reference medicinal material of Eupolyphaga sinensis (methanol extraction), 2 is petroleum ether extraction, 3 is toluene extraction, 4 is ethyl acetate extraction, 5 is anhydrous ethanol extraction, 6 is methanol extraction, and 7 is negative (methanol extraction). The results show that when methanol is used as the solvent for extraction, the spots in the chromatogram of the test sample at the corresponding position of the reference medicinal material are not clear. The main spots of Eupolyphaga sinensis can be extracted by using other solvents, but the colors of the main spots are all lighter, and the samples need to be further purified. At the same time, when the extraction solvents are petroleum ether, toluene and ethyl acetate, the other spots of the chromatogram have less interference, which can be used as solvents for enrichment of Eupolyphaga sinensis.

[0093] The product was subjected to silica gel column chromatography and petroleum ether extraction to prepare the test solution. Thin layer chromatography was performed according to Example 4 and the results were shown in Table 1. Figure 7 , Figure 7 1 is the reference medicinal material of Eupolyphaga sinensis, 2 is the toluene layer of column chromatography, 3 is the ethyl acetate 1 of column chromatography, 4 is the ethyl acetate 2 of column chromatography, 5 is the reference medicinal material of Eupolyphaga sinensis, 6 is the petroleum ether extraction, and 7 is the ethyl acetate 1 of column chromatography. Note: For the convenience of comparison, two thin layer plates are placed on one picture for side-by-side comparison, so Figure 7 1 and 5, 3 and 7 are actually the same sample located on two thin layer plates.

[0094] Compared with the petroleum ether extraction, the extraction spots of the ethyl acetate layer by column chromatography were clearer and the operation was simpler. The treatment method of the test sample was selected as methanol ultrasonic extraction followed by petroleum ether extraction.

[0095] Example 7

[0096] This example is used to select vinegar frankincense developing agent, and the specific operating steps are the same as those in Example 4 to examine the development system and its influence 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-ether-acetone-acetic acid (7:3.5:2:0.1);

[0099] Developing agent 3: petroleum ether (60-90°C)-cyclohexane-ethyl acetate-formic acid (2:6:3:0.2)

[0100] Perform thin layer chromatography according to Example 4 and check the results. Figure 8 , Figure 8 (a), (b), and (c) are the thin layer chromatograms corresponding to developing agent 1, developing agent 2, and developing agent 3, respectively; Figure 81 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; (a), (b), and (c) are chromatograms at 254 nm on the left and daylight chromatograms on the right.

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

[0102] Example 8

[0103] This example is used to investigate the durability of identifying ground beetles. According to the identification method of Example 4, the identification effects of different time, different temperature, different humidity and different thin layer plates were investigated respectively. Figure 9-12 ; Among them, Fig. 9 This is a diagram showing the identification effect of earthworms after the test solution has been placed for different periods of time;

[0104] Fig. 9 1 is the control medicinal material of Eupolyphaga sinensis, 2 is 0 days, 3 is 1 day, 4 is 2 days, and 5 is 3 days; Fig.10 This is the effect diagram of identifying earthworms on different thin layer boards. Fig.10 1 is German Merck silica gel G, 2 is Qingdao Si Chuang silica gel G plate, 3 is Qingdao Ocean silica gel G plate, 4 is Qingdao Ocean high efficiency G, 5 is German Merck gaoxiao G; Fig.11 This is the effect diagram of identifying earthworms at different unfolding temperatures. Fig.11 1 is 10℃, 2 is 20℃, 3 is 30℃; Fig.12 This is the effect diagram of identifying ground beetles at different humidity levels. Fig.12 Among them, 1 is RH33%, 2 is RH60%, and 3 is RH92.5%.

[0105] illustrate: Fig.10 , Fig.11 and Fig.12 In 1, 2, and 3, the left side is the control medicinal material solution of Eupolyphaga sinensis, and the right side is the test sample solution.

[0106] The experimental results showed that the main spot separation effect was good under all conditions, indicating that the identification method was highly operable, repeatable and durable.

[0107] Example 9

[0108] This example is a study on the durability of vinegar frankincense identification. According to the identification method of Example 5, the identification effects of different time, different temperature, different humidity and different thin layer plates were investigated respectively. Figure 13-Figure 16 .

[0109] in, Fig.13 This is a diagram showing the identification effect of vinegar frankincense stored for different periods of time; Fig.13 (a) is placed for 0 days, (b) is placed for 1 day, (c) is placed for 2 days, and (d) is placed for 3 days. Fig.13 In the middle, 1 is mixed reference solution 1, 2 is control medicinal material solution, 3 is test solution, 4 is negative test solution, and 5 is mixed reference solution 2;

[0110] Fig.14 This is the effect diagram of vinegar frankincense identification using different silica gel plates. Fig.14 (a) is GF254 silicone plate, (b) is high-efficiency GF254 silicone plate, and (c) is high-efficiency GF254 silicone plate from Core Silicon Valley. Fig.14 In the middle, 1 is mixed reference solution 1, 2 is control medicinal material solution, 3 is test solution, 4 is negative test solution, and 5 is mixed reference solution 2;

[0111] Fig.15 This is the effect diagram of identifying vinegar frankincense at different development temperatures. Fig.15 (a) is 10°C, (b) is 20°C, and (c) is 30°C. Fig.15 In the middle, 1 is mixed reference solution 1, 2 is control medicinal material solution, 3 is test solution, 4 is negative test solution, and 5 is mixed reference solution 2;

[0112] Fig.16 This is the effect diagram of identifying vinegar frankincense with different humidity. Fig.16 (a) is RH33%, (b) is RH60%, (c) is RH92.5%, Fig.16 Among them, 1 is mixed reference solution 1, 2 is control medicinal material solution, 3 is test solution, 4 is negative test solution, and 5 is mixed reference solution 2.

[0113] illustrate: Figure 13-Figure 16 The left sides of (a), (b), and (c) are all chromatograms at 254 nm, and the right sides are all daylight chromatograms.

[0114] Depend on Figure 13-Figure 16 The experimental results show that the main spot separation effect is good under each condition, indicating that the identification method has strong operability, good repeatability and good durability.

[0115] Example 10

[0116] This example is a verification of the content determination methodology of ginsenoside Rg1, ginsenoside Rb1, and notoginsenoside R1.

[0117] Linearity and range: Take ginsenoside Rg 1About 12 mg was placed in a 20 mL volumetric flask and weighed accurately. Notoginseng saponin R was also accurately drawn. 1 (1.031mg / mL) and ginsenoside Rb 1 4 mL of each reference stock solution (2.020 mg / mL) was placed in a 20 mL volumetric flask at the same time, dissolved and diluted to the scale, shaken, and the solution was diluted step by step to prepare five linear solutions with concentration gradients. The determination was carried out according to the proposed chromatographic conditions, and the standard curve was drawn with concentration as the horizontal axis X and peak area as the vertical axis Y, and the regression equation was obtained. The results are shown in Table 1.

[0118] Table 1 Investigation of the linear relationship between the three components

[0119] Element Regression equation R Linear range <![CDATA[Notoginsenoside R 1 > Y=3.0538X+4.061 0.9997 20.6μg / mL206.2μg / mL <![CDATA[Ginsenoside Rg 1 > Y=3.4466X+3.9939 0.9996 60.5μg / mL605.0μg / mL <![CDATA[Ginsenoside Rb 1 > Y=2.3185X+1.9134 0.9997 40.4μg / mL404.4μg / mL

[0120] The results showed that notoginsenoside R 1 , Ginsenoside Rg 1 , Ginsenoside Rb 1 In the concentration ranges of 20.6-206.2 μg / mL, 60.5-605.0 μg / mL, and 40.4-404.0 μg / mL, the linear relationship between the concentration C and its peak area A was good, and the external standard one-point method could be used to quantify the concentration of notoginseng saponin R 1 , Ginsenoside Rg 1 , Ginsenoside Rb 1 Carry out content determination and calculation.

[0121] Injection precision: Take notoginsenoside R 1 , Ginsenoside Rg 1 , Ginsenoside Rb 1 The mixed reference solution was sampled 5 times continuously and measured according to the method. The RSD value of the peak area of ​​each reference substance was calculated to be less than 2.0%.

[0122] Repeatability: Take the contents of Huoxue Zhitong Capsule (190501), prepare the test solution according to the method under Example 1, and make 6 test solutions in parallel. 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 for content determination methodology verification.

[0123] Table 2 Repeatability test results (n=6)

[0124]

[0125] Intermediate precision: Another analyst re-prepared 6 test sample solutions according to the method under the repeatability test on different dates, injected the samples for measurement, and compared the measurement results with the repeatability test results. The measurement results of a total of 12 samples are shown in Table 3. The results show that the RSD of the 12 measurement results in both groups is less than 2.0%, and the method precision is good.

[0126] Table 3 Intermediate precision test results

[0127]

[0128]

[0129] Stability: Take the contents of Huoxue Zhitong Capsule (190503) and prepare the test solution according to the method in Example 3. After standing at room temperature for 0, 2, 4, 8, 12 and 24 hours, inject the sample respectively and record the value of Panax notoginsenoside R 1 , Ginsenoside Rg 1 , Ginsenoside Rb 1 The chromatographic peak area of ​​each peak was calculated, and the RSD value of the peak area of ​​each peak was less than 2.0%. The results are shown in Table 4. The results show that the chromatographic peak area RSD values ​​of Panax notoginseng saponin R1, ginsenoside Rg1, and ginsenoside Rb1 in the chromatogram of the test sample were all less than 2.0%, indicating that the test sample solution was stable within 24 hours at room temperature.

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

[0131]

[0132] Accuracy: Take about 1g of the contents of Huoxue Zhitong Capsule (190503) (the content has been measured) and place it in a 50mL conical flask, accurately weigh it, and at the same time accurately draw the reference substance reserve solution Panax notoginseng saponin R 1 (1.031mg / mL), ginsenoside Rb 1 (2.022mg / mL), ginsenoside Rg 1 (1.420mg / mL) 0.6mL, 1.3mL, 2mL were placed in the above conical flask, 16mL 70% methanol was added, weighed, ultrasonicated for 30min, cooled, and re-weighted. Six portions were prepared in parallel, injected into HPLC chromatograph, measured according to the law, recorded the chromatogram, and calculated the recovery rate according to the following formula (Table 5). The results showed that notoginsenoside R 1 , Ginsenoside Rg 1 Ginsenoside Rb 1 The recovery rates were between 90% and 108%, which met the requirements of methodological validation and the method had good accuracy.

[0133] Table 5. Sample recovery test results

[0134]

[0135]

[0136] Embodiment 11

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

[0138] Linearity and range: Take a mixed reference solution of ferulic acid (53.8 μg / mL) and 11-carbonyl-β-acetyl boswellic acid (254.5 μg / mL), dilute the solution step by step, and prepare five linear solutions with concentration gradients. According to the proposed chromatographic conditions, the standard curve is drawn with concentration as the horizontal coordinate X and peak area as the vertical coordinate Y, and the regression equation is obtained. The results are shown in Table 6.

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

[0140] Element Regression equation R Linear range Ferulic acid Y=48.651X-27.557 0.9995 5.38 μg / mL~53.80 μg / mL 11-Carbonyl-β-acetylboswellic acid Y=31.523X-34.601 0.9994 25.45 μg / mL~254.50 μg / mL

[0141] The results showed that when the concentrations of ferulic acid and 11-carbonyl-β-acetylboswellic acid were 5.38μg / mL~53.8μg / mL and 25.45μg / mL~254.5μg / mL, respectively, the concentration C had a good linear relationship with the peak area A. The external standard one-point method can be used to determine and calculate the contents of ferulic acid and 11-carbonyl-β-acetylboswellic acid.

[0142] Injection precision: Take linear solutions of ferulic acid and 11-carbonyl-β-acetylboswellic acid with concentrations of 26.90μg / mL and 127.25μg / mL respectively, inject them continuously for 5 times, determine according to the method, calculate the RSD value of the peak area of ​​each reference substance, and the results are all less than 2.0%.

[0143] Repeatability: Take the contents of Huoxue Zhitong Capsule (190503), prepare the test solution according to the method under Example 2, prepare 6 copies in parallel, and measure continuously. 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 methodology verification.

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

[0145]

[0146] Intermediate precision: Another analyst re-prepared 6 test sample solutions according to the method under the repeatability test on different dates, injected the samples for measurement, and compared the measurement results with the repeatability test results. The measurement results of a total of 12 samples are shown in Table 8. The results show that the RSD of the 12 measurement results in both groups was less than 2.0%, and the method precision was good.

[0147] Table 8 Intermediate precision test results

[0148]

[0149] Stability: Take the contents of Huoxue Zhitong Capsule (190503), prepare the test solution according to the method under Example 2, and inject the samples after standing at room temperature for 0, 2, 4, 8, 12 and 24 hours, record the chromatographic peak areas of ferulic acid and 11-carbonyl-β-acetyl boswellic acid, and calculate the peak area RSD value of each peak to be less than 2.0%. The results are shown in Table 9. The results show that the chromatographic peak area RSD values ​​of ferulic acid and 11-carbonyl-β-acetyl boswellic acid in the test chromatogram are less than 2.0%, indicating that the test solution is stable within 24 hours at room temperature.

[0150] Table 9 Stability test results

[0151]

[0152] Accuracy: Take about 1 g of the contents of Huoxue Zhitong Capsule (190503) (the content has been measured), place it in a 50 mL conical flask, accurately weigh it, accurately pipette 2 mL of ferulic acid reference substance stock solution (538 μg / mL) into a 20 mL volumetric flask and dilute to the scale to obtain ferulic acid reference substance solution (53.8 μg / mL), take about 10 mg of 11-carbonyl-β-acetylboswellic acid reference substance, accurately weigh it, place it in a 5 mL volumetric flask, add methanol to dissolve and dilute to the scale to obtain 11-carbonyl-β-acetylboswellic acid reference substance solution (2.036 mg / mL), take 3 mL and 1 mL of ferulic acid and 11-carbonyl-β-acetylboswellic acid reference substance solutions respectively, place them in the above-mentioned conical flask, add 16 mL of 70% methanol, weigh the weight, ultrasonically treat for 30 min, cool, and reweight. Six samples were prepared in parallel, injected into HPLC chromatograph, measured according to the law, recorded chromatograms, and calculated the recovery rate according to the following formula (Table 10). The results showed that the recovery rates of ferulic acid and 11-carbonyl-β-acetyl boswellic acid were between 90% and 108%, which met the requirements of methodological validation and the method had good accuracy.

[0153] Table 10 Sample recovery test results

[0154]

[0155]

[0156] Example 12 Methodological Verification of Determination of Borneol and Isoborneol Content

[0157] Linearity and range: Take isoborneol (1.517 mg / mL) and borneol (2.043 mg / mL) reference stock solutions, dilute the solutions step by step, and prepare five linear solutions with concentration gradients. According to the proposed chromatographic conditions, the standard curve is drawn with concentration as the abscissa X and peak area as the ordinate Y, and the regression equation is obtained. The results are shown in Table 11.

[0158] Table 11 Investigation of the linear relationship between the two components

[0159] Element Regression equation R Linear Range Isoborneol 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 when the concentrations of isoborneol and borneol were 151.7μg / mL~1517μg / mL and 204.3μg / mL~2043μg / mL, respectively, the concentration C had a good linear relationship with its peak area A, and the external standard one-point method could be used to determine and calculate the contents of isoborneol and borneol.

[0161] Injection precision: Take a mixed reference solution of isoborneol (0.7585 mg / mL) and borneol (1.0215 mg / mL), inject it continuously 5 times, determine it according to the law, calculate the RSD value of the peak area of ​​each reference substance, and the results are all less than 2.0%.

[0162] Repeatability: Take the contents of Huoxue Zhitong Capsule (190503), prepare the test solution according to the method under Example 3, prepare 6 copies in parallel, and measure continuously. The content of each index component and its RSD value are shown in Table 12 The test results showed that the RSD values ​​of each peak were less than 2.0%, which met the requirements for content determination methodology verification.

[0163] Table 12 Repeatability test results (n=6)

[0164]

[0165] Intermediate precision: Another analyst re-prepared 6 test sample solutions according to the method under the repeatability test on different dates, injected the samples for measurement, and compared the measurement results with the repeatability test results. The measurement results of a total of 12 samples are shown in Table 13. The results show that the RSD of the 12 measurement results in both groups was less than 2.0%, and the method precision was good.

[0166] Table 13 Intermediate precision test results

[0167]

[0168] Stability: Take the contents of Huoxue Zhitong Capsule, prepare the test solution according to the method under Example 3, and inject the samples after standing at room temperature for 0, 2, 4, 8, 12 and 24 hours, record the chromatographic peak areas of isoborneol and borneol, and calculate the peak area RSD value of each peak to be less than 2.0%. The results are shown in Table 14. The results show that the chromatographic peak area RSD values ​​of isoborneol and borneol in the test chromatogram are less than 2.0%, indicating that the test solution is stable within 24 hours at room temperature.

[0169] Table 14 Stability test results

[0170]

[0171] Accuracy: Take about 1g of the contents of Huoxue Zhitong Capsule (the content has been measured) and place it in a 50mL conical flask, accurately weigh it, take about 8mg of isoborneol reference substance and about 10mg of borneol reference substance, accurately weigh it, place it in a 50mL conical flask, add 20mL of ethyl acetate, weigh it, ultrasonicate it for 30min, let it cool, and make up the weight. Prepare 6 parts in parallel, inject them into GC chromatograph respectively, determine them according to the law, record the chromatogram, and calculate the recovery rate of sample addition according to the following formula (Table 15). The results show that the recovery rates of isoborneol and borneol are between 92% and 105%, which meets the requirements of methodological validation, and the method has good accuracy.

[0172] Table 15 Sample recovery test results (n = 6)

[0173]

[0174] Example 13

[0175] This example is an application of quality control of Huoxue Zhitong Capsules. The content determination method established in Example 4-5 was used to test 10 batches of Huoxue Zhitong Capsules. The batch numbers are shown in Table 16, the content determination results are shown in Table 4, and the thin layer identification results of Eupolyphaga sinensis and Olibanum vulgaris in 10 batches of Huoxue Zhitong Capsules are shown in Table 16. Figure 17-Figure 18 .

[0176] exist Fig.17 In the table, 1 is the control medicinal material solution of Eupolyphaga sinensis, 2-11 are 10 batches of test solution (batch numbers are: 221001, 221002, 221003, 221004, 221005, 221006, 221007, 220707, 220805, 220909), 12-negative test solution;

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

[0178] illustrate: Fig.18 The top is the chromatogram at 254 nm, and the bottom is the daylight chromatogram.

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

[0180]

[0181] The identification results of 10 batches of Huoxue Zhitong Capsules showed that the established identification method of Eupolyphaga in Huoxue Zhitong Capsules can identify Eupolyphaga in Huoxue Zhitong Capsules; the identification spots of Eupolyphaga in 10 batches of Huoxue Zhitong Capsules were clear, the negative had no interference, and the quality was stable; the identification results of frankincense in 10 batches of Huoxue Zhitong Capsules showed that the established identification method of frankincense in Huoxue Zhitong Capsules can evaluate the quality of frankincense in Huoxue Zhitong Capsules more comprehensively; the frankincense identification spots of 10 batches of Huoxue Zhitong Capsules were clear, the negative had no interference, 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 method can well control the quality of Huoxue Zhitong Capsules, provide a reference basis for ensuring its stable, uniform and controllable quality, realize quality monitoring of preparations, and ensure the efficacy of drugs.

[0182] The above descriptions are only preferred examples of the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for detecting a blood-activating and analgesic compound preparation, characterized in that: The detection method comprises: The contents of ginsenoside Rg1, ginsenoside Rb1 and notoginsenoside R1 in the blood-activating and analgesic compound preparation are simultaneously detected by high performance liquid chromatography; High performance liquid chromatography is used to simultaneously detect the contents of ferulic acid and 11-carbonyl-β-acetylboswellic acid in the blood-activating and analgesic compound preparation; The content of borneol and isoborneol in the blood-activating and analgesic compound preparation is detected by gas chromatography; The TLC method is used to qualitatively identify the Eupolyphaga sinensis and the vinegar frankincense in the blood-activating and analgesic compound preparation; Among them, the qualitative identification of the earthworm in the said blood-activating and analgesic compound preparation uses the earthworm as the reference medicinal material; the qualitative identification of the vinegar frankincense in the said blood-activating and analgesic compound preparation uses frankincense as the reference medicinal material, and 11-carbonyl-β-boswellic acid, 11-carbonyl-β-acetylboswellic acid, β-boswellic acid, and 3-acetyl-β-boswellic acid are used as reference substances.

2. The method for detecting a blood-activating and analgesic compound preparation according to claim 1, characterized in that: The liquid chromatography conditions for simultaneous detection of ginsenoside Rg1, ginsenoside Rb1, and notoginsenoside R1 include: C18 column; mobile phase A is acetonitrile, mobile phase B is water; detection wavelength is 203 nm; injection volume is 10 μL; flow rate is 1 mL / min; column temperature is 30°C; The mobile phase gradient elution program was as follows: 0 min to 12 min, mobile phase A was 20%, mobile phase B was 80%; 12 min to 40 min, mobile phase A was 20%→30%, mobile phase B was 80%→70%; 40 min to 55 min, mobile phase A was 30%→36%, mobile phase B was 70%→64%; 55 min to 60 min, mobile phase A was 36%→47%, mobile phase B was 64%→53%.

3. The method for detecting a blood-activating and analgesic compound preparation according to claim 1, characterized in that: The liquid chromatography conditions for simultaneous detection of ferulic acid and 11-carbonyl-β-acetyl boswellic acid include: C18 chromatographic column; mobile phase A is acetonitrile, mobile phase B is 0.1% phosphoric acid water; the detection wavelength is 316nm from 0min to 8min, and the detection wavelength is 250nm from 8min to 37min; the injection volume is 10μL; the flow rate is 1mL / min; the column temperature is 30°C; The mobile phase gradient elution program was as follows: 0 min to 8 min, mobile phase A was 27% → 30%, mobile phase B was 73% → 70%; 8 min to 15 min, mobile phase A was 30% → 80%, mobile phase B was 70% → 20%; 15 min to 37 min, mobile phase A was 80%, mobile phase B was 20%.

4. The method for detecting a blood-activating and analgesic compound preparation according to claim 1, characterized in that: The gas chromatography conditions for detecting borneol and isoborneol include: a quartz capillary column; the detector is FID; the detector temperature is 250°C; the injection port temperature is 220°C; the column temperature is programmed, with an initial temperature of 90°C, rising to 105°C at a rate of 3°C / min, and rising to 220°C at a rate of 10°C / min, and maintaining for 5 minutes; the injection volume is 1 μL; and the split ratio is 20:

1.

5. The method for detecting a blood-activating and analgesic compound preparation according to claim 1, characterized in that: The specific operation of qualitative identification of Eupolyphaga sinensis in the blood-activating and analgesic compound preparation is as follows: The contents of the compound preparation for promoting blood circulation and relieving pain were used as the test sample, and methanol was used as the solvent. The residue was subjected to ultrasonic treatment, filtration, and evaporation to obtain the residue. The residue was extracted with petroleum ether, and the petroleum ether liquid was separated, evaporated to dryness, and then dissolved in methanol to prepare the test sample solution. Using Eupolyphaga sinensis as a control medicinal material, repeat the preparation steps of the test solution to prepare a control medicinal material solution; The test sample solution and the control medicinal material solution are respectively spotted on the same silica gel G thin layer plate, developed with a developing agent, taken out, dried, and examined under 365nm ultraviolet light; in the test sample chromatogram, a main fluorescent spot of the same color appears at the corresponding position in the control medicinal material chromatogram.

6. The method for detecting a blood-activating and analgesic compound preparation according to claim 5, characterized in that: The developing agent used in the qualitative identification process of Eupolyphaga sinensis in the blood-activating and analgesic compound preparation is toluene-dichloromethane-acetone-formic acid.

7. The method for detecting a blood-activating and analgesic compound preparation according to claim 5, characterized in that: The temperature of the petroleum ether extraction is 60°C to 90°C.

8. The method for detecting a blood-activating and analgesic compound preparation according to claim 1, characterized in that: The specific operation of qualitative identification of vinegar frankincense in the compound preparation for promoting blood circulation and relieving pain is as follows: The contents of the compound preparation for promoting blood circulation and relieving pain were used as the test sample, methanol was used as the solvent, and the filtrate was used as the test sample solution after ultrasonic treatment and filtration. Frankincense was used as the control medicinal material, methanol was used as the solvent, and the filtrate was treated with ultrasound and filtered, and the filtrate was the test solution; Separately, 11-keto-β-boswellic acid and 11-carbonyl-β-acetylboswellic acid were mixed and methanol was added to prepare mixed reference solution 1; separately, β-boswellic acid and 3-acetyl-β-boswellic acid were mixed and methanol was added to prepare mixed reference solution 2; Pipette the test sample solution, control medicinal material solution, mixed reference solution 1 and mixed reference solution 2, spot them on the same silica gel GF254 thin layer plate respectively, develop with a developing agent, take out and dry, examine under 254nm ultraviolet light, spray with sulfuric acid ethanol solution, and then heat until the spots are clearly colored, and examine under sunlight; in the test sample chromatogram, spots of the same color or fluorescent spots appear at the corresponding positions of the mixed reference sample and control medicinal material chromatograms.

9. The detection method of the blood-activating and analgesic compound preparation according to claim 8, characterized in that: The developing agent in the qualitative identification process of vinegar frankincense in the blood-activating and analgesic compound preparation is cyclohexane-ethyl acetate-formic acid.

10. Application of the detection method of the blood-activating and analgesic compound preparation according to any one of claims 1 to 9 in the detection field of blood-activating and analgesic capsules.

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