A quality control method for Yuxuebi Capsules and its application

Abstract

The present invention relates to the technical field of quality control, and particularly to a quality control method for Yuxuebi Capsules and its application. The method includes: performing liquid chromatography detection on a Yuxuebi Capsule sample to be detected, comparing the detection result with the fingerprint spectrum of Yuxuebi Capsules, and judging the Yuxuebi Capsule sample to be detected according to the comparison result. Based on a specific fingerprint spectrum of Yuxuebi Capsules, the present invention constructs a quality control method for Yuxuebi Capsules, which can effectively ensure the stability of the overall quality of Yuxuebi Capsules, and has the advantages of simple operation, high precision, good stability, good repeatability, and high accuracy, providing an effective basis for the quality control of Yuxuebi Capsules.

Description

Technical Field

[0001] The present invention relates to the technical field of quality control, and in particular to a quality control method for Yuxuebi Capsules and its application. Background Art

[0002] Yuxuebi Capsules is a traditional Chinese medicine preparation commonly used for promoting blood circulation to remove blood stasis and dredging collaterals to relieve pain. It is usually composed of 11 herbs, namely, frankincense (processed), myrrh (processed), clematis root, safflower, salvia miltiorrhiza, cyathula root, ligusticum wallichii, angelica sinensis, turmeric, nutgrass flatsedge rhizome (processed), and roasted astragalus membranaceus.

[0003] Yuxuebi Capsules is mainly used for treating arthralgia syndrome caused by blood stasis blocking collaterals, with symptoms such as severe pain in muscles and joints, pain at the affected area being tender to pressure, fixed without migration, and there may be hard nodules or ecchymosis. In the prescription, frankincense and myrrh promote blood circulation to remove blood stasis and dredge collaterals to relieve pain, and the two are often used in combination. Safflower and ligusticum wallichii promote blood circulation to remove blood stasis and relieve pain; nutgrass flatsedge rhizome soothes the liver and regulates qi, taking the meaning of "qi moving promotes blood circulation"; angelica sinensis and roasted astragalus membranaceus supplement qi and nourish blood. Clematis root dispels wind and dampness, dredges collaterals to relieve pain; cyathula root promotes blood circulation to dredge menstruation and relieve pain; turmeric breaks blood and promotes qi movement, dredges collaterals to relieve pain. All the herbs are used together to achieve the effects of promoting blood circulation to remove blood stasis and dredging collaterals to relieve pain. Yuxuebi Capsules is a new drug for treating arthralgia.

[0004] Existing clinical studies have shown that Yuxuebi Capsules is a safe and effective drug for treating rheumatoid arthritis (type of blood stasis blocking collaterals), and the curative effect on improving traditional Chinese medicine syndromes is 82.5%. It is widely popular because of its definite clinical curative effect and small toxic and side effects. However, at present, the quality control research on Yuxuebi Capsules is only limited to thin-layer identification and content determination. Summary of the Invention

[0005] In order to solve the problems existing in the prior art, the present invention provides a quality control method for Yuxuebi Capsules and its application. By using the fingerprint spectrum of Yuxuebi Capsules for quality control of Yuxuebi Capsules, the accuracy of the results can be effectively improved.

[0006] In the first aspect, the present invention provides a quality control method for Yuxuebi Capsules, including:

[0007] Performing liquid chromatography detection on the Yuxuebi Capsules sample to be detected, comparing the detection result with the fingerprint spectrum of Yuxuebi Capsules, and judging the Yuxuebi Capsules sample to be detected according to the comparison result;

[0008] The fingerprint spectrum of Yuxuebi Capsules includes the following chromatographic information:

[0009] The chromatographic peak of hydroxysafflor yellow A appeared at the retention time of 15.242-18.629, the chromatographic peak of ferulic acid appeared at the retention time of 16.602-20.292, the chromatographic peak of calycosin glucoside appeared at the retention time of 29.390-35.922, the chromatographic peak of rutin appeared at the retention time of 31.406-38.386, the chromatographic peak of cyasterone appeared at the retention time of 38.141-46.617, and the chromatographic peak of salvianolic acid B appeared at the retention time of 50.229-61.391. The chromatographic peaks of lingxianxin appeared at the retention time of 58.564-71.578, the chromatographic peak of kaempferol appeared at the retention time of 61.141-74.727, the chromatographic peak of curcumin appeared at the retention time of 75.750-92.584, the chromatographic peak of cryptotanshinone appeared at the retention time of 85.190-104.122, the chromatographic peak of tanshinone I appeared at the retention time of 85.928-105.024, and the chromatographic peak of tanshinone IIA appeared at the retention time of 93.682-114.500.

[0010] Furthermore, the fingerprint of the Yuxuebi capsule includes the following chromatographic information:

[0011] The chromatographic peak of hydroxysafflor yellow A appeared at the retention time of 16.935, the chromatographic peak of ferulic acid appeared at the retention time of 18.447, the chromatographic peak of calycosin glucoside appeared at the retention time of 32.656, the chromatographic peak of rutin appeared at the retention time of 34.896, the chromatographic peak of cyperasterone appeared at the retention time of 42.379, the chromatographic peak of salvianolic acid B appeared at the retention time of 55.810, the chromatographic peak of linaloside appeared at the retention time of 65.071, the chromatographic peak of kaempferol appeared at the retention time of 67.934, the chromatographic peak of curcumin appeared at the retention time of 84.167, the chromatographic peak of cryptotanshinone appeared at the retention time of 94.656, the chromatographic peak of tanshinone I appeared at the retention time of 95.476, and the chromatographic peak of tanshinone IIA appeared at the retention time of 104.091.

[0012] Furthermore, the Yuxuebi Capsule sample is a sample extracted from the Yuxuebi Capsule by a reflux extraction method.

[0013] Furthermore, the solvent used in the reflux extraction method is 75-85% methanol solution; and / or the extraction time is 50-70 minutes.

[0014] Furthermore, the conditions for the liquid chromatography detection are as follows:

[0015] Acetonitrile was used as mobile phase A, 0.01-1% phosphoric acid was used as mobile phase B, the flow rate was 0.5-1.5 ml / min, the column temperature was 28-32° C., and the detection wavelength was 200-210 nm.

[0016] Further, the elution process of the liquid chromatography detection is as follows:

[0017] From 0 to 10 minutes, mobile phase A is maintained at 10% and mobile phase B is maintained at 90%;

[0018] From 10 to 40 minutes, mobile phase A ranges from 10% to 22% and mobile phase B ranges from 90% to 78%;

[0019] From the 40th to the 50th minute, mobile phase A is maintained at 22% and mobile phase B is maintained at 78%;

[0020] From the 50th to the 80th minute, mobile phase A ranges from 22% to 50% and mobile phase B ranges from 78% to 50%;

[0021] From the 80th to the 115th minute, mobile phase A ranges from 50% to 75% and mobile phase B ranges from 50% to 25%.

[0022] Further, the chromatographic column used for the liquid chromatography detection is Omega C18 chromatographic column.

[0023] Further, the detection result is the chromatogram of the liquid chromatography detection from the 100th to the 120th minute.

[0024] Further, the determination of the sample of the Yuxuebi Capsule to be detected according to the comparison result is as follows:

[0025] When the similarity between the retention times and chromatographic peaks in the detection result and the fingerprint spectrum of the Yuxuebi Capsule is not less than 90%, it is determined that the sample of the Yuxuebi Capsule to be detected is an effective Yuxuebi Capsule product.

[0026] As a preferred specific implementation manner, the present invention provides a quality control method for Yuxuebi Capsule, including:

[0027] 1. For Yuxuebi Capsule, a sample of Yuxuebi Capsule is obtained by reflux extraction; the extraction solvent is 75-85% methanol solution, and the extraction is carried out for 50-70 minutes;

[0028] 2. Perform liquid chromatography detection on the sample of the Yuxuebi Capsule to be detected, using Omega C18 chromatographic column, with acetonitrile as mobile phase A and 0.01-1% phosphoric acid as mobile phase B, the flow rate is 0.5-1.5 ml / min, the column temperature is 28-^32 °C, and the detection wavelength is 200-210 nm. The elution process is as follows:

[0029] From 0 to 10 minutes, mobile phase A is maintained at 10% and mobile phase B is maintained at 90%;

[0030] From 10 to 40 minutes, mobile phase A ranges from 10% to 22%, and mobile phase B ranges from 90% to 78%.

[0031] From 40 to 50 minutes, mobile phase A remains at 22%, and mobile phase B remains at 78%.

[0032] From 50 to 80 minutes, mobile phase A ranges from 22% to 50%, and mobile phase B ranges from 78% to 50%.

[0033] From 80 to 115 minutes, mobile phase A ranges from 50% to 75%, and mobile phase B ranges from 50% to 25%.

[0034] 3. Use the chromatogram from the 100th to 120th minute as the result and compare it with the fingerprint of Yuxuebi Capsules. When the similarity between the retention times and chromatographic peaks in the test result and the fingerprint of Yuxuebi Capsules is not less than 90%, it is determined that the sample of the Yuxuebi Capsules to be tested is an effective Yuxuebi Capsules product.

[0035] The present invention further provides the application of Yuxuebi Capsules in the preparation of drugs for treating rheumatoid arthritis or heart damage. It may relieve heart damage by inhibiting the expression of NLRP3 inflammasome.

[0036] As a preferred specific embodiment, the present invention provides a preparation method of Yuxuebi Capsules, including:

[0037] (1) Take Cyathulae Radix and half of the amount of Salviae Miltiorrhizae Radix and Astragali Radix Praeparata from the raw materials, and pass through a 100-200 mesh sieve to obtain medicinal powder. [[ID=X]]

[0038] (2) Decoct the raw materials of Frankincense, Myrrh, Carthami Flos, Clematidis Radix, Cyathulae Radix, Curcumae Longae Rhizoma, Angelicae Sinensis Radix, Chuanxiong Rhizoma and half of the amount of Salviae Miltiorrhizae Radix and Astragali Radix Praeparata twice in the form of boiling first and then adding materials. The first time is for 2-3 hours, and the second time is for 1-2 hours. Then combine the medicinal liquids, filter, and let stand for 12-14 hours; take the supernatant and concentrate it (below -0.06 Mpa) to a thick paste with a relative density of 1.25-1.30.

[0039] (3) Mix the thick paste and medicinal powder in steps (1) and (2), and then granulate, dry, size, and sieve.

[0040] The present invention further provides the application of the quality control method in improving the accuracy of quality detection of Yuxuebi Capsules.

[0041] The present invention has the following beneficial effects:

[0042] The present invention established a fingerprint of Yuxuebi Capsule based on high performance liquid chromatography and conducted the detection of the fingerprint to overall control the main components of Yuxuebi Capsule. The characteristic peaks in the obtained fingerprint of Yuxuebi Capsule are rich in information. When in use, the obtained results are compared with the fingerprint of Yuxuebi Capsule, and it can be judged whether the sample can effectively ensure the stability of the overall quality of Yuxuebi Capsule. The relevant research results are conducive to providing an effective basis for the quality control of Yuxuebi Capsule quickly and effectively on the premise of ensuring the efficacy of Yuxuebi Capsule.

[0043] The present invention further studied Yuxuebi Capsule and found that in addition to having a certain relieving effect on rheumatoid arthritis, it may also effectively relieve heart damage by regulating the NLRP3 pathway, which is of great significance for the study of the function of Yuxuebi Capsule. Description of the Drawings

[0044] Figure 1 It is a schematic diagram of the detection results of different detection wavelengths for Yuxuebi Capsule provided in Example 2 of the present invention.

[0045] Figure 2 It is a schematic diagram of the liquid chromatography detection results of different mobile phase systems provided in Example 2 of the present invention.

[0046] Figure 3 It is a schematic diagram of the liquid chromatography detection results of different flow rates provided in Example 2 of the present invention.

[0047] Figure 4 It is a schematic diagram of the liquid chromatography detection results of different column temperatures provided in Example 2 of the present invention.

[0048] Figure 5 It is a schematic diagram of the liquid chromatography detection results of different chromatographic columns provided in Example 2 of the present invention.

[0049] Figure 6 It is a schematic diagram of the liquid chromatography detection results of different chromatographs provided in Example 2 of the present invention.

[0050] Figure 7 It is a schematic diagram of the liquid chromatography detection results of different extraction methods provided in Example 3 of the present invention.

[0051] Figure 8 It is a schematic diagram of the liquid chromatography detection results of different extraction solvents provided in Example 3 of the present invention.

[0052] Figure 9 It is a schematic diagram of the liquid chromatography detection results of different sampling amounts provided in Example 3 of the present invention.

[0053] Figure 10 It is a schematic diagram of the liquid chromatography detection results of different extraction times provided in Example 3 of the present invention.

[0054] Figure 11 Schematic diagram of the stability test results for liquid chromatography detection provided in Example 4 of the present invention.

[0055] Figure 12 Schematic diagram of the precision test results for liquid chromatography detection provided in Example 4 of the present invention.

[0056] Figure 13 Schematic diagram of the repeatability test results for liquid chromatography detection provided in Example 4 of the present invention.

[0057] Figure 14 Schematic diagram of the fingerprint test results for 10 batches of Yuxuebi Capsules provided in Example 4 of the present invention; among them, the upper figure is the blank test sample spectrum, the middle figure is the original spectrum, and the lower figure is the spectrum after matching.

[0058] Figure 15 Schematic diagram of the fingerprint of Yuxuebi Capsules provided in Example 4 of the present invention; among them, 1 is the chromatographic peak of hydroxysafflor yellow A; 2 is the chromatographic peak of ferulic acid; 3 is the chromatographic peak of calycosin-7-O-β-D-glucoside; 4 is the chromatographic peak of rutin; 5 is the chromatographic peak of cyasterone; 6 is the chromatographic peak of salvianolic acid B; 7 is the chromatographic peak of clematoside A; 8 is the chromatographic peak of kaempferide; 9 is the chromatographic peak of curcumin; 10 is the chromatographic peak of cryptotanshinone; 11 is the chromatographic peak of tanshinone I; 12 is the chromatographic peak of tanshinone IIA.

[0059] Figure 16 Schematic diagram of the corresponding results of each chromatographic peak in the fingerprint provided in Example 4 of the present invention and the related medicinal materials.

[0060] Figure 17 Statistical chart of the effect of Yuxuebi Capsules on the ejection fraction of rats provided in Experimental Example 1 of the present invention.

[0061] Figure 18 Photograph of HE staining of the mouse heart provided in Experimental Example 1 of the present invention; among them, the arrow indicates the infiltration of inflammatory cells in the myocardial interstitium.

[0062] Figure 19 Schematic diagram of the changes in the expression levels of NLRP3, Caspase-1, IL-1β, and eleaved-GSDMD in the heart caused by arthritis inhibited by Yuxuebi Capsules provided in Experimental Example 1 of the present invention; among them, A is the normal group, B is the model group, and C-F are the Yuxuebi Capsule groups. Detailed implementation manners

[0063] The following examples are used to illustrate the present invention, but are not used to limit the scope of the present invention.

[0064] Example 1

[0065] This embodiment studies the preparation process of Yuxuebi Capsule, which is as follows:

[0066] 1. Formula:

[0067] Frankincense (processed) 60g, Myrrh (processed) 60g, Carthamus tinctorius 100g, Clematidis 150g, Cyperus rotundus 150g, Cyperus rotundus (processed) 120g, Curcuma longa 100g, Angelica sinensis 100g, Salvia miltiorrhiza 200g, Ligusticum chuanxiong 150g, and roasted Astragalus membranaceus 150g.

[0068] 2. The preparation method is as follows:

[0069] Crush Achyranthes bidentata and half amount of Salvia miltiorrhiza and Astragalus membranaceus, pass through a 100-mesh sieve and set aside; take half amount of Salvia miltiorrhiza, Astragalus membranaceus and the remaining medicinal materials, add water and boil twice (boiling water for 2 hours for the first time and 1.5 hours for the second time), combine the medicinal liquids, filter, and let stand for 12 hours, take the supernatant and concentrate (below -0.06) to a thick paste with a relative density of 1.25-1.30 (50°C) for set aside, mix the thick paste and the medicinal powder, granulate, dry, shape the particles, and sieve to obtain the product.

[0070] 3. This example uses the salvianolic acid B in the formula as a reference indicator and adjusts the preparation method (small-scale process investigation) as follows:

[0071] 3.1 Preparation Specifications of Salvia miltiorrhiza

[0072] Three sizes of Danshen preparation were selected for investigation: thick slices 2-4mm, 0.5-1.5cm, and 2-6cm. The results are shown in the table below:

[0073] Table 1 Effects of Danshen processing specifications on salvianolic acid B

[0074]

[0075] The results showed that different processing specifications of Salvia miltiorrhiza had a greater impact on the extraction rate of salvianolic acid B, but had little effect on the dry paste yield. Therefore, it can be determined that the processing specifications of Salvia miltiorrhiza medicinal materials are 2-4mm.

[0076] 3.2 Extraction method

[0077] Two methods were selected for investigation: adding materials first and then boiling, and boiling and then adding materials. The results are shown in the table below:

[0078] Table 2 Effect of extraction method on salvianolic acid B content

[0079]

[0080] The results showed that the feeding method had no significant effect on the content of salvianolic acid B and the yield of dry paste, so boiling was selected before feeding.

[0081] 3.3 Concentration Temperature and Time

[0082] Two concentration temperatures, 60°C and 80°C, were selected, and samples were taken at 1 h, 3 h, 4 h, 5 h, and 6 h for investigation. The results are shown in the following table:

[0083] Table 3 Effects of Concentration Temperature and Time on Salvianolic Acid B Content

[0084]

[0085] The results showed that within the concentration temperature range of 60°C and 80°C and the concentration time of 6 h, there was no effect on the content of salvianolic acid B. Therefore, 60°C was selected for concentration for 1 h.

[0086] 4. Further pilot-scale process investigations were carried out in this example as follows:

[0087] 4.1 Extraction Conditions

[0088] The effects of different feeding methods (normal feeding and wastewater feeding) and insulation steam pressures (0.02 Mpa and 0.04 Mpa) on the transfer rate of salvianolic acid B were compared:

[0089] Table 4 Effects of Different Feeding Methods and Insulation Steam Pressures on the Transfer Rate of Salvianolic Acid B

[0090]

[0091] The results showed that the transfer rate of salvianolic acid B was higher under boiling water feeding. Therefore, boiling water feeding was selected, and the insulation steam pressure was selected as 0.02 Mpa.

[0092] 4.2 Concentration Conditions

[0093] The effects of different vacuum degrees (-0.04 Mpa, -0.06 Mpa, and -0.08 Mpa) and concentration temperatures (1.5 h, 2.0 h, and 3.0 h) on the transfer rate of salvianolic acid B were compared:

[0094] Table 5 Effects of Different Vacuum Degrees and Concentration Times on the Transfer Rate of Salvianolic Acid B

[0095]

[0096] The results showed that when the vacuum degree was -0.06 Mpa and the concentration temperature was 57.4 - 74.3°C, a relatively high transfer rate of salvianolic acid B was obtained.

[0097] 4.3 Belt Drying Experiment Investigation

[0098] The effects of different belt drying heating temperatures on the transfer rate of salvianolic acid B were compared:

[0099] Table 6 Effects of Different Belt Drying Heating Temperatures on the Transfer Rate of Salvianolic Acid B

[0100]

[0101] The results showed that the transfer rate of salvianolic acid B was higher when the temperature of zone 1 was 120℃ and that of zone 2 was 110℃.

[0102] Example 2

[0103] This embodiment studies the liquid chromatography detection method of Yuxuebi Capsule, which is as follows:

[0104] 1. Chromatographic detection wavelength:

[0105] The present invention detects chromatograms at multiple wavelengths by full wavelength scanning (the results are as follows Figure 1 The results showed that each peak was well reflected at 205 nm, and the baseline was stable, so 205 nm was selected as the detection wavelength of the Yuxuebi Capsule fingerprint spectrum.

[0106] 2. Mobile phase

[0107] The present invention compares three mobile phase systems: methanol-water gradient system, acetonitrile-water gradient system and acetonitrile-0.1% phosphoric acid gradient system. The chromatographic detection results are as follows: Figure 2 As shown, the results showed that the acetonitrile-0.1% phosphoric acid gradient system was the best, with good separation of each chromatographic peak and moderate retention time, so the acetonitrile-0.1% phosphoric acid gradient system was selected as the mobile phase.

[0108] 3. Flow rate

[0109] The present invention compares the effect of flow rate on separation, and the chromatographic detection results are as follows: Figure 3 As shown, the results show that the flow rate is more suitable at 1.0 mL / min.

[0110] 4. Column temperature

[0111] The present invention compares the effect of different column temperatures on separation, and the chromatographic detection results are as follows: Figure 4 As shown in the results, the column temperature of 30℃ is more suitable.

[0112] 5. Chromatographic column

[0113] The present invention compares different chromatographic columns (Agilent HC-C 18 Chromatographic columns, OmegaC 18 Chromatographic columns and thermoscientificBDSH Hypersil TM C 18 Chromatographic column), the chromatographic results are as follows Figure 5 As shown, OmegaC18 The chromatographic column has good separation effect and peak shape, so OmegaC 18 chromatographic column is selected.

[0114] 6. Chromatograph

[0115] The present invention compares two chromatographs, Shimadzu LC-20AT and Waters 2695. The chromatographic detection results are as Figure 6 shown. The results show that the peak emergence of Waters 2695 chromatograph is more appropriate.

[0116] Example 3

[0117] This example studies the extraction conditions of the active ingredients of Yuxuebi Capsule as follows:

[0118] 1. Extraction method

[0119] The present invention compares two extraction methods: ultrasonic method and reflux extraction method. The chromatographic detection results are as Figure 7 shown. Among them, more active ingredients are obtained by reflux extraction method, and the peak area is larger. Therefore, the reflux method is selected for extraction.

[0120] 2. Extraction solvent

[0121] The present invention compares three solvents: water, 50% methanol and 80% methanol solution. The chromatographic detection results are as Figure 8 shown. The most active ingredients are extracted by 80% methanol solution, and the peak area is larger. Therefore, 80% methanol solution is selected as the extraction solvent.

[0122] 3. Sampling amount

[0123] The present invention compares three different sampling amounts (1 g, 3 g, 5 g). The chromatographic detection results are as Figure 9 shown. When the sampling amount is 3 g, the extraction is sufficient and the chromatographic peak is more appropriate. Therefore, the sampling amount of 3 g is selected.

[0124] 4. Extraction time

[0125] The present invention compares three different extraction times (30 min, 60 min, 120 min). The chromatographic detection results are as Figure 10 shown: As the time prolongs, the peak area first increases and then decreases. The results of the extraction time of 60 min show that the peak areas of most of the extracted active ingredients are the largest. Therefore, the reflux time of 60 min is selected.

[0126] Example 4

[0127] Based on the research of Examples 1-3, the present invention adopts the following process for detection:

[0128] Treatment of test sample: Take about 3.0 g of the content of this product, weigh accurately, accurately add 30 ml of 80% methanol solution, weigh, reflux for 1 h, take out and cool, weigh again, make up the lost weight with 80% methanol, shake well, filter, and obtain the solution.

[0129] Chromatographic conditions and system suitability test: Use octadecylsilane chemically bonded silica as the filler; use acetonitrile as mobile phase A and 0.1% phosphoric acid as mobile phase B, and perform gradient elution according to the following table; flow rate is 1.0 ml / min; column temperature is 30 °C; detection wavelength is 205 nm.

[0130] Table 7 Gradient elution process

[0131]

[0132] Determination method: Accurately pipette 10 μl of the test sample solution, inject it into the liquid chromatograph, determine, and record the chromatogram for 115 minutes to obtain.

[0133] Based on the above method, the sample of Yuxuebi Capsule prepared by the optimized steps as in Example 1 of the present invention is used for stability test, precision test and reproducibility test by the above liquid chromatographic detection method, as follows:

[0134] 1. Stability test

[0135] Take the sample of Yuxuebi Capsule, detect the fingerprint chromatogram at 0, 4, 8, 12, and 24 hours respectively. The similarity calculation results are shown in Figure 11 and Table 8 (time window width: 0.20; method for generating reference fingerprint chromatogram: average value). The results show that the similarities of each chromatographic peak meet the requirements of the fingerprint chromatogram.

[0136] Table 8 Stability test results

[0137]

[0138] 2. Precision test

[0139] Take the sample of Yuxuebi Capsule, detect it according to the aforementioned method, inject samples continuously for 5 times, detect the fingerprint chromatogram. The similarity calculation results are shown in Figure 12 and Table 9 (time window width: 0.10; method for generating reference chromatogram: average value). The results show that the similarities of each chromatographic peak meet the requirements of the fingerprint chromatogram.

[0140] Table 9 Precision test results

[0141]

[0142] 3. Reproducibility test

[0143] Take the Yuxuebi capsule sample, prepare 5 samples in parallel, and test them according to the above method. The fingerprint spectrum is tested and the similarity calculation results are shown in Figure 13 and Table 10. The results showed that the relative retention time of each chromatographic peak and the similarity of the chromatographic peaks met the requirements of the fingerprint.

[0144] Table 10 Reproducibility test results

[0145]

[0146]

[0147] 4. Sample determination

[0148] The present invention further conducted fingerprint detection on 10 batches of Yuxuebi capsules (prepared by the optimized method in Example 1), and the results were shown in Table 1. Figure 14 and Table 11. Figure 14 The upper picture is the blank test sample spectrum, the middle picture is the original spectrum, and the lower picture is the matched spectrum.

[0149] Table 11 Fingerprint test results of 10 batches of Yuxuebi capsules

[0150]

[0151] Fingerprint analysis of 10 batches of Yuxuebi Capsules revealed that all 10 batches were relatively similar, with no significant differences. Typical chromatographic peaks that appeared in the fingerprints of each batch were identified as common fingerprint peaks. Twelve peaks were identified, of which peak 6 was the peak for salvianolic acid B, designated "S."

[0152] The results also showed that the similarity between the fingerprints of the ten Yuxuebi capsules and the common pattern was greater than 90% (see Figure 14 ), and generate a control fingerprint ( Figure 15 ), so it is stipulated that the fingerprint similarity of Yuxuebi Capsule should be no less than 90%; Figure 15 Among them, 1. Hydroxysafflor yellow A; 2. Ferulic acid; 3. Calycosin isoflavone glucoside; 4. Rutin; 5. Cyperasterone; 6. Salvianolic acid B; 7. Polygoni multiflorifolia glycosides; 8. Kaempferol; 9. Curcumin; 10. Cryptotanshinone; 11. Tanshinone I; 12. Tanshinone IIA.

[0153] In addition, in order to clarify the source of the material basis of Yuxuebi Capsule, the present invention conducted a comparative study on the fingerprint of Yuxuebi Capsule finished product and the corresponding fingerprints of 11 medicinal materials in the prescription composition. The results showed that the chromatographic peaks of the fingerprint of Yuxuebi Capsule were well reflected in the corresponding medicinal materials. Figure 16 .

[0154] Experimental Example 1

[0155] 1. Materials and Instruments

[0156] 1.1 Materials

[0157] Samples of Yuxuebi Capsules (S1 among 10 batches of Yuxuebi Capsules in Part 4 of Example 4), Bovine Type II Collagen Solution (from Chondrex, USA), Incomplete Freund's Adjuvant (IFA, from Chondrex, USA), IL-1β, TNF-α and NLRP3 detection kits (from R&D), NLRP3, Caspase-1, IL-1β and eleaved-GSDMD rabbit anti-human polyclonal antibodies (from Cell Signaling Technology (USA)), Sodium Carboxymethylcellulose (from Beijing Bio-optotech Co., Ltd.), Chloral Hydrate (from Shanghai Sangon Biotech), Paraformaldehyde Fixative (from Wuhan Boster Biological Technology Co., Ltd. (China)).

[0158] 1.2 Instruments

[0159] High-speed low-temperature centrifuge (from Eppendorf), Nucleic Acid and Protein Detector, Full-wavelength Microplate Reader, -80°C Ultra-low Temperature Refrigerator (from Thermo), Electronic Analytical Balance (from Mettler-Toledo).

[0160] 1.3 Animals

[0161] Healthy male Wistar rats, SPF grade, weighing 210±20 g, provided by Liaoning Changsheng Biotechnology Co., Ltd. They were housed in the specific pathogen-free (SPF) animal house of Liaoning Changsheng Biotechnology Co., Ltd. for at least seven days before the study. During the feeding period, they had free access to food and water, with a 12-hour light time per day.

[0162] 2. Experimental Methods

[0163] 2.1 Establishment of CIA Rat Model

[0164] An appropriate amount of Bovine Type II Collagen Solution was thoroughly mixed and emulsified with an equal volume of Incomplete Freund's Adjuvant, and 0.2 mL of the above emulsion was injected intradermally at the base of the tail of each rat. Seven days after the first immunization, the second injection was carried out according to the previous method, 0.1 mL per rat for booster immunization. Compared with the normal group, when obvious redness, swelling, deformity and other changes appeared in the joints of the rats in the model group, it was considered that the CIA model was successfully prepared.

[0165] 2.2 Grouping and Administration

[0166] SD rats were randomly divided into a normal group, a model group, and a Yuxuebi Capsule group (0.94 g / kg, once a day, equivalent to 1 time the clinical equivalent dose). The number of rats in each group was 10 in the normal group, 10 in the model group, and 10 in each subgroup of the Yuxuebi Capsule group. Since the first immunization, the normal group and the model group were intragastrically administered 0.3% sodium carboxymethylcellulose (CMC-Na) according to body weight, and the administration groups were intragastrically administered the required dose of the drug according to body weight. The administration continued for 30 days.

[0167] 2.3 Evaluation of the incidence of arthritis in rats

[0168] After the first immunization of rats in each group, the disease onset of rats was observed every 3 days, and the incidence was statistically calculated at the same time. Incidence = number of diseased rats in each group / total number of rats in that group × 100%.

[0169] 2.4 Measurement of ejection fraction

[0170] The ejection fraction of rats in each group was measured using echocardiography. By measuring the end-systolic volume and end-diastolic volume, the ejection fraction was calculated.

[0171] 2.5 Hematoxylin-eosin staining (HE staining) of the heart

[0172] The heart was placed in paraformaldehyde for fixation for 1 day, and after fixation, it was sent to Liaoning University of Traditional Chinese Medicine for HE staining.

[0173] 2.6 Enzyme-linked immunosorbent assay (ELISA)

[0174] Precisely weigh 20 mg of heart tissue, cut it into pieces and put it into a 1.5 mL EP tube. Add sterile PBS according to the ratio of 1 g∶10 mL, shake well, and then ultrasonically crush it on ice at 4℃. Centrifuge at 14000 r / min for 20 min, and take the supernatant. Treat the synovial tissue samples according to the ELISA kit instructions, and use a multifunctional microplate reader to detect the contents of TNF-α, IL-1β, and NLRP3 in the samples at a wavelength of 450 nm.

[0175] 2.7 Western blot (WB) detection of the protein expression level in the heart

[0176] Take out and weigh the heart tissues of each group stored in liquid nitrogen, cut the tissues into small pieces with surgical scissors and place them in EP tubes for standby. According to the instructions, add RIPA lysis buffer and phenylmethylsulfonyl fluoride to each EP tube at a ratio of 100:1, mix well and homogenize with a homogenizer for more than 15 times, paying attention to operating at low temperature. Transfer the tissue homogenate to a 1.5 mL pre-cooled centrifuge tube, centrifuge at 10000 revolutions per minute at 4℃ for 5 minutes. Transfer the supernatant to a new EP tube, which is the protein extract, and then perform protein quantification.

[0177] 2.8 Results

[0178] 2.8.1 Effect on the incidence of CIA rats

[0179] The rats in the model group started to develop the disease on the 12th day after the first immunization, and the incidence rate was as high as about 70% and continued to increase, reaching 100% on the 15th day after administration; although the rats in the Yuxuebi Capsule group developed the disease at the same time as the model group, the incidence rate was always significantly lower than that of the model group. After 30 days of administration, the incidence rates of each dose group were about 57%, indicating that Yuxuebi Capsule can delay the development of arthritis in CIA rats. The results are shown in the following table.

[0180] Table 12 Effect of Yuxuebi Capsule on the incidence of arthritis in CIA rats

[0181]

[0182] 2.8.2 Yuxuebi Capsule has a protective effect on the cardiac function of CIA rats

[0183] The results are shown in Figure 17 As shown. Compared with the normal group, the ejection fraction of the model group continued to decline. Compared with the normal group, the ejection fraction of the Yuxuebi Capsule group tended to be that of the normal group rats as the time of rats taking Yuxuebi Capsule increased. Yuxuebi Capsule has a protective effect on the cardiac function of CIA rats.

[0184] 2.8.3 Yuxuebi improves the pathological changes of the cardiac tissue of CIA rats

[0185] Observation Figure 18 It was found by observing the HE staining results of the heart that the myocardial cells of the control group mice were arranged neatly and densely, and the myocardial muscle bundles were in complete form; a large number of inflammatory cells were infiltrated in the myocardial interstitium of the model group mice, and the myocardial cells were arranged disorderly; compared with the model group, the degree of inflammatory cell infiltration in the Yuxuebi Capsule group mice was reduced.

[0186] 2.8.4 Expression of TNF-α, IL-1β and NLRP3 in the cardiac tissue of CIA rats

[0187] Compared with the normal group, the expressions of IL-1β, TNF-α and NLRP3 in the model group were significantly increased (P<0.01); compared with the model group, Yuxuebi Capsule could significantly reduce the expressions of IL-1β and NLRP3 in the cardiac tissue of CIA rats (P<0.01). The results are shown in Table 13.

[0188] Table 13 Comparison of the expressions of IL-1β, TNF-α and NLRP3 in the cardiac tissue of rats in each group ( pg / mL)

[0189]

[0190] Note: Compared with the normal group, *P<0.05, **P<0.01; compared with the model group, #P<0.05, ##P<0.01.

[0191] 2.8.5 Effects of CIA rat heart tissue on the expression of protein molecules in the heart

[0192] Compared with the control group, the expression levels of NLRP3, Caspase-1, IL-1β, and eleaved-GSDMD in the heart tissue of the model group were significantly increased ( Figure 19 Table 14). Compared with the model group, the expression levels of NLRP3, Caspase-1, IL-1β, and eleaved-GSDMD in the mice of the Yuxuebi Capsule administration group were significantly decreased ( Figure 19 Table 14).

[0193] Table 14 Comparison of the expression of related proteins in the heart tissue of rats in each group (times)

[0194]

[0195] Note: Compared with the normal group, **P<0.01; compared with the model group, ##P<0.01.

[0196] 3. Conclusions

[0197] In summary, the present invention proves the Yuxuebi Capsule sample through the overall model of CIA rats, which has a significant inhibitory effect on experimental RA. From the research results, it can be seen that Yuxuebi Capsule can significantly reduce the incidence of CIA in rats and delay the development of the disease; and by inhibiting the infiltration of inflammatory cells in the myocardial interstitium and fibrosis, it can improve heart damage. Yuxuebi Capsule can effectively inhibit the expression of NLRP3 inflammasome, reduce pyroptosis of cardiomyocytes and improve myocardial damage, and its mechanism may be related to the regulation of the NLRP3 pathway.

[0198] Although the present invention has been described in detail with general descriptions and specific embodiments above, based on the present invention, some modifications or improvements can be made, which are obvious to those skilled in the art. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection required by the present invention.

Claims

1. A detection method for Yuxue Bi Capsules, characterized in that, Including: Performing liquid chromatography detection on the sample of Yuxuebi Capsule to be detected, comparing the detection result with the fingerprint of Yuxuebi Capsule, and judging whether the sample of Yuxuebi Capsule to be detected is an effective Yuxuebi Capsule product according to the comparison result; The fingerprint of Yuxuebi Capsule includes the following chromatographic information: The chromatographic peak of hydroxysafflor yellow A appears at the retention time of 15.242 - 18.629, the chromatographic peak of ferulic acid appears at the retention time of 16.602 - 20.292, the chromatographic peak of calycosin-7-O-β-D-glucoside appears at the retention time of 29.390 - 35.922, the chromatographic peak of rutin appears at the retention time of 31.406 - 38.386, the chromatographic peak of cyasterone appears at the retention time of 38.141 - 46.617, the chromatographic peak of salvianolic acid B appears at the retention time of 50.229 - 61.391, the chromatographic peak of clematoside A appears at the retention time of 58.564 - 71.578, the chromatographic peak of kaempferide appears at the retention time of 61.141 - 74.727, the chromatographic peak of curcumin appears at the retention time of 75.750 - 92.584, the chromatographic peak of cryptotanshinone appears at the retention time of 85.190 - 104.122, the chromatographic peak of tanshinone I appears at the retention time of 85.928 - 105.024, and the chromatographic peak of tanshinone IIA appears at the retention time of 93.682 - 114.500; The sample of Yuxuebi Capsule is a sample obtained by extracting Yuxuebi Capsule with 75 - 85% methanol solution; The conditions of the liquid chromatography detection include: Using acetonitrile as mobile phase A and 0.01 - 1% phosphoric acid as mobile phase B, with a detection wavelength of 200 - 210 nm; The elution process of the liquid chromatography detection is as follows: From 0 to ******** minutes, mobile phase A is maintained at 10% and mobile phase B is maintained at 90%; From 10 to 40 minutes, mobile phase A increases from 10% to 22% and mobile phase B decreases from 90% to 78%; At the 40 - 50 minute mark, mobile phase A is maintained at 22% and mobile phase B is maintained at********%; At the 50 - 80 minute mark, mobile phase A increases from 22% to 50% and mobile phase B decreases from 78% to 50%; At the 80 - 115 minute mark, mobile phase A increases from 50% to 75% and mobile phase B decreases from 50% to 25%.

2. The detection method according to claim 1, wherein The fingerprint of Yuxuebi Capsule includes the following chromatographic information: Note: There seems to be a number missing in line 8 and 10 of the original text. I've marked them with ******* for you to fill in. If there is no missing information, please correct me. The chromatographic peak of hydroxysafflor yellow A appears at the retention time of 16.935, the chromatographic peak of ferulic acid appears at the retention time of 18.447, the chromatographic peak of calycosin-7-O-β-D-glucoside appears at the retention time of 32.656, the chromatographic peak of rutin appears at the retention time of 34.896, the chromatographic peak of cyasterone appears at the retention time of 42.379, the chromatographic peak of salvianolic acid B appears at the retention time of 55.810, the chromatographic peak of clematoside A appears at the retention time of 65.071, the chromatographic peak of kaempferide appears at the retention time of 67.934, the chromatographic peak of curcumin appears at the retention time of 84.167, the chromatographic peak of cryptotanshinone appears at the retention time of 94.656, the chromatographic peak of tanshinone I appears at the retention time of 95.476, and the chromatographic peak of tanshinone IIA appears at the retention time of 104.

091.

3. The detection method according to claim 1 or 2, characterized in that, The sample of the Yuxuebi Capsule is a sample obtained by reflux extraction for the Yuxuebi Capsule.

4. The detection method according to claim 3, characterized in that, The extraction time of the reflux extraction method is 50 - 70 min.

5. The detection method according to claim 1, wherein The flow rate of the liquid chromatography detection is 0.5 - 1.5 ml / min, and the column temperature is 28 - 32 °C.

6. The detection method according to claim 1, wherein The detection result is the chromatogram of the liquid chromatography detection from the 100th to the 120th minute.

7. The detection method according to claim 1, wherein The judgment of whether the sample of the Yuxuebi Capsule to be detected is an effective Yuxuebi Capsule product according to the comparison result is as follows: When the similarity between the retention times and chromatographic peaks in the detection result and the fingerprint of the Yuxuebi Capsule is not less than 90%, it is judged that the sample of the Yuxuebi Capsule to be detected is an effective Yuxuebi Capsule product.

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