A traditional Chinese medicine composition for treating primary dysmenorrhea, its Babu plaster and preparation method

Abstract

The present invention belongs to the field of traditional Chinese medicine, and specifically relates to a traditional Chinese medicine composition for treating primary dysmenorrhea, a cabu plaster thereof, and a preparation method. The traditional Chinese medicine composition for treating primary dysmenorrhea comprises vinegar-soaked Corydalis yanhusuo, salvia miltiorrhiza, cinnamon bark, Chuanxiong rhizome, vinegar-soaked Cyperus rotundus, Evodia rutaecarpa, and ginger. By preparing the above-mentioned traditional Chinese medicine composition into a cabu plaster for acupoint application on patients with primary dysmenorrhea, the preparation exhibits excellent quality, controllable quality, good in vitro transdermal absorption, and a particularly significant analgesic effect.

Description

Technical Field

[0001] The present invention belongs to the technical field of traditional Chinese medicine, and particularly relates to a traditional Chinese medicine composition for treating primary dysmenorrhea, a Babu plaster thereof and a preparation method thereof. Background Art

[0002] Primary dysmenorrhea (PD) is the most common gynecological disorder affecting women during menstruation. According to a 2006 systematic epidemiological review by the World Health Organization, the prevalence of dysmenorrhea in menstruating women ranges from 17% to 81%, an underestimated rate because only a minority of women seek treatment. The prevalence of primary dysmenorrhea ranges from 45% to 97% across age and nationality groups. Primary dysmenorrhea falls under the categories of "dysmenorrhea" and "menstrual abdominal pain" in traditional Chinese medicine. In Traditional Chinese Medicine (TCM), the cold-coagulation and blood stasis syndrome is the most common syndrome.

[0003] Current treatments for primary dysmenorrhea involve personal care, improving unhealthy lifestyle habits, and applying uterine warming patches. These only alleviate dysmenorrhea but fail to effectively improve the symptoms. Currently, medications are also available to alleviate pain, including commonly used prostaglandin synthase inhibitors and birth control pills. However, these medications require strict adherence to doctor's orders and are associated with potential side effects, potentially harming patients and even interfering with gastrointestinal function.

[0004] Traditional Chinese Medicine (TCM) addresses the two fundamental pathogenesis of primary dysmenorrhea: "pain due to lack of nourishment" or "pain due to obstruction." Treatments generally focus on warming the meridians and dispelling cold, promoting qi and blood circulation, and removing dampness, removing blood stasis, and alleviating pain. Treatment options include oral Chinese medicine, acupuncture, topical medications, acupoint application, and other internal and external therapies. These treatments offer unique advantages in treating primary dysmenorrhea without affecting a woman's menstrual cycle and promoting ovarian development. While these various traditional Chinese medicine treatments demonstrate significant clinical efficacy, some have limitations. For example, oral Chinese medicine requires decocting before use, and long-term use often lacks palatability, making it difficult for patients to tolerate. Acupuncture is difficult to administer on their own and is unsuitable for patients with needle phobias. Topical medications can be applied to a variety of sites, ranging from rectal administration to widespread, haphazard application. Umbilical therapy has been recorded in many medical books of doctors throughout the ages. It is one of my country's traditional and unique methods of treating diseases. It has the characteristics of reliable efficacy, simple operation, no pain, safety, no adverse reactions, and is easy for patients to accept.

[0005] As a new transdermal drug delivery formulation in Traditional Chinese Medicine (TCM), Babu plasters are a hot topic for development in the domestic and international pharmaceutical industries due to their advantages, including high drug loading capacity, easy peelability, repeatable application, good skin biocompatibility, high transdermal drug absorption efficiency, high bioavailability, and reduced gastrointestinal irritation. However, the preparation of Babu plasters for different TCM products is affected by product quality attributes, process parameters, and molding techniques, resulting in fluctuations in the transdermal absorption of the active ingredients. This results in limited quality controllability and poor therapeutic efficacy. Summary of the Invention

[0006] The purpose of the present invention is to provide a traditional Chinese medicine composition for treating primary dysmenorrhea, its Babu plaster and preparation method. The drug formula is scientifically formulated. By preparing the Babu plaster and optimizing the process, the prepared preparation has excellent quality, controllable quality, good in vitro transdermal absorption performance, and particularly significant analgesic effect, thereby solving the problems in the prior art.

[0007] The present invention provides one of the following technical solutions:

[0008] A traditional Chinese medicine composition for treating primary dysmenorrhea, comprising the following raw materials in parts by weight:

[0009] 30-45 parts of vinegar-cooked Corydalis yanhusuo, 25-35 parts of salvia miltiorrhiza, 15-20 parts of cinnamon bark, 25-35 parts of chuanxiong, 22-26 parts of vinegar-cooked Cyperus rotundus, 8-12 parts of Evodia rutaecarpa, and 16-24 parts of ginger.

[0010] Furthermore, the invention comprises the following raw materials in parts by weight: 40 parts of vinegared Corydalis yanhusuo, 30 parts of salvia miltiorrhiza, 18 parts of cinnamon bark, 30 parts of chuanxiong, 24 parts of vinegared Cyperus rotundus, 10 parts of Evodia rutaecarpa, and 20 parts of ginger.

[0011] The present invention provides the following technical solution:

[0012] A traditional Chinese medicine Babu plaster for treating primary dysmenorrhea is prepared from the above traditional Chinese medicine composition for treating primary dysmenorrhea.

[0013] Furthermore, the above-mentioned traditional Chinese medicine Babu plaster for treating primary dysmenorrhea also includes excipients, which are NP-700, glycerin, aluminum glycolate, PVA, PVP K-90, micropowder silica gel, azone and water.

[0014] Furthermore, the dosage of each auxiliary material is: 4-5 parts of NP-700, 30-35 parts of glycerol, 0.3-0.5 parts of aluminum glycolate, 0.4-0.5 parts of PVA, 2-4 parts of PVP K-90, and 3-5 parts of micropowder silica gel; the mass dosage of azone is 3% of the total amount of the auxiliary materials and the extracted thick paste.

[0015] Furthermore, the amounts of the auxiliary materials are: 4.5-4.6 parts g of NP-700, 32-32.5 parts of glycerol, 0.39-0.42 parts of aluminum glycolate, 0.45 parts of PVA, 3 parts of PVP K-90, and 4 parts of micro powder silica gel.

[0016] The present invention provides the following technical solution three:

[0017] The preparation method of the above-mentioned traditional Chinese medicine Babu plaster for treating primary dysmenorrhea comprises the following steps:

[0018] (1) Extract preparation

[0019] a. Weigh vinegar Corydalis yanhusuo and Danshen, extract with alcohol, filter, combine the three filtrates, recover ethanol under reduced pressure, and concentrate into a thick paste with a relative density of 1.25-1.30 (50°C). The residue is set aside;

[0020] b. Weigh cinnamon bark, Chuanxiong rhizome, vinegar-cured cyperus, evodia rutaecarpa, and ginger, add 8 times the amount of water, and extract the total volatile oil by steam distillation. Separate the total volatile oil, the aqueous extract, and the medicinal residue for later use;

[0021] C, step b is extracted volatile oil after the medicinal residues and step a alcohol extraction after the medicinal residues are merged, add 8 times of water, decocted 1.5 hours, filtered, the gained medicinal residues added 6 times of water again, decocted 1.5 hours, merged 2 decoctions and the aqueous extract after extracting total volatile oil, concentrated under reduced pressure into the medicinal liquid with a relative density of 1.06~1.10 (50 ℃), slowly added ethanol under sufficient stirring, making the medicinal liquid contain alcohol amount is 60%, continued stirring 30min, left standstill 24 hours at 0~4 ℃, the supernatant was filtered, and the decompression recovery ethanol was also concentrated into the thick paste with a relative density of 1.25~1.30 (50 ℃);

[0022] d. Combine the thick pastes of step a and step c, heat slightly, and stir thoroughly to obtain a thick paste, and set aside;

[0023] (2) Preparation of Babu Plaster

[0024] a. Place NP-700 in glycerol, add aluminum glycolate, total volatile oil and azone, and stir evenly to prepare phase A. Dissolve PVA in water at 75°C, add PVP K-90, stir evenly, and ultrasonicate (250W, 33kHz) for 15 minutes. Add micropowdered silica gel and stir evenly to prepare phase B.

[0025] b. Add phase A to phase B, stirring slowly in a 60°C water bath until the mixture is evenly mixed. This will serve as the base of the babu paste.

[0026] c. Add the thick paste extracted in step (1), continue stirring in a 60°C water bath until uniform, and ultrasonically treat for 20 minutes;

[0027] d. Apply it on a non-woven fabric backing while it is still hot, place it in an oven at 40°C to dry for 12 hours, take it out, cover it with a protective film, and seal it for storage.

[0028] Furthermore, the alcohol extraction operation in step (1) a is as follows: adding 8 times the amount of 60% ethanol of vinegar Corydalis and Salvia miltiorrhiza, reflux extraction for 2 hours, filtering, adding 6 times the amount of 60% ethanol to the residue, reflux extraction twice, each time for 1.5 hours; b in step (1) is steam distilled and extracted for 5 hours.

[0029] Furthermore, in step (2), the amount of water added in a is 3 times the mass of the auxiliary material in phase B; in step (2), the weight ratio of the thick paste c to the matrix is ​​1:5; in step (2), the coating temperature of d is 45° C. and the thickness is 2.0 mm.

[0030] Furthermore, the above-mentioned phase A is made of NP-700, glycerol, aluminum glycolate, total volatile oil and azone; item B is made of PVA, water, PVP K-90 and micro-powder silica gel, and the amount of water added in step (2) is 3 times the mass of the auxiliary material component of item B.

[0031] Beneficial effects of the present invention:

[0032] 1. The present invention's formula is derived from a modified version of Shaofu Zhuyu Decoction, a book published in the Qing Dynasty's "Medical Corrections." It consists of seven Chinese herbs, including vinegar-soaked Corydalis, Chuanxiong, Danshen, and cinnamon bark. This formula is formulated as a topical plaster applied to the Shenque acupoint, directly reaching the lesion through the acupoint-meridian-uterine network. Studies and clinical applications in rats with a cold-coagulation and blood-stasis PD model have demonstrated that this formula warms the meridians and dispels cold, resolves blood stasis and dredges the meridians, and promotes blood circulation and relieves pain. The analgesic effect is particularly significant, and patient compliance is good. The formula is also convenient to carry and use.

[0033] 2. The Chinese medicine composition of the present invention is prepared into a Babu plaster, and the process parameters are optimized to make the drug easy to penetrate and absorb, and can directly reach the Chong and Ren meridians to regulate the disharmony of qi and blood in the two meridians. The aforementioned prescription drugs with the functions of warming the meridians, removing blood stasis, promoting blood circulation and relieving pain are selected to treat primary dysmenorrhea, and have achieved good clinical efficacy.

[0034] 3. In the Babu plaster matrix formula of the present invention, NP-700 is a skeleton material, aluminum glycolate is a cross-linking agent, and the two are cross-linked to form a network three-dimensional structure, PVP K90 is an adhesive, and PVA is a viscosity enhancer, so that the paste produces sufficient cohesion, high elasticity and a certain strength; micropowder silica gel is a filler, and glycerin is a moisturizer, which can adjust its formability and spreadability and increase the strength of the paste. At the same time, the micropowder silica gel structure is loose and porous, and can adsorb oily substances such as volatile oils in the extract, so that it is evenly mixed with the extract of the Chinese medicine composition, which can improve the uniformity and efficacy of the preparation. The selection of excipients and their dosage better improves the molding of the Chinese medicine Babu plaster of the present invention, and has a favorable effect on the appearance properties of the Babu plaster uniformity, spreadability, film residue, skin followability, and excipient properties, as well as key quality attributes such as adhesion and peel strength. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] The drawings described herein are used to provide a further understanding of the present invention and constitute a part of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0036] Figure 1 UHPLC charts of the reference substance and the transdermal receiving solution of the present invention;

[0037] Figure 2 The cumulative permeation amount-time curves of each active ingredient of Example 1 and Comparative Examples 3 and 4 of the present invention (n=3);

[0038] Figure 3 To test the effects of drugs on the four coagulation functions of PD rats with cold coagulation and blood stasis type;

[0039] Figure 4 To investigate the effect of experimental drugs on plasma PGF in PD rats with cold coagulation and blood stasis 2α , the influence of PGE2 levels;

[0040] Figure 5 To correspond Figure 2 Color view of .

[0041] in, Figure 1 Figure a is the UHPLC graph of the reference solution. Figure 1 b is a UHPLC graph of the scalp test solution of the test sample of Example 1 of the present invention, in which 1-5 are tetrahydropalmatine, ferulic acid, cinnamaldehyde, salvianolic acid A, and salvianolic acid B, respectively;

[0042] Figure 2 AE are the cumulative permeation amount-time curves of tetrahydropalmatine, ferulic acid, cinnamaldehyde, salvianolic acid A and salvianolic acid B respectively; in the figure, no azone label corresponds to Example 3, 3% azone label corresponds to Example 1, and 5% azone label corresponds to Example 4). DETAILED DESCRIPTION

[0043] To clearly illustrate the technical features of this solution, the present invention is described in detail below through specific embodiments with reference to the accompanying drawings. The scope of the present invention is not limited to the following embodiments. Those skilled in the art will appreciate that various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention.

[0044] The following instruments were used in the following examples: Ultimate 3000 high performance liquid chromatograph, DAD detector, Chromeleon 7 workstation (Thermo Scientific, USA), Thermo Scientific Syncronis C18 chromatographic column, SB-5200DTS ultrasonic cleaning machine (Ningbo Xinzhi Biotechnology Co., Ltd.), AE224C electronic balance (Shanghai Sunny Hengping Scientific Instrument Co., Ltd.), MS105 electronic balance (Mettler, Switzerland). Toledo Co., Ltd.), CZY-G initial adhesion tester, CZY-6S holding adhesion tester, BLD-200N electronic peel tester (Jinan Languang Electromechanical Technology Co., Ltd.), T-3000 coating machine (Beijing Tongde Chuangye Technology Co., Ltd.), LHP-250 constant temperature and humidity incubator (Shanghai Sanfa Scientific Instrument Co., Ltd.), HH-4 digital display constant temperature water bath (Changzhou Boyuan Experimental Analytical Instrument Factory); YBP6 intelligent transdermal tester (Tianjin Pharmacopoeia Standard Instrument Factory), N-2110 rotary evaporator (Tokyo Rika Instrument Co., Ltd.), MTN-2800D nitrogen blowdown apparatus (Tianjin Aotesense Instrument Co., Ltd.).

[0045] The reagents involved in the following examples: NP-700 (sodium polyacrylate-700) (ISPTECHNOLOGIESINC, batch number 470370); aluminum glycolate (Shanghai McLean Biochemical Technology Co., Ltd.); PVPK90 (Shanghai Dexiang Pharmaceutical Technology Co., Ltd., batch number: 03000260334); azone (Hubei Kejie Pharmaceutical Co., Ltd., batch number 220201); glycerol (Jiangxi Yipsheng Pharmaceutical Co., Ltd., batch number 20220401); polyvinyl alcohol (PVA, Jiangxi Alpha High-tech Pharmaceutical Co., Ltd., batch number: 20211002); micropowder silica gel (Qingdao Heyu Technology Co., Ltd., batch number: 220810).

[0046] Salvia miltiorrhiza (batch number 220203), Ligusticum chuanxiong (batch number 211201), Evodia rutaecarpa (batch number 201101), and cinnamon bark (batch number 200601) were purchased from Shandong Baiweitang Chinese Medicine Pieces Co., Ltd., and vinegar-cured Corydalis yanhusuo (batch number 20200401) and vinegar-cured Cyperus rotundus (batch number 2220401) were purchased from the Chinese Medicine Pieces Factory of Shandong Jianlian Shengjia Chinese Medicine Co., Ltd. They were identified as authentic by Researcher Jin Guangqian from the Institute of Chinese Medicinal Resources, Shandong Academy of Traditional Chinese Medicine.

[0047] Tetrahydropalmatine (purity: 99.3%, batch number: 110726-202020), cinnamaldehyde (batch number: 110710-202223), ferulic acid (purity: 99.4%, batch number: 110773-201915), and salvianolic acid B (purity: 96.6%, 111562-201917) were purchased from the China Food and Drug Inspection Institute, and salvianolic acid A (purity: 99.1%, batch number: MUST-18082810) was purchased from Chengdu Munster Biotechnology Co., Ltd.

[0048] Methanol (Shanghai Xingke High Purity Solvent Co., Ltd., chromatographic grade), distilled water (Guangzhou Watsons Food and Beverage Co., Ltd.), and other reagents were of analytical grade.

[0049] The experimental animals used in the following examples were SPF-grade healthy male Sprague-Dawley rats, weighing (200 ± 20) g, purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd., license number: SCXK(Lu)20190003. The animal experiments were approved by the Animal Experiment Management Committee of Shandong Academy of Traditional Chinese Medicine, approval number: SYXK(Lu)20230004.

[0050] Unless otherwise specified, the experimental methods and detection methods involved in the following embodiments are all conventional experimental methods and detection methods in the prior art.

[0051] Example 1

[0052] A traditional Chinese medicine Babu plaster for treating primary dysmenorrhea, the effective raw material ingredients and dosages of the traditional Chinese medicine used are as follows: 40 parts of vinegar-soaked Corydalis yanhusuo, 30 parts of salvia miltiorrhiza, 18 parts of cinnamon bark, 30 parts of Chuanxiong rhizome, 24 parts of vinegar-soaked Cyperus rotundus, 10 parts of Evodia rutaecarpa, and 20 parts of ginger.

[0053] The preparation method of the above-mentioned Chinese medicine Babu plaster for treating primary dysmenorrhea is as follows:

[0054] (1) Extract preparation

[0055] Weigh 40g of vinegared Corydalis yanhusuo and 30g of salvia miltiorrhiza, add 8 times the amount of 60% ethanol, reflux extract for 2 hours, filter, add 6 times the amount of 60% ethanol to the residue, reflux extract twice, each time for 1.5 hours, filter, combine the three filtrates, recover ethanol under reduced pressure, and concentrate into a thick paste with a relative density of 1.25-1.30 (50°C). The residue is set aside;

[0056] Weigh 18g of cinnamon bark, 30g of Chuanxiong, 24g of vinegar-cured cyperus, 10g of Evodia rutaecarpa, and 20g of ginger, add 8 times the amount of water, and extract the total volatile oil by steam distillation for 5h. Separate the total volatile oil, the aqueous extract, and the medicinal residue for later use;

[0057] Combine the residue after extracting the volatile oil with the residue after alcohol extraction, add 8 times the amount of water, decoct for 1.5 hours, filter, add 6 times the amount of water to the residue, decoct for 1.5 hours, combine the two decoctions and the water after extracting the volatile oil, and concentrate under reduced pressure to a liquid with a relative density of 1.06-1.10 (50°C). Slowly add ethanol under sufficient stirring to make the alcohol content of the liquid 60%, continue stirring for 30 minutes, set at 0-4°C and let stand for 24 hours, filter the supernatant, recover the ethanol under reduced pressure and concentrate to a thick paste with a relative density of 1.25-1.30 (50°C). Combine the water extract paste with the above-mentioned alcohol extract paste, heat slightly, and stir thoroughly to extract the thick paste for later use;

[0058] (2) Preparation of Babu Plaster

[0059] Place the prescribed amount of NP-700 in glycerol, add aluminum glycolate, the prescribed total volatile oil and azone, and stir evenly to prepare phase A; dissolve the prescribed amount of PVA in water at 75°C (the amount of water added is 3 times the total mass of the excipients in item B), add PVP K-90, stir evenly, and ultrasonically treat (250W, 33kHz) for 15 minutes, add micropowder silica gel, and stir evenly to prepare phase B.

[0060] Add phase A to phase B, stirring slowly in a 60°C water bath until uniformly mixed. This will serve as the base for the diaper paste. Add the thick extract paste (thick paste / base ratio is 1:5), continue stirring in a 60°C water bath until uniform, and ultrasonicate for 20 minutes. While hot, apply the coating to a non-woven fabric backing at 45°C to a thickness of approximately 2.0 mm. Place in an oven at 40°C to dry for 12 hours, remove, cover with protective film, and seal for storage.

[0061] In the above step (2), the amount of NP-700 used in the prescription is 4.6 g, the amount of glycerol used is 32 g, the amount of aluminum glycolate used is 0.42 g, the amount of PVA used is 0.45 g, the amount of PVP K-90 used is 3 g, the amount of micropowder silica gel used is 4 g, and azone is added at 3% of the total amount of the matrix (excipient) and the extracted thick paste.

[0062] Example 2

[0063] The effective ingredient composition of the traditional Chinese medicine Babu plaster for treating primary dysmenorrhea is the same as that of the traditional Chinese medicine Babu plaster in Example 1, and the overall steps of the preparation method are the same as the preparation process in Example 1, except that the amount of NP-700 used is 5g, the amount of glycerol used is 35g, and the amount of aluminum glycolate used is 0.44g.

[0064] Example 3

[0065] The effective ingredient composition of the traditional Chinese medicine Babu plaster for treating primary dysmenorrhea is the same as that of the traditional Chinese medicine Babu plaster in Example 1, and the overall steps of the preparation method are the same as the preparation process in Example 1, except that the amount of NP-700 used is 4.5g, the amount of glycerol used is 32.5g, and the amount of aluminum glycolate used is 0.4g.

[0066] Comparative Example 1

[0067] The effective ingredient composition of the traditional Chinese medicine Babu plaster for treating primary dysmenorrhea is the same as that of the traditional Chinese medicine Babu plaster in Example 1, and the overall steps of the preparation method are the same as the preparation process in Example 1, except that the amount of NP-700 used is 6g, the amount of glycerol used is 40g, and the amount of aluminum glycolate used is 0.44g.

[0068] Comparative Example 2

[0069] The effective ingredient composition of the traditional Chinese medicine Babu plaster for treating primary dysmenorrhea is the same as that of the traditional Chinese medicine Babu plaster in Example 1, and the overall steps of the preparation method are the same as the preparation process in Example 1, except that the amount of NP-700 used is 5.25g, the amount of glycerol used is 30g, and the amount of aluminum glycolate used is 0.53g.

[0070] Comparative Example 3

[0071] The effective component composition of the traditional Chinese medicine Babu plaster for treating primary dysmenorrhea is the same as that of the traditional Chinese medicine Babu plaster in Example 1, and the overall steps of the preparation method are the same as those in Example 1, except that azone is not added.

[0072] Comparative Example 4

[0073] The effective component composition of the traditional Chinese medicine Babu plaster for treating primary dysmenorrhea is the same as that of the traditional Chinese medicine Babu plaster in Example 1, and the overall steps of the preparation method are the same as those in Example 1, except that the amount of azone added is 5%.

[0074] 1. Key Quality Attributes and Quality Evaluation Criteria

[0075] The adhesion, peel strength, and comprehensive appearance properties of the Babu plaster samples, including paste uniformity, spreadability, and film residue, were used as evaluation indicators. The sum of the scores of each indicator was the comprehensive score, with a total score of 100 points. The evaluation method and scoring criteria are shown in Table 1.

[0076] Table 1 Indicators, methods and scoring criteria for the molding process of the Babu plaster of the present invention

[0077]

[0078] The evaluation results of the above embodiments are shown in Table 2 below.

[0079] Table 2 Scoring results

[0080]

[0081]

[0082] As can be seen from the results in Table 2, the amount of NP-700, glycerol, and aluminum glycolate in the excipients significantly affects the quality of the Babu plaster product of the present invention. The Babu plaster product of the present invention has advantages in terms of appearance indicators such as uniformity, spreadability, film residue, skin followability, and excipient properties, as well as adhesion and peel strength, providing an effective basic guarantee for the use of the drug. Based on the active ingredients and excipients, the addition of azone as a penetration enhancer has limited effect on the quality of the Babu plaster.

[0083] 2. Determination of the content of transdermal components in vitro

[0084] 2.1 Preparation of test solution: Accurately pipette 1 mL of transdermal receiving solution prepared using the drug of Example 1, blow dry with nitrogen at 37°C, dissolve in 200 μL of methanol, centrifuge at 3000 rpm for 10 min, and collect the supernatant.

[0085] 2.2 Preparation of reference solution Accurately weigh appropriate amounts of tetrahydropalmatine, ferulic acid, cinnamaldehyde, salvianolic acid A, and salvianolic acid B reference substances, dissolve them in methanol to prepare mixed solutions with mass concentrations of 25.57, 10.05, and 5.025 μg / mL, respectively.

[0086] 2.3 Chromatographic Conditions: A Thermo Scientific Syncronis C18 column (100 mm × 2.1 mm, 1.7 μm) was used with tetrahydrofuran-acetonitrile (25:15) as mobile phase A and 0.2% formic acid solution as mobile phase B. Gradient elution was performed with the following elution program: 0 min, 90% B; 20 min, 60% B; 22 min, 45% B; 47 min, 10% B; 55 min, 10% B. The flow rate was 0.3 mL / min; the column temperature was 30°C; and the detection wavelength was 275 nm. See the chromatogram for details. Figure 2 .

[0087] 2.4 Linear Relationship Investigation: Mixed reference substance stock solutions with mass concentrations of 51.14, 19.64, 10.05, 48.39, and 15.26 μg / mL, respectively, were prepared by serial dilution with methanol to obtain a series of mixed reference substance solutions of varying concentrations. Accurately pipette 5 μL of each solution and perform the assay according to the chromatographic conditions described in 2.3. The peak areas of tetrahydropalmatine, ferulic acid, cinnamaldehyde, salvianolic acid A, and salvianolic acid B were recorded. Linear regression was performed using the peak areas (Y) of each of the three solutions against their mass concentrations (X). The results are shown in Table 3.

[0088] Table 3 Linear relationship between peak area and mass concentration of active ingredients

[0089]

[0090] 2.5 Precision investigation: 5 μL of mixed reference solution with mass concentrations of 5.114, 1.964, 1.005, 4.839 and 1.526 μg / mL of tetrahydropalmatine, ferulic acid, cinnamaldehyde, salvianolic acid A and salvianolic acid B were accurately aspirated, and the sample was injected and measured 6 times according to the chromatographic conditions under item 2.3. The RSDs of the peak areas of tetrahydropalmatine, ferulic acid, cinnamaldehyde, salvianolic acid A and salvianolic acid B were measured to be 0.23%, 0.34%, 0.38%, 0.42% and 0.31%, respectively, indicating that the instrument has good precision.

[0091] 2.6 Stability Study Accurately aspirate 5 μL of the same test solution and inject the sample for determination at 0, 2, 4, 8, 12, and 24 h after preparation according to the chromatographic conditions under 2.3. The RSDs of the peak areas of tetrahydropalmatine, ferulic acid, cinnamaldehyde, salvianolic acid A, and salvianolic acid B were 0.86%, 1.12%, 0.79%, 1.23%, and 1.36%, respectively, indicating that the test solution had good stability within 24 h.

[0092] 2.7 Repeatability Test Take the transdermal receiving solution at the same time point and mix it. Prepare 6 test solutions according to the preparation method of test solution in 2.1. Inject the sample according to the chromatographic conditions in 2.3. The average contents of tetrahydropalmatine, ferulic acid, cinnamaldehyde, salvianolic acid A and salvianolic acid B were 5.510, 1.415 and 1.616, respectively.

[0093] The concentrations of the two compounds were 1.114, 6.055, and 6.544 μg / mL, and the RSDs were 1.29%, 1.32%, 0.98%, 0.86%, and 1.21%, respectively, indicating that the method had good repeatability.

[0094] 2.8 Sample addition recovery test Take the transdermal absorption medium mixed under 2.7, accurately aspirate 0.5 mL, accurately add appropriate amounts of tetrahydropalmatine, ferulic acid, cinnamaldehyde, salvianolic acid A, and salvianolic acid B reference substances, prepare the test solution according to the preparation method of the test solution under 2.1, operate 6 times in parallel, and inject and determine according to the chromatographic conditions under 2.3. The average sample addition recoveries of tetrahydropalmatine, ferulic acid, cinnamaldehyde, salvianolic acid A, and salvianolic acid B were 101.23%, 98.45%, 99.67%, 102.37%, and 101.56%, respectively, and the RSDs were 1.19%, 1.07%, 1.34%, 0.89%, and 1.21%, respectively.

[0095] 3. In vitro transdermal test

[0096] 3.1 Preparation of ex vivo mouse skin The rats were depilated with 8% sodium sulfide after cutting off the abdominal hair and fasting for 24 h. The rats were then killed by cervical dislocation. The skin was removed and the subcutaneous fat was removed. The mucous tissue was washed with saline to ensure the integrity of the skin. The skin was then stored in a -20°C refrigerator.

[0097] 3.2 Transdermal Experiment: The mouse skin described in 3.1 was thawed in 37°C physiological saline. The plaster of the present invention was tightly attached to the mouse skin and fixed between the supply tank and the receiving tank of the Franz transdermal diffusion device, with the epidermis facing upwards to the supply tank and the dermis facing downwards to the receiving tank. The volume of the receiving tank was 17.5 mL, and the effective diffusion area was 2.54 cm. 2 Each batch of samples was run in triplicate. The receiving cell was filled with physiological saline solution containing 20% ​​ethanol as the receiving medium. Air bubbles were removed and the cells were kept constant at 32°C in a water bath with constant stirring at 300 rpm. 1 mL of transdermal receiving solution was removed at 2, 4, 6, 8, 10, 12, and 24 hours. Simultaneously, an equal volume of receiving medium was added to the receiving cell at this temperature and air bubbles were removed.

[0098] The contents of tetrahydropalmatine, ferulic acid, cinnamaldehyde, salvianolic acid A and salvianolic acid B in the receiving solution were determined by UHPLC, and the cumulative permeation capacity per unit area Qn was calculated according to the following formula.

[0099] Qn=(V 总 C n +ΣC n-1 V 取 ) / A

[0100] Where V 总 is the volume of the receiving pool, C n is the component content at the nth sampling point, C n-1 is the component content at the n-1th sampling point, V 取 is the sampling volume, and A is the permeation receiving area.

[0101] Q nQ for time t n-t The figure is the cumulative permeation-time curve, and the results are shown in Figure 2 , and Q n-t The data were subjected to linear regression, and the slope of the line was the steady-state transdermal rate (J).

[0102] Depend on Figure 2 It can be seen that the addition of azone to the Babu plaster of the present invention can accelerate the transdermal absorption of tetrahydropalmatine, ferulic acid, cinnamaldehyde, salvianolic acid A and salvianolic acid B. The transdermal absorption rate and cumulative permeation amount are significantly improved within 6 hours. As time goes by, the permeation-enhancing effect of azone becomes more and more obvious. The amount of azone added has a significant effect on the transdermal absorption effect of the product, and the effect on the transdermal absorption rate and cumulative permeation amount of different components is also different. The permeation-enhancing effect of 3% azone in Example 1 is significantly better than that of 5% azone in Comparative Example 4.

[0103] IV. Efficacy Test

[0104] 1) Experimental materials and methods

[0105] 4.1 Materials

[0106] Forty SPF-grade female Sprague-Dawley rats, 8 weeks old, weighing 200-220 g, were purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd. (Production License No. SCXK(Lu)20190003) and housed in the animal laboratory of Shandong Institute of Traditional Chinese Medicine (SYXK(Lu)20230004). They were housed at a room temperature of 22-25°C, a relative humidity of 50%-60%, and a 12-hour light-dark cycle. All animal experimental procedures in this study were approved by the Laboratory Animal Welfare and Ethics Committee of Shandong Institute of Traditional Chinese Medicine (Approval No. SDZYY20210925001). The experimental procedures adhered to the principles of replacement, reduction, and optimization to minimize animal use and alleviate animal suffering. Babu plaster (size: 2 cm in diameter), composed of vinegar-soaked Corydalis yanhusuo, salvia miltiorrhiza, cinnamon bark, Chuanxiong rhizome, vinegar-soaked Cyperus rotundus, Evodia rutaecarpa, and ginger, was provided by the Institute of Traditional Chinese Medicine Preparations, Shandong Academy of Traditional Chinese Medicine; Wenjing Zhitong Plaster (batch number: 20221001, size: 4 cm × 6 cm), was provided by Jiaozuo Lianmeng Medical Materials Co., Ltd.; Rat prostaglandin E2 enzyme-linked immunosorbent assay kit (batch number: 202308), rat prostaglandin F 2α Enzyme-linked immunosorbent assay kit (Batch No. 202308) was purchased from Suzhou Xunjie Biotechnology Co., Ltd.

[0107] 4.2 Methods

[0108] 4.2.1 Animal grouping and model preparation

[0109] Forty female SD rats were adaptively fed for one week, fasted for 12 hours but not water, and then their body weights were measured. The rats were then randomly divided into four groups according to their body weights: a blank control group (CG), a primary dysmenorrhea group (PD), a warming and analgesic plaster group (WJZT), and a Babu plaster group (WTT) of the present invention (corresponding to the drug in Example 1), with 10 rats in each group.

[0110] A cold stimulation combined with estradiol benzoate and oxytocin was used to establish a dysmenorrhea model. Except for the blank group, all rats in the remaining groups were placed on ice for 20 minutes daily at a fixed time for 12 consecutive days. Simultaneously, rats received subcutaneous injections of estradiol benzoate (0.4 mg / rat) daily, with 0.8 mg / rat on days 1 and 12. Oxytocin (2 IU / rat) was injected intraperitoneally 1 hour after the last estradiol benzoate injection. The primary dysmenorrhea model of cold coagulation and blood stasis syndrome was established by demonstrating chills, curling up, lack of movement, sluggish reflexes, dark purple paws and tails, dark red ears, weak breathing, a tendency to huddle together, dull fur, a dark purple tongue, thickened and enlarged sublingual veins, and writhing reactions.

[0111] 4.2.2 Animal Dosing Methods

[0112] Starting from the 7th day of modeling, the blank control group and the model control group were given a blank matrix patch, while the Wenjing Zhitong Ointment group and the Babu Plaster group were given the Wenjing Zhitong Ointment and the Babu Plaster of the present invention, respectively, on the Shenque acupoint of the rat abdomen (Shenque acupoint location: based on the analogy principle, it is located at the junction of the 3 / 4 and the lower 1 / 4 of the line connecting the upper edge of the sternum manubrium to the external genitalia on the midline of the rat's chest and abdomen). The drug was administered once a day for 6 consecutive days.

[0113] 4.2.3 Pharmacodynamic evaluation index detection method

[0114] 4.2.3.1 Evaluation of animal writhing reactions

[0115] Two hours after the last dose, administer 2 units of oxytocin per rat intraperitoneally. Immediately, a stopwatch was used to time the rat's writhing response (abdomen contraction and inward concavity, trunk and hind limb extension, buttocks and one limb internal rotation) as an indicator of uterine contraction and dysmenorrhea (Note: The latency period for rats without writhing was calculated as 30 minutes). Observe and record the time to pain onset (latency period) and the number of writhings within 30 minutes. The writhing inhibition rate was calculated.

[0116] The writhing inhibition rate = [(the number of writhings in the model control group - the number of writhings in the drug-treated group) / the number of writhings in the model control group] × 100%.

[0117] 4.2.3.2 Evaluation of blood circulation and stasis-removing effects

[0118] Four hours after the last administration, the rat plasma was collected and the four coagulation function parameters of the rats in each group [prothrombin time (PT), fibrinogen (FIB), activated partial thromboplastin time (APTT), and thrombin time (TT)] were detected.

[0119] 4.2.3.3 Plasma PGF 2α , PGE2 level determination

[0120] Four hours after the last administration, blood was collected from the abdominal aorta under isoflurane anesthesia. Whole blood samples were allowed to rest at room temperature for 30 minutes, then centrifuged at 3000 rpm for 10 minutes at 4°C. The supernatant was collected and stored at -20°C until further use. Plasma PGF2α and PGE2 levels in each group were measured using ELISA.

[0121] 4.2.4 Statistical analysis

[0122] All data are expressed as mean ± standard error Statistical analysis was performed using SPSS 21.0 and GraphPad Prism 9.0 statistical software. One-way analysis of variance was used for comparison between groups, and p < 0.05 was considered statistically significant.

[0123] 2) Results

[0124] 4.3 Effects on writhing reaction in PD rats with cold coagulation and blood stasis

[0125] Compared with the blank control group, the latency of the writhing reaction in the rats in the primary dysmenorrhea group was significantly shortened, and the number of writhing reactions within 30 minutes was significantly increased, indicating that the strong uterine contractions caused by oxytocin acting on the uterus can cause abdominal pain in rats and writhing reactions, suggesting that the rat dysmenorrhea model established by cold stimulation combined with estradiol benzoate and oxytocin was successfully replicated. Compared with the primary dysmenorrhea group, the latency of the writhing reaction in the rats in the Babu plaster group of the present invention was significantly prolonged (P < 0.05), and the number of writhing reactions was significantly reduced (P < 0.01), suggesting that the Babu plaster of the present invention has a significant analgesic effect on oxytocin-induced dysmenorrhea in rats. The results are shown in Table 4.

[0126] Table 4 Effects on writhing reaction in PD rats with cold coagulation and blood stasis ( n=10)

[0127]

[0128] Note: Compared with the PD group, *p<0.05, **p<0.01

[0129] 4.4 Effects on the four coagulation function parameters in PD rats with cold coagulation and blood stasis

[0130] Compared with the blank control group, the thrombin time (TT) and activated partial thromboplastin time (APTT) of the four plasma coagulation indicators of rats in the primary dysmenorrhea group were significantly reduced (P < 0.01), while there was no significant effect on fibrinogen (FIB) and prothrombin time (PT), indicating that the activity of coagulation factors increased, coagulation function was abnormal, and the cold coagulation and blood stasis rat model was successfully established. Compared with the primary dysmenorrhea group, the Babu plaster group of the present invention significantly increased the rat thrombin time (TT) (P < 0.05), reduced the prothrombin time (PT) and increased the activated partial thromboplastin time (APTT), but there was no difference. See Figure 3 (Compared with the PD group, *p<0.05, **p<0.01). This indicates that the drug of the present invention alleviates the hypercoagulable state of the blood in rats with cold coagulation and blood stasis to a certain extent.

[0131] 4.5 Plasma PGF in PD rats with cold coagulation and blood stasis 2α , the effect of PGE2 levels

[0132] Compared with the blank control group, the plasma PGF 2α Compared with the primary dysmenorrhea group, the plasma PGF2α content in the Babu plaster group of the present invention was significantly reduced (P < 0.01), the PGE2 content was significantly increased (P < 0.05), and the PGF 2α The ratio of PGE2 / PGE2 was significantly reduced (P<0.05). This suggests that the Babu plaster of the present invention can reduce the PGF of dysmenorrhea model rats. 2α The content of PGE2 is increased, thereby reducing the degree of smooth muscle contraction and achieving analgesic effect. The results are as follows Figure 4 (*p<0.05, **p<0.01 compared with the PD group).

[0133] 3) Discussion

[0134] The present study found that the latency of the writhing reaction of the rats in the Babu Plaster group of the present invention was significantly prolonged (P < 0.05), and the number of writhing times was significantly reduced (P < 0.01), indicating that the Babu Plaster of the present invention has a significant analgesic effect on dysmenorrhea in PD rats with cold coagulation and blood stasis type; the thrombin time (TT) of the rats in the Babu Plaster group of the present invention was significantly increased (P < 0.05), the prothrombin time (PT) was reduced, and the activated partial thromboplastin time (APTT) was increased, but there was no difference, indicating that the administration of the Babu Plaster of the present invention alleviated the hypercoagulable state of the blood in the rats with cold coagulation and blood stasis type to a certain extent; the plasma PGF .... 2α The content of PGE2 was significantly decreased (P<0.01), the content of PGE2 was significantly increased (P<0.05), and the content of PGF 2αThe ratio of PGE2 / PGE2 was significantly reduced (P<0.05). 2α Increased content causes uterine spasmodic contraction, local ischemia and hypoxia, and dysmenorrhea, while PGE2 can inhibit the spontaneous contraction of uterine smooth muscle, thereby reducing the degree of smooth muscle contraction to achieve analgesic effect. In summary, after administration of the Babu plaster of the present invention, it may relieve the hypercoagulable state of the blood in PD rats with cold coagulation and blood stasis, reduce the prostaglandin PGF 2α The secretion of prostaglandins PGE2 is increased, thereby reducing the degree of smooth muscle contraction and achieving analgesic effect.

[0135] The foregoing is merely an embodiment of the present application, and the scope of protection of the present application is not limited by these specific embodiments, but is determined by the claims of the present application. For those skilled in the art, various modifications and variations can be made to the present application. Any modifications, equivalent substitutions, improvements, etc. made within the technical ideas and principles of the present application should be included in the scope of protection of the present application.

Claims

1. A Chinese medicine Babu plaster for treating primary dysmenorrhea, characterized in that: Made of traditional Chinese medicine composition and excipients; The Chinese medicine composition comprises the following raw materials, calculated by weight: 30-45 parts of vinegared Corydalis yanhusuo, 25-35 parts of salvia miltiorrhiza, 15-20 parts of cinnamon bark, 25-35 parts of chuanxiong rhizome, 22-26 parts of vinegared cyperus rotundus, 8-12 parts of evodia rutaecarpa, and 16-24 parts of ginger; the excipients comprise 4-5 parts of NP-700, 30-35 parts of glycerin, 0.3-0.5 parts of aluminum glycolate, 0.4-0.5 parts of PVA, 2-4 parts of PVP K-90, 3-5 parts of micro-powdered silica gel, azone, and water. The amount of azone is 3% of the total amount of the excipients and the thick paste extracted from the Chinese medicine composition. The preparation method of the Chinese medicine Babu plaster comprises the following steps: (1) Extract preparation a. Weigh vinegar Corydalis yanhusuo and Danshen, extract with alcohol, filter, combine the three filtrates, recover ethanol under reduced pressure, and concentrate into a thick paste with a relative density of 1.25 to 1.30 at 50°C. The residue is set aside; b. Weigh cinnamon bark, Chuanxiong rhizome, vinegar-cured cyperus, evodia rutaecarpa, and ginger, add 8 times the amount of water, and extract the total volatile oil by steam distillation. Separate the total volatile oil, the aqueous extract, and the medicinal residue for later use; C, step b is extracted volatile oil after the medicinal residues and step a alcohol extraction after the medicinal residues are merged, add 8 times the amount of water, decocted 1.5 hours, filtered, the gained medicinal residues are added 6 times the amount of water again, decocted 1.5 hours, merged 2 water decoctions and the water extract after extracting total volatile oil, concentrated under reduced pressure to a medicinal liquid with a relative density of 1.06 to 1.10 at 50 ℃, slowly added ethanol under sufficient stirring, making the medicinal liquid contain alcohol amount is 60%, continued stirring for 30 min, left standstill at 0 to 4 ℃ for 24 hours, the supernatant was filtered, and the ethanol was recovered under reduced pressure and concentrated into a thick paste with a relative density of 1.25 to 1.30 at 50 ℃; d. Combine the thick pastes of step a and step c, heat slightly, and stir thoroughly to obtain a thick paste, and set aside; (2) Preparation of Babu ointment a. Place NP-700 in glycerol, add aluminum glycolate, total volatile oil and azone, and stir evenly to prepare phase A. Dissolve PVA in water at 75°C, add PVP K-90, stir evenly, and sonicate at 250 W, 33 kHz for 15 min. Add micropowdered silica gel and stir evenly to prepare phase B. b. Add phase A to phase B, stirring slowly in a 60°C water bath until the mixture is evenly mixed. This will serve as the base of the babu paste. c. Add the thick paste extracted in step (1), continue stirring in a 60°C water bath until uniform, and ultrasonically treat for 20 min; d. Apply it on a non-woven fabric backing while it is still hot, place it in an oven at 40°C to dry for 12 hours, take it out, cover it with a protective film, and seal it for storage.

2. The Chinese medicine Babu plaster for treating primary dysmenorrhea according to claim 1, characterized in that The Chinese medicine composition is composed of the following raw materials calculated by weight: 40 parts of vinegared Corydalis yanhusuo, 30 parts of salvia miltiorrhiza, 18 parts of cinnamon bark, 30 parts of Chuanxiong rhizome, 24 parts of vinegared Cyperus rotundus, 10 parts of Evodia rutaecarpa and 20 parts of ginger.

3. The preparation method of the Chinese medicine Babu plaster for treating primary dysmenorrhea according to claim 1, characterized in that, The alcohol extraction operation of step a in step (1) is as follows: adding 8 times the amount of 60% ethanol to vinegar Corydalis yanhusuo and Salvia miltiorrhiza, reflux extraction for 2 hours, filtering, adding 6 times the amount of 60% ethanol to the residue, and reflux extraction twice, each time for 1.5 hours; the steam distillation extraction of step b in step (1) is performed for 5 hours.

4. The preparation method of the Chinese medicine Babu plaster for treating primary dysmenorrhea according to claim 1, characterized in that, In step (2), the amount of water added to a is 3 times the mass of the auxiliary material of phase B; in step (2), the weight ratio of the thick paste c to the matrix is ​​1:5; in step (2), the coating temperature of d is 45°C and the thickness is 2.0 mm.

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