She medicine twelve-hour compound percolation extract and preparation method thereof

Abstract

The invention discloses a She medicine twelve-hour compound percolation extract and a preparation method thereof, and relates to the technical field of traditional Chinese medicine extracts.The She medicine twelve-hour compound percolation extract is prepared from 20 parts of twelve-hour, 1 part of ligusticum wallichii, 1.25 parts of pseudo-ginseng, 1.25 parts of safflower, 1.5 parts of honeysuckle and 1.25 parts of borneol; wetting the obtained coarse powder with an ethanol solution with the volume fraction of 30%-70%; filling the wetted coarse powder into a percolator, adding an ethanol solution with the volume fraction of 30-70%, and percolating at room temperature at the flow rate of 0.5-1.5 mL / (min.kg of medicinal materials); and stirring until the mixture is completely dissolved to obtain the She medicine twelve-hour compound percolation extract. According to the method, the extraction efficiency and process stability of the oleanolic acid and the extract are remarkably improved; the quality controllability of the product is ensured; the traditional Chinese medicine composition fully embodies the synergistic advantage of compound compatibility, can avoid gastrointestinal toxicity, liver toxicity and kidney toxicity of oral medicines as an external preparation, and provides an efficient, stable and safe new choice for treating rheumatoid arthritis.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine extraction technology, and in particular to a percolation extract of a She ethnic minority medicine formula prepared according to the twelve-hour cycle, and its preparation method. Background Technology

[0002] Rheumatoid arthritis (RA) is a systemic autoimmune disease characterized by chronic inflammation of the synovial membrane of the joints. It is characterized by joint swelling, pain, and stiffness, ultimately leading to joint deformities and loss of function. Currently, clinical treatments for RA mainly include nonsteroidal anti-inflammatory drugs (NSAIDs), glucocorticoids, and disease-modifying antirheumatic drugs (DMARDs). However, these drugs often have adverse effects on the gastrointestinal tract, liver, and kidneys, and some patients do not respond well to treatment. "Twelve Hours" refers to the double-flowered Clematis genus of the Ranunculaceae family. Clematis florida var The dried root of *Plena* is a traditional medicine used in She medicine. According to *Fu'an She Medicine*, it is warm in nature and pungent in taste, and has the effects of clearing the meridians, opening the orifices, dispelling wind and dampness, and relieving pain. Modern research shows that oleanolic acid is one of its main active ingredients. Based on its good pharmacological activity and folk application experience, local She medicine has developed a compound preparation with the twelve two-hour periods as the main medicine, combined with other medicinal materials such as chuanxiong, safflower, notoginseng, honeysuckle, and borneol. This compound has been used in Fu'an for a long time. The combined use of these medicines has the effects of promoting blood circulation, removing blood stasis, reducing swelling and relieving pain, and relaxing muscles and tendons. It significantly enhances the anti-inflammatory, analgesic, and anti-swelling effects. Percolation is a common method for extracting Chinese medicine and is suitable for medicinal materials with high heat sensitivity and low content of effective ingredients.

[0003] However, the common solutions currently available have many drawbacks, including: the traditional percolation method has problems such as slow extraction speed, long operation time, large solvent consumption, and difficulty in industrialization, which often become the speed limit link in the overall production process. At present, there are no systematic research reports on the extraction process of the twelve-hour compound formula, and there is a lack of an efficient and stable extraction method to support its modern development. Summary of the Invention

[0004] The purpose of this section is to outline some aspects of embodiments of the present invention and to briefly describe some preferred embodiments. Simplifications or omissions may be made in this section, as well as in the abstract and title of this application, to avoid obscuring the purpose of these documents; however, such simplifications or omissions should not be construed as limiting the scope of the invention.

[0005] In view of the problems existing in the above-mentioned She medicine twelve-hour compound percolation extract and its preparation method, the present invention is proposed.

[0006] Therefore, the purpose of this invention is to provide a percolation extract of the She ethnic medicine Twelve Hours Compound and its preparation method. It is applicable to solving the problems of slow extraction speed, long operation time, large solvent consumption, and difficulty in industrialization of traditional percolation methods, which often become the speed-limiting link in the overall production process. At present, there are no systematic research reports on the extraction process of this Twelve Hours Compound, and there is a lack of an efficient and stable extraction method to support its modern development.

[0007] To solve the above-mentioned technical problems, the present invention provides the following technical solution: In a first aspect, embodiments of the present invention provide a percolating extract of a She ethnic minority medicine formula based on the Twelve Hours of the Day and its preparation method, comprising taking the following raw materials: 20 parts of the Twelve Hours of the Day, 1 part of Ligusticum chuanxiong, 1.25 parts of Panax notoginseng, 1.25 parts of Carthamus tinctorius, 1.5 parts of Lonicera japonica, and 1.25 parts of Borneol. The raw materials, except for Borneol, are pulverized into coarse powder that passes through a No. 2 sieve. The obtained coarse powder is moistened with a 30%–70% (v / v) ethanol solution, the amount of ethanol being twice the total weight of the medicinal materials, and left in a sealed container for 12–16 hours. The moistened coarse powder is then placed in a percolator, and a 30%–70% (v / v) ethanol solution is added. The percolator is then incubated at room temperature at a flow rate of 0.5–1.5%. Percolation was performed at a flow rate of mL / (min·kg medicinal material), with the total amount of ethanol being 8 to 12 times the total weight of the medicinal material. The percolate was collected until percolation was complete. Borneol was dissolved in a small amount of anhydrous ethanol and then added to the percolate. The mixture was stirred until completely dissolved to obtain the She medicine twelve-hour compound percolate extract.

[0008] As a preferred embodiment of the preparation method of the She ethnic medicine Twelve Hours Compound Percolation Extract of the present invention, wherein: the Twelve Hours refers to the dried root of Clematis florida, a plant of the Ranunculaceae family; the borneol is synthetic borneol; and the volume fraction of the ethanol solution is the same, ranging from 30% to 70%.

[0009] As a preferred embodiment of the preparation method of the She medicine twelve-hour compound percolation extract described in this invention, the volume fraction of the ethanol solution is 48%, and this concentration is optimized by Box-Behnken response surface methodology with oleanolic acid yield and extract yield as response values.

[0010] As a preferred embodiment of the preparation method of the She medicine twelve-hour compound percolation extract of the present invention, wherein: the total amount of ethanol used is 8 to 12 times the total weight of the medicinal materials, the percolation flow rate is 0.5 to 1.5 mL / (min·kg medicinal materials), and the percolation process is carried out at room temperature.

[0011] As a preferred embodiment of the preparation method of the She medicine twelve-hour compound percolation extract of the present invention, wherein: the total amount of ethanol used is 10 times the total weight of the medicinal materials, and the percolation flow rate is 1 mL / (min·kg medicinal materials).

[0012] As a preferred embodiment of the preparation method of the She medicine twelve-hour compound percolation extract described in this invention, the coarse powder is powder that has passed through a No. 2 sieve and meets the powdering requirements of the General Rules of the 2020 edition of the Chinese Pharmacopoeia.

[0013] In a preferred embodiment of the preparation method of the She medicine twelve-hour compound percolation extract of the present invention, the amount of the ethanol solution used for wetting is twice the total weight of the medicinal materials, by weight.

[0014] Secondly, in order to further solve the above-mentioned technical problems, the present invention provides a She medicine twelve-hour compound percolation extract, which includes: the extract contains oleanolic acid and extract, wherein the yield of oleanolic acid is 0.50%~0.80% and the yield of extract is 28%~38%.

[0015] As a preferred embodiment of the She medicine twelve-hour compound percolation extract of the present invention, wherein: oleanolic acid is used as a quality control indicator component of the extract, and its content is determined by high performance liquid chromatography.

[0016] As a preferred embodiment of the She medicine twelve-hour compound percolation extract described in this invention, the medicine is a topical preparation with anti-inflammatory and analgesic effects, capable of inhibiting xylene-induced ear swelling in mice, reducing the number of acetic acid-induced writhing responses in mice, and inhibiting primary and secondary paw swelling in adjuvant-induced arthritis rats.

[0017] The beneficial effects of this invention are as follows: This invention scientifically optimized the percolation process using the Box-Behnken response surface methodology, determining the optimal parameters for 48% ethanol, 10 times the solvent dosage, and a percolation flow rate of 1 mL / (min·kg), significantly improving the extraction efficiency and process stability of oleanolic acid (yield of 0.50%~0.80%) and extract (yield of 28%~38%). Simultaneously, an HPLC quality control system centered on oleanolic acid was established, ensuring the controllability of product quality. Pharmacodynamic studies confirmed that the extract exhibited significant anti-inflammatory and analgesic effects in xylene-induced mouse ear swelling, acetic acid-induced mouse writhing, and adjuvant-induced arthritis rat models. Its effects were significantly superior to single-herb formulas and formulas without specific ingredients, fully demonstrating the synergistic effect of compound formulations. Furthermore, as a topical preparation, it avoids the gastrointestinal and hepatotoxicity associated with oral medications, providing a highly efficient, stable, and safe new option for treating rheumatoid arthritis. Attached Figure Description

[0018] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort. Wherein: Figure 1 This is a flowchart illustrating the implementation of the present invention in Example 1.

[0019] Figure 2 This is a contour map (AB contour map) showing the effect of ethanol concentration and liquid-to-solid ratio on the overall score.

[0020] Figure 3 The response surface plot (AB response surface plot) shows the effect of ethanol concentration and liquid-to-solid ratio on the overall score.

[0021] Figure 4 This is a contour map (AC contour map) showing the effect of ethanol concentration and percolation rate on the overall score.

[0022] Figure 5 The response surface plot (AC response surface plot) shows the effect of ethanol concentration and percolation rate on the overall score.

[0023] Figure 6 This is a contour map (BC contour map) showing the influence of liquid-to-material ratio and percolation rate on the overall score.

[0024] Figure 7 This is a response surface plot (BC response surface plot) showing the influence of liquid-to-material ratio and percolation rate on the overall score. Detailed Implementation

[0025] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings.

[0026] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0027] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in different places in this specification does not necessarily refer to the same embodiment, nor is it a single or selective embodiment that is mutually exclusive with other embodiments.

[0028] Example 1 Reference Figure 1 This is the first embodiment of the present invention, which provides a She ethnic medicine twelve-hour compound percolation extract and its preparation method, including the following steps: S1: Take the following raw materials: 20 parts of the twelve-hour cycle, 1 part of Ligusticum chuanxiong, 1.25 parts of Panax notoginseng, 1.25 parts of Carthamus tinctorius, 1.5 parts of Lonicera japonica, and 1.25 parts of Borneol. Except for Borneol, all raw materials are crushed into coarse powder that passes through a No. 2 sieve.

[0029] Preferably, the twelve two-hour periods refer to the dried root of Clematis florida, a plant belonging to the Ranunculaceae family and the Clematis genus; it has been identified by the Fujian Academy of Traditional Chinese Medicine as meeting the medicinal standards of the twelve two-hour periods in "Fu'an She Medicine and Herbal Remedies", and the authentic specimen is preserved in the Institute of Materia Medica of the Academy.

[0030] Furthermore, the borneol is synthetic borneol, which complies with the provisions of the "Borneol" item in Part I of the 2020 edition of the Chinese Pharmacopoeia, and its borneol content shall not be less than 98.0%. Compared with natural borneol, synthetic borneol has more stable quality and smaller batch-to-batch variation, making it more suitable for industrial production.

[0031] Furthermore, the pulverization is carried out using a traditional Chinese medicine pulverizer. After pulverization, the powder is passed through a No. 2 sieve (24 mesh, sieve hole inner diameter 850 μm±29 μm). The powder that passes through the sieve is collected, and the powder that does not pass through is pulverized again to ensure that all coarse powder meets the pulverization requirements of the General Rules of the 2020 Edition of the Chinese Pharmacopoeia.

[0032] Specifically, the "coarse powder that passes through the No. 2 sieve" refers to powder that passes through the No. 2 sieve completely, but contains no more than 40% of powder that can pass through the No. 4 sieve. This particle size range ensures that the active ingredients are fully dissolved, while also preventing the powder from being too fine and causing blockage during percolation.

[0033] S2: Moisten the obtained coarse powder with an ethanol solution of 30% to 70% by volume, the amount of ethanol being twice the total weight of the medicinal material, and place it in a sealed container for 12 to 16 hours.

[0034] Preferably, the volume fraction of the ethanol solution is the same, ranging from 30% to 70%, and the ethanol used for wetting and percolation uses the same concentration, which can avoid changes in osmotic pressure or precipitation caused by sudden changes in solvent concentration.

[0035] Furthermore, the volume fraction of the ethanol solution is 48%. This concentration was optimized using the Box-Behnken response surface methodology with oleanolic acid yield and extract yield as response values. The specific optimization process is as follows: Based on single-factor experiments, ethanol concentration (A), liquid-to-solid ratio (B), and percolation rate (C) were selected as the factors to be investigated. The comprehensive score of oleanolic acid yield (weight 60%) and extract yield (weight 40%) was used as the response value. After analysis using Design-Expert software, 48% was determined to be the optimal ethanol concentration.

[0036] Specifically, the coarse powder is powder that has passed through a No. 2 sieve and meets the pulverization requirements of the General Rules in the 2020 edition of the Chinese Pharmacopoeia. When wetting, the ethanol solution is slowly added to the coarse powder while stirring, until the coarse powder is evenly moistened, there are no dry powder lumps, it can be formed into a ball by hand, and it can be easily dispersed by light pressure.

[0037] Furthermore, the amount of the ethanol solution used for wetting is twice the total weight of the medicinal material. For example, 200 mL of ethanol solution is used for wetting every 100 g of coarse powder. This amount has been proven by experiments to be sufficient to allow the medicinal material to swell fully, which is beneficial for subsequent percolation.

[0038] S3: Load the moistened coarse powder into a percolator, add an ethanol solution with a volume fraction of 30% to 70%, and percolate at a flow rate of 0.5 to 1.5 mL / (min·kg medicinal material) at room temperature. The total amount of ethanol used should be 8 to 12 times the total weight of the medicinal material. Collect the percolate until percolation is complete.

[0039] Preferably, the total amount of ethanol used is 8 to 12 times the total weight of the medicinal materials, the percolation flow rate is 0.5 to 1.5 mL / (min·kg medicinal materials), and the percolation process is carried out at room temperature, which refers to 15 to 25°C. This temperature condition can avoid the loss of volatile components such as borneol caused by high temperature.

[0040] Specifically, the total amount of ethanol used is 10 times the total weight of the medicinal materials, and the percolation flow rate is 1 mL / (min·kg medicinal materials). These parameters are the optimal process conditions optimized by response surface methodology. It has been verified that under these conditions, the extraction rate of oleanolic acid and the yield of extract both reach a high level.

[0041] Furthermore, when filling the container, the moistened coarse powder should be added in several batches. Each layer should be gently pressed down with a flat rod to ensure even distribution and avoid the formation of channels or cracks. This will ensure uniform solvent penetration. After adding the solvent, the bottom opening should be opened first to expel air bubbles. Once the solvent has filled the drug layer, the bottom opening should be closed. Continue adding solvent until it is about 3-5 cm above the drug surface. Cover and soak for 12-16 hours before starting percolation.

[0042] Specifically, during the percolation process, the solvent should always be kept above the drug surface to prevent the drug layer from drying out. After collecting the percolate to the specified amount, 1 mL of the last flowing percolate can be taken, evaporated to dryness, and the residue should not exceed 5 mg. Alternatively, thin-layer chromatography can be used to detect that oleanolic acid has no obvious spots, which can be used as the criterion for judging complete percolation.

[0043] Furthermore, the percolator is made of stainless steel or glass, and has a sieve plate and degreased cotton or filter paper at the bottom as a filter layer to prevent the powder from clogging the outlet. The collected percolate can be concentrated under reduced pressure below 60°C as needed, but the temperature must be controlled to avoid damage to the components.

[0044] Specifically, the percolation flow rate is calculated as follows: based on each kilogram of medicinal material, for example, if the total weight of the medicinal material is 1 kg, the flow rate is controlled at 0.5 to 1.5 mL / min. The actual flow rate can be controlled by adjusting the valve at the bottom of the percolator and is periodically checked with a stopwatch and a measuring cylinder.

[0045] S4: Add borneol to the percolate after dissolving it with a small amount of anhydrous ethanol, and stir until completely dissolved to obtain the She medicine twelve-hour compound percolate extract.

[0046] Specifically, place borneol in a dry container, add 1 mL of anhydrous ethanol, and gently stir or shake until the borneol is completely dissolved to obtain a clear solution. Slowly add the borneol ethanol solution to the percolate obtained in S3 under stirring, and continue stirring for 15 to 30 minutes to mix evenly. Let it stand until there is no precipitation or layering, and you have obtained the solution.

[0047] Furthermore, the obtained extract is a clear, brownish-yellow to brownish-red liquid with the characteristic aroma of borneol. It can be used directly as a medicinal solution as needed for formulation, or concentrated under reduced pressure to a specified volume at a temperature not exceeding 40°C, and further formulated into topical preparations such as creams, gels, and liniments.

[0048] Specifically, the yield of oleanolic acid in the extract is 0.50%~0.80% (determined by high performance liquid chromatography), and the yield of the extract is 28%~38% (calculated by taking an appropriate amount of extract, evaporating it in a water bath, and drying it at 105℃ to constant weight).

[0049] In summary, this invention scientifically optimized the percolation process using the Box-Behnken response surface methodology, determining the optimal parameters for 48% ethanol, 10 times the solvent volume, and a percolation flow rate of 1 mL / (min·kg). This significantly improved the extraction efficiency and process stability of oleanolic acid (yield of 0.50%~0.80%) and extract (yield of 28%~38%). Simultaneously, an HPLC quality control system centered on oleanolic acid was established to ensure product quality controllability. Pharmacodynamic studies confirmed that the extract exhibited significant anti-inflammatory and analgesic effects in xylene-induced mouse ear swelling, acetic acid-induced mouse writhing, and adjuvant-induced arthritis rat models. Its effects were significantly superior to those of single-herb formulas and formulas without specific ingredients, fully demonstrating the synergistic effect of compound formulations. Furthermore, as a topical preparation, it avoids the gastrointestinal and hepatotoxicity associated with oral medications, providing a highly efficient, stable, and safe new option for treating rheumatoid arthritis.

[0050] Example 2, an embodiment of the present invention, provides a She medicine twelve-hour compound percolation extract, comprising: the extract contains oleanolic acid and extract, wherein the yield of oleanolic acid is 0.50%~0.80% and the yield of extract is 28%~38%.

[0051] Furthermore, the content of oleanolic acid, which is used as a quality control indicator component of the extract, is determined by high performance liquid chromatography.

[0052] Example 3: Optimization of Percolation Extraction Process Using Box-Behnken Response Surface Methodology 3.1 Experimental Design Based on the previous single-factor experiments, three factors were selected: ethanol concentration (A), liquid-to-solid ratio (B), and percolation rate (C). The yield of oleanolic acid and the yield of extract were used as response values ​​for optimization experiments. The factor levels are shown in Table 1.

[0053] Table 1. Factor Levels Table for Box-Behnken Response Surface Methodology ; 3.2 Evaluation Indicators and Methods (1) Extract yield: Take 5 mL of percolate and place it in an evaporating dish that has been dried to constant weight. Evaporate to dryness in a water bath, dry to constant weight at 60℃, cool to room temperature in a desiccator, accurately weigh the mass, and calculate the extract yield.

[0054] (2) Oleanolic acid yield: determined by high performance liquid chromatography. Accurately weigh 2 mg of oleanolic acid reference standard, place it in a 10 mL volumetric flask, dissolve it in methanol and dilute to the mark to obtain a 0.2 mg / mL reference solution. Dilute 1 / 2 times to obtain a series of concentrations, inject 20 μL, and perform linear regression on the peak area integral value (Y) and oleanolic acid mass concentration (X). The regression equation is Y = 451.65X + 1.0913, r = 0.9998, with a linear range of 0.00625–0.2 mg / mL. Take 1 mL of percolate, add 15 mL of 2 mol / L hydrochloric acid solution, heat under reflux for 2 h, cool, transfer to a separatory funnel, and extract twice with ethyl acetate, 15 mL each time. Combine the ethyl acetate extracts, concentrate to near dryness below 70 °C, dissolve in methanol and dilute to 10 mL, inject for determination, and calculate the yield.

[0055] 3.3 Experimental Results The experimental design and results of the Box-Behnken response surface methodology are shown in Table 2.

[0056] Table 2 Experimental Design and Results of Box-Behnken Response Surface Methodology ; Note: Weighted sum = Oleanolic acid yield × 60% + Extract yield × 40%.

[0057] 3.4 Analysis of Variance Using Design-Expert software, a multiple regression was performed on the data to obtain the regression equation: Y = 15.16 - 0.3928A + 0.3618B + 0.4188C + 0.3063AB + 0.8035AC +0.5394BC - 1.53A 2 - 2.08B 2 - 1.41C 2 .

[0058] The results of the analysis of variance are shown in Table 3.

[0059] Table 3. Analysis of Variance for Regression Models ; Note: **P<0.01, *P<0.05.

[0060] The results showed that the model was highly significant (P<0.0001), the lack-of-fit term was not significant (P=0.1923), and R0 was [value missing]. 2 =0.9859, indicating that the model fits well and can be used for analysis and prediction.

[0061] 3.5 Optimal Process Validation According to the regression model, the optimal process conditions were: ethanol volume fraction 48.407%, liquid-to-solid ratio 1:10.201, and percolation flow rate 1.073 mL / (min·kg). For ease of implementation, the conditions were adjusted to: ethanol volume fraction 48%, liquid-to-solid ratio 1:10, and percolation flow rate 1 mL / (min·kg). Three parallel validation experiments were conducted under these conditions, with comprehensive scores of 15.0871, 14.7824, and 15.1123, and an average of 14.9939 (predicted value 15.221), with a relative error of 1.49%, indicating that the optimized process is stable and feasible.

[0062] 3.6 Response Surface Analysis Diagram Based on the regression model, Design-Expert software was used to draw contour plots and response surface plots showing the impact of the interactions of various factors on the overall score. The results are shown in [Figure number missing]. Figures 2-7 .

[0063] Figure 2 and Figure 3 The interaction between ethanol concentration (A) and liquid-to-solid ratio (B) was shown. When the percolation rate was fixed at the center level (1 mL / min·kg), the overall score first increased and then decreased with the increase of ethanol concentration and liquid-to-solid ratio, and the contour lines were elliptical, indicating that there was a certain interaction between the two, but the significance was low (P=0.0925), which is consistent with the results of the analysis of variance.

[0064] Figure 4 and Figure 5The interaction between ethanol concentration (A) and percolation rate (C) was shown. When the liquid-to-solid ratio was fixed at the center level (10 times), the response surface slope was steep, the contour lines were dense and clearly elliptical, indicating that the interaction between the two was significant (P=0.0014), and the matching of ethanol concentration and percolation rate had a significant impact on extraction efficiency.

[0065] Figure 6 and Figure 7 The interaction between liquid-to-solid ratio (B) and percolation rate (C) is shown. When the ethanol concentration is fixed at the central level (50%), the response surface shows a distinct convexity, and the contour lines are elliptical, indicating a significant interaction between the two (P=0.0110). Appropriately increasing the liquid-to-solid ratio and decreasing the percolation rate helps to improve extraction efficiency.

[0066] Comprehensive response surface methodology revealed that the optimal process region was concentrated within the range of ethanol concentration of 45%–55%, liquid-to-solid ratio of 9.5–10.5, and percolation flow rate of 0.9–1.1 mL / (min·kg), which is consistent with the optimal process conditions predicted by the regression model (48.4%, 10.2, and 1.07 mL / min·kg), further verifying the reliability of the optimization results.

[0067] Further, based on the optimal extraction conditions obtained by response surface methodology, the percolate of single herbs, compound herbs, and herbs without the twelve-hour cycle was extracted to evaluate the pharmacodynamics of xylene-induced ear swelling and acetic acid-induced writhing analgesia.

[0068] Example 4: Pharmacodynamic Study 4.1 Anti-inflammatory experiment on xylene-induced ear swelling in mice Sixty male ICR mice were randomly divided into four groups: a blank control group, a model control group, a positive control group (diclofenac), a twelve-hour cycle group (crude drug concentration 1.2 g / mL), a twelve-hour cycle compound group (crude drug concentration 1.5 g / mL), and a group without a twelve-hour cycle compound group (crude drug concentration 0.3 g / mL), with 10 mice in each group. After drug administration, xylene was applied to the right ear to induce inflammation, and the degree of ear swelling and inhibition rate were calculated. The results are shown in Table 4.

[0069] Table 4. Effects of the She medicine twelve-hour compound on ear swelling in mice of different groups (x±s, n=10) ; Note: Compared with the model control group, ***P<0.001.

[0070] 4.2 Acetic acid-induced writhing analgesia experiment in mice Fifty male ICR mice were randomly divided into four groups: a model control group, a positive control group (diclofenac), a twelve-hour cycle group (crude drug concentration 1.2 g / mL), a twelve-hour cycle compound group (crude drug concentration 1.5 g / mL), and a group without a twelve-hour cycle compound group (crude drug concentration 0.3 g / mL), with 10 mice in each group. Acetic acid was injected intraperitoneally after drug administration, and the number of writhing movements was recorded. The inhibition rate was calculated, and the results are shown in Table 5.

[0071] Table 5. Effects of the She medicine twelve-hour compound on acetic acid writhing response in mice of different groups (x±s, n=10) ; Note: Compared with the model control group, P<0.001, P<0.01, P<0.05.

[0072] 4.3 Anti-inflammatory effect experiment in adjuvant-induced arthritis (AA) rats Thirty-six SD rats were randomly divided into four groups: a blank control group, a model control group, a positive control group (diclofenac), a twelve-hour cycle group (crude drug concentration 1.2 g / mL), a twelve-hour cycle compound group (crude drug concentration 1.5 g / mL), and a group without a twelve-hour cycle compound group (crude drug concentration 0.3 g / mL), with six rats in each group. Except for the blank control group, all other groups of rats were injected intradermally with Freund's complete adjuvant into the left hind paw to induce inflammation. Drug administration began the day after model establishment and continued for 28 days. The swelling degree of the primary and secondary paws was measured on days 7, 14, 21, and 28. The results are shown in Tables 6 and 7.

[0073] Table 6. Effects of the She medicine twelve-hour compound formula on the swelling of the primary lateral paw in AA rats (x±s, n=6) ; Note: Compared with the blank group, ###P<0.001, ##P<0.05; compared with the model control group, *P<0.01.

[0074] Table 7. Effects of the She medicine twelve-hour compound on secondary lateral paw swelling in AA rats (x±s, n=6) ; Note: Compared with the control group, ###P<0.001, #P<0.05; compared with the model control group, **P<0.001, P<0.01.

[0075] 4.4 Conclusion Pharmacodynamic results showed that the extract of the Twelve Hours Compound exhibited significant anti-inflammatory and analgesic effects in xylene-induced inflammation, acetic acid-induced writhing, and AA rat models, with significantly better effects than the single-herb Twelve Hours Compound and the Twelve Hours Compound without Twelve Hours, proving that the compound has a synergistic effect.

[0076] It should be noted that the drug is a topical preparation with anti-inflammatory and analgesic effects. It can inhibit xylene-induced ear swelling in mice, reduce the number of writhing responses induced by acetic acid in mice, and inhibit primary and secondary paw swelling in adjuvant-induced arthritis rats.

Claims

1. A method for preparing a compound percolation extract of She ethnic medicine based on the twelve hours of the day, characterized in that: include: Take the following raw materials: 20 parts of the twelve-hour cycle, 1 part of Ligusticum chuanxiong, 1.25 parts of Panax notoginseng, 1.25 parts of Carthamus tinctorius, 1.5 parts of Lonicera japonica, and 1.25 parts of Borneol. Except for Borneol, all raw materials are crushed into coarse powder that passes through a No. 2 sieve. The obtained coarse powder is moistened with an ethanol solution with a volume fraction of 30% to 70%, the amount of ethanol being twice the total weight of the medicinal material, and then sealed and left for 12 to 16 hours. The moistened coarse powder is placed into a percolator, and an ethanol solution with a volume fraction of 30% to 70% is added. Percolation is carried out at room temperature at a flow rate of 0.5 to 1.5 mL / (min·kg medicinal material). The total amount of ethanol used is 8 to 12 times the total weight of the medicinal material. The percolate is collected until percolation is complete. After dissolving borneol in a small amount of anhydrous ethanol, add it to the percolate and stir until completely dissolved to obtain the She medicine twelve-hour compound percolate extract.

2. The preparation method of the She ethnic medicine twelve-hour compound percolation extract as described in claim 1, characterized in that: The twelve two-hour periods mentioned refer to the dried roots of Clematis florida, a plant belonging to the Ranunculaceae family and the Clematis genus. The borneol is synthetic borneol; The volume fraction of the ethanol solutions is the same, ranging from 30% to 70%.

3. The preparation method of the She ethnic medicine twelve-hour compound percolation extract as described in claim 2, characterized in that: The volume fraction of the ethanol solution was 48%, which was optimized using the Box-Behnken response surface methodology with oleanolic acid yield and extract yield as response values.

4. The preparation method of the She ethnic medicine twelve-hour compound percolation extract as described in claim 1, characterized in that: The total amount of ethanol used is 8 to 12 times the total weight of the medicinal materials, the percolation flow rate is 0.5 to 1.5 mL / (min·kg medicinal materials), and the percolation process is carried out at room temperature.

5. The preparation method of the She ethnic medicine twelve-hour compound percolation extract as described in claim 4, characterized in that: The total amount of ethanol used is 10 times the total weight of the medicinal materials, and the percolation flow rate is 1 mL / (min·kg medicinal materials).

6. The method for preparing a compound percolation extract of She ethnic medicine according to claim 1, characterized in that: The coarse powder is powder that has passed through a No. 2 sieve.

7. The method for preparing a compound percolation extract of She ethnic medicine according to claim 1, characterized in that: The amount of the ethanol solution used for wetting is twice the total weight of the medicinal materials, by weight.

8. A compound percolation extract of She ethnic medicine prepared by the method according to any one of claims 1 to 7, characterized in that, The extract contains oleanolic acid and an extract, wherein the yield of oleanolic acid is 0.50% to 0.80% and the yield of the extract is 28% to 38%.

9. The She ethnic medicine twelve-hour compound percolation extract according to claim 8, characterized in that, Oleanolic acid, used as a quality control indicator for the extract, was determined by high performance liquid chromatography.

10. The use of the She ethnic medicine twelve-hour compound percolation extract as described in claim 9 in the preparation of a medicament for treating rheumatoid arthritis, characterized in that, The drug is a topical preparation with anti-inflammatory and analgesic effects. It can inhibit xylene-induced ear swelling in mice, reduce the number of writhing responses induced by acetic acid in mice, and inhibit primary and secondary paw swelling in adjuvant-induced arthritis rats.

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