Preparation method and application of zhuang medicine luojianyuba plaster for treating knee arthritis

Abstract

The present invention discloses a preparation method of a Zhuang medicine Luoqianyu cataplasm and its application in the treatment of knee arthritis. The preparation method includes extracting effective medicinal components such as volatile oils, alkaloids, organic acids, and flavonoids from the Zhuang medicine Luoqianyu formula, mixing the extracted medicinal components with a cataplasm forming matrix and stirring evenly, then coating on non-woven fabric, and drying and forming, etc. The Zhuang medicine Luoqianyu cataplasm prepared by the method of the present invention for treating knee arthritis not only has a smooth surface without granular feeling, a soft paste body, good quality, but also has a large drug loading capacity, fast onset, good curative effect, high bioavailability, convenient use, easy preservation, can continuously exert the medicinal effect for a long time, is suitable for long-term use by middle-aged and elderly people, and can better exert the curative effect of the Zhuang medicine Luoqianyu formula.

Description

Technical Field

[0001] This invention relates to a preparation method and application of a traditional Chinese medicine, Luoqianyu poultice, belonging to the field of traditional Chinese medicine technology. Background Technology

[0002] Knee osteoarthritis (KOA) is a chronic degenerative osteoarthritis of the knee that primarily affects middle-aged and older adults. It is characterized by degeneration of the articular cartilage and reactive hyperplasia of the subchondral bone, and is marked by joint pain, deformity, and limited mobility. It is a complex, multifactorial pathogenesis and is one of the most common disabling diseases in clinical practice. Studies have found that it primarily affects the elderly, with a gradually decreasing age of onset and an increasing incidence rate with age. Patients suffer from long-term physical pain and psychological distress, severely impacting their quality of life and work.

[0003] In traditional Chinese medicine, knee osteoarthritis falls under the category of "Bi syndrome" and is primarily treated with medication. Emphasis is placed on syndrome differentiation and treatment, focusing on dispelling wind and dampness, warming the meridians and dispersing cold, supplemented by tonifying Qi and blood, and nourishing the liver and kidneys. This approach effectively eliminates knee joint inflammation, achieving significant results and ultimately curing the root cause. Traditional Chinese medicine treatment for KOA has proven effective, with a lower recurrence rate compared to Western medicine. It combines the accumulated experience of predecessors with extensive modern research technology. Therefore, leveraging the unique advantages of traditional Chinese medicine in treating knee osteoarthritis, it is essential to develop safe, inexpensive, and effective new drugs for this condition.

[0004] The Zhuang medicine Luoqian Yufang is a prescription developed by the Guangxi University of Traditional Chinese Medicine Pharmaceutical Factory based on the research and summarization of traditional Zhuang medicine for treating knee osteoarthritis, and formulated according to Zhuang medical theory. It has shown significant clinical efficacy in treating knee osteoarthritis. The prescription consists of 19 herbs, including *Manshanxiang* (Kehanbo), *Qingfengteng* (Gourongqiang), *Shenjincao* (Keyanyin), and *Huzhang* (Ketiangang). It has the effects of removing dampness and toxins, dispelling cold and toxins, dispelling wind and toxins, and clearing the meridians. In Zhuang medicine, it is believed to have the functions of clearing the meridians and relieving pain, dispelling wind and dampness, and resolving blood stasis and relieving numbness. It is mainly used in folk medicine for knee arthritis, which is the modern clinically common condition of knee osteoarthritis (KOA), degenerative knee arthritis, and primary proliferative arthritis.

[0005] The Zhuang medicine Luoqian Yufang formula contains many herbs and has a complex composition. The alkaloids, organic acids, flavonoids, and volatile oils it contains are all closely related to the efficacy of the drug preparations made using this formula. The traditional dosage form of Zhuang medicine Luoqian Yufang is a wine-based preparation. Wine-based preparations are inconvenient to use, requiring application to the affected area several times a day. They have a small drug-carrying capacity, a short duration of action, and poor absorption, thus failing to fully exert their efficacy and affecting treatment results. A poultice refers to a plaster made by uniformly mixing medicinal extracts, medicinal materials, or chemical drugs with a suitable hydrophilic matrix and then applying the mixture to a backing material. Traditional Chinese medicine poultices refer to poultices made from Chinese herbs that have been extracted to form a herbal extract or by ultra-finely pulverizing Chinese herbs and then mixing them with a matrix and stirring evenly. Poultices can directly act on the target site to exert therapeutic effects, avoiding the first-pass effect of the liver and gastrointestinal tract and the irritation of the gastrointestinal tract by the drug. They maintain a constant blood drug concentration for a longer period of time, reduce adverse drug reactions, and can reduce the frequency of administration. They are convenient to use, can be repeatedly applied, have good breathability, and a large drug loading capacity. Therefore, developing a new dosage form of Zhuang medicine Luoqianyu Fang—Zhuang medicine Luoqianyu poultice—that is convenient to use, has good efficacy, is easy to store, and can exert its efficacy for a longer period of time will help to better exert the therapeutic effect of Zhuang medicine Luoqianyu Fang. Summary of the Invention

[0006] The purpose of this invention is to provide a method for preparing a Zhuang medicine, Luoqianyu poultice, for the prevention and treatment of knee osteoarthritis, and its application. The Zhuang medicine, Luoqianyu poultice prepared using this method has good efficacy, rapid onset of action, high bioavailability, is convenient to use, easy to store, and can exert its efficacy for a relatively long period, making it suitable for long-term use by middle-aged and elderly people, and better leveraging the therapeutic effects of the Zhuang medicine, Luoqianyu poultice.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] A method for preparing a traditional Chinese medicine ointment, Luo Qian Yu Ba Bao, includes the following steps:

[0009] (1) Take the ingredients of each ingredient in the prescription of Zhuangyao Luoqianyufang according to the weight proportions, as well as 0.196 parts of borneol, 0.196 parts of menthol, 0.196 parts of camphor, 0.5 parts of Tween-80, and 0.223 parts of methyl salicylate, and set aside for later use;

[0010] (2) Take five medicinal materials: Manshanxiang, Shenjincao, Sifangmupi, Yinxiangpi, and Shanju, and steam distill them for 8 hours to obtain volatile oil. Alternatively, add 12-14 times the amount of water and use steam distillation for 8-10 hours each time, extracting 1-2 times to obtain volatile oil components for later use.

[0011] (3) Mix the residue from which the volatile oil has been extracted with the remaining medicinal materials of the Zhuang medicine Luoqianyufang (excluding dragon's blood, frankincense, and myrrh), add 8-10 times the amount of ethanol with a volume concentration of 55-65%, and reflux extract for 1.5-2 hours each time, extract twice, filter out the residue and combine the extracts; recover the ethanol from the obtained extract under reduced pressure until there is no alcohol odor, concentrate it to a relative density of 1.20-1.25, and obtain clear extract A;

[0012] (4) Powder the dragon's blood, frankincense and myrrh, dissolve them with an appropriate amount of ethanol and water, add clear paste A and mix well to obtain thick paste B, set aside; add borneol, menthol and camphor to an appropriate amount of ethanol to dissolve, then add Tween-80, methyl salicylate and the volatile oil obtained in step (2) and stir evenly to obtain mixture C.

[0013] (5) According to the weight ratio: NP-700: micronized silica gel: aluminum hydroxyl: carbomer 940: CMC-Na: glycerol: water: thick paste B = (0.95~1.65): (0.70~1.30): (0.40~0.80): 0.5: 0.5: (7.50~14.2): 10:12 and 1% of the total matrix weight of EDTA-2Na; take each matrix component for later use; first take NP-700, micronized silica gel and aluminum hydroxyl and place them in glycerol to swell as phase A, dissolve EDTA-2Na in water, add carbomer 940 and CMC-Na to fully swell as phase B, mix phase B and phase A evenly to prepare the matrix, then add thick paste B and mixed solution C, stir with a mixer at 400r / min for 15 min, after stirring evenly, coat it on non-woven fabric, dry and shape it, add a cover, cut and package it to obtain the product.

[0014] In the above-mentioned preparation method of Zhuang medicine Luoqianyu poultice, the prescription of the raw materials of Zhuang medicine Luoqianyu formula is as follows: by weight, 8.55 parts of Manshanxiang, 4.27 parts of Qingfengteng, 4.27 parts of Fenfangji, 3.85 parts of Shenjincao, 4.27 parts of Huzhang, 2.14 parts of Feilongzhangxue, 2.14 parts of Haitongpi, 3.42 parts of Sanbaicao, 3.42 parts of Sanjiapi, 3.42 parts of Hongchuanposhi, 3.42 parts of Sifangmupi, 2.14 parts of Niuerfeng, 2.14 parts of Yinxiangpi, 2.14 parts of Tenghuangtan, 2.14 parts of Shanju, 1.28 parts of Changchunweimao, 0.85 parts of Ruxiang (vinegar-processed), 0.85 parts of Moyao (vinegar-processed), and 1.28 parts of Longxuejie.

[0015] Furthermore, the Zhuang medicine Luoqianyu Babu prepared using the above-mentioned method can be used to prevent and treat knee osteoarthritis. This Zhuang medicine Luoqianyu Babu can be directly used to treat knee osteoarthritis.

[0016] The Zhuang medicine theory of Luoqian Yufang is explained as follows: Kehanbo (Manshanxiang) and Gou Rongqiang (Qingfengteng) are the main medicines, which are dedicated to dispelling wind and dampness toxins, clearing the meridians and relieving pain. Keyanyin (Shenjincao), Ketiangang (Huzhang), Kesanpang (Sanbaicao), Wenxiao (Feilongzhangxue), Goupenglou (Shanju), Gouwenlie (Sanjiapi), Meitong (Haitongpi), Meilangma (Sifangmupi), Meiximei (Niuerfeng), Goulerong (Hongchuanposhi), Meizhongtun (Yinxiang), Meilelong (Longxuejie), Tongbiling (Tenghuangtan), Gouyao (Changchunweimao), and Fenfangji are the auxiliary medicines, which help the main medicine to clear the meridians and relieve pain, resolve blood stasis and reduce swelling, and eliminate dampness and wind toxins. Borneol, menthol, and camphor are the guiding medicines, which can drive the main and auxiliary medicines directly to the lesion, so as to achieve the effects of clearing the meridians and relieving pain, dispelling wind and dampness toxins, resolving blood stasis and eliminating numbness.

[0017] The Zhuang medicine formula Luoqian Yufang contains many herbs and has a complex composition. The main components of the formula, such as Qingfengteng, Fenfangji, Huzhang, Feilongzhangxue, Haitongpi, Shenjincao, and Shanju, are alkaloids. The main components of Sanjiapi, Niuerfeng, Sanbaicao, and Changchunweimao are organic acids. Manshanxiang, Huzhang, Shenjincao, Niuerfeng, Sanbaicao, Tenghuangtan, and Changchunweimao all contain flavonoids. In addition, Manshanxiang, Shenjincao, Shanju, Sifangmupi, and Yinxiang contain a large amount of volatile oils. Alkaloids, organic acids, flavonoids, and volatile oils all have anti-inflammatory and analgesic effects. All of these components are closely related to the efficacy of the medicinal preparations made using this formula.

[0018] The Zhuang medicine Luoqian Yufang contains five herbs: *Manshanxiang*, *Shenjincao*, *Shanju*, *Sifangmupi*, and *Yinxiang*, all of which contain abundant volatile oils. These volatile oils are among the main active ingredients, possessing anti-inflammatory and analgesic effects, and also promoting transdermal absorption. Therefore, in this invention, the preparation of the Zhuang medicine Luoqian Yufang poultice first involves extracting the volatile oil components from these five herbs, and then using the residue from which the volatile oils have been extracted for the next step of extracting the active ingredients. Simultaneously, in the research experiments, the inventors used the content of sinomenine, tetrandrine, lindane, polydipsia glycoside, and chlorogenic acid, the yield of the extract, and the relative total peak area as evaluation indicators, employing a star-point design-response surface optimization method to optimize the alcohol extraction process of the Zhuang medicine Luoqian Yufang. By using the alcohol extraction process steps defined in this invention, alkaloids, organic acids, flavonoids, and other active ingredients in the Zhuang medicine Luoqian Yufang can be effectively extracted, thus obtaining the effective active ingredients of the Zhuang medicine Luoqian Yufang.

[0019] The beneficial effects of this invention are as follows:

[0020] The present invention provides a preparation method of Zhuangyao Luoyan Yu cataplasm and its application in the treatment of knee arthritis. First, the present invention obtains the optimal extraction process for extracting the effective pharmacodynamic components in the Zhuangyao Luoyan Yu formula through research. The extraction method can effectively extract the volatile oil, alkaloids, organic acids, flavonoids and other effective pharmacodynamic components in the Zhuangyao Luoyan Yu formula, which is beneficial to improving the efficacy of the Zhuangyao Luoyan Yu formula. Then, the star point design-response surface optimization method is used to optimize the较佳 process parameters for preparing the Zhuangyao Luoyan Yu cataplasm, and further obtain the stable and feasible preparation method of the Zhuangyao Luoyan Yu cataplasm of the present invention.

[0021] Compared with the traditional dosage form of the Zhuangyao Luoyan Yu formula - wine agent, the Zhuangyao Luoyan Yu cataplasm prepared by the method of the present invention contains the effective pharmacodynamic components of the Zhuangyao Luoyan Yu formula, has a large drug loading capacity, quick onset, good absorption effect, high bioavailability, is easy to store, can continuously exert the drug effect for a long time, has good curative effect, is convenient to use, is suitable for long-term use by middle-aged and elderly people, and can better exert the curative effect of the Zhuangyao Luoyan Yu formula. Moreover, when the method of the present invention is used to prepare the Zhuangyao Luoyan Yu cataplasm, the matrix has good formability, good compatibility with the Luoyan Yu medicinal paste, the prepared cataplasm has a smooth surface without granular feeling, good skin following property and peeling property, the paste body is soft, and the quality is good. The preparation method of the Zhuangyao Luoyan Yu cataplasm provided by the present invention for preventing and treating knee arthritis can provide a reliable reference basis for the clinical pharmacodynamic effect of the Zhuangyao Luoyan Yu formula and the change of the dosage form. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Contour plots and three-dimensional surface plots of the OD value with respect to extraction time and solvent dosage;

[0023] Figure 2 Contour plots and three-dimensional surface plots of the OD value with respect to ethanol concentration and solvent dosage;

[0024] Figure 3 Contour plots and three-dimensional surface plots of the OD value with respect to ethanol concentration and extraction time;

[0025] Figure 4 Contour plots and three-dimensional surface plots of the OD value with respect to the dosage of NP-700 and the dosage of micro-powdered silica gel;

[0026] Figure 5 Contour plots and three-dimensional surface plots of the OD value with respect to the dosage of NP-700 and the dosage of glyceryl aluminum hydroxide;

[0027] Figure 6 Contour plots and three-dimensional surface plots of the OD value with respect to the dosage of NP-700 and the dosage of glycerol;

[0028] Figure 7 Contour plots and three-dimensional surface plots of the OD value with respect to the dosage of glyceryl aluminum hydroxide and the dosage of micro-powdered silica gel;

[0029] Figure 8 Contour lines and 3D surface plots showing the effects of micronized silica gel dosage and glycerin dosage on OD;

[0030] Figure 9 Contour lines and 3D surface plots showing the effects of aluminum hydroxyacetate dosage and glycerol dosage on OD;

[0031] Figure 10 HE staining of the left knee joint of a rat (×50 magnification);

[0032] Figure 11 Toluidine blue staining of the left knee joint of a rat (×100x);

[0033] Figure 12 TRAP staining of the left knee joint of a rat (×200 magnification);

[0034] Figures 10-12 Among them, A, sham surgery group; B, KOA model group; C, swelling-reducing and pain-relieving tincture group; D, high-dose group of Zhuang medicine Luoqian formula; E, medium-dose group of Zhuang medicine Luoqian formula; and F, low-dose group of Zhuang medicine Luoqian formula. Detailed Implementation

[0035] Example 1

[0036] one, Research on the alcohol extraction process of Zhuang medicine Luoqian Yufang

[0037] 1. Instruments and Materials

[0038] 1.1 Instruments: Watres C18 (2.1 × 100 mm column), PEG-20 capillary column (30 m × 0.25 mm, 0.25 μm), Agilent 1290 ultra-high performance liquid chromatograph (including quaternary pump, autosampler, column oven, DAD detector, Agilent Technologies, USA); Agilent 7820B gas chromatograph, METTLER TOLEDO XS105 balance; SOP balance, Sartorius Scientific Instruments Ltd.; 0.5 μl - 10 μl manual pipettes, Eppendorf AG, Germany; 20 μl - 200 μl pipettes, SXKW heating mantle, SHIMADZU 10 μl microsyringe; constant temperature water bath.

[0039] 1.2 Materials: Eucalyptol (batch number: 110788-201707), Limonene (batch number: 100470-201503), Methyl salicylate (batch number: 110707-2011815), α-terpineol (batch number: 111859-201804), Tetrandrine (batch number: 110774-201808), Zanthoxylum bungeanum extract (batch number: 111531-201603), Tetrandrine (batch number: 110711-201801) Chlorogenic acid (batch number: 110753-201415) and polygalactoside (batch number: 111575-201603) were purchased from the National Institutes for Food and Drug Control; n-decanoic acid (batch number: AF20050451, purity: 98%) was purchased from Chengdu Efa Biotechnology Co., Ltd.; acetonitrile (chromatographic grade: Fisher Scientific), methanol, acetic acid were analytical grade, ethanol was food-grade alcohol, and water was purified water; all experimental medicinal materials were identified by Professor Zhu Yilin of Guangxi University of Traditional Chinese Medicine. Information on medicinal materials and samples is shown in Table 1.

[0040]

[0041]

[0042] 2. Prescription for Zhuang medicine Luoqian Yufang: 8.55g of Manshanxiang, 4.27g of Qingfengteng, 4.27g of Fenfangji, 3.85g of Shenjincao, 4.27g of Huzhang, 2.14g of Feilongzhangxue, 2.14g of Haitongpi, 3.42g of Sanbaicao, 3.42g of Sanjiapi, 3.42g of Hongchuanposhi, 3.42g of Sifangmupi, 2.14g of Niuerfeng, 2.14g of Yinxiangpi, 2.14g of Tenghuangtan, 2.14g of Shanju, 1.28g of Changchunweimao, 0.85g of Ruxiang (vinegar-processed), 0.85g of Moyao (vinegar-processed), and 1.28g of Longxuejie.

[0043] 3. This experiment optimized the extraction process parameters of the effective components in the Zhuang medicine Luoqian Yufang. The content of sinomenine, tetrandrine, styracin, styracin, chlorogenic acid, extract yield, and relative total peak area were used as evaluation indicators. The star point design-response surface optimization method was adopted, and the experiment was arranged according to a 3-factor, 5-level star point design to select the best alcohol extraction process.

[0044] 3.1 Experimental methods and results of alkaloid components

[0045] 3.1.1 Preparation of test solution: Weigh about 30g of medicinal material according to the prescription ratio, place it in a stoppered Erlenmeyer flask, extract according to the experimental protocol, filter, combine the filtrates, accurately transfer 5ml of the filtrate into an evaporating dish with a pipette, evaporate to dryness, dilute to 5ml with methanol, shake well, centrifuge, and filter through a microporous membrane to obtain the test solution.

[0046] 3.1.2 Preparation of the mixed reference solution: Accurately weigh appropriate amounts of sinomenine, zeaxanthin, tetrandrine, and reference standards, dissolve them in methanol, and prepare reference stock solutions with concentrations of 1.23 mg / ml, 1.01 mg / ml, and 0.92 mg / ml, respectively. Separately, take appropriate amounts of the above sinomenine, zeaxanthin, and tetrandrine solutions, place them in the same volumetric flask, and dilute with methanol to prepare a mixed alkaloid reference solution containing sinomenine, zeaxanthin, and tetrandrine concentrations of 0.072 mg / ml, 0.065 mg / ml, and 0.025 mg / ml, respectively.

[0047] 3.1.3 Preparation of negative control solution

[0048] 3.1.3.1 Preparation of negative control solution for lack of Sinomenium acutum: Weigh all medicinal materials except Sinomenium acutum according to the prescription ratio, and prepare negative control solution for lack of Sinomenium acutum according to the test sample method in section “3.1.1”.

[0049] 3.1.3.2 Preparation of negative control solution for lack of *Pterocarya stenoptera* blood: Weigh all medicinal materials except *Pterocarya stenoptera* blood according to the prescription ratio, and prepare negative control solution for lack of *Pterocarya stenoptera* blood according to the test sample method in section “3.1.1”.

[0050] 3.1.3.3 Preparation of negative control solution for Stephania tetrandra deficiency: Weigh all medicinal materials except Stephania tetrandra according to the prescription ratio, and prepare negative control solution for Stephania tetrandra deficiency according to the test sample method in section “3.1.1”.

[0051] 3.1.4 Determination of Chromatographic Conditions

[0052] 3.1.5 Chromatographic conditions and system suitability test: A Watres ACQUITY UPLC BEH C18 (2.1 × 100 mm Column) column was used; the mobile phase was acetonitrile (B) - 0.1 mol ammonium acetate solution (D), gradient elution is shown in Table 2, flow rate: 0.2 mL / min; detection wavelength: 280 nm; column temperature: 40 ℃; injection volume: 2 μL.

[0053] Under these chromatographic conditions, reference solutions and sample solutions were injected for analysis and detected using DAD. The results showed that the retention times and UV spectra of the chromatographic peaks of sinomenine, buergerianin, and tetrandrine were consistent with those of the corresponding chromatographic peaks in the samples, and their matching purity reached over 900%. The resolution of each peak was good, and the tailing factors were all between 0.95 and 1.05. Other components in the samples did not interfere with the determination of these three index components. The theoretical plate number of the chromatographic column, calculated based on sinomenine, was 8000.

[0054]

[0055] 3.1.6 Methodological Examination

[0056] 3.1.6.1 Linearity Range Study: Accurately weigh appropriate amounts of sinomenine, zeaxanthin, tetrandrine, and reference standards, dissolve them in methanol, and prepare reference stock solutions with concentrations of 1.23 mg / ml, 1.01 mg / ml, and 0.92 mg / ml, respectively. Separately, take appropriate amounts of the above sinomenine, zeaxanthin, and tetrandrine solutions, place them in the same volumetric flask, and dilute with methanol to prepare a mixed alkaloid reference solution containing sinomenine, zeaxanthin, and tetrandrine concentrations of 0.072 mg / ml, 0.065 mg / ml, and 0.025 mg / ml, respectively. Using an autosampler, precise amounts of 0.5, 1, 2, 3, 4, and 5 µL were injected and analyzed according to the chromatographic conditions described in section "3.1.5". The peak areas of each index component were recorded. The linear regression equations for sinomenine (Y) were calculated as follows: Y = 3878.3X - 3.62 r = 1; tschonoside (Y = 3230.3X - 1.1416 r = 1); and tetrandrine (Y = 3444.1 - 2.4633 r = 1). The results showed that sinomenine, tschonoside, and tetrandrine exhibited good linear relationships with the injection volume (µg) within the ranges of 0.036 µg–0.36 µg, 0.0325 µg–0.325 µg, and 0.0125 µg–0.125 µg, respectively (n = 6).

[0057] In addition, the inventors conducted precision tests, stability tests, repeatability tests, and sample recovery tests during the methodological investigation. The results showed that the instrument had good precision, the sample solution was stable within 24 hours, and the method had good repeatability and accuracy.

[0058] 3.2 Experimental methods and results for other components

[0059] 3.2.1 Preparation of test solution: Weigh about 30g of medicinal material according to the prescription ratio, place it in a stoppered Erlenmeyer flask, extract according to the experimental protocol, filter, combine the filtrates, accurately transfer 5ml of the filtrate into an evaporating dish with a pipette, evaporate to dryness, dilute to 10ml with methanol, shake well, centrifuge, and filter through a microporous membrane to obtain the test solution.

[0060] 3.2.2 Preparation of the mixed reference solution: Accurately weigh appropriate amounts of polygalactoside and chlorogenic acid reference standards, dissolve them in methanol, and prepare reference stock solutions with concentrations of 0.98 mg / ml and 1.27 mg / ml, respectively. Separately, take appropriate amounts of the above polygalactoside and chlorogenic acid solutions to prepare a mixed reference solution containing polygalactoside and chlorogenic acid concentrations of 0.049 mg / ml and 0.096 ml / ml, respectively.

[0061] 3.2.3 Preparation of negative control solution

[0062] 3.2.3.1 Preparation of negative control solution for Polygonum cuspidatum deficiency: Weigh all medicinal materials except Polygonum cuspidatum according to the prescription ratio, and prepare the negative control solution for Polygonum cuspidatum deficiency according to the test sample method in section "3.2.1". 3.2.3.2 Preparation of negative control solution for Acanthopanax senticosus deficiency: Weigh all medicinal materials except Acanthopanax senticosus according to the prescription ratio, and prepare the negative control solution for Acanthopanax senticosus deficiency according to the test sample method in section "3.2.1".

[0063] 3.2.4 Determination of Chromatographic Conditions

[0064] 3.2.5 Chromatographic conditions and system suitability test: A Watres C18 (2.1 × 100 mm Column) was used; the mobile phase was acetonitrile (B) - 0.1% acetic acid aqueous solution (D), gradient elution is shown in Table 3, flow rate: 0.3 mL / min; detection wavelength: 310 nm; column temperature: 25℃; injection volume: 2 μL.

[0065] Under these chromatographic conditions, reference solution and sample solution were injected for analysis and detected by DAD. The results showed that the retention times and UV spectra of the chromatographic peaks of polydipsia glycoside and chlorogenic acid were consistent with those of the corresponding chromatographic peaks in the sample, and the matching purity reached over 900%. The resolution of each peak was good, and the tailing factor was between 0.95 and 1.05. Other components in the sample did not interfere with the determination of this indicator component. The theoretical plate number of the chromatographic column was 5000 based on polydipsia glycoside.

[0066]

[0067] 3.2.6 Methodological Examination

[0068] 3.2.6.1 Linearity Range Study: Accurately weigh appropriate amounts of polygalactoside and chlorogenic acid reference standards, dissolve them in methanol, and prepare reference stock solutions with concentrations of 0.98 mg / ml and 1.27 mg / ml, respectively. Separately, take appropriate amounts of the above polygalactoside and chlorogenic acid solutions to prepare mixed reference solutions containing polygalactoside and chlorogenic acid concentrations of 0.06 mg / ml and 0.096 mg / ml, respectively. Separately, take appropriate amounts of the above polygalactoside and chlorogenic acid solutions, place them in the same volumetric flask, dilute with methanol, and prepare mixed alkaloid reference solutions containing polygalactoside and chlorogenic acid concentrations of 0.06 mg / ml and 0.096 mg / ml, respectively. Using an autosampler, precise amounts of 0.3, 1, 2, 3, 4, and 6 µL were injected and analyzed under the chromatographic conditions described in section "3.2.5". The peak areas of each index component were recorded. The linear regression equations for polydipsia glycoside (Y) and chlorogenic acid (Y) were calculated as follows: Y = 13521x - 9.0838r = 1; Y = 8114.2x - 13.398r = 1. The results showed that polydipsia glycoside and chlorogenic acid exhibited good linear relationships with the injection volume (µg) within the ranges of 0.03 µg to 0.3 µg and 0.048 µg to 0.48 µg, respectively (n = 6).

[0069] In addition, the inventors conducted precision tests, stability tests, repeatability tests, and sample recovery tests during the methodological investigation. The results showed that the instrument had good precision, the sample solution was stable within 24 hours, and the method had good repeatability and accuracy.

[0070] 3.3 Single-factor analysis

[0071] This study employed a point-response surface methodology (PSM) to optimize the ethanol extraction process of the effective components of the traditional Chinese medicine Luoqian Yufang. Since this method involves continuous variation, extraction time, solvent volume, and ethanol concentration were selected as influencing factors. The medicinal materials were weighed according to the prescription proportions. Single-factor experiments were conducted on extraction time, solvent volume, and ethanol concentration using sinomenine, zeaxanthin, tetrandrine, glutinosa glycoside, and chlorogenic acid as single-factor evaluation indicators to determine the optimal point-response surface methodology levels for each factor.

[0072] 3.3.1 Extraction Time Investigation: The Zhuang herbal medicine *Luoqianyufang* was weighed according to the prescription ratio, and 10 times the amount of 60% ethanol was added. Different extraction times were selected (see Table 4), and extraction was performed twice. The results are shown in Table 4. The content increased significantly between 30 and 90 minutes of extraction, and the content of some samples decreased significantly after 90 minutes.

[0073]

[0074] 3.3.2 Solvent Combination Test: The Zhuang medicine Luoqianyufang was weighed according to the prescription ratio, and 60% ethanol was added at different solvent combinations (see Table 5). Extraction was performed for 90 minutes, twice. The results are shown in Table 5. The content of some indicators showed significant changes when the solvent combination ranged from 6 to 12 times.

[0075]

[0076] 3.3.3 Ethanol Concentration Investigation: The Zhuang medicine Luoqianyufang was weighed according to the prescription ratio, and 10 times the amount of ethanol of different concentrations was added (see Table 6). Extraction was performed for 90 minutes, twice. The results are shown in Table 6. The ethanol concentration increased significantly between 30% and 70%, and the increase slowed down after 70%.

[0077]

[0078] 3.4 Star Point Design – Response Surface Experiment

[0079] 3.4.1 Response Surface Design: This experiment used UPLC to determine the total area of ​​characteristic peaks in the extract and the yields of sinomenine, zeaxanthin, tetrandrine, chlorogenic acid, polygalactoside, and the dry extract as indicators for response surface design. Based on these seven indicators, and in conjunction with preliminary and single-factor experiments, the main factors affecting the ethanol extraction process of the Zhuang medicine *Luoqianyufang* were determined to be solvent volume (A), extraction time (B), and ethanol concentration (C). Since the number of extractions was a non-continuous variable and could not be regressed, the number of extractions was fixed at two. Following the principle of star point design, a three-factor, five-level experiment was adopted, with level codes of -α, -1, 0, 1, and α. The factor levels are shown in Table 7.

[0080]

[0081] 3.4.2 Total Peak Area Determination: Test samples treated according to the method in section "3.2.1" were taken, and the UPLC chromatograms of the extracts were determined according to the chromatographic conditions in section "3.2.5". Thirty common characteristic peaks were identified. The relative areas of the common characteristic peaks were statistically analyzed, as shown in Table 8.

[0082] 3.4.3 Determination of dry extract yield: Take the extract, shake well, accurately take 5 ml (1-20) of the extract with a pipette, place it in an evaporating dish that has been dried to constant weight, evaporate to dryness in a water bath, dry at 105℃ for 3 h, cool in a desiccator for 30 min, quickly and accurately weigh the mass, and calculate the dry extract yield.

[0083] 3.5 Results and Data Analysis

[0084] 3.5.1 Standardization of indicator data

[0085] This experiment used UPLC to determine the total area of ​​characteristic peaks in the extract and the contents of seven indicators, including sinomenine, zeaxanthin, tetrandrine, chlorogenic acid, polygalactoside, and dry extract yield, to optimize the ethanol extraction process of the Zhuang medicine Luoqianyufang. Directly adding indicators of different properties cannot accurately reflect the combined effect of different factors. In order to accurately describe the relationship between the indicators and the ethanol extraction process, the total evaluation normalization method was used.

[0086] The Hassan method was used to standardize the numerical values ​​to a normalized value (d, desirability) between 0 and 1. In this experiment, a larger value was considered better for the index, and the formula dmax = (Yi - Ymin) / (Ymax - Ymin) was used, where Ymax represents the maximum value of the index and Ymin represents the minimum value. The arithmetic and total score normalized value (ODs) = (d1 + d2 + d3 + ... + dk) / k, i = 1, 2, 3...n; (k is the number of indices). The data of the seven indices were standardized according to the formula to eliminate the influence of different units and dimensions of the indices and the large differences in the range of the indicated quantities, so as to make a comprehensive evaluation. The experimental scheme designed according to DesignExpert 8.0.6 software, the measured values ​​of each index and the arithmetic total score normalized value are shown in Table 8.

[0087]

[0088] 3.5.2 Analysis of the results of the star-point design

[0089] This experiment used solvent volume (A), extraction time (B), and ethanol concentration (C) as independent variables, and the arithmetic total score normalization value (ODs) as the dependent variable. Multiple linear regression and binomial fitting were performed, and the results are as follows:

[0090]

[0091] From the p-values ​​of the equations, the p-values ​​for the multiple linear model are greater than 0.05, while those for the quadratic polynomial fitting model are less than 0.01, indicating that the quadratic polynomial fitting model has higher significance. The correlation coefficients r of the fitting equations show that the correlation coefficient of the binomial fitting equation is significantly higher than that of the multiple linear fitting equation. Specifically, the multiple correlation coefficient r of the quadratic polynomial fitting model is... 2 =0.9298, close to 1, the multiple linear correlation coefficient r 2 =0.2576, indicating a poor linear correlation coefficient between independent variables A, B, and C and the dependent variable OD. Therefore, a linear regression model cannot be used. The binomial fitting equation, however, has a higher correlation coefficient and a better fit, suggesting that a quadratic polynomial equation is more appropriate for fitting the various indicators of the extraction process. Therefore, a quadratic polynomial fitting equation is chosen to optimize the process parameters. An F-test was used to analyze the variance of the model, as shown in Table 9.

[0092]

[0093] 3.5.3 Optimization of alcohol extraction process and model prediction: Using Design-Expert 8.0.6 software, based on the regression analysis results, a three-dimensional surface and contour plot of the dependent variable changing with the independent variable were plotted (see...). Figures 1-3 As shown in the figure, the interactions between extraction time, solvent volume, and ethanol concentration on the response value were not significant (P>0.05). After analysis and screening using Design Expert 8.0.6 software, the optimal extraction parameters for the Zhuang medicine *Ligusticum striatum* were determined to be: solvent volume 6–14 times, extraction time 90.24–109.4 min, and ethanol concentration 56.66–63.61%. For ease of production, the optimal extraction process was selected with a solvent volume of 8 times, an extraction time of 90 min, and an ethanol concentration of 60%, with a predicted OD value of 0.917.

[0094] 3.5.4 Confirmatory Test: The medicinal materials were weighed according to the prescription ratio of Zhuang medicine Luoqian Yufang, and a Chinese-style test was conducted by taking samples at a magnified scale. Three batches were extracted repeatedly under the optimal extraction conditions (A: 8 times, B: 90 min, C: 60%). The total peak area, the content of sinomenine, styracin, tetrandrine, chlorogenic acid, and glutinosa glycoside, as well as the yield of dry extract were measured. The ODs value was calculated to be 0.806. Compared with the predicted value (0.917) calculated according to the fitted equation, the deviation was -0.111%, indicating that the predicted value of the optimized extraction process was good.

[0095] II. Research on the Forming Process of Zhuang Medicine Luoqianyu Poultice

[0096] Zhuang medicine Luoqianyu poultice has the effects of removing dampness, dispelling cold, and dispelling wind, as well as clearing the meridians and promoting blood circulation. In Zhuang medicine, it is used to relieve pain, dispel wind and dampness, and resolve blood stasis and numbness. It is mainly used for knee arthritis, a common clinical condition. The poultice matrix is ​​mainly composed of cross-linking agents, fillers, moisturizers, and transdermal penetration enhancers. The variety of matrix types makes selection difficult. In previous experiments on the molding process of poultices based on traditional Chinese medicine compound formulas, the evaluation was often limited to a weighted comprehensive score based on adhesion and appearance indicators. However, due to the uncertainty of the weighting coefficients, this method has certain limitations. Therefore, this experiment, based on single-factor experiments, used a star-point design-response surface optimization method to optimize the best process parameters for Zhuang medicine Luoqianyu poultice, providing a reliable reference for clinical efficacy and changes in dosage form.

[0097] 4. Instruments and Materials

[0098] 4.1 Instruments: CZY-Z type initial tack tester, LX-402A type holding power tester (Jinan Langguang Electromechanical Technology Co., Ltd.), LHP-250 type constant temperature and humidity incubator (Shanghai Sanfa Scientific Instrument Co., Ltd.), CHTB-03 type coating machine (Jinan Chuchuang Electromechanical Equipment Co., Ltd.), digital display constant speed mixer (Hunan Lichen Technology Co., Ltd.), DZKW-S-6 type electric thermostatic water bath (Beijing Yongguangming Medical Instrument Co., Ltd.), KQ5200B type ultrasonic cleaner (Kunming Ultrasonic Instrument Co., Ltd.).

[0099] 4.1.2 Materials: Blank matrix: glycerol (Tianjin Fuyu Fine Chemical Co., Ltd.), sodium polyacrylate-700 (NP-700) and aluminum hydroxyl (Shanghai Dexiang Pharmaceutical Technology Co., Ltd.), micronized silica gel (Shandong Liaocheng Ahua Pharmaceutical Co., Ltd.), carbomer, glycerol, sodium carboxymethyl cellulose, EDTA-2Na (Chengdu Huayi Pharmaceutical Excipients Manufacturing Co., Ltd.), Tween-80, and pure water.

[0100] Transdermal absorption agents: methyl salicylate, camphor, borneol, and menthol. The drug paste used was prepared in-house by our research group. The water was self-prepared purified water, and the reagents were of chromatographic or analytical grade.

[0101] 4.2 Methods and Results

[0102] 4.2.1 Preparation of Volatile Oil and Volatile Transdermal Absorbent: In this study, the transdermal absorbent was dissolved in an appropriate amount of water and mixed evenly with the volatile oil to form an emulsion. An appropriate amount of Tween-80 was then added to dissolve the emulsion, forming a transparent micelle compatibilization system. This method employs micelle compatibilization, using the surfactant Tween-80 as a carrier for the volatile oil and the volatile transdermal absorbent in the poultry. This allows the emulsion to enter the liquid space of the micelle core and disperse evenly within it, forming a transparent, thermodynamically and kinetically stable micelle compatibilization system. This system does not exhibit oil-water stratification over time and is less prone to diffusion, volatilization, or oxidation reactions, thus facilitating the control and improvement of the poultry's quality and stability.

[0103] 4.2.2 Preparation of Zhuang medicine Luoqianyu poultice: Through preliminary experiments, the preparation process of the poultice matrix was determined as follows: Take the prescribed amount of aluminum hydroxide, NP-700, and micronized silica gel, mix them evenly, and disperse them in the prescribed amount of glycerin as phase A; put the prescribed amount of carbomer 940, CMC-Na, and 1% EDTA-2Na in an appropriate amount of water and let them swell fully as phase B; add phase B to phase A in small amounts several times and stir evenly, add volatile oil, transdermal absorbent micelle compatibilizer, and the prescribed amount of thick paste (about 12g) (relative density 1.07), stir evenly with a mixer (400r / min), coat it on non-woven fabric, and let it dry and solidify at room temperature to obtain the poultice.

[0104] 4.2.3 Selection and determination of evaluation indicators: In this study, the adhesion determination method under the plaster section of the 2015 edition of the Chinese Pharmacopoeia was used to determine the initial tack, holding power and comprehensive sensory evaluation (plaster shape, residue and skin adherence) as comprehensive evaluation indicators to score the plaster. The evaluation criteria are shown in Table 10.

[0105]

[0106] 4.2.4 Single-factor experiment: A single-factor experiment was conducted, keeping other dosages constant, and changing the dosage of one matrix component. The poultice matrix was prepared according to the method described in section "4.2.2", and the initial tack and holding power of the matrix were tested. The higher the score, the better the matrix's formability. The optimal dosages for each matrix component were determined to be 1.6g NP-700, 0.5g Carbomer 940, 0.5g Aluminum Hydroxide, 1g Micronized Silica Glycol, 0.5g Carbomer 940, 0.5g CMC-Na, 1% EDTA-2Na, 10g Glycerin, and 10mL Water. The results showed that the dosages of Carbomer 940, CMC-Na, and 1% EDTA-2Na had relatively small effects on this matrix formulation, while the dosages of Aluminum Hydroxide, NP-700, Micronized Silica Glycol, and Glycerin had relatively large effects. Therefore, Aluminum Hydroxide, NP-700, Micronized Silica Glycol, and Glycerin were selected as the main factors to be investigated.

[0107] 4.2.5 Star Design – Response Surface Methodology

[0108] 4.2.5.1 Response Surface Design: This experiment selected the following factors that significantly affect the performance of the Zhuang medicine Luoqian Yuba plaster: the dosage of NP-700 (X1), the dosage of micronized silica gel (X2), the dosage of aluminum hydroxyacetate (X3), and the dosage of glycerin (X4). Based on the star point design principle, a 4-factor, 5-level experiment was adopted, with level codes represented by -α, -1, 0, 1, and α. The factor levels are shown in Table 11.

[0109]

[0110] 4.2.6 Results and Data Analysis

[0111] 4.2.6.1 Standardization of indicator data: The indicator data was standardized according to the method in section “3.5.1”, and the results are shown in Table 12.

[0112]

[0113] 4.2.6.2 Analysis of Results of the Star Point Design: This experimental design used the dosage of NP-700 (X1), the dosage of micronized silica gel (X2), the dosage of aluminum hydroxyl (X3), and the dosage of glycerol (X4) as independent variables, and the arithmetic total score normalization value (ODs) as the dependent variable. Multiple linear regression and binomial fitting were performed, and the results are as follows:

[0114]

[0115] For the multiple linear model, P > 0.05, r 2 =0.0813; P < 0.01 for the quadratic polynomial fitting model, r 2 =0.9132, indicating that the multiple linear regression fitting equation has a low correlation coefficient, and the linear correlation between the independent and dependent variables is poor. Therefore, it is not suitable to use the linear regression model for prescription optimization analysis. The quadratic polynomial fitting equation has a high correlation coefficient, close to 1, and the linear correlation between the independent and dependent variables is significant, making it suitable as a model for analysis and prediction. An F-test was used to analyze the variance of the model. The model P < 0.01, and the lack-of-fit term P > 0.05, showing no significant difference, indicating that the equation fits well and the unknown factors have little impact on the experimental results. The influence of the four factors on the molding process is A > C > D > B, as shown in Table 13.

[0116]

[0117] 4.2.6.3 Molding process optimization and model prediction:

[0118] Using Design-Expert 8.0.6 software, based on the regression analysis results, a three-dimensional surface and contour plot of the dependent variable as a function of the independent variable were plotted (see...). Figures 4-9 As shown in the figure, the interactions between NP-700 and aluminum hydroxyl, micronized silica gel and aluminum hydroxyl, and aluminum hydroxyl and glycerol on the response values ​​were not significant (P>0.05). After analysis and screening using Design Expert 8.0.6 software, the optimal dosage parameters for NP-700, micronized silica gel, aluminum hydroxyl, and glycerol were determined to be: NP-700 0.95g–1.65g, micronized silica gel 0.70g–1.30g, aluminum hydroxyl 0.40g–0.80g, and glycerol 7.50g–14.2g.

[0119] 4. Discussion: In the matrix of Zhuangyao Luoyan Yu cataplasm, NP-700 is used as the skeleton material, glyceryl aluminum hydroxide is used as the cross-linking agent, microsilica is used as the filler, carbomer 940 and CMC-Na are used as thickeners, and glycerol is used as the humectant. On the basis of single-factor experiments, the types of matrices and the preliminary ratio of the dosage of ointment to matrix were screened. The 4-factor 5-level star point-response surface method was used to optimize the ratio of ointment to matrix of Zhuangyao Luoyan Yu cataplasm, avoiding the compatibility problems between the matrix and Luoyan Yu ointment when adding the ointment after screening the blank prescription matrix. In addition to the preparation process of Zhuangyao Luoyan Yu cataplasm being related to the dosage of each matrix, it is also related to the stirring time, speed, and drying environment. During the stirring process, in this experiment, it is appropriate to stir at 400 r / min for 15 minutes. If the speed is too fast or the time is too long, the cataplasm will become disconnected. If it is too slow or the time is too short, the stirring will be uneven.

[0120] Example 2

[0121] Study on the Therapeutic, Anti-inflammatory and Analgesic Effects of Zhuangyao Luoyan Prescription on Knee Osteoarthritis (KOA) in Rats

[0122] 1. Materials

[0123] 1.1 Animals: 182 SPF-grade SD rats, half male and half female, with a body weight of 200±20 g, and 60 SPF-grade KM mice, half male and half female, with a body weight of 20±2 g. They were all purchased from Changsha Tianqin Biotechnology Co., Ltd. The production license number of experimental animals is: SCXK(Xiang)2019-0014, and the license number for the use of experimental animals is: SYXK(Gu)2019-0001.

[0124] 1.2 Drugs and reagents: The concentrated thick paste of the extract of Luoyan Yufang herbs in this experiment was prepared by adding 70% ethanol (5.8 g of crude drug / g of paste); Xiaozhong Zhitong Tincture (Flower Red Pharmaceutical Group Co., Ltd., Guangxi Zhuang Autonomous Region, national drug approval number Z45021991, batch number: 20210103); Rat TNF-α ELISA kit (Shanghai Fanke Industrial Co., Ltd., batch number: F3056-A); Rat IL-6 ELISA kit (Shanghai Fanke Industrial Co., Ltd., batch number: F3066-A); Rat IL-17A ELISA kit (Shanghai Fanke Industrial Co., Ltd., batch number: F40260-A); Rat IL-1β ELISA kit (Shanghai Fanke Industrial Co., Ltd., batch number: F2923-A); Rat VEGF ELISA kit (Shanghai Fanke Industrial Co., Ltd., batch number: F3055-A); H&E staining solution (Beijing Regen Biotechnology Co., Ltd., batch number: 0603A20); Acid phosphatase staining solution (Beijing Regen Biotechnology Co., Ltd., batch number: 0428A21); Toluidine blue staining solution (Beijing Regen Biotechnology Co., Ltd., batch number: 0413A19); Carrageenan (Shanghai Ruji Biotechnology Development Co., Ltd., batch number: 09016); Sodium pentobarbital (Chengdu Kelong Chemical Co., Ltd., batch number: 2021062221), Chloral hydrate (Chengdu Kelong Chemical Co., Ltd., batch number: 2021062801). The wet compress plaster concentrations of the high, medium, and low dose groups of the Zhuang medicine Luoyan formula were wet wipes containing the medicinal liquid of the Zhuang medicine Luoyan formula with concentrations of 2.32, 1.16, and 0.58 g of crude drug / mL respectively.

[0125] The preparation method of the concentrated thick paste of the above-mentioned extract of Luoyan Yufang herbs: Take each raw material medicinal herb of the prescription amount of the Zhuang medicine Luoyan Yufang and 0.196 parts of borneol, 0.196 parts of menthol, 0.196 parts of camphor, 0.5 parts of Tween-80, and 0.223 parts of methyl salicylate by weight, and set aside; Take the five medicinal herbs of Manshanxiang, Shenjincao, Sifangmupi, Yinxiangpi, and Shanju, and directly steam distill for 8 h to obtain volatile oil, and set aside; Mix the medicinal residues after extracting the volatile oil with the remaining eleven medicinal herbs of the Zhuang medicine Luoyan Yufang except for dragon's blood, frankincense, and myrrh, add 10 times the amount of 60% ethanol, extract for 90 min, extract twice, filter, and recover the ethanol under reduced pressure until there is no alcohol smell, and concentrate to a relative density of 1.20 - 1.25 to obtain clear paste A; Crush frankincense, myrrh, and dragon's blood, dissolve them with appropriate amounts of ethanol and water, add them to clear paste A and mix evenly to obtain thick paste B; Dissolve borneol, menthol, and camphor in a small amount of ethanol, then add volatile oil, Tween-80, and methyl salicylate and mix evenly to obtain mixed solution C; Then mix thick paste B and mixed solution C evenly to obtain the final thick paste.

[0126] 1.3 Instruments: FA1004 electronic balance for plate mounting, 320R benchtop refrigerated centrifuge, MultiskanGo full-wavelength fully automatic multifunctional microplate reader, Leica EG1150 H paraffin embedding machine, Leica RM2255 microtome, Leica DM2500 microscope, IITC520 swelling / volume meter (USA), DHG-9070A vertical forced-air drying oven.

[0127] 2 Methods

[0128] 2.1 Effects on knee osteoarthritis (KOA) in rats

[0129] 2.1.1 Replication of the Knee Osteoarthritis Model and Grouping and Drug Administration: Several SPF-grade SD rats were randomly divided into a sham-operated group (n=12, half male and half female) and a surgically created traumatic knee osteoarthritis model group (n=60, half male and half female) after stratification by body weight. Rats in both groups were anesthetized by intraperitoneal injection of sodium pentobarbital at a concentration of 4 mg / mL (40 mg / kg). In the surgically created traumatic knee osteoarthritis model group, a modified Hulth method was used to create the model. The joint cavity was opened from the medial side of the left knee joint, the meniscus cartilage surface was scraped with a small scraper, the anterior cruciate ligament was cut with ophthalmic scissors, the distal end of the knee joint was pulled to confirm the ligament was severed, the knee joint was reduced, and the joint cavity was sutured closed. In the sham-operated group, only the joint cavity was opened; the meniscus cartilage surface was not scraped, and the anterior cruciate ligament was not cut. Postoperatively, each rat received an intramuscular injection of 105 U of penicillin twice daily for three consecutive days. Six weeks post-surgery, 10% of the postoperative knee joints were randomly selected for histopathological examination. The model of traumatic knee arthritis was established by the proliferation of meniscal chondrocytes and the roughening of the cartilage surface. The success of this batch of model rats was determined by the fact that 80% of the examined knee joints were successful.

[0130] The day after successful modeling, except for the sham-operated group, rats with traumatic knee arthritis were randomly divided into three groups: a KOA model group, a positive control drug (anti-inflammatory and analgesic tincture) group, and high, medium, and low dose groups of the traditional Chinese medicine Luoqianfang. Each group consisted of 10 rats, half male and half female. Hair was removed from the area around the knee joint of each group, approximately 2×2 cm. 2 Apply a wet compress 4cm 2 Medicated wipes (containing 2 mL of drug; based on an average rat weight of 400 g, the high, medium, and low doses of Zhuang medicine Luoqian Yuba plaster were 11.60, 5.80, and 2.90 g of raw drug / kg, respectively; the medium dose is equivalent to 6.15 times the human clinical dose, the same below) were wrapped with non-breathable adhesive tape, keeping the medicated solution in close contact with the skin surface of the knee joint for more than 4 hours. The sham-operated group and the KOA model group received medicated wipes containing 2 mL of physiological saline; the positive control group received medicated wipes containing 2 mL of anti-inflammatory and swelling-reducing tincture, once daily for 8 consecutive weeks.

[0131] 2.1.2 Effects of Zhuang medicine Luoqianfang on serum inflammatory factors and vascular endothelial growth factor levels in rats with knee osteoarthritis

[0132] Following the last administration, rats in each group were anesthetized by intraperitoneal injection of sodium pentobarbital at a concentration of 4 mg / mL (40 mg / kg). Blood was collected from the abdominal aorta, centrifuged at 3000 r / min, and serum was separated. Serum levels of inflammatory factors TNF-α, IL-6, IL-17A, IL-1β, and vascular endothelial growth factor (VEGF) were measured by ELISA.

[0133] 2.1.3 Effects of Zhuang medicine Luoqianfang on the histology of rats with knee osteoarthritis

[0134] Left knee joints of rats from each group were removed, fixed in 4% paraformaldehyde, decalcified, embedded in paraffin, and sectioned at 4µm. HE staining and microscopic examination were performed to observe chondrocyte proliferation and cartilage surface roughness in the meniscus, synovial proliferation within the joint cavity, inflammatory cell infiltration, and angiogenesis. The Mankin score for cartilage damage was also assessed, with specific scoring criteria referenced in the literature. Scoring criteria: 0 points for normal cartilage structure, normal chondrocyte count, normal matrix staining, and relatively intact tide lines; 1 point for irregular fissures on the cartilage surface, diffuse increase in chondrocyte count, decreased matrix staining, and multiple tide lines; 2 points for cartilage fissures extending into the muscle layer, clustered chondrocyte growth, significantly decreased matrix staining, and subchondral blood vessels infiltrating the muscle layer; 3 points for cartilage fissures extending into the radial layer, significantly reduced chondrocyte count, and significantly decreased matrix staining; 4 points for cartilage fissures extending into the calcified layer and complete disappearance of matrix staining; and 5 points for cartilage detachment. A higher score indicates more severe cartilage damage.

[0135] Toluidine blue staining was used to observe cartilage damage. The thickness of the cartilage layer was measured using the Image-Pro Plus 6.0 fully automated image analysis system. The thickness of the cartilage layer was measured at three different locations for each specimen, and the average value was recorded as the result for that specimen. TRAP staining was used to observe the effect of osteoclasts on bone erosion, and the number of osteoclasts in each field of view for each specimen was recorded.

[0136] 2.2 Anti-inflammatory effects

[0137] 2.2.1 Effects of Zhuang medicine Luoqian formula on cotton pellet granuloma in rats

[0138] Sixty SD rats were randomly divided into six groups: a normal control group, a model control group, a positive control group (anti-swelling and analgesic tincture), and high, medium, and low dose groups of the traditional Chinese medicine Luoqianfang, with 10 rats in each group (half male and half female). Except for the normal control group, rats were anesthetized with 4% chloral hydrate before drug administration. Under aseptic conditions, a 1cm incision was made in the axilla, and a pre-weighed, smooth cotton ball (50 mg, accurately weighed and autoclaved) was implanted subcutaneously in the left axilla using ophthalmic forceps. The skin was then sutured, and the area was disinfected with iodine solution for three consecutive days. Rats were then fed as usual. Drug intervention began on the fourth day post-surgery, with a 1cm dressing applied to the implanted cotton ball site. 2 Medicated wipes (containing 2 mL of medication) were applied and bandaged with non-breathable adhesive tape, maintaining close contact between the medication and the skin surface at the site of the cotton ball for at least 4 hours. Normal control and model control groups received medicated wipes (containing 2 mL of normal medication); the positive control group received medicated wipes containing anti-inflammatory and swelling-reducing tincture (containing 2 mL of medication), once daily for 7 consecutive days. On day 8, rats were anesthetized and euthanized. The cotton ball, along with connective tissue, was removed, adipose tissue was removed, and the weight was measured. The net weight of the granuloma was obtained by subtracting the original weight of the cotton ball from the obtained weight. The inhibition rate (%) was calculated as follows: (average weight of granuloma in control group - average weight of granuloma in experimental group) / average weight of granuloma in control group × 100%. Simultaneously, the thymus, spleen, and adrenal glands of the rats were weighed upon removal of the cotton ball, and the coefficients of granulomas and organs in each group were compared.

[0139] 1.2.2.2 Effect of Zhuang medicine Luoqian formula on carrageenan-induced paw edema in rats

[0140] Fifty SD rats were randomly divided into five groups: a normal control group, a positive control group, a high-dose group, a medium-dose group, and a low-dose group, with ten rats in each group (half male and half female). A 1cm layer of the drug was applied to the right hind paw of each rat in each group. 2 Medicated wipes (containing 2 mL of medication) were wrapped with non-breathable adhesive tape, keeping the medication in close contact with the skin of the right hind limb sole for at least 4 hours. The normal control group received normal wipes (containing 2 mL of normal medication); the positive control group received wipes containing medicated anti-swelling and anti-inflammatory tincture (containing 2 mL of medication); the high, medium, and low dose groups of Zhuang medicine Luoqianfang received wipes containing medicated plaster with concentrations of 2.32, 1.16, and 0.58 g crude drug / mL, respectively, once daily for 5 consecutive days. On day 6, 0.1 mL of 1% carrageenan saline solution was injected subcutaneously into the right hind limb of rats to induce inflammation. The paw volume was measured before and 1, 2, 3 and 6 hours after the induction of inflammation using a rat paw volume analyzer. The swelling degree of the right paw was calculated at different time points (paw swelling degree = paw volume at different time points after the induction of inflammation - paw volume before the induction of inflammation).

[0141] 2.3 Statistical Methods

[0142] Quantitative data results are expressed as mean ± standard deviation. The data analysis was performed using SPSS 16.0 software, and one-way ANOVA was used for comparisons between groups. P <0.05, P <0.01 indicates that the difference is statistically significant.

[0143] 3. Results

[0144] 3.1 Effects on knee osteoarthritis (KOA) in rats

[0145] 3.1.1 Effects of Zhuang medicine Luoqian formula on serum inflammatory factors and vascular endothelial growth factor levels in rats with knee osteoarthritis

[0146]

[0147] The serum levels of inflammatory factors and vascular endothelial growth factor (VEGF) in each group of rats are detailed in Table 14. As shown in Table 14, compared with the sham-operated group, the serum levels of inflammatory factors TNF-α, IL-6, IL-17A, IL-1β, and VEGF in the KOA model group were significantly elevated. P <0.01). Compared with the KOA model group, the anti-inflammatory and analgesic tincture could reduce the serum levels of TNF-α, IL-6 and VEGF in KOA rats ( P <0.05 or P <0.01); Zhuang medicine Luoqianfang at high, medium, and low doses can also reduce the serum levels of TNF-α, IL-6, IL-17A, and VEGF in KOA rats, with high and medium doses showing more significant and stable effects. P <0.05 or P <0.01).

[0148] 3.1.2 Effects of Zhuang medicine Luoqianfang on the histology of rats with knee osteoarthritis

[0149] Left knee joints of rats in each group were harvested, fixed in 4% paraformaldehyde, decalcified, embedded in paraffin, and sectioned. HE staining was used to observe synovial hyperplasia, inflammatory cell infiltration, and angiogenesis. Toluidine blue staining was used to observe cartilage damage, and TRAP staining was used to observe the effect of osteoclasts on bone erosion. HE staining results (×50x) of ankle joint morphology in each group of rats are detailed below. Figure 10 Mankin scoring results are detailed in Table 15; toluidine blue staining results (×100) are detailed in Table 15. Figure 11 The cartilage layer thickness results are detailed in Table 16; the TRAP staining results (×200) are detailed in Table 16. Figure 12 The osteoclast count results are detailed in Table 17.

[0150]

[0151] Figure 10 As can be seen, the knee articular surface and synovial membrane structure of rats in the sham-operated group were intact, chondrocytes were horizontally arranged, and the articular cartilage edge was smooth; the edge of the knee articular cartilage of rats in the KOA model group was severely damaged, with cartilage hyperplasia, and disordered arrangement of synovium and chondrocytes; the cartilage structure of rats in the high-dose group of Luoqianfang and the swelling-reducing and analgesic tincture group was closer to normal, with occasional uneven distribution of chondrocytes and a less smooth articular cartilage surface; the articular cartilage edge of the medium- and low-dose groups of Luoqianfang was uneven, and the synovium and chondrocytes were disordered. As shown in Table 15, the Mankin score of the knee articular cartilage of rats in the KOA model group was the highest, and the difference was statistically significant compared with the sham-operated group. P <0.01); the Mankin scores of cartilage decreased in all dosage groups of Zhuang medicine Luoqianfang and the swelling-reducing and analgesic tincture group. P <0.01), and the Mankin score of cartilage was < high dose group of Zhuang medicine Luoqianfang < swelling and pain relief tincture group < medium dose group of Zhuang medicine Luoqianfang < low dose group of Zhuang medicine Luoqianfang.

[0152]

[0153] In toluidine blue staining, the tissue background appears pale blue, while cartilage and osteoblasts appear purplish-blue. Figure 11 As can be seen, the cartilage layer in the normal control group was lighter in color and the chondrocytes were arranged neatly; the cartilage hyperplasia in the knee joint of the KOA model group was darker in color and the chondrocytes were arranged disorderly, indicating that the cartilage in the model group had hyperplasia after traumatic injury; the knee joint cartilage layer of rats in the high, medium and low dose groups of Xiaozhong Zhitong Ding and Zhuangyao Luoqian Fang showed varying degrees of thickening, among which the high and medium doses of Xiaozhong Zhitong Ding and Zhuangyao Luoqian Fang had better cartilage protection effects. As shown in Table 16, the knee joint cartilage of rats in the KOA model group was the thickest, and the difference was statistically significant compared with the sham-operated group. P <0.01); the cartilage thickness decreased in all dosage groups of Zhuang medicine Luoqianfang and in the swelling-reducing and analgesic tincture group ( P <0.01), and the cartilage layer thickness of the high-dose group of Zhuang medicine Luoqianfang < the medium-dose group of Zhuang medicine Luoqianfang < the swelling and pain relief tincture group < the low-dose group of Zhuang medicine Luoqianfang.

[0154]

[0155] TRAP staining results showed osteoclasts distributed within and around the bone marrow cavity, appearing as irregular, purplish-red cells. Figure 12It was observed that no osteoclasts were found at the edge of the bone marrow cavity in the sham-operated group, while osteoclasts were distributed in the cartilage and growth plate of the KOA model group. Numerous osteoclasts eroded bone structure at the edge of the bone marrow cavity beneath the cartilage, resulting in fewer bone trabeculae and an enlarged bone marrow cavity. No osteoclasts were found at the edge of the bone marrow cavity in the high-dose groups of the anti-inflammatory and analgesic tincture and the Zhuang medicine Luoqianfang, while the osteoclast-induced bone erosion effect was reduced in the medium- and low-dose groups of the Zhuang medicine Luoqianfang. As shown in Table 17, the number of osteoclasts in the knee joint of the KOA model group was the highest, with a statistically significant difference compared to the sham-operated group. P <0.01); The number of osteoclasts in the knee joint field of each dose group of Zhuang medicine Luoqianfang and the swelling-reducing and pain-relieving tincture group decreased ( P <0.05 or P <0.01), and the number of osteoclasts in the field of vision was < high dose of Zhuangyao Luoqianfang group < swelling and pain relief tincture group < medium dose of Zhuangyao Luoqianfang group < low dose of Zhuangyao Luoqianfang group.

[0156] 3.2 Anti-inflammatory effects

[0157] 3.2.1 Effects of Zhuang medicine Luoqian formula on cotton pellet granuloma in rats

[0158] As shown in Table 18, compared with the model control group, the cotton pellet granuloma mass of rats in the high, medium, and low dose groups of the swelling-reducing and analgesic tincture and the Zhuang medicine Luoqianfang was significantly reduced, and the differences were statistically significant. P <0.05 or P <0.01). As shown in Table 19, compared with the normal control group, the spleen and thymus indices of rats in the model control group with cotton balls embedded were increased, suggesting that embedding cotton balls can cause enlargement of the immune organs spleen and thymus. The results of cotton ball granuloma weight and inhibition rate in each treatment group are detailed in Table 18, and the results of organ indices of thymus, spleen, adrenal gland, and granuloma in each group are detailed in Table 19.

[0159]

[0160]

[0161] The spleen and granuloma index of rats in the high, medium and low dose groups of the anti-inflammatory and analgesic tincture and the Zhuang medicine Luoqianfang were reduced to varying degrees. P <0.05 or P <0.01), indicating that the Zhuang medicine Luoqianfang can inhibit the formation of cotton pellet granulomas in rats and has an inhibitory effect on non-specific inflammatory responses caused by foreign body stimulation.

[0162] 3.2.3 Effect of Zhuang medicine Luoqian formula on carrageenan-induced paw edema in rats

[0163] Carrageenan induced persistent paw swelling in rats, with the strongest swelling occurring in the first 2 hours, followed by a decrease in swelling over time. Anti-inflammatory and analgesic tinctures could alleviate carrageenan-induced paw swelling. P<0.05 or P <0.01%, the effect can last for 6 hours, but the effect weakens over time. The high dose of Zhuang medicine Luoqianfang can reduce carrageenan-induced toe swelling for up to 3 hours. P <0.05 or P <0.01); the effects of medium and low doses of the Zhuang medicine Luoqianfang can last for 2 hours ( P <0.05 or P <0.01). The results of right paw swelling in rats in each treatment group are detailed in Table 20. This suggests that topical application of the Zhuang medicine Luoqianfang for prophylactic and therapeutic purposes can inhibit carrageenan-induced paw swelling in rats.

[0164]

[0165] Example 3

[0166] A method for preparing a traditional Chinese medicine ointment, Luo Qian Yu Ba Bao, includes the following steps:

[0167] (1) Take the following raw medicinal materials according to the prescribed amount by weight: 8.55 parts of *Lysimachia christinae*, 4.27 parts of *Sinomenium acutum*, 4.27 parts of *Stephania tetrandra*, 3.85 parts of *Lycopodium clavatum*, 4.27 parts of *Polygonum cuspidatum*, 2.14 parts of *Pterocarya stenoptera*, 2.14 parts of *Erythrina variegata*, 3.42 parts of *Saururus chinensis*, 3.42 parts of *Acanthopanax senticosus*, 3.42 parts of *Smilax china*, 3.42 parts of *Smilax china*, 3.42 parts of *Smilax china* bark, 2.14 parts of *Acer buergerianum*, 2.14 parts of *Cinnamomum camphora* bark, 2.14 parts of *Dalbergia odorifera*, 2.14 parts of *Piper kadsura*, 1.28 parts of *Euonymus fortunei*, 0.85 parts of frankincense (processed with vinegar), and 0.85 parts of myrrh (processed with vinegar). 1.28 parts of dragon's blood; and 0.196 parts of borneol, 0.196 parts of menthol, 0.196 parts of camphor, 0.5 parts of Tween-80, and 0.223 parts of methyl salicylate, for later use;

[0168] (2) Take five medicinal materials, namely, Manshanxiang, Shenjincao, Sifangmupi, Yinxiangpi, and Shanju, add 14 times the amount of water, and use steam distillation to extract for 8 hours each time, and extract twice to obtain volatile oil components for later use;

[0169] (3) The residue from which the volatile oil has been extracted is mixed with the remaining medicinal materials of the Zhuang medicine Luoqianyufang except for dragon's blood, frankincense and myrrh. Eight times the amount of ethanol with a volume concentration of 60% is added for reflux extraction, each extraction lasting 1.5 hours, for two extractions. The residue is filtered out and the extracts are combined. The ethanol in the obtained extract is recovered under reduced pressure until there is no alcohol odor, and concentrated to a relative density of 1.20~1.25 to obtain clear extract A.

[0170] (4) Powder the dragon's blood, frankincense and myrrh, dissolve them with an appropriate amount of ethanol and water, add clear paste A and mix well to obtain thick paste B (relative density 1.07), set aside; add borneol, menthol and camphor to an appropriate amount of ethanol to dissolve, then add Tween-80, methyl salicylate and the volatile oil obtained in step (2) and stir evenly to obtain mixture C.

[0171] (5) According to the weight ratio: NP-700: micronized silica gel: aluminum hydroxyl: carbomer 940: CMC-Na: glycerol: water: thick paste B = 1.6: 0.70: 0.80: 0.5: 0.5: 9: 10: 12 and 1% of the total matrix weight of EDTA-2Na; take each matrix component for later use; first take NP-700, micronized silica gel and aluminum hydroxyl and place them in glycerol to swell as phase A, dissolve EDTA-2Na in water, add carbomer 940 and CMC-Na to fully swell as phase B, mix phase B and phase A evenly to prepare the matrix, then add thick paste B and mixed solution C, stir with a mixer at 400r / min for 15 min, after stirring evenly, coat it on non-woven fabric, dry and shape it, add a cover, cut and package it to obtain the final product.

Claims

1. A method for preparing a traditional Chinese medicine plaster containing *Luoqianyu* for treating knee arthritis, characterized in that... The medicinal materials for the Zhuang medicine Luoqian Yufang formula are as follows (by weight): 8.55 parts of *Manshanxiang*, 4.27 parts of *Qingfengteng*, 4.27 parts of *Fenfangji*, 3.85 parts of *Shenjincao*, 4.27 parts of *Huzhang*, 2.14 parts of *Feilongzhangxue*, 2.14 parts of *Haitongpi*, 3.42 parts of *Sanbaicao*, 3.42 parts of *Sanjiapi*, 3.42 parts of *Hongchuanposhi*, 3.42 parts of *Sifangmupi*, 2.14 parts of *Niuerfeng*, 2.14 parts of *Yinxiangpi*, 2.14 parts of *Tenghuangtan*, 2.14 parts of *Shanju*, 1.28 parts of *Changchunweimao*, 0.85 parts of *Ruxiang*, 0.85 parts of *Moyao*, and 1.28 parts of *Longxuejie*. The preparation method includes the following steps: (1) Take the raw materials of Zhuangyao Luoqianyufang and 0.196 parts of borneol, 0.196 parts of menthol, 0.196 parts of camphor, 0.5 parts of Tween-80, and 0.223 parts of methyl salicylate according to the weight proportions, and set aside; (2) Take five medicinal materials: Manshanxiang, Shenjincao, Sifangmupi, Yinxiangpi, and Shanju, and steam distill them for 8 hours to obtain volatile oil. Alternatively, add 12-14 times the amount of water and use steam distillation for 8-10 hours each time, extracting 1-2 times to obtain volatile oil components for later use. (3) Mix the residue from which the volatile oil has been extracted with the remaining medicinal materials of the Zhuang medicine Luoqianyufang (excluding dragon's blood, frankincense, and myrrh), add 8-10 times the amount of ethanol with a volume concentration of 55-65%, and reflux extract for 1.5-2 hours each time, extract twice, filter out the residue and combine the extracts; recover the ethanol from the obtained extract under reduced pressure until there is no alcohol odor, and concentrate it to a relative density of 1.20-1.25; to obtain clear extract A; (4) Powder the dragon's blood, frankincense and myrrh, dissolve them with an appropriate amount of ethanol and water, add clear paste A and mix well to obtain thick paste B, set aside; add borneol, menthol and camphor to an appropriate amount of ethanol to dissolve, then add Tween-80, methyl salicylate and the volatile oil obtained in step (2) and stir evenly to obtain mixture C. (5) According to the weight ratio: NP-700: micronized silica gel: aluminum hydroxyl: carbomer 940: CMC-Na: glycerol: water: thick paste B = (0.95~1.65): (0.70~1.30): (0.40~0.80): 0.5: 0.5: (7.50~14.2): 10: 12, and 1% of the total matrix weight of EDTA-2Na; take each matrix component for later use; first take NP-700, micronized silica gel and aluminum hydroxyl in glycerol to swell as phase A, dissolve EDTA-2Na in water, add carbomer 940 and CMC-Na to fully swell as phase B, mix phase B and phase A evenly to prepare the matrix, then add thick paste B and mixture C, stir with a mixer at 400r / min for 15 min, after stirring evenly, coat on non-woven fabric, dry and shape, add cover, cut and package to obtain the product.

2. The application of the poultice obtained by the preparation method according to claim 1 in the preparation of drugs for the prevention and treatment of knee arthritis.

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