Tongla-5 barbus agent, its preparation process and application in anti-rheumatoid arthritis

Abstract

This invention belongs to the field of traditional Chinese medicine. It relates to a Tonglaga-5 poultice, its preparation process, and its application in treating rheumatoid arthritis. Tonglaga-5 powder is extracted with ethanol to prepare a concentrated Tonglaga-5 solution. The concentrated solution is then freeze-dried to obtain a lyophilized Tonglaga-5 powder, which is further used to prepare a Tonglaga-5 gel ointment and a Tonglaga-5 poultice. Specifically, the optimal extraction process for the Tonglaga-5 concentrate is 77% ethanol (volume fraction), a material-to-liquid ratio of 1:20, and an extraction time of 1 hour. The optimal formulation for the Tonglaga-5 gel is a ratio of carbomer-941: Tonglaga-5 lyophilized powder: sodium polyacrylate: glycerin: aluminum hydroxyl group of 1:2:4:20:5. Pharmacological experiments show that the prepared Tonglaga-5 gel ointment has a uniform and fine appearance, and the Tonglaga-5 poultice prepared using this as a matrix can effectively inhibit the inflammatory response in CIA rats and delay the progression of bone damage.

Description

Technical Field

[0001] This invention belongs to the field of medicine. It relates to a tonlagar-5 poultice, its preparation process, and its application in treating rheumatoid arthritis. Background Technology

[0002] TLG-5, also known as Wuwei Qingzhuo San in the 2020 edition of the Chinese Pharmacopoeia, is a mixture of five medicinal herbs—pomegranate, safflower, cinnamon, cardamom, and long pepper—in a specific ratio. It not only aids digestion and warms the stomach but also provides some protection to the stomach. Clinically, it is used to treat symptoms such as loss of appetite, indigestion, abdominal distension, and diarrhea (National Pharmacopoeia Commission. Pharmacopoeia of the People's Republic of China: Part I [M]. Beijing: China Medical Science and Technology Press. 2020: 648.). Modern research has largely focused on the chemical composition and pharmacological effects of single herbs in the Tonglaga-5 formula. For example, pomegranate mainly contains flavonoids, phenolic acids, and fatty acids, among which punicin has anti-inflammatory and antioxidant effects; safflower mainly contains flavonoids, alkaloids, and polyacetylenes, which have anti-inflammatory, analgesic, and cardiovascular protective effects; cinnamon mainly contains volatile oils, polysaccharides, and sesquiterpenes, which have anti-tumor, anti-inflammatory, and lipid-lowering effects; cardamom mainly contains lignans, volatile oils, and polyphenols, which have liver and intestinal protective, anti-inflammatory, and hypoglycemic effects; and long pepper mainly contains alkaloids, terpenes, and volatile oils, which have lipid-lowering, anti-inflammatory, and anti-tumor effects. However, research on its compound components is limited, and the exploration of its material basis and mechanism of action remains insufficient.

[0003] Modern research has confirmed that Tonglaga-5 has a clear therapeutic effect on osteoporosis by regulating the osteoblast-osteoclast balance (Wu Yihan. Clinical study on the treatment of osteoporosis with Mongolian medicine "Jiawei Tonglaga-5" [D]. Inner Mongolia Medical University, 2024.). It has shown significant therapeutic effects in bone repair, bone formation and reducing inflammation levels. The essence of bone destruction in rheumatoid arthritis (RA) is that the inflammatory microenvironment disrupts the dynamic balance between bone formation and bone resorption. The active ingredients in Tonglaga-5, such as ellagic acid and gallic acid, may reduce synovial inflammation levels by inhibiting inflammatory pathways and prolong its osteoprotective effect in osteoporosis, thus doubly regulating osteoblast-osteoclast function and blocking the pathological progression of RA from inflammation to bone structure destruction. It addresses the core clinical needs of controlling inflammation and protecting bone structure in the treatment of RA, making up for the limitations of traditional nonsteroidal anti-inflammatory drugs that only relieve pain and cannot reverse bone destruction, and also provides a new direction to avoid the infection risks of long-term use of biological agents. Summary of the Invention

[0004] Based on the aforementioned existing technologies, this invention makes two innovations on the basis of previous osteoporosis research: First, it breaks through the limitations of traditional indications and verifies for the first time the effect of Tonglaga-5 on improving synovial inflammation, cartilage degradation and immune disorders in RA through a collagen-induced arthritis (CIA) model; Second, in response to the gastrointestinal side effects of oral preparations, a novel patch formulation is developed, which achieves high concentration accumulation and sustained drug release of the drug in the joint through transdermal drug delivery technology, forming a long-term targeted drug effect at the lesion site.

[0005] This invention, through dual innovation in pharmacodynamics and dosage form, not only provides a scientific basis for the clinical application of Tonglaga-5 in the treatment of RA, but also explores a new path for the modernization of ethnic medicine development, and has important demonstrative significance for promoting the standardization of Mongolian external medicine preparations.

[0006] This invention employs the following method: Based on single-factor experiments, the Box-Behnken response surface methodology is used, with ethanol volume fraction, liquid-to-solid ratio, and extraction time as the factors under investigation, and the comprehensive score of chromatographic peak area of ​​gallic acid, punicalin, and ellagic acid, and the yield of lyophilized powder as the evaluation index, to optimize the ethanol extraction process. A literature review of gel ointment preparation formulations and processes is conducted, and the optimal excipient ratio is determined using single-factor experiments based on the gel ointment's initial tack, holding power, peel strength, appearance evaluation, viscosity, spreadability, and sensory evaluation indicators, for the preparation of the topical formulation. Thirty-six rats were randomly divided into NC group, CIA group, low-dose (TLG-5-L) and high-dose (TLG-5-H) groups of Tungrag-5 ointment, with eight rats in each group. Pharmacodynamic studies are conducted using paw swelling, arthritis index score, immune organ index, micro-CT analysis of ankle joint tissue, and enzyme-linked immunosorbent assay (ELISA) to detect changes in serum inflammatory factor levels as indicators. Results: The optimal extraction process for TLG-5 was 77% ethanol (v / v), a solid-liquid ratio of 1:20, and an extraction time of 1 hour. The optimal formulation for the poultice was 0.3g carbomer-941, 6.0g glycerin, 1.2g sodium polyacrylate, and 0.15g aluminum hydroxyl. Pharmacological experiments showed that compared with the CIA group, the TLG-5-L and TLG-5-H groups showed significant improvements in foot swelling and immune organ indices, and significantly reduced serum TNF-α levels. The study reduced IL-17 inflammatory factor levels, increased IL-10 levels, and elevated BMD, BV / TV, and Tb.N levels in the rat ankle joint, while decreasing Tb.Sp levels. Conclusion: The prepared Tonraga-5 gel ointment has a uniform and fine appearance, providing a reference for further research on this drug. Tonraga-5 poultice effectively inhibits the inflammatory response in CIA rats and delays the progression of bone damage, showing potential therapeutic value.

[0007] Specifically, the present invention provides the following technical solution.

[0008] This invention provides a method for preparing Tonragar-5 poultice, the method comprising: preparing phase A: dissolving carbomer-941 in distilled water overnight, then adding Tonragar-5 lyophilized powder and mixing evenly; preparing phase B: mixing glycerin and sodium polyacrylate evenly; preparing phase C: dissolving aluminum hydroxyl, tartaric acid, and azone in distilled water; adding phase A to phase B and mixing, and adding phase C in multiple equal portions to the above mixture, stirring to obtain Tonragar-5 gel poultice.

[0009] In the preparation method described above, the ratio of carbomer-941:sodium polyacrylate:glycerol:aluminum hydroxyl is 0.5-2:2-8:10-40:2.5-10, preferably 1:4:20:5; and / or, the ratio of carbomer-941:tonragar-5 lyophilized powder:sodium polyacrylate:glycerol:aluminum hydroxyl is 0.5-2:1-4:2-8:10-40:2.5-10, preferably 1:2:4:20:5.

[0010] In the preparation method described above, the ratio of carbomer-941 to its swollen distilled water is 0.5-2g:25-100ml, preferably 1g:50ml; the ratio of aluminum hydroxyl, tartaric acid, azone to its dissolved distilled water is 1.5-6g:50-200ml, preferably 3g:100ml.

[0011] The preparation method described above, wherein the lyophilized Tonlagar-5 powder is prepared by the following method: taking Tonlagar-5 powder raw material, extracting it with ethanol to prepare Tonlagar-5 extract, wherein the volume fraction of ethanol is about 70-90%, preferably 80%, the material-to-liquid ratio is 1:15-1:30, preferably 1:20, and the extraction time is 0.5-2 hours, preferably 1 hour; and then freeze-drying the Tonlagar-5 extract to obtain lyophilized Tonlagar-5 powder.

[0012] The preparation method described above further includes heating and softening the stirred Tonglaga-5 gel paste, coating it on a base material, drying it, and cutting it to obtain the Tonglaga-5 poultice.

[0013] This invention provides a Tonglaga-5 poultice prepared by the aforementioned preparation method.

[0014] This invention provides a tonlagar-5 gel ointment, the preparation method of which includes: preparing phase A: adding carbomer-941 to distilled water and swelling overnight, then adding tonlagar-5 lyophilized powder and mixing evenly; preparing phase B: mixing glycerin and sodium polyacrylate evenly; preparing phase C: dissolving aluminum hydroxyl, tartaric acid, and azone in distilled water; adding phase A to phase B and mixing, and adding phase C in multiple equal portions to the above mixture and stirring to obtain the final product.

[0015] The present invention provides a pharmaceutical preparation, which is further prepared from the Tonglaga-5 poultice and the Tonglaga-5 gel ointment.

[0016] The present invention provides a medicine box comprising the aforementioned Tonglaga-5 poultice, the aforementioned Tonglaga-5 gel ointment, or the pharmaceutical preparation of claim 8.

[0017] The use of the above-described Tonglaga-5 poultice, Tonglaga-5 gel ointment, and pharmaceutical preparation in the preparation of a medicament for treating arthritis and / or reducing immune indices, preferably, the arthritis being rheumatoid arthritis.

[0018] For topical formulations, many factors influence their efficacy, with transdermal absorption having a particularly significant impact. Furthermore, many factors also affect transdermal absorption to some extent, such as physiological factors, where the degree and rate of absorption vary among different species, genders, and ages. Secondly, the physicochemical properties of the drug, such as pH, solubility, molecular weight, and permeability, also significantly affect the absorption process. Finally, different topical dosage forms also influence the transdermal process. Therefore, in the development of topical formulations, determining the best methods to promote transdermal absorption has become crucial in the formulation screening process. Currently, commonly used methods to promote transdermal absorption include chemical, physical, and pharmaceutical methods. Commonly used transdermal absorption enhancers include azone, oleic acid, alcohols, and surfactants. Research shows that the penetration-enhancing strength is in the order of azone > menthol > oleic acid, and the combined use of azone and oleic acid exhibits the strongest penetration-enhancing effect, indicating a synergistic effect among different penetration enhancers. Therefore, this experiment selected azone as the transdermal absorption enhancer. The amount of thickeners, binders, and humectants used is crucial to the preparation of gel pastes. Too much thickener will make the base too dense, making it difficult to spread or absorb, while too little will make the base too fluid, affecting the molding and user experience. Too much binder will make the gel paste too viscous, making it difficult to spread, while too little will result in insufficient adhesion, affecting its adhesion during use. Too much humectant will make the paste too wet, causing penetration or ingredient loss, while too little may cause the paste to dry out, affecting the comfort and moisturizing effect during use.

[0019] Inhibiting bone and joint destruction is key to treating rheumatoid arthritis (RA). A complex relationship exists between bone and RA; the immune and inflammatory responses triggered by RA are the cause, while bone and joint destruction is the effect. During the pathogenesis, the inflammatory cytokine IL-17 produced by T cells stimulates other related inflammatory factors (such as IL-1, IL-6, TNF-α). The secretion of TNF-α (such as TNF-α) leads to an inflammatory state in the joint microenvironment, ultimately resulting in osteoclast differentiation, accelerated bone resorption, and bone erosion and destruction in the joint. This invention uses H&E staining to observe the effects of the drug on joint destruction and cartilage improvement. Results show that Tonglaga-5 poultice effectively inhibits synovitis, inhibits bone erosion, and repairs cartilage damage. It is considered an autocrine stimulant of IL-17 and a paracrine inducer of granulocyte-macrophage colony-stimulating factor, increasing its secretion through positive feedback of its own gene expression. Previous reports have shown that TNF-α... It is an effective pro-inflammatory cytokine that promotes the occurrence and development of RA-related inflammation. Simultaneously, TNF-α... It can also stimulate the biosynthesis of IL-6, IL-8, and GM-CSF, and induce the secretion of adhesion molecules VCAM-1 and ICAM-1.73, thereby causing damage to bones, joints, and cartilage. (This is in contrast to TNF-α.) Similarly, IL-17 is also a pro-inflammatory cytokine, which interacts with TNF-α. IL-17 levels are typically elevated in RA patients with poor prognosis. Furthermore, IL-17 promotes bone erosion and angiogenesis. IL-10, a pleiotropic cytokine, stimulates B cell survival, proliferation, differentiation, and antibody isotype conversion, playing a crucial role in the pathogenesis of RA. IL-10 levels are negatively correlated with RA susceptibility and disease severity; therefore, increasing IL-10 levels can help improve inflammatory states and alleviate disease progression. In this invention, tonragar-5 poultice reduced TNF-α levels in RA rats. The increase in IL-17 levels and the increase in IL-10 levels indicate that the drug has a relieving effect on RA.

[0020] This invention optimizes the coating matrix formulation and preparation process of Tonglaga-5 poultice using Box-Behnken response surface methodology, successfully constructing a transdermal drug delivery system with a hydrophilic polymer framework. This system combines drug sustained-release properties with skin compatibility, significantly improving the penetration efficiency and retention time of active ingredients at the joint site, providing key technical support for targeted drug delivery strategies. Pharmacodynamic evaluation shows that the optimized poultice effectively inhibits foot swelling in rheumatoid arthritis model animals and reduces the expression of pro-inflammatory cytokines. Future preclinical pharmacokinetic and safety evaluations can be conducted to lay the foundation for the clinical translation of this dosage form and provide new ideas for the standardized development of topical formulations of traditional Chinese medicine. Attached Figure Description

[0021] Figure 1 The effect of ethanol volume fraction on the overall score.

[0022] Figure 2 The effect of the feed-to-liquid ratio on the overall score.

[0023] Figure 3 The impact of extraction time on the overall score.

[0024] Figure 4 The impact of the number of extractions on the overall score.

[0025] Figure 5 Response surface plot of each factor and the overall score.

[0026] Figure 6 Changes in body weight of rats in each group. NC: Normal control group; CIA: CIA model group; TLG-5-L: Low-dose tonolacrifice group; TLG-5-H: High-dose tonolacrifice group; MTX: Methotrexate positive control group; Compared with the NC group... P<0.05, P<0.01; compared with the CIA group, P<0.05, P<0.01; compared with the MTX group, P<0.05, P < 0.01; all data are expressed as mean ± standard deviation (n=8)

[0027] Figure 7 Comparison of paw joint width and scores among different groups of rats. NC: normal control group; CIA: CIA model group; TLG-5-L: low-dose tonolagra-5 poultice group; TLG-5-H: high-dose tonolagra-5 poultice group; MTX: methotrexate positive control group; compared with the NC group... P<0.05, P<0.01; compared with the CIA group, P<0.05, P<0.01; compared with the MTX group, P<0.05, P<0.01; all data are expressed as mean ± standard deviation (n=8).

[0028] Figure 8 Serum IL-10, IL-17, and TNF-α levels in each group of rats And a comparison of RF content. NC: normal control group, CIA: CIA model group, TLG-5-L: low-dose tonolagra-5 patch group, TLG-5-H: high-dose tonolagra-5 patch group; MTX: methotrexate positive control group; compared with the NC group, P<0.05, P<0.01; compared with the CIA group, P<0.05, P<0.01; compared with the MTX group, P<0.05, P<0.01; all data are expressed as mean ± standard deviation (n=8). Detailed Implementation

[0029] 1. Experimental Materials

[0030] 1.1 Experimental Apparatus

[0031] Table 1. Experimental Instruments

[0032]

[0033] 1.2 Reagents and Medicinal Materials

[0034] Table 2. Reagents and Medicinal Materials

[0035]

[0036] 2. Methods and Results

[0037] 2.1 Comprehensive Scoring Method

[0038] 2.1.1 Preparation of reference solution

[0039] Accurately weigh punicin, ellagic acid, and piperine reference standards using an analytical balance, dissolve them in 75% methanol, and prepare a reference solution of a certain concentration.

[0040] 2.1.2 Preparation of Tonlagar-5 test solution

[0041] Take 1.0 g of lyophilized Tonlagar-5 powder obtained by ethanol extraction and freeze-drying, place it in an Erlenmeyer flask, add 75% methanol, weigh it with a balance, sonicate for 30 minutes, remove it and cool it in a cool place, weigh it again with a balance, and finally make up the weight loss with 75% methanol. Use a pipette to take a certain amount of supernatant, filter it through a 0.45 μm microporous membrane into a sample vial to obtain the Tonlagar-5 test solution.

[0042] 2.1.3 Chromatographic conditions

[0043] The above-mentioned Tonglaga-5 test sample solution was subjected to chromatographic separation to obtain the Tonglaga-5 test sample extract. The chromatographic conditions were as follows: ThermoODS-2HYPERSILC18 column (250mm×4.6mm, 5μm); mobile phase: acetonitrile (A)-0.5% formic acid aqueous solution (B); gradient elution program: 0.0-10.0min, 7% (A); 10.1-30.0min, 7%-20% (A); 30.1-45.0min, 20%-45% (A); 45.1-60.0min, 45%-100% (A); 60.1-70.0min, 100%-7% (A); flow rate: 1mL / min; injection volume: 10μL; column temperature: 35℃; detection wavelength: 240nm.

[0044] 2.1.4 Determination of the yield of Tonragar-5 freeze-dried powder

[0045] The above-mentioned extract of Tonglaga-5 was concentrated under reduced pressure to a Tonglaga-5 concentrate with a relative density of 1.1-1.3. The Tonglaga-5 concentrate was freeze-dried to obtain a purified freeze-dried powder of Tonglaga-5. The mass of the purified freeze-dried powder of Tonglaga-5 was calculated by the weight loss method.

[0046] 2.1.5 Comprehensive scoring calculation method

[0047] Using the peak areas of gallic acid, punicalin, and ellagic acid, as well as the yield of Tonaga-5 lyophilized powder, the comprehensive score was calculated using the following formula: M = (punicalin peak area / maximum punicalin peak area). 0.2+ (Ellagic acid peak area / maximum ellagic acid peak area) 0.2+ (piperine peak area / maximum piperine peak area) 0.2+ (Yield of purified lyophilized powder / Maximum yield of purified lyophilized powder) 0.4.

[0048] 2.2 Single-factor experiment

[0049] The 2.1.5 comprehensive scoring method was used to evaluate the effects of different extraction factors on the extraction efficiency of Tonglaga-5 crude freeze-dried powder.

[0050] 2.2.1 Ethanol volume fraction

[0051] First, 3g of Tonragar-5 powder was accurately weighed using a balance and placed in a clean 250mL round-bottom flask. Ethanol was added at a volume of 30 times the powder volume, and the mixture was heated under reflux for 1.5 hours to extract the Tonragar-5 extract. Then, single-factor experiments were conducted to investigate the effect of different ethanol volume fractions on the overall extraction score of the Tonragar-5 lyophilized powder. Figure 1The highest overall score was achieved when the volume fraction of added ethanol was 80%. Therefore, ethanol volumes of 60%, 80%, and 100% were selected for subsequent response surface methodology experiments.

[0052] 2.2.2 Material-to-liquid ratio

[0053] 3 g of Tonraga-5 powder was accurately weighed using a balance and placed in a 250 mL round-bottom flask. Extraction was carried out under the conditions of 75% ethanol concentration and 1.5 h to obtain Tonraga-5 extract, which was then freeze-dried to prepare Tonraga-5 lyophilized powder. Single-factor experiments were conducted to investigate the effect of different material-to-liquid ratios on the overall extraction score of Tonraga-5 lyophilized powder. Figure 2 The highest overall score was achieved when the liquid-to-material ratio was 1:20. Therefore, liquid-to-material ratios of 1:15, 1:20, and 1:25 were selected for subsequent response surface methodology experiments.

[0054] 2.2.3 Extraction Time

[0055] Accurately weigh 3g of Tonragar-5 powder raw material and place it in a 250mL round-bottom flask. Add 75% ethanol to make the material-to-liquid ratio 1:30. Measure the Tonragar-5 extract obtained at different extraction times (1h, 2h, 3h), and freeze-dry to prepare Tonragar-5 lyophilized powder. Figure 3 As shown, the comprehensive score is highest when the extraction time is 1 hour, so 0.5 hours, 1 hour, and 1.5 hours were selected for subsequent response surface methodology experiments.

[0056] 2.2.4 Number of extractions

[0057] Three portions (3g each) of tonlagar-5 powder were accurately weighed and placed in 250mL volumetric flasks. 75% ethanol solution was added to achieve a solid-liquid ratio of 1:30. The tonlagar-5 extracts obtained after one, two, and three extractions were measured, and the extracts were then freeze-dried to prepare tonlagar-5 lyophilized powder. The experimental results are as follows: Figure 4 As shown.

[0058] 2.2.5 Response Surface Methodology

[0059] Using Design-Expert 13.0 software, based on the results of single-factor experiments, the factors to be investigated were determined to be ethanol volume fraction (X1), liquid-to-solid ratio (X2), and extraction time (X3). The factor levels are shown in Table 3.

[0060] Table 3. Factor Levels in Response Surface Methodology

[0061]

[0062] Table 4 Response Surface Methodology Design and Results

[0063]

[0064] Table 5. Analysis of variance and significance test

[0065]

[0066] A multivariate fitting analysis was performed on the results in Table 4, yielding the equation: M = 0.8888 - 0.0480X1 + 0.0255X2 - 0.0169X3 + 0.0376X1X2 - 0.0118X1X3 + 0.0061X2X3 - 0.1135X 12 -0.0455X2 2 -0.0255X3 2 .

[0067] Analysis of variance (Table 5) shows that the p-value = 0.0120 < 0.05, and the lack-of-fit term is not significant (p = 0.0601 > 0.05), indicating that the model is realistic and reliable. Since PX1 < PX2 < PX3, the influence of each factor on the overall score, in descending order, is X1 (ethanol volume fraction) > X2 (liquid-to-solid ratio) > X3 (extraction time), R0. 2 =0.8900, Radj=0.7486, indicating that the model has a good fit and can be used to analyze and predict the optimal extraction process of Tonlagar-5.

[0068] Analysis using Design-Expert 13.0 software yielded the following results: Figure 5 The steepness of the response surface methodology directly reflects the strength of the interaction between various influencing factors. As shown in the graph, the steepness of X1X2 is greater than that of X1X3 and X2X3, indicating a stronger interaction between X1X2 and X2, consistent with the significance results in Table 3. Finally, the optimal ethanol extraction process for Tonaga-5 was determined to be: 76.696% ethanol (v / v), a solid-liquid ratio of 1:20.970, and an extraction time of 0.865 h. Considering the convenience of practical operation, the process was modified to: 80% ethanol (v / v), a solid-liquid ratio of 1:20, and an extraction time of 1 h.

[0069] 2.3 Optimal Process Validation Test

[0070] The optimal extraction process obtained by response surface methodology was used in three parallel experiments. The comprehensive score was calculated according to the formula in section 2.2.5, and the results are shown in Table 6. The average comprehensive score of the three validation experiments was 0.9184, which deviated from the predicted value of 0.8980 by 2.27%, indicating that the process has good stability, high reliability, and can be used for the extraction of this drug.

[0071] Table 6. Results of the verification experiment (n=3)

[0072]

[0073] 2.4 Preparation methods of Tonglaga-5 gel ointment and Tonglaga-5 poultice

[0074] A literature review of the preparation formula and process of the gel ointment was conducted. The preparation formula is shown in Table 7. The specific preparation method is as follows: Phase A: Weigh a certain amount of carbomer-941 and add it to a beaker of 15 mL distilled water to swell overnight. Then add 2 g of Tonragar-5 lyophilized powder and mix evenly. Phase B: Mix a certain amount of glycerin and sodium polyacrylate evenly in a mortar. Phase C: Weigh aluminum hydroxyl, tartaric acid, and azone and dissolve them in a beaker of 5 mL distilled water. Add Phase A to Phase B and mix. Add Phase C in small amounts to the mortar and stir to obtain Tonragar-5 gel ointment. After softening the prepared ointment by heating it in a water bath, apply it to a non-woven fabric, dry it, and cut it to obtain Tonragar-5 patch.

[0075] Table 7. Formulation for preparing Tonglaga-5 gel ointment

[0076]

[0077] 2.4.1 Evaluation Indicators for Gel Ointments

[0078] Referring to the adhesive performance testing methods for gel patches specified in Part IV of the 2020 edition of the Chinese Pharmacopoeia, the key adhesive performance indicators of the prepared gel patches were examined, focusing on initial tack, holding power, and peel strength. Simultaneously, a quantitative scoring system was constructed, using the appearance, tackiness, and spreadability of the gel patches as core sensory indicators. The overall score was used as the core basis for evaluating the overall quality of the gel patch. A higher overall score indicates better gel patch performance. Based on practical application, initial tack accounted for 30 points, holding power and peel strength each accounted for 20 points. Therefore, the initial tack score = (measured value / maximum value) × 30, the holding power (peel strength) score = (measured value / maximum value) × 20, and the sensory evaluation included 10 points for appearance, 10 points for tackiness, and 10 points for spreadability. The overall score (Y) = initial tack + holding power + peel strength + sensory score.

[0079] 2.4.1.1 Method for determining initial viscosity

[0080] The specific steps are as follows: Refer to Section IV, 0952, Method I (Determination of Initial Tack), of the 2020 edition of the Chinese Pharmacopoeia for determination of adhesion. The initial tack is determined using the rolling ball ramp stop method, and the largest steel ball number that can adhere to the viscous surface of the test sample is recorded.

[0081] 2.4.1.2 Method for determining holding power

[0082] To measure the holding power of TLG-5 using a holding power tester: Place it parallel to the middle of adjacent test plates and loading plates, and press it back and forth with a pressure roller (without air bubbles); suspend it vertically with a weight at the bottom, and record the time it takes to slide off completely when released. Repeat 3 times, and evaluate the holding power by the time it takes to slide off.

[0083] 2.4.1.3 Method for determining peel strength

[0084] Cut the gel paste into 7 cm × 5 cm pieces, with the top 1 cm × 5 cm as the free end and the bottom 6 cm × 5 cm as the experimental area. Peel off the release liner and attach it to a smooth stainless steel plate. Roll it back and forth 10 times with a 500 g metal cylinder. Place the plate vertically for 20 minutes, then hook a 20 g weight onto the free end boundary and measure the time to complete peeling. The minimum value is scored as 10 points, and the rest are scored as (measured value / minimum value) × 10.

[0085] 2.4.1.4 Sensory Evaluation Indicators

[0086] In the single-factor experiment for screening blank matrices, the appearance, viscosity, and spreadability of the matrices were used as sensory evaluation indicators to score the prepared matrices. The scoring details are shown in Table 8.

[0087] Table 8. Sensory Evaluation Criteria

[0088]

[0089] 2.5 Single-factor experiment on the preparation of Tonglaga-5 gel ointment

[0090] 2.5.1 Investigation into the Dosage of Carbomer-941

[0091] Using the appearance, viscosity, and spreadability of the gel ointment as evaluation indicators, and keeping the amounts of sodium polyacrylate (1.2g), glycerin (6.0g), and aluminum hydroxyl (0.15g) constant, the effect of carbomer-941 dosage on sensory scores was investigated. The results are shown in Table 9. The sensory score was highest when the amount of carbomer-941 was 0.3g, so the dosage of carbomer-941 was selected as 0.3g.

[0092] Table 9. Effect of Carbomer-941 Dosage on Tonglaga-5 Gel Ointment

[0093]

[0094] 2.5.2 Investigation on Glycerin Dosage

[0095] With sodium polyacrylate 1.2g, carbomer-941 0.3g, and aluminum hydroxyl 0.15g kept constant, the effect of glycerol dosage (4.0g, 6.0g, 8.0g) on ​​sensory scores was investigated. The results are shown in Table 10. The sensory score was highest when the glycerol dosage was 6g, so the glycerol dosage of 6.0g was selected.

[0096] Table 10. Effect of glycerin dosage on Tonglagar-5 gel ointment

[0097]

[0098] 2.5.3 Investigation on the dosage of sodium polyacrylate

[0099] With the dosages of carbomer-941 0.3g, glycerin 6.0g, and aluminum hydroxyl 0.15g kept constant, the effect of sodium polyacrylate dosage (1.0g, 1.2g, 1.4g) on ​​sensory scores was investigated. The results are shown in Table 11. The sensory score was highest when the sodium polyacrylate dosage was 1.2g, so the dosage of sodium polyacrylate was selected as 1.2g.

[0100] Table 11. Effect of sodium polyacrylate dosage on Tonglagar-5 gel ointment

[0101]

[0102] The matrix formulation selected in this experiment is 1.2g sodium polyacrylate, 0.3g carbomer-941, 6.0g glycerin, and 0.15g aluminum hydroxyl. The Tonglaga-5 gel ointment prepared using this matrix has a uniform and delicate appearance, fewer bubbles, and good spreadability.

[0103] 3. Study on the efficacy of Tonglaga-5 patch in treating rheumatoid arthritis

[0104] 3.1 Animals

[0105] Forty SPF-grade female SD rats weighing (200±20) g were purchased from SPAF (Beijing) Biotechnology Co., Ltd. (SCXK(Mongolia)2020-0003). They were housed in a 12-hour day-night cycle at a temperature of (20±2)℃ and a relative humidity of (55±5)%, with free access to food and water. The animal experiments were approved by the Medical Ethics Committee of Inner Mongolia Medical University (Approval No.: YKD202301198).

[0106] 3.2 Establishment of the CIA Model

[0107] Based on the required number of animals to be immunized, the mass of Collagen II was determined and dissolved in 1.50 mM glacial acetic acid to a final concentration of 3 mg / ml. The solution was homogenized overnight to obtain a non-clare solution. An equal volume of complete Freund's adjuvant was added to the prepared collagen solution in small, repeated applications. The collagen was emulsified using a homogenizer for approximately 15 minutes to prepare an emulsion (viscous, which did not spread when dropped into water). The entire process was performed on ice, protected from light, and prepared fresh for each use. Immediately after emulsification, the injection was performed. All rats were anesthetized with isoflurane, and 200 μL of the collagen emulsion was injected subcutaneously at a single point at the base of the tail (2-3 cm from the body). Seven days later, 100 μL of the collagen emulsion was injected at the same location. The animals were then observed daily for disease progression. After 14 days, the mice showed decreased activity and varying degrees of swelling and erythema in their paws, indicating successful establishment of the CIA animal model.

[0108] 3.3 Grouping and Dosing

[0109] Rats were randomly divided into 5 groups: a blank control group (Normal control, NC), a CIA group, a TLG-5-L group (0.81 g / kg / d, equivalent to 1 times the clinical equivalent dose), a TLG-5-H group (3.24 g / kg / d, 4 times the clinical equivalent dose), and a methotrexate (MTX) group (administered at a dose of 5 mg / kg for 7 days). -1 Eight rats were placed in each group. Administration of the drug via gavage began on day 7 after booster immunization. The calculated dosage of the drug solution was prepared, and the gavage volume was based on the rat's body weight (1 mL / 100 g). The CIA and NC groups received the same volume of solution. All groups received the drug continuously for 35 days.

[0110] 3.4 General Condition Observation

[0111] During the experiment, the animals' food intake, behavior, condition, coat color, and other general characteristics were observed and recorded daily. Rats' weight was measured and recorded before and every 3 days after the initial immunization. After the drug administration was completed, the thymus and spleen of the rats were weighed, and organ indices were calculated. Results showed that by observing the clinical manifestations and weight changes of rats in each group after the initial immunization, the results were as follows: Figure 7 Following the initial immunization, rats gradually developed symptoms such as swollen and red joints, ataxia, ruffled fur, decreased appetite, and weight loss. Except for the NC group, all other groups exhibited paw swelling and reddened, congested skin on the claws, with the most severe symptoms appearing on day 24. Furthermore, due to decreased appetite after the initial immunization, weight gain was generally slow in all groups except the NC group; from day 12 onwards, the weight of rats in all groups was lower than that of the NC group, and the difference was statistically significant. Figure 6As shown, compared with the NC group, the CIA group rats had increased spleen and thymus wet weight and elevated immune organ index, with statistically significant differences (P<0.05). Compared with the CIA group, the TLG-5-L and TLG-5-H groups rats had significantly decreased spleen and thymus wet weight and significantly reduced immune organ index, with statistically significant differences (P<0.05). Compared with the MTX group, the TLG-5-L group had a higher arthritis score after day 36 (P<0.05).

[0112] 3.5 Evaluation of joint swelling degree and arthritis index

[0113] Before and every 3 days after the initial immunization, rat paw joint swelling was measured using vernier calipers. The arthritis index of each group of rats was evaluated according to the arthritis scoring criteria. The scoring criteria are as follows:

[0114] 0 points: No red spots or swelling in the joint area;

[0115] 1 point: Red spots or mild swelling in the joint area;

[0116] 2 points: Moderate swelling in the joint area;

[0117] 3 points: Severe swelling in the joint area;

[0118] 4 points: The joint area is severely swollen and affects normal activities.

[0119] Each rat's score is the sum of the scores for its four limbs and feet, with an upper limit of 16 points. A higher score indicates more severe arthritis symptoms.

[0120] The results showed that from day 12 onwards, except for the NC group, rats in all other groups successively developed paw joint swelling, with the paw joint width significantly increased compared to the NC group (P<0.05). The paw joint swelling was most severe on day 24 after the initial immunization, and the paw joint width in each group was significantly wider than in the NC group (P<0.05). After drug intervention, the paw joint width in the TLG-5-L and TLG-5-H groups gradually decreased with prolonged administration. After day 24, the paw joint width in the TLG-5-L group was higher than that in the MTX group (P<0.05); on days 36, 42, and 45, the paw joint width in the TLG-5-H group was higher than that in the MTX group (P<0.05). See details below. Figure 7 .

[0121] 3.6 Inflammatory marker detection

[0122] After the last administration, rats in each group were fasted for 12 hours. After anesthetizing the rats, blood was collected from the abdominal aorta, and the centrifuged serum was stored at -80℃. Serum IL-10, IL-17, TNF-α, and RF levels were measured using ELISA. The instruments and kits were used strictly according to the instructions. Compared with the control group, the CIA group showed a significantly decreased IL-10 level and significantly increased levels of IL-17, TNF-α, and RF (P<0.01). Compared with the CIA group, the TLG-5-L and TLG-5-H groups showed significantly increased IL-10 levels and significantly decreased levels of IL-17 and TNF-α (P<0.05). There was no statistically significant difference in serum inflammatory factors (RF) between the MTX and TLG-5-H groups (P>0.05). See below for detailed results. Figure 8 .

Claims

1. A method for preparing a Tonglaga-5 poultice, characterized in that, The method includes: preparing phase A: adding carbomer-941 to distilled water and swelling overnight, then adding tollaga-5 lyophilized powder and mixing evenly; preparing phase B: mixing glycerol and sodium polyacrylate evenly; preparing phase C: dissolving aluminum hydroxyl, tartaric acid, and azone in distilled water; adding phase A to phase B and mixing, and adding phase C in multiple equal portions to the above mixture, stirring to obtain tollaga-5 gel ointment.

2. The preparation method according to claim 1, characterized in that, The ratio of Carbomer-941: Sodium polyacrylate: Glycerin: Aluminum hydroxyl is 0.5-2:2-8:10-40:2.5-10, preferably 1:4:20:5; and / or the ratio of Carbomer-941: Tonragar-5 lyophilized powder: Sodium polyacrylate: Glycerin: Aluminum hydroxyl is 0.5-2:1-4:2-8:10-40:2.5-10, preferably 1:2:4:20:

5.

3. The preparation method according to claim 1 or 2, characterized in that, The ratio of carbomer-941 to its swollen distilled water is 0.5-2g:25-100ml, preferably 1g:50ml; the ratio of aluminum hydroxyl, tartaric acid, azone to its dissolved distilled water is 1.5-6g:50-200ml, preferably 3g:100ml.

4. The preparation method according to any one of claims 1-3, characterized in that, The lyophilized Tonlagar-5 powder is prepared by the following method: Tonlagar-5 powder raw material is taken and extracted with ethanol to prepare Tonlagar-5 extract, wherein the volume fraction of ethanol is about 70-90%, preferably 80%, the material-to-liquid ratio is 1:15-1:30, preferably 1:20, and the extraction time is 0.5-2 hours, preferably 1 hour; the Tonlagar-5 extract is then freeze-dried to obtain lyophilized Tonlagar-5 powder.

5. The preparation method according to any one of claims 1-4, characterized in that, The method further includes heating and softening the stirred Tonglaga-5 gel paste, coating it on a base material, drying it, and cutting it to obtain Tonglaga-5 poultice.

6. A Tonglaga-5 poultice prepared by the preparation method according to any one of claims 1-5.

7. A tonlagar-5 gel ointment, characterized in that, The preparation method includes: preparing phase A: adding carbomer-941 to distilled water and swelling overnight, then adding tollaga-5 lyophilized powder and mixing evenly; preparing phase B: mixing glycerol and sodium polyacrylate evenly; preparing phase C: dissolving aluminum hydroxyl, tartaric acid, and azone in distilled water; adding phase A to phase B and mixing, and adding phase C in multiple portions to the above mixture and stirring to obtain the final product.

8. A pharmaceutical preparation, characterized in that, The pharmaceutical preparation is further made from the Tonglaga-5 poultice of claim 6 and the Tonglaga-5 gel ointment of claim 7.

9. A medicine box comprising the Tonglaga-5 poultice of claim 6, the Tonglaga-5 gel ointment of claim 7, or the pharmaceutical preparation of claim 8.

10. Use of the tonlagar-5 poultice of claim 6, the tonlagar-5 gel ointment of claim 7, and the pharmaceutical preparation of claim 7 in the preparation of a medicament for treating arthritis and / or reducing immune indices, preferably, the arthritis being rheumatoid arthritis.

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