Bacteriostatic Tibetan medicine preparation and preparation method thereof

Abstract

This invention discloses an antibacterial Tibetan medicine preparation and its preparation method, belonging to the field of pharmaceutical preparation technology. The preparation formulation disclosed in this invention exhibits good antibacterial and anti-inflammatory activity, containing a large amount of polyphenolic substances with good water solubility. Analysis identified 104 compounds, with polyphenols (gallic acid and its derivatives), anthraquinones (emodin, etc.), and alkaloids (benzoyl aconitine, etc.) as the main active substances. Simultaneously, no aconitine, hypoaconitine, or neoaconitine (diester aconitines requiring content limits) were detected, reflecting the scientific nature of Tibetan medicine's "processing to reduce toxicity." Furthermore, this preparation is effective against methicillin-resistant Staphylococcus aureus (MRSA)... MRSA The presence of distinct inhibition zones against Candida albicans and Escherichia coli indicates that the preparation has a significant antibacterial effect, providing scientific support for the optimization of Tibetan medicine preparation processes, quality control, and standardized clinical application.

Description

Technical Field

[0001] This invention belongs to the field of drug preparation technology, specifically relating to an antibacterial Tibetan medicine preparation and its preparation method. Background Technology

[0002] Xiuqiong Enbao Powder is a traditional Tibetan medicine preparation composed of nine herbs: sickle-shaped radix, rhubarb, iron bark, chebula, hairy chebula, amla, myrrh, broad-leaved vine, and artificial musk. Among these, sickle-shaped radix, myrrh, musk, and iron bark all have the effects of dispelling wind and dampness, reducing swelling and relieving pain. Combined with chebula, hairy chebula, amla, rhubarb, and broad-leaved vine, it promotes qi circulation, removes blood stasis, and harmonizes the three factors (blood, qi, and blood). It not only dispels wind and dampness, reduces swelling and relieves pain, but also regulates imbalances in the three factors of Long, Chiba, and Peigen. This powder has been a traditional treatment for gout, dampness-related arthralgia, swelling, pain, fever, herpes, and epidemic fever caused by "Gamba" and "yellow water" diseases throughout Tibetan medicine history. However, due to the lack of optimization of various process parameters in the original preparation process, the consistency of quality and safety could not be guaranteed, affecting the promotion and clinical application of this preparation. Summary of the Invention

[0003] The technical problem to be solved by the present invention is to provide an antibacterial Tibetan medicine preparation and its preparation method, so as to solve the technical problems of safety and unstable process of existing Tibetan medicine preparations.

[0004] To achieve the above objectives, the technical solution adopted by the present invention is as follows: providing an antibacterial Tibetan medicine preparation (XQEB), the raw materials used including, by mass parts: 80-120 parts of *Hylocereus zebrina*, 20-30 parts of *Rheum palmatum*, 40-60 parts of *Impatiens balsamina*, 50-80 parts of *Terminalia chebula*, 50-80 parts of *Terminalia chebula*, 70-100 parts of *Phyllanthus emblica*, 20-30 parts of *Myrrha mukur*, 80-120 parts of *Tinospora sinensis*, and 0.6-1 parts of artificial musk.

[0005] Based on the above technical solution, the present invention can be further improved as follows: Furthermore, the raw materials used, by weight, include: 100 parts of sickle-shaped yam, 25 parts of rhubarb, 50 parts of iron bark, 65 parts of Terminalia chebula, 65 parts of Terminalia chebula var. pubescens, 85 parts of Phyllanthus emblica, 25 parts of myrrh, 100 parts of broad-skinned vine, and 0.8 parts of artificial musk.

[0006] The present invention also discloses a method for preparing the above-mentioned antibacterial Tibetan medicine preparation (XQEB), which includes the following steps: except for artificial musk, the remaining raw materials are crushed, then water is added and decocted 1-3 times, each time for 2-5 hours, the decoction is combined and concentrated, then mixed with artificial musk, filled and sterilized to obtain the product.

[0007] Based on the above technical solution, the present invention can be further improved as follows: Furthermore, the ratio of ingredients to liquid during decoction is 1g:10-14mL.

[0008] Furthermore, the decoction is performed twice, with each decoction lasting 3 hours, and the ratio of the decoction to the liquid is 1g:12mL.

[0009] The beneficial effects of this invention are as follows: The antibacterial Tibetan medicine preparation (XQEB) disclosed in this invention has good antibacterial and anti-inflammatory activity. It contains a large amount of polyphenols and has good water solubility, specifically manifested in: 1. UPLC-QTOF-MS / MS component analysis revealed a total of 104 compounds, with polyphenols (gallic acid and its derivatives), anthraquinones (emodin, etc.), and alkaloids (benzoyl aconitine, etc.) as the main active substances. At the same time, no toxic diester aconitines such as aconitine, hypoaconitine, and neoaconitine, which require content restrictions, were detected. Therefore, this preparation has good safety and reflects the scientific nature of Tibetan medicine's "processing to reduce toxicity".

[0010] 2. Antibacterial Tibetan medicine preparation (XQEB) is effective against methicillin-resistant Staphylococcus aureus (MRSA). MRSA The presence of distinct inhibition zones against Candida albicans and Escherichia coli indicates that the preparation has a significant antibacterial effect, providing scientific support for the optimization of Tibetan medicine preparation processes, quality control, and standardized clinical application.

[0011] 3. Animal experiments have confirmed that XQEB's therapeutic effect on MV model mice exhibits a significant dose-dependent effect: the high-dose group can significantly inhibit the growth of Candida albicans and Escherichia coli in the vagina, repair vaginal mucosal damage, reduce inflammatory cell infiltration, and has no significant toxicity to major organs such as the heart, liver, spleen, lungs, and kidneys, demonstrating good safety. Mechanistic studies have shown that XQEB can alleviate inflammatory responses by inhibiting the expression of inflammatory factors such as IL-6 and TNF-α; at the same time, it can regulate the vaginal flora structure, increase the richness and diversity of the flora, restore the Firmicutes / Bacteroidetes (F / B) ratio to a healthy level, promote the growth of beneficial bacteria (such as Lactobacillus), inhibit the proliferation of pathogenic bacteria (such as Escherichia coli and Shigella), and improve the dysbiosis state. Attached Figure Description

[0012] Figure 1 Standard curve of gallic acid; Figure 2 The effect of decoction time on the total phenol extraction yield; Figure 3 The effect of the solid-liquid ratio on the total phenol extraction yield; Figure 4 The effect of the number of decoctions on the total phenol extraction yield; Figure 5 To investigate the effect of decoction time and liquid-to-solid ratio on phenol content; Figure 6Contour plot showing the interaction between decoction time and liquid-to-solid ratio on phenol content; Figure 7 To investigate the effects of decoction time and number of decoctions on phenol content; Figure 8 Contour plot showing the interaction between decoction time and number of decoctions on phenol content; Figure 9 To investigate the effect of the number of decoctions and the liquid-to-material ratio on the phenol content; Figure 10 Contour plot showing the interaction between the number of decoctions and the liquid-to-solid ratio on phenol content; Figure 11 The total ion chromatogram of the detergent in UPLC-QTOF-MS positive ion mode; Figure 12 The total ion chromatogram of the detergent in UPLC-QTOF-MS negative ion mode; Figure 13 Macroscopic observation of the external vagina of mice; Figure 14 Analysis of vaginal flora in mice; Figure 15 Sections of mouse vaginal tissue for pathological analysis; Figure 16 Pathological sections of mouse internal organs; Figure 17 To detect the expression of TNF-α and IL-6 proteins in each group using Western blotting; Figure 18 The relative expression levels of TNF-α protein in each group; Figure 19 The relative expression levels of TNF-α protein in each group are shown. Detailed Implementation

[0013] The specific embodiments of the present invention are described below to facilitate understanding of the invention by those skilled in the art. Unless otherwise specified, specific conditions are applied according to conventional conditions or the manufacturer's recommendations. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products. However, it should be understood that the present invention is not limited to the scope of the specific embodiments. For those skilled in the art, various modifications are obvious as long as they fall within the spirit and scope of the invention as defined and determined by the appended claims. All inventions utilizing the concept of this invention are protected.

[0014] Gallic acid reference standard (Chengdu Pusi Biotechnology Co., Ltd., batch number: PS014298); tryptone powder, yeast extract powder, and agar were all purchased from Beijing Aoboxing Biotechnology Co., Ltd., and glucose and sodium chloride were all purchased from Tianjin Aopusheng Chemical Co., Ltd.

[0015] Escherichia coli CMCC(B)44102, Staphylococcus aureus CMCC(B)26003, methicillin-resistant Staphylococcus aureus ( MRSA ATCC43300, Staphylococcus epidermidis CMCC(B)26069, and Candida albicans CMCC(F)98001 were all purchased from Shanghai Luwei Technology Co., Ltd.

[0016] Example 1 An antibacterial Tibetan medicine preparation, the raw materials of which include: 100 parts of sickle-shaped thorn bean, 25 parts of rhubarb, 50 parts of iron rod hammer, 65 parts of Terminalia chebula, 65 parts of hairy Terminalia chebula, 85 parts of Phyllanthus emblica, 25 parts of myrrh, 100 parts of broad-skinned vine, and 0.8 parts of artificial musk.

[0017] The preparation method of the above-mentioned antibacterial Tibetan medicine preparation includes the following steps: except for artificial musk, the other raw materials are crushed, and then water is added at a ratio of 1g:12mL to decoct twice, each time for 3 hours. The decoctions are combined and concentrated, and then mixed with artificial musk to obtain the final product.

[0018] Example 2 An antibacterial Tibetan medicine preparation, the raw materials of which include: 80g of sickle-shaped thorn bean, 20g of rhubarb, 60g of iron rod hammer, 50g of Terminalia chebula, 80g of Terminalia chebula, 100g of Phyllanthus emblica, 20g of myrrh, 120g of broad-skinned vine and 0.6g of artificial musk.

[0019] The preparation method of the above-mentioned antibacterial Tibetan medicine preparation includes the following steps: except for artificial musk, the remaining raw materials are crushed, and then water is added at a ratio of 1g:10mL to decoct once, each time for 5 hours. The decoctions are combined and concentrated, and then mixed with artificial musk to obtain the final product.

[0020] Example 3 An antibacterial Tibetan medicine preparation, the raw materials of which include: 120g of sickle-shaped thorn bean, 30g of rhubarb, 40g of iron rod hammer, 80g of chebula, 50g of hairy chebula, 70g of amla, 30g of myrrh, 80g of broad-skinned vine and 1g of artificial musk.

[0021] The preparation method of the above-mentioned antibacterial Tibetan medicine preparation includes the following steps: except for artificial musk, the remaining raw materials are crushed, and then water is added at a ratio of 1g:14mL to decoct three times, each time for 2 hours. The decoctions are combined and concentrated, and then mixed with artificial musk to obtain the final product.

[0022] The following experiment uses the formulation components of Example 1 as an example.

[0023] Experimental Example 1: Determination of Polyphenol Content and Calculation of Dry Extract Yield 1. Construction of the standard curve for gallic acid Accurately weigh 10.0 mg of gallic acid standard, dissolve it in distilled water, and dilute to a 100 mL volumetric flask to prepare a standard solution with a concentration of 0.1 mg / mL. Then, sequentially measure 0, 0.1, 0.2, 0.3, 0.4, 0.5, and 0.6 mL into 10 mL volumetric flasks. Add 0.5 mL of Folin-phenol reagent and react for 5 min. Then, add 1.5 mL of 20% sodium carbonate solution to each volumetric flask and dilute to the mark with distilled water. Transfer the diluted solution to a 10 mL centrifuge tube, incubate at 75°C for 10 min, and let stand at room temperature for 2 h. Measure the absorbance of gallic acid at 760 nm. Plot a standard curve with gallic acid concentration on the x-axis and absorbance at 760 nm on the y-axis, as shown below. Figure 1 As shown, the linear regression equation obtained is: Y = 1.6417X + 0.0096, with a correlation coefficient R² = 0.9993. The results indicate that the absorbance value and gallic acid concentration exhibit a good linear relationship within the concentration range of 0.1-0.5 mg / mL, and this method can be used to analyze the total phenol content in the preparation.

[0024] The raw materials were sampled and decocted according to Example 1. The decoction was concentrated to a viscous state and transferred to an evaporating dish that had been dried and accurately weighed. The evaporating dish was placed in a 60°C oven to dry. After cooling to room temperature, an appropriate amount of the dried product was accurately weighed. The test solution (prepared by the same method as above) was taken and the absorbance was measured at a wavelength of 760 nm. The polyphenol content in the sample was calculated according to the gallic acid standard curve equation.

[0025] 2. Calculation method for dry extract yield Collect all extracts and filter them. Concentrate the filtrate to a viscous consistency and pour it into a constant-weight evaporating dish. Dry the dish in a 60°C oven until constant weight. Remove the dish and cool it to room temperature in a desiccator. Weigh the dish precisely and record the relevant data. Calculate the yield of the dry extract using the following formula: ; Where a is the dry extract yield (%), b is the extract mass (g), and c is the total mass of raw materials (g).

[0026] Experimental Example 2: Study on Preparation Process 1. Single-factor experiment The three factors of decoction time, material-to-liquid ratio, and number of decoctions all had significant effects on the polyphenol extraction rate and the yield of dry extract. Single-factor experiments were conducted sequentially, with five levels of decoction time (1h, 2h, 3h, 4h, 5h), six levels of material-to-liquid ratio (1:4, 1:6, 1:8, 1:10, 1:12, and 1:14, g / mL), and three levels of decoctions (1 time, 2 times, and 3 times).

[0027] (1) Simmering time Five portions of the formulation raw materials were prepared according to the formula disclosed in Example 1. The decoction time for each batch was set to 1, 2, 3, 4 and 5 hours, respectively. The raw materials were then concentrated, dried, and the yield and polyphenol content of the dry extract were calculated.

[0028] (2) Material-liquid ratio Prepare six portions of the formulation raw materials according to the formula disclosed in Example 1. Add distilled water at material-to-liquid ratios (g / mL) of 1:4, 1:6, 1:8, 1:10, 1:12 and 1:14 respectively. After decoction, concentrate and dry the raw materials, and calculate the yield of dry extract and polyphenol content.

[0029] (3) Number of times to boil Prepare six portions of the formulation raw materials according to the formula disclosed in Example 1, decoct them 1, 2 and 3 times respectively, combine and concentrate them, dry them, and calculate the yield of dry extract and polyphenol content.

[0030] The results are as follows Figures 2-4 As shown, the product had the highest phenol content when the decoction time was 3 hours, the material-to-liquid ratio was 1 g: 12 mL, and the decoction was performed twice.

[0031] 2. Response surface methodology Based on the single-factor experiments, response surface methodology was used to further optimize the process parameters. This experiment identified decoction time (A), material-to-liquid ratio (B), and number of decoctions (C) as the three main influencing factors, and set three levels for each factor based on the results of the single-factor experiments. Referring to the Box-Behnken central composite design method, a three-factor, three-level response surface experiment with 17 experimental points was designed, using polyphenol content and dry extract yield as evaluation indicators, and then calculating the comprehensive score.

[0032] Based on the suitable level range determined by the three single-factor experiments of material-liquid ratio, number of decoctions, and decoction time, a three-factor, three-level experimental condition was set up, with a total of 17 experimental points and response surface analysis was carried out. The experimental combinations and corresponding results of each factor are detailed in Table 1.

[0033] Table 1 Results of Response Surface Methodology Experiments

[0034] Table 1 shows that the highest yield of dry extract was achieved when the decoction was simmered for 3 hours, the liquid-to-solid ratio was 12:1, and the decoction was performed twice.

[0035] 3. Interaction analysis of various factors The interaction effects of various factors on polyphenol content are as follows: Figures 5-10 As shown. By Figure 5It can be seen that, under the interactive influence of decoction time and liquid-to-solid ratio, the phenol content generally shows a trend of first increasing and then decreasing with increasing decoction time, and also shows a change of first increasing and then decreasing with increasing liquid-to-solid ratio. However, the overall increase and decrease of polyphenol content under the influence of the two factors are different within the same level range. With the increase of the former, the overall increase is less than the decrease, while with the increase of the latter, the overall increase is greater than the decrease. The phenol content reaches its maximum peak point when the decoction time is about 3 hours, with a value of about 0.75 mg / g. The phenol content reaches its maximum when the liquid-to-solid ratio is about 12 g / mL, with a maximum value of about 0.75 mg / g. The response surface is generally convex, and the interaction effect between the two factors is significant.

[0036] according to Figure 7 It can be seen that under the interaction of decocting time and number of decocting times, the phenol content reaches its peak when the number of decocting times reaches about 2. As both increase, the phenol content shows a trend of first increasing and then decreasing. Moreover, the overall change of phenol content around the peak point is similar for both. The tangent plane of the response surface is first gentle and then steep, and the tangent slope first decreases and then increases.

[0037] according to Figure 9 It can be seen that, under the interaction of the number of decoctions and the liquid-to-material ratio, the phenol content is more affected by the liquid-to-material ratio than by the number of decoctions, and the interaction effect between the two is significant.

[0038] According to the contour map ( Figure 6 , Figure 8 and Figure 10 As can be seen, the interactive contour lines are elliptical, and the maximum phenol content can be predicted within the red area of ​​the response point map. Therefore, the optimal extraction process parameters are determined to be: a material-to-liquid ratio of 1g:12mL, a single decoction time of 3h, and two decoction cycles.

[0039] Experimental Example 3: Identification of Chemical Composition The components were identified using UPLC-QTOF-MS technology, and preliminary antibacterial and other follow-up studies were conducted on the formulation samples prepared in Example 1.

[0040] 1. Preparation of the test solution Take 1 mL of the preparation sample liquid and add 1 mL of methanol. Extract by sonication for 10 min. Place the mixture in a centrifuge and centrifuge at 13000 r / min for 15 min. Take the supernatant and filter it through a 0.22 μm filter membrane. Collect the filtrate as the test solution.

[0041] 2. Chromatographic conditions A Waters ACQUITY UPLC HSS T3 2.1×100mm×1.8μm (LC-226) was used; acetonitrile (A) and 0.1% formic acid solution (B) were used as the mobile phases, and the gradient elution program was as follows: 0-40 min, 15% A; 40-55 min, 15% A→40% A; 55-60 min, 40% A→90% A; 60-65 min, 90% A; 65-65.1 min, 90% A→0 A; 65.1-70 min, 0% A; the injection volume was 1 μL, the flow rate was 0.3 mL / min, and the column temperature was 35℃.

[0042] 3. Mass spectrometry conditions The ion source was electrospray ionization (ESI); the electrospray voltage was 3500V (positive) and -3500V (negative); the sheath gas temperature was 350℃; the sheath gas flow rate was 8L / min; the drying gas flow rate was 11L / min; the drying gas temperature was 350℃; the scanning mode was full scan; the scanning range was 100~1700m / z; the cone voltage was 100V; and the collision energy gradients were 10eV, 20eV and 40eV, respectively.

[0043] 4. Chemical composition analysis The chemical composition of the formulation was analyzed using UPLC-QTOF-MS in both positive and negative ion modes. The total ion current (TIC) of the formulation samples in both modes is shown below. Figure 11 and Figure 12 A total of 104 compounds were detected, and the main compounds are shown in Table 2.

[0044] Table 2. Identification Results of Main Chemical Components

[0045] The table shows that this preparation contains a large number of polyphenolic derivatives such as gallic acid, chebulic acid, and ellagic acid, anthraquinone components such as emodin and aloe-emodin, and alkaloids such as benzoyl aconitine. These components are the material basis for its anti-inflammatory and antibacterial effects. Based on the above results, it is speculated that the anti-inflammatory and antibacterial activity of this preparation originates from a synergistic mechanism involving multiple components, multiple targets, and multiple pathways. In addition, combined with the analysis of the detection results in the table, diester-type aconitines (such as aconitine, hypoaconitine, and neoaconitine), which are the focus of quality control, were not detected, indicating that these components have undergone hydrolysis during the processing of medicinal materials and the decoction of the prescription, transforming into monoester-type alkaloids.

[0046] Experiment Example 4: Antibacterial Activity Test 1. Preparation of culture medium Preparation of liquid culture medium: Weigh 2.50g tryptone powder, 1.25g yeast extract and 2.50g sodium chloride into a 500mL Erlenmeyer flask and mix well. Add 250mL of pure water and sterilize at 121℃ for 30min. After cooling, place in a clean bench for later use.

[0047] Preparation of Sabouraud broth: Weigh 2.50g of tryptone powder and 10g of glucose into a 500mL Erlenmeyer flask and mix well. Add 250mL of pure water and adjust the pH to 5.6±0.2 (adjust with 1mol / L hydrochloric acid or sodium hydroxide). Sterilize at 121℃ for 30min. After cooling, place in a clean bench for later use.

[0048] Preparation of solid culture medium: Weigh 2.50g tryptone powder, 1.25g yeast extract powder, 2.50g sodium chloride and 3.75g agar, add them to a 500mL Erlenmeyer flask and mix well. Add 250mL of pure water and sterilize in a high-temperature sterilizer at 121℃ for 30min. After removing from the sterile environment, pour the mixture into petri dishes while hot to make nutrient agar plates. Each petri dish contains 12-15mL. Let the plates cool and solidify for later use.

[0049] Preparation of Sabouraud dextrose agar (SDA): Weigh 10.0g glucose, 2.50g tryptone powder and 5.0g agar, add to a 500mL Erlenmeyer flask and mix well. Add 250mL pure water, adjust the pH to 5.6, sterilize at 121℃ for 30min, and pour into petri dishes while hot to make Sabouraud dextrose agar plates. Each petri dish contains 12-15mL. Let cool and solidify for later use.

[0050] 2. Preparation of bacterial suspension Inside the laminar flow hood, take a flame-sterilized inoculation loop, pick a single colony of the target bacterial species, and quickly transfer it to a pre-sterilized 15mL centrifuge tube (containing 8mL of sterile liquid culture medium). Maintain aseptic operation throughout the process to avoid contamination. After sealing with high-temperature sealing adhesive, place the centrifuge tube in a 37°C constant temperature shaker and continuously shake at 100rpm for 18-24 hours to ensure sufficient dispersion and efficient proliferation of the bacteria, obtaining a high-density bacterial suspension, i.e., a turbid liquid visible to the naked eye. Measure its OD value with a UV spectrophotometer to ensure a final concentration of 1×10⁻⁶. 8 Prepare a bacterial suspension of CFU / mL for later use.

[0051] 3. Antibacterial test The antibacterial activity of the preparation was determined using the filter paper diffusion method. Qualitative filter paper discs were prepared in batches using a stainless steel punch with an inner diameter of 6.00 mm. These discs were then placed in glass containers and autoclaved at 121°C for 20 min, before being transferred to a clean bench for cooling. Sterilized filter paper discs were thoroughly soaked with the sample solutions to be tested for 24 h. The bacterial suspension was then evenly spread onto the surface of an SDA plate. After complete absorption of the bacterial suspension, the sample-soaked filter paper discs were flattened onto the agar surface and gently pressed to ensure full contact and prevent displacement. The inoculated plates were then inverted and placed in a CO2 incubator and incubated at 37°C for 24 h. After incubation, the diameter of the inhibition zone was measured and the average value was calculated. The antibacterial effect is determined based on the diameter of the diffusion zone (d): if the diameter of the antibacterial zone d≤6mm (consistent with the diameter of the filter paper), it indicates that the sample does not have significant antibacterial activity; 6mm<d≤10mm reflects that the sample has a detectable antibacterial effect, which is low sensitivity; 10mm<d≤14mm is moderate sensitivity, and d>14mm is high sensitivity.

[0052] The results of the inhibition zone diameter determination are shown in Table 3. The preparation showed different degrees of antibacterial effect against each test species, including methicillin-resistant Staphylococcus aureus (MRSA). MRSA The formulation showed good antibacterial activity against Escherichia coli and Candida albicans, and the antibacterial activity gradually increased with increasing concentration.

[0053] Table 3. Results of the measurement of the diameter of the inhibition zone

[0054] Experimental Example 5: The therapeutic effect of the formulation on mixed-infection vaginitis in vivo. To further study the efficacy of the preparation, this experiment used Fuyanjie wash as a positive control and evaluated the antibacterial effect of the antibacterial Tibetan medicine preparation prepared in Example 1 of this invention on vaginitis caused by Candida albicans and Escherichia coli in mice.

[0055] 1. Culture of experimental strains Inside a clean bench, single colonies of *Candida albicans* and *Escherichia coli* were picked up separately using a sterile inoculation loop and inoculated into 10 mL centrifuge tubes containing 5 mL of sterile liquid culture medium. The tubes were then incubated at 37°C and 100 rpm for 6 hours. The OD value was measured to be 10. 8 CFU / ml, for later use.

[0056] 2. Animal grouping and modeling A vaginitis model was established in mice, with the mice specifically grouped as follows: (1) Blank group: 0.01M PBS buffer; (2) Model group: inoculated with Candida albicans and Escherichia coli; (3) Control group, inoculated with Candida albicans and Escherichia coli. After successful modeling, commercially available Fuyanjie diluted 10 times was administered for 8 consecutive days, once a day. (4) Drug administration group: High-dose group: After inoculation with Candida albicans and Escherichia coli, and successful modeling, Tibetan medicine preparations were administered at a concentration of 4.8 g / mL for 8 consecutive days, once a day. Medium-dose group: After inoculation with Candida albicans and Escherichia coli, and successful modeling, the patient was given a Tibetan medicine preparation at a concentration of 2.4 g / mL for 8 consecutive days, once a day. Low-dose group: After inoculation with Candida albicans and Escherichia coli, and successful modeling, Tibetan medicine preparation at 1.2 g / mL was administered continuously for 8 days, once a day.

[0057] Except for the control group, all other groups of mice were used to establish a pseudoestrus model by subcutaneous injection of 0.2 mL of estradiol benzoate injection (2 mg / mL). The administration frequency was once every 2 days for 6 consecutive days. On the 7th day of pseudoestrus, mice were inoculated vaginally with Candida albicans and Escherichia coli in sequence, once a day, to establish the model. The observation indicators were vulvar redness and swelling with a large amount of purulent discharge. The appearance of the above signs was considered as successful model establishment.

[0058] like Figure 13 As shown, after successful modeling, vulvar redness and swelling with copious discharge were observed in mice on day 0. No significant improvement was observed in the model group under naked-eye observation on days 3 and 8. Neither the control group nor the drug-treated group showed significant improvement on day 3. Subsequent observation revealed that by day 8, the vulvar swelling in the medium- and low-dose groups had somewhat subsided, but still showed significant swelling compared to the blank group. The high-dose group showed significant symptom improvement on day 3, and by day 8, the swelling had largely subsided, with a condition consistent with the blank group.

[0059] The vaginal wash fluid from each group of mice was counted using the plate count method, and the results are as follows: Figure 14 As shown in the figure. On day 8, the colony count in the model group showed no significant change. After 7 days of treatment with the control group and the high-dose group, the colony count decreased significantly. Furthermore, there were significant differences between the medium- and low-dose groups and the control group, as well as between the high-dose group and the blank group. These results confirm that the antibacterial Tibetan medicine preparation prepared in this invention has a clear intervention effect on vaginitis caused by mixed infection of Candida albicans and Escherichia coli.

[0060] 3. Pathological and histological analysis Heart, liver, spleen, lung, kidney, and vaginal tissues from mice in each experimental group were collected after fixation. The tissues were then dehydrated, cleared, and embedded sequentially to prepare 4μm paraffin sections. After dewaxing, HE staining, gradient dehydration, clearing, and mounting, the sections were observed and analyzed under an optical microscope to examine the pathological morphological changes of each tissue.

[0061] Histological staining analysis of mouse vaginal tissue ( Figure 15 In the model group, vaginal mucosa sloughed off, inflammatory cells infiltrated the epithelial layer, and various inflammatory factors were present in the muscle layer. In the control and drug-treated groups, mucosal hyperplasia was reduced, and epithelial inflammatory cell infiltration was decreased. Histochemical analysis ( Figure 16 The results showed that after 7 days of continuous vaginal administration, no abnormal changes were observed in the major organs of mice in each administration group, including the heart, liver, spleen, lungs, and kidneys, indicating good safety.

[0062] 4. Western blot analysis Equal amounts of protein were separated by SDS-polyacrylamide gel electrophoresis and then transferred to a PVDF filter membrane. The membrane was blocked with 5% milk and TBST for 0.5 h, incubated overnight at 4°C with primary antibodies (IL6, TNF-α) using a shaker, and washed three times with TBST. Secondary antibody was then incubated for 0.5 h using a shaker and washed three times with TBST. β-actin was used as an internal control. Band intensity was detected using an imaging system with ECL reagent.

[0063] Quantitative proteomics detection results ( Figure 17 The results showed that the β-actin bands in each group had uniform brightness, indicating no deviation in sample loading and reliable results. The band brightness in the model group was significantly higher than that in the normal group, indicating successful modeling. Figure 18 and Figure 19 As shown, as the dosage changes from high to medium-low, the band brightness of TNF-α and IL-6 gradually increases, indicating that the agent prepared in this invention can reduce the expression of TNF-α and IL-6 inflammatory factors, and the effect is more obvious at high doses.

[0064] In summary, the antibacterial Tibetan medicine preparation prepared by this invention treats vaginitis through a synergistic effect of "antibacterial-anti-inflammatory" multiple pathways. This invention optimizes the preparation process of Xiuqiong Enbao lotion through experimental research, clarifies its antibacterial and anti-inflammatory effects, provides sufficient scientific basis for its clinical application, and provides a feasible technical route for the modernization research of Tibetan medicine compound preparations.

Claims

1. An antibacterial Tibetan medicine preparation, characterized in that, The raw materials used, by weight, include: 80-120 parts of sickle-shaped yam, 20-30 parts of rhubarb, 40-60 parts of iron bark, 50-80 parts of Terminalia chebula, 50-80 parts of Terminalia chebula, 70-100 parts of Phyllanthus emblica, 20-30 parts of myrrh, 80-120 parts of broad-skinned vine, and 0.6-1 part of artificial musk.

2. The antibacterial Tibetan medicine preparation according to claim 1, characterized in that, The raw materials used, by weight, include: 100 parts of sickle-shaped yam, 25 parts of rhubarb, 50 parts of iron bark, 65 parts of Terminalia chebula, 65 parts of Terminalia chebula var. chinensis, 85 parts of Phyllanthus emblica, 25 parts of myrrh, 100 parts of broad-skinned vine, and 0.8 parts of artificial musk.

3. The method for preparing the antibacterial Tibetan medicine preparation according to claim 1 or 2, characterized in that, Includes the following steps: Except for artificial musk, the remaining raw materials are crushed, then boiled with water 1-3 times, each time for 2-5 hours. The decoctions are combined and concentrated, and then mixed with artificial musk to obtain the final product.

4. The method for preparing the antibacterial Tibetan medicine preparation according to claim 3, characterized in that, The ratio of ingredients to liquid during decoction is 1g:10-14mL.

5. The method for preparing the antibacterial Tibetan medicine preparation according to claim 4, characterized in that, The decoction was performed twice, with each decoction lasting 3 hours, and the ratio of the decoction material to the liquid was 1g:12mL.

Images