Application of pulsatilla saponin B5 in treatment of vaginitis and / or cervicitis

Abstract

The invention discloses an application of pulsatilla saponin B5 with a structure shown as a formula I in preparation of a medicine for vaginitis and / or cervicitis. The vaginitis is bacterial vaginitis, and the cervicitis is bacterial inflammation of the cervix and the vagina. The invention relates to a spray for vaginitis and / or cervicitis, in particular to bacterial vaginitis and / or bacterial cervix and vagina inflammation and application thereof. The spray is prepared from the following components in parts by weight: 0.5 to 5 parts of pulsatilla saponin B5, 0.05 to 1 part of sucralose, 0.01 to 0.05 part of benzalkonium chloride and 100 parts of water.

Description

[0001] Cross-reference to related applications

[0002] This patent application claims priority to Chinese Patent Application No. CN202510301325.1, filed on March 14, 2025, the entire contents of which are incorporated herein by reference. Technical Field

[0003] This invention belongs to the field of medicine, specifically relating to a new medicinal use of Pulsatilla saponin B5. Background Technology

[0004] Vaginitis is the most common infectious disease of the female reproductive system. The upper part of the vagina surrounds the vaginal portion of the cervix, so vaginitis often affects the cervix, causing inflammation of the vaginal portion of the cervix. Based on the presence or absence of a specific causative pathogen, vaginitis can be classified into vulvovaginal candidiasis (VVC; also known as yeast infection or candidal vaginitis), trichomonal vaginitis (TV), bacterial vaginosis (BV; also known as nonspecific vaginitis), aerobic vaginitis (AV), and atrophic vaginitis. Bacterial vaginosis and aerobic vaginitis are pathological manifestations of the vagina caused by the abnormal proliferation of pathogenic bacteria due to various factors. Atrophic vaginitis, also known as senile vaginitis, is often caused by decreased ovarian function, reduced estrogen secretion, and weakened immunity in older women, manifesting as changes in the morphology of the vulva and vagina and abnormal vaginal flora distribution.

[0005] In Traditional Chinese Medicine (TCM), vaginitis falls under the categories of "vaginal itching" and "leukorrhea." Throughout history, physicians have considered leukorrhea to be persistent and difficult to cure, a view shared by modern medicine that vaginitis is characterized by a long course and frequent recurrence. Western medicine treatments for vulvovaginal candidiasis and trichomoniasis often lead to drug resistance; and for bacterial vaginosis and aerobic vaginitis, Western medicine lacks effective treatments. TCM can avoid the overuse and abuse of antibiotics in clinical practice, supplementing the shortcomings of Western medicine in treating vaginitis.

[0006] Pulsatilla chinensis, first recorded in the *Shennong Bencao Jing* (Divine Farmer's Materia Medica), is the dried root of *Pulsatilla chinensis* (Bge.) Regel, a plant belonging to the genus *Pulsatilla* in the Ranunculaceae family. It is harvested in spring and autumn, cleaned of dirt, and then dried. Pulsatilla chinensis is bitter and cold in nature, and enters the stomach and large intestine meridians. It has the effects of clearing heat and detoxifying, cooling blood and stopping dysentery. Clinically, it is often combined with *Sanguisorba officinalis*, *Dryopteris crassirhizoma*, and *Sophora flavescens* to treat gynecological inflammation. The main components of Pulsatilla chinensis include volatile oils, phenolic acids, flavonoids, and glycosides. Pulsatilla saponin B5, with the structural formula shown in Figure I, is a pentacyclic triterpenoid saponin isolated from Pulsatilla chinensis.

[0007]

[0008] The inventors discovered in their research that the compound has a good therapeutic effect on rats with nonspecific vaginitis, thus providing a new option for the clinical treatment of nonspecific vaginitis. Summary of the Invention

[0009] In view of the problems existing in the prior art, the present invention provides a new pharmaceutical use for Pulsatilla saponin B5.

[0010] Therefore, the present invention adopts the following technical solution:

[0011] The use of Pulsatilla saponin B5, with the structure shown in I, in the preparation of drugs for vaginitis and / or cervicitis;

[0012]

[0013] Preferably, the vaginitis is bacterial vaginosis, and the cervicitis is bacterial cervical vaginitis.

[0014] Preferably, the pulsatilla saponin B5 is the sole active ingredient of the drug.

[0015] Preferably, the drug may further include pharmaceutically acceptable excipients.

[0016] Preferably, the drug is a non-oral formulation.

[0017] More preferably, the non-oral preparation is selected from one or more of vaginal suppositories, vaginal gels, and vaginal sprays.

[0018] The vaginal suppositories, vaginal gels, and vaginal sprays include pulsatilla saponin B5 and a suitable matrix, and may also include one or more excipients selected from surfactants, diluents, lubricants, and antibacterial agents.

[0019] Sprays offer significant advantages in both local and systemic drug delivery due to their convenient administration, good uniformity, rapid absorption, and quick onset of action.

[0020] Therefore, as a preferred embodiment, the present invention provides a spray for vaginitis and / or cervicitis, particularly for bacterial vaginitis and / or bacterial cervical vaginitis, comprising:

[0021] 0.5–5 parts by weight of Pulsatilla saponin B5, 0.05–1 part by weight of sucralose, 0.01–0.05 parts by weight of benzalkonium chloride, and 100 parts by weight of water.

[0022] Preferably, the composition of the spray is as follows:

[0023] 1-5 parts by weight of Pulsatilla saponin B5, 0.05-1 part by weight of sucralose, 0.01-0.02 parts by weight of benzalkonium chloride, and 100 parts by weight of water.

[0024] The present invention also provides a method for preparing the spray, comprising the following steps:

[0025] Prepare the raw materials according to the weight proportions, then add Pulsatilla saponin B5, sucralose and benzalkonium chloride to the water in sequence while stirring, stir to dissolve, filter to remove bacteria, and package to obtain the final product.

[0026] Preferably, the non-oral formulation is administered to mammals, more preferably to humans.

[0027] When applied to humans, the daily dose of Pulsatilla saponin B5 is 4–100 mg / kg body weight, preferably 6–50 mg / kg, and most preferably 8–20 mg / kg.

[0028] Experiments have shown that Pulsatilla saponin B5 can significantly reduce leukocyte levels in rats with phenol-induced vaginitis, alleviate vaginal edema, reduce inflammatory infiltration in vaginal tissues, and lower IL-6 levels. Therefore, this invention can provide a new option for the clinical treatment of bacterial vaginosis, thereby helping patients recover from this persistent condition and improve their quality of life.

[0029] In this specification, unless otherwise stated, "water" refers to purified water that meets specified requirements, such as distilled water, double-distilled water, deionized water, etc.

[0030] In this specification, the term "parts by weight" does not refer to the actual mass (or weight) of the substance, but rather to the relative proportions between the components in the composition. Depending on the specific circumstances, one part by weight can be any mass (or weight), such as 1g, 5g, 250g, 500g, 1kg, etc. Attached Figure Description

[0031] The present invention will be further described below with reference to the accompanying drawings.

[0032] Figure 1 The image shows the HPLC chromatogram of total saponins from Pulsatilla chinensis prepared in Example 1, where peak number 7 is the chromatographic peak of Pulsatilla chinensis saponin B5.

[0033] Figure 2 This is the high-resolution mass spectrum of Pulsatilla saponin B5 prepared in Example 1.

[0034] Figure 3 The 1H NMR spectrum of Pulsatilla saponin B5 prepared in Example 1.

[0035] Figure 4 The image shows the carbon NMR spectrum of Pulsatilla saponin B5 prepared in Example 1.

[0036] Figure 5 These are photographs of vaginal secretion smears from rats in each experimental group in Experiment Example 1.

[0037] Figure 6 These are photographs of the ovaries, uterus, cervix, and vagina of rats in each experimental group in Experiment Example 1.

[0038] Figure 7 These are photographs of vaginal sections from rats in each experimental group in Experiment Example 1.

[0039] Figures 5-7 Among them, (1): normal group; (2): model group; (3) total saponins of Pulsatilla chinensis group; (4) saponin B4 group of Pulsatilla chinensis group; (5) low dose group of saponin B5 of Pulsatilla chinensis group; (6) medium dose group of saponin B5 of Pulsatilla chinensis group; (7) high dose group of saponin B5 of Pulsatilla chinensis group; (8) matrine group. Detailed Implementation

[0040] The present invention will be described below with reference to specific embodiments. Those skilled in the art will understand that these embodiments are for illustrative purposes only and do not limit the scope of the invention in any way.

[0041] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, all medicinal materials and reagents used in the following examples are commercially available products.

[0042] Example 1 Preparation of Pulsatilla saponin B5

[0043] Take an appropriate amount of Pulsatilla chinensis herb, add 10 times (w / w) of 95% ethanol and reflux twice, 2 hours each time. Combine the extracts and concentrate until no alcohol odor remains to obtain an extract. Add 10 times (w / w) of water to the extract, stir well and filter. Concentrate the filtrate to a relative density of 1.02-1.06 at 40-50℃ to obtain Pulsatilla chinensis extract. Adsorb the Pulsatilla chinensis extract onto an AB-8 macroporous resin column, eluing successively with 2-3 times the volume of water, 20% ethanol, 70% ethanol, and 90% ethanol, collecting the 70% ethanol eluent. Concentrate the 70% ethanol eluent to a relative density of 1.02-1.06 at 40-50℃ to obtain total saponins from Pulsatilla chinensis. HPLC-UV determination (detection wavelength 201nm) showed that the relative content of Pulsatilla chinensis saponin B5 was 48.59%. The HPLC chromatogram of total saponins from Pulsatilla chinensis is shown below. Figure 1 The retention time of Pulsatilla saponin B5 was 6.179 min. Figure 1 Peak 7 in the chromatogram). The relative area (%) of each chromatographic peak was calculated by integrating the area of ​​each peak and normalizing the area. The results are shown in Table 1.

[0044] Table 1. HPLC chromatographic peaks and relative areas of total saponins from Pulsatilla chinensis.

[0045] Detector A 201nm

[0046] Peak Retention time area high concentration area% 1 1.701 370314 24864 46.250 46.250 2 2.110 2967 407 0.371 0.371 3 2.400 10949 2002 1.367 1.367 4 4.226 4106 322 0.513 0.513 5 5.112 8328 549 1.040 1.040 6 5.606 444 48 0.055 0.055 7 6.179 389065 24357 48.592 48.592 8 7.569 5836 295 0.729 0.729 9 9.246 2454 126 0.307 0.307 10 13.427 4621 179 0.577 0.577 11 14.486 1601 71 0.200 0.200 total 800683 53220 100.000

[0047] Then, the total saponins of Pulsatilla chinensis were further analyzed by preparative HPLC. First, a coarse fraction was obtained using 50%-55% (v / v) methanol-water as the mobile phase. Then, the fraction with a Pulsatilla chinensis saponin B5 content greater than 98% was collected using 25%-30% (v / v) acetonitrile-water as the mobile phase. The collected fraction was concentrated and dried under reduced pressure to obtain a white amorphous powder. The powder was then analyzed by melting point, mass spectrometry, and nuclear magnetic resonance (NMR). 1 H and 13 C) Testing to identify the structure of the powder.

[0048] Its melting point is 222℃.

[0049] The molecular ion peak is [M–H]. - 1219.6102, the predicted molecular formula is C 59 H 96 O 26 (Calculated value: 1220.6190, theoretical value in negative ion mode: 1219.6117); High-resolution mass spectrum shown below. Figure 2 .

[0050] 1H-NMR(600MHz,C5D5N)δ:0.84(3H,s,H-29),0.85(3H,s,H-30),0.96(3H,s,H-26),1.06(3H,s,H-27), 1.10(3H,s,H-24),1.14(3H,s,H-25),1.62(3H,d,J=6.0Hz,6′-H3ofrha),1.69(3H,d,J=6.6Hz,6-H3of rha),6.23(1H,brs,1-H of rha),6.24(1H,d,J=6Hz,1′-H of glc),5.09(1H,brs,1′-H of rha),4.98(1H,d,J=6Hz,1″-H of glc), 5.09 (1H, d, J = 6Hz, 1-H of ara). The proton NMR spectrum is shown below. Figure 3 .

[0051] 13 C-NMR (150MHz, C5D5N): Data are shown in Table 2, and spectra are shown in […]. Figure 4 .

[0052] Table 2. Pulsatilla saponin B5 13 C-NMR data

[0053]

[0054] The above data can identify the compound as Pulsatilla saponin B5.

[0055] Example 2 A spray

[0056] The formulation of the spray in this embodiment is shown in Table 3.

[0057] Table 3. Formulation of the spray (100ml)

[0058]

[0059] Preparation method

[0060] Add an appropriate amount of deionized water to the mixing tank, add the prescribed amount of Pulsatilla saponin B5 (prepared in Example 1), stir to dissolve, then add the prescribed amount of flavoring agent and antibacterial agent, stir to dissolve, add deionized water to the prescribed amount, stir, filter with a 0.22μm polytetrafluoroethylene filter membrane for sterilization, bottle, and screw on the cap to obtain the product.

[0061] Study Example 1 Optimization study of spray formulation

[0062] 1. Study on the solubility of Pulsatilla saponin B5

[0063] B5 was added to 20 ml of different media (pH values) (purified water, pH 1.2, pH 4.0, pH 6.8, pH 9.0), shaken at room temperature, and samples were taken at different times (16 h, 48 h, 120 h) to determine the B5 content in the solution. The results are shown in Table 4.

[0064] Table 4. Results of solubility study of Pulsatilla saponin B5 in different media

[0065]

[0066] The results show that the solubility of B5 in different media ranges from 117 to 134 mg / ml, indicating good water solubility.

[0067] 2. Study on the oil-water partition coefficient of Pulsatilla saponin B5

[0068] Water was mixed with different volumes of n-octanol, and the mixture was shaken at a constant temperature of (37±0.5)℃ for 48 hours. The mixture was then allowed to stand and separate into layers for later use. The upper layer was designated as the saturated oil phase, and the lower layer as the saturated water phase. An excess of B5 raw material was added to fully wet the mixture, which was then placed in a constant temperature shaker and shaken at (37±0.5)℃ for 48 hours. The concentration of B5 in both phases was measured, and the oil-water partition coefficient was calculated. The results are shown in Table 5.

[0069] Table 5. Investigation of oil-water distribution coefficients

[0070] Two-phase ratio (oil phase: water phase) Time (hours) lg P 1：1 48 -0.33 1：2 48 -1.06 2：1 48 -0.15

[0071] The results show that the solubility of Pulsatilla saponin B5 in the aqueous phase is greater than that in the oil, indicating that it has good water solubility.

[0072] 3. Stability of B5 active pharmaceutical ingredient

[0073] Take an appropriate amount of B5 raw material and place it at 60℃ under light for 10 days. Take samples on the 5th and 10th days, determine and calculate the percentage content of B5. The results are shown in Table 6.

[0074] Table 6. Results of the stability study of Pulsatilla saponin B5

[0075]

[0076] Table 6 shows that the raw material of Pulsatilla saponin B5 remained stable under both heating and light conditions.

[0077] 4. Stability of solution B5 (60℃, light exposure)

[0078] An appropriate amount of B5 active pharmaceutical ingredient was dissolved in different media to obtain a solution with a concentration of 10 mg / ml. The solution was placed at 60℃ and under light for 10 days. Samples were taken on the 5th and 10th days to determine the content of B5 in the active pharmaceutical ingredient (expressed as a percentage). The results are shown in Table 7.

[0079] Table 7. Stability Study of Pulsatilla Saponin B5 Solution

[0080] solvent 0 days (%) High temperatures for 5 days (%) 10 days of high temperatures (%) water 99.59 99.36 99.45 pH 1.2 99.37 62.41 0 pH 4.0 99.85 98.15 98.46 pH 6.8 99.51 97.84 97.02 pH 9.18 99.72 97.65 97.15

[0081] In neutral water, the content of B5 remained relatively stable for 10 days at 60°C; however, it was unstable under both acidic and alkaline conditions.

[0082] 5. Stability study of B5 active pharmaceutical ingredient in different media after high temperature and high pressure sterilization

[0083] Dissolve an appropriate amount of B5 active pharmaceutical ingredient in different media to obtain solutions with a concentration of 10 mg / ml; sterilize each solution under the following conditions:

[0084] 1) 121℃, 15min; 2) 121℃, 8min; 3) 115℃, 30min.

[0085] After cooling to room temperature, the concentration of B5 was measured. The relative content of B5 in the solution (expressed as a percentage) was calculated based on the initial concentration before sterilization. The results are shown in Table 8.

[0086] Table 8. Relative content (%) of B5 in the sterilized solution.

[0087]

[0088] As shown in Table 8, Pulsatilla saponin B5 remains stable after high-temperature sterilization in neutral water, but it is not heat-resistant in acidic and alkaline media. Considering the potential adverse effects of the media on Pulsatilla saponin B5 during high-temperature sterilization, filtration sterilization was chosen.

[0089] 6. The effect of different microporous membranes on the drug solution

[0090] Sterilization filtration effectively removes microorganisms from pharmaceutical solutions, playing a crucial role in product microbial control. In the production process, sterilization filters with a pore size of 0.22 μm are generally selected, typically using composite filter membranes or microporous filter cartridges. An appropriate amount of B5 raw material was dissolved in water to obtain a solution with a concentration of 10 mg / ml. Several filter cartridges (0.22 μm pore size) of different materials were used to filter the solution. The compatibility of different filter materials with the pharmaceutical solution was investigated based on the appearance of the filtrate and the relative content of B5 in the filtrate (compared to the initial B5 concentration). The results are shown in Table 9.

[0091] Table 9. Compatibility Study Results of Different Filter Materials with Drug Solution

[0092] Filter membrane material Appearance B5 relative content (%) Polyethersulfone filter membrane Clear solution 99.45 Nylon filter membrane Clear solution 99.28

[0093] As shown in Table 9, different filter membranes have little effect on the drug solution. The drug solution has good compatibility with filter membranes of different materials and no obvious adsorption effect. This can provide a reference for the microbial control of the formulation in the later stage.

[0094] 7. Selection of Preservatives

[0095] The spray matrix of this invention is water, therefore it is necessary to add preservatives to inhibit bacterial growth. In preliminary studies, different types of antibacterial agents, including potassium sorbate and benzalkonium chloride, were investigated. The studies found that sprays using potassium sorbate as the antibacterial agent showed bacterial colonies and failed to achieve an antibacterial effect, possibly because the pH of the solution did not meet the suitable pH for potassium sorbate, resulting in poor antibacterial performance. Therefore, benzalkonium chloride was ultimately used. To screen the optimal concentration of the antibacterial agent and evaluate its antibacterial efficacy, the antibacterial effect of B5 sprays containing different concentrations of the antibacterial agent was examined according to the antibacterial efficacy testing method in Part IV of the 2020 edition of the Chinese Pharmacopoeia, and the most suitable concentration of the antibacterial agent was selected.

[0096] (1) Preparation of bacterial culture

[0097] Bacterial suspensions of Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Candida albicans, and Aspergillus niger were prepared according to the antibacterial efficacy test method in General Chapter 1211 of Part IV of the 2020 edition of the Chinese Pharmacopoeia.

[0098] (2) Antibacterial agent dosage screening design

[0099] The antibacterial effect of benzalkonium chloride at different concentrations on the test bacteria was investigated to screen the optimal dosage of the antibacterial agent. Referring to relevant formulations and literature, the maximum dosage of benzalkonium bromide as an antibacterial agent is generally 0.02%. Therefore, the antibacterial concentrations of benzalkonium bromide were designed to be 0.005%, 0.01%, and 0.02%.

[0100] (3) Applicability test of counting method

[0101] The culture medium used in the microbial counting of the test sample should be tested for suitability, and the microbial counting method of the test sample should be tested for method suitability to confirm that the method used is suitable for the microbial counting of the product.

[0102] (4) Antibacterial efficacy of test sample inoculation

[0103] Positive control: Use the test solution without the addition of the antibacterial agent benzalkonium bromide as a positive control.

[0104] Inoculation of test solutions: Divide the prepared test solutions and positive control solutions of different concentrations of antibacterial agents into 5 portions, each 9.9 mL. Inoculate each portion with bacterial suspensions of Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Candida albicans, and Aspergillus niger, respectively. The inoculation amount per 1 mL of test solution is 10. 5 ~10 6 CFU, the volume of the inoculum (0.1 mL) should not exceed 1% of the volume of the test sample. Mix thoroughly to ensure even distribution of the test bacteria, and store at 20–25°C in the dark.

[0105] (5) Antibacterial efficacy results

[0106] According to the regulations under the "Test Method for Antibacterial Efficacy" in Part IV of the 2020 edition of the Chinese Pharmacopoeia, samples of the test samples with added bacterial solutions were taken at 2, 7, 14, and 28 days to determine the number of surviving bacteria. The results were compared with the initial bacterial dosage and analyzed. The results are shown in the table below. The antibacterial efficacy of dermal preparations was judged according to the criteria for judging antibacterial efficacy in Part IV of the 2020 edition of the Chinese Pharmacopoeia. The results are shown in Table 10.

[0107] Table 10 Results of Antibacterial Efficacy Evaluation

[0108]

[0109] The antibacterial efficacy test results show that benzalkonium chloride concentration ≥0.01% achieves Grade A antibacterial efficacy against both bacteria and fungi. Therefore, the optimal concentration of the antibacterial agent is not less than 0.01%.

[0110] 8. Compatibility study of raw materials and auxiliary materials

[0111] Based on the principles of pharmaceutical excipient use, the selected flavoring agents and preservatives were subjected to compatibility tests with the active pharmaceutical ingredient (Pulsatilla saponin B5). Referring to the "Basic Technical Guidelines for Research on Chemical Drug Preparations," B5 was mixed with each excipient at a ratio of 1:20 and then placed under high temperature (60℃) and light (4500Lx±500Lx) conditions. Samples were taken on days 0, 5, and 10 to determine the percentage content of B5 and calculate it. The results are shown in Table 11.

[0112] Table 11 Results of the Compatibility Study of Raw Materials and Auxiliary Materials

[0113]

[0114] As shown in Table 11, the B5 content of raw materials and auxiliary materials did not change significantly under high temperature, high humidity and light conditions, indicating that the raw materials and auxiliary materials have good compatibility.

[0115] 9. Conclusion

[0116] Based on the above systematic research, the optimal composition of the Pulsatilla saponin B5 spray is as follows:

[0117] Raw material: Pulsatilla saponin B5; Flavoring agent: Sucralose; Antibacterial agent: Benzalkonium chloride; Solvent: Water.

[0118] The weight parts of each component are as follows:

[0119] 0.5-5 parts by weight of Pulsatilla saponin B5, 0.05-1 parts by weight of sucralose, 0.01-0.05 parts by weight of benzalkonium chloride, and 100 parts by weight of water.

[0120] Preferably, the weight parts of each component are:

[0121] Pulsatilla saponin B5 1-5 parts by weight, sucralose 0.05-1 parts by weight, benzalkonium chloride 0.01-0.02 parts by weight, and water 100 parts by weight.

[0122] The preparation method of Pulsatilla saponin B5 spray is as follows:

[0123] Add an appropriate amount of water to the mixing tank, add the prescribed amount of Pulsatilla saponin B5, stir to dissolve, then add the prescribed amount of flavoring agent and antibacterial agent, stir to dissolve, add water to the prescribed amount, stir, bottle, and screw on the cap to obtain the product.

[0124] Example 3 A suppository

[0125] The prescription for Pulsatilla chinensis saponin B5 vaginal suppositories in this embodiment is as follows:

[0126]

[0127] Take the prescribed amount of mixed fatty acid glycerides 36 # Mixed fatty acid glycerides 38 # (1:1) Heat and melt in a water bath at 65-70℃. After it is completely melted, add an appropriate amount of Pulsatilla saponin B5 that has passed through an 80-mesh sieve, stir evenly to disperse Pulsatilla saponin B5 evenly, pour into a mold at 50-55℃ in one go, and scrape off the excess after cooling at 10℃. Remove from the mold.

[0128] Example 4 A gel

[0129] The formulation of the Pulsatilla saponin B5 in situ gel in this embodiment is as follows:

[0130]

[0131] Preparation method: Weigh out the prescribed amount of Pulsatilla saponin B5, place it in a beaker, add an appropriate amount of distilled water, and sonicate it until completely dissolved. Under ice-water bath, continuously stir and slowly add the prescribed amounts of poloxamer 407, poloxamer 188 and hydroxypropyl methylcellulose. After stirring evenly, place it in a refrigerator at 4°C for 24 hours to allow it to fully swell, so as to obtain a clear, lumpy, uniformly dispersed in-situ gel solution for later use.

[0132] Example 5 A gel

[0133] The formulation of the Pulsatilla saponin B5 in situ gel in this embodiment is as follows:

[0134]

[0135] Preparation method: Same as in Example 4.

[0136] Experimental Example 1 The therapeutic effect of Pulsatilla saponin B5 on a rat model of vaginitis induced by phenol gel.

[0137] (I) Experimental Materials and Instruments

[0138] 1. Drugs, reagents and animals

[0139] Total saponins of Pulsatilla chinensis, Pulsatilla saponin B4 (batch number: 0320240101, Jiangxi Bencao Tiangong Technology Co., Ltd.), Pulsatilla saponin B5; positive control drug: matrine, batch number: 20230310, Wuhan Nengren Pharmaceutical Chemical Co., Ltd. Among them, total saponins of Pulsatilla chinensis and Pulsatilla saponin B5 were prepared in Example 1.

[0140] Pulsatilla chinensis total saponins, Pulsatilla chinensis saponin B4 and matrine were prepared into Pulsatilla chinensis total saponins spray (40 mg / ml), Pulsatilla chinensis saponin B4 spray (40 mg / ml) and matrine spray (2 mg / ml) respectively according to the method in Example 2.

[0141] Pulsatilla saponin B5 was prepared into three spray formulations of 40 mg / ml, 20 mg / ml and 10 mg / ml according to the method in Example 2.

[0142] Phenol (AR), specification: 500g / bottle, batch number: 170902, Xilong Scientific Co., Ltd. Astragalus gum, specification: BR 99% 100g, batch number: F8745, Shandong Xiya Chemical Co., Ltd. Glycerol (AR), specification: 500ml / bottle, batch number: 170414, Xilong Scientific Co., Ltd. Wright-Gymsa staining reagent, Nanchang Yulu Experimental Co., Ltd. Rat IL-6 detection kit, Thermo Fisher Scientific, USA.

[0143] SPF grade, SD rats, female, 64 rats at 8 weeks old, purchased from Nanjing K瑞斯 Animal Co., Ltd., license number SCXK(Beijing)2019-0010, animal certificate number: 110324231107030223.

[0144] Preparation of phenol mucilage: Take 10 mL of phenol, 2 g of tragacanth gum, 8 mL of glycerol, add distilled water to 22 mL, grind and mix evenly with a mortar to obtain 25% phenol mucilage.

[0145] 2. Experimental instruments

[0146]

[0147] (2) Experimental methods

[0148] 1. Model establishment, grouping and administration

[0149] Number the animal tails with a marker pen and weigh each animal to obtain the initial weight on the first day. Set 8 rats in the normal group. For the remaining rats, slowly inject 0.2 mL of 25% phenol mucilage into the vagina of the rats about 1.5 cm deep using a 1 mL syringe with a blunt intragastric needle once a day according to the above method for 3 consecutive days (D1-D3). After the model establishment (D4), randomly divide the modeled rats into: model group (spray normal saline), total saponins of Pulsatilla chinensis spray group, B4 spray group, high-dose B5 spray group, medium-dose B5 spray group, low-dose B5 spray group, matrine spray group, a total of 7 groups, with 8 rats in each group. Start administration on the day when the grouping ends on the 4th day. Spray the vagina with the spray, 0.15 mL each time, and the single-dose administration doses are respectively:

[0150] Total saponins of Pulsatilla chinensis: 24 mg / kg,

[0151] Pulsatilla saponin B4: 24 mg / kg;

[0152] High-dose Pulsatilla saponin B5: 24 mg / kg, medium-dose: 12 mg / kg, low-dose: 6 mg / kg;

[0153] Matrine: 1.25 mg / kg.

[0154] All administration groups are continuously administered for three days (D4-D6). Among them, on the 4th day (D4) and the 5th day (D5), spray four times a day, with an interval of 3 hours each time; on the 6th day (D6), administer once, and collect samples 2 hours after administration. 2. Observation of general status of animals.

[0155] Observe the general symptoms of the rats every day, including whether there are abnormalities in morphological appearance, behavioral activities, diet, drinking water, and autonomous activity functions, etc., and weigh and record the weight changes of the rats.

[0156] 3. Observation of vaginal secretion smear of rats.

[0157] On day 6, before sampling, vaginal secretions from rats were collected using sterile cotton swabs, smeared onto glass slides, and allowed to air dry. After fixation with 95% ethanol for half an hour, Wright staining was performed. Changes in inflammatory cells in the vaginal secretions of rats in each group were observed under an optical microscope.

[0158] 4. Detection of white blood cell count in vaginal irrigation fluid.

[0159] After collecting vaginal secretions, 150 μL of physiological saline was pipetted into the rat's vagina and repeatedly blown and rinsed 5 times to obtain vaginal irrigation fluid in a centrifuge tube. After diluting the vaginal fluid 5 times with physiological saline, the white blood cell count was detected using a blood cell counter. The remaining irrigation fluid was stored at -80°C.

[0160] 5. Organize photography and weighing.

[0161] After collecting the vaginal irrigation fluid, the rats were anesthetized and euthanized. The ovaries, uterus, cervix, and vaginal tissues were then photographed and recorded using a gross microscopy system.

[0162] 6. Observation of pathological tissue morphology

[0163] A portion of the vaginal and cervical tissue was fixed with 4% paraformaldehyde, dehydrated, embedded, and stained with hematoxylin and eosin (HE). The effect of the drug on the pathology of the vaginal and cervical tissue was evaluated by observing the pathological morphology of the tissue. Another portion of the tissue was flash-frozen in liquid nitrogen and stored at -80°C for later use.

[0164] 7. Detection of IL-6 levels in vaginal douches

[0165] Take 20 μl of the reconstituted vaginal irrigation solution, dilute it with physiological saline to 100 μl, and then perform the test according to the kit instructions.

[0166] 8. Statistical Analysis

[0167] All data is based on This indicates that a statistically significant result was achieved using one-way ANOVA or a t-test, with a p-value < 0.05.

[0168] (III) Experimental Results

[0169] 1. The body weights of rats in groups D1, D3 and D5 are shown in Table 12.

[0170] Table 12 Changes in rat body weight (g) in each experimental group ( n=8)

[0171]

[0172] Table 12 shows that, compared with the normal group, the body weight of rats in the model group did not decrease significantly after phenol gel modeling. Compared with the model group, the body weight of rats in each dose group of Pulsatilla chinensis total saponins, Pulsatilla chinensis saponin B4, and Pulsatilla chinensis saponin B5 sprays did not change significantly, and matrine also had no significant effect on rat body weight. The results indicate that Pulsatilla chinensis saponin B5 spray had no effect on the body weight of rats with vaginitis induced by phenol gel, showing good safety.

[0173] 2. Effects of Pulsatilla saponin B5 spray on vaginal secretions in rats with phenol-induced vaginitis; results are shown in […]. Figure 5 .

[0174] Figure 5 The results showed that, compared with the normal group, the vaginal smears of rats in the model group had a large number of white blood cells; the number of white blood cells in the total saponins of Pulsatilla chinensis group did not change significantly, while the number of white blood cells in the vaginal smears of the B4 group was significantly reduced; the number of white blood cells in the medium-dose and high-dose groups of B5 was significantly reduced, while the number of white blood cells in the low-dose group showed a decreasing trend; the number of white blood cells in the matrine group also decreased significantly, and the number of white blood cells decreased more in the high-dose group of B5 than in the matrine group.

[0175] The results showed that Pulsatilla saponin B5 could reduce the number of inflammatory cells in rat vaginal secretions induced by phenol gel.

[0176] 3. Effect of Pulsatilla saponin B5 on the number of white blood cells in vaginal irrigation fluid of rats with vaginitis induced by phenol gel. The results are shown in Table 13.

[0177] Table 13 Changes in white blood cells in vaginal irrigation fluid of rats in each experimental group (10^9 / L) n=8)

[0178]

[0179] Note: # Compared with the normal group, # P < 0.05 ## P < 0.01; *Compared with the model group, * P < 0.05 ** P < 0.01.

[0180] Vaginal lavage fluid was analyzed using a five-part differential blood test. The results showed that, compared to the normal group, the model group rats had significantly increased numbers of leukocytes, neutrophils, lymphocytes, and monocytes in their vaginal lavage fluid. Compared to the model, the total saponins of Pulsatilla chinensis group showed a decrease primarily in neutrophils and eosinophils. The B4 group showed a significant decrease in neutrophils, lymphocytes, monocytes, and eosinophils. The high and medium doses of B5 showed a significant decrease in leukocytes, neutrophils, and monocytes, with a more significant decrease in neutrophils and monocytes. The low dose of B5 showed only a significant decrease in neutrophils. Compared to the model group, the matrine group also showed a significant decrease in leukocytes and neutrophils (P<0.05). Although the difference was not statistically significant, the high dose of B5 showed a greater decrease in leukocytes, neutrophils, lymphocytes, and monocytes compared to the matrine group.

[0181] 4. Effects of Pulsatilla saponin B5 spray on vaginal tissue morphology in rats with phenol-induced vaginitis. Results are shown in […]. Figure 6 .

[0182] like Figure 6 As shown, after phenol gel modeling, the model group rats exhibited significant vaginal and cervical swelling, while the total saponins of Pulsatilla chinensis group showed some relief from vaginal and cervical swelling. The B4 group showed a more significant relief from vaginal and cervical swelling. The medium-dose and high-dose B5 groups also showed significant improvement in vaginal and cervical swelling, significantly more so than the matrine group, especially the high-dose B5 group. However, there was no significant difference between the B4 group and the matrine group.

[0183] 5. Effects of Pulsatilla saponin B5 on the histopathological changes of rat vaginal tissue induced by phenol gel. Results are shown in […]. Figure 7 .

[0184] like Figure 7 As shown, the vaginal layer structure of the normal group rats was clear and intact, with the mucosal layers arranged neatly. After phenol gel modeling, the vaginal mucosal epithelium of the model group was severely sloughed off, with a large amount of inflammatory infiltration in the submucosa and thickening of the lamina propria. The submucosal inflammation in the total saponins of Pulsatilla chinensis group was slightly improved. The vaginal mucosal epithelial damage in the B4 group, the medium-dose B5 group, and the matrine group was improved to some extent, and the inflammatory infiltration in the submucosa was significantly reduced. The improvement was most significant in the high-dose B5 group.

[0185] 6. Effect of Pulsatilla saponin B5 on IL-6 levels in rat vaginal irrigation fluid induced by phenol gel. The results are shown in Table 14.

[0186] Table 14. Changes in IL-6 levels in vaginal irrigation fluid of rats in each experimental group (pg / ml) n=8)

[0187]

[0188] Note:# Compared with the normal group, # P < 0.05 ## P < 0.01; * Compared with the model group, * P < 0.05 ** P < 0.01.

[0189] Table 14 shows that the IL6 content in the vaginal irrigation fluid of the model group rats was significantly higher than that in the normal group; the total saponins of Pulsatilla chinensis showed a decreasing trend; the IL6 content in the B4 group decreased significantly; the IL6 levels in the high- and medium-dose B5 groups were significantly lower than those in the model group, and showed a dose-response relationship; in addition, the IL6 level in the high-dose B5 group was lower than that in the matrine group.

[0190] (IV) Experimental Conclusions

[0191] Phenol gel, when applied topically for three consecutive days, effectively established a rat model of nonspecific vaginitis and cervicitis. All doses of Pulsatilla saponin B5 significantly reduced leukocyte levels in rats with phenol gel-induced vaginitis and cervicitis, alleviated vaginal and cervical edema, reduced inflammatory infiltration in vaginal tissue, and decreased IL-6 levels, exhibiting a dose-response relationship. The medium and high doses of Pulsatilla saponin B5 showed stronger therapeutic effects on nonspecific vaginitis and cervicitis than total Pulsatilla saponins, while the effect of high-dose B5 was not statistically different from that of matrine. Therefore, Pulsatilla saponin B5 has a therapeutic effect on vaginitis and / or cervicitis.

[0192] Finally, it should be noted that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. The use of Pulsatilla saponin B5, with the structure shown in I, in the preparation of drugs for vaginitis and / or cervicitis; 2. The application according to claim 1, characterized in that, The vaginitis mentioned is bacterial vaginosis, and the cervicitis mentioned is bacterial inflammation of the vaginal portion of the cervix.

3. The application according to claim 1 or 2, characterized in that, The pulsatilla saponin B5 is the sole active ingredient of the drug.

4. The application according to any one of claims 1 to 3, characterized in that, The drug may also include pharmaceutically acceptable excipients.

5. The application according to any one of claims 1 to 4, characterized in that, The drug is a non-oral preparation.

6. The application according to claim 5, characterized in that, The non-oral preparation is selected from one or more of vaginal suppositories, vaginal gels, and vaginal sprays.

7. The application according to claim 6, characterized in that, The vaginal suppositories, vaginal gels, and vaginal sprays include pulsatilla saponin B5 and a suitable matrix, and may also include one or more selected from surfactants, diluents, lubricants, and antibacterial agents.

8. A spray for vaginitis and / or cervicitis, particularly for bacterial vaginitis and / or bacterial cervical vaginitis, comprising: 0.5–5 parts by weight of Pulsatilla saponin B5, 0.05–1 part by weight of sucralose, 0.01–0.05 parts by weight of benzalkonium chloride, and 100 parts by weight of water.

9. The spray according to claim 8, characterized in that, The composition of the spray is as follows: 1-5 parts by weight of Pulsatilla saponin B5, 0.05-1 part by weight of sucralose, 0.01-0.02 parts by weight of benzalkonium chloride, and 100 parts by weight of water.

10. The use of the spray according to claim 8 or 9 in the preparation of a medicament for vaginitis and / or cervicitis.

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