Traditional Chinese medicine composition for treating vagina inflammation as well as preparation method and application of traditional Chinese medicine composition

Abstract

The invention belongs to the technical field of traditional Chinese medicine compositions, and particularly relates to a traditional Chinese medicine composition for treating vagina inflammation and a preparation method and application thereof, the preparation method comprises the following steps: S1, preparing a prepared rehmannia root extract; s2, preparing a salted golden cypress extract and a salted rhizoma anemarrhenae extract; s3, preparing a radix paeoniae alba extract; s4, crushing tortoise-shell glue to obtain tortoise-shell glue powder; and S5, uniformly mixing the prepared rehmannia root extract, the salted golden cypress and rhizoma anemarrhenae extract, the white peony root extract and the tortoise-shell glue powder to obtain the traditional Chinese medicine composition. The traditional Chinese medicine composition prepared by a specific method on the basis of a formula of Guanhuang mother granules has a remarkable effect of improving vagina inflammation, and all the components have a synergistic effect, so that the vagina environment is improved, vagina epithelium repair is promoted, the local resistance of the vagina is enhanced, and the effect is remarkable.

Description

Technical Field

[0001] The present invention belongs to the technical field of traditional Chinese medicine compositions, and in particular relates to a traditional Chinese medicine composition for treating vaginitis, a preparation method thereof and an application thereof. Background Art

[0002] Vaginitis is a common gynecological disease in women, characterized by abnormal changes in the color, odor, or volume of vaginal discharge, accompanied by vaginal itching or burning. Vaginitis can be caused by microbial infections such as bacteria and fungi. Under normal circumstances, aerobic and anaerobic bacteria reside in the vagina, forming the normal vaginal flora. Any disruption of the ecological balance between the vagina and the flora can also lead to the formation of opportunistic pathogens. Vaginitis can also be associated with poor hygiene (such as irritation from underwear materials) and hormonal changes (such as decreased estrogen after menopause). Common types of vaginitis in clinical practice include Trichomonas vaginitis, vulvovaginal candidiasis, bacterial vaginosis, and atrophic vaginitis.

[0003] Atrophic vaginitis, also known as senile vaginitis, is a common disease in perimenopausal and postmenopausal women. It is primarily caused by a significant decline in ovarian function, decreased estrogen levels, vaginal atrophy, thinning of the vaginal mucosa, increased vaginal pH, and decreased local immune function. This weakens the immune system against vaginal pathogens, allowing them to easily multiply and invade, leading to inflammation. Its primary clinical manifestations include vaginal dryness and burning pain, dyspareunia, vulvar itching, and foul-smelling vaginal discharge, often accompanied by urinary symptoms such as frequent urination, urgency, and pain. The recurrence of these symptoms severely impacts the quality of life and physical and mental well-being of perimenopausal women.

[0004] The use of local antibiotic suppositories is a traditional method for treating atrophic vaginitis. Although antibiotics can inhibit bacterial growth, the cure rate of the treatment method using only antibiotics is low, and the disease is prone to relapse after being cured. The use of estrogen combined with antibiotics can reduce recurrence. Estrogen can increase local vaginal resistance, increase vaginal glycogen, and lower vaginal pH. However, the long-term use of estrogen is bound to have certain risks, and most patients, especially elderly patients, often have concerns about the use of estrogen. Therefore, developing a drug for the treatment of vaginitis with few side effects, safety and effectiveness is a research hotspot in this field.

[0005] Chinese patent application CN103301353A discloses a pharmaceutical composition for treating atrophic vaginitis and its preparation method, aiming to solve the problem of treating atrophic vaginitis. It is characterized by being prepared from the following raw materials: Pittosporum tobira bark, Triangle Herb, Herba Amygdaluse var. serrata, Magnolia tianmu, Mulberry Parasite, Herba Cat's Claw, Scorched Malt, Scorched Shenqu, Ephedra Root, Sophora japonica, Peach Gum, and Cinnamon Twig; the preparation method comprises: Pittosporum tobira bark, Triangle Herb, Herba Amygdaluse var. serrata, Magnolia tianmu, Mulberry Parasite, Herba Cat's Claw, Scorched Malt, Scorched Shenqu, Ephedra Root, Sophora japonica, Peach Gum, and Cinnamon Twig are mixed and boiled with water, the filtrate is collected after 30 minutes, the residue is boiled with water for another 20 minutes, the filtrate is collected, and the two filtrates are combined for external washing. However, the formula has a complex composition.

[0006] Based on this, developing a new Chinese medicine composition with simple components and the ability to significantly improve vaginal inflammation has become the research focus of researchers in this field.

[0007] Guanhuangmu Granules are a drug with marketing authorization held by the applicant. Its formula is Rehmannia glutinosa, tortoise shell glue, salt-peony root, salt-peony root, and white peony root. Its indications are to tonify the liver and kidneys, nourish yin and reduce internal heat. It is used for female menopausal syndrome (pre- and post-menopausal symptoms) characterized by liver and kidney yin deficiency according to Traditional Chinese Medicine, with symptoms such as hot flashes, sweating, dizziness, tinnitus, soreness of the waist and knees or heel pain, insomnia, frequent dreams, and irritability. The applicant discovered during post-marketing research on Guanhuangmu Granules that it has a good effect in improving vaginitis, and therefore proposed this invention. Summary of the Invention

[0008] In response to the above problems, the present invention provides a traditional Chinese medicine composition for treating vaginitis and a preparation method thereof. By optimizing the extraction method of Rehmannia glutinosa, tortoise shell glue, Phellodendron chinense, Anemarrhena asphodeloides and Paeonia lactiflora, the prepared traditional Chinese medicine composition can significantly improve vaginitis.

[0009] To achieve the above object, the technical solution adopted by the present invention is as follows: The first aspect of the present invention provides a method for preparing a traditional Chinese medicine composition for treating vaginitis, comprising the following steps: S1. Grind Rehmannia root, add to ethanol aqueous solution, heat and reflux extraction, filter to obtain alcohol filtrate and filter residue, add water to the filter residue, heat and reflux extraction to obtain water filtrate, combine the alcohol filtrate and water filtrate, concentrate, and dry to obtain Rehmannia root extract; S2. Grind the salt-guan Huangcork and salt-guan Huangcork, add them to a deep eutectic solvent aqueous solution, perform ultrasonic extraction, centrifuge, collect the supernatant, and concentrate the supernatant to obtain the salt-guan Huangcork and salt-guan Huangcork extract; S3, taking white peony root, crushing it, adding it to an aqueous solution containing a complex enzyme, heating for enzymolysis, adding anhydrous ethanol to make the ethanol concentration reach 50wt%-80wt%, heating for reflux extraction, filtering to obtain a filtrate, concentrating the filtrate to obtain an extract, and drying to obtain a white peony root extract; the complex enzyme is a mixture of cellulase, papain and pectinase; S4, crushing the tortoise shell glue to obtain tortoise shell glue powder; S5. Evenly mix the Radix Rehmanniae Preparata extract, Phellodendron chinense and Anemarrhena Rhizoma extracts, Paeonia lactiflora extract and tortoise shell glue powder to obtain a traditional Chinese medicine composition.

[0010] In some preferred embodiments, in step S1, the ratio of the Radix Rehmanniae Preparata, ethanol aqueous solution and water is 1 g:8-20 mL:8-20 mL, preferably 1 g:15 mL:15 mL.

[0011] In some preferred embodiments, step S1 is specifically: crushing Rehmannia glutinosa, adding it to a 50wt%-90wt% ethanol aqueous solution, heating and refluxing for extraction 2-3 times, each time for 1-2 hours, filtering, combining the filtrate to obtain an alcohol filtrate and a filter residue, adding water to the filter residue, heating and refluxing for extraction 2-3 times, each time for 1-2 hours, combining the filtrate to obtain a water filtrate, combining the alcohol filtrate and the water filtrate, concentrating, and drying to obtain a Rehmannia glutinosa extract.

[0012] Preferably, step S1 is specifically as follows: crushing Rehmannia glutinosa, adding it to a 75wt% ethanol aqueous solution, heating and refluxing for extraction 3 times, each time for 1.5 hours, filtering, combining the filtrate to obtain an alcohol filtrate and a filter residue, adding water to the filter residue, heating and refluxing for extraction 2 times, each time for 1.5 hours, combining the filtrate to obtain a water filtrate, combining the alcohol filtrate and the water filtrate, concentrating, and drying to obtain a Rehmannia glutinosa extract.

[0013] In some preferred embodiments, in step S2, the ratio of the total mass of the salt-treated Phellodendron chinense and the salt-treated Anemarrhena asphodeloides to the low eutectic solvent aqueous solution is 1 g:10-25 mL, preferably 1 g:20 mL.

[0014] In some preferred embodiments, in step S2, the preparation method of the deep eutectic solvent aqueous solution is: mixing choline chloride and citric acid, stirring to obtain a deep eutectic solvent, and mixing the deep eutectic solvent and water to obtain a deep eutectic solvent aqueous solution.

[0015] Preferably, the molar ratio of choline chloride to citric acid is 1:1-2, preferably 1:2.

[0016] Preferably, the volume ratio of the deep eutectic solvent to water is 60-80:20-40, preferably 70:30.

[0017] Preferably, in step S2, the preparation method of the deep eutectic solvent aqueous solution is: mixing choline chloride and citric acid, heating and stirring at 60-100° C. to obtain a deep eutectic solvent, and mixing the deep eutectic solvent and water to obtain a deep eutectic solvent aqueous solution; Further preferably, in step S2, the preparation method of the deep eutectic solvent aqueous solution is: mixing choline chloride and citric acid, stirring at 80° C. until a transparent and uniform liquid is formed to obtain a deep eutectic solvent, and mixing the deep eutectic solvent and water to obtain a deep eutectic solvent aqueous solution.

[0018] In some preferred embodiments, in step S2, the conditions for ultrasonic extraction are: temperature of 30-70°C, power of 100-300W, and time of 30-90 min.

[0019] Preferably, the ultrasonic extraction conditions are: temperature of 50° C., power of 250 W, and time of 60 min.

[0020] In some preferred embodiments, in step S3, the material-liquid ratio of the white peony root and the aqueous solution is 1 g:4-12 mL, preferably 1 g:6 mL.

[0021] Preferably, in step S3, the mass ratio of the cellulase, papain and pectinase is 0.8-1.2:0.3-0.7:0.5-1.

[0022] Further preferably, in step S3, the mass ratio of the cellulase, papain and pectinase is 1:0.5:0.8.

[0023] Preferably, in step S3, the mass of the complex enzyme in the aqueous solution containing the complex enzyme is 1%-3% of the mass of white peony root. Further preferably, in step S3, the mass of the complex enzyme in the aqueous solution containing the complex enzyme is 1.5% of the mass of white peony root.

[0024] Preferably, the step S3 is specifically as follows: The white peony root is crushed, added to an aqueous solution containing a complex enzyme, heated at 30-60°C for enzymatic hydrolysis for 1-3 hours, and then anhydrous ethanol is added to make the ethanol concentration reach 50wt%-80wt%, heated under reflux for extraction 1-3 times, filtered to obtain a filtrate, concentrated the filtrate to obtain an extract, and dried to obtain the white peony root extract. The complex enzyme is a mixture of cellulase, papain and pectinase.

[0025] Further preferably, the step S3 is specifically as follows: The white peony root is crushed, added to an aqueous solution containing a complex enzyme, heated at 30-50°C for enzymatic hydrolysis for 1-2 hours, and then anhydrous ethanol is added to make the ethanol concentration reach 60wt%-80wt%, heated under reflux for extraction 2-3 times, filtered to obtain a filtrate, concentrated the filtrate to obtain an extract, and dried to obtain the white peony root extract. The complex enzyme is a mixture of cellulase, papain and pectinase.

[0026] Preferably, in step S4, the particle size of the tortoise shell glue powder is 100-200 μm. Further preferably, in step S4, the particle size of the tortoise shell glue powder is 120-180 μm.

[0027] In some preferred embodiments, the Chinese medicine composition is composed of 150-170 parts of Rehmannia root, 30-40 parts of tortoise shell glue, 80-120 parts of salt-guan Huangbai, 80-120 parts of salt-guan Anemarrhena and 120-140 parts of white peony root, calculated by mass.

[0028] Preferably, the Chinese medicine composition is composed of 166 parts of Rehmannia root, 35 parts of tortoise shell glue, 100 parts of salt-guan Huangbai, 100 parts of salt-guan Anemarrhena and 125 parts of white peony root, calculated by mass.

[0029] The second aspect of the present invention provides a traditional Chinese medicine composition obtained by the above preparation method.

[0030] The third aspect of the present invention provides the use of the traditional Chinese medicine composition obtained by the above preparation method in the preparation of a medicine for treating vaginitis.

[0031] Preferably, the drug is an oral or external preparation.

[0032] Preferably, the vaginal inflammation comprises atrophic vaginitis (senile vaginitis) and / or bacterial vaginosis.

[0033] More preferably, the vaginal inflammation is atrophic vaginitis (senile vaginitis).

[0034] Compared with the prior art, the present invention has the following beneficial effects: The present invention is based on the formula of Guanhuangmu granules and is a traditional Chinese medicine composition obtained by mixing Rehmannia glutinosa extract, salt Guan Huangbai and salt Anemarrhena extract, white peony root extract and tortoise shell glue prepared by a specific method. The traditional Chinese medicine composition has a significant effect of improving vaginitis, and the components synergistically enhance the effect, improve the vaginal environment, regulate relevant hormones, promote vaginal epithelial repair, and enhance local vaginal resistance, with significant effects. DETAILED DESCRIPTION

[0035] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0036] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each smaller range between any intermediate value within a stated value or stated range and any other stated value or intermediate value within the stated range is also encompassed by the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0037] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0038] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be illustrative only.

[0039] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0040] Example 1 The preparation method of the Chinese medicine composition for treating vaginitis is as follows: S1. Crush 150 g of Rehmannia glutinosa, add it to a 75% by mass ethanol aqueous solution, heat and reflux and extract it 3 times, each time for 1.5 h, filter, combine the filtrates to obtain an alcohol filtrate and a filter residue, add water to the filter residue, heat and reflux and extract it 2 times, each time for 1.5 h, combine the filtrates to obtain a water filtrate, combine the alcohol filtrate and the water filtrate, concentrate, and dry to obtain a Rehmannia glutinosa extract; The ratio of the prepared rehmannia root, 75% ethanol aqueous solution and water is 1 g: 15 mL: 15 mL; S2, 80g of salt-guan Huangbai and 120g of salt-guan Huangbai were crushed, added to a deep eutectic solvent aqueous solution, and ultrasonically extracted (temperature 50°C, power 250W, time 60min), centrifuged, and the supernatant was collected. The supernatant was concentrated and dried to obtain salt-guan Huangbai and salt-guan Huangbai extracts; The ratio of the total amount of salt-peppermint bark and salt-peppermint anemarrhena to the deep eutectic solvent aqueous solution is 1 g: 20 mL; The preparation method of the deep eutectic solvent aqueous solution comprises: mixing choline chloride and citric acid (in a molar ratio of 1:2), stirring at 80° C. until a transparent and uniform liquid is formed to obtain a deep eutectic solvent, and mixing the deep eutectic solvent and water (in a volume ratio of 70:30) to obtain a deep eutectic solvent aqueous solution; S3, 140g of white peony root was crushed, added to an aqueous solution containing 1.8g of complex enzyme (the complex enzyme is a mixture of cellulase, papain and pectinase), heated at 40°C for 1.5h, and then anhydrous ethanol was added to make the ethanol mass fraction reach 70%, heated and refluxed for 3 times, filtered to obtain a filtrate, concentrated the filtrate to obtain an extract, and dried to obtain a white peony root extract; the mass ratio of the cellulase, papain and pectinase was 1:0.5:0.8; The material-liquid ratio of the white peony root and the aqueous solution is 1g:6mL; S4, crushing 40g of tortoise shell glue to obtain tortoise shell glue powder with a particle size of 120-180μm; S5. Evenly mix the Radix Rehmanniae Preparata extract, Phellodendron chinense and Anemarrhena Rhizoma extracts, Paeonia lactiflora extract and tortoise shell glue powder to obtain a traditional Chinese medicine composition.

[0041] Example 2 The preparation method of the Chinese medicine composition for treating vaginitis is as follows: The only difference from Example 1 is that the amount of Rehmannia root is 170 g, the amount of tortoise shell glue is 30 g, the amount of salt-cured Phellodendron chinense is 100 g, the amount of salt-cured Anemarrhena asphodeloides is 80 g, and the amount of white peony root is 120 g; the rest are the same.

[0042] Example 3 The preparation method of the Chinese medicine composition for treating vaginitis is as follows: The only difference from Example 1 is that the raw materials consist of 166g of Rehmannia root, 35g of tortoise shell glue, 100g of salt-guan Huangbai, 100g of salt-guan Anemarrhena and 125g of white peony root; the rest are the same.

[0043] Comparative Example 1 The preparation method of the Chinese medicine composition for treating vaginitis is as follows: The only difference from Example 3 is that step S1 is: Rehmannia root prepared with 15 times the amount of 92% ethanol solution by mass, extracted three times at 30° C. for 3 h each time, filtered, recovered the filtrate, concentrated and dried to obtain Rehmannia root prepared extract; the rest are the same.

[0044] Comparative Example 2 The preparation method of the Chinese medicine composition for treating vaginitis is as follows: The only difference from Example 3 is that the amount of Rehmannia root is 100 g, the amount of tortoise shell glue is 50 g, the amount of salt-cured Phellodendron chinense is 150 g, the amount of salt-cured Anemarrhena asphodeloides is 150 g, and the amount of white peony root is 83.3 g; the rest are the same.

[0045] Comparative Example 3 The preparation method of the Chinese medicine composition for treating vaginitis is as follows: The difference from Example 3 is that in step S2, the method for preparing the deep eutectic solvent aqueous solution is: mixing lactic acid and D-proline (molar ratio of 1:2), stirring at 80°C for 1 hour to obtain a deep eutectic solvent, and mixing the deep eutectic solvent and water (volume ratio of 70:30) to obtain a deep eutectic solvent aqueous solution; the rest are the same.

[0046] Comparative Example 4 The preparation method of the Chinese medicine composition for treating vaginitis is as follows: The only difference from Example 3 is that step S2 is: S2.1. Chop the salt-cured cork into 1.5cm pieces 3 Afterwards, the extract was mixed with 15 times the amount of acidic ethanol solution and soaked for 4 hours, and then subjected to 50°C heating-assisted ultrasonic extraction twice, each extraction for 2 hours, and the filtrate was decompressed to recover the ethanol, and sodium chloride was added for salting out, and the extract was dried to obtain the salt-guan Huangbai extract; the volume concentration of the ethanol solution was 60%; the acid added was phosphoric acid, and the volume content of phosphoric acid in the acidic ethanol solution was 0.5%; S2.2. Mix the salt anemarrhena with 10 times the amount of water and decoct for 2 h; filter the residue and decoct again; combine the decoctions and concentrate to obtain a salt anemarrhena concentrate; add ethanol to the salt anemarrhena concentrate to an ethanol concentration of 65%, stir evenly, and let stand for 12 h. Recover the supernatant, concentrate the supernatant under reduced pressure at 50°C, remove the ethanol, and dry to obtain a salt anemarrhena extract; S2.3. Mix the extract of Phellodendron chinense and the extract of Anemarrhena asphodeloides to obtain the extract of Phellodendron chinense and Anemarrhena asphodeloides; the rest are the same.

[0047] Comparative Example 5 The preparation method of the Chinese medicine composition for treating vaginitis is as follows: The only difference from Example 3 is that step S3 is: White peony root was crushed and added to an aqueous solution containing 1.8 g of cellulase. The mixture was heated at 40°C for 1.5 h for enzymatic hydrolysis. Anhydrous ethanol was then added to make the ethanol concentration reach 70%. The mixture was heated under reflux for extraction three times. The mixture was filtered to obtain a filtrate. The filtrate was concentrated to obtain an extract, which was then dried to obtain the white peony root extract. The material-liquid ratio of the white peony root to the aqueous solution was 1 g:6 mL. Comparative Example 6 The preparation method of the Chinese medicine composition for treating vaginitis is as follows: The preparation method of a traditional Chinese medicine composition for treating vaginitis is as follows: 166g of Rehmannia root, 35g of tortoise shell glue, 100g of salt-guan Huangbai, 100g of salt-guan Anemarrhena and 125g of white peony root are added with water and decocted twice, 10 times the amount of water is added for the first time and decocted for 2h, 8 times the amount of water is added for the second time and decocted for 2h, the decoctions are combined, concentrated and dried to obtain the traditional Chinese medicine composition.

[0048] Test Example 1 Treatment of atrophic vaginitis Experimental Animals: 88 healthy, clean-grade female Sprague-Dawley rats were selected for this study. They weighed 201-275 g (mean weight: 237.05 ± 8.03 g). The rats were randomly divided into a sham-operated group (8 rats), a model group (8 rats), and groups 1-3 (72 rats, 8 rats per group) and groups 1-6 (8 rats per group).

[0049] All rats were reared for 7 days after purchase and fed a standard diet. Water and food were available ad libitum. The 11 groups showed no significant differences in basic information, including gender, age, and weight, as well as feeding conditions (P>0.05), indicating comparability.

[0050] Animal Model: Rats in the model group, Example 1-3 groups, and Comparative Example 1-6 groups were anesthetized intraperitoneally with 2% sodium pentobarbital and placed in the supine position on a fixed table. Hair was clipped slightly below the lower rib and thoroughly disinfected with iodine. A 1-2.5 cm incision was made along the midline of the back from the lumbar vertebrae downward. After the skin was opened, round-headed scissors were inserted between the muscle and skin. Dissection was performed while expanding the skin to reveal the ovaries and their associated uterine horns. The fallopian tubes and surrounding blood vessels were then ligated. The uterine horns were then severed with ophthalmic scissors, the ovaries removed, and the peritoneal and muscular incisions sutured. The skin was inspected for bleeding and sutured. Postoperatively, all rats received 20,000 units of penicillin intraperitoneally for 3 consecutive days. Vaginal smears were performed 5 days after surgery, once daily for 5 consecutive days. The absence of an estrus response was considered a successful animal model. Rats in the sham-operated group also underwent ovariectomy, but the ovaries were not removed and the ovaries were sutured directly. Postoperatively, 20,000 units of penicillin were administered intraperitoneally. All rats were fed naturally for 20 days after surgery to allow the reproductive organs of the rats in the model group, Example 1-3 groups, and Comparative Example 1-6 groups to reach an aging state.

[0051] Medication method: Rats in Example 1-3 and Comparative Example 1-6 groups were given the prepared Chinese medicine composition at a dose of 2.8 g / kg / d. -1 The rats in the sham operation group and the model group were gavaged with an equal volume of normal saline.

[0052] Observational indicators: All rats were treated with the drug for 30 consecutive days. Vaginal pH, uterine and vaginal body mass index, malondialdehyde, reactive oxygen species, and superoxide dismutase levels were compared among the rats in each group. After 30 days of drug treatment, vaginal pH was measured using pH test paper. After anesthesia, the rats were opened abdominally and 5 mL of arterial blood was collected. After standing for 30 minutes, the blood was centrifuged at 3000 rpm for 10 minutes. After separation, serum was collected and stored at -20°C for testing.

[0053] Malondialdehyde and superoxide dismutase levels were measured using chemiluminescence assays, with kits provided by Nanjing Baishijin Biotechnology Co., Ltd. Reactive oxygen species levels were measured using enzyme-linked immunosorbent assays, with kits provided by Hairenjie Biotechnology Co., Ltd. The rat uterus and vagina were excised and accurately weighed, and the uterine and vaginal weight index was calculated as organ weight (mg) / body weight (mg).

[0054] Statistical analysis: The statistical software used for data processing was SPSS 22.0. Count data were expressed as (%) and the independent sample x 2 Inspection; measurement data passed ( ), multiple group comparisons were performed with the F test, and pairwise comparisons were performed with the t test. P < 0.05 indicated a significant difference.

[0055] The results are shown in Tables 1 and 2 below: Table 1. Comparison of vaginal pH, uterine and vaginal mass index in rats

[0056] Note: Compared with the sham operation group, △ P < 0.05, △△ P<0.01; compared with the model group, * P < 0.05, ** P<0.01.

[0057] Table 2. Comparison of serum malondialdehyde, reactive oxygen species, and superoxide dismutase levels in rats

[0058] Note: Compared with the sham operation group, △ P < 0.05, △△ P<0.01; compared with the model group, * P < 0.05, ** P<0.01.

[0059] As shown in Tables 1 and 2, the vaginal pH value of the modeled rats increased significantly, the uterine and vaginal weight index decreased significantly, the levels of malondialdehyde and reactive oxygen species increased significantly, and the superoxide dismutase decreased significantly. This suggests that the modeled rats have genital atrophy, endocrine disorders, and aggravated aging, and the model can simulate the pathological state of atrophic vaginitis (P < 0.01).

[0060] Genital atrophy and vaginal pH values of rats with atrophic vaginitis were improved after medication, and the effects of the traditional Chinese medicine compositions prepared in Examples 1 to 3 were significantly better than those prepared in Comparative Examples 1 to 6.

[0061] This indicates that the traditional Chinese medicine composition prepared by the specific preparation method of the present invention has a significant effect on the treatment of atrophic vaginitis.

[0062] Test Example 2 The Chinese medicine compositions prepared in Examples 1 to 3 and Comparative Examples 1 to 6 were diluted with 15-fold volume of water, respectively, and the inhibitory effects of the different dilutions of the Chinese medicine compositions on Escherichia coli (purchased from Ningbo Testo Biotechnology Co., Ltd., No. TS337391) and β-hemolytic Streptococcus (purchased from Wuhan Warner Biotechnology Co., Ltd., No. N-86604) were tested; The specific test method is: 1. Preparation 1) Bacterial strains and culture medium Escherichia coli: inoculated into LB liquid medium (purchased from Beijing Solebow Technology Co., Ltd.).

[0063] β-Hemolytic Streptococcus: Inoculated on blood agar plates containing 5% defibrinated sheep blood (Shanghai Chunshi Biotechnology Co., Ltd.).

[0064] 2) Sample processing The Chinese medicine composition was diluted with sterile water at 15 times its volume (v / v), mixed thoroughly, and then sterilized through a 0.22 μm filter membrane for later use.

[0065] 3) Control group setting Negative control: Sterile water was used instead of the Chinese medicine diluent.

[0066] ‌ 2. Detection of antibacterial activity using plate count method‌ ‌(1)E. coli inhibition experiment: Take 100 μL of E. coli solution (concentration ≈ 10 6 CFU / mL) was mixed with 900 μL of Chinese medicine diluent and incubated at 37℃ for 24 h. The mixed solution was serially diluted (10 -1 -10 -3 ), spread onto LB agar plates, and incubate at 37°C for 24 h. Count the colonies.

[0067] (2) β-hemolytic streptococcus inhibition test: Take 100 μL of β-hemolytic streptococcus suspension (concentration ≈ 10 6 CFU / mL) was mixed with 900 μL of Chinese medicine diluent; after incubation at 37°C for 24 h, the mixture was diluted serially (10 -1 -10 -5 ); Spread 100 μL of the dilution onto a blood agar plate containing 5% sheep blood and culture anaerobically for 24-48 h; count the colonies with a typical β-hemolytic ring (off-white colonies + completely transparent hemolytic ring).

[0068] (3) Calculation formula for antibacterial rate: Inhibition rate (%) = [(number of colonies in the negative control group - number of colonies in the experimental group) / number of colonies in the negative control group] × 100.

[0069] The results are shown in Table 3.

[0070] Table 3. Antibacterial rate of different dilutions of Chinese medicine compositions (%)

[0071] The core pathological basis of atrophic vaginitis is ovarian failure or estrogen deficiency (e.g., menopause, oophorectomy), which leads to vaginal mucosal atrophy, decreased epithelial cell glycogen, and elevated vaginal pH (>4.5). This disruption of the vaginal acidic environment reduces local resistance, leading to abnormal proliferation of aerobic bacteria, such as Streptococcus and Escherichia coli, and triggering an inflammatory response characterized by neutrophil infiltration. As shown in Table 3, the traditional Chinese medicine compositions prepared in the examples of the present invention exhibit significant antibacterial effects against Streptococcus and Escherichia coli, with an inhibition rate exceeding 91%. Compared with Comparative Examples 1-6, it is clear that only the traditional Chinese medicine compositions prepared using the specific preparation method of the present invention exhibit superior antibacterial properties.

[0072] The above further describes the present invention in conjunction with specific embodiments. However, these embodiments are merely exemplary and do not constitute any limitation on the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solutions of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, and such modifications and replacements shall fall within the scope of protection of the present invention.

Claims

1. A method for preparing a traditional Chinese medicine composition for treating vaginitis, characterized in that: The following steps are involved: S1. Grind Rehmannia root, add to ethanol aqueous solution, heat and reflux extraction, filter to obtain alcohol filtrate and filter residue, add water to the filter residue, heat and reflux extraction to obtain water filtrate, combine the alcohol filtrate and water filtrate, concentrate, and dry to obtain Rehmannia root extract; S2. Grind the salt-guan Huangcork and salt-guan Huangcork, add them to a deep eutectic solvent aqueous solution, perform ultrasonic extraction, centrifuge, collect the supernatant, and concentrate the supernatant to obtain the salt-guan Huangcork and salt-guan Huangcork extract; S3, taking white peony root, crushing it, adding it to an aqueous solution containing a complex enzyme, heating for enzymolysis, adding anhydrous ethanol to make the ethanol concentration reach 50wt%-80wt%, heating for reflux extraction, filtering to obtain a filtrate, concentrating the filtrate to obtain an extract, and drying to obtain a white peony root extract; the complex enzyme is a mixture of cellulase, papain and pectinase; S4, crushing the tortoise shell glue to obtain tortoise shell glue powder; S5. Mix the Radix Rehmanniae Preparata extract, Phellodendron chinense and Anemarrhena Rhizoma extracts, Paeonia lactiflora extract and tortoise shell glue powder to obtain a traditional Chinese medicine composition.

2. The preparation method according to claim 1, characterized in that Step S1 is specifically as follows: crushing Rehmannia glutinosa, adding it to an ethanol aqueous solution, heating and refluxing for 2-3 times, each time for 1-2 hours, filtering, combining the filtrate to obtain an alcohol filtrate and a filter residue, adding water to the filter residue, heating and refluxing for 2-3 times, each time for 1-2 hours, combining the filtrate to obtain a water filtrate, combining the alcohol filtrate and the water filtrate, concentrating, and drying to obtain a Rehmannia glutinosa extract.

3. The preparation method according to claim 1, characterized in that In step S2, the preparation method of the deep eutectic solvent aqueous solution is: mixing choline chloride and citric acid, stirring to obtain a deep eutectic solvent, and mixing the deep eutectic solvent and water to obtain a deep eutectic solvent aqueous solution.

4. The preparation method according to claim 3, characterized in that The molar ratio of the choline chloride to the citric acid is 1:1-2; the volume ratio of the deep eutectic solvent to water is 60-80:20-40.

5. The preparation method according to claim 3, characterized in that In step S2, the preparation method of the deep eutectic solvent aqueous solution is: mixing choline chloride and citric acid, heating and stirring at 60-100° C. to obtain a deep eutectic solvent, and mixing the deep eutectic solvent and water to obtain a deep eutectic solvent aqueous solution; In step S2, the ratio of the total mass of the salt-cured Phellodendron chinense and the salt-cured Anemarrhena asphodeloides to the low eutectic solvent aqueous solution is 1 g: 10-25 mL; the conditions of the ultrasonic extraction are: temperature of 30-70° C., power of 100-300 W, and time of 30-90 min.

6. The preparation method according to claim 1, characterized in that The step S3 specifically comprises: crushing white peony root, adding the powder to an aqueous solution containing a complex enzyme, heating and enzymolysis at 30-60° C. for 1-3 hours, then adding anhydrous ethanol to make the ethanol concentration reach 50wt%-80wt%, heating and reflux extraction at 40-85° C. for 1-3 times, filtering to obtain a filtrate, concentrating the filtrate to obtain an extract, and drying to obtain the white peony root extract; the complex enzyme is a mixture of cellulase, papain, and pectinase.

7. The preparation method according to claim 1, characterized in that The Chinese medicine composition comprises, by weight, 150-170 parts of Radix Rehmanniae Preparata, 30-40 parts of tortoise shell glue, 80-120 parts of Phellodendron chinense, 80-120 parts of Anemarrhena Rhizoma and 120-140 parts of White Peony Root.

8. The preparation method according to claim 7, characterized in that The Chinese medicine composition is composed of 166 parts of Radix Rehmanniae Preparata, 35 parts of tortoise shell glue, 100 parts of Phellodendron chinense, 100 parts of Anemarrhena Rhizoma and 125 parts of White Peony Root, calculated by mass.

9. A Chinese medicine composition obtained by the preparation method according to any one of claims 1 to 8.

10. Use of the traditional Chinese medicine composition obtained by the preparation method according to any one of claims 1 to 8 in preparing a medicament for treating vaginitis.

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