Traditional Chinese medicine composition for treating spleen deficiency and dampness stagnation type candida vaginitis

Abstract

The invention relates to the technical field of traditional Chinese medicine compositions, and discloses a traditional Chinese medicine composition for treating spleen deficiency and dampness obstruction type monilial vaginitis, which is prepared from 10-60 parts of rhizoma smilacis glabrae, 1-24 parts of cortex phellodendri, 3-36 parts of herba agrimoniae, 2-30 parts of zaocys dhumnade and 3-36 parts of cortex dictamni. The traditional Chinese medicine composition disclosed by the invention can be applied in a lavage or oral administration manner to play a role, can improve and treat symptoms of a spleen deficiency and dampness obstruction type candida vaginitis model mouse, can improve the weight of the mouse, can reduce the inflammatory factor level and can reduce the vaginal index, and has great popularization and application values.

Description

Technical Field

[0001] The invention relates to the technical field of Chinese medicine compositions, and in particular to a Chinese medicine composition for treating spleen deficiency and dampness obstruction type candidal vaginitis. Background Art

[0002] The statements in this section merely provide background information related to the present disclosure and may not constitute prior art.

[0003] Vulvovaginal candidiasis ( vulvovaginal candidiasis , VVC), also known as candidal vaginitis, is mainly caused by Candida albicans ( Candida albicans ) is a common gynecological fungal infection, with an infection rate of up to 11.8%. VVC patients experience vulvar itching, burning pain, sexual intercourse pain, urinary pain, etc., accompanied by curd-like or tofu-like vaginal discharge, which is very painful. Although it is not life-threatening, it seriously affects the quality of life of patients. At present, the treatment of VVC mainly relies on azole antifungal drugs such as fluconazole, but the drug resistance of strains has greatly reduced the effectiveness of such drugs, which has brought great challenges to the clinical treatment of candidal vaginitis. Considering that in traditional Chinese medicine theory, VVC belongs to the category of "leucorrhea and vaginal itching", it is believed that "leucorrhea is all dampness syndrome", and the main cause of the disease is caused by dampness, heat, and insects. The basic pathogenesis is the dysfunction of the liver, spleen, and kidney. The dampness damages the Ren and Dai meridians, and the dampness and heat are injected downward. The Ren meridian is not solid and the Dai meridian fails to meet the contract, which is a leucorrhea disease. Therefore, we use the thinking of TCM to find the corresponding Chinese medicine and compound prescriptions. Through the trinity of antibacterial-repair-recovery treatment of TCM, we can avoid the dysbacteriosis caused by the simple use of antibiotics, improve the vaginal flora, reduce the recurrence of infection, and improve the local immunity of the vagina and the human body. It provides a new treatment method for the clinical treatment of vaginal microecological disorders. Therefore, it is of great significance to conduct in-depth research on the treatment of candidal vaginitis with TCM. Summary of the invention

[0004] The purpose of the present invention is to provide a Chinese medicine composition for treating candidal vaginitis of spleen deficiency and dampness stagnation type in view of the defect of the current prior art that there is a lack of Chinese medicine formula. On the basis of simple vulvovaginal candidal infection, a "disease-syndrome" combined mouse model is constructed, and modern medical technology is combined with changes in relevant immune indicators to verify that the formula has a significant therapeutic effect on candidal vaginitis of spleen deficiency and dampness stagnation type.

[0005] The technical solution of the present invention is as follows: On one hand, the present invention provides a traditional Chinese medicine composition for treating spleen deficiency and dampness obstruction type candidal vaginitis, comprising 10-60 parts of smilax glabra, 1-24 parts of phellodendron chinense, 3-36 parts of agrimony, 2-30 parts of garter snake, and 3-36 parts of difficile cortex.

[0006] Preferably, the Chinese medicine composition comprises 25-35 parts of Smilax glabra, 7-17 parts of Phellodendron amurense, 13-23 parts of Agrimoniae, 10-20 parts of Agrimoniae urticae, and 13-23 parts of Dictamni cortex.

[0007] Preferably, the Chinese medicine composition comprises 27-33 parts of Smilax glabra, 9-15 parts of Phellodendron amurense, 15-21 parts of Agrimoniae, 13-17 parts of Agrimoniae urticae, and 15-21 parts of Dictamni cortex.

[0008] Preferably, the Chinese medicine composition comprises 30 parts of Smilax glabra, 12 parts of Phellodendron amurense, 18 parts of Agrimoniae scabra, and 18 parts of Dictamni cortex.

[0009] The Chinese medicine composition of the present application is composed of five Chinese medicinal materials, namely, Smilax glabra, Phellodendron amurense, Herba Agrimoniae, Snakehead Snake, and Dictamni Cortex. The whole formula is matched and suitable for both internal and external use. The combination of these medicines has the effects of drying dampness and clearing heat, killing insects and relieving itching, and ultimately achieves the therapeutic purpose of removing dampness but not completely and promoting spleen function.

[0010] Preferably, the Candida albicans is Candida tropicalis, Candida glabrata, Candida parapsilosis, Candida krusei, or Candida dubliniensis.

[0011] Preferably, the Candida albicans is ATCC14053.

[0012] According to a preferred embodiment, the Chinese medicine composition is a water-extracted concentrate.

[0013] According to a preferred embodiment, the Chinese medicine composition is administered externally or orally.

[0014] According to a preferred embodiment, the oral dosage of the Chinese medicine composition is: 10g / kg-20g / kg. The external dosage is: 1.25g / kg-2.5g / kg.

[0015] Compared with the prior art, the present invention has the following beneficial effects: 1. A Chinese medicine composition for treating candidal vaginitis of spleen deficiency and dampness obstruction type, the prescription uses Smilax glabra as the main ingredient for detoxification and dehumidification, and is effective in removing dampness and removing heat, and is mainly used to treat various symptoms of leucorrhea caused by damp-heat accumulation and toxicity; Phellodendron chinense is bitter and cold in nature, and can clear away heat and dry dampness, detoxify and relieve itching, and detoxify and treat sores, and has a broad-spectrum antifungal effect; Herba Agrimoniae is astringent and hemostatic, tonifying deficiency, killing insects and replenishing, and detoxifying, and is suitable for people with symptoms of vaginal itching, sores, tinea, and eczema, and is a minister with Phellodendron chinense; Snakehead snake is good at running around, enters the network to search for wind and stop itching, and helps the main ingredient Smilax glabra to search and remove dampness and toxicity in the network, and Dictamni is bitter and dry in nature, and can clear away heat and dry dampness, dispel wind and detoxify, and is a medicine for removing dampness from the Taiyin and Yangming meridians of the foot, and the two are adjuvant drugs. The whole prescription is matched, and it is suitable for both internal and external use, and is used in combination with dehumidifying, dispelling wind, and unblocking meridians to achieve the therapeutic purpose of not exhausting dampness and exhausting the spleen.

[0016] 2. A Chinese medicine composition for treating candidal vaginitis of spleen deficiency and dampness stagnation type. The compound composition provided by the present invention can effectively treat and relieve candidal vaginitis of spleen deficiency and dampness stagnation type both for oral and external use, has great clinical application value, provides new anti-vaginitis drugs, provides a better basis for the discovery of new drug targets, and also provides a reference for the treatment of VVC with natural antifungal drugs such as traditional Chinese medicine and compound prescriptions.

[0017] 3. A traditional Chinese medicine composition for treating candidal vaginitis of spleen deficiency and dampness obstruction type, based on the addition of the key component, black-necked snake, which works together with phellodendron chinense, herbaceous agrimony, cortex dasyphyllae and smilax glabra, exerts excellent effects in treating candidal vaginitis of spleen deficiency and dampness obstruction type, breaking the technical prejudice that traditional Chinese medicine has a slow onset of effect through oral administration, and the oral effect is close to the duration of effect of irrigation. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The basic conditions and appearance changes of each group of mice in Example 2; Figure 2 is the weight change of mice in Example 2; Figure 3 The local inflammatory changes in the vagina of mice in Example 2; Figure 4 The change of fungal load in the vaginal lavage fluid of mice in Example 2; Figure 5 The fungal morphological changes in the vaginal lavage fluid of mice in Example 2; Figure 6 The change of vaginal index of mice in Example 2; Figure 7 is the level of inflammatory factors in the vaginal lavage fluid of mice in Example 2, Figure 7 A is TNF-α, Figure 7 B is IL-1β, Figure 7 C is IL-18, Figure 7 D is IL-8, Figure 7 E is IL-6; Figure 8 The basic conditions and appearance changes of the mice in Example 4; Fig. 9 The local inflammatory changes in the vagina of mice in Example 4; Fig.10 The change of fungal load in the vaginal lavage fluid of mice in Example 4. Note: Compared with the BLANK group, **** P <0.0001,*** P <0.001,** P <0.01,*< P 0.05,ns P >0.05; compared with the Model group, #### P <0.0001,###P <0.001,## P <0.01, # <P0.05,ns P >0.05; Fig.11 Changes in the appearance of vaginal tissue in mice in Example 4; Fig.12 The change of vaginal index of mice in Example 4; Fig.13 The changes in the colonization of Candida albicans in the vaginal tissue of mice in Example 4; Fig.14 The basic conditions, appearance changes and fungal load of each group of mice in Comparative Example 2; Fig.15 The weight changes of mice in each group in Comparative Example 2; Fig.16 The change of vaginal index of mice in comparative example 2; Fig.17 This is the numerical change of fungal load in the vaginal lavage fluid of mice in Comparative Example 2. DETAILED DESCRIPTION

[0019] The specific embodiments listed in the present invention are only examples of the present invention, and the present invention is not limited to the specific embodiments described below. For those skilled in the art, any equivalent modifications and substitutions to the embodiments described below are also within the scope of the present invention. Therefore, the equal transformations and modifications made without departing from the spirit and scope of the present invention should be included in the scope of the present invention. If the specific conditions are not specified in the embodiments, they are carried out according to the conventional conditions or the conditions recommended by the manufacturer. If the manufacturers are not specified for all reagents or instruments, they are all conventional products that can be purchased commercially. In order to better illustrate the present invention, numerous specific details are given in the specific embodiments below. It should be understood by those skilled in the art that the present invention can also be implemented without certain specific details. In other embodiments, the methods, means, equipment and steps well known to those skilled in the art are not described in detail in order to highlight the purport of the present invention.

[0020] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art. Unless otherwise specified, the units used in this specification are all international standard units, and the numerical values ​​and numerical ranges appearing in the present invention should be understood to include the inevitable systematic errors in industrial production.

[0021] Candida albicans strains were purchased from ATCC, USA; Sabouraud liquid medium (SDB) was purchased from Guangdong Huankai Microbiological Technology Co., Ltd. Sabouraud solid medium (SDA) was purchased from Guangdong Huankai Microbiological Technology Co., Ltd. Fluconazole (FLC) solution was purchased from Nanchang Hongyi Pharmaceutical Co., Ltd. Estradiol benzoate was purchased from Shanghai Quanyu Biotechnology Co., Ltd.

[0022] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0023] Example 1 A Chinese medicine composition for treating spleen deficiency and dampness obstruction type candidal vaginitis It is composed of 30g of Smilax glabra, 12g of Phellodendron, 18g of Agrimoniae, 15g of Snakehead, and 18g of Dictamni. The decoction pieces of Smilax glabra, Phellodendron, Agrimoniae, Snakehead, and Dictamni were purchased from the University Hospital of Yunnan University of Traditional Chinese Medicine. All Chinese medicine pieces were identified as authentic by Professor Pu Shibiao of Yunnan University of Traditional Chinese Medicine and placed in a cool and dry place. Referring to the decoction method in the textbook of Chinese Pharmacy, add 10 times the amount of water to soak for 30 min, heat, concentrate and filter, add 10 times the amount of water to soak for 30 min, heat, concentrate and filter, mix the two filtrates thoroughly, heat and concentrate to a final concentration of 1g / ml (specific gravity measurement), and obtain the water-extracted concentrate of the Chinese medicine composition of the present application, which is placed in a 4°C refrigerator for standby use.

[0024] Example 2 Experimental study on the treatment of spleen deficiency and dampness obstruction type candidal vaginitis with the aqueous extract concentrate of the Chinese medicine composition of the present application 1. Experimental Materials 1. Experimental strains The Candida albicans strain was used after recovery, subculture and activation.

[0025] 2. Experimental drugs For the positive control drug fluconazole (FLC) solution, grind the tablets into fine powder, dissolve in DMSO, mix and sonicate for 20 min, centrifuge and take the supernatant, the storage concentration is 50 mg / mL, and seal and store in a refrigerator at 4°C for later use. On the day of use, estradiol benzoate was added with sesame oil to prepare a solution with a concentration of 1 mg / mL, stored in a refrigerator at 4°C, and prepared before each use.

[0026] Experimental drug: Chinese medicine composition prepared in Example 1 3. Culture medium Sabouraud Broth (SDB): Dissolve 32.55 g of liquid culture medium in 500 mL of ultrapure water, sterilize at 115°C for 15 minutes, place on a clean bench for 30 minutes, and store in a refrigerator at 4°C.

[0027] Sabouraud solid culture medium (SDA): Dissolve 32.55 g of solid culture medium in 500 mL of ultrapure water, sterilize at 115°C for 15 minutes, pour into a culture dish on a clean bench and wait for solidification, and store in a refrigerator at 4°C.

[0028] 2. Experimental Methods 1. Animal Grouping SPF Balb / c mice, female, 6-8 weeks old, 16g-20g (animal certificate number: SYXK (Yunnan) K2017-0005), after one week of adaptive feeding, were randomly divided into 8 groups, namely, blank group (Blank), spleen deficiency and dampness resistance Candida infection model group (Model), high-dose group of Chinese medicine composition lavage of the present application (XWN-H (perf)), low-dose group of Chinese medicine composition lavage of the present application (XWN-L (perf)), high-dose group of Chinese medicine composition gavage of the present application (XWN-H (ig)), low-dose group of Chinese medicine composition gavage of the present application (XWN-L (ig)), fluconazole lavage group (FLC (perf)), fluconazole gavage group (FLC (ig)). Before the experiment, the vaginal lavage fluid of the mice was collected for culture to observe whether Candida albicans grew, and mice without Candida albicans growth were selected for the experiment.

[0029] 2. Construction of mouse model of Candida vaginitis caused by spleen deficiency and dampness obstruction The blank group was fed normally for the first 14 days, and the other groups were set up with spleen deficiency and dampness resistance model, that is, they were kept in a climate box (humidity 90%, temperature 35±2℃) during the day, and fed with high-fat and high-sugar feed (80% ordinary feed + 12% lard + 8% honey); they were kept in a normal environment (humidity 60%, temperature 20±2℃) at night, and the model was set up alternately for 14 days. From the 15th day, except for the blank group, the other groups were subcutaneously injected with 0.025mL of estradiol benzoate E2 (1mg / mL) in the neck, once every 1 day, and a total of 4 injections. On the 22nd day, 20μL of Candida albicans suspension was sucked with a micropipette and injected into the vagina of mice except the blank group, and stayed in situ for 5 minutes (at least) to prevent bacterial liquid from overflowing. The blank group was injected with an equal amount of sterile PBS. The mice were inoculated once every 2 days for a total of 3 times. At the same time, the vagina of the mice was irrigated with sterile PBS on the 3rd and 7th days of modeling. The vaginal lavage fluid was collected for plate smear and Gram staining. Finally, the success of the vaginal model was comprehensively evaluated based on the results of lavage fluid smear microscopy, vaginal fungal CFU count, etc.

[0030] 3. Drug administration After the model was successfully established, the dosage was given by gavage and vaginal lavage according to the drug instructions and the dosage of "Pharmacological Experimental Methodology". The high-dose lavage group was 2.5g / kg; the low-dose lavage group was 1.25g / kg, the low-dose gavage group was 10g / kg, and the high-dose gavage group was 20g / kg, once a day, for 7 consecutive days. The general state and signs of mice in each group were observed daily, and the drug efficacy was evaluated according to the test indicators every day.

[0031] 4.Indicator detection 4.1 Examination of mouse survival status: The survival status of the mice was observed throughout the experiment, the overall condition of the mice was observed, the body surface state and stress response of the mice were recorded, and whether the mice had behaviors such as poor appetite, depression, and susceptibility to fright was observed.

[0032] 4.2 Mouse weight recording The weight of mice was recorded during the experiment, and the weight changes of mice were sorted out after the experiment.

[0033] 4.3 Observation and scoring of local conditions of mouse vulva During the experiment, the whole body was observed, and local conditions such as swelling and discharge of the vaginal opening were observed; local symptoms of the vulva were scored after administration. The scoring standard was 0 points: no swelling and discharge of the vulva and vagina; 1 point: slight swelling and discharge of the vulva and vagina, no discharge; 2 points: obvious swelling and discharge of the vulva and vagina with discharge; 3 points: obvious swelling and discharge of the vulva and vagina, ulcers, and discharge; 4 points: death.

[0034] 4.4 Microscopic examination of mouse vaginal lavage fluid smear and determination of fungal load At 1, 3, and 7 days after modeling and 1 day after drug treatment, the vagina of each group of mice was slowly irrigated with 100 μL sterile PBS. The irrigating fluid was placed in a clean and sterile EP tube. 10 μL of the irrigating fluid was taken onto a glass slide and evenly spread on the slide using a pipette. The slide was stained according to the instructions of the Gram staining kit and the fungal morphology was observed under a microscope. Another irrigating fluid was diluted 10 times with sterile PBS, and 10 μL was inoculated into Sabouraud agar medium. After culturing at 30°C for 48 hours, the colonies were observed and counted.

[0035] 4.5 Mouse vaginal index detection After the last administration, the mice were killed, the vagina was removed and the surface liquid was sucked dry, and the vaginal weight was weighed to calculate the vaginal index (vaginal index = vaginal weight / animal body weight).

[0036] 4.6 Detection of cytokines in mouse vaginal lavage fluid: After the treatment, vaginal lavage fluid from mice was collected and centrifuged at 1000 g for 20 min at 4°C according to the instructions of the ELISA kit. The supernatant was collected and the contents of inflammatory factors such as TNF-α, IL-6, IL-18, IL-1β, and IL-8 were detected.

[0037] 3. Experimental Results 1. Effect of the Chinese medicine composition of the present application on the basic conditions of VVC mice with spleen deficiency and dampness resistance The results are as follows Figure 1As shown, compared with the blank group, the mice in the model group had poor appetite, hair loss on the body surface, were timid, easily gathered in groups, were easily frightened, and had redness and swelling at the vaginal opening visibly, and increased vaginal secretions; compared with the model group, the Chinese medicine composition treatment group (lavage and gavage) of the present application significantly improved the body surface symptoms of the mice and the redness and swelling of the vaginal opening.

[0038] 2. Effect of the Chinese medicine composition of the present application on body weight changes of VVC mice with spleen deficiency and dampness obstruction like Figure 2 As shown, the experimental results showed that compared with the blank group, the body weight of mice in the model group decreased significantly 5 weeks after modeling (P < 0.001); compared with the model group, the body weight of the group treated with the Chinese medicine composition of the present application increased after administration (P < 0.001, P < 0.01), and there was no statistically significant difference in body weight between the mice in the fluconazole group and the model group.

[0039] 3. Effect of the Chinese medicine composition of the present application on local inflammation of the vulva of VVC mice with spleen deficiency and dampness obstruction The experimental results are as follows Figure 3 As shown, the blank group mice had no vaginal discharge, no obvious redness and swelling of the vulva, and a smooth surface; the model group mice had increased discharge, obvious vaginal redness and swelling after inoculation with Bacillus subtilis, and the vaginal symptom score increased significantly (P < 0.001); compared with the model group, the vaginal redness and swelling of the mice were relieved after administration, and the local inflammation score decreased (P < 0.01, P < 0.05).

[0040] 4. Effect of the Chinese medicine composition of the present application on fungal load in vaginal lavage fluid of VVC mice with spleen deficiency and dampness resistance like Figure 4 As shown, the experimental results showed that there was no Candida albicans colonization in the blank group mice. Compared with the blank group, on the 14th day, a large amount of Candida albicans was present in the vaginal lavage fluid of the model group mice (P < 0.001); compared with the model group, the fungal load in the drug-treated group was significantly decreased, and the downward trend in the Chinese medicine composition group of the present application was more obvious (P < 0.001).

[0041] 5. Effect of the Chinese medicine composition of the present application on the strain morphology of the vaginal lavage fluid of VVC mice with spleen deficiency and dampness resistance like Figure 5 As shown, the experimental results showed that no Candida albicans hyphae were found in the vaginal lavage fluid of the blank group mice 3, 7, and 14 days after the administration of bacteria; a large number of slender, intricately crossed hyphae were observed in the other groups after the administration of bacteria; after the administration, the model group still had slender hyphae, but no hyphae were found in the high-dose group of the Chinese medicine composition of the present application, the number of hyphae in the low-dose group was reduced and the length was shortened, and the fluconazole group still had longer hyphae. (Gram staining, ×200, black arrows point to hyphae) 6. Effect of the Chinese medicine composition of the present application on the vaginal index of VVC mice with spleen deficiency and dampness obstruction like Figure 6 As shown, the experimental results showed that the vagina of the blank group was slender and the vaginal index was low; compared with the blank group, the vaginal index of the mice in the model group was significantly increased (P < 0.001); compared with the model group, the vaginal index of the high-dose group of lavage and oral gavage of the Chinese medicine composition of the present application was significantly reduced (P < 0.001), and the low-dose group of oral gavage was also significantly reduced (P < 0.05).

[0042] 7. Effect of the Chinese medicine composition of the present application on inflammatory factors in vaginal lavage fluid of VVC mice with spleen deficiency and dampness obstruction like Figure 7 As shown, the experimental results showed that compared with the blank group, the levels of inflammatory factors TNF-α, IL-1β, IL-18, IL-8, and IL-6 in the vaginal lavage fluid of mice in the model group were significantly increased (P < 0.001, P < 0.01); compared with the model group, the lavage and gavage groups of the Chinese medicine composition of the present application could significantly reduce the levels of inflammatory factors TNF-α, IL-1β, IL-18, IL-8, and IL-6 (P < 0.001, P < 0.01, P < 0.05).

[0043] IV. Conclusion In summary, the Chinese medicine composition of the present application can improve the body surface state of VVC mice with spleen deficiency and dampness resistance type by oral and external administration; reduce local inflammation; reduce the fungal load in vaginal lavage fluid, reduce the number of hyphae and shorten the length of hyphae; improve the redness and swelling of vaginal tissue, reduce the vaginal index; reduce the level of inflammatory factors in vaginal lavage fluid, thereby achieving a therapeutic effect.

[0044] Example 3 Preparation of a Chinese medicine composition for treating spleen deficiency and dampness obstruction type candidal vaginitis The traditional Chinese medicine composition comprises phellodendron, smilax glabra, agrimony, snakehead and dittany.

[0045] Preparation of the lyophilized powder of the Chinese medicine composition of the present application: Weigh 36g of Phellodendron chinense, 90g of Smilax glabra, 54g of Herba Agrimoniae, 45g of Agrimoniae, and 54g of Dictamni, place the pieces in a sand pot, add about 10 times the amount of water, soak for 30min, boil over high heat, turn to low heat, keep boiling for 30min, pour out the filtrate after the first decoction, add appropriate amount of water, and decoct again for 30min. Combine the decoctions of the first and second decoctions, concentrate, and freeze-dry to obtain 50.4g of freeze-dried powder, which is stored in an ultra-low temperature refrigerator for standby use. The extraction rate of the Chinese medicine composition of the present application = crude drug weight / freeze-dried powder weight, which is calculated to be 18.06%.

[0046] Example 4 Experimental study on the treatment of spleen deficiency and dampness obstruction type candidal vaginitis using the Chinese medicine composition of the present application 1. Experimental Materials 1. Experimental strains The Candida albicans strain was used after recovery, subculture and activation.

[0047] Experimental drugs The lyophilized powder of the traditional Chinese medicine composition of the present application is calculated based on the equivalent dose of the drug to the mouse. 2.2 g of the lyophilized powder is weighed and ground into fine powder with a mortar. 10 mL of pure aqueous solution is added in small amounts and multiple times to fully dissolve it. The powder is prepared before each administration.

[0048] Prepare estradiol benzoate by adding sesame oil on the day of use to prepare a solution with a concentration of 1 mg / mL, store in a refrigerator at 4°C, and prepare it fresh before each use.

[0049] Culture medium Sabouraud liquid medium (SDB): Dissolve 32.55 g of liquid medium in 500 mL of ultrapure water, sterilize at 115°C for 15 minutes, place on a clean bench for 30 minutes, and store in a refrigerator at 4°C.

[0050] Sabouraud solid culture medium (SDA): Dissolve 32.55 g of solid culture medium in 500 mL of ultrapure water, sterilize at 115°C for 15 minutes, pour into a culture dish on a clean bench and wait for solidification, and store in a refrigerator at 4°C.

[0051] 2. Experimental Methods 1. Animal Grouping SPF Balb / c mice, female, 6-8 weeks old, 16g-20g (animal qualification certificate number: SYXK (Yunnan) K2017-0005), were randomly divided into 3 groups after one week of adaptive feeding, namely, blank group (Blank), spleen deficiency and dampness obstruction Candida infection model group (Model), and the Chinese medicine composition group of the present application (XWN). Before the experiment, the vaginal lavage fluid of the mice was collected for culture to observe whether Candida albicans grew, and mice without Candida albicans growth were selected for the experiment.

[0052] 2. Construction of mouse model of Candida vaginitis caused by spleen deficiency and dampness obstruction The modeling method is consistent with that in Example 2.

[0053] 3. Drug administration After the model was successfully established, the drug was administered by gavage at a dose of 204.4 g / kg, once a day, for 7 consecutive days, with reference to the drug instructions and the dosage of "Pharmacological Experimental Methodology". The general state and signs of mice in each group were observed daily, and the drug efficacy was evaluated according to the test indicators every day.

[0054] 4.Indicator detection 4.1 The following indicators of mice were evaluated: survival status, observation and scoring of local vulva conditions, determination of fungal load in vaginal lavage fluid, and detection of vaginal index. The evaluation method was consistent with that in Example 2.

[0055] 4.2 Periodic acid hexaammine silver (PASM) staining of mouse vaginal tissue (1) After the vaginal tissue was fixed with 4% paraformaldehyde overnight, it was dehydrated, embedded, sliced, and stained with PASM according to the following steps. (2) The fixed tissue was dehydrated in an automatic dehydrator (dehydration time: 75% alcohol for 3 hours, 85% alcohol for 1 hour, 95% alcohol for 1 hour and 20 minutes, 100% alcohol I for 25 minutes, 100% alcohol II for 20 minutes, 100% alcohol III for 20 minutes, 100% alcohol: xylene mixed in a ratio of 1:1 for 20 minutes, xylene I for 15 minutes, xylene II for 15 minutes, paraffin I for 1 hour, paraffin II for 2 hours, paraffin III for 3 hours), embedded, and sliced, and then the following operations were performed: ① Dewax the sections to water. Specific steps: xylene I 5-10 min, xylene II 5-10 min, anhydrous ethanol I 5 min, anhydrous ethanol II 5 min, 95% alcohol 5 min, 85% alcohol 5 min, 75% alcohol 5 min, UP water soaking 5 min.

[0056] ② Hexamine silver staining; ③ Gradient alcohol dehydration; ④ Xylene is transparent; ⑤Seal with neutral gum.

[0057] (3) Conduct microscopic examination and take photos under a microscope at 10× or 20×.

[0058] 3. Experimental Results 1. Effect of the Chinese medicine composition of the present application on the basic condition and vaginal appearance of VVC mice with spleen deficiency and dampness obstruction The results are as follows Figure 8 As shown, compared with the blank group, the mice in the model group had severe hair choking phenomenon on their body surface, were timid and easily frightened, turned their heads back to touch their vagina from time to time, the vaginal opening was red and swollen, vaginal secretions were visible, and the vaginal tissue was red, swollen and short; compared with the model group, there was no obvious improvement in the mice in the group treated with the traditional Chinese medicine composition of the present application.

[0059] 2. Effect of the Chinese medicine composition of the present application on local inflammation of the vulva of VVC mice with spleen deficiency and dampness obstruction The results are as follows Figure 8 and Fig. 9 As shown, the vaginal opening of the blank group mice was pink and white, and the vaginal symptom score was 0 (not shown in the figure); after the model group mice were inoculated with white pills, the secretions increased, the vagina was obviously red and swollen, and the vaginal symptom score increased significantly (P < 0.001); compared with the model group, the vaginal redness and swelling of the mice were slightly relieved after administration, and the local inflammation score decreased slightly, but there was no significant difference.

[0060] 3. Effect of the Chinese medicine composition of the present application on fungal load in vaginal lavage fluid of VVC mice with spleen deficiency and dampness resistance The results are as follows Fig.10 As shown, there was no Candida albicans colonization in the blank group mice (not shown in the figure); compared with the blank group, on the 14th day, a large amount of Candida albicans was present in the vaginal lavage fluid of the model group mice (P < 0.001); compared with the model group, there was no significant change in the colony count of the group administered with the traditional Chinese medicine composition of the present application.

[0061] 4. Effect of the Chinese medicine composition of the present application on the vaginal index of VVC mice with spleen deficiency and dampness obstruction The results are as follows Fig.11 and Fig.12 As shown, the vaginal tissue of the blank group was slender and smooth, and the vaginal index was low; compared with the blank group, the vaginal tissue of the mice in the model group was short and thick, and the index was significantly increased (P < 0.001); compared with the model group, the vaginal tissue of the Chinese medicine composition group of the present application had no obvious improvement.

[0062] 5. Effect of the Chinese medicine composition of the present application on Candida albicans colonization in vaginal tissues of VVC mice with spleen deficiency and dampness obstruction The results are as follows Fig.13 As shown, the vagina of mice in the blank group was normal, without Candida albicans colonization; the vagina of mice in the Model group was colonized with a large number of dark brown Candida albicans hyphae; a large number of hyphae colonization was also observed in the XWN group, and there was no significant improvement compared with the Model group. (The black arrow points to the hyphae) 4. Experimental Conclusion In summary, the freeze-dried powder of the Chinese medicine composition of the present application has no obvious improvement effect on VVC mice with spleen deficiency and dampness resistance type.

[0063] It is possible that moisture is not completely removed during freeze-drying or storage; the process is long and the drug activity is lost; the drug structure is unstable, affecting drug release.

[0064] Example 5 Evaluation of the minimum inhibitory concentration (MIC) of the Chinese medicine composition of the present application against Candida albicans in vitro The in vitro antibacterial activity of the Chinese medicine composition prepared in Example 1 of the present application against Candida albicans was tested according to the microdilution method recommended by the Clinical and Laboratory Standards Institute (CLSI) M27-A3. The minimum inhibitory concentration (MIC50) of the Chinese medicine composition prepared in Example 1 of the present application against Candida albicans was determined. Candida albicans in the logarithmic growth phase was adjusted to a concentration of 1×10 5CFU / mL. The Chinese medicine composition prepared in Example 1 of the present application was diluted 5 times with SDB in a 96-well culture plate to a final concentration of 0.032 mg / mL-100 mg / mL, with a total of 100 mg / mL, 20 mg / mL, 4 mg / mL, 0.8 mg / mL, 0.16 mg / mL and 0.032 mg / mL, 6 concentration gradients. A growth control group (drug-free and bacteria-free) and a blank control group (drug-free and sterile) were also set up. Three replicate wells were set in parallel for each drug and each concentration, and the final volume of each well was 200 µL. The 96-well culture plate was placed in a 30°C constant temperature incubator and cultured for 24 h, and the OD value was detected at 630 nm using a multifunctional microplate reader. The MIC50 value of each drug was calculated. The experiment was repeated 3 times.

[0065] Inhibition rate = 1 - (OD experimental - OD blank) / (OD control - OD blank) × 100%; MIC50: represents the lowest concentration of antifungal drug that inhibits 50% fungal growth compared to the growth control group.

[0066] The experimental results are shown in Table 1:

[0067] As can be seen from Table 1, the compound of Example 1 of the present application does not directly act on in vitro bacteria, but regulates the body's immune system through in vivo effects, regulates immune cells, improves immune response, and improves the body's immunity, thereby enhancing the body's ability to fight foreign pathogens, exerting an immunotherapeutic effect, and restoring vaginal mucosal damage.

[0068] Comparative Example 1 A Chinese medicine composition for treating spleen deficiency and dampness obstruction type candidal vaginitis It is composed of 4 Chinese medicinal herbs: Phellodendron amurense, Smilax glabra, Herba Agrimoniae, and Cortex Dictamni. Phellodendron amurense 12g, Smilax glabra 30g, Herba Agrimoniae 18g, Cortex Dictamni 18g, refer to the decoction method in the textbook of Chinese Materia Medica, add 10 times the amount of water to soak for 30 minutes, heat, concentrate and filter, add water to soak for 30 minutes, heat, concentrate and filter, mix the two filtrates thoroughly, heat and concentrate to a final concentration of 1g / ml, and place in a 4 ℃ refrigerator for later use.

[0069] Comparative Example 2 Effect of the Chinese medicine composition of Comparative Example 1 in treating spleen deficiency and dampness obstruction type candidal vaginitis 1. Experimental Materials 1. Experimental strains The Candida albicans strain was used after recovery, subculture and activation.

[0070] 2. Experimental drugs Comparative Example 1 Chinese medicine composition (without black snake) decoction 3. Culture medium Sabouraud liquid medium (SDB): Dissolve 32.55 g of liquid medium in 500 mL of ultrapure water, sterilize at 115°C for 15 minutes, place on a clean bench for 30 minutes, and store in a refrigerator at 4°C.

[0071] Sabouraud solid culture medium (SDA): Dissolve 32.55 g of solid culture medium in 500 mL of ultrapure water, sterilize at 115°C for 15 minutes, pour into a culture dish on a clean bench and wait for solidification, and store in a refrigerator at 4°C.

[0072] 2. Experimental Methods 1. Animal Grouping SPF Balb / c mice, female, 6-8 weeks old, 16g-20g (animal certificate number: SYXK (Yunnan) K2017-0005), were randomly divided into 3 groups after one week of adaptive feeding, namely, blank group (Blank), spleen deficiency and dampness obstruction Candida infection model group (Model), and the Chinese medicine composition (without black-banded snake) group of comparative example 1 (XWN (without black-banded snake)). Before the experiment, the vaginal lavage fluid of mice was collected for culture to observe whether Candida albicans grew, and mice without Candida albicans growth were selected for the experiment.

[0073] 2. Construction of mouse model of Candida vaginitis caused by spleen deficiency and dampness obstruction The blank group was fed normally for the first 14 days, and the other groups were set up with spleen deficiency and dampness resistance model, that is, they were kept in a climate box (humidity 90%, temperature 35±2℃) during the day, and fed with high-fat and high-sugar feed (80% ordinary feed + 12% lard + 8% honey); they were kept in a normal environment (humidity 60%, temperature 20±2℃) at night, and the model was set up alternately for 14 days. From the 15th day, except for the blank group, the other groups were subcutaneously injected with 0.025mL of estradiol benzoate E2 (1mg / mL) in the neck, once every 1 day, and a total of 4 injections. On the 22nd day, 20μL of Candida albicans suspension was sucked with a micropipette and injected into the vagina of mice except the blank group, and stayed in situ for 5 minutes (at least) to prevent bacterial liquid from overflowing. The blank group was injected with an equal amount of sterile PBS. The mice were inoculated once every 2 days for a total of 3 times. At the same time, the vagina of the mice was irrigated with sterile PBS on the 3rd and 7th days of modeling. The vaginal lavage fluid was collected for plate smear and Gram staining. Finally, the success of the vaginal model was comprehensively evaluated based on the results of lavage fluid smear microscopy, vaginal fungal CFU count, etc.

[0074] 3. Drug administration After the model was successfully established, the drug was administered by gavage at a dose of 20 ml / kg, once a day, for 7 consecutive days, with reference to the drug instructions and the dosage of "Pharmacological Experimental Methodology". The general state and signs of mice in each group were observed daily, and the drug efficacy was evaluated according to the test indicators every day.

[0075] 4.Indicator detection 4.1 Examination of mouse survival status: The survival status of the mice was observed throughout the experiment, the overall condition of the mice was observed, the body surface state and stress response of the mice were recorded, and whether the mice had behaviors such as poor appetite, depression, and susceptibility to fright was observed.

[0076] 4.2 Mouse weight recording The weight of mice was recorded every week during the experiment, and the weight changes of mice in each group were observed. After the experiment, the weight change curve of mice was compiled.

[0077] 4.3 Observation and scoring of local conditions of mouse vulva During the experiment, the whole body was observed, and local conditions such as swelling and discharge of the vaginal opening were observed; local symptoms of the vulva were scored after administration. The scoring standard was 0 points: no swelling and discharge of the vulva and vagina; 1 point: slight swelling and discharge of the vulva and vagina, no discharge; 2 points: obvious swelling and discharge of the vulva and vagina with discharge; 3 points: obvious swelling and discharge of the vulva and vagina, ulcers, and discharge; 4 points: death.

[0078] 4.4 Determination of fungal load in mouse vaginal lavage fluid At 7 days after modeling and 1 day after drug treatment, the lavage fluid was diluted 10 times with sterile PBS, and 10 μL was inoculated into Sabouraud agar medium. After culturing at 30°C for 48 hours, the colonies were observed and counted.

[0079] 3. Experimental Results 1. Effect of the Chinese medicine composition of comparative example 1 (without snake) on the basic condition and vaginal appearance of VVC mice with spleen deficiency and dampness obstruction The results are as follows Fig.14 As shown, the vagina of the mice in the blank group appeared white and translucent with a smooth surface. Compared with the blank group, the mice in the model group were sluggish in movement, with hairless bodies, obviously red and swollen vulva, and vaginal secretions were visible. Compared with the model group, there was no significant improvement in the mice in the group treated with the Chinese medicine composition of Comparative Example 1 (without black-necked snake).

[0080] 2. Effect of the Chinese medicine composition of comparative example 1 (without snake) on the body weight of VVC mice with spleen deficiency and dampness resistance The results are as follows Fig.15 As shown, compared with the blank group, the body weight of the mice in the model group decreased significantly 5 weeks after modeling (P < 0.001); compared with the model group, the body weight of the group treated with the Chinese medicine composition of Comparative Example 1 (without black-striped snake) increased slightly after administration, but there was no significant difference.

[0081] 3. Effect of the Chinese medicine composition of comparative example 1 (without black-striped snake) on local inflammation of the vulva in VVC mice of spleen deficiency and dampness obstruction type.

[0082] The results are as follows Fig.14 and Fig.16 As shown, the vaginal opening of mice in the blank group was pink and white, with no secretions and a vaginal symptom score of 0 (not shown in the figure); the secretions of mice in the model group increased, the vaginal opening was red and swollen, and the vaginal symptom score increased significantly (P <0.001); compared with the model group, the vaginal redness and swelling of mice were slightly relieved after administration, and the local inflammation score decreased slightly, but there was no significant difference.

[0083] 4. Effect of the Chinese medicine composition of comparative example 1 (without snake) on the fungal load in the vaginal lavage fluid of VVC mice with spleen deficiency and dampness resistance The results are as follows Fig.14 and Fig.17 As shown, there was no Candida albicans colonization in the blank group mice (not shown in the figure); compared with the blank group, a large amount of Candida albicans was present in the vaginal lavage fluid of the model group mice on the 14th day ( P <0.001); compared with the model group, the bacterial colony count in the vaginal lavage fluid of the mice in the drug-treated group did not improve significantly.

[0084] In summary, the Chinese medicine composition of Comparative Example 1 (without black-banded snake) had no significant improvement effect on VVC mice with spleen deficiency and dampness obstruction type.

[0085] The above-mentioned embodiments only express the specific implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the protection scope of the present application. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the technical solution concept of the present application, and these all belong to the protection scope of the present application.

Claims

1. A Chinese medicine composition for treating candidal vaginitis of spleen deficiency and dampness obstruction type, comprising 10-60 parts of Smilax glabra, 1-24 parts of Phellodendron chinense, 3-36 parts of Agrimoniae, 2-30 parts of Snakehead Snake, and 3-36 parts of Dictamni Cortex.

2. A Chinese medicine composition for treating spleen deficiency and dampness obstruction type candidal vaginitis according to claim 1, characterized in that: The traditional Chinese medicine composition comprises 25-35 parts of Rhizoma Smilacis Glabrae, 7-17 parts of Phellodendron Amurense, 13-23 parts of Herba Agrimoniae, 10-20 parts of Snakehead Snake, and 13-23 parts of Cortex Dictamni.

3. A Chinese medicine composition for treating spleen deficiency and dampness obstruction type candidal vaginitis according to claim 1, characterized in that: The traditional Chinese medicine composition comprises 27-33 parts of Rhizoma Smilacis Glabrae, 9-15 parts of Phellodendron Amurense, 15-21 parts of Herba Agrimoniae, 13-17 parts of Snakehead Snake, and 15-21 parts of Cortex Dictamni.

4. A Chinese medicine composition for treating spleen deficiency and dampness obstruction type candidal vaginitis according to claim 3, characterized in that: The traditional Chinese medicine composition comprises 30 parts of Rhizoma Smilacis Glabrae, 12 parts of Cortex Phellodendri, 18 parts of Herba Agrimoniae and 18 parts of Cortex Dictamni.

5. A Chinese medicine composition for treating spleen deficiency and dampness obstruction type candidal vaginitis according to claim 1, characterized in that: The pathogenic bacteria of the spleen deficiency and dampness obstruction type candidal vaginitis are Candida albicans, Candida tropicalis, Candida glabrata, Candida parapsilosis, Candida krusei or Candida dubliniensis.

6. A Chinese medicine composition for treating spleen deficiency and dampness obstruction type candidal vaginitis according to claim 5, characterized in that: The Candida albicans is ATCC14053.

7. A Chinese medicine composition for treating spleen deficiency and dampness obstruction type candidal vaginitis according to any one of claims 1 to 6, characterized in that: The traditional Chinese medicine composition is a water-extracted concentrated solution.

8. A Chinese medicine composition for treating spleen deficiency and dampness obstruction type candidal vaginitis according to claim 7, characterized in that: It is administered externally or orally.

9. A Chinese medicine composition for treating spleen deficiency and dampness obstruction type candidal vaginitis according to claim 8, characterized in that: The dosage for external use is: 1.25g / kg-2.5g / kg.

10. A Chinese medicine composition for treating spleen deficiency and dampness obstruction type candidal vaginitis according to claim 8, characterized in that: The oral dosage is: 10g / kg-20g / kg.

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