A traditional Chinese medicine composition for treating candidal vaginitis of spleen deficiency and dampness obstruction type

Abstract

The present invention relates to the technical field of traditional Chinese medicine compositions. The present invention discloses a traditional Chinese medicine composition for treating candidal vaginitis of the spleen deficiency and dampness obstruction type, which comprises 10 parts - 60 parts of Smilax glabra, 1 part - 24 parts of Phellodendron amurense, 3 parts - 36 parts of Agrimonia pilosa, 2 parts - 30 parts of Zaocys dhumnades, and 3 parts - 36 parts of Dictamnus dasycarpus Turcz. The traditional Chinese medicine composition of the present invention can be administered by irrigation or oral administration to exert its effects, can improve and treat the symptoms of model mice with candidal vaginitis of the spleen deficiency and dampness obstruction type, increase the body weight of the mice, reduce the levels of inflammatory factors, and reduce the vaginal index, and has great popularization and application value.

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:

[0006] 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 pygmy serpentine, and 3-36 parts of difficile cortex.

[0007] Preferably, the traditional Chinese medicine composition comprises 25 parts - 35 parts of Smilax glabra Roxb., 7 parts - 17 parts of Phellodendron amurense Rupr., 13 parts - 23 parts of Agrimonia pilosa Ledeb., 10 parts - 20 parts of Zaocys dhumnades (Cantor), and 13 parts - 23 parts of Dictamnus dasycarpus Turcz.

[0008] Preferably, the traditional Chinese medicine composition comprises 27 parts - 33 parts of Smilax glabra Roxb., 9 parts - 15 parts of Phellodendron amurense Rupr., 15 parts - 21 parts of Agrimonia pilosa Ledeb., 13 parts - 17 parts of Zaocys dhumnades (Cantor), and 15 parts - 21 parts of Dictamnus dasycarpus Turcz.

[0009] Preferably, the traditional Chinese medicine composition comprises 30 parts of Smilax glabra Roxb., 12 parts of Phellodendron amurense Rupr., 18 parts of Agrimonia pilosa Ledeb., and 18 parts of Dictamnus dasycarpus Turcz.

[0010] The traditional Chinese medicine composition of the present application is composed of 5 traditional Chinese medicines, namely Smilax glabra Roxb., Phellodendron amurense Rupr., Agrimonia pilosa Ledeb., Zaocys dhumnades (Cantor), and Dictamnus dasycarpus Turcz. With the overall compatibility, it is suitable for both oral and external use. The combination of various medicines together exerts the effects of drying dampness and clearing heat, killing insects and relieving itching, and ultimately achieves the therapeutic purpose of removing dampness without completely exhausting it and promoting the transportation function of the spleen when dampness is removed.

[0011] Preferably, the candida is Candida albicans, Candida tropicalis, Candida glabrata, Candida parapsilosis, Candida krusei, or Candida dubliniensis.

[0012] Preferably, the Candida albicans is ATCC14053.

[0013] According to a preferred embodiment, the traditional Chinese medicine composition is a water extract concentrate.

[0014] According to a preferred embodiment, the traditional Chinese medicine composition is administered by external use or oral administration.

[0015] According to a preferred embodiment, the oral administration dose of the traditional Chinese medicine composition is: 10 g / kg - 20 g / kg. The external use administration dose is: 1.25 g / kg - 2.5 g / kg.

[0016] Compared with the existing technology, the beneficial effects of the present invention are:

[0017] 1. A traditional Chinese medicine composition for treating candidal vaginitis of spleen deficiency and dampness obstruction type. In this formula, Smilax glabra Roxb. is used as the monarch drug to detoxify and remove dampness, promote diuresis and clear heat, and mainly treat various syndromes of abnormal vaginal discharge caused by dampness-heat toxin accumulation; Phellodendron amurense Rupr. is bitter and cold in nature, clearing heat and drying dampness, relieving itching and detoxifying, and has a broad-spectrum antifungal effect; Agrimonia pilosa Ledeb. can arrest bleeding, tonify deficiency, kill insects and detoxify, and is suitable for people with symptoms such as vulvar pruritus, sores, and eczema, and together with Phellodendron amurense Rupr., it serves as the ministerial drug; Zaocys dhumnades (Cantor) is good at moving around and searching for wind and relieving itching in the collaterals, assisting the monarch drug Smilax glabra Roxb. to search for and remove dampness toxin in the collaterals. Dictamnus dasycarpus Turcz. is bitter and dry in nature, clearing heat and drying dampness, dispelling wind and detoxifying, and is a drug for removing dampness in the spleen and stomach meridians of the foot taiyin and yangming. The two are adjuvant drugs. With the overall compatibility, it is suitable for both oral and external use. By combining drugs for removing dampness, expelling wind, and dredging collaterals, it achieves the therapeutic purpose of removing dampness without completely exhausting it and promoting the transportation function of the spleen when dampness is removed.

[0018] 2. A traditional Chinese medicine composition for treating candidal vaginitis with spleen deficiency and dampness obstruction. The compound composition provided by the present invention can effectively treat and relieve candidal vaginitis with spleen deficiency and dampness obstruction both orally and topically, 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 anti-fungal drugs such as traditional Chinese medicines and compound preparations.

[0019] 3. A traditional Chinese medicine composition for treating candidal vaginitis with spleen deficiency and dampness obstruction. Based on the addition of the key component Zaocys dhumnades, it acts together with Phellodendron chinense, Agrimonia pilosa, Dictamnus dasycarpus, and Smilax glabra to exert excellent effects in treating candidal vaginitis with spleen deficiency and dampness obstruction, breaking the technical prejudice that traditional Chinese medicines have a slow onset of effect when taken orally, and the oral effect is close to the onset time of douching. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 For the basic conditions and appearance changes of mice in each group in Example 2;

[0021] Figure 2 For the body weight changes of mice in Example 2;

[0022] Figure 3 For the local vaginal inflammation changes of mice in Example 2;

[0023] Figure 4 For the changes in the fungal load in the vaginal lavage fluid of mice in Example 2;

[0024] Figure 5 For the changes in the fungal morphology in the vaginal lavage fluid of mice in Example 2;

[0025] Figure 6 For the vaginal index changes of mice in Example 2;

[0026] Figure 7 For the levels 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;

[0027] Figure 8 For the basic conditions and appearance changes of mice in Example 4;

[0028] Figure 9 For the local vaginal inflammation changes of mice in Example 4;

[0029] Figure 10 For the changes in the 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;

[0030] Figure 11 Appearance changes of the vaginal tissue of mice in Example 4;

[0031] Figure 12 For the change of vaginal index of mice in Example 4;

[0032] Figure 13 For the change of the colonization of Candida albicans in the vaginal tissue of mice in Example 4;

[0033] Figure 14 For the basic conditions, appearance changes and fungal load of mice in each group of Comparative Example 2;

[0034] Figure 15 For the change of body weight of mice in each group of Comparative Example 2;

[0035] Figure 16 For the change of vaginal index of mice in Comparative Example 2;

[0036] Figure 17 For the numerical change of fungal load in the vaginal lavage fluid of mice in Comparative Example 2. Detailed implementation manners

[0037] The specific examples listed in the present invention are only examples of the present invention, and the present invention is not limited to the specific examples described below. For those skilled in the art, any equivalent modifications and substitutions to the examples described below are also within the scope of the present invention. Therefore, equivalent transformations and modifications made without departing from the spirit and scope of the present invention should be covered within the scope of the present invention. For those conditions not specified in the examples, they are carried out according to the conventional conditions or the conditions recommended by the manufacturer. For all reagents or instruments without indicating the manufacturer, 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 following detailed implementation manners. Those skilled in the art should understand that the present invention can still be implemented without some specific details. In other embodiments, methods, means, equipment and steps well-known to those skilled in the art are not described in detail in order to highlight the gist of the present invention.

[0038] 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.

[0039] Candida albicans strains were purchased from ATCC, USA;

[0040] Sabouraud liquid medium (SDB) was purchased from Guangdong Huankai Microbiological Technology Co., Ltd.

[0041] Sabouraud solid medium (SDA) was purchased from Guangdong Huankai Microbiological Technology Co., Ltd.

[0042] Fluconazole (FLC) solution was purchased from Nanchang Hongyi Pharmaceutical Co., Ltd.

[0043] Estradiol benzoate was purchased from Shanghai Quanyu Biotechnology Co., Ltd.

[0044] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0045] Example 1 A Chinese medicine composition for treating spleen deficiency and dampness obstruction type candidal vaginitis

[0046] 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.

[0047] 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

[0048] 1. Experimental Materials

[0049] 1. Experimental strains

[0050] The Candida albicans strain was used after recovery, subculture and activation.

[0051] 2. Experimental drugs

[0052] Positive control drug fluconazole (FLC) solution: The tablets were ground into fine powder, dissolved in DMSO, mixed well by ultrasonic treatment for 20 min, centrifuged, and the supernatant was taken. The storage concentration was 50 mg / mL, and it was stored in a sealed manner in a 4°C refrigerator for later use. Estradiol benzoate was added to sesame oil on the day of use to prepare a solution with a concentration of 1 mg / mL, stored in a 4°C refrigerator, and freshly prepared before each use.

[0053] Experimental drug: The traditional Chinese medicine composition prepared in Example 1

[0054] 3. Culture medium

[0055] Sabouraud dextrose broth (SDB):

[0056] 32.55 g of liquid medium was dissolved in 500 mL of ultrapure water, sterilized at 115°C for 15 minutes, placed in a laminar flow hood for 30 minutes, and stored in a 4°C refrigerator.

[0057] Sabouraud dextrose agar (SDA): 32.55 g of solid medium was dissolved in 500 mL of ultrapure water, sterilized at 115°C for 15 minutes, poured into petri dishes in a laminar flow hood and allowed to solidify, and stored in a 4°C refrigerator.

[0058] II. Experimental methods

[0059] 1. Animal grouping

[0060] SPF-grade Balb / c mice, female, 6 - 8 weeks old, 16 g - 20 g (animal certificate number: SYXK (Yunnan) K2017 - 0005). After one week of adaptive feeding, they were randomly divided into 8 groups, namely the blank group (Blank), the Candida albicans infection model group with spleen deficiency and dampness obstruction (Model), the high-dose lavage group of the traditional Chinese medicine composition of this application (XWN-H (perf)), the low-dose lavage group of the traditional Chinese medicine composition of this application (XWN-L (perf)), the high-dose intragastric administration group of the traditional Chinese medicine composition of this application (XWN-H(ig)), the low-dose intragastric administration group of the traditional Chinese medicine composition of this application (XWN-L(ig)), the fluconazole lavage group (FLC(perf)), and the fluconazole intragastric administration group (FLC(ig)). Before the experiment, the vaginal lavage fluid of the mice was collected for culture to observe the growth of Candida albicans. Mice without the growth of Candida albicans were selected for the experiment.

[0061] 2. Establishment of a mouse model of Candida albicans vaginitis with spleen deficiency and dampness obstruction

[0062] In the first 14 days, the blank group was fed normally, and the other groups were made into the spleen deficiency and dampness obstruction models, that is, they were raised in a climate chamber (humidity 90%, temperature 35±2°C) during the day, and at the same time fed a high-fat and high-sugar diet (80% ordinary feed + 12% lard + 8% honey); they were raised in a normal environment (humidity 60%, temperature 20±2°C) at night, and the modeling was carried out alternately for 14 days. Starting from the 15th day, except for the blank group, the other groups were subcutaneously injected with estradiol benzoate E2 (1mg / mL) 0.025mL in the neck, injected once every other day, and injected 4 times in total. On the 22nd day, 20 μL of Candida albicans suspension was aspirated with a micropipette and injected into the vagina of mice except the blank group, and stayed in situ for at least 5 minutes to prevent the overflow of the bacterial solution. The blank group was injected with an equal amount of sterile PBS. It was inoculated once every 2 days, and inoculated 3 times in total. At the same time, on the 3rd and 7th days of modeling, the vaginas of mice were lavaged with sterile PBS, the vaginal lavage fluid was collected, and plate coating and Gram staining microscopy were performed. Finally, the success of the vaginal model was comprehensively evaluated according to the results of smear microscopy of the lavage fluid and the counting of vaginal fungal CFU.

[0063] 3. Drug administration

[0064] After the successful establishment of the model, referring to the drug dosage in the drug instruction manual and "Pharmacological Experiment Methodology", intragastric administration and vaginal lavage administration were adopted. The high-dose group for lavage was 2.5 g / kg; the low-dose group for lavage was: 1.25 g / kg, the low-dose group for intragastric administration was 10 g / kg, and the high-dose group for intragastric administration was 20 g / kg. It was administered 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 detection indexes every day.

[0065] 4. Index detection

[0066] 4.1 Examination of the survival status of mice:

[0067] The survival status of mice was observed throughout the experiment, the general condition of mice was observed, the surface state and stress response of mice were recorded, and whether there were behaviors such as reduced food intake, depression, and being easily frightened in mice was observed.

[0068] 4.2 Recording of the body weight of mice

[0069] The body weight of mice was recorded during the experiment, and the change in the body weight of mice was sorted out after the experiment.

[0070] 4.3 Observation and scoring of the local condition of the vulva of mice

[0071] During the experiment, the general condition of the mice was observed, and local conditions such as vulvar swelling and secretions were observed; the local symptoms of the vulva were scored after administration. The scoring criteria were as follows: 0 points: no swelling or increased secretions in the vulva and vagina; 1 point: slight swelling in the vulva and vagina, no increased secretions; 2 points: obvious swelling in the vulva and vagina accompanied by increased secretions; 3 points: obvious swelling in the vulva and vagina, ulcers appeared, and accompanied by increased secretions; 4 points: death.

[0072] 4.4 Microscopic examination of mouse vaginal lavage fluid smear and determination of fungal load

[0073] On the 1st, 3rd, and 7th days after modeling, and 1 day after drug treatment, the vaginas of mice in each group were slowly irrigated with 100 μL of sterile PBS. The lavage fluid was placed in a clean and sterile EP tube. Take 10 μL of the lavage fluid onto a glass slide, and use a pipette to evenly smear it on the glass slide. Stain according to the instructions of the Gram staining kit, and observe the fungal morphology under the microscope; another 10 μL of the lavage fluid was diluted 10 times with sterile PBS, and 10 μL was inoculated on Sabouraud agar medium. After culturing at 30 °C for 48 h, colony observation and counting were performed.

[0074] 4.5 Detection of mouse vaginal index

[0075] The mice were sacrificed after the last administration. The vagina was taken out, the surface liquid was blotted dry, and then the weight of the vagina was measured. The vaginal index was calculated (vaginal index = vaginal weight / animal body weight).

[0076] 4.6 Detection of cytokines in mouse vaginal lavage fluid:

[0077] After the treatment was completed, the vaginal lavage fluid of the mice was taken. According to the instructions of the ELISA kit, it was centrifuged at 1000 g for 20 minutes at 4 °C, 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.

[0078] III. Experimental results

[0079] 1. Effects of the traditional Chinese medicine composition of the present application on the basic conditions of mice with spleen deficiency and dampness obstruction type VVC

[0080] The results were as Figure 1 shown. Compared with the blank group, the mice in the model group had reduced food intake, hair loss and rough hair on the body surface, timidity, tendency to huddle together, easy to be frightened, and visible swelling of the vaginal orifice and increased vaginal secretions; compared with the model group, the traditional Chinese medicine composition treatment group (irrigation and gavage) of the present application significantly improved the body surface symptoms of the mice and the swelling of the vaginal orifice.

[0081] 2. Effects of the traditional Chinese medicine composition of the present application on the body weight change of mice with spleen deficiency and dampness obstruction type VVC

[0082] As Figure 2As shown, the experimental results indicate that, compared with the blank group, after 5 weeks of modeling, the body weight of the mice in the model group decreased significantly (P < 0.001); compared with the model group, the body weight of the mice in the treatment group with the traditional Chinese medicine composition of the present application increased after administration (P < 0.001, P < 0.01), and there was no significant difference in the body weight of the mice in the fluconazole group and the model group.

[0083] 3. Influence of the traditional Chinese medicine composition of the present application on local vulvar inflammation in mice with spleen deficiency and dampness obstruction type VVC

[0084] The experimental results are as Figure 3 shown, indicating that there was no vaginal secretion in the mice of the blank group, no obvious swelling of the vulva, and the surface was smooth; after inoculating Candida albicans in the mice of the model group, the secretion increased, the vagina was significantly swollen, and the vaginal symptom score increased significantly (P < 0.001); compared with the model group, after administration, the vaginal swelling of the mice was relieved and the local inflammation score decreased (P < 0.01, P < 0.05).

[0085] 4. Influence of the traditional Chinese medicine composition of the present application on fungal load in vaginal lavage fluid of mice with spleen deficiency and dampness obstruction type VVC

[0086] As Figure 4 shown, the experimental results indicate that there was no colonization of Candida albicans in the mice of the blank group. Compared with the blank group, on the 14th day, a large number of Candida albicans were present in the vaginal lavage fluid of the mice in the model group (P < 0.001); compared with the model group, the fungal load in the administration group decreased significantly, and the decreasing trend in the group with the traditional Chinese medicine composition of the present application was more obvious (P < 0.001).

[0087] 5. Influence of the traditional Chinese medicine composition of the present application on the morphology of strains in vaginal lavage fluid of mice with spleen deficiency and dampness obstruction type VVC

[0088] As Figure 5 shown, the experimental results indicate that no hyphae of Candida albicans were found in the vaginal lavage fluid of the mice in the blank group on the 3rd, 7th, and 14th days after inoculating the bacteria; a large number of long and slender, intricately crossed hyphae could be observed in other groups after inoculating the bacteria; after administration, long and slender hyphae were still visible in the model group, no hyphae were seen in the high-dose group with the traditional Chinese medicine composition of the present application, the number of hyphae decreased and the length shortened in the low-dose group, and relatively long hyphae were still present in the fluconazole group. (Gram staining, ×200, black arrow points to the hyphae)

[0089] 6. Influence of the traditional Chinese medicine composition of the present application on vaginal index in mice with spleen deficiency and dampness obstruction type VVC

[0090] As Figure 6As shown, the experimental results indicate that the vagina of the blank group is slender and the vaginal index is relatively low; compared with the blank group, the vaginal index of the mice in the model group is significantly increased (P < 0.001); compared with the model group, the vaginal index of the high-dose group of the traditional Chinese medicine composition of the present application by lavage and intragastric administration is significantly decreased (P < 0.001), and that of the low-dose intragastric administration group is also significantly decreased (P < 0.05).

[0091] 7. Influence of the traditional Chinese medicine composition of the present application on inflammatory factors in the vaginal lavage fluid of mice with VVC of spleen deficiency and dampness obstruction type

[0092] As Figure 7 shown, the experimental results show 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 the mice in the model group are significantly increased (P < 0.001, P < 0.01); compared with the model group, both the lavage and intragastric administration groups of the traditional Chinese medicine composition of the present application can 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).

[0093] IV. Conclusion

[0094] In summary, both oral and topical administration of the traditional Chinese medicine composition of the present application can improve the body surface state of mice with VVC of spleen deficiency and dampness obstruction type; reduce local inflammation; reduce the fungal load in the vaginal lavage fluid, reduce the number of hyphae and shorten the hyphal length; improve the red and swollen phenomenon of vaginal tissue and reduce the vaginal index; reduce the level of inflammatory factors in the vaginal lavage fluid, thereby achieving the therapeutic effect.

[0095] Example 3 Preparation of a traditional Chinese medicine composition for treating candidal vaginitis of spleen deficiency and dampness obstruction type

[0096] The traditional Chinese medicine composition includes Phellodendri Chinensis Cortex, Smilacis Glabrae Rhizoma, Agrimonia Pilosa Ledeb., Zaocys, and Dictamni Cortex.

[0097] Preparation of the freeze-dried powder of the traditional Chinese medicine composition of the present application:

[0098] Weigh 36 g of Phellodendri Chinensis Cortex, 90 g of Smilacis Glabrae Rhizoma, 54 g of Agrimonia Pilosa Ledeb., 45 g of Zaocys, and 54 g of Dictamni Cortex. Place the cut herbs in a sand pot, add about 10 times the amount of water, soak for 30 minutes, bring to a boil over high heat, turn to low heat, and keep boiling gently for 30 minutes. After the first decoction, pour out the filtrate, add an appropriate amount of water again, and decoct for another 30 minutes. Combine the decoctions of the first and second decoctions, concentrate, and freeze-dry to obtain 50.4 g of freeze-dried powder, which is stored in an ultra-low temperature refrigerator for later use. The extraction rate of the traditional Chinese medicine composition of the present application = weight of crude drug / weight of freeze-dried powder, and the calculated value is 18.06%.

[0099] Example 4 Experiment on treating candidal vaginitis of spleen deficiency and dampness obstruction type with the traditional Chinese medicine composition of the present application

[0100] I. Experimental Materials

[0101] 1. Experimental Strains

[0102] The Candida albicans strains were used after resuscitation, subculture and activation.

[0103] Experimental Drugs

[0104] For the freeze-dried powder of the traditional Chinese medicine composition of this application, according to the conversion of the equivalent dose of the drug and mice, 2.2 g of the freeze-dried powder was weighed and ground into fine powder in a mortar, and 10 mL of pure aqueous solution was added in small amounts and multiple times to dissolve it fully. It was prepared immediately before each administration.

[0105] On the day of use, estradiol benzoate was added to sesame oil to prepare a solution with a concentration of 1 mg / mL, stored in a refrigerator at 4°C, and prepared immediately before each use.

[0106] Culture Media

[0107] Sabouraud Dextrose Broth (SDB): 32.55 g of liquid medium was dissolved in 500 mL of ultrapure water, sterilized at 115°C for 15 minutes, placed in a laminar flow hood for 30 minutes, and stored in a refrigerator at 4°C.

[0108] Sabouraud Dextrose Agar (SDA): 32.55 g of solid medium was dissolved in 500 mL of ultrapure water, sterilized at 115°C for 15 minutes, poured into petri dishes in a laminar flow hood and allowed to solidify, and stored in a refrigerator at 4°C.

[0109] II. Experimental Methods

[0110] 1. Animal Grouping

[0111] SPF-grade Balb / c mice, female, 6 - 8 weeks old, 16 g - 20 g (animal certificate number: SYXK (Yunnan) K2017 - 0005). After one week of adaptive feeding, they were randomly divided into 3 groups, namely the blank group (Blank), the Candida infection model group with spleen deficiency and damp obstruction (Model), and the traditional Chinese medicine composition group of this application (XWN). Before the experiment, vaginal lavage fluid of the mice was collected for culture to observe the growth of Candida albicans, and mice without the growth of Candida albicans were selected for the experiment.

[0112] 2. Establishment of the Mouse Model of Candidal Vaginitis with Spleen Deficiency and Damp Obstruction

[0113] The modeling method was the same as that in Example 2.

[0114] 3. Administration

[0115] After the successful establishment of the model, referring to the dosage in the drug instruction manual and "Pharmacological Experiment Methodology", intragastric administration was carried out. The intragastric administration dosage was 204.4 g / kg, once a day, and continuous administration was carried out for 7 days. The general state and signs of mice in each group were observed daily, and the drug efficacy was evaluated according to the detection indexes every day.

[0116] 4. Index Detection

[0117] 4.1 The following indexes 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 the same as that in Example 2.

[0118] 4.2 Periodic acid hexamine silver (PASM) staining of mouse vaginal tissue

[0119] (1) After the vaginal tissue was fixed with 4% paraformaldehyde overnight, dehydration, embedding, sectioning, and PASM staining were carried out according to the following steps

[0120] (2) The fixed tissue was dehydrated by an automatic dehydrator (dehydration duration: 75% alcohol for 3 h, 85% alcohol for 1 h, 95% alcohol for 1 h 20 min, 100% alcohol I for 25 min, 100% alcohol II for 20 min, 100% alcohol III for 20 min, 100% alcohol: xylene 1:1 mixture for 20 min, xylene I for 15 min, xylene II for 15 min, paraffin I for 1 h, paraffin II for 2 h, paraffin III for 3 h), embedded, and sectioned, and then the following operations were carried out:

[0121] ① The sections were dewaxed to water. The specific operation was: xylene I for 5 - 10 min, xylene II for 5 - 10 min, absolute ethanol I for 5 min, absolute ethanol II for 5 min, 95% alcohol for 5 min, 85% alcohol for 5 min, 75% alcohol for 5 min, and soaked in UP water for 5 min.

[0122] ② Hexamine silver staining;

[0123] ③ Gradient alcohol dehydration;

[0124] ④ Xylene transparency;

[0125] ⑤ Sealing with neutral gum.

[0126] (3) Microscopic examination and photographing observation were carried out under 10× and 20× of the microscope.

[0127] III. Experimental Results

[0128] 1. Effects of the traditional Chinese medicine composition of the present application on the basic conditions and vaginal appearance of VVC mice with spleen deficiency and dampness obstruction

[0129] The results are as Figure 8As shown, compared with the blank group, the mice in the model group had severe hair bristling on the body surface, were timid and easily frightened, often turned back to touch the vagina, the vaginal orifice was red and swollen, vaginal secretions were visible, and the vaginal tissue was red, swollen, thick and short; compared with the model group, there was no obvious improvement in the mice in the traditional Chinese medicine composition treatment group of this application.

[0130] 2. Influence of the traditional Chinese medicine composition of this application on the local inflammation of the vulva of mice with spleen deficiency and damp obstruction type VVC

[0131] The results are as Figure 8 and Figure 9 shown. The vaginal orifice of the mice in the blank group was pinkish white, and the vaginal symptom score was 0 (not shown in the figure); after the mice in the model group were inoculated with Candida albicans, the secretions increased, the vagina was significantly red and swollen, and the vaginal symptom score increased significantly (P < 0.001); compared with the model group, after administration, the redness and swelling of the vagina of the mice were slightly relieved, and the local inflammation score decreased slightly, but there was no significant difference.

[0132] 3. Influence of the traditional Chinese medicine composition of this application on the fungal load in the vaginal lavage fluid of mice with spleen deficiency and damp obstruction type VVC

[0133] The results are as Figure 10 shown. There was no colonization of Candida albicans in the bodies of the mice in the blank group (not shown in the figure); compared with the blank group, on the 14th day, there were a large number of Candida albicans in the vaginal lavage fluid of the mice in the model group (P < 0.001); compared with the model group, there was no significant change in the number of colonies in the traditional Chinese medicine composition administration group of this application.

[0134] 4. Influence of the traditional Chinese medicine composition of this application on the vaginal index of mice with spleen deficiency and damp obstruction type VVC

[0135] The results are as Figure 11 and Figure 12 shown. The vaginal tissue of the mice in 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 increased significantly (P < 0.001); compared with the model group, there was no obvious improvement in the vaginal tissue of the traditional Chinese medicine composition group of this application.

[0136] 5. Influence of the traditional Chinese medicine composition of this application on the colonization of Candida albicans in the vaginal tissue of mice with spleen deficiency and damp obstruction type VVC

[0137] The results are as Figure 13 shown. The vagina of the mice in the blank group was normal, without colonization of Candida albicans; there were a large number of dark brown hyphae of Candida albicans colonized in the vagina of the mice in the Model group; a large number of hyphae were also visible in the XWN group, and there was no obvious improvement compared with the Model group. (The black arrow points to the hyphae)

[0138] IV. Experimental conclusions

[0139] In summary, the freeze-dried powder of the traditional Chinese medicine composition of the present application has no obvious improvement effect on VVC mice with spleen deficiency and dampness obstruction.

[0140] It may be that the moisture removal during freeze-drying or storage is not thorough; the process is long, and the drug activity is lost; the drug structure is unstable, affecting drug release.

[0141] Example 5 Evaluation of the minimum inhibitory concentration (MIC) of the traditional Chinese medicine composition of the present application against Candida albicans in vitro

[0142] For the in vitro antibacterial activity of the traditional Chinese medicine composition prepared in Example 1 of the present application against Candida albicans, it was carried out according to the microdilution method recommended by the Clinical and Laboratory Standards Institute (CLSI) M27-A3. The minimum inhibitory concentration (MIC50) of the traditional 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 5 CFU / mL with SDB. The traditional Chinese medicine composition prepared in Example 1 of the present application was serially diluted 5-fold with SDB in a 96-well culture plate to a final concentration of 0.032 mg / mL - 100 mg / mL, and a total of 6 concentration gradients of 100 mg / mL, 20 mg / mL, 4 mg / mL, 0.8 mg / mL, 0.16 mg / mL, and 0.032 mg / mL were set. At the same time, a growth control group (with bacteria but no drug) and a blank control group (no drug and no bacteria) were set. Each drug had three parallel replicates for each concentration, and the final volume of each well was 200 µL. The 96-well culture plate was placed in an incubator at 30°C for 24 h, and the OD value was detected at 630 nm with a multifunctional microplate reader. The MIC50 value of each drug was calculated. The experiment was repeated 3 times.

[0143] Bacteriostatic rate = 1 - (ODexperiment - ODblank) / (ODcontrol - ODblank) × 100%;

[0144] MIC50: It represents the lowest concentration of an antifungal drug that inhibits 50% of fungal growth compared with the growth control group.

[0145] The experimental results are shown in Table 1 below:

[0146]

[0147] As can be seen from Table 1, the compound in Example 1 of the present application does not directly act on the in vitro bacteria, but regulates the body's immune system through in vivo action, regulates immune cells, improves the immune response, improves the body's immunity, thereby enhancing the body's ability to resist foreign pathogens, plays an immunotherapeutic role, and restores vaginal mucosal damage.

[0148] Comparative Example 1 A traditional Chinese medicine composition for treating candidal vaginitis with spleen deficiency and dampness obstruction

[0149] It is composed of 4 traditional Chinese medicines, namely Phellodendri Chinensis Cortex, Smilacis Glabrae Rhizoma, Agrimonia Pilosa Ledeb., and Dictamni Cortex. 12 g of Phellodendri Chinensis Cortex, 30 g of Smilacis Glabrae Rhizoma, 18 g of Agrimonia Pilosa Ledeb., and 18 g of Dictamni Cortex. Referring to the decocting method in the textbook of Chinese Materia Medica, add 10 times the amount of water and soak for 30 min, heat and concentrate, filter, then add water and soak for 30 min again, heat and concentrate, filter. Mix the two filtrates thoroughly, heat and concentrate to a final concentration of 1 g / ml, and store in a refrigerator at 4 °C for later use.

[0150] The effect of the traditional Chinese medicine composition of Comparative Example 2 on the treatment of candidal vaginitis with spleen deficiency and damp obstruction

[0151] I. Experimental materials

[0152] 1. Experimental strains

[0153] The Candida albicans strain is used after resuscitation, subculture, and activation.

[0154] 2. Experimental drugs

[0155] The decoction of the traditional Chinese medicine composition of Comparative Example 1 (without Zaocys dhumnades)

[0156] 3. Culture media

[0157] 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 in a laminar flow hood for 30 minutes, and store in a refrigerator at 4 °C.

[0158] Sabouraud solid medium (SDA): Dissolve 32.55 g of solid medium in 500 mL of ultrapure water, sterilize at 115 °C for 15 minutes, pour into petri dishes in a laminar flow hood until solidified, and store in a refrigerator at 4 °C.

[0159] II. Experimental methods

[0160] 1. Animal grouping

[0161] SPF-grade Balb / c mice, female, 6 - 8 weeks old, 16 g - 20 g (animal certificate number: SYXK (Yunnan) K2017 - 0005). After one week of adaptive feeding, they are randomly divided into 3 groups, namely the blank group (Blank), the spleen deficiency and damp obstruction with candidal infection model group (Model), and the traditional Chinese medicine composition of Comparative Example 1 (without Zaocys dhumnades) group (XWN (without Zaocys dhumnades)). Before the experiment, collect the vaginal lavage fluid of the mice for culture, and observe the growth of Candida albicans. Select the mice without the growth of Candida albicans for the experiment.

[0162] 2. Construction of the mouse model of candidal vaginitis with spleen deficiency and damp obstruction

[0163] In the first 14 days, the blank group was fed normally, and the other groups were made with the spleen deficiency and dampness obstruction model, that is, they were raised in a climate chamber (humidity 90%, temperature 35±2°C) during the day, and at the same time fed with a high-fat and high-sugar diet (80% ordinary feed + 12% lard + 8% honey); they were raised in a normal environment (humidity 60%, temperature 20±2°C) at night, and the modeling was carried out alternately for 14 days. Starting from the 15th day, except for the blank group, the other groups were subcutaneously injected with estradiol benzoate E2 (1mg / mL) 0.025mL under the neck, injected once every other day, and accumulated 4 injections. On the 22nd day, 20 μL of Candida albicans suspension was aspirated with a micropipette and injected into the vagina of mice except the blank group, staying in situ for at least 5 minutes to prevent the overflow of the bacterial solution. The blank group was injected with an equal amount of sterile PBS. Inoculation was carried out once every 2 days, and accumulated 3 inoculations. At the same time, on the 3rd and 7th days of modeling, the vaginas of mice were lavaged with sterile PBS, the vaginal lavage fluid was collected, and plate coating and Gram staining microscopy were performed. Finally, the vaginal model was comprehensively evaluated based on the results of smear microscopy of the lavage fluid and the counting of fungal CFU in the vagina.

[0164] 3. Drug administration

[0165] After the successful establishment of the model, referring to the drug dosage in the drug instruction manual and "Pharmacological Experiment Methodology", intragastric administration was adopted. The intragastric administration dose was 20 ml / kg, once a day, and continuous drug treatment was carried out for 7 days. The general state and signs of mice in each group were observed daily, and the drug efficacy was evaluated according to the detection indexes every day.

[0166] 4. Index detection

[0167] 4.1 Examination of the survival status of mice:

[0168] The survival status of mice was observed throughout the experiment, the general condition of mice was observed, the surface state and stress response of mice were recorded, and whether mice had behaviors such as reduced food intake, depression, and being easily frightened was observed.

[0169] 4.2 Recording of mouse body weight

[0170] The body weight of mice was recorded weekly during the experiment, the body weight changes of mice in each group were observed, and the mouse body weight change curve was sorted out after the experiment.

[0171] 4.3 Observation and scoring of the local condition of the vulva of mice

[0172] The general condition was observed during the experiment, and the local conditions such as redness and swelling of the vaginal orifice and secretions were observed; the local symptoms of the vulva were scored after drug administration. The scoring criteria were 0 point: no redness and swelling of the vulva and vagina and no increase in secretions; 1 point: slight redness and swelling of the vulva and vagina, no increase in secretions; 2 points: obvious redness and swelling of the vulva and vagina accompanied by an increase in secretions; 3 points: obvious redness and swelling of the vulva and vagina, ulcers appeared, and accompanied by an increase in secretions; 4 points: death.

[0173] 4.4 Determination of fungal load in vaginal lavage fluid of mice

[0174] On the 7th day after modeling and 1 day after drug treatment, the lavage fluid was diluted 10-fold with sterile PBS, and 10 μL was inoculated onto Sabouraud agar medium. After culturing at 30 °C for 48 h, colony observation and counting were performed.

[0175] III. Experimental results

[0176] 1. Effects of the traditional Chinese medicine composition of Comparative Example 1 (without Zaocys dhumnades) on the basic conditions and vaginal appearance of mice with spleen deficiency and dampness obstruction type VVC

[0177] The results are as Figure 14 shown. The vaginal appearance of mice in the blank group was white and translucent, with a smooth surface. Compared with the blank group, the mice in the model group were dull in movement, had bristled hair on the body surface, obvious swelling and redness of the vulva, and vaginal secretions were visible. Compared with the model group, there was no obvious improvement in the mice in the treatment group of the traditional Chinese medicine composition of Comparative Example 1 (without Zaocys dhumnades).

[0178] 2. Effects of the traditional Chinese medicine composition of Comparative Example 1 (without Zaocys dhumnades) on the body weight of mice with spleen deficiency and dampness obstruction type VVC

[0179] The results are as Figure 15 shown. Compared with the blank group, the body weight of the mice in the model group decreased significantly (P < 0.001) 5 weeks after modeling; compared with the model group, the body weight of the mice in the treatment group of the traditional Chinese medicine composition of Comparative Example 1 (without Zaocys dhumnades) increased slightly after administration, but there was no significant difference.

[0180] 3. Effects of the traditional Chinese medicine composition of Comparative Example 1 (without Zaocys dhumnades) on local vulvar inflammation in mice with spleen deficiency and dampness obstruction type VVC.

[0181] The results are as Figure 14 and Figure 16 shown. The vaginal orifice of the mice in the blank group was pink and white, without secretions, and the vaginal symptom score was 0 (not shown in the figure); the secretions of the mice in the model group increased, the vaginal orifice was swollen and red, and the vaginal symptom score increased significantly (P < 0.001); compared with the model group, after administration, the vaginal swelling of the mice was slightly relieved, and the local inflammation score decreased slightly, but there was no significant difference.

[0182] 4. Effects of the traditional Chinese medicine composition of Comparative Example 1 (without Zaocys dhumnades) on the fungal load in vaginal lavage fluid of mice with spleen deficiency and dampness obstruction type VVC

[0183] The results are as Figure 14 and Figure 17 shown. There was no Candida albicans colonization in the mice in the blank group (not shown in the figure); compared with the blank group, on the 14th day, there were a large number of Candida albicans in the vaginal lavage fluid of the mice in the model group( P(<0.001); compared with the model group, the number of colonies in the vaginal lavage fluid of the mice in the administration group did not improve significantly.

[0184] In summary, the traditional Chinese medicine composition in Comparative Example 1 (without Zaocys dhumnades) has no obvious improvement effect on mice with VVC of spleen deficiency and dampness obstruction type.

[0185] The above-described embodiments merely represent the specific implementation manners of the present application, and the description thereof is relatively specific and detailed, but it should not be construed as a limitation to the protection scope of the present application. It should be noted that for those of ordinary skill in the art, without departing from the concept of the technical solution of the present application, several deformations and improvements can still be made, 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; the Chinese medicine composition is a water extract concentrate.

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 consists of 25-35 parts of Rhizoma Smilacis Glabrae, 7-17 parts of Cortex Phellodendri, 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 consists of 27-33 parts of Rhizoma Smilacis Glabrae, 9-15 parts of Cortex Phellodendri, 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 consists of 30 parts of Rhizoma Smilacis Glabrae, 12 parts of Cortex Phellodendri, 18 parts of Herba Agrimoniae, 13 parts to 17 parts of Snakehead Snake, and 18 parts of Cortex Dictamni.

5. A Chinese medicine composition for treating spleen deficiency and dampness obstruction type candidal vaginitis according to any one of claims 1 to 4, characterized in that: The pathogenic bacteria of the candidal vaginitis of spleen deficiency and dampness obstruction type is Candida albicans.

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 4, characterized in that: It is administered externally or orally.

8. A Chinese medicine composition for treating spleen deficiency and dampness obstruction type candidal vaginitis according to claim 7, characterized in that: The dosage for external use is: 1.25g / kg-2.5g / kg.

9. A Chinese medicine composition for treating spleen deficiency and dampness obstruction type candidal vaginitis according to claim 7, characterized in that: The oral dosage is: 10g / kg-20g / kg.

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