Application of L-theanine in preparing preparations for treating vaginitis

Abstract

The present invention provides the use of L-theanine in preparing a preparation for treating vaginitis. The L-theanine of the present invention can significantly reduce vaginal pH, improve tissue pathology scores, and is significantly effective for vaginitis.

Description

Technical Field

[0001] The present invention belongs to the technical field of new medical applications, and particularly relates to the application of L-theanine in preparing a preparation for treating vaginitis. Background Art

[0002] The vaginal microbiome of healthy women is dominated by Lactobacilli. The presence of Lactobacilli varies across regions and individuals, primarily including L. crispatus, L. iners, and G. vaginalis. L. crispatus provides the greatest protection for the host, while L. iners and G. vaginalis offer relatively weaker protection. Both L. iners and G. vaginalis exhibit lower cholesterol-dependent cytolysin expression when coexisting with Lactobacilli, but higher expression when coexisting with other facultative and obligate anaerobes.

[0003] In a healthy state, the richness of vaginal microbial colonies is low, lactic acid bacteria are dominant, lactic acid is the main vaginal acidifying substance, and protonated lactic acid can make the vaginal pH <4.5 (average ~3.5) without inflammation.

[0004] In vaginitis, Lactobacilli lose their dominant position, the vaginal microbiome becomes more diverse, and anaerobic and facultative anaerobes increase in number. Short-chain fatty acids (formic, acetic, propionic, and butyric acid) serve as the primary acidifying substances, but their acidifying capacity is relatively weak, resulting in a vaginal pH > 4.5. Pathogenic bacteria decompose immature epithelial cells, producing biogenic amines that cause vaginal inflammation and odor. Common pathogens include Gardnerella vaginalis, Group B Streptococcus, and Staphylococcus aureus.

[0005] Antimicrobial therapy is the standard treatment for vaginitis. While highly effective, pathogenic bacteria have developed varying degrees of resistance to these treatments. Exogenous lactobacilli and lactic acid supplementation have also been attempted, but both methods have limitations: Lactobacilli can only successfully colonize when the vaginal pH is below 4.5, and exogenous lactic acid is not protonated, making it less effective at maintaining a pH below 4.5 and exhibiting a weak antibacterial effect. Therefore, lactobacilli and lactic acid therapy are often used sequentially with antimicrobial therapy.

[0006] Existing literature 1 (Xu Wei, Effects and Mechanisms of L-theanine on the Metabolism of Short-Chain Fatty Acids Related to Intestinal Mucosal Immune [D], Hunan Agricultural University, 2020) disclosed that L-theanine can promote the growth of intestinal probiotics, but at the same time it can also promote the growth of intestinal pathogenic bacteria, indicating that L-theanine is not a broad-spectrum antibacterial drug, but a microbial community regulator.

[0007] Existing literature 2 (Ge Yan, Extraction, Isolation, and Purification of Theanine from Summer and Autumn Green Tea and Its Effects on the Intestinal Microbiome [D]. Nanjing Agricultural University, 2014) reveals that L-theanine can increase the richness of intestinal flora and the content of short-chain fatty acids. This suggests that intravaginal administration of L-theanine may not be suitable for the treatment of vaginitis. As previously mentioned, a healthy vagina is dominated by Lactobacilli and has a low richness of flora. Increased bacterial richness and an increase in short-chain fatty acids are manifestations of vaginitis.

[0008] Existing literature 2 also discloses that L-theanine can promote intestinal lactic acid secretion, but the number of lactic acid bacteria does not change significantly.

[0009] Existing literature 3 (SAEED Effects of ML-theanine dietary supplementation levels on broiler growth performance, meat quality and immune response [D], Northwest Agriculture and Forestry University, 2018) disclosed that L-theanine increased the phylum, order and genus (Lactobacillus) of Lactobacillus in the ileum and colon of chickens, but did not disclose the bacterial strains, and the vaginal and intestinal vaginal strains were different.

[0010] Chinese patent CN202310412887.4 discloses an L-theanine fecal bacteria liquid. The fecal bacteria liquid containing L-theanine contains Lactobacillus reuteri, Johnsonill, and Lactobacillus rhamnosus, which are not common vaginal colonizing bacteria.

[0011] Existing document 4 (Qu, Qing-Yun et al. “L-Theanine Modulates Intestine-Specific Immunity by Regulating the Differentiation of CD4+T Cells in Ovalbumin-Sensitized Mice.” Journal of agricultural and food chemistry vol.70, 47(2022):14851-14863.doi:10.1021 / acs.jafc.2c06171) discloses that L-theanine reduces L.iners in a dose-dependent manner, and L.iners is the most abundant lactobacillus in the vagina and has a protective effect on the vagina.

[0012] Existing literature 5 (France, Michael T et al. “Insight into the ecology of vaginal bacteria through integrative analyses of metagenomic and metatranscriptomic data.” Genome biology vol. 23, 166.1Mar. 2022, doi: 10.1186 / s13059-022-02635-9) revealed that the transcriptional activity of L. crispatus and L. iners varies with the taxonomic composition of the community in which they are located.

[0013] As can be seen, the existing literature does not reveal whether L-theanine can promote the growth of vaginal lactobacilli in vitro, whether it can promote the growth of lactobacilli in vivo, or whether L-theanine can be used to treat vaginitis. Therefore, the existing art does not disclose the use of L-theanine in vaginitis. Summary of the Invention

[0014] To this end, the present inventors conducted a pharmacodynamic study of L-theanine in vaginitis models (bacterial vaginitis, aerobic vaginitis, Candida albicans vaginitis, and atrophic vaginitis). The inventors unexpectedly discovered that L-theanine can significantly reduce vaginitis symptoms, including the disappearance of odor and the restoration of normal vaginal discharge.

[0015] The present invention provides the use of L-theanine in preparing a preparation for treating vaginitis.

[0016] In the preferred technical solution of the application of the present invention, preferably, the vaginitis includes bacterial vaginitis, aerobic vaginitis, fungal vaginitis, atrophic vaginitis, and mixed vaginitis.

[0017] In the preferred technical solution of the application of the present invention, preferably, L-theanine is administered vaginally.

[0018] In the preferred technical solution of the application of the present invention, preferably, the effective therapeutic concentration of L-theanine in vaginal administration is 0.1 w / w% to 2.5 w / w%.

[0019] In the preferred technical solution of the application of the present invention, preferably, the effective therapeutic amount of L-theanine for vaginal administration is 10 mg to 250 mg per day.

[0020] Experimental results show that the L-theanine of the present invention can significantly reduce vaginal pH, improve tissue pathology scores, and is significantly effective in treating vaginitis. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1The effect of L-theanine aqueous solution on vaginal bacterial colonization and cleanliness in a rat mixed vaginitis model (×100);

[0022] Figure 2 Effects of L-theanine aqueous solution on vaginal pathological changes (HE, ×200). DETAILED DESCRIPTION

[0023] The present invention is further described below through cases, but it should be understood that the cases of the present invention are only used to illustrate rather than to limit the present invention.

[0024] Example 1: Pharmacodynamic Study on Mixed Vaginitis in Rats with L-Theanine Aqueous Solution

[0025] The L-theanine preparations proposed in the present invention can be in the form of solutions, vaginal gels, vaginal suppositories, vaginal tablets, and capsules. The preparation methods of the preparations are well known to those skilled in the art.

[0026] experimental animals

[0027] 70 SPF SD rats, female, weighing 193.7-254.4 g.

[0028] Preparation of infection solution

[0029] Three strains of Candida albicans (CMCC(F)98001), Group B Streptococcus (ATCC12386) and Gardnerella vaginalis (BNCC337545) were revived and cultured in sterile nutrient broth, passaged, and prepared at a concentration of 2.0×10 9 The three bacterial solutions were then mixed at a ratio of 1:1:1 to prepare the infection bacterial solution for later use.

[0030] Animal model establishment

[0031] 70 SPF female SD rats, weighing 193.7 to 254.4, were selected as the normal control group. The remaining rats were anesthetized by isoflurane inhalation. The ovaries of the rats were removed on a sterile operating table. After surgery, 200,000 units of penicillin were injected intramuscularly for 3 consecutive days. Starting from the 4th day after surgery, estradiol benzoate was injected subcutaneously at a dose of 10 mg / kg every day for 4 days. After that, hydrocortisone injection was injected subcutaneously at a dose of 60 mg / kg every other day for 3 days to replicate the rat immunodeficiency model. After 2 days, 50 μL of infection solution (1:1:1) was injected into the rat vagina. The rat tail was lifted and kept for about 2 minutes to prevent the bacteria from overflowing. After 1 day, the infection was repeated once in the same way, and then the subcutaneous injection was alternated every other day. Estradiol benzoate and hydrocortisone injections were administered once each. After modeling, the vulva of the rats was observed. If swelling, redness, and white secretions were exuded, a small amount of secretions was smeared on blood agar medium and incubated at 37°C. After 72 hours, colonies of varying sizes were observed on the culture medium with the naked eye, indicating successful model establishment. Fifty rats with successful models were randomly divided into a model control group, a compound metronidazole vaginal suppository group, and a (0.5 w / w%, 1.25 w / w%, or 2.5 w / w%) L-theanine aqueous solution group, with 10 rats in each group. Except for the normal and model control groups, which were given 0.9% sodium chloride injection intravaginally, all other groups received intravaginal administration of 0.1 g / rat once daily for 14 consecutive days. The vaginal secretions of the rats were observed on D0 and on D5, D10, and D14 after administration, and photographs were taken. Vaginal pH was tested once a week. After the last administration, vaginal smears and vaginal wash culture were performed to calculate the negative conversion rate and colony counts. Vaginal tissue pathology scores were performed to evaluate pathological changes.

[0032] Trial Groups and Dosages

[0033] Table 1. Trial grouping and dosage design

[0034]

[0035] Detection indicators

[0036] Vaginal smears: After the final dose, vaginal secretions were collected from each group of rats using a sterile cotton swab and subjected to Wright and Gram stains. The smears were then smeared onto blood agar and incubated at 37°C. After 24 hours, the smears were visually inspected for the presence of bacterial colonies. The absence of bacterial colonies on two consecutive occasions, 12 hours apart, indicated a negative result. The negative conversion rate was calculated as follows: Negative conversion rate = number of rats with bacterial translocation / total number of rats in each group × 100%.

[0037] Vaginal secretion and cleanliness examination: The vaginal secretions of rats were observed (vaginal smear) on D0 and D5 / D10 / D14 after administration, and photos were taken.

[0038] Vaginal pH test: Perform a vaginal pH test weekly

[0039] Colony count of vaginal lavage fluid: After the last administration, the vaginal lavage fluid of each group of rats was collected with a sterile cotton swab (strictly quantified during the operation), smeared on blood agar medium (sterile operation), and placed in a 37°C incubator for culture. Colonies were counted after 24 hours.

[0040] Vaginal histopathological scoring: Vaginal tissue was collected for HE staining. The degree of inflammation was scored based on the pathological changes of vaginal mucosal tissue smoothness, congestion, edema, inflammatory cell infiltration, etc. in the lower layer and muscle layer. 1. Degree of degeneration and necrosis of vaginal mucosal epithelial cells: normal, intact (0 points); mild (1 points); moderate (2 points); severe (3 points); serious (4 points); 2. Degree of inflammatory cell infiltration (each point) Under high-power microscope field of view): none (recorded as "0" score); very few <25 (recorded as "1" score); mild 26-50 (recorded as "2" score); moderate 51-100 (recorded as "3" score); severe >100 (recorded as "4" score); 3. Degree of vascular congestion and edema: none (recorded as "0" score); very few (recorded as "1" score); mild (recorded as "2" score); moderate (recorded as "3" score); severe (recorded as "4" score) and add up the scores of each animal to get the total score.

[0041] Experimental results

[0042] Effects of L-theanine aqueous solution on vaginal pH value in a rat model of mixed vaginitis

[0043] As shown in Table 2, compared with the normal control group, the vaginal pH values of the model control group rats before and 1 week after administration were significantly increased (P ≤ 0.01). Compared with the model control group, the vaginal pH values of the rats in the 0.1 w / w%, 0.5 w / w%, and 2.5 w / w% L-theanine aqueous solution groups and the compound metronidazole vaginal suppository groups were significantly decreased 1 week after administration (P ≤ 0.01). Compared with the compound metronidazole vaginal suppository group, the vaginal pH values of the 0.5 w / w% and 2.5 w / w% L-theanine aqueous solution groups were significantly decreased 1 week after administration (P ≤ 0.01).

[0044] Table 2. Effect of L-theanine aqueous solution on vaginal pH value ( n=10)

[0045]

[0046] Note: Compared with the normal control group, ++ P≤0.01; compared with the model control group, ** P≤0.01; compared with the compound metronidazole vaginal suppository group, ▲▲P≤0.01.

[0047] Effects of L-theanine aqueous solution on vaginal bacterial colonization and cleanliness in a rat model of mixed vaginitis

[0048] As shown in Table 3, compared with the normal control group, the number of vaginal bacterial colonization in the model control group before administration was significantly increased (P ≤ 0.01). Compared with the model control group, the number of vaginal bacterial colonization in the rats in the D14 compound metronidazole vaginal suppository group was significantly reduced (P ≤ 0.05). The negative conversion rates of the model control group, compound metronidazole vaginal suppository group, 0.1 w / w% L-theanine aqueous solution group, 0.5 w / w% L-theanine aqueous solution group, and 2.5 w / w% L-theanine aqueous solution group were 30%, 40%, 20%, 20%, and 20%, respectively.

[0049] As shown in Table 4, Figure 1 As shown, compared with the normal control group, the number of vaginal Gram bacteria colonization in rats in the model control group on D0 and D5 was significantly increased (P≤0.01). Compared with the model control group, the number of vaginal Gram bacteria colonization in rats in the 0.1w / w% L-theanine aqueous solution group was significantly reduced on D5 after administration (P≤0.05), the number of vaginal Gram bacteria colonization in rats in the 2.5w / w% L-theanine aqueous solution group was significantly reduced on D10 and D14 after administration (P≤0.05 or P≤0.01), and the number of vaginal Gram bacteria colonization in rats in the compound metronidazole vaginal suppository group was significantly reduced on D5, D10, and D14 after administration (P≤0.05). Compared with the compound metronidazole vaginal suppository group, the number of Gram bacteria colonizing in the vagina of rats was significantly increased on D14 after administration in the 0.1w / w% L-theanine aqueous solution group, D10 before administration in the 0.5w / w% L-theanine aqueous solution group, and D5 after administration in the 2.5w / w% L-theanine aqueous solution group (P≤0.05).

[0050] As shown in Table 5, Figure 1 As shown, compared with the normal control group, the vaginal cleanliness level of rats in the model control group was significantly increased from D0 to D14 after administration (P ≤ 0.01). Compared with the model control group, the vaginal cleanliness level of rats in the 0.1 w / w%, 0.5 w / w%, and 2.5 w / w% L-theanine aqueous solution groups was significantly decreased from D5 to D14 after administration (P ≤ 0.05 or P ≤ 0.01). Compared with the compound metronidazole vaginal suppository group, the vaginal cleanliness level of rats in the 2.5 w / w% L-theanine aqueous solution group was significantly increased from D10 to D14 after administration, and in the 0.1 w / w% and 0.5 w / w% L-theanine aqueous solution groups was significantly increased from D14 after administration (P ≤ 0.05). Lactobacillus isolation and culture results showed that the increased colonies in the L-theanine aqueous solution group were mainly Lactobacillus.

[0051] Table 3. Effects of L-theanine aqueous solution on vaginal bacterial colonization ( n=10)

[0052]

[0053] Note: Compared with the normal control group, ++ P≤0.01, compared with the model control group, * P≤0.05.

[0054] Table 4. Effect of L-theanine aqueous solution on vaginal Gram bacteria ( n=10)

[0055]

[0056]

[0057] Note: Compared with the normal control group, ++ P≤0.01; compared with the model control group, * P≤0.05, ** P≤0.01; compared with the compound metronidazole vaginal suppository group, ▲ P≤0.05.

[0058] Table 5. Effect of L-theanine aqueous solution on vaginal cleanliness ( n=10)

[0059]

[0060] Note: Compared with the normal control group, ++ P≤0.01; compared with the model control group, * P≤0.05, ** P≤0.01; compared with the compound metronidazole vaginal suppository group, ▲ P≤0.05.

[0061] Effects of L-theanine aqueous solution on vaginal pathological changes in a mixed vaginitis model in rats

[0062] As shown in Table 6, Figure 2 As shown, compared with the normal control group, the model control group showed significantly increased vaginal epithelial cell degeneration and necrosis, inflammatory cell infiltration, and total pathological scores (P ≤ 0.01). Compared with the model control group, the 0.1 w / w%, 0.5 w / w%, and 2.5 w / w% L-theanine aqueous solution groups and the compound metronidazole vaginal suppository groups showed significantly decreased vaginal epithelial cell degeneration and necrosis, inflammatory cell infiltration, and total pathological scores (P ≤ 0.05 or P ≤ 0.01). Compared with the compound metronidazole vaginal suppository group, no significant differences were observed among the test groups.

[0063] Table 6. Effect of L-theanine aqueous solution on vaginal pathology score ( n=10)

[0064]

[0065]

[0066] Note: Compared with the normal control group, ++ P≤0.01; compared with the model control group, * P≤0.05, ** P≤0.01.

[0067] From the above Figure 1 、 Figure 2 As can be seen from Tables 1, 2, 3, 4, 5 and 6, L-theanine aqueous solution has a significant therapeutic effect on the mixed vaginitis model in rats.

[0068] It should be noted that the above invention content and specific embodiments are intended to demonstrate the practical application of the technical solutions provided by the present invention and should not be interpreted as limiting the scope of protection of the present invention. Those skilled in the art will be able to make various modifications, equivalent substitutions, or improvements within the spirit and principles of the present invention.

Claims

1. Use of L-theanine as the sole active ingredient in the preparation of a preparation for treating vaginitis, characterized in that: The vaginitis is bacterial vaginosis, fungal vaginitis, atrophic vaginitis, or mixed vaginitis; the preparation is a vaginal administration preparation containing L-theanine as the only active ingredient, and the effective therapeutic concentration of L-theanine in vaginal administration is 0.1 w / w% to 2.5 w / w%.

2. The use according to claim 1, wherein: The vaginitis is bacterial vaginosis, atrophic vaginitis, or mixed vaginitis; and the effective therapeutic amount of L-theanine administered intravaginally is 10 mg to 250 mg per day.

3. Use of L-theanine as the sole active ingredient in the preparation of a preparation for treating vaginitis, characterized in that: The vaginitis is aerobic bacterial vaginitis; the preparation is a vaginal administration preparation containing L-theanine as the only active ingredient, and the effective therapeutic concentration of L-theanine in vaginal administration is 0.1w / w%~2.5w / w%.

4. The use according to claim 2, characterized in that The effective therapeutic concentration of L-theanine for vaginal administration is 0.1w / w%.

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