A pharmaceutical composition for killing trichomonas vaginalis and use thereof

Abstract

The application discloses a medicine composition for killing trichomonas vaginalis, and the active ingredients of the medicine composition are composed of fibraureus and metronidazole; the mass ratio of the fibraureus and the metronidazole is 1:1-8:1; in the process of killing the trichomonas vaginalis, the concentration of the fibraureus is 0.01-0.04 g / L, and the concentration of the metronidazole is 0.005-0.02 g / L. The medicine composition can effectively kill the trichomonas vaginalis, the fibraureus combined with the metronidazole can effectively reduce the medication time and the dosage of the metronidazole, can avoid the risk of premature delivery of pregnancy medication, and can reduce the health problems of breast-feeding infants during the medication period; the problems of adverse reactions and drug resistance of the metronidazole and other nitroimidazole drugs in treating trichomonas vaginalis disease are solved; the application provides a theoretical basis for treating drug-resistant trichomonas vaginalis vaginitis by combining traditional Chinese medicine with western medicine, and further expands the clinical application of the fibraureus.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of medicine, and particularly relates to a medicine composition for killing Trichomonas vaginalis and application thereof. BACKGROUND

[0002] Trichomonas vaginalis (Tv) is the most common non-viral parasite in the world today, with more than 270 million people infected globally each year, mainly through sexual transmission, and also through indirect transmission such as using public bath facilities and toilets. Trichomonas vaginalis damages the urogenital system and causes inflammation, is easily co-infected with Chlamydia trachomatis and Neisseria gonorrhoeae, increases the susceptibility to human immunodeficiency virus (HIV) infection, and can cause pelvic inflammatory disease, infertility, premature birth, premature rupture of membranes, and low birth weight, and is a global pathogen of great concern.

[0003] Currently, the main treatment for trichomonas vaginitis is nitroimidazole drugs such as metronidazole, tinidazole or ornidazole. Nitroimidazole drugs are the only drugs that can be used for oral or parenteral treatment of trichomoniasis. Adverse reactions of oral drugs may include nausea, vomiting, gastrointestinal reactions, headache, skin rash, leukopenia, and potential carcinogenic, teratogenic, and mutagenic effects. Metronidazole is the first choice for treating trichomoniasis and has a high success rate. The 5-nitro group on metronidazole is reduced by obtaining an electron donor through ferredoxin (Fd) mediated electron transfer in the worm body, thereby activating metronidazole and exerting its pharmacological effects. If the Fd electron donor is blocked, the drug cannot be activated. However, with the increasing number of metronidazole-resistant strains, the drug resistance of Trichomonas vaginalis to metronidazole has reached 9.6%. Tv contains various cysteine proteinases (CPs), which are important virulence markers, and directly act on vaginal and cervical cells, causing them to shed and causing symptoms of vaginitis. Compared with other protozoa, the Tv genome contains more histone genes. Histone refers to a special protein that binds to DNA to form chromatin in cells. The amino acids at the N-terminus of histone are modified in different ways to regulate the loose or dense structure of chromatin, thereby promoting or inhibiting gene expression.

[0004] Chinese herbal medicine is a traditional medicine in China, although some western medicine is not as fast as some western medicine, but because it is pure natural, less toxic and side effects, more and more people's favor. Palmatine is a natural Chinese herbal medicine ingredient developed by China, also known as palmatine, which is a kind of quaternary amine salt isoquinoline alkaloid. Because of its exact effect, it was recorded in the Chinese Pharmacopoeia in 1977, and its use is very wide, and there are or are applying for registration of palmatine tablets, capsules, granules, injection, suppositories, effervescent tablets and other dosage forms, which are applied to treat gynecological inflammation, surgical infection, bacillary dysentery, enteritis, gastritis, conjunctivitis, respiratory tract and urinary tract infection and other diseases, Chen Xi et al. reported that the in vitro activity of palmatine against Trichomonas vaginalis was equivalent to that of metronidazole, but the mechanism of action was not clear. In order to solve the adverse reactions and drug resistance problems of metronidazole and other nitroimidazole drugs in the treatment of trichomonas vaginalis, especially the reports that the use of drugs during pregnancy increases the risk of premature birth, and breastfeeding affects the health of infants, the present application proposes to use palmatine and metronidazole combination to kill trichomonas vaginalis, which has a significant effect, and no related literature has been reported.

[0005] The disclosure of the above background art content is only used to assist in understanding the inventive concept and technical solutions of the present application, and it does not necessarily belong to the prior art of the present patent application. In the absence of explicit evidence that the above content has been disclosed on the filing date of the present patent application, the above background art should not be used to evaluate the novelty and inventiveness of the present application. SUMMARY

[0006] The purpose of the present application is to provide a kind of medicine composition for killing trichomonas vaginalis and its application, to solve the deficiencies existing in the prior art above.

[0007] To achieve the above purpose, the technical scheme adopted by the present application is as follows:

[0008] A kind of medicine composition for killing trichomonas vaginalis, the active ingredient of the medicine composition is composed of palmatine and metronidazole.

[0009] Palmatine is recorded in the first volume of Chinese Pharmacopoeia 2020 edition on page 433, which is extracted from the alkaloid of the dry stem of the Fibraurea plant Fibraurea recisa Pierre. Its molecular formula is C 21 H 22 ClNO4, molecular weight 387.86, and the structural formula is:

[0010]

[0011] The medicine composition for killing trichomonas vaginalis described above, the mass ratio of palmatine and metronidazole is preferably 1:1 to 8:1, and further preferably 2:1 to 8:1.

[0012] The drug composition for killing Trichomonas vaginalis has the following advantages: the concentration of fibrous element is preferably 0.01-0.04 g / L, and further preferably 0.02-0.04 g / L; and the concentration of metronidazole is preferably 0.005-0.02 g / L, and further preferably 0.005-0.01 g / L.

[0013] The drug composition for killing Trichomonas vaginalis comprises fibrous element, metronidazole and other pharmaceutically acceptable excipients, and can be prepared into any dosage form permitted by drugs, such as tablets, capsules, oral liquids, syrup, granules, injection, suppositories, effervescent tablets and the like, and is preferably prepared into suppositories, effervescent tablets or sustained-release tablets.

[0014] The drug composition for killing Trichomonas vaginalis is used for preparing a drug for killing Trichomonas vaginalis.

[0015] The drug composition for killing Trichomonas vaginalis has the following advantages:

[0016] 1. The drug composition can effectively kill Trichomonas vaginalis, and the use of fibrous element in combination with metronidazole can significantly reduce the dosage of metronidazole and the medication time. In the process of killing Trichomonas vaginalis, the dosage of metronidazole can be reduced to 1 / 8-1 / 2, and the insecticidal rate in 4 hours is significantly higher than that of metronidazole with the same concentration, and the insecticidal rate in 8 hours can reach 98-100%. The insecticidal rate of 0.04 g / L fibrous element and 0.005 g / L metronidazole in 48 hours can reach 100%, but in Example 3, 0.04 g / L fibrous element combined with 0.005 g / L metronidazole: the insecticidal rate in 8 hours is 98%, and the insecticidal rate in 12 hours is 100%, and the insecticidal time is reduced by half. The present application significantly reduces the dosage of metronidazole, and solves the problem of adverse reactions in the treatment of trichomoniasis by conventional use of metronidazole and other nitroimidazole drugs.

[0017] 2. The drug composition can destroy the nuclear structure of Trichomonas vaginalis, has a stronger regulatory effect on Fd gene, can significantly reduce the transcription of CP protein gene and H3 histone gene, and further promotes the deformation, rupture and apoptosis of the worm body, so as to effectively kill drug-resistant strains of Trichomonas vaginalis. The mechanism of the drug composition for killing Trichomonas vaginalis is completely different from that of metronidazole, which avoids the drug resistance problem caused by the conventional use of metronidazole, provides a theoretical basis for the combined treatment of drug-resistant trichomonas vaginitis in traditional Chinese medicine and Western medicine, and further expands the clinical application of fibrous element.

[0018] 3. The drug composition for killing Trichomonas vaginalis uses fibrous element in combination with metronidazole, which can avoid the risk of premature delivery of pregnant women, reduce the health problems of breastfed infants during medication, and provide safer drugs for pregnant women and lactating women. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 Graph of growth curve of Trichomonas vaginalis

[0020] Figure 2 Micrograph of Trichomonas vaginalis morphology for the blank control group

[0021] Figure 3 Micrograph of Trichomonas vaginalis morphology for the metronidazole group

[0022] Figure 4 Micrograph of Trichomonas vaginalis morphology for the fibrauretin group

[0023] Figure 5 Micrograph of Trichomonas vaginalis morphology for Example 1

[0024] Figure 6 Micrograph of Trichomonas vaginalis morphology for Example 2

[0025] Figure 7 Micrograph of Trichomonas vaginalis morphology for Example 3

[0026] Figure 8 Micrograph of Trichomonas vaginalis morphology for Example 4

[0027] Figure 9 Micrograph of Trichomonas vaginalis acridine orange fluorescent staining for the blank control group

[0028] Figure 10 Micrograph of Trichomonas vaginalis acridine orange fluorescent staining for the metronidazole group

[0029] Figure 11 Micrograph of Trichomonas vaginalis acridine orange fluorescent staining for the fibrauretin group

[0030] Figure 12 Micrograph of Trichomonas vaginalis acridine orange fluorescent staining for Example 1

[0031] Figure 13 Micrograph of Trichomonas vaginalis acridine orange fluorescent staining for Example 2

[0032] Figure 14 Micrograph of Trichomonas vaginalis acridine orange fluorescent staining for Example 3

[0033] Figure 15 Micrograph of Trichomonas vaginalis acridine orange fluorescent staining for Example 4

[0034] Figure 16 Figure of Venn analysis of different gene sets

[0035] Figure: Control is the blank group, HTS is the fibrauretin group, and MDZ is the metronidazole group

[0036] Figure 17Figure 3. GO annotation of differentially expressed genes between Control and MDZ groups

[0037] Figure: Control is blank group, MDZ is metronidazole group

[0038] Figure 18 Figure 4. GO annotation of differentially expressed genes between Control and HTS groups

[0039] Figure: Control is blank group, HTS is fibrauretin group

[0040] Figure 19 Figure 5. GO annotation of differentially expressed genes between Control vs MDZ, Control vs HTS and MDZ vs HTS groups

[0041] Figure: Control is blank group, HTS is fibrauretin group, MDZ is metronidazole group

[0042] Figure 20 Figure 6. KEGG pathway annotation of differentially expressed genes between Control and MDZ groups

[0043] Figure: Control is blank group, MDZ is metronidazole group

[0044] Figure 21 Figure 7. KEGG pathway annotation of differentially expressed genes between Control and HTS groups

[0045] Figure: Control is blank group, HTS is fibrauretin group

[0046] Figure 22 Figure 8. The effect of Example 3 on the transcription of Fd gene of Trichomonas vaginalis

[0047] Figure: Control is blank group, MDZ is metronidazole group, HTS is fibrauretin group, MDZ+HTS is Example 3 group

[0048] Figure 23 Figure 9. The effect of Example 3 on the transcription of CP protein gene of Trichomonas vaginalis

[0049] Figure: Control is blank group, MDZ is metronidazole group, HTS is fibrauretin group, MDZ+HTS is Example 3 group

[0050] Figure 24 Figure 10. The effect of Example 3 on the transcription of H3 histone gene of Trichomonas vaginalis

[0051] Figure: Control is blank group, MDZ is metronidazole group, HTS is fibrauretin group, MDZ+HTS is Example 3 group DETAILED DESCRIPTION

[0052] The application will be further described in conjunction with specific examples, but not limit the scope of protection and application range of the application: I. Experimental materials

[0053] 1. Insect strain

[0054] The test insect strain was taken from the vaginal secretions of patients with positive results in the Trichomonas vaginalis microscopy test at the First Affiliated Hospital of Guangxi University of Chinese Medicine, Ren'ai Branch.

[0055] 2. Drugs and reagents

[0056] Fresh beef liver was purchased; proteose peptone, maltose, sodium chloride, L-cysteine hydrochloride were purchased from Sigma Company, USA; Hyclone fetal bovine serum was purchased from GE Company, USA; ferulaeine (Cas: 3486-67-7) and metronidazole (Cas: 443-48-1) were purchased from Shanghai McLean Biochemical Technology Co., Ltd.

[0057] RNA extraction kit (TIANGEN, DP431); II Q RT SuperMix for qPCR (+gDNAwiper) (Vazyme, R223); ChamQTM Universal qPCR Master Mix (Vazyme, Q711).

[0058] 3. Instruments

[0059] Super-clean workbench (Suzhou Air Purification Equipment Co., Ltd.), electric heating incubator (Beijing Fuyilian Electric Appliance Co., Ltd.), solid microscope (Japan Nikon), high-pressure steam sterilizer (Shanghai Sanshen Medical Instrument Co., Ltd.), centrifuge (China Feige Company), electric heating constant temperature air drying oven (Shanghai Boxin Industry Co., Ltd.), blood cell counting plate (Shanghai Qiexing Biochemical Reagent Instrument Co., Ltd.).

[0060] II. Culture of Trichomonas vaginalis

[0061] Preparation of culture medium: take fresh beef liver 15 g, cut into millet size, immerse in 100 mL distilled water, place in refrigerator for 24 h, boil for 30 min, filter with 4-6 layers of gauze, and make up to 100 mL with distilled water; add proteose peptone 2.0 g, maltose 1.0 g, sodium chloride 0.5 g and L-cysteine hydrochloride 0.2 g, heat to promote dissolution, adjust pH to 5.8. Sterilize at 100°C for 20 min, cool to 37°C in a constant temperature incubator for 24 h, and store at 4°C after proving sterile. Add sterile newborn calf serum according to 10% volume ratio, add 1×10 5 U / L.

[0062] Collect Trichomonas vaginalis that has been stably cultured for 3 generations, centrifuge at 1500 r / min for 5 min, discard the supernatant, wash 2-3 times with culture medium, discard the supernatant again, then add culture medium containing 10% (v / v) newborn calf serum and mix well. Count the number of worms using a hemocytometer. If the viability rate is greater than 95%, it is usable. Adjust the worm density to 1×10⁻⁶. 5 Concentration per mL, for later use.

[0063] III. In vitro anti-trichomonal test

[0064] 1. Grouping:

[0065] Blank control group: Add 100 μL of physiological saline.

[0066] Metronidazole group: 100 μL of the above Trichomonas vaginalis strain was inoculated into the metronidazole group (0.04 g / L, 0.02 g / L, 0.01 g / L and 0.005 g / L respectively).

[0067] Berberine group: 100 μL of the above Trichomonas vaginalis strain was inoculated into the berberine group (0.04 g / L, 0.02 g / L and 0.01 g / L respectively).

[0068] Example group: 100 μL of the above-mentioned Trichomonas vaginalis strain was inoculated into the drug composition for killing Trichomonas vaginalis of the present invention, namely Example 1 (0.04 g / L berberine + 0.02 g / L metronidazole), Example 2 (0.04 g / L berberine + 0.01 g / L metronidazole), Example 3 (0.04 g / L berberine + 0.005 g / L metronidazole), and Example 4 (0.02 g / L berberine + 0.02 g / L metronidazole).

[0069] 2. Experimental Procedure: 5 mL of the culture medium was cultured in 15 mm × 150 mm test tubes, which were sealed and incubated at 37°C. At 2, 4, 8, 12, 24, 36, and 48 hours after drug administration, 10 μL of the suspension was collected and inoculated into a hemocytometer for counting live worms under a microscope. The live worms in the blank control group and the metronidazole group were observed first; the results for both groups were correct, and the entire experimental procedure was meaningful. The total mitochondrial (TV) mortality rate was recorded for each group, and the average of the three parallel tubes from each group was used for calculation. This experiment was repeated three times.

[0070] Trichomonas mortality rate = (number of dead trichomonas / total number of counted trichomonas) × 100%.

[0071] 3. Experimental Results: The insecticidal rates of the metronidazole group at the same concentration after 2h, 4h, 8h, and 12h of treatment were significantly higher than those of the berberine group at the same concentration (P < 0.05); however, in Examples 1 to 4 of this invention, the insecticidal rate of berberine combined with metronidazole after 4h of treatment was significantly higher than that of metronidazole at the same concentration (P < 0.05). The insecticidal rate of 0.04g / L berberine and 0.005g / L metronidazole after 48h of treatment could both reach 100%, but in Example 3, the insecticidal rate of 0.04g / L berberine combined with 0.005g / L metronidazole after 12h of treatment could reach 100%; there was no significant difference in the insecticidal rate of 8h of treatment in Examples 1-4 compared to 8h of treatment with 0.04g / L metronidazole (P > 0.05). The results showed that berberine could effectively kill Trichomonas vaginalis. When used in combination with metronidazole, the insecticidal rate was significantly higher than that of metronidazole at the same concentration at the same treatment time (4h, 8h). This indicates that the combination of berberine and metronidazole in Examples 1 to 4 of this invention can effectively reduce the treatment time and concentration of metronidazole, as shown in Table 1.

[0072] Table 1. Anti-trichomoniasis activity

[0073]

[0074] Note: Different letters within the same column represent significant differences (P < 0.05).

[0075] IV. Microscopic observation of Trichomonas vaginalis

[0076] Blank control group: Trichomonas vaginalis exhibited a normal morphology, mostly pear-shaped, and was actively motile, with constantly waving anterior flagella and undulating membranes on the sides of the body. The density of the parasites gradually increased over time. Berberine group, metronidazole group, and groups 1-4 of this invention: The morphology of Trichomonas vaginalis showed significant changes; the parasites became rounder, with blurred outlines and irregular edges. Flagellar movement weakened or stopped, and the parasites contained numerous particles or ruptured. With increasing drug concentration and duration of action, the number of dead Trichomonas vaginalis gradually increased. At the same time and concentration, the berberine group showed a higher number of live parasites and slower morphological changes compared to the metronidazole group and groups 1-4 of this invention. There was no significant difference in the number of dead parasites or the morphology of the parasites between the metronidazole group and groups 1-4 of this invention. See Figures 2-8 .

[0077] V. Acridine orange fluorescent staining method for detecting apoptosis in Trichomonas vaginalis

[0078] 1. Acridine orange fluorescence staining method: Take 5 μL of each tube of the 2h treatment group prepared in the above "in vitro anti-trichomonal test" onto a clean glass slide, fix with 95% ethanol for 5 min, and after it is completely dry, add 0.01% acridine orange staining solution prepared with PBS for 5 min, absorb excess liquid with absorbent paper, and examine with a fluorescence microscope. The excitation wavelength is 490 nm and the receiving wavelength is 520 nm.

[0079] 2. Microscopic observation results: Acridine orange can bind DNA yellow-green and RNA orange-yellow or orange-red through the complete membrane structure. The acridine orange staining results are shown in Figures 9-15 .

[0080] The blank control group: The Trichomonas vaginalis has normal morphology, and the acridine orange binds the cytoplasmic RNA to orange-red.

[0081] The metronidazole group, the fibrauretin group and the example 1-4 group of the present application: The Trichomonas vaginalis is ruptured and apoptotic, and the released nuclear DNA binds to acridine orange to yellow-green; the acridine orange binds the cytoplasmic RNA to orange-yellow.

[0082] The test results show that the metronidazole group, the fibrauretin group and the example 1-4 group of the present application can make the Trichomonas vaginalis cell nucleus rupture, and the DNA spills out and turns yellow-green; under the microscope, the same time and concentration, the fibrauretin group has less yellow-green proportion than the metronidazole group and the example 1-4 group of the present application, and there is no obvious difference between the metronidazole group and the example 1-4 group of the present application.

[0083] Six, RNA-seq detects the mRNA transcriptome level of Trichomonas vaginalis

[0084] The RNA micro kit is used to extract the RNA of Trichomonas vaginalis, and after identification, the Illumina Hiseq 2500 platform is used for transcriptome sequencing; then the Blast2GO program is used to compare the genes on the alignment with the GO (gene ontology) database, and then the Goatools program is used to perform GO classification annotation on the main biological functions involved in the genes; finally, by comparing with the KEGG (Kyoto encyclopedia of genes and genomes) database, the signal pathways or metabolic pathways involved in the genes are analyzed; the information annotation, Venn analysis, COG classification, GO function classification and enrichment, and KEGG pathway enrichment analysis of the differentially expressed genes in each group are performed; the construction, sequencing and analysis of the transcriptome library are completed by Shanghai Meiji Biological Co., Ltd.

[0085] The Venn analysis results show that, compared with the blank control, the differential genes of the metronidazole group are 355, and the differential genes of the fibrauretin group are as many as 3847; compared with the metronidazole group, the differential genes of the fibrauretin group are 3235; these differential genes all include 64 common genes. Among them, there are 6 Fd genes: Fd1, Fd2, Fd3, Fd4, Fd5, Fd6; 4 CP genes: CP1, CP2, CP3, CP4; 5 H3 histone genes: H3-1, H3-2, H3-3, H3-4, H3-5. See Figure 16 .

[0086] GO analysis was used to annotate the functions of the differentially expressed genes. Metronidazole mainly affected the functions of the differentially expressed genes of Tv as small molecule metabolic process, see Figure 17 ; while fibraurin mainly affected the functions of the differentially expressed genes of Tv as cellular_component, see Figure 18 The differentially expressed genes were classified and annotated, involving 20 small categories of biological processes (BP), cellular components (CC) and molecular functions (MF), and the number of the regulated genes of the 20 categories by fibraurin was higher than that by metronidazole, see Figure 19 .

[0087] KEGG was used to annotate the signal pathways of the differentially expressed genes. Metronidazole affected 85 genes of Tv, mainly enriched in Metabolism and Genetic Information Process signal pathways, involving 15 signal pathways, see Figure 20 ; fibraurin affected as many as 562 genes of Tv, mainly enriched in Metabolism and Genetic Information Process signal pathways, involving 18 signal pathways, see Figure 21 .

[0088] Seven, RT-qPCR detection of Trichomonas vaginalis mRNA expression

[0089] The blank control group, metronidazole group (0.005 g / L), fibrauretin group (0.04 g / L), and fibrauretin + metronidazole group (0.04 g / L + 0.005 g / L) of the "in vitro Trichomonas vaginalis test" described above were subjected to reverse transcription of Trichomonas vaginalis RNA into cDNA, and the expression level of the target gene was detected by RT-qPCR. 16s was used as an internal reference gene, and the upstream primer sequence was 5'-TCATAGTTCTTGGGCTCTGG-3', and the downstream primer was 5'-TAAGTTTCCCCGTGTTGATT-3'. The primer sequences of the target gene were as follows: the upstream primer sequence of Fd1 was 5'-CTGTTTCAGGCACAAAAGC-3', and the downstream primer sequence was 5'-AGTTCCTTCTCGTCAGCATC-3'; the upstream primer sequence of Fd2 was 5'-TCGGTACTTTCCTTCCTTAACA-3', and the downstream primer sequence was 5'-TATATTCTGCCTTGGTTCCTTC-3'; the upstream primer sequence of Fd3 was 5'-CTCTTTGATGCCGTTTCTG-3', and the downstream primer sequence was 5'-AGGCGAGATGTTGGTGTAA-3'; the upstream primer sequence of Fd4 was F: 5'-GAACAAACACTCTTCACAGTCCT-3', and the downstream primer sequence was R: 5'-AATTCCTTCTCTTCCTCATCG-3'; the upstream primer sequence of Fd5 was F: 5'-ACACTTGCTTGCTCGCTGAT-3', and the downstream primer sequence was R: 5'-CTTCTTGATGCCGTGGTTGATG-3'; and the upstream primer sequence of Fd6 was 5'-ATCCCTTCGTATGCTTGCT-3', and the downstream primer sequence was R: 5'-GCTCATCCATTGTTTCCTG-3'.

[0090] The upstream primer sequence of CP1 was F: 5'-AAGGGAGTCGTCAATCCTATC-3', and the downstream primer sequence was R: 5'-GCTTCTGCTTCTGCTTCTGCT-3'.

[0091] The upstream primer sequence of CP2 was F: 5'-GCTCAAGAATCTGCTTATGCTA-3', and the downstream primer sequence was R: 5'-CGTAAACGTAATCCATCCATC-3'.

[0092] The upstream primer sequence of CP3 was F: 5'-GCTTCTGCTTCTGCTTCTGCT-3', and the downstream primer sequence was R: 5'-GCTTCTGCTTCTGCTTCTGCT-3'. The upstream primer sequence of CP4 was F: 5'-GCATTACCAGGCAGTCAAA-3', and the downstream primer sequence was R: 5'-GTAGAGATGGAAGGAATGCTG-3'.

[0093] GAGAAAGCCCAGCAAGAAC-3 '.

[0094] H3-1 upstream primer sequence is F: 5 '-AAGTCAACAGGTGGCAAGA-3 ', and downstream primer sequence is R: 5 '-TACGGATTTCACGAAGAGC-3 '.

[0095] H3-2 upstream primer sequence is F: 5 '-AAGACAACAGGTGGCAAGAC-3 ', and downstream primer sequence is R: 5 '-TTACAAGACGCTGGAATGGAAG-3'; H3-3 upstream primer sequence is F: 5 '-AAGACAACAGGTGGCAAGAC-3 ', and downstream primer sequence is R: 5 '-TTACAAGACGCTGGAATGGAAG-3 '.

[0096] H3-4 upstream primer sequence is F: 5 '-GGGTGCTAAGAAGCAACATC-3 ', and downstream primer sequence is R: 5 '-TCACCACGGAAGCCAGAAG-3'; H3-5 upstream primer sequence is F: 5 '-AAGTCAACAGGTGGCAAGA-3 ', and downstream primer sequence is R: 5 '-TACGGATTTCACGAAGAGC-3 '.

[0097] H3-4 upstream primer sequence is F: 5 '-GGGTGCTAAGAAGCAACATC-3 ', and downstream primer sequence is R: 5 '-TCACCACGGAAGCCAGAAG-3'; H3-5 upstream primer sequence is F: 5 '-AAGTCAACAGGTGGCAAGA-3 ', and downstream primer sequence is R: 5 '-TACGGATTTCACGAAGAGC-3 '.

[0098] The RT-qPCR test results show that, relative to the blank control group, the metronidazole group significantly reduces the expression of Fd1 and Fd3 mRNA (P<0.05) and significantly increases the expression of Fd6 mRNA (P<0.05) ; the fibrauretin group significantly increases the expression of Fd1, Fd2, Fd3, Fd4, Fd5 and Fd6 mRNA (P<0.05) ; and the example 3 group significantly reduces the expression of Fd1 and Fd3 mRNA (P<0.05) but has no significant difference in the expression of Fd2, Fd4, Fd5 and Fd6 mRNA (P>0.05). Figure 22 .

[0099] The RT-qPCR test results show that, relative to the blank control group, the metronidazole group significantly reduces the expression of CP1, CP3 and CP4 mRNA (P<0.05) ; the fibrauretin group and the example 3 group significantly reduce the expression of CP1, CP2, CP3 and CP4 mRNA (P<0.05). Figure 23 .

[0100] The results of RT-qPCR test show that, relative to the blank control group, the metronidazole group has no significant difference in the expression of H3-1, H3-2, H3-3, H3-4 and H3-5 mRNA (P>0.05); the fibrauretin group and the example 3 group significantly reduce the expression of H3-1, H3-2, H3-3, H3-4 and H3-5 mRNA (P<0.05). See Figure 24 .

[0101] The prior art metronidazole is the first choice for treating trichomoniasis and has a high success rate, and its mechanism of action is that the 5-nitro group on the metronidazole is reduced by obtaining an electron body through iron-sulfur protein-mediated electron transfer in the worm body, so that the metronidazole enters an activated state and exerts a pharmacological effect. It is found that the Fd in the Trichomonas vaginalis is closely related to the resistance of metronidazole, and if the electron transfer of Fd is blocked, the drug cannot be activated. The metronidazole-resistant strain of Trichomonas vaginalis has a decrease or absence of Fd, and the level of Fd mRNA is reduced by 50%-65%, and the gene transcription rate is reduced by 40%-65%. However, with the continuous increase of metronidazole-resistant strains, the resistance of Trichomonas vaginalis to metronidazole reaches 9.6%.

[0102] Tv contains various cysteine proteases (CP), which are important virulence markers. Through the secretion of CP toxic factors, Tv directly acts on the cells of the vagina and cervix, causing them to fall off and causing symptoms of vaginitis. Compared with other protozoans, the genome of Tv contains more histone genes. Histone refers to a special protein that binds with DNA to form chromatin in cells. Two molecules of histone H2A, H2B, H3 and H4 form a histone octamer, which is coiled with DNA to form a nucleosome, and is the basic component of chromatin. The amino acids at the N-terminus of the histone are modified in different ways to regulate the structure (loose or dense) of chromatin, thereby promoting the expression (promotion or inhibition) of genes.

[0103] Fibrauretin is the main active pharmacological component of Fibraurea, which has the pharmacological effects of anti-oxidation, anti-inflammation, bacteriostasis, anti-virus, enhancing immunity, improving memory, etc. and is praised as "plant antibiotic" by modern medicine. Fibrauretin is now commonly applied to treat gynecological inflammation, surgical infection, bacillary dysentery, enteritis, gastritis, conjunctivitis, respiratory tract infection, urinary tract infection, Candida vaginitis and other diseases. Our research shows that fibrauretin can destroy the nuclear structure of Trichomonas vaginalis, and then cause the deformation, rounding, rupture and apoptosis of the worm body, and the combination of fibrauretin and metronidazole can significantly reduce the dosage and time of metronidazole. The present application further studies the mechanism of killing Trichomonas vaginalis, and the RNA-seq results show that the differential genes of the fibrauretin group are 3847, which are significantly more than the differential genes of the metronidazole group, which are 355, and 64 differential genes are commonly targeted and regulated, including 6 Fd genes: Fd1, Fd2, Fd3, Fd4, Fd5 and Fd6; 4 CP genes: CP1, CP2, CP3 and CP4; and 5 H3 histone genes: H3-1, H3-2, H3-3, H3-4 and H3-5. GO analysis of the differential expression genes shows that the main function of the differential genes affected by metronidazole is small molecule metabolic process, while the main function of the differential genes affected by fibrauretin is cellular_component, which involves 20 small categories of biological processes (BP), cellular components (CC) and molecular functions (MF), and the number of regulated function genes of the 20 categories in the fibrauretin group is higher than that in the metronidazole group. The KEGG signal pathway annotation results show that the differential genes affected by metronidazole and fibrauretin are 85 and 562 respectively, the regulated signal pathways involved are 15 and 18 respectively, and they are mainly enriched in Metabolism and Genetic Information Proces signal pathways. The RT-qPCR test results show that the expression of Fd1 and Fd3 mRNA is significantly reduced, and the expression of Fd6 mRNA is significantly increased in the metronidazole group; the expression of Fd1, Fd2, Fd3, Fd4, Fd5 and Fd6 mRNA is significantly increased in the fibrauretin group; the expression of Fd1 and Fd3 mRNA is significantly reduced, but the expression of Fd2, Fd4, Fd5 and Fd6 mRNA has no significant difference in the example 1-4 group. The expression of CP1, CP3 and CP4 mRNA is significantly reduced in the metronidazole group; the expression of CP1, CP2, CP3 and CP4 mRNA is significantly reduced in the fibrauretin group and the example 1-4 group. The expression of H3-1, H3-2, H3-3, H3-4 and H3-5 mRNA has no significant difference in the metronidazole group; the expression of H3-1, H3-2, H3-3, H3-4 and H3-5 mRNA is significantly reduced in the fibrauretin group and the example 1-4 group.

[0104] In summary, compared with metronidazole, the number of Trichomonas vaginalis differential genes regulated by ferula ferruginea is more, the number of 20 functional genes regulated is more, and the number of signal pathways regulated is more; ferula ferruginea and its combination with metronidazole can significantly reduce the transcription of Fd gene, CP protein gene and H3 histone gene. Therefore, the application of ferula ferruginea combined with metronidazole can effectively reduce the dosage and reduce the medication time, solve the clinical treatment problem of drug resistance caused by the lack of Fd target point of Trichomonas vaginalis, avoid the risk of premature delivery of pregnant medication, reduce the health problems of breastfed infants during medication, and provide safer drugs for pregnant women and lactating women.

Claims

1. A pharmaceutical composition for killing Trichomonas vaginalis, characterized in that, The active ingredient of the pharmaceutical composition is composed of questrin and metronidazole; The mass ratio of the questrin and metronidazole is 8:1; In the process of killing Trichomonas vaginalis, the concentration of the questrin is 0.04 g / L, and the concentration of the metronidazole is 0.005 g / L.

2. The pharmaceutical composition for killing Trichomonas vaginalis according to claim 1, characterized by The pharmaceutical composition comprises questrin and metronidazole and other pharmaceutically acceptable adjuvants.

3. The use of a pharmaceutical composition for killing Trichomonas vaginalis according to claim 1, wherein The pharmaceutical composition is applied to the preparation of a medicine for killing Trichomonas vaginalis.

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