CN102743432B - Application of patchouli oil in preparation of medicines used for treating colpitis - Google Patents

Abstract

The invention discloses the application of Huoxiang oil in the preparation of medicine for treating vaginitis. The invention also discloses a pharmaceutical composition for treating vaginitis, which is a medicament prepared by taking Huoxiang oil as an active ingredient and adding pharmaceutically acceptable auxiliary materials or auxiliary ingredients. Huoxiang oil has remarkable curative effect in treating vaginitis, and has strong practical application value.

Description

Application of Huoxiang oil in the preparation of medicine for treating vaginitis

technical field

The invention relates to a new application of Huoxiang oil, which belongs to the field of medicine and health.

Background technique

Vaginitis is an inflammation of the vaginal mucosa and submucosal connective tissue, and is a common disease in gynecological clinics. For normal healthy women, due to anatomical and biochemical characteristics, the vagina has a natural defense function against the invasion of pathogens. When the natural defense function of the vagina is destroyed, pathogens are easy to invade and cause vaginal inflammation. At present, the treatment of vaginitis is mainly based on anti-infection and anti-inflammation, supplemented by physical therapy. The commonly used treatment methods include medicinal vaginal douching, local drug delivery, and oral medication. Common vaginitis includes bacterial vaginosis, candida vaginitis, trichomonas vaginitis, and senile vaginitis.

Bacterial vaginosis is a mixed infection caused by the imbalance of normal vaginal flora. It is a group of syndromes with a large number of bacteria in the vagina accompanied by changes in the nature of vaginal secretions. The main symptoms are increased vaginal secretions, which are thin Homogeneous or thin paste, off-white or gray-yellow, fishy smell, aggravated after sexual intercourse, may be accompanied by mild genital itching or burning sensation, and some people may experience lower abdominal pain, dyspareunia or dysuria. Common drugs for the treatment of bacterial vaginosis are antibiotics such as tetracycline and broad-spectrum antibacterial chemicals such as sulfomenidazole.

Candida vaginitis is vaginitis caused by Candida infection, and the main pathogenic bacteria is Candida albicans. Candida is a yeast and an conditional pathogenic fungus. Its pathogenicity is relative, and whether it will develop depends on the level of human immunity, the number and virulence of the infected bacteria. In a normal body, its number is small, oval in shape, in a symbiotic state with the body, and does not cause disease. When the body's resistance or immunity is reduced under the influence of certain physiological and pathological factors, the balance state is destroyed, and Candida changes from yeast phase to hyphae phase, and grows and reproduces in large numbers locally, causing vaginitis. Manifested as increased leucorrhea, vulva, vaginal itching, burning sensation, painful urination, often redness and edema around the vulva, and various changes in the epidermis; very shallow blisters and papules may appear in groups; eczema-like erosions may also form, Confined to the vulva or extended to the perineum, around the anus, and femoral genital folds, until the inner thigh and outer appearance, it is completely similar to acute or subacute eczema; the mucous membranes near the labia and clitoris are thickened, and the skin surfaces that are in contact with each other are flushed and eroded; some can cause Tiny white pustules, ulcers, vulvar pain, and local lymphadenopathy occur in severe cases. Common drugs for the treatment of candidal vaginitis are antibiotics such as nystatin and broad-spectrum antifungal chemicals such as fluconazole and itraconazole.

Trichomonas vaginitis is a common vaginitis caused by Trichomonas vaginalis. Clinically, the main manifestations are increased leucorrhea, rare foam, foul smell, and vaginal itching. During the examination, vaginal mucosal hyperemia can be seen. In severe cases, there are scattered bleeding spots. There is a lot of leucorrhea in the posterior fornix, which is gray-yellow, yellow-white thin liquid or yellow-green purulent secretion, often in the form of foam. Common treatment drugs are insecticides such as metronidazole.

Senile vaginitis is common in postmenopausal elderly women. Due to ovarian function decline, estrogen level decreases, vaginal wall atrophy, mucous membrane becomes thinner, glycogen content in epithelial cells decreases, vaginal pH value increases, and local resistance decreases, resulting in Bacteria are easy to invade and multiply and cause inflammation. The main symptoms are increased vaginal discharge and genital itching and burning sensation. Examination showed that the vagina showed senile changes, epithelial atrophy, folds disappeared, and the epithelium became smooth and thin. Vaginal mucosal hyperemia, small bleeding spots, sometimes superficial ulcers, if the ulcer surface adheres to the opposite side, the adhesions may be separated during vaginal examination and cause bleeding, severe adhesions may cause vaginal stenosis or even atresia, and poor drainage of inflammatory secretions Can form pyometra or even pyometra. Supplementing a small amount of estrogen is the principle of treatment for senile vaginitis, which can thicken the vaginal mucosa and enhance resistance.

At present, most of the drugs commonly used to treat vaginitis are antibiotics and broad-spectrum antibacterial drugs, which have large side effects and will produce drug resistance when widely used. Traditional Chinese medicine has a wide range of sources, low toxicity and long drug effect. Therefore, it is very necessary to seek new medicines for treating vaginitis from Chinese herbal medicines.

Patchouli Pogostemon cablin (Blanco) Benth. is a perennial herb of the Lamiaceae, also known as Zhixiang, which is native to the Philippines and is now widely distributed in China, India, Japan, Indonesia, Malaysia, Madagascar, Brazil, Paraguay and Russia. Patchouli oil is extracted from the leaves, branches and roots of Pogostemon cablin (Blanco) Benth., a perennial herb of the Labiatae family, through steam distillation or fermented dry leaves. Agastache rugosa (Fisch.EtMey.) O.Ktze. is a labiatae plant, containing volatile oil, and has the same effect as patchouli.

Patchouli oil mainly contains patchouli alcohol and patchouli ketone (accounting for more than 40% of the total), which has anti-inflammatory, anti-allergic, immune-enhancing, antibacterial, analgesic, anti-spasmodic, anti-oxidant, and antiemetic effects. It mainly contains methyl piperonol (accounting for more than 80%), which has antibacterial, antispasmodic, sedative and leukocyte-increasing effects (Liu Liangfeng et al., "Research Overview of Patchouli Oil and Huoxiang Oil", China Journal of Traditional Chinese Medicine Information, 2009 2 Volume 16, Issue 2, July). Among them, there are many literature reports on patchouli oil and Huoxiang oil in vitro antibacterial, such as Mo Xiaolu et al., "Research on the Antibacterial Activity of Patchouli Essential Oil on Phytopathogenic Fungi", Chinese Medicinal Materials, Volume 27, No. 11, November 2004 It was reported that patchouli oil has different degrees of inhibitory effects on 13 kinds of plant pathogenic fungi, among them, the inhibitory effect on Polychaete sandalwood, tomato early blight, and sclerotinia is stronger; Mo Xiaolu et al., "Two kinds of broad Antibacterial Effect of Patchouli Oil", China Modern Chinese Medicine, Volume 13, No. 9, September 2011, disclosed that patchouli oil can inhibit Escherichia coli, Staphylococcus epidermidis, Staphylococcus aureus, Proteus vulgaris, Bacillus subtilis, Pseudomonas aeruginosa Bacillus, Enterococcus faecalis 8 species of bacteria, and 3 species of fungi Penicillium chrysogenum, Aspergillus niger and Candida albicans; Yang Depo et al., "Study on the anti-skin bacterial activity and chemical constituents of the volatile oils of Huoxiang and Patchouli", Microbiology Magazine December 1998, Volume 18, No. 4, disclosed the in vitro inhibitory effect of Agastache and Patchouli volatile oil on 16 kinds of skin bacteria, such as Corynebacterium sicca, Micrococcus sessileus, etc.; Inhibitory effect of dermatophytes and conditionally curative fungi", Chinese Journal of Pharmaceutical Sciences, Volume 35, No. 1, January 2000, reported that ageratum oil and patchouli oil have inhibitory effects on dermatophytes such as rubrum rubrum and molds such as Aspergillus fumigatus; Zhang Guangwen et al., "Analysis of the Chemical Components of Patchouli Essential Oil and Its Antibacterial Activity (II)", Chinese Herbal Medicine, Volume 33, Issue 3, 2002, disclosed the effect of patchouli oil on pathogenic fungi such as Aspergillus niger and Aspergillus flavus and bacteria such as Escherichia coli. Antibacterial effect. Existing studies on the antibacterial and antibacterial effects of patchouli oil and Huoxiang oil mostly focus on plant pathogenic bacteria and skin pathogens, and the main purpose is to provide new antibacterial drugs for external use.

Contents of the invention

The object of the present invention is to provide a new application of the patchouli oil and a pharmaceutical composition using the patchouli oil as an active ingredient.

Firstly, the invention provides the use of Huoxiang oil in the preparation of medicine for treating vaginitis.

Wherein, the medicine is a medicine for treating candidal vaginitis or trichomonas vaginitis. Preferably, the medicine is for the treatment of candidal vaginitis caused by Candida albicans.

Wherein, the patchouli oil is derived from the volatile oil extracted from Pogostemon cablin (Blanco) Benth. or Agastache rugosa (Fisch. Et Mey.) O.Ktze.

Wherein, the patchouli oil is derived from the volatile oil extracted from Pogostemon cablin (Blanco) Benth., and the volatile oil contains not less than 26.0% w/w of bocicol.

Wherein, described ageratum oil is prepared by the following method: get the whole herb of Pogostemoncablin (Blanco) Benth. or Agastache rugosa (Fisch.Et Mey.) O.Ktze., be ground into coarse powder, Add water, soak, and extract by steam distillation to obtain Huoxiang oil.

The invention also provides a pharmaceutical composition for treating vaginitis, which is a medicament prepared by taking Huoxiang oil as an active ingredient and adding pharmaceutically acceptable auxiliary materials or auxiliary ingredients.

Wherein, the medicament is an external preparation, an oral preparation or an injection preparation. Preferably, the external preparation is lotion, suppository, film coating or liniment.

Wherein, the content of the patchouli oil in the preparation is 0.1%-100% w/w.

The Huoxiang oil of the invention has the effect of treating vaginitis, especially has definite curative effect on candida vaginitis and trichomonas vaginitis, and has good clinical application prospect.

Apparently, according to the above content of the present invention, according to common technical knowledge and conventional means in this field, without departing from the above basic technical idea of the present invention, other various forms of modification, replacement or change can also be made.

The above-mentioned content of the present invention will be further described in detail below through specific implementation in the form of examples. However, this should not be construed as limiting the scope of the above-mentioned subject matter of the present invention to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention.

Description of drawings

Fig. 1 the influence of medicine of the present invention on the ultrastructure of Trichomonas vaginalis. Figure A is the normal ultrastructure of Trichomonas vaginalis without any drug action, in which 1 is the nucleus, 2 is the rough endoplasmic reticulum, 3 is the perhydrogenase body, and 4 is the cross-section of the flagella; Figures B to H The ultrastructural changes of Trichomonas vaginalis after drug action, a: vacuoles, b: autophagic vacuoles, c: perinuclear space widening, d: rough endoplasmic reticulum dilation, e: chromatin accumulation, f: Ribosome disappears, g: nuclear membrane disappears, the nucleus gradually disintegrates, h: nuclear pyknosis, i: organelle disintegration, j: cell membrane damage, cytoplasm leaks out, and the parasite dies.

Detailed ways

The preparation of embodiment 1 Huoxiang oil of the present invention

Take the whole herb of Pogostemon cablin (Blanco) Benth., crush it through a 20-40 mesh sieve, add 10-14 times the weight of distilled water, soak it for 1-5 hours, and extract it by steam distillation for 2-6 hours, that is The patchouli oil of the present invention is obtained, which contains not less than 26.0% of bocicol (C ₁₅ H ₂₆ O).

The preparation of embodiment 2 Huoxiang oil of the present invention

Take the whole herb of Agastache rugosa (Fisch.Et Mey.) O.Ktze., crush it through a 20-40 mesh sieve, add 10-14 times the weight of distilled water, soak for 1-5 hours, and extract 2 by steam distillation. -6 hours, to obtain Huoxiang oil of the present invention.

Patchouli oil of the present invention also can be extracted according to Pharmacopoeia (2005 edition) appendix XD volatile oil determination method, can also adopt organic solvent extraction, supercritical _CO Extraction, or obtain by purchasing commercially available products.

Prove the beneficial effects of the present invention below by drug efficacy test:

Experimental Example 1 The activity of Huoxiang oil of the present invention in the treatment of candida vaginitis

1. Materials and instruments

1.1 Experimental strains

① Standard strain: Candida albicans standard strain (ATCC14053), from American Clinical Laboratory Culture Collection Center; Candida albicans standard strain (CICC32819) and Candida albicans standard strain (CICC31284), purchased from China Industrial Microbiology Culture Collection Management center.

②Clinical isolates: 14 strains of Candida albicans, which were isolated from the vaginal secretions of patients in the gynecology clinic of Sichuan Provincial Maternal and Child Health Hospital from September to December 2011, and identified by KC-16 of BIO-KONT Company and drug sensitivity The disk was identified as Candida albicans.

1.2 Drugs

Test drug: Huoxiang oil of the present invention (prepared according to the method of Example 1).

Positive drugs: clotrimazole vaginal tablets (Jinan Limin Pharmaceutical Co., Ltd., batch number: 1108148), Jieeryin lotion (Sichuan Enwei Pharmaceutical Co., Ltd., batch number: 1107102).

1.3 Experimental animals

Mice (SPF grade) [Experimental Animal Research Center, Chengdu University of Traditional Chinese Medicine, production license number: SCXK (Chuan) 2008-11, use certificate number: SCXK (Chuan) 2008-049].

1.4 Media, reagents and consumables

Sabouraud agar medium (0.1 g chloramphenicol per liter, Guangdong Huankai Microbial Technology Co., Ltd.).

Estradiol Benzoate Injection (Tianjin Jinyao Amino Acid Co., Ltd., batch number: 1007061), disposable sterile petri dishes (Jiangsu Kangjian Biological Co., Ltd.), etc.

1.5 Main Instruments

Multi-point inoculation instrument (Japan Sakuma Works, SAKUMA MIT-P type), laboratory autoclave (SANYO, MLS-3780 type), CO2INCUBATOR (SANYO, MOC-15A), energy-saving purification workbench (Chengdu Xinguang Feilante Purification Engineering Co., Ltd.).

2. Experimental method

2.1 The in vivo activity of medicine treatment candidal vaginitis of the present invention

2.1.1 Establishment of mouse candida vaginitis model

Activation of test bacteria: Inoculate Candida albicans standard strain CICC32819 on Sabouraud agar medium, culture at 37°C for 48 hours, and activate it for later use.

Model preparation: through pre-experiments, various factors affecting the establishment of the model are optimized, and the establishment method of the candida vaginitis model is determined. In the formal experiment, 70 SPF Kunming mice were selected, weighing (20±2) g, 60 of which were started 6 days before infection, and each mouse was subcutaneously injected with 0.1ml of 1mg/ml estradiol benzoate oil on the back every other day , and inoculated with Candida albicans 1.5×10 ⁷ CFU per mouse after the last injection, and re-infected once 24 hours later. At 48h and 72h after the last infection, the vaginal secretions were taken for smear microscopy to observe the formation of hyphae and blastospores, and inoculated on Sabouraud agar plates to observe the growth of the strains; and to observe the vaginal secretions Wait for changes. 48 hours after the last infection, the vaginal secretions of the mice were inoculated on the Sabouraud agar plate with an inoculation loop for 24 hours to 48 hours, and the colony growth of Candida albicans on the plate was observed. If typical Candida albicans grows on the plate, and mycelia and blastospores are observed under the microscope, it indicates that the candida vaginitis model mouse has been successfully prepared.

2.1.2 Experimental grouping

After the mouse model is successfully established, each mouse is rinsed with 0.1ml sterile normal saline, and the rinse solution is counted for colonies. High, medium and low dose groups, positive drug clotrimazole group and Jieeryin group, model group, 10 rats in each group. In addition, 10 mice not infected with Candida albicans were used as blank control. The analysis of intravaginal infection of Candida albicans in each group of mice is shown in Table 1.

Table 1: Analysis of vaginal infection of Candida albicans in each group of mice

Note: ^A indicates extremely significant statistical significance compared with the blank group (P<0.01).

2.1.2 Treatment of Candida Vaginitis Model Mice

The candida vaginitis model mice were treated by intravaginal administration of drugs, that is, the infected model mice were treated with high, medium and low doses of the invented drug every day, once a day, for 20 consecutive days. Clotrimazole and Jieeryin were used as positive drugs, and normal saline was used in the model group. Wash the vagina with 0.1ml sterile normal saline before administration (0d), and 6h after administration on the 3rd, 5d and 7th day, count the colonies of Candida albicans in the washing liquid, and statistically analyze before administration (0d) , After 3 days of administration, after 5 days of administration and after 7 days of administration, the changes of the number of colonies in the vagina of mice were observed, and the effect of the drug on killing or inhibiting Candida albicans in the vagina of infected mice was observed. After the 7th day of administration, inoculate and observe the hyphae and blastospore growth of the vaginal secretions and the growth in the Sabouraud plate every day, and observe the changes of the vaginal hyperemia, redness and secretions of the mice every day. The cure time and cure rate of administration 14d and administration 20d were counted respectively. No Candida albicans growth was found in the vaginal contents for 3 consecutive days on the Sabouraud agar plate, and no vaginal secretions, no obvious congestion, swelling and other clinical symptoms were judged to be cured, and the cured mice were no longer treated with drugs.

2.2 Anti-Candida albicans in vitro activity of the medicament of the present invention

Preparation of the drug-containing plate: the drug Huoxiang oil of the present invention is diluted by the doubling dilution method, and diluted into 11 gradients of 1:2~1:2048 respectively, and drug solutions of different concentration gradients are respectively added to disposable sterile culture dishes 1ml and 14ml of sterilized Sabouraud agar medium, that is, the dilution gradient of each drug in the plate is 35.00mg/ml~34.18μg/ml, mix well, dry and set aside, add the same amount of normal saline to the plate Prepare a positive control plate in place of the drug.

Bacteria solution configuration: adjust the Candida albicans standard strain and clinical isolates with sterile saline to a concentration of 1.5×10 ⁶ CFU/ml.

Determination of the minimum inhibitory concentration (MIC): Add the bacterial solution of the experimental strain to the drug-containing plate and positive control plate with different concentration gradients using a multi-point inoculator, and use normal saline instead of the bacterial solution as a negative control. Cultivate at a constant temperature of 37°C for 24h~48h, and observe the growth of each strain on the drug plate containing different concentration gradients.

Result judgment: the minimum inhibitory concentration of this medicine is this medicine to this bacterial strain with the medicine minimum concentration that does not have Candida albicans growth in the flat plate.

3. Experimental results

3.1 In vivo activity of the drug of the present invention

See Table 2 for the effect of the medicine of the present invention on the growth of Candida albicans in the vagina of the candida vaginitis model mice, and see Table 3 for the analysis of the cure time and cure rate.

Table 2: Effect of the medicine of the present invention on the growth of Candida albicans in the vagina of the candidal vaginitis model mice

Note: 0d, 3d, 5d, 7d represent before administration, the 3rd day, the 5th day and the 7th day after administration respectively.

Compared with the blank group, ^** P<0.01; compared with before administration, ^▲ P<0.05, ^▲▲ P<0.01; compared with the 3rd day after administration, ^★ P<0.05.

It can be seen from Table 2 that before administration, compared with the blank group, the number of bacterial colonies in the vagina of the model group, the positive drug group and the high, medium and low dose groups of the present invention increased significantly (P<0.01), while the model group There was no statistical significance in the comparison of the number of colonies between the experimental group and the experimental group (P>0.05).

After administration, compared with the model group, the number of vaginal colonies in the high, medium and low dose groups of the drug of the present invention and the clotrimazole group decreased significantly; compared with the positive group Jieeryin, the high, medium and low doses of the drug of the present invention group and the clotrimazole group vaginal colony count significantly decreased; compared with the positive drug clotrimazole, the high, middle and low dose groups of the present invention have similar anti-Candida albicans activity in the body, and with the administration time Prolonged, the number of colonies showed an obvious downward trend, the condition gradually improved, and the curative effect was better than that of Jieeryin.

Comparing the changes in the number of colonies in each dosage group before and after administration and at different administration times, compared with before administration, the number of colonies in the high-dose group decreased significantly on the 3d, 5d, and 7d after administration (P<0.05); The number of colonies in the low-dose group decreased significantly on the 3d, 5d, and 7d after administration (P<0.05); Very significantly decreased (P <0.01). Compared with the 3rd day of administration, the number of colonies on the 5th day and the 7th day after administration in the middle dose group were significantly reduced (P<0.05).

Experimental results show that the drug Huoxiang oil of the present invention can significantly kill Candida albicans in the vagina of candidal vaginitis.

Table 3: The cure situation analysis of medicine of the present invention to candidal vaginitis

Note: In the statistical results of 14 days of medication, the healing time of mice that were not cured within 14 days was recorded as 15 days.

In the statistical results of 20 days of medication, the healing time of uncured mice within 20 days was recorded as 21 days.

Compared with the model group, ^* P<0.05, ^** P<0.01.

It can be seen from Table 3:

When taking medicine for 14 days, the average cure times of the high, middle and low dose groups of the present invention were 11.50 days, 11.70 days and 13.10 days respectively within 14 days for vaginitis model mice, and the cure rates were 70.00%, 60.00% and 40.00% respectively ;Compared with the model group (the average self-healing time is 14.30d, and the self-healing rate is 10%), the healing time of the high-dose drug group of the present invention is significantly reduced (P<0.05), and the high-, medium-, and low-dose groups of the present invention have significant healing rates Compared with the positive drug Jieeryin, there was no significant difference; the comparison within each dose group showed a certain degree of dose-effect relationship.

During medication 20d, the average cure time of the high, middle and low dose groups of the medicine of the present invention is respectively 12.90d, 13.20d and 15.40d in 20d to the vaginitis model mice, and the cure rate is 80%; The self-healing time is 18.90d, and the self-healing rate is 30%). By comparison, the healing time of the high-dose and middle-dose groups of the present invention is significantly reduced (P<0.01), and the cure rate of the high-dose, medium-dose, and low-dose groups of the present invention is significantly improved. ; Compared with the positive drug Jieeryin, there was no significant difference; there was no significant statistical significance among the comparisons within each dose group.

Experimental results illustrate that Huoxiang oil of the present invention has the effect of treating candidal vaginitis.

3.2 In vitro activity of the drug of the present invention

The minimum inhibitory concentration of the invention drug, the positive drug clotrimazole and Jieeryin, on 3 Candida albicans standard strains and 14 clinical isolates from patients with vaginitis infection was determined by using the agar plate double dilution method. The results are shown in Table 4.

Table 4: The in vitro antibacterial activity of the medicine of the present invention to Candida albicans

Note: "-" indicates that the maximum concentration of the drug in the plate has no obvious antibacterial activity against the corresponding Candida albicans. Among them, the maximum concentration of the invented drug in the plate is 35.00mg/ml; the maximum concentration of clotrimazole in the plate is 1.67mg/ml; Jieeryin is diluted 15 times with the original drug in the plate.

As can be seen from the above table, the medicine of the present invention has stronger antifungal activity to 3 strains of Candida albicans standard strains and clinical isolates, wherein the minimum inhibitory concentration to 3 strains of Candida albicans standard strains is 0.07mg/ml. The minimum inhibitory concentration of isolates from patients with clinical vaginitis infection was 0.07~0.27mg/ml (the average minimum inhibitory concentration was 0.10±0.06mg/ml). The positive drug, clotrimazole, had no obvious antibacterial activity against 1 Candida albicans standard strain ATCC14053 and 2 clinical isolates, indicating that clotrimazole had a certain degree of drug resistance to some Candida albicans strains. However, Jieeryin had no obvious antibacterial activity against Candida albicans in vitro. Experiment shows that the medicine Huoxiang oil of the present invention has obvious antibacterial effect in vitro to Candida albicans.

The above in vivo and in vitro test results show that Huoxiang oil of the present invention can significantly inhibit Candida albicans, and has the effect of treating candida vaginitis.

Experimental Example 2 The activity of Huoxiang oil of the present invention in the treatment of trichomonal vaginitis

1 Materials and Instruments

1.1 Experimental strains

Trichomonas vaginalis: 2 strains (numbered Tv1 and Tv2 respectively). The experimental strain was obtained by using OXOID Trichomonas No. 2 culture medium to isolate, purify, culture and microscopically identify the vaginal secretions of patients in the gynecology clinic of Sichuan Provincial Maternal and Child Health Hospital in April 2012.

1.2 Drugs

Test drug: drug of the present invention: Huoxiang oil (prepared according to the method of Example 1).

Positive drugs: metronidazole (Sichuan Kelun Pharmaceutical Co., Ltd., batch number: M11120602), Jieeryin lotion (Sichuan Enwei Pharmaceutical Co., Ltd., batch number: 1107102).

1.3 Media, reagents and consumables

TRICHOMONAS MEDIUM No.2 (OXOID Ltd. batch number: 1180675), PBS buffer, 96-well cell culture plate, cell counting plate, etc.

1.4 Main Instruments

Hitachi transmission electron microscope (H-600IV type), ultra-thin slicer (ULTRACUT-E type), laboratory autoclave (SANYO, MLS-3780 type), CO ₂ INCUBATOR (SANYO, MOC-15A), energy-saving purification Workbench (Chengdu Xinguang Feilante Purification Engineering Co., Ltd.), high-speed centrifuge (HC-3518, Keda Innovation Co., Ltd.), low-speed centrifuge (model KDC-40, Keda Innovation Co., Ltd.).

2 Experimental methods

2.1 Research on anti-Trichomonas vaginalis activity of the drug of the present invention

Preparation of worm suspension: Take the identified Trichomonas vaginalis and culture them in No. 2 Trichomonas culture medium at 37°C for 48 hours, shake and mix well as the original culture, and count the density and live worm rate with a cell counting plate. The live worm rate of the original culture was >95%, the cell shape was intact, the movement was active, and the worm density was 2.0×10 ⁶ worms/ml. Take 1ml of the original culture, centrifuge at 500rpm for 10min, and resuspend the worm bodies with 10ml of pre-warmed new culture medium to make the worm density 2.0×10 ⁵ worms/ml.

Medicine preparation: first carry out emulsification treatment to medicine Huoxiang oil of the present invention, prepare 10% emulsion. The prepared inventive drug was cultured with No. 2 Trichomonas and diluted into 10 gradients of 1:2~1:1024 respectively.

Determination of the minimum insecticidal concentration (MLC): Add 100 μl of insect suspension and 100 μl of the drug of the present invention with different concentration gradients to each well of a 96-well plate, culture in a wet box at 37°C for 48 hours, and use a capillary tube to take a smear from each drug hole by hole Microscopic examination, statistical dead insect rate. Metronidazole was set as the positive drug, and the well containing only the culture medium and Trichomonas vaginalis without the drug was used as the negative control. The minimum lethal concentration (MLC) of the drug against Trichomonas vaginalis was defined as the minimum drug concentration with a death rate >95%. Experiments were repeated three times.

2.2 The influence of medicine of the present invention on the ultrastructure of Trichomonas vaginalis

Preparation and fixation of worm strains: Add the drug of the present invention to the trichomonas medium containing Tv2 strain, wherein the density of worms is 2.0×10 ⁶ worms/ml, and the concentration of the drug of the present invention is 1/2 MLC. In a _CO2 incubator at 37°C for 1h, 3h and 5h, centrifuge at 2000rpm for 10min, discard the supernatant; slowly add 1ml of cold 0.5% glutaraldehyde along the tube wall, let stand at 4°C for 30min, centrifuge at 12000rpm for 10min, discard the supernatant Slowly add 1ml of cold 3% glutaraldehyde along the tube wall for pre-fixation, and 1% osmium tetroxide for fixation. The Tv2 worm strain that can replace the medicine of the present invention with physiological saline is used as the normal control group.

Ultra-thin section and staining: Dehydrate the fixed sample step by step with acetone, embed in Epon812, optically locate the semi-thin section, ultra-thin section, double stain with uranyl acetate and lead citrate.

Ultrastructural analysis: Hitachi H-600IV transmission electron microscope was used to observe the ultrastructural changes of the Trichomonas strain after 1/2 MLC of the drug of the present invention was used for 1 hour, 3 hours and 5 hours respectively, and its mechanism of action was explored.

3 Experimental results

3.1 The insecticidal activity of the medicine of the present invention to Trichomonas vaginalis

The insecticidal activity of the medicament of the present invention on Trichomonas isolated from patients with nosocomial vaginitis infection was determined by using a 96-orifice plate microdilution method, and the results are shown in Table 5.

Table 5: The minimum insecticidal concentration of the medicine of the present invention to Trichomonas vaginalis

It can be seen from the above table that the drug of the present invention has obvious insecticidal activity against Trichomonas vaginalis, and the minimum insecticidal concentration (MLC) for two strains of Trichomonas vaginalis in the secretions of patients with clinical vaginitis is 0.41mg/ml , illustrate that medicine of the present invention has the effect of killing Trichomonas.

3.2 The mechanism of action of the medicine of the present invention to Trichomonas vaginalis

The ultrastructural changes of the Tv2 strain were observed and analyzed respectively after 1/2MLC of the drug of the present invention was used for 1h, 3h and 5h by using a transmission electron microscope. As a result, it was found that under different action time points of the drug of the present invention, the same ultrastructural changes occurred in the strains, indicating that the duration of action of the drug of the present invention has no significant difference on the ultrastructure and mechanism of action of the worm.

Among them, the ultrastructural changes after 1 hour of drug action of the present invention are shown in Fig. 1, and the ultrastructural changes of Trichomonas vaginalis are obvious compared with the normal control. The changes from the inside to the outside mainly include: multiple changes in the nucleus, such as widening of the perinuclear space (Figure D), nuclear pyknosis (Figure G), chromatin accumulation (Figure E), and finally the disappearance of the nuclear membrane (Figure G) , the nuclei gradually dissolve. A large number of vacuoles appeared in the cytoplasm (Panels B and C), rough endoplasmic reticulum expanded (Panel D), ribosomes were reduced to disappear (Panels E and F), and autophagic vacuoles appeared (Panels C, E and Panel F), organelle disintegration (panel G). Partial cell membrane damage (Panel H), cytoplasm leakage (Panel H), and finally cell disintegration and necrosis (Panel H). It shows that the medicine of the present invention finally kills parasites by destroying the nucleus and organelle structure.

According to the above experimental results of the influence of the medicament of the present invention on the insecticidal activity and ultrastructure of Trichomonas vaginalis, the medicament of the present invention has the effect of killing Trichomonas vaginalis and can be used for treating trichomonas vaginitis.

In summary, Huoxiang oil has the effect of treating vaginitis, especially for candida vaginitis and trichomonas vaginitis, with strong pharmacological effects and good market application prospects.

Claims (10)

1. the purposes of oil of Herba Pogostemonis in the colpitic medicine of preparation treatment.

2. purposes according to claim 1, is characterized in that: described medicine is the medicine for the treatment of monilial vaginitis or trichomonal vaginitis.

3. purposes according to claim 2, is characterized in that: described medicine is the monilial vaginitis that treatment Candida albicans causes.

4. according to the purposes described in claim 1～3 any one, it is characterized in that: described oil of Herba Pogostemonis derives from the volatile oil of Herba Pogostemonis Pogostemon cablin (Blanco) Benth. or Herba Pogostemonis Agastache rugosa (Fisch.Et Mey.) O.Ktze. extraction.

5. according to the purposes described in claim 1～3 any one, it is characterized in that: described oil of Herba Pogostemonis derives from the volatile oil that Herba Pogostemonis Pogostemon cablin (Blanco) Benth. extracts, must not be lower than 26.0%w/w containing patchouli alcohol in this volatile oil.

6. according to the purposes described in claim 1～3 any one, it is characterized in that: described oil of Herba Pogostemonis is prepared by following methods: the herb of getting Herba Pogostemonis Pogostemon cablin (Blanco) Benth. or Herba Pogostemonis Agastache rugosa (Fisch.Et Mey.) O.Ktze., be ground into coarse powder, add water, soak, adopt extraction by steam distillation, obtain oil of Herba Pogostemonis.

7. purposes according to claim 1, is characterized in that: described medicine is taking oil of Herba Pogostemonis as active component, adds the medicament that pharmaceutically acceptable adjuvant or complementary composition are prepared from.

8. purposes according to claim 7, is characterized in that: described medicament is external preparation, oral formulations or ejection preparation.

9. purposes according to claim 8, is characterized in that: described external preparation is lotion, suppository, liniment or liniment.

10. purposes according to claim 7, is characterized in that: in described preparation, the content of oil of Herba Pogostemonis is 0.1%～100%w/w.