CN102716407A - Application of amomum tsao-ko oil on preparing medicaments with functions of restraining or killing candida albicans - Google Patents

Abstract

The invention discloses application of amomum tsao-ko oil on preparing medicaments with functions of restraining or killing candida albicans. The invention further discloses a medicine composite with the functions of restraining or killing the candida albicans. The medicine composite enables the amomum tsao-ko oil to serve as an active ingredient, and auxiliary materials or auxiliary ingredients which are acceptable on the pharmacy are added to prepare. The amomum tsao-ko oil has the functions of restraining or killing the candida albicans, can treat candida albicans diseases, and has strong practical application value.

Description

Use of grass fruit oil in the preparation of medicines capable of inhibiting or killing Candida albicans

technical field

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

Background technique

Candida albicans, also known as Candida albicans, widely exists in nature and is one of the normal flora of the human body. It usually lives in the mouth, skin, mucous membranes, digestive tract, vagina and other organs of the human body. Candida albicans is an opportunistic pathogenic fungus, and its pathogenicity is relative. Whether it develops 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 albicans changes from the yeast phase to the hyphae phase, and it grows and reproduces in a large number locally, causing skin and mucous membrane infection. Even systemic candidiasis.

Candida albicans can invade many parts of the human body, causing a variety of candidiasis albicans, such as: 1. Skin candidiasis, which is prone to occur in skin folds (organ fossa, groin, under the breasts, around the anus, nail groove, between fingers ), the skin is flushed, moist, shiny, sometimes covered with a layer of white or cracked, and there are small blisters around the lesion. 2. Mucosal candidiasis, thrush, angular cheilitis, and vaginitis are the most common. The surface of the mucous membrane is covered with a white film of varying sizes. After peeling off, a flushed base is left, and fissures and superficial ulcers are formed. 3. Visceral and central nervous candidiasis, which can be caused by the spread of bacteria on the mucous membranes and skin, including pneumonia, gastroenteritis, endocarditis, meningitis, encephalitis, etc., and occasionally sepsis can also occur.

Mycotic vaginitis caused by Candida albicans is the most common candidiasis albicans, manifested as increased leucorrhea, vulvar and vaginal itching, burning sensation, painful urination, often redness and edema around the vulva, and various changes in the epidermis; Very shallow vesicular papules may appear in groups; eczema-like erosions may also be formed, confined to the vulva or extending to the perineum, around the anus, and femoral genital folds, until the inner thighs and appearance, completely similar to acute or subacute eczema; The mucous membranes near the labia and clitoris are thickened, and the skin surfaces in contact with each other are flushed and eroded; some may cause tiny white pustules, and in severe cases, ulcers, vulvar pain, and local lymph node enlargement may occur.

Common drugs for the treatment of candidiasis are antibiotics such as nystatin and broad-spectrum antifungal chemicals such as fluconazole and itraconazole. Antibiotics and azole anti-candida drugs have the characteristics of broad antifungal spectrum, rapid and complete oral absorption, and high blood-brain barrier permeability, but their side effects are large, and extensive use will cause Candida to develop drug resistance. Traditional Chinese medicine antifungal agents have a wide range of sources, low toxicity, broad spectrum and long efficacy, and produce various pharmacological effects in fungal cells. Therefore, it is necessary to seek new anti-Candida drugs from Chinese herbal medicines.

Caoguo is the mature fruit of Amomum tsa-ko Crevost et Lemaire, a plant of the genus Cardamom in the ginger family, and is mainly produced in Yunnan, Guangxi, Guizhou and other places. According to traditional Chinese medicine, it is warm in nature, pungent in taste, and belongs to the spleen and stomach meridian. It has the effect of drying dampness and warming the middle. Inverse nausea, choking diaphragm, regurgitation and accumulation of phlegm and fluid, etc. Cao Guo contains volatile oil and flavonoids, and its volatile oil is a yellow oily transparent substance with a special pungent smell.

Tsaoguo oil is widely used in food and pharmaceutical industries, and it has anti-oxidation, antimutagenic, calm, anti-hepatitis B virus and anti-mold effects (Feng Xue, etc., research progress of tsaoguo volatile oil in seasoning spices, "Chinese seasoning Products”, No. 8, 2009.) In addition, grass fruit oil also has anti-inflammatory, anthelmintic, antitussive, gastrointestinal and bacterial infectious diseases, such as patent application number: 200410022726.1, invention name: a kind of The patent application for the preparation, preparation method and application of Tsaoguo volatile oil discloses that the traditional Chinese medicine preparation prepared by extracting volatile oil from the kernel, fruit, stem, leaf and root of the ginger plant Amomum tas-ko crevost etlemaire can be used to treat gastrointestinal diseases Medicine for disease; patent application number: 200410008132.5, name of invention: Tsaoguo powder and its preparation method patent application, which discloses that Tsaoguo powder prepared from Tsaoko volatile oil can be used as food flavoring seasoning, anti-inflammatory and insect repellent , antitussive, phlegm-resolving, cold-dispelling and other drugs. Patent application number: 201010622746.8, invention name: the patent application for the use of grass fruit oil in the preparation of medicines for treating bacterial infectious diseases, which discloses that grass fruit oil can be used to treat bacterial infectious diseases.

Contents of the invention

The purpose of the present invention is to provide a new application of grass fruit oil, that is, a new use in the preparation of drugs capable of inhibiting or killing Candida albicans, and also provide a pharmaceutical composition with grass fruit oil as an active ingredient.

The invention provides the use of grass fruit oil in the preparation of medicines capable of inhibiting or killing Candida albicans.

Wherein, the medicine is a medicine for treating candidiasis albicans.

Wherein, the drug is a drug for treating skin candidiasis, mucosal candidiasis, visceral candidiasis or central nervous system candidiasis.

Wherein, the medicine is a medicine for treating mycotic vaginitis.

Wherein, the grass fruit oil is derived from the volatile oil extracted from the mature fruit of the plant Amomum tsa-ko Crevostet Lemaire of the genus Zingiberaceae Cardamom.

Wherein, the grass fruit oil is prepared by the following method: take grass fruit, crush it into a coarse powder, add 10 to 14 times the weight of distilled water, soak for 1 to 5 hours, and extract 2 to 6 hours, that is, grass fruit oil.

The present invention also provides a pharmaceutical composition capable of inhibiting or killing Candida albicans, which is a medicament prepared by taking grass fruit oil as an active ingredient and adding pharmaceutically acceptable auxiliary materials or auxiliary ingredients.

Wherein, the grass fruit oil is prepared by the following method: take grass fruit, crush it into a coarse powder, add 10 to 14 times the weight of distilled water, soak for 1 to 5 hours, and extract 2 to 6 hours, that is, grass fruit oil. Wherein, the medicament is an external preparation, an oral preparation or an injection preparation. Preferably, the external preparation is lotion, film coating, gargle or liniment.

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

The medicine of the invention has good antibacterial and antibacterial effects on Candida albicans, can effectively treat various diseases caused by Candida albicans infection, especially has definite curative effect on 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.

Detailed ways

The preparation of embodiment 1 grass fruit oil of the present invention

Take the grass fruit, crush it through a 20-40 mesh sieve, add 10-14 times the weight of distilled water, soak for 1-5 hours, and extract it by steam distillation for 2-6 hours to obtain the grass fruit oil of the present invention.

The grass fruit oil of the present invention can also be extracted according to the pharmacopoeia (2005 edition) appendix XD volatile oil determination method, can also adopt organic solvent extraction, supercritical CO Extraction and other existing technologies to extract, or obtain by purchasing commercially available products.

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

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, which were isolated from the respiratory tract, skin, vagina, etc. of the gynecological outpatients of Sichuan Maternal and Child Health Hospital from September to December 2011, and were identified by KC-16 of BIO-KONT Company. The sensitive paper was identified as Candida albicans.

1.2 Drugs

Experimental drug: the drug grass fruit oil of the present invention.

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 Manufacturing Co., Ltd., SAKUMA MIT-P type), laboratory autoclave (SANYO, MLS-3780 type), CO ₂ INCUBATOR (SANYO, MOC-15A), energy-saving purification bench ( Chengdu Xinguang Feilante Purification Engineering Co., Ltd.).

Experimental Example 1 In vitro experiment of Tsaoko oil of the present invention against Candida albicans

1 Test method

Preparation of the drug-containing plate: the grass fruit oil of the present invention is diluted by the doubling dilution method, and diluted into 11 gradients of 1:2~1:1024 respectively, and drug solutions of different concentration gradients are added to the disposable sterile petri dish 1ml and 14ml of sterilized Sabouraud agar medium, that is, the dilution gradient of each drug in the plate is 30.70mg/ml~59.95μg/ml, mix well, dry and set aside, add an equal 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 determination: the minimum inhibitory concentration of the drug to the bacterial strain is the minimum concentration of the drug without the growth of Candida albicans in the flat plate.

2 Experimental results

The minimum inhibitory concentrations of herbal fruit oil, clotrimazole and Jieeryin to 3 Candida albicans standard strains and 14 clinical isolates were measured by the agar plate double dilution method, and the results are shown in Table 1.

Table 1: In vitro antibacterial activity of grass fruit oil of the present invention against 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 30.70mg/ml; the maximum concentration of clotrimazole in the plate is 1.67mg/ml; Jieeryin is the original drug and is diluted 15 times in the plate.

It can be seen from Table 1 that the positive drug clotrimazole has no obvious antibacterial activity against the standard strain of Candida albicans ATCC14053 and two clinical isolates, indicating that clotrimazole has produced a certain degree of drug resistance against some Candida albicans strains. It has no obvious antibacterial activity against Candida albicans in vitro. And grass fruit oil of the present invention all has stronger antibacterial activity to 3 strains of Candida albicans standard strains and 14 strains of clinical isolates, the minimum inhibitory concentration to 3 strains of Candida albicans standard strains is 0.48mg/ml, to clinical The minimum inhibitory concentration of isolates was 0.12~0.96mg/ml (the average value of minimum inhibitory concentration was 0.44±0.21mg/ml).

The experiment shows that the medicine of the present invention has obvious antibacterial and bactericidal effect on Candida albicans in vitro, and can treat diseases caused by Candida albicans infection.

Experimental Example 2 In vivo activity of grass fruit oil of the present invention in treating mycotic vaginitis caused by Candida albicans

1 Experimental method

(1) Establishment of a mouse model of mycotic vaginitis caused by Candida albicans

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 method for establishing the model of mycotic vaginitis caused by Candida albicans 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 1 mg/ml estradiol benzoate oil 0.1 ml, and inoculate Candida albicans 1.5×10 ⁷ CFU per mouse after the last injection, and re-infect once 24 hours later. And 48h and 72h after the last infection, the vaginal secretions were taken for smear microscopy to observe the production of hyphae and blastospores. At the same time, they were inoculated on Sabouraud agar plates to observe the growth of the strains; and observed vaginal congestion, Changes such as redness and discharge. 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. If typical Candida albicans grows on the plate, hyphae and blastospores are observed under the microscope, it indicates that the fungal vaginitis model mouse has been successfully prepared.

(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. Three groups of high, medium and low doses of grass fruit oil, 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. Wherein the situation analysis of the vaginal infection of Candida albicans in each group of mice is shown in Table 2.

Table 2: 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).

(3) Treatment of mycotic vaginitis model mice caused by Candida albicans

The fungal vaginitis model mice caused by Candida albicans are treated by intravaginal administration of drugs, that is, the grass fruit oil of the present invention with high, medium and low doses is used to treat the infected model mice every day. 1 time, 20 days in a row. 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) , Administration 3d, administration 5d and administration 7d the change situation of the bacterial colony number in the vagina of mice, observe the killing or inhibitory action of medicine to infection mouse intravaginal Candida albicans. 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 on the Sabouraud agar plate with vaginal secretions for 3 consecutive days, and no vaginal secretions, no obvious congestion, swelling and other clinical symptoms were judged as cured, and the cured mice were no longer treated with drugs. The one-way analysis of variance method in SPSS 17.0 software was used to compare and analyze the significance of the differences among the groups, and the paired T test was used to analyze the significance of the differences of each dose group of the inventive drug before and after administration and at different administration times.

2 Experimental results

Wherein the colony count analysis results are shown in Table 3, and the cure time and cure rate analysis are shown in Table 4.

Table 3: Effect of grass fruit oil of the present invention on the growth of Candida albicans in the vagina of 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 3 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 drug of the present invention increased significantly, which was statistically significant (P<0.01); Compared with the model group, the number of colonies in the positive drug group and the high, medium and low doses of the drug of the present invention had no statistical significance (P>0.05).

After administration, compared with the model group, the number of vaginal colonies in the high, middle and low dose groups of the drug of the present invention and the clotrimazole group decreased significantly; Group and clotrimazole group significantly decreased the number of vaginal colonies. Compared with the positive group clotrimazole, the high, medium and low dose groups of the present invention have similar in vivo anti-Candida albicans activity. With the prolongation of administration time, the number of colonies showed a significant downward trend, and the condition gradually improved . Paired T test analysis finds that compared with positive drug clotrimazole, three dosage groups of the present invention's medicine high, middle and low have similar anti-candida albicans fungal vaginitis activity in the body: with the prolongation of administration time , the number of colonies showed an obvious downward trend, and the condition gradually improved. Analysis of the changes in the number of colonies in each dosage group before and after administration, compared with before administration, the number of colonies in the high-dose group decreased significantly on the 3rd, 5th and 7th day after administration (P<0.05); in the middle-dose group The number of colonies decreased significantly on the 3d, 5d and 7d after administration (P<0.01); the number of colonies in the low-dose group decreased significantly on the 3d, 5d and 7d after administration (P<0.05). Compared with the 3rd day after administration, the number of colonies in the high-dose group decreased significantly after 7 days of administration (P<0.05).

The above experimental results show that the herb fruit oil of the present invention can significantly kill Candida albicans in vivo.

Table 4: The effect of grass fruit oil of the present invention on the recovery time of vaginitis model mice

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<0.01. Compared with the positive group, ^▲ P<0.05, ^▲▲ P<0.01. Compared with Jieeryin, ^☆☆ P<0.01. Compared with high dose, ^★ P＜0.05, ^★★ P＜0.01.

It can be seen from Table 4:

When taking medicine for 14 days, the average healing time of the grass fruit oil high, medium and low dose groups of the present invention to the vaginitis model mice within 14 days were 6.67d, 10.10d and 10.70d respectively, and the cure rates were 100.00%, 80.00% and 10.00% respectively. 70.00%. Compared with the model group (the average healing time is 14.30d, and the curing rate is 10.00%), the healing time of the three dosage groups of grass fruit oil of the present invention is significantly reduced (P<0.01), and the curing rate is significantly improved; Compared with azole (average healing time 5.90d), the healing time (average healing time 6.67d) of grass fruit oil high dose group of the present invention had no significant statistical significance (P>0.05). Compared with the positive medicine Jieeryin (the average healing time is 11.30 days), the healing time of the high-dose grass fruit oil group of the present invention is significantly reduced (P<0.01), and there is no significant difference in the healing time of the grass fruit oil medium and low dose groups of the present invention. According to comparative analysis within each dosage group of Caoguo of the present invention, compared with the high dosage, the healing time of the middle dosage is significantly prolonged (P<0.05); the healing time of the low dosage is extremely significantly prolonged (P<0.01), showing an obvious dose-effect relationship.

When taking medicine for 20 days, the average healing time of the grass fruit oil high, medium and low dose groups of the present invention to the vaginitis model mice within 20 days were 6.67d, 10.30d and 12.00d respectively, and the cure rates were 100.00%, 100.00% and 100.00% respectively. 90.00%. Compared with the model group (the average healing time is 18.90d, and the curing rate is 30.00%), the healing time of the three dosage groups of grass fruit oil of the present invention is significantly reduced (P<0.01), and the curing rate is significantly improved. Compared with the positive drug clotrimazole, the healing time of the grass fruit oil high-dose group of the present invention has no significant statistical significance (P>0.05). Compared with the positive medicine Jieeryin, the healing time of the grass fruit oil high dose group of the present invention was significantly reduced (P<0.01), and the healing time of the grass fruit oil medium and low dose groups of the present invention had no significant difference. According to the comparative analysis within each dosage group of Tsaoguo of the present invention, compared with the high dosage, the healing time of the middle dosage has no obvious statistical significance, and the healing time of the low dosage is significantly prolonged (P<0.05), showing an obvious dose-effect relationship.

The experimental results show that the grass fruit oil of the present invention has the effect of treating mycotic vaginitis caused by Candida albicans. When administered in high doses, the curative effect is equivalent to that of the positive drug clotrimazole, and significantly better than the positive traditional Chinese medicine Jieeryin.

In summary, grass fruit oil can inhibit the growth of Candida albicans, and can be used to treat diseases caused by Candida albicans infection, especially for the treatment of mycotic vaginitis caused by Candida albicans. The development and application of drugs for diseases has a good market prospect.

Claims (11)

1. Fructus Tsaoko oil has the purposes in the medicine that suppresses or kill the Candida albicans effect in preparation.

2. purposes according to claim 1 is characterized in that: said medicine is the medicine of treatment candidiasis.

3. purposes according to claim 2 is characterized in that: said medicine is treatment dermatocandidiasis, candidiasis of the mucous membranes, internal organs candidiasis or the oidiomycotic medicine of nervus centralis.

4. according to claim 2 or 3 described purposes, it is characterized in that: said medicine is the medicine of treatment colpitis mycotica.

5. according to any described purposes of claim 1 ~ 4, it is characterized in that: described Fructus Tsaoko oil derives from the volatile oil of the mature fruit extraction of Zingiberaceae Amomum plant Fructus Tsaoko Amomum tsa-ko Crevost et Lemaire.

6. according to any described purposes of claim 1 ~ 4; It is characterized in that: described Fructus Tsaoko oil prepares through following method: get Fructus Tsaoko; Be ground into coarse powder, add the distilled water of 10 ~ 14 times of weight, soak after 1 ~ 5 hour; Adopt steam distillation to extract 2 ~ 6 hours, promptly get Fructus Tsaoko oil.

7. one kind has the pharmaceutical composition that suppresses or kill the Candida albicans effect, it is characterized in that: it is to be active component with Fructus Tsaoko oil, adds the medicament that acceptable accessories or complementary composition are prepared from.

8. pharmaceutical composition according to claim 7 is characterized in that: described Fructus Tsaoko oil prepares through following method: get Fructus Tsaoko, be ground into coarse powder; The distilled water that adds 10 ~ 14 times of weight; Soak after 1 ~ 5 hour, adopt steam distillation to extract 2 ~ 6 hours, promptly get Fructus Tsaoko oil.

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

10. pharmaceutical composition according to claim 9 is characterized in that: said external preparation is lotion, liniment, gargarism or liniment.

11. pharmaceutical composition according to claim 7 is characterized in that: the content that contains Fructus Tsaoko oil in the said preparation is 0.1%～100%w/w.