CN112891405A

CN112891405A - Anti-candida traditional Chinese medicine composition and preparation method and application thereof

Info

Publication number: CN112891405A
Application number: CN202110291660.XA
Authority: CN
Inventors: 刘清泉; 岳慧珍; 徐霄龙; 郭玉红; 赵京霞; 崔旭然
Original assignee: Beijing Hospital of Traditional Chinese Medicine Affiliated to Capital University of Medicine Sciences
Current assignee: Beijing Hospital of Traditional Chinese Medicine Affiliated to Capital University of Medicine Sciences
Priority date: 2021-03-18
Filing date: 2021-03-18
Publication date: 2021-06-04

Abstract

The invention discloses an anti-candida traditional Chinese medicine composition as well as a preparation method and application thereof, belonging to the technical field of medicines. The traditional Chinese medicine composition is prepared from the following raw materials in parts by weight: 5-15 parts of rheum officinale, 10-20 parts of liquorice, 5-15 parts of cinnamon and 10-20 parts of agastache rugosus, and the prepared traditional Chinese medicine preparation can effectively kill candida albicans and has a certain antibacterial effect on non-candida albicans and fluconazole resistant bacteria. Thus, the Chinese medicinal composition can be used for treating diseases caused by anti-candida infection, such as skin infection, thrush, urogenital and gastrointestinal candida infection, candidemia and the like.

Description

Anti-candida traditional Chinese medicine composition and preparation method and application thereof

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to an anti-candida traditional Chinese medicine composition and a preparation method and application thereof.

Background

In recent years, with the development and application of new medical technologies, such as organ transplantation and cancer treatment, the use of indwelling catheters, and the abuse of antibiotics, there has been an increase in the population with immunodeficient conditions, as well as an increase in the incidence and prevalence of pathogenic fungal infections. Among the clinical pathogenic fungal infections, candida is the most common and most major opportunistic pathogenic fungus. The candida can coexist in the skin, the oral cavity, the gastrointestinal tract, the genitourinary tract and other parts of a human body for a long time without causing any diseases; when the immune competence of the human host is reduced, candida rapidly proliferate as a dominant flora and cause common epidermal or mucosal infections, as well as severe systemic invasive infections. Candida epidermidis or mucosae infection has low fatality rate, but has high morbidity and seriously affects the life quality of patients. Research data show that 50-70% of women have at least one candida vaginitis in their lifetime with 5-8% recurrent vaginal candida infections. However, candidemia, caused by candidemia infection, is the most common invasive candida infection, with mortality rates as high as 30% -50% in ICU patients, and becomes the second most common cause of nosocomial infectious death. Studies have shown that blood infections caused by candida in the united states are as high as 22%, and candidemia mortality is as high as 46% -75% in some developed countries and regions.

Clinically, fungal infectious diseases are often accompanied by long-term hospitalization, high medical expenses and high mortality, so that the fungal infectious diseases become important factors threatening human health, and meanwhile, the fungal infectious diseases attract extensive attention of clinicians and scientific researchers. Clinically, antifungal drugs mainly include azole drugs, amphotericin B and echinocandin drugs. In the treatment process of critically ill patients, surgical patients and cancer chemotherapy patients, azole drugs are often applied to the preventive treatment of fungal infection on a large scale, and cause the frequent emergence of drug-resistant strains of candida albicans, thereby bringing greater challenges to the treatment of diseases.

Disclosure of Invention

In order to solve the technical problems, one of the purposes of the invention is to provide an anti-candida traditional Chinese medicine composition, which is composed of the following raw materials in parts by weight: 5-15 parts of rheum officinale, 10-20 parts of liquorice, 5-15 parts of cinnamon and 10-20 parts of agastache rugosus.

Further, the traditional Chinese medicine composition is prepared from the following raw materials in parts by weight: 10 parts of rhubarb, 15 parts of liquorice, 10 parts of cinnamon and 15 parts of wrinkled gianthyssop herb.

The invention also aims to provide a traditional Chinese medicine preparation with anti-candida function, and the main active component of the traditional Chinese medicine preparation is the extract of the traditional Chinese medicine composition.

Further, the dosage form of the pharmaceutical preparation is any one of the common oral dosage forms.

Furthermore, the oral administration dosage form is one of capsules, tablets and granules.

The invention also aims to provide a preparation method of the traditional Chinese medicine preparation, which is characterized in that the preparation method of the capsule comprises the following steps:

(1) preparing an extract of the Chinese medicinal composition;

(2) adding common medicinal adjuvants, and making into capsule by conventional method;

the preparation method of the tablet comprises the following steps:

(1) preparing an extract of the Chinese medicinal composition;

(2) adding common pharmaceutical adjuvants for tablet, and making into tablet according to conventional production method;

the preparation method of the granules comprises the following steps:

(1) preparing an extract of the Chinese medicinal composition;

(2) adding common pharmaceutical adjuvants, and making into granule by conventional method.

The fourth purpose of the invention is to provide the application of the traditional Chinese medicine composition in preparing anti-candida medicaments.

The fifth purpose of the invention is to provide the application of the traditional Chinese medicine preparation in preparing anti-candida medicaments.

Compared with the prior art, the invention has the following beneficial effects:

the antibacterial traditional Chinese medicine composition can be applied to preparation of medicines for diseases caused by candida, such as skin infection, thrush, urogenital and gastrointestinal candida infection, candidemia and other diseases. The antibacterial traditional Chinese medicine composition can also be used as an active ingredient to be added into medicines for treating corresponding diseases.

Drawings

FIG. 1 is the MIC values of the herbal cinnamomum japonicum perfume decoction and the herbal cinnamomum japonicum extract to Candida albicans SC5314 strain of example 2.

FIG. 2 is a graph showing the in vitro killing results of the herbal eau de toilette decoction on Candida albicans SC5314 in example 3.

FIG. 3 is a graph showing the in vitro killing results of the cinnamomum japonicum extracts of example 3 against Candida albicans SC 5314.

FIG. 4 is a graph showing the effect of the Pistacia lentiscus extract on the morphological transformation of Candida albicans SC5314 strain in example 4.

FIG. 5 is a graph showing the effect of cinnamomum japonicum extracts on the biofilm formation of Candida albicans SC5314 of example 5.

FIGS. 6a-6c are graphs showing the results of the stable antibacterial activity of the Pimenta racemosa extract to Candida albicans SC5314 of example 6.

FIGS. 7a-7b are graphs showing the results of the antibacterial effect of the herbal and cinnamomum cassia compound of example 7 on non-candida albicans and fluconazole resistant bacteria.

FIG. 8 is a graph showing the result of PI staining after Candida albicans induction by the herbal and cinnamomum cassia presl compound of example 8.

FIG. 9 is a graph showing the results of example 9, wherein the composition of F.lutescens induces Candida albicans to produce ROS.

Detailed Description

Example 1

Weighing 200g of liquorice, rhubarb, cinnamon and agastache rugosus according to the mass ratio of 15:10:10:15, and adding ddH with the volume being 6 times that of the liquorice, the rhubarb, the cinnamon and the agastache rugosus₂Soaking for 30 minutes in O; boiling, decocting with slow fire for 30 min, and filtering to obtain medicinal liquid; add a further 4 volumes of ddH₂Continuously decocting for 20 minutes, and filtering out liquid medicine; mixing the twice decocted liquid medicines, and concentrating to obtain 1g crude drug/ml compound water decoction of grass yellow osmanthus fragrans. Freeze drying the above compound decoction to obtain the final productAn extract of compound Huanggui Xiang.

Example 2

In this example, the minimum inhibitory concentration of the herbal and cinnamomum japonicum perfume decoction and the herbal and cinnamomum japonicum extract prepared in example 1 on candida albicans SC5314 was determined according to the CLSI-M27-A3 protocol, and the specific steps were as follows: respectively dissolving the decoction and the extract of the grass yellow osmanthus perfume in RPMI1640 culture solution to prepare 51.2 percent (volume fraction) decoction of the grass yellow osmanthus perfume and 80mg/ml extract of the grass yellow osmanthus perfume; adding 200ul of the liquid medicine into a first hole of a 96-hole plate, adding 100ul of the liquid medicine from the first hole into a second hole, diluting by a 2-time gradient method for 9 times in sequence, and setting the last hole as an no-medicine group control; adding 100ul Candida albicans suspension (about 500 Candida albicans cells) into corresponding pore plate, and mixing; after culturing at 37 ℃ for 24 hours, interpretation was carried out, and amphotericin B was used as a control, and the results are shown in FIG. 1. As can be seen from FIG. 1, MIC values of the Osmanthus fragrans perfume decoction (CHGX) and the Osmanthus fragrans extract (CHGXE) against Candida albicans SC5314 strain were 12.8% and 20mg/ml, respectively.

Example 3

In this example, the in vitro killing effect of the herbal osmanthus fragrans perfume decoction and the herbal osmanthus fragrans extract prepared in example 1 on candida albicans SC5314 is detected, and the specific steps are as follows:

selecting a single clone of candida albicans SC5314 to be cultured in a 3ml YPD liquid culture medium test tube at 30 ℃ by shaking overnight; 1ml of the strain solution was transferred into a 20ml YPD liquid medium Erlenmeyer flask and the shake culture was continued until the logarithmic phase (OD 600. about.1.2). Collecting thallus, washing with double distilled water for 2 times, suspending in Lee's glucose culture medium, and performing gradient dilution counting to obtain 2 × 10 thallus⁵cells/ml bacterial suspension; adding herbal osmanthus perfume decoction (final volume fraction of 0%, 5%, 10% and 15%) and herbal osmanthus extract (final concentration of 0mg/ml, 10mg/ml, 20mg/ml and 30mg/ml), wherein 32ug/ml Fluconazole (FLC) is drug control group. Continuing shake culture at 30 ℃, and setting three repeats in each group; respectively sucking 100ul of mixed liquor at different time points, and coating the mixed liquor on a YPD plate after gradient dilution; after 2 days of culture at 37 ℃, the number of plate colonies was counted and the cell viability was calculated.

As shown in FIGS. 2 and 3, it can be seen from FIGS. 2 and 3 that 10% and 15% of the herbal cinnamon perfume decoctions have significant killing effect on Candida albicans, 20mg/ml and 30mg/ml of the herbal cinnamon extract also have significant killing effect on Candida albicans, and 10mg/ml of the herbal cinnamon extract can not effectively kill Candida albicans, but can effectively inhibit the growth of Candida albicans. In addition, the killing effect of the herbal and cinnamomum cassia twig perfume decoction and the extract thereof on candida albicans shows concentration dependence.

Example 4

In this example, the effect of the saffron extract prepared in example 1 on the morphological transformation of candida albicans SC5314 was examined, which specifically includes the following steps:

activating candida albicans SC5314 yeast cells, collecting thalli, washing the thalli for 2 times by double distilled water, suspending the thalli in a Lee's glucose culture medium, diluting and counting; adding the bacterial suspension into a culture medium of Lee's glucose + 10% serum to a final concentration of 2 × 10⁶cells/ml; adding the extracts of grass yellow and cassia twig with final concentrations of 0mg/ml, 10mg/ml and 20mg/ml respectively, setting three times for each group, and performing shake culture at 37 ℃ for 24 hours; sucking 100ul of mixed bacteria liquid at different time points, enriching the bacteria, photographing to observe the cell morphology, and counting the proportion of mycelium cells, wherein the result is shown in figure 4.

Yeast-hyphal morphology switching plays an important role in Candida albicans pathogenic processes. The yeast state is favorable for the candida albicans to adhere to the surface of host tissue cells and hypha growth; the hypha state is favorable for the candida albicans to colonize and penetrate through host tissue cells to enter blood and be converted into yeast cells; the yeast cells diffuse and spread the candida albicans in each part of the body along with blood circulation; when the host's immunity is reduced, invasive candida infections are produced in different parts of the human body. In this embodiment, the antibacterial mechanism of the compound herbal and cinnamomum cassia is further explained by observing the influence of the compound herbal and cinnamomum cassia on the development of candida albicans hyphae. Serum is a key factor for inducing the growth of candida albicans hyphae. As can be seen from FIG. 4, in Lee's glucose medium containing 10% serum, Candida albicans yeast cells were rapidly transformed into hyphal cells on a large scale (within 3 hours); under the same culture conditions, the addition of 10mg/ml and 20mg/ml of the grass yellow cinnamon extract can effectively inhibit yeast-strain conversion, and the hypha inhibition phenomenon shows concentration dependence. Therefore, the compound of the grass yellow osmanthus fragrans has the effect of inhibiting the growth of candida albicans hyphae, and the potential pharmacodynamic effect of the compound in the treatment of candida albicans infection is suggested.

Example 5

This example examined the effect of the cinnamomum japonicum thunb extract prepared in example 1 on the biofilm formation of candida albicans SC5314, and the specific steps were as follows:

activating Candida albicans SC5314 yeast cells, collecting thallus, washing with double distilled water for 2 times, re-suspending in Spider culture medium, diluting, counting, and configuring to 1 × 10⁶cells/ml bacterial suspension; mu.l of the bacterial suspension was added to a 96-well plate containing 100. mu.l of the grass yellow osmanthus fragrans extract with a gradient concentration (20mg/ml-0.078mg/ml), after being beaten and mixed well, the mixture was left to stand at 37 ℃ for 3 days, and then biofilm formation was observed, and the results are shown in FIG. 5.

The candida albicans biofilm can effectively prevent antibacterial drugs and harmful substances from entering, maintain the stability of internal nutrient substances and escape the immune killing effect of host cells. The biofilm formation creates favorable conditions for the propagation and infection of candida albicans, and simultaneously brings great challenges for clinically preventing and treating candida infection. As can be seen from FIG. 5, 2.5mg/ml HGRHE has a certain inhibitory effect on the formation of a Candida albicans biofilm, and the Candida albicans cannot form a complete biofilm under the action of HGRHE of more than 5 mg/ml.

Example 6

In this example, whether the cinnamomum japonicum thunb extract prepared in example 1 has stable antibacterial activity on candida albicans SC5314 is determined, and the specific steps are as follows:

activating Candida albicans yeast or hypha cells, collecting thallus, washing with double distilled water for 2 times, suspending in YPD or Lee's glucose culture medium, gradient diluting and counting to 2 × 10⁵cells/ml bacterial suspension; adding grass yellow osmanthus fragrans extracts with different concentrations (the final concentration is 10mg/ml and 20mg/ml), and continuously performing shake culture at 30 ℃ or 37 ℃, wherein each group is provided with three repeats; respectively sucking 100ul of mixed solution at different time points, and coating after gradient dilutionSpread on YPD plates; after 2 days of culture at 37 ℃, the number of plate colonies was counted and the cell viability was calculated. The temperature and nutrition are closely related to the growth and reproduction of candida albicans. Among them, 37 ℃ is the optimum growth temperature of Candida albicans, and YPD medium is a typical nutrient-rich medium. As shown in FIGS. 6a to 6c, it can be seen from FIGS. 6a to 6c that 20mg/ml and 30mg/ml of the Pimenta racemosa extract have significant killing activity against Candida albicans at 37 ℃ or YPD; hypha development is an important characteristic of candida albicans virulence expression, and 20mg/ml and 30mg/ml of the chamaemelo basilica extract can effectively kill candida albicans hypha type cells. The results show that the compound of the grass yellow cinnamon has stable activity of resisting the candida albicans.

Example 7

In this example, the antibacterial effect of the herbal and cinnamomum cassia presl compound prepared in example 1 on non-candida albicans and fluconazole resistant bacteria is detected, and the specific steps are as follows:

activating cells of different strains, collecting thallus, washing with double distilled water for 2 times, suspending in Lee's glucose liquid culture medium, diluting and counting in gradient, and configuring to 2 × 10⁵cells/ml bacterial suspension; adding the extract of grass yellow osmanthus fragrans with the final concentration of 20mg/ml, setting three times for each group, and continuously performing shake culture for 4 hours at the temperature of 30 ℃; respectively sucking 100ul of mixed solution dilution at different time points, and coating the mixed solution dilution on a YPD plate after gradient dilution; after 2 days of culture at 37 ℃, the number of plate colonies was counted and the cell viability was calculated.

As shown in FIG. 7a, it is known that Candida albicans P37005, Candida dublin d172, Candida glabrata JX1002 and Candida auriculata BJCA001 all showed significant cell death phenomenon after the 20mg/ml extract of Pistacia strumaria was treated for 4 hours; therefore, the compound of the grass yellow and the cassia twig has broad-spectrum bactericidal action on candida. We found that about 70% of the cells in resistant strains BMW00716 and 17# were killed after 4 hours treatment of fluconazole resistant candida albicans strain with 20mg/ml of cinnamomum cassia presl extract (fig. 7 b). Although the 20mg/ml chamaemelo cayratia extract has no remarkable killing effect on the drug-resistant bacteria CAR (G5), the growth of cells of the chamaemelo cayratia extract can be effectively inhibited. The results show that the herbal and cinnamomum cassia presl compound also has certain antibacterial and bactericidal activity on fluconazole resistant bacteria.

Example 8

In this example, the mechanism of inducing cell death of candida albicans by the herbal, yellow and osmanthus fragrans compound prepared in example 1 is studied, and the specific steps are as follows:

activating Candida albicans SC5314, collecting thallus, washing with double distilled water for 2 times, resuspending in Lee's glucose culture medium, diluting, counting, and preparing 2 × 10⁶cells/ml bacterial suspension; adding the extract of the grass yellow osmanthus fragrans prescription with the final concentration of 20mg/ml, performing shake culture at 30 ℃ for 3 hours, collecting thalli, and washing with PBS for three times; the cells were resuspended in PBS, PI was added to a final concentration of 50. mu.g/ml, mixed well, and subjected to light-shielding treatment at 4 ℃ for 30 minutes, followed by observation under a fluorescence microscope. Amphotericin B treated groups were set as controls. The structural integrity of the cell membrane is critical to maintaining the growth and reproduction of fungal cells. Clinically, commonly used azole and polyene medicaments mainly influence the integrity of the cell membrane structure of candida albicans and promote the overflow of substances in cells, thereby causing cell death and achieving the antibacterial and bactericidal effects. In order to further and deeply research the mechanism of the compound of the grass-yellow cinnamon for resisting the candida albicans, the influence of the compound of the grass-yellow cinnamon on the cell membrane of the candida albicans is detected by PI staining and flow cytometry. The results showed that about 68% of the cells produced PI fluorescence signals after 3 hours of treatment of Candida albicans with 20mg/ml Pimenta fragrans extract at 30 ℃ under Lee's glucose, as shown in FIG. 8. Amphotericin B (2ug/ml) as a positive drug control, produced PI fluorescent signal in about 32% of cells 1 hour after treatment of Candida albicans; in contrast, in the negative control group without drug treatment, only 3.6% of the cells produced PI fluorescence signals. In addition, we observed further by fluorescence microscopy, which is consistent with the flow assay results. The results show that the destruction of the integrity of the cell membrane of the candida albicans is an important mechanism for the compound herbal and cinnamomum cassia to play an antibacterial role.

Example 9

In this example, whether the compound herbal and cinnamomum cassia prepared in example 1 has the effect of inducing candida albicans to generate ROS is determined, and the specific steps are as follows:

activating Candida albicans SC5314, collecting thallus, washing with double distilled water for 2 times, and resuspendingDiluting and counting in Lee's glucose medium, and configuring 2x10⁶cells/ml bacterial suspension; adding the extract of the grass yellow osmanthus fragrans prescription with the final concentration of 20mg/ml, performing shake culture at 30 ℃ for 3 hours, collecting thalli, and washing with PBS for three times; suspending the thallus in PBS, adding DCFDA needle control, mixing uniformly, and processing at 37 ℃ for 30 minutes in a dark place; PBS was washed once and observed under a fluorescent microscope.

The production and accumulation of intracellular ROS can cause severe oxidative damage and cytotoxicity, leading to cell death, which is also a killing mechanism for antimicrobial peptides. To verify whether the herbal-huang-gui-xiang compound affects the production of ROS in candida albicans cells, we examined the change in ROS content in cells treated with herbal-huang-gui-xiang extract. As shown in FIG. 9, after 3 hours of Candida albicans treatment with 20mg/ml of the Osmanthus fragrans champ extract, a fluorescence signal was detected in about 52% of the cells; while the negative control group without drug treatment did not detect a significant fluorescence signal. Also, the fluorescence microscope observation result was consistent with the flow detection result. The results show that the killing effect of the epimedium compound on the candida albicans is related to the generation of intracellular ROS.

The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.

Claims

1. The anti-candida traditional Chinese medicine composition is characterized by comprising the following raw materials in parts by weight: 5-15 parts of rheum officinale, 10-20 parts of liquorice, 5-15 parts of cinnamon and 10-20 parts of agastache rugosus.

2. The traditional Chinese medicine composition according to claim 1, which is prepared from the following raw materials in parts by weight: 10 parts of rhubarb, 15 parts of liquorice, 10 parts of cinnamon and 15 parts of wrinkled gianthyssop herb.

3. A traditional Chinese medicine preparation with anti-candida, which is characterized in that the main active ingredient of the traditional Chinese medicine preparation is the extract of the traditional Chinese medicine composition of claim 1 or 2.

4. The Chinese medicinal preparation according to claim 3, wherein the dosage form of the pharmaceutical preparation is any one of the conventional oral dosage forms.

5. The Chinese medicinal preparation according to claim 4, wherein the oral dosage form is one of capsule, tablet and granule.

6. The method for preparing a Chinese medicinal preparation according to claim 5, wherein the capsule is prepared by the steps of:

(1) preparing an extract of the Chinese medicinal composition;

the preparation method of the tablet comprises the following steps:

(1) preparing an extract of the Chinese medicinal composition;

the preparation method of the granules comprises the following steps:

(1) preparing an extract of the Chinese medicinal composition;

7. Use of the Chinese medicinal composition of claim 1 or 2 for the preparation of an anti-candida medicament.

8. Use of a Chinese medicinal preparation according to any one of claims 3 to 5 for the manufacture of an anti-candida medicament.

9. The use of claim 7, wherein the medicament is a Chinese medicinal formulation of any one of claims 3-5.