CN104857116A - Applications of traditional Chinese medicine sanguisorba officinalis in preparing antifungal drug synergist - Google Patents

Abstract

The invention relates to the technical field of medicine, in particular to the application of traditional Chinese medicine Burnet eucalyptus in the preparation of synergists for antifungal drugs. The present invention has been confirmed by in vitro experiments that Burnet has synergistic effects on conventional antifungal drugs such as fluconazole, miconazole, caspofungin, and amphotericin B. For drug-resistant fungi, when antifungal drugs are used in combination with Burnet , can significantly reduce the drug dosage of conventional antifungal drugs, can improve the inhibitory effect of commonly used antifungal drugs on fungi to varying degrees, and can restore the effect of antifungal drugs on drug-resistant fungi, and enhance the inhibitory effect on drug-resistant fungi. The invention opens up a new application for the traditional Chinese medicine Sanguis Burnet, and can reduce the dosage of antifungal drugs under the condition that the drug resistance of clinical fungi is becoming more and more common and the degree of drug resistance is becoming more and more serious, which saves medical expenses for patients and reduces the cost of drugs. toxic side effects.

Description

Application of traditional Chinese medicine Burnet in the preparation of antifungal drug synergists

technical field

The invention relates to the technical field of medicine, in particular to the application of traditional Chinese medicine Burnet eucalyptus in the preparation of synergists for antifungal drugs. the

Background technique

Burnet is the dry root of Sanguisorba officinalis L or Sanguisorba officinalis L.var.longifolia (Bert.) Yüet Li, a plant of the family Rosaceae. It is produced all over the country, mainly in Zhejiang, Anhui, Shandong, Hunan, Hubei and other places. Burnet eucalyptus is slightly cold in nature, bitter, astringent, and sour. It returns to the liver, stomach, and large intestine meridian. Treatment of diseases such as swollen sores (see: Qing Sun Xingyan, Sun Xingyi Series. Shennong's Herbal Classic [M]. Beijing: Commercial Press, 1955.94.). the

Burnet also has antibacterial and anti-inflammatory effects, but it is mainly concentrated on Staphylococcus aureus, Pseudomonas aeruginosa and other bacteria (see literature: Wu Kaiyun, Huang Xuefang, Peng Xuanxian. Experimental research on the antibacterial effects of borneol, Polygonum cuspidatum, and Burnet [J ]. Jiangxi Medical College Journal, l996,36(2):53-55.). Burnet is mainly used raw or fried charcoal. Raw Burnet is unprocessed Burnet decoction pieces. It is often ground and used externally to treat water and fire burns, eczema, skin ulcers and other diseases. It has the effect of astringent and moisture absorption. Burnet charcoal is a decoction piece of Burnet that is prepared by frying charcoal. It has a strong hemostatic effect, and its curative effect is better than that of raw Burnet. The raw Burnet crude extract was extracted with petroleum ether, ethyl acetate, n-butanol, and water to detect its antifungal activity, and it was found that the antibacterial activity of the ethyl acetate extract was the best. the

At present, there is no report about the antifungal effects of Burnet, nor is there any report about it as a synergistic antifungal synergistic drug. the

Contents of the invention

The purpose of the present invention is to provide a new medical application of traditional Chinese medicine Sangui. the

The invention provides a new application of the traditional Chinese medicine Burnet eucalyptus as an antifungal drug synergist, that is, the application of Burnet eucalyptus in the preparation of an antifungal drug synergist. the

The traditional Chinese medicine Burnet (burnet) of the present invention includes two different medicinal materials of Burnet—raw Burnet and Burnet charcoal. It also includes the active ingredient of Burnet extracted by ethyl acetate extraction and other methods. the

The antifungal drugs described in the present invention are conventional antifungal drugs, including but not limited to fluconazole, caspofungin, terbinafine, amphotericin B and the like. the

The present invention has been confirmed by in vitro experiments that Burnet has synergistic effects on conventional antifungal drugs such as fluconazole, miconazole, caspofungin, and amphotericin B. For drug-resistant fungi, when antifungal drugs are used in combination with Burnet , can significantly reduce the drug dosage of conventional antifungal drugs, can improve the inhibitory effect of commonly used antifungal drugs on fungi to varying degrees, and can restore the effect of antifungal drugs on drug-resistant fungi, and enhance the inhibitory effect on drug-resistant fungi. the

The invention opens up a new application for the traditional Chinese medicine Burnet elata, and using it as a synergist of antifungal drugs can not only improve the antifungal effect of the drug, but also become more common and more serious in clinical fungal drug resistance. Under the circumstances, the antifungal drugs can restore the effect on drug-resistant fungi, which can reduce the dosage of antifungal drugs, save medical expenses for patients, and reduce the toxic and side effects of drugs. the

The traditional Chinese medicine Burnet eucalyptus of the present invention is a traditional medicine in my country and has the advantages of abundant resources, low price and less side effects, and the combined use of Burnet eucalyptus and antifungal drugs can also reduce the toxic and side effects of drugs and save the medical expenses of patients. the

Detailed ways

Now, the present invention will be described in detail in conjunction with the embodiments, but the implementation of the present invention is not limited thereto. the

Embodiment 1: The effect of raw Burnet and antifungal drugs combined with 7 strains of Candida

1. Materials and methods

1. Reagents

Raw Burnet: Department of Traditional Chinese Medicine, Shanghai Leiyunshang Pharmaceutical Co., Ltd.

Burnet Charcoal: Department of Traditional Chinese Medicine, Shanghai Leiyunshang Pharmaceutical Co., Ltd.

Fluconazole injection: Pfizer Pharmaceutical Co., Ltd.

Anhydrous ethanol: Shanghai Sinopharm Group Co., Ltd.

Petroleum ether: Shanghai Sinopharm Group Co., Ltd.

Ethyl acetate: Shanghai Sinopharm Group Co., Ltd.

Butanol: Shanghai Sinopharm Group Co., Ltd.

Preparation of traditional Chinese medicine extract: Take 5g of raw Burnet and Burnet charcoal Chinese medicine powder respectively, add 50ml of 70% ethanol solution, ultrasonicate at 50KHz for 40 minutes at room temperature, repeat ultrasonication twice after cooling, centrifuge at 3000×g for 5min, and take The supernatant, the crude drug concentration of which is 0.1g/mL, is stored in a 4°C refrigerator for later use. the

Preparation of the ethyl acetate extract of Raw Burnet: Dry 40ml of the preserved traditional Chinese medicinal material, add water to suspend, add 1 to 1.5 times the volume of the extract (petroleum ether, ethyl acetate, n-butanol) respectively, and immediately Shake and mix well, then put the separatory funnel aside, and after the liquid is layered, take the extract from the outlet at the lower end, and repeat the extraction for each extract until the extract is colorless or the color becomes lighter. The extracts were combined, evaporated to dryness, and a small amount was prepared into a 5.12 mg/mL solution with DMSO. the

2. Strains

Strains: Candida albicans 103 (abbreviated as 103), Candida albicans SC5314 (abbreviated as sc5314), Candida albicans 100 (abbreviated as 100), Candida krusei ATCC2159 (abbreviated as Kerou ATCC2159), Candida tropicalis ATCC087 (abbreviated as tropical ATCC087) , Candida glabrata ATCC28226 (abbreviated as Smooth ATCC28226), and Candida parapsilosis 22090 (abbreviated as Jinping 22090), all provided by the Fungi Department of Shanghai Changhai Hospital, collected from clinical samples from different departments of Changhai Hospital, and identified by morphology and biochemistry , and can also be purchased from the Institute of Cells, Chinese Academy of Sciences or the ATCC depository, etc. the

All experimental strains were activated on sandcastle dextrose agar medium (SDA). After Candida albicans was cultured at 30°C for 2 weeks, single clones were picked and activated again, and the single clones obtained for the second time were placed on the SDA slope. , cultured by the above method and stored at 4°C for future use. the

3. Culture medium

Sandcastle dextrose agar solid medium (SDA):

Add 10g of peptone, 40g of glucose, and 18g of agar, add 900ml of triple-distilled water to dissolve, add 50ml of 2mg/ml chloramphenicol aqueous solution, adjust the pH to 7.0, dilute to 1000ml with triple-distilled water, and sterilize under high pressure (121°C for 15min). Store at 4°C for later use. the

YEPD medium:

Yeast extract 10g, peptone 20g, glucose 20g, add 900ml of triple distilled water to dissolve, add 50ml of 2mg/m chloramphenicol aqueous solution, dilute to 1000ml with triple distilled water, autoclave (121°C, 15min) and store at 4°C spare. the

RPMI1640 liquid culture medium:

RPMI1640 (Gibco BRL) 10g, NaHCO ₃ 2.0g, morpholine propanesulfonic acid (MOPS) (Sigma) 34.5g (0.165M), add three distilled water 900ml to dissolve, 1N NaOH to adjust the pH to 7.0 (25°C), three distilled Dilute the water to 1000ml, filter and sterilize with a 0.22μm microporous membrane, and store it at 4°C after aliquoting.

4. Instruments and equipment

MDF1-100 Portable High Speed Traditional Chinese Medicine Grinder (Shanghai Minde Machinery Manufacturing Co., Ltd.) 

DF-101S collector type constant temperature heating magnetic stirrer (Gongyi Yingyu Maohua Instrument Factory)

SHZ-D(Ⅲ) Circulating Water Vacuum Pump (Gongyi Yingyu Maohua Instrument Factory) 

RE-52C rotary evaporator (Shanghai Qingpu Huxi Instrument Factory)

KQ-C Airflow Dryer for Glass Instruments (Gongyi Yingyu Maohua Instrument Factory)

Multiskan MK3 enzyme label detector (product of Finnish Labsystems)

Full temperature shaking incubator (Peiying, HZQ-F160)

MJX Intelligent Mold Incubator (Ningbo Jiangnan Instrument Factory) 

SW-CT-IF single-person double-sided purification workbench (Suzhou Purification Co., Ltd.)

Biological Microscope (Chongqing Optical Instrument Factory XSZ-G)

5. Preparation of bacterial solution

Before the experiment, Candida was picked from the SDA medium stored at 4°C, inoculated into 1ml of YEPD medium, and cultured at 30°C with shaking at 200rpm. After two activations, the fungus was in the late exponential growth phase. Take 10ul of the bacteria solution into 1ml YEPD culture solution, and reactivate it by the above method. After 16 hours, count with a hemocytometer, and adjust the concentration of the bacteria solution to 1×10 ³ -5×10 ³ cells/ml with RPMI1640 culture solution.

6. Preparation of DSR plate

Take a sterile 96-well plate, and add 100 μl of RPMI1640 liquid medium to No. 1 well of each row as a blank control; add 100 μl of freshly prepared bacterial solution to each of No. 3 to No. 12 wells; add 198 μl of bacterial solution and test compound solution to No. 2 wells respectively 2 μl; No. 12 well does not contain drugs, only 100 μl of bacterial solution is added as a positive growth control. Doubling dilutions were carried out in wells 2 to 11, so that the final drug concentrations in each well were 1000, 500, 250, 125, 62.5, 31.25, 15.625, 7.8125, 3.906 and 1.953 μg/ml, and the ethanol content in each well was lower than 1%. A drug-sensitivity plate of quality control bacteria was prepared each time the drug-sensitivity plate was prepared, and each drug-sensitivity plate was cultured in a 30°C incubator. the

7. Determination of minimum inhibitory concentration (MIC value)

Candida albicans was cultured in an incubator at 30°C for 48 hours, and the OD value of each well was measured at 630 nm with an enzyme label analyzer. Compared with the positive control wells, the drug concentration in the lowest concentration well whose OD value drops by more than 80% is MIC ₈₀ (drug concentration when 80% of fungal growth is inhibited).

When the MIC ₈₀ value of the drug exceeds the concentration range of the determination, the following methods are used for statistics: when the MIC ₈₀ value is higher than the highest concentration of 1000 μg/ml, it is counted as “>1000 μg/ml”; the MIC ₈₀ value of fluconazole is higher than the highest concentration of 64 μg /ml, it is counted as ">64μg/ml"; when the drug MIC ₈₀ value is the lowest concentration or below the lowest concentration, it is counted as "≤1.953μg/ml" without distinction; the fluconazole MIC ₈₀ value is the lowest concentration Or when it is below the minimum concentration, no distinction shall be made, and it shall be counted as "≤0.125μg/ml". The above experiments were performed 2 to 3 times in parallel. When the MIC ₈₀ value can be accurately repeated or only differs by one concentration, it is accepted, and a higher concentration is used as the MIC ₈₀ value; when the MIC ₈₀ value differs by more than two concentrations, it is required to Experiment again until you meet the requirements.

2. The experimental results are shown in Table 1. the

Table 1 The antifungal effect of the traditional Chinese medicine Burnet eucalyptus extract combined with fluconazole on 7 strains of Candida (crude drug concentration)

It can be seen from Table 1 that raw Burnet can produce synergistic effects on Candida albicans SC5314, 100, and 103, and the combination of Burnet and fluconazole can significantly reduce the drug concentration of the two drugs, allowing drug-resistant bacteria 100, 103 to re-affect Sensitive to fluconazole. It also exhibits antifungal effects on non-albicans Candida krusei ATCC2159, Jinping 22090 and other bacteria. This experiment shows that the traditional Chinese medicine Burnet has a synergistic antifungal effect. the

Embodiment 2: Raw Burnet, the effect of Burnet charcoal and antifungal drugs on Candida albicans 103, SC5314

1. Materials and methods

1. Drugs

Fluconazole Injection Pfizer Pharmaceutical Co., Ltd.

Caspofungin Acetate Merck sharp&Dohme (I.A.) CORP Singapore

Terbinafine Hydrochloride Xi'an Kaihong Biotechnology Co., Ltd.

Amphotericin B Pfizer Pharmaceuticals LLC

Dimethyl sulfoxide Shanghai Sinopharm Group Co., Ltd.

Caspofungin acetate, terbinafine, and amphotericin B were dissolved in dimethyl sulfoxide to form a 6.4 mg/ml solution, and each reagent was stored at -20°C. Before the experiment, the drug was taken out and placed in a 35°C incubator to melt, mixed well, and pharmacodynamic tests were carried out respectively. the

Other experimental steps and method are with embodiment 1

2. The experimental results are shown in Table 2, Table 3, Table 4, and Table 5. the

Table 2 The effect of Burnet charcoal and antifungal drugs on drug-resistant Candida albicans 103 (crude drug concentration)

Table 3 The effect of Burnet charcoal and antifungal drugs on Candida albicans SC5314 (crude drug concentration)

Table 4 The effect of some drugs extracted from raw Burnet ethyl acetate and antifungal drugs on Candida albicans 103

The concentration of the extract is displayed, and the concentration of the crude drug is in brackets

Table 5 The effect of some drugs extracted from raw Burnet urnica ethyl acetate and antifungal drugs on Candida albicans SC5314

The concentration of the extract is displayed, and the concentration of the crude drug is in brackets

It can be seen from Table 2, Table 3, Table 4, and Table 5 that after combining the extracts of raw Burnet and Burnet charcoal with fluconazole, it was found that both raw Burnet and Burnet charcoal It can be used in combination with antifungal fluconazole to reduce the drug concentration of fluconazole, making fluconazole re-sensitized to drug-resistant Candida albicans100,103, and it is compatible with commonly used antifungal agents such as caspofungin, terbinafine, and amphotericin B. All fungal drugs can produce synergistic effects. Except for caspofungin, which has a relatively poor synergistic antifungal effect, most other drugs can significantly reduce the concentration of antifungal drugs. the

The above experiments all prove that the traditional Chinese medicine Burnet has a synergistic antifungal effect and can reduce the dosage of antifungal drugs, so it can be used as a synergist for antifungal drugs. the

The preferred embodiments of the present invention have been specifically described above, but the present invention is not limited to the described embodiments, and those skilled in the art can also make various equivalents without violating the spirit of the present invention. Modifications or replacements, these equivalent modifications or replacements are all included within the scope defined by the claims of the present application. the

Claims (4)

1. Radix Sanguisorbae is preparing the application in antifungal medicine synergist.

2. Radix Sanguisorbae according to claim 1 is preparing the application in antifungal medicine synergist, it is characterized in that, described Radix Sanguisorbae is Radix Sangusorbae or charred Radix Sanguisorbae.

3. Radix Sanguisorbae according to claim 1 is preparing the application in antifungal medicine synergist, it is characterized in that, described Radix Sanguisorbae is the active ingredient that Radix Sanguisorbae is extracted by extraction into ethyl acetate method.

4. the Radix Sanguisorbae according to claim 1,2 or 3, preparing the application in antifungal medicine synergist, is characterized in that, described antifungal drug is fluconazol, Caspofungin, terbinafine, or amphotericin B.