CN113082150B

CN113082150B - Application of effective part of deer medicine in preparing antifungal medicine and synergist thereof

Info

Publication number: CN113082150B
Application number: CN202110485227.XA
Authority: CN
Inventors: 刘伟; 赵然然; 张梦莎; 罗凯英; 王胜正
Original assignee: Shaanxi University of Science and Technology
Current assignee: Shaanxi University of Science and Technology
Priority date: 2021-04-30
Filing date: 2021-04-30
Publication date: 2022-10-04
Anticipated expiration: 2041-04-30
Also published as: CN113082150A

Abstract

The invention provides application of effective parts of a deer drug in preparing antifungal drugs and synergists thereof, in-vitro cell experiments show that after the deer drug is combined with amphotericin B or anidulafungin, the sensitivity of drug-resistant candida albicans 103, sensitive candida albicans SC5314, cryptococcus neoformans 32609 and drug-resistant candida tropicalis to amphotericin B or anidulafungin can be obviously reduced, so that the compound can be used as an antifungal drug synergist to open up new application for the deer drug.

Description

Application of effective part of deer medicine in preparing antifungal medicine and synergist thereof

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to application of effective parts of a deer medicine in preparation of antifungal medicines and synergists thereof.

Background

The deer medicine belongs to one of Qinling mountain seven medicines, belongs to Liliaceae deer medicine, and belongs to dried root and rhizome of perennial herb deer medicine (English is called Smilacina japonica A.Gray). The deer herbs are warm in nature, sweet and bitter in flavor, and enter kidney and liver meridians. The deer medicine has the effects of tonifying kidney and strengthening yang, activating blood circulation to dissipate blood stasis, and dispelling wind and relieving pain, belongs to yang-tonifying medicines classified under deficiency-tonifying medicines, and can be used for treating diseases such as kidney deficiency and impotence, irregular menstruation, migraine, headache, rheumatic arthralgia, carbuncle swelling and sore, traumatic injury and the like.

According to previous reports, the deer medicine has antifungal and even fungicidal activity, but no report of the deer medicine for preparing antifungal medicine synergist exists at present.

Disclosure of Invention

Aiming at the problems in the prior art, the invention provides the application of the effective part of the deer medicine in preparing antifungal medicines and synergists thereof.

The invention is realized by the following technical scheme:

the application of the effective part of the deer medicine in the antifungal medicine synergist.

Preferably, the root and rhizome of the deer medicine are selected for extraction to obtain the effective part of the deer medicine.

Further, the effective part of the deer medicine is obtained by the following steps:

pulverizing root and rhizome of deer medicine into powder, and mixing the powder according to the weight ratio of 1g:20mL of material-liquid ratio, removing residues in the reflux liquid after condensation reflux extraction by using ethanol to obtain a primary extracted liquid medicine, dissolving the primary extracted liquid medicine by using ultrapure water after concentration and drying, extracting for 3-5 times by using n-butyl alcohol with the same volume as the ultrapure water, collecting an extract liquid to obtain an extracted liquid medicine, and concentrating and drying the extracted liquid medicine to obtain the deer medicine effective part.

Preferably, the antifungal drug comprises amphotericin B and anidulafungin.

Preferably, the fungi include sensitive and resistant fungal strains.

Further, the sensitive fungus strains comprise sensitive candida albicans SC5314 and cryptococcus neoformans 32609.

Further, the drug-resistant fungal strains include drug-resistant Candida albicans 103 and drug-resistant Candida tropicalis.

A preparation method of antifungal medicine comprises mixing deer effective component with medicinal adjuvants, and making into tablet, capsule or injection.

Further, the pharmaceutical excipients comprise one or more of a stabilizer, a solubilizer, a lubricant and a disintegrant.

An antifungal medicine obtained by the preparation method of the antifungal medicine.

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

the application of the effective part of the deer drug in the antifungal drug synergist shows that after the deer drug is used with amphotericin B or anidulafungin, the sensibility of drug-resistant candida albicans 103, sensitive candida albicans SC5314, cryptococcus neoformans 32609 and drug-resistant candida tropicalis to amphotericin B or anidulafungin can be obviously improved, so that the deer drug can be used as the antifungal drug synergist, a new application is developed for the deer drug, the application of the deer drug in preparing the antifungal drug synergist not only provides a new treatment strategy for treating fungal infection, but also improves the antifungal effect of the existing drug, and under the condition that the clinical fungal drug resistance is increasingly common and the drug resistance degree is increasingly serious, the antifungal drug can recover the effect on drug-resistant fungi, and the dosage of the antifungal drug is reduced, so that the toxic and side effects and the medical cost of the drug are reduced.

The preparation method of the antifungal drug provided by the invention is characterized in that the effective part of the deer drug is mixed with pharmaceutic adjuvant, and the mixture can be prepared into tablets, capsules or injections, so that the application is convenient, the antifungal effect of the drug is improved, and the application range of the deer drug is further widened.

Drawings

FIG. 1 is a graph showing the susceptibility of Candida albicans to various antifungal drugs after the action of the deer drug with different concentrations according to the present invention.

Detailed Description

The present invention will now be described in further detail with reference to specific examples, which are intended to be illustrative, but not limiting, of the invention.

1. The combination of the effective part of the deer drug n-butanol and the anidulafungin or amphotericin B has the effect on different fungi

1. Reagent

Deer medicine: purchased from dell Kang Yiyao linkage (fourth linkage of sophora bud).

Anidulafungin: MCE, lot 011155125.

Amphotericin B: acros corporation, lot number P1434922.

Dimethylsulfoxide (DMSO): tianjin, nature chemical reagents, inc.

The reagents are stored at-20 ℃. Before the experiment, all the medicines are taken out and melted, fully and uniformly mixed, and pharmacodynamic tests are respectively carried out.

2. Bacterial strains

A strain for use comprising:

1) Sensitive fungal strains: sensitive candida albicans (SC 5314) and cryptococcus neoformans (32609);

2) Drug-resistant fungal strains: drug-resistant Candida albicans (103) and drug-resistant Candida tropicalis.

The above strains are all provided by fungus houses in Shanghai Changhai Hospital and are identified by morphology and biochemistry.

All strains for experiments are subjected to paddle activation in a sandcastle glucose agar (SDA) culture medium, and after candida albicans, candida tropicalis, cryptococcus neoformans and other cocci are cultured for 2-3 days at 35 ℃, monoclones are respectively picked and subjected to paddle activation again, and the monoclones obtained in the second time are taken and placed on an SDA inclined plane, and are cultured by the method and then stored at 4 ℃ for later use.

3. Culture medium/media

1) RPMI 1640 liquid culture solution

RPMI1640(Gibco BRL)10g，NaHCO ₃ 2.0g and 34.5g (0.165 mol) of morpholine propanesulfonic acid (Sigma), adding 900mL of triple distilled water for dissolution, adjusting the pH to 7.0 by 1mol/L NaOH, fixing the volume of the triple distilled water to 1000mL, filtering and sterilizing by a 0.22 mu m microporous membrane, subpackaging and storing at 4 ℃ for later use.

2) Sambo glucose agar solid medium (SDA)

10g of peptone, 40g of glucose and 18g of agar, adding 900mL of triple distilled water for dissolving, adjusting the pH to 7.0, using the triple distilled water for constant volume to 1000mL, sterilizing under high pressure (121 ℃,20 min), and storing at 4 ℃ for later use.

3) YEPD culture solution

Dissolving yeast extract 10g, peptone 20g and glucose 20g in 900mL of triple distilled water, diluting to 1000mL of constant volume, sterilizing under high pressure (121 ℃,20 min), and storing at 4 ℃ for later use.

4. Instrumentation apparatus

GHP-9080 type water-proof constant temperature incubator (Shanghai-Hengscientific instruments Co., ltd.);

THZ-98C type constant temperature shaking box (Shanghai-Hengchun scientific instruments Co., ltd.);

SW-CT-IF model superclean bench (Suzhou Antai air technologies, inc.).

5. Deer medicine n-butanol effective part, amphotericin B and anidulafungin stock solution

Crushing the purchased dry roots and rhizomes of the deer medicine into fine powder, weighing 5g, carrying out condensation reflux extraction for 2 hours by using ethanol with 20 times volume (namely 100 mL) and 75% volume fraction, cooling, filtering to remove residues, carrying out rotary evaporation on the liquid medicine, concentrating to dryness, dissolving in 100mL of purified water, carrying out two-phase repeated extraction for 3-5 times by using n-butyl alcohol liquid with the same volume, collecting n-butyl alcohol extract phase liquid medicine, concentrating and drying to obtain the n-butyl alcohol effective part containing the deer medicine with high activity. Taking 15mL DMSO to dissolve completely, preparing a mother solution with a concentration of 0.3333g/mL (calculated by 5g fine powder and 15mL DMSO), subpackaging, and storing at-20 ℃ for later use.

Commercially available anidulafungin and amphotericin B were dissolved in DMSO to prepare solutions having concentrations of 1mg/mL and 4mg/mL, respectively, and stored at-20 ℃ for further use.

6. Preparation of bacterial liquid

Before the experiment, a small amount of each globular fungus is picked from an SDA culture medium stored at 4 ℃ by using an inoculation ring, and is respectively inoculated into 1mL of YEPD culture solution, and is subjected to shaking culture at the speed of 200rpm at the temperature of 35 ℃ for 16h, so that the fungus is in the later period of exponential growth. Then adding each bacterial liquid to 1mL YEPD culture solution, activating again by the above method, culturing for 16h, counting by using a blood cell counting plate, and adjusting each bacterial liquid concentration to 1 × 10 with RPMI 1640 culture solution ³ ～5×10 ³ CFU/mL。

7. Preparing a drug sensitive plate:

taking each strain separately one sterile 96-well plate, and adding 100 μ L of RPMI 1640 liquid culture medium into each row of No. 1 wells as blank control; adding 100 mu L of the freshly prepared bacterial liquid into each of the No. 3 to No. 12 holes; no. 2 Kong Jiajun liquid 198 μ L; no. 12 wells contained no drug, and 100. Mu.L of inoculum was added as a positive growth control. mu.L of each of the deer drug stock solution with a concentration of 0.3333g/mL, the amphotericin B solution with a concentration of 4mg/mL or the anidulafungin solution with a concentration of 1mg/mL was added to well No. 2 of each row so that the deer drug concentrations in the wells of rows 2 to 6 were 208.1. Mu.g/mL, 104.1. Mu.g/mL, 52.0. Mu.g/mL, 26.0. Mu.g/mL and 13.0. Mu.g/mL, respectively. Then, the wells 2-11 were diluted 10-fold to achieve final synergistic deer drug concentrations of 208.1. Mu.g/mL, 104.1. Mu.g/mL, 52.0. Mu.g/mL, 26.0. Mu.g/mL, and 13.0. Mu.g/mL, anidulafungin drug concentrations of 1. Mu.g/mL, 0.5. Mu.g/mL, 0.25. Mu.g/mL, 0.125. Mu.g/mL, 0.0625. Mu.g/mL, 0.03125. Mu.g/mL, 0.0156. Mu.g/mL, 0.0078. Mu.g/mL, 0.0039. Mu.g/mL, and 0.002. Mu.g/mL, amphotericin B drug concentrations of 4. Mu.g/mL, 2. Mu.g/mL, 1. Mu.g/mL, 0.5. Mu.g/mL, 0.25. Mu.g/mL, 0.125. Mu.0075. Mu.g/mL, 0.06256. Mu.25. Mu.g/mL, 0318. Mu.g/mL, and 0.0078. Mu.25. Mu.g/mL, respectively. The DMSO content in each well was below 1%. The drug sensitive plates were cultured in a thermostat at 35 ℃.

8. And (3) determining an MIC value:

culturing sensitive candida albicans (SC 5314) for 24 hours and cryptococcus neoformans (32609) for 72 hours in a 35 ℃ incubator, observing the experimental result, and determining the MIC value of the experimental result. After the observation is finished, the culture medium is placed back into the constant temperature incubator for continuous culture. When the MIC value of the drug exceeds the measured concentration range, counting is carried out according to the following method: MIC value was > 4. Mu.g/mL when higher than the highest concentration of 4. Mu.g/mL "; when the MIC value is the lowest concentration or below, the MIC value is calculated to be less than or equal to 0.0078 mu g/mL without distinction. The experiments are all operated for 3 times in parallel, the MIC value is accepted when the MIC value can be accurately repeated or only differs by one concentration, and the higher concentration is taken as the MIC value; when the MIC values differ by more than two concentrations, re-experiments are required until the requirements are met.

Evaluation of the effect of drug combination: the main parameter for evaluating the interaction mode of two drugs for combined administration is the FIC Index for Combination (FICI). The bacteriostatic concentration Fraction (FIC) is the ratio of the minimum bacteriostatic concentration (MIC) required when each drug is combined for bacteriostasis to the MIC when the drug is used singly, and the FIC index (FICI) is the sum of FICs of the two drugs. When the FICI is less than or equal to 0.5, the interaction is a synergistic effect, and the smaller the FICI is, the stronger the synergistic effect is; the additive effect is when FICI is more than 0.5 and less than or equal to 1; when FICI is more than 1 and less than or equal to 4, the effect is irrelevant; when FICI is greater than 4, the two drugs are antagonistic.

The experimental results are shown in tables 1 and 2 below:

TABLE 1 MIC value (μ g/ml) of effective fraction of N-butanol of deer drug in combination with anidulafungin against fungi

As can be seen from Table 1, the combination of the effective part of the deer drug n-butanol and the antifungal drug anidulafungin has antibacterial and synergistic effects. The MIC values of anidulafungin for sensitive candida albicans SC5314 and drug-resistant candida albicans 103 when used alone are 0.125 mu g/mL and 0.0625 mu g/mL respectively, and the MIC values of the effective parts of the deer drug n-butanol are 416 mu g/mL respectively. After the two medicines are used together, the MIC values of anidulafungin are respectively reduced to 0.03125 mu g/mL and less than or equal to 0.0078 mu g/mL, the MIC values of the deer medicine n-butanol effective parts are reduced to 52 mu g/mL and 26 mu g/mL, similarly, the MIC value of the anidulafungin to drug-resistant candida tropicalis is 1 mu g/mL when the anidulafungin is used alone, and the MIC value of the deer medicine n-butanol effective part is 1560 mu g/mL. After the two medicines are used together, the MIC value of anidulafungin is reduced to 0.125 mu g/mL, and the effective part of the deer medicine n-butanol is reduced to 52 mu g/mL. Shows the synergistic effect of the effective part of the deer drug n-butanol on anidulafungin.

TABLE 2 MIC values (μ g/ml) of the effective fraction of N-butanol of deer drug in combination with amphotericin B against fungi

As can be seen from Table 2, the combination of the effective part of the deer drug n-butanol and the antifungal drug amphotericin B has antibacterial and synergistic effects. When the amphotericin B is singly used, the MIC values of sensitive Candida albicans SC5314, drug-resistant Candida albicans 103 and cryptococcus neoformans are all 0.25 mu g/mL, and the MIC values of the effective parts of the deer drug n-butanol are all 416 mu g/mL. After the two medicines are used together, the MIC values of amphotericin B are respectively reduced to 0.0625 mu g/mL, 0.125 mu g/mL and 0.0313 mu g/mL, the effective part of the deer medicine n-butyl alcohol is reduced to 52 mu g/mL, and similarly, the MIC value of the amphotericin B for drug-resistant candida tropicalis is 0.5 mu g/mL when the amphotericin B is used alone, and the MIC value of the effective part of the deer medicine n-butyl alcohol is 1560 mu g/mL. After the two medicines are used together, the MIC value of amphotericin B is reduced to 0.125 mu g/mL, and the effective part of the deer medicine n-butanol is reduced to 52 mu g/mL. Shows the synergistic effect of the effective part of the deer medicine n-butanol on amphotericin B.

2. The sensitivity of the sensitive candida albicans acted by the deer drugs with different concentrations to different antifungal drugs.

The drugs, strains and experimental materials used were as described above.

The SC5314 strain is inoculated into 1mL YEPD culture solution, and is subjected to shaking culture at 200rpm at 35 ℃ for 16h, so that the fungus is in the later exponential phase of growth. Diluting and adjusting the concentration of the bacterial liquid to 1 × 10 ⁵ ～5×10 ⁵ CFU/mL, adding deer medicine mother liquor with different concentrations respectively, and grouping the medicines as follows: performing reaction for 1 hr on blank group (containing no medicine), deer medicine (208 μ g/mL), deer medicine (416 μ g/mL), and deer medicine (832 μ g/mL), collecting bacterial solution, washing with PBS buffer solution for three times, and diluting to 1 × 10 ⁷ CFU/mL、2×10 ⁶ CFU/mL、4×10 ⁵ CFU/mL、8×10 ⁴ CFU/mL、1.6×10 ⁴ CFU/mL, 3. Mu.L of each of the YPD plates (i.e., normal YPD plate, YPD plate containing 0.025. Mu.g/mL anidulafungin, and YPD plate containing 0.25. Mu.g/mL amphotericin B) was spotted, incubated for 48 hours, and photographed.

As shown in FIG. 1, it can be seen from FIG. 1 that after 48h of culture, on normal YPD plates, the susceptible Candida albicans treated with different concentrations of deer drug (208. Mu.g/mL, 416. Mu.g/mL, 832. Mu.g/mL) had viable clones, but on YPD plates containing anidulafungin 0.025. Mu.g/mL and amphotericin B0.25. Mu.g/mL, the blank group still had viable clones, and there were few viable clones of deer drug-treated bacteria, further showing the potentiating effect of deer drug on anidulafungin and amphotericin B.

The effective part of the deer medicine can be further prepared into antifungal medicines, and specifically, the effective part of the deer medicine is mixed with pharmaceutic adjuvants, and can be prepared into tablets, capsules or injections according to the existing method, and the pharmaceutic adjuvants can be one or more of a stabilizer, a solubilizer, a lubricant and a disintegrant, so that the application is convenient, the antifungal effect of the medicines is improved, and the application range of the deer medicine is further widened.

In conclusion, in vitro cell experiments show that after the effective part of the deer-medicine n-butanol is combined with the anidulafungin or the amphotericin B, the sensitivity of drug-resistant candida albicans 103, sensitive candida albicans SC5314, cryptococcus neoformans (32609) and drug-resistant candida tropicalis to the two medicines can be obviously enhanced, so that the deer-medicine can be used as a synergist of antifungal medicines. The invention opens up a new application for the deer medicine, and the deer medicine is used as the synergist of the antifungal medicine, thereby not only providing a new candidate treatment strategy for the fungal treatment, but also improving the antifungal effect of the existing medicine, restoring the effect of the antifungal medicine on the drug-resistant fungi under the conditions that the clinical fungal drug resistance is increasingly common and the drug-resistant degree is increasingly serious, and reducing the dosage of the antifungal medicine, thereby saving the medical cost for patients and reducing the toxic and side effect of the medicine.

Claims

1. The application of the effective part of the deer medicine in the preparation of the antifungal medicine synergist is characterized in that the root and rhizome of the deer medicine are selected for extraction to obtain the effective part of the deer medicine;

the deer medicine effective part is obtained by the following steps:

pulverizing root and rhizome of deer drug into powder, mixing the powder according to a weight ratio of 1g:20mL of material-liquid ratio, removing residues in reflux liquid after condensation reflux extraction by using 75% ethanol in volume fraction to obtain preliminarily extracted liquid medicine, concentrating and drying the preliminarily extracted liquid medicine, dissolving the preliminarily extracted liquid medicine by using ultrapure water, extracting 3~5 times by using n-butyl alcohol with the same volume as the ultrapure water, collecting extract liquid to obtain extracted liquid medicine, concentrating and drying the extracted liquid medicine to obtain the deer medicine effective part;

the fungus is sensitive fungus strain or drug-resistant fungus strain, the sensitive fungus strain is sensitive candida albicans SC5314, and the drug-resistant fungus strain is drug-resistant candida albicans 103 or drug-resistant candida tropicalis;

the antifungal medicine is amphotericin B or anidulafungin.