CN111494447B

CN111494447B - Drug-resistant fungus resistant pharmaceutical composition and preparation thereof

Info

Publication number: CN111494447B
Application number: CN202010577399.5A
Authority: CN
Inventors: 王睿睿; 其他发明人请求不公开姓名
Original assignee: Yunnan University of Traditional Chinese Medicine TCM
Current assignee: Yunnan University of Traditional Chinese Medicine TCM
Priority date: 2020-06-23
Filing date: 2020-06-23
Publication date: 2022-05-27
Anticipated expiration: 2040-06-23
Also published as: CN111494447A

Abstract

The invention belongs to the technical field of biological medicines, and discloses a pharmaceutical composition with drug-resistant fungi, which consists of a panax notoginseng n-butyl alcohol extract and an antifungal drug. Cleaning radix Panacis Quinquefolii, sun drying, pulverizing, extracting with n-butanol, and distilling under reduced pressure to obtain radix Panacis Quinquefolii n-butanol extract. The n-butyl alcohol extract of the panax notoginseng can obviously reduce the efflux function of an efflux pump of a fungal cell, so that the expression level of drug-resistant related genes is obviously reduced, and the mitochondrial membrane potential level in the fungal cell is reduced; the pharmaceutical composition consisting of the n-butyl alcohol extract of the panax stipuleanatus and the antifungal drug has good inhibition effect on drug-resistant fungi.

Description

Drug-resistant fungus resistant pharmaceutical composition and preparation thereof

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to a drug-resistant fungus pharmaceutical composition consisting of a panax stipuleanatus n-butanol extract and an antifungal drug, and a preparation thereof.

Background

Candida albicans (C., (C.))Candida albicans) Is an opportunistic pathogenic fungus existing on human skin, mucous membrane and the like, and generally does not cause diseases in normal organisms. In recent years, the incidence of systemic candida albicans infection has rapidly increased, with an infection rate of 46.36%, due to the extensive use of chemoradiotherapy in cancer patients, the use of a large amount of immunosuppressive agents in organ transplant patients, and the large-scale use of broad-spectrum antibiotics.

At present, few medicines for clinically resisting the deep infection of candida albicans are available, and amphotericin B, miconazole, fluconazole, voriconazole, itraconazole and the like are mainly available. Among them, fluconazole is widely used for clinical antifungal infection diseases due to less side effects and better bioavailability. Although fluconazole is a preferential drug for treating candida albicans infection, fluconazole is only a bacteriostatic agent for most fungi and has no direct killing effect, and the bacteriostatic but non-bactericidal effect provides a powerful condition for the survived strains to generate drug resistance. In the long-term treatment process, the repeated administration aggravates the drug resistance of the candida albicans fluconazole, most fungi which have drug resistance to the fluconazole also have cross drug resistance to other antifungal drugs, and the current stage treatment of fungal infectious diseases by the antifungal drugs falls into a bottleneck in the face of the occurrence of a large number of drug-resistant strains.

Radix Notoginseng (semen Sinapis Albae)Panax stipuleanatus H.T. Tsai et K.M. Feng) is a plant of Panax of Araliaceae, also called XIANGCI and YESANG, which is national sumThe second-level wild protection material produced in southeast of Yunnan province, Notoginseng radix (Notoginseng radix)Panax notoginseng(Burk) F.H. Chen) is a closely related species, but the main component of the (Burk) F.H. Chen) is different from that of pseudo-ginseng, and the main component of the (Burk) F.H. Chen) is oleanane type pentacyclic triterpene saponin, but the pharmacological activity of the (Burk) F.H. Chen) is not much researched at present, and the (Burk) F.H. Chen) F.Chen) is mainly anti-inflammatory and anticancer activity. In the research process, the n-butyl alcohol extract of panax notoginseng has the synergistic effect of fluconazole in resisting drug-resistant candida albicans, and has the prospect of preparing drugs for resisting drug-resistant fungi.

Disclosure of Invention

The invention aims to provide a pharmaceutical composition with drug-resistant fungal action, which consists of an n-butyl alcohol extract of panax notoginseng and an antifungal drug.

The purpose of the invention is realized as follows:

the n-butyl alcohol extract of the panax stipuleanatus in the pharmaceutical composition is obtained by the following steps:

(1) cleaning radix Stephaniae Sinicae, air drying, and pulverizing into coarse powder;

(2) adding ethanol into the coarse powder for refluxing for 2 times, wherein the dosage of the ethanol is 5-15 times of the mass volume (kg/L) of the pseudo-ginseng screening coarse powder each time, and the dosage of the ethanol is 1-3 hours each time, filtering after refluxing, merging filtrate, and concentrating the extracting solution under reduced pressure until no ethanol smell exists to obtain pseudo-ginseng screening ethanol extract;

(3) suspending ethanol extract of radix Panacis Quinquefolii with water, sequentially extracting with petroleum ether and n-butanol for 3-6 times; mixing n-butanol extractive solutions, and concentrating under reduced pressure until no alcohol smell exists to obtain n-butanol extract of radix Schefflerae Arboricolae.

The mass concentration of the ethanol in the step (2) is 80-90%; the dosage of the suspension water in the step (3) is 1-3 times of the ethanol extract of the panax stipuleanatus, and the dosages of the oil ether and the n-butanol are 1/6-1/2 of the volume (L) of the water phase in each extraction.

The antifungal agent is azole antifungal agent.

The antifungal drug is fluconazole, itraconazole or fluconazole.

The mass ratio of the n-butyl alcohol extract of the panax stipuleanatus to the antifungal medicine is 2: 1.

The drug-resistant fungi is clinical fluconazole drug-resistant strains of candida albicans.

The n-butyl alcohol extract of the panax stipuleanatus remarkably reduces the function of an efflux pump of the drug-resistant fungi.

The n-butyl alcohol extract of the panax notoginseng can obviously reduce the expression levels of three main efflux pump genes of CDR1, CDR2 and MDR1 of the drug-resistant fungi.

The pharmaceutical composition is added with pharmaceutically acceptable auxiliary materials to prepare tablets, hard capsules, soft capsules, powder, pills and granules.

The invention has the advantages that:

the pharmaceutical composition provided by the invention has good drug-resistant fungus resistance.

Drawings

FIG. 1 is a time-growth graph;

FIG. 2 is a graph showing the survival and weight changes of mice infected with systemically resistant Candida albicans;

wherein, A is survival curve of the mouse, B is survival rate of the mouse, C is weight change of the mouse; blank-blank group, model-model group, FLC-fluconazole-only group, Panax-Panax notoginseng n-butanol extract-only group, FLC + Panax low-Panax notoginseng n-butanol extract-combination fluconazole-low-dose group, FLC + Panax high-Panax notoginseng n-butanol extract-combination fluconazole-high-dose group;

FIG. 3 is a graph of the dirty coefficients;

wherein, A is spleen organ coefficient, B is thymus organ coefficient; blank-blank group, model-model group, FLC-fluconazole single-use group, Panax-Panax stipuleanus n-butanol extract single-use group, FLC + Panax low-Panax stipuleanus n-butanol extract combined with fluconazole low-dose group, FLC + Panax high-Panax stipuleanus n-butanol extract combined with fluconazole high-dose group;

FIG. 4 is a graph showing the distribution of the number of colonies in the organ;

wherein, the A-lung colony number and the B-kidney colony number; blank-blank group, model-model group, FLC-fluconazole single-use group, Panax-Panax stipuleanus n-butanol extract single-use group, FLC + Panax low-Panax stipuleanus n-butanol extract combined with fluconazole low-dose group, FLC + Panax high-Panax stipuleanus n-butanol extract combined with fluconazole high-dose group;

FIG. 5 is a graph of pathological section HE staining of lung tissue;

wherein, the group a comprises a blank group, a model group, a single-use group of c-fluconazole, a single-use group of d-n-butanol extract of panax notoginseng, a low-dose group of e-n-butanol extract of panax notoginseng combined with fluconazole, and a high-dose group of f-n-butanol extract of panax notoginseng combined with fluconazole;

FIG. 6 is a graph of staining of kidney histopathology sections HE;

FIG. 7 is a graph of liver histopathology section HE staining;

wherein, the group a is a blank group, the group b is a model group, the group c is a single-use group of fluconazole, the group d is a single-use group of n-butyl alcohol extract of panax notoginseng, the group e is a low-dose group of n-butyl alcohol extract of panax notoginseng combined with fluconazole, and the group f is a high-dose group of n-butyl alcohol extract of panax notoginseng combined with fluconazole.

FIG. 8 is a graph of HE staining of rectal histopathology sections;

FIG. 9 is a graph of the pump-out fluorescence intensity of Candida albicans efflux pump;

wherein the fluorescence intensity of the pump output by the A-non-energy-dependent efflux pump is changed, and the fluorescence intensity of the pump output by the B-energy-dependent efflux pump is changed; control-growth control, Pannx-panax stipuleanus n-butanol extract treated group;

FIG. 10 is a graph of efflux pump-associated gene expression levels;

wherein, control-growth control, Pannx-setsusan n-butanol extract treated group;

FIG. 11 is a graph of mitochondrial membrane potential levels within fungi;

of these, control-growth control, Pannx-panax stipuleanus n-butanol extract treated group.

Detailed Description

The present invention is further illustrated but not limited in any way by the following examples, and any modifications made thereto are intended to fall within the scope of the present invention.

A pharmaceutical composition with antifungal effect comprises n-butanol extract of radix Panacis Quinquefolii and antifungal agent.

The antifungal agent is azole antifungal agent.

The antifungal drug is fluconazole, itraconazole or fluconazole.

The n-butanol extract of the panax notoginseng can obviously reduce the expression levels of three main efflux pump genes of CDR1, CDR2 and MDR1= of the drug-resistant fungi.

Example 1

Cleaning radix Stephaniae Sinicae, air drying, and pulverizing into coarse powder. 1100g of coarse powder is taken, and 95% ethanol is added for reflux for 2 times, 2 hours for the 1 st time and 1.5 hours for the 2 nd time, and the dosage of the 95% ethanol for each time is 22L. Filtering after refluxing, merging the filtrate, and obtaining 342.8g of extract after reduced pressure distillation. Dissolving the extract with 1L water, extracting with petroleum ether and n-butanol for 4 times, wherein the solvent consumption is 0.3L each time; and (3) mixing the n-butyl alcohol extract, and distilling under reduced pressure to obtain an n-butyl alcohol part extract: 151.90g, namely the n-butyl alcohol extract of the radix cynanchi hancei.

Example 2

(1) Cleaning radix Stephaniae Sinicae, air drying, and pulverizing into coarse powder.

Test example:

the n-butanol extracts of Panax notoginseng (F.B.H.) F.H. Chen in examples 3 to 5 of the experimental examples were obtained from example 1. The drugs and reagents were respectively:

fluconazole dispersible tablet (batch No. 181001) purchased from Nanchang Hongyao pharmaceutical Co Ltd; cyclophosphamide (lot X22D8Y 51136), a source leaf organism; 0.5% sodium carboxymethylcellulose (CMC-Na) (batch 130812), Guangfu Density Fine chemical research institute; 4% paraformaldehyde (batch 183608), Wuhan Severer Biotech Ltd; sa's (SDA) agar medium (lot 1076641), Sa's (SDA) liquid medium (lot 1069251), Kyork Kai Microbiol technologies, Inc.; 0.9% sodium chloride injection (batch No. C180707C), Kunming south Jiang pharmaceutical Co., Ltd; dimethyl sulfoxide (DMSO) (batch 67-68-5), Biotoped; rhodamine 6G (lot number C10074770), shanghai maclin; 0.1MPBS buffer (pH 7.4) was purchased from Biosharp; tryptone was purchased from Guangdong Huanji Microbiol technologies, Inc.; the yeast extract is obtained from Kyork, Guangdong, Microbiol technologies, Inc.; glucose was purchased from pioneer pegmatization glass ltd, luoyang; chloroform (analytically pure) was purchased from Yunnan pharmaceutical Co., Ltd; absolute ethanol was purchased from the national pharmaceutical group; isopropyl alcohol was purchased from Tianjin, Feng boat chemical reagent science and technology, Inc.; 75% ethanol was purchased from Kunming, Disinfection products, Inc.; trizol lysate was purchased from Ambion; cDNA reverse transcription kit was purchased from Thermo Scientific; the real-time fluorescent quantitative PCR kit is purchased from Thermo Scientific; 1% DEPC treated water was purchased from Beijing Lei Gen Biotechnology Ltd; 2 × SYBR available from Thermo Scientific; JC-1 fluorescent probe (batch No. J58070), Shanghai assist in san Francisco Biotech, Ltd.

The source and pretreatment of the strain:

clinical candida albicans fluconazole resistant strain 23^#The skin disease professor Liyuye of the first subsidiary hospital of Kunming medical university gives a gift and completes the identification; the Candida albicans standard strain ATCC10231 was a gift from snow experimenter, Kunming plant, Chinese academy of sciences. Before the experiment, the strain frozen at-80 ℃ is placed in a constant temperature and humidity box to be thawed and revived at 37 ℃, after being cultured for 24-48h, a proper amount of bacterial colony is taken to be resuspended in YPD liquid culture medium, and is incubated for 16h by a constant temperature shaking table to reach logarithmic phase for later use.

Experimental animals:

84 inbred ICR mice, 18-22 g in each half of male and female, 6-8 weeks old, purchased from Kunming medical university, cleaning grade (license number: SCXK (Yunnan) K2015-0002).

Statistical detection method

Experimental dataUsing the mean. + -. standard deviation

Showing that the data is processed by adopting Graphpad prism7 software, the staining scan is checked by CaseViewer software, the data is analyzed by adopting an independent sample t test method, and the results take p less than 0.05, p less than 0.01, p less than 0.001 and p less than 0.0001 as reference standards, have statistical significance, and the # is compared with a blank group and is compared with a model group.

Example 3

In vitro drug-resistant Candida albicans resistance assay

1-fold microdilution method

Dissolving 0.1g of n-butanol extract of radix Panacis Quinquefolii in 1mL of dimethyl sulfoxide (DMSO) to obtain 100mg/mL stock solution, and refrigerating at 4 deg.C; grinding a piece of fluconazole dispersible tablet into powder, adding 1mL of DMSO to prepare a stock solution with the concentration of 50mg/mL, and refrigerating at 4 ℃.

Diluting the prepared n-butanol extract stock solution (100 mg/mL) and fluconazole stock solution (50 mg/mL) of radix Selaginellae Doederleinii to 1mg/mL and 0.5mg/mL respectively to obtain initial concentrations, diluting by 5-fold dilution with micro-scale dilution method, and setting 6 concentration gradients in each experiment^[19]Setting a single use group of the n-butyl alcohol extract of the radix cynanchi bungei, a single use group of fluconazole and a combination fluconazole group of the n-butyl alcohol extract of the radix cynanchi bungei, respectively diluting the single use group, the single use group and the combination fluconazole group on a 96-well plate, setting a blank control group (without medicine and bacteria) and a growth control group (without medicine and bacteria), and setting 3 multiple wells in each group. The bacteria regulating concentration is 1 × 10⁵CFU/mL, adding the bacterial liquid into corresponding hole, culturing at 37 deg.C for 24 hr in constant temperature and humidity chamber, measuring absorbance (absorbance) value at 625nm wavelength with microplate reader, and calculating inhibition rate and Minimum Inhibitory Concentration (MIC)₈₀) And a joint index (FICI), using the formula:

the bacteriostasis rate is 1- (experiment group OD-blank group OD)/(control group OD-blank group OD)

MIC₈₀=10^（LOG(A)+(a-80)/(a-b)×LOG(1/5))

Wherein A is the minimum concentration of the gradient concentration corresponding to the bacteriostasis rate of more than 80%, a is the bacteriostasis rate corresponding to A, and b is the bacteriostasis rate only smaller than a.

FICI = (M IC for combination of A drugs/M IC for single A drugs) and (M IC for combination of B drugs/M IC for single B drugs); when no MIC value was used, twice the maximum concentration of the drug was used instead of the MIC value for calculation.

The experimental results are as follows:

the single use of the n-butyl alcohol extract of the panax stipuleanatus and the single use of the fluconazole have no effect on drug-resistant strains, the combination of the n-butyl alcohol extract of the panax stipuleanatus and the fluconazole has obvious bacteriostatic effect, and the average MIC of the n-butyl alcohol extract of the panax stipuleanatus is the combination₈₀85.99 mu g/ml is achieved, the two medicines show synergistic effect (Table 1), and the n-butyl alcohol extract of the panax notoginseng and fluconazole are combined to have synergistic effect on the standard strain (Table 2).

TABLE 1 MIC of Selaginella hindsii in combination with fluconazole for drug resistant strains₈₀And FICI

Note: FICI is less than or equal to 0.5; 0.5-4 independently; > 4 antagonism

TABLE 2 MIC of drug alone₈₀MIC for Standard Strain in combination₈₀And FICI

Note: FICI is less than or equal to 0.5; 0.5-4 independently; > 4 antagonism

2 time-growth Curve plotting

Inoculating the drug-resistant strain in YEPD liquid culture medium, culturing at 30 deg.C and 115rpm in constant temperature shaking table for 16h, and adjusting the bacteria concentration to 1 × 10 with YEPD liquid culture medium in centrifuge tube⁵CFU/mL, adding the medicines into groups, dividing the experiment into blank group (blank), growth control group (control), fluconazole single-use group (FLC), radix Notoginseng n-butanol extract single-use group (Panax) and radix Notoginseng n-butanol extract combined with fluconazoleGroup (FLC + Panax); adding the solutions into a 96-well plate at 0h, 4h, 8h, 12h, 24h and 48h, respectively, wherein each well is 100 μ L, each group is provided with three multiple wells, measuring the absorbance of each well at the wavelength of 625nm, taking out the wells, and putting the centrifuge tubes back into a shaker for continuous culture; the obtained data of each group were plotted.

The drawing results are shown in fig. 1, compared with the model group, the absorbance (OD) of the fluconazole single-use group and the absorbance (OD) of the n-butanol extract of the panax stipuleanatus is not obviously changed, the OD value of the fluconazole combined n-butanol extract of the panax stipuleanatus is obviously reduced, and p is less than 0.0001 at three time points of 24h, 36h and 48 h. Showing that the growth of the combined group of candida albicans was significantly inhibited after 24 hours.

Example 4

In vivo detection of drug resistance of Candida albicans

1 preparation of drugs and grouping of Experimental animals

Preparing appropriate amount of extract with sodium carboxymethylcellulose (0.5% CMC-Na) into 2.5mg/mL and 9mg/mL solutions, and refrigerating at 4 deg.C; grinding fluconazole dispersible tablets into powder, adding 1mL of sodium carboxymethylcellulose (0.5% of CMC-Na) to prepare a storage solution with the concentration of 50mg/mL, refrigerating at 4 ℃, and diluting to 0.1mg/mL when in use; appropriate amount of cyclophosphamide powder is weighed to prepare 10mg/mL solution which is prepared as it is.

84 inbred line ICR mice, half of male and female, weighing and grouping to make the weight of each group evenly distributed, 42 female ICR mice divided into blank group, fungal infection model group, single-use group of fluconazole, single-use group of n-butyl alcohol extract of radix cynanchi glaucescentis, low-dose group of n-butyl alcohol extract of radix cynanchi glaucescentis combined with fluconazole, and high-dose group of n-butyl alcohol extract of radix cynanchi glaucescentis combined with fluconazole; male ICR mice 42 were the same as above.

2 ICR mouse immunodeficiency model establishment

The cyclophosphamide solution (with the concentration of 10mg/mL and the dosage of 100 mg/kg) is injected into the abdominal cavity according to the body weight for three days continuously to cause an ICR mouse immunodeficiency model, and the cyclophosphamide solution is injected once every three days after the success of constructing the mouse systemic drug-resistant fungal infection model, and then the mice are weighed and observed for the state every day.

Establishment of 3 ICR mouse systemic drug-resistant Candida albicans infection model

Adjusting the concentration of the bacterial liquid to 1 × 10 by using 0.9% sodium chloride injection⁶CFU/mL, the bacterial liquid is injected into the tail vein one day after the last cyclophosphamide injection, the dosage is 0.2 mL/body, the bacterial liquid is divided into a blank group (without bacteria injection, 0.9% sodium chloride with the same volume is injected), a fungal infection model group, a fluconazole single-use group, a panax notoginseng n-butyl alcohol extract combined fluconazole low-dose group and a panax notoginseng n-butyl alcohol extract combined fluconazole high-dose group, and an ICR mouse systemic drug-resistant fungal infection model is created.

4 dosing and body weight and survival recording

According to blank group, fungal infection model group and fluconazole single-use group (2 mg/kg)^[20]) The single use group (179.4 mg/kg) of the n-butanol extract of radix cynanchi bungei, the group of low dose (53.82 mg/kg) of the n-butanol extract of radix cynanchi bungei combined with fluconazole (2 mg/kg), and the group of high dose (179.4 mg/kg) of the n-butanol extract of radix cynanchi bungei combined with fluconazole (2 mg/kg) were weighed and subjected to intragastric administration at a weight of 0.2mL/10 g. The concentration is selected according to the formula of clinical administration of panax notoginseng, 3-10 g of panax notoginseng is decocted and taken for an adult, 3g and 10g of panax notoginseng are respectively taken, the extraction rate of the n-butyl alcohol extract of panax notoginseng is 13.8 percent, and the formula is calculated according to the administration amount of a mouse: the dosage per kilogram is multiplied by 9.1 multiplied by 13.8 percent, and the low dose and the high dose of the in vivo experiment administration are calculated. Performing intragastric administration in two steps, wherein in the first step, the blank group, the fungal infection group and the single fluconazole group are subjected to intragastric administration by using 0.5 percent CMC-Na solution, and the single n-butyl alcohol extract of the panax stipuleanatus, the low-dose group and the high-dose group of the fluconazole are subjected to intragastric administration by using prepared n-butyl alcohol extract liquid medicine of the panax stipuleanatus; after one hour interval, the blank group, the fungal infection group and the n-butanol extract of radix cynanchi bungei were gavaged with 0.5% CMC-Na solution alone, and the fluconazole alone group, the n-butanol extract of radix cynanchi bungei in combination with the fluconazole low-dose group and the high-dose group were gavaged with the prepared fluconazole liquid medicine according to the body weight. Once daily for 10 consecutive days, body weights were recorded daily, mice were observed for mortality, survival counts were recorded, and survival rates for each group were calculated and plotted.

The survival condition of the mice infected by the systemic drug-resistant candida albicans is shown in fig. 2-A, B, compared with the blank group, the survival rate of the model group is obviously reduced from 4 days, and the model construction success of the systemic drug-resistant candida albicans infection model is shown. Compared with the model group, the survival rate of the fluconazole single-use group and the survival rate of the n-butanol extract of the panax notoginseng are obviously reduced, the survival curve is obviously reduced, and the difference between the survival rate and the survival curve is almost zero; the survival rates of the n-butanol extract of the panax stipuleanatus and the fluconazole combined low-dose group (p is less than 0.05) and the high-dose group (p is less than 0.01) are obviously higher than those of the single-use groups, wherein the survival rate of the combined low-dose group is as high as 78.5 percent, the survival rate of the combined high-dose group is as high as 85.7 percent, but the difference between the combined low-dose group and the combined high-dose group is not obvious.

The body weight change is shown in fig. 2-C, and compared with the blank group, the body weight of the model group is obviously reduced from day 3, which indicates that the model of systemic drug-resistant candida albicans infection is successfully molded. Compared with the model group, the single fluconazole group and the single n-butanol extract of the panax stipuleanatus have no obvious difference and show a descending trend; the weight of the fluconazole combined with the n-butanol extract of the panax stipuleanatus in the low-dose group (p < 0.001) and the high-dose group (p < 0.0001) is not obviously reduced, and the average weight value is higher than that of each single-use group, but the two combined groups have no obvious difference.

5 obtaining experimental animal materials

The administration was stopped after 10 days, and the surviving mice were anesthetized with 0.5% sodium pentobarbital 24 hours after the last administration, and the thymus, spleen, lung, kidney, liver and rectum of the mice were taken, and cut-off portions from the taken kidney and lung were weighed with an analytical balance.

6 detection of spleen and thymus organ coefficients

Weighing the thymus and spleen of the mouse, calculating the organ coefficients of the thymus and spleen, mapping, and calculating according to a formula:

organ coefficient (mg/g) = [ organ mass (mg)/body mass (g) ] × 100%

The result of organ coefficients of the spleen of the mouse infected by the systemic drug-resistant candida albicans is shown in figure 3-A, compared with a blank group, the organ coefficients of the model group are obviously reduced, and the modeling is successful; edge of screen compared to model groupThe organ coefficients of the single pseudo-ginseng n-butanol extract and the single fluconazole group are increased but have no significant difference, and the n-butanol extract of the pseudo-ginseng was combined with the low-dose fluconazole group (P< 0.05) and high dose group (P< 0.05) was significantly higher than the model group. The result of the organ coefficient of the thymus is shown in figure 3-B, and compared with the blank group, the organ coefficient of the model group is obviously reduced; compared with the model group, the organ coefficients of the n-butyl alcohol extract of the panax notoginseng used alone and the organ coefficients of the fluconazole used alone are increased, but no obvious difference exists, and the n-butyl alcohol extract of the panax notoginseng combined with the fluconazole low dose group (the group of the n-butyl alcohol extract of the panax notoginseng combined with the fluconazole: (the group of the n-butyl alcohol extract of the panax notoginseng combined with the fluconazole of the model group) ((the group of the n-butyl alcohol extract of the panax notoginseng combined with the group of the fluconazole low dose group)P< 0.05) and high dose group (PLess than 0.05) is obviously increased compared with the organ coefficient of the model group, and the change trend of the organ coefficient of the model group is consistent with that of the organ coefficient of the spleen, but the combined low dose and high dose groups have no obvious difference.

7 Lung and Kidney colony count assay

Taking the weighed lung and kidney, adding 0.9% sodium chloride injection according to 10 mul/mg, grinding with a homogenizer, spreading 10 mul each on SDA solid medium, culturing in an incubator at 37 ℃ for 24h, counting colony number, and calculating log₁₀CFU/g is plotted in units.

The statistical result of the number of the colonies of the mouse lung infected by the systemic drug-resistant candida albicans is shown in figure 4-A, compared with a blank group, the number of the colonies of the model group is obviously increased, and the modeling is successful; compared with the model group, the colony numbers of the n-butanol extract of the panax notoginseng used alone and the colony numbers of the fluconazole used alone have no obvious difference, and the n-butanol extract of the panax notoginseng combined with the fluconazole low dose group (a)P< 0.05) and high dose group (PLess than 0.01) is obviously increased compared with the colony number of the model group. The statistical result of the kidney colony number is shown in figure 4-B, and compared with the blank group, the colony number of the model group is obviously increased; compared with the model group, the single use group of the n-butanol extract of the panax notoginseng has no significant difference with the single use group of the fluconazole, and the n-butanol extract of the panax notoginseng is combined with the low dose group of the fluconazole: (P< 0.0001) and high dose group(s) <P< 0.0001) is obviously improved compared with the model group, shows the same trend with the statistical result of the lung colony number, but has no obvious difference between the combined low-dose group and the high-dose group.

Histopathological examination of 8 organs

The lung, kidney, liver and rectum tissues of the mice are fixed in 4% paraformaldehyde solution, embedded in paraffin, sliced, and subjected to hematoxylin-eosin staining (HE).

The HE staining result of a lung tissue section of a mouse infected by systemic drug-resistant Candida albicans is shown in figure 5 (multiplied by 200), compared with a blank group, the alveolar wall capillary of a model group is obviously expanded, and peripheral inflammatory exudates are obviously increased; the parts of alveoli of the single-use group of fluconazole and the single-use group of n-butyl alcohol extract of radix cynanchi bungei are ruptured and fused into large vesicles, the alveolar wall has obvious telangiectasia, and compared with a model group, the pathological forms of the n-butyl alcohol extract of radix cynanchi bungei combined with the low-dose group and the high-dose group of fluconazole are obviously improved, the telangiectasia is obviously reduced, inflammatory exudates are obviously reduced, and the alveoli are complete and have no rupture. The HE staining result of the kidney tissue section is shown in figure 6, and compared with the blank group, the glomerular cells of the model group are obviously increased and infiltrated by the neutrophils; the glomeruli of the single-use group of fluconazole and the glomerulus of the single-use group of n-butyl alcohol extract of radix cynanchi bungei are obviously enlarged and infiltrated by neutral granulocytes, and compared with the model group, the glomerulus cells of the low-dose group and the high-dose group of the n-butyl alcohol extract of radix cynanchi bungei combined with fluconazole are obviously reduced, and the pathological form is obviously improved. The HE staining result of the liver tissue section is shown in FIG. 7, and compared with the blank group, the liver cells of the model group are scattered and distributed with necrotic cells and inflammatory cell infiltration; obvious increase of hepatocyte necrosis can be observed in the single-use group of fluconazole and the single-use group of n-butyl alcohol extract of radix notoginseng, and the necrosis degree of hepatocytes is obviously reduced by the n-butyl alcohol extract of radix notoginseng and the combination of the low-dose group and the high-dose group of fluconazole. The HE staining result of the rectal tissue section is shown in figure 8, and compared with the blank group, the model group has partial cytopathy and changes the morphology; compared with the model group, the combination of the n-butyl alcohol extract of the panax stipuleanatus and the fluconazole low-dose group and the high-dose group can relieve the necrosis of cells, but the effect is not obvious.

Example 5

Mechanism research of drug-resistant candida albicans resistance

Study on influence of n-butanol extract of panax stipuleanatus on efflux pump function of drug-resistant candida albicans

Weighing a proper amount of rhodamine 6G powder, dissolving the rhodamine 6G powder by DMSO to obtain a rhodamine 6G solution with the concentration of 10mmol/L for later use; weighing 5g of glucose powder, adding PBS buffer solution to 100mL, fully dissolving, autoclaving at 115 deg.C for 15min, cooling, and storing at 4 deg.C.

Inoculating the drug-resistant strain in a YEPD liquid culture medium, culturing for 16h at the temperature of 115r/min in a constant-temperature shaking table at 30 ℃ to enable the strain to reach an exponential growth phase, centrifugally collecting cells, and re-suspending the collected cells in the YEPD culture medium. In order to understand the influence of the n-butyl alcohol extract of the panax stipuleanatus on the active efflux and the passive efflux of cells, two groups are arranged, wherein one group is not added with glucose; each group is divided into two groups according to bacteria, one group is treated with solution of radix Schefflerae Arboricolae (86 μ g/mL), the other group is growth control group, i.e. four groups, and bacteria concentration is adjusted to 1 × 10 by using blood cell counting plate⁷And (3) treating the cells at the temperature of 30 ℃ and 115r/min for 5 hours by using a CFU/mL shaking table, taking out the cells after the cells are treated for 5 hours, washing the cells for 3 times by using PBS (phosphate buffer solution), then re-suspending the cells by using the PBS, and treating the cells for 1 hour at the temperature of 115r/min by using the shaking table to fully exhaust the glucose in the cells. After being taken out, adding rhodamine 6G (10 mu mol/L) into a corresponding group, setting a negative control without adding rhodamine 6G, treating for 50min at the temperature of 30 ℃ in a shaking table at 115r/min, taking out and placing into an ice water bath to stop absorption, centrifugally washing for 3 times at 3000r/min in PBS buffer solution for 5min, and removing the exogenous rhodamine 6G. Resuspending the raw materials by using PBS buffer solution and 5% glucose PBS buffer solution according to the components, taking 1mL of the supernatant in an EP tube at 9000r/min, centrifuging the supernatant for 5min, taking 100mL of the supernatant in a 96-pore plate blackboard, sampling the supernatant at 0, 30, 60, 90, 120 and 150min respectively, repeating three multiple pores in each group, repeating the experiment for three times, and detecting the fluorescence value (FI) at the excitation wavelength of 488nm and the emission wavelength of 525nm by using a multifunctional microplate reader.

The detection result of the function of the efflux pump is shown in figure 9, in figure 9-A, glucose is not added, the change trends of the fluorescence intensity pumped by the efflux pump of the growth control group and the efflux pump of the panax notoginseng n-butyl alcohol extract treatment group along with time show consistency, and no obvious difference exists, which indicates that the effect of the panax notoginseng n-butyl alcohol extract on the function of the passive efflux pump is small; in fig. 9-B, the fluorescence intensity pumped out by the efflux pump of the growth control group showed an increasing trend with time and the fluorescence intensity pumped out by the n-butanol extract of panax notoginseng was shown to show a significantly decreasing trend with time, indicating that the n-butanol extract of panax notoginseng can significantly inhibit the function of the active efflux pump.

Influence of n-butanol extract of radix Trifolii Pratentis on level of drug-resistant Candida albicans efflux pump related gene

Selecting appropriate amount of drug-resistant Candida albicans with inoculating loop, inoculating into YEPD liquid culture medium in two groups, treating one group with n-butanol extract solution of radix Schefflerae Arboricolae (86 μ g/mL), and treating in a shaker at 30 deg.C and 115r/min for 16h to make the strain reach logarithmic phase.

And (3) grinding by liquid nitrogen to extract RNA:

and centrifuging and cleaning the collected cells for 3 times at 3000r/min for 5min by using PBS buffer solution, transferring fresh thalli obtained after centrifugation into a mortar by using a cell scraper, adding liquid nitrogen, rapidly and forcibly grinding until the thalli are white powder.

(1) Adding an appropriate amount of Trizol lysate into the well-ground thalli, slightly grinding to ensure that the lysate is fully contacted with the thalli, transferring the homogenate into a 1.5mL enzyme-free centrifuge tube, and standing for 5min at room temperature.

(2) Adding 200 mu L chloroform into 1mL Trizol lysate, shaking vigorously for 15s, standing at room temperature for 3min, centrifuging at 4 ℃ and 12000g for 15min, and dividing the sample into three layers: the bottom layer is a pink organic phase, the upper layer is a colorless aqueous phase, and the middle layer is a white precipitate.

(3) The upper aqueous phase was aspirated and transferred to a new 1.5mL enzyme-free centrifuge tube, 500. mu.L isopropanol was added to each 1mL Trizol lysate and gently shaken, RNA was precipitated, left to stand at room temperature for 10min, and centrifuged at 12000g for 10min at 4 ℃.

(4) Discarding supernatant, slowly adding 75% ethanol along the tube wall, gently inverting to clean the tube wall, discarding ethanol, adding 75% ethanol, adding at least 1mL ethanol per 1mL Trizol lysate, performing brief vortex, and centrifuging at 4 deg.C and 12000g for 10 min.

(5) Carefully sucking the supernatant by using a pipette gun, and drying the centrifuge tube on an ultraclean workbench for 5 min; adding 60 μ L of enzyme-free 0.1% DEPC water to completely dissolve RNA precipitate, and storing at-80 deg.C; placing 0.8 mu LRNA solution in ultramicro ultraviolet spectroscopyMeter sample port, measure RNA concentration, measure RNA sample concentration and A₂₆₀/A₂₈₀。

(6) Synthesis of reverse transcription cDNA:

reverse transcription reaction solution was added according to the system in Table 3, and both the tip and the PCR tube used were operated on ice without enzyme.

TABLE 3 reverse transcription reaction System

After the sample adding is finished, a small-sized low-speed centrifuge is used for short-time centrifugation, so that liquid on the tube wall is sunk to the bottom of the tube, and the PCR tube is placed into a common PCR instrument and is set according to the conditions in the table 4 for reaction.

TABLE 4 reverse transcription reaction conditions

Real-time fluorescent quantitative PCR:

after obtaining the above synthesized cDNA, the corresponding components were added to the PCR tube according to the following reaction system, all the operations were performed on ice, and the primer sequences (synthesized in Shanghai) and the reaction system are shown in tables 5, 6 and 7.

TABLE 5 primer sequences

Note: f is an upstream primer, and R is a downstream primer.

TABLE 6 real-time fluorescent quantitative PCR experiment reaction system

After the sample is added, the PCR tube is put into a small-sized low-speed centrifuge for short-time centrifugation to ensure that the liquid on the tube wall is sunk into the tube bottom, and the PCR tube is put into a real-time fluorescence quantitative PCR instrument for reaction according to the set conditions in the table 6.

TABLE 7 reaction conditions for real-time fluorescent quantitative PCR experiments

After obtaining the expression of each gene, use 2^-ΔΔCtThe method carries out relative quantitative calculation, and the calculation formula is as follows:

ct values of the respective genes obtained by real-time fluorescence quantitative PCR were corrected by ACT1 to obtain Δ Ct [ Δ Ct = Ct (target gene) -Ct (ACT 1)]Taking the average delta Ct of the samples as a reference; comparison of each sample with the average Δ Ct yielded Δ Δ Ct [ Δ Δ Ct = Δ Ct (experimental strain) - Δ Ct (average)](ii) a Final calculation 2^-ΔΔCt

The expression of each gene is shown in FIG. 10, and the growth control group is used as reference, and the Panax notoginseng n-butanol extract treatment group CDR1 (C.F.)P＜0.0001），CDR2（P＜0.001），MDR1（P< 0.01) three efflux pump major genes (CDR 1 and CDR2 are active efflux pump genes; MDR1 is a passive efflux pump gene) transcription level is obviously reduced, and ergosterol synthesis target enzyme ERG11 is obviously increased (P＜0.01）。

Influence of n-butanol extract of radix Stephaniae Sinicae on mitochondrial membrane potential in drug-resistant Candida albicans cells

Inoculating drug-resistant Candida albicans into YEPD liquid culture medium, dividing into two groups, treating one group with 2mg/mL n-butanol extract solution of radix Notoginseng, placing in constant temperature shaking table, culturing at 30 deg.C and 150r/min for 16h, centrifuging at 3000r/min for 5min, collecting thallus, washing with PBS buffer solution for 3 times, centrifuging, re-suspending in PBS buffer solution, adjusting bacteria concentration to 1 × 10⁷CFU/mL, adding JC-1 fluorescent probe to make the final concentration of the CFU/mL to be 10 mu g/mL, incubating for 10min in dark and dark places, washing the thalli with PBS buffer solution for 2 times, then re-suspending with PBS buffer solution, adding the re-suspended bacteria solution into a black 96-well plate according to the quantity of 100 mu L without holes, immediately using a multifunctional microplate reader to measure red fluorescence at the excitation wavelength of 550nm and the emission wavelength of 600 nm; at excitation wavelength of 485nm, emitting waveMeasurement of Green fluorescence at 535nm, Membrane potential (. DELTA.. psi.)_m) Represented by the ratio of red and green fluorescence.

The membrane potential results are shown in FIG. 11, mitochondrial membrane potential Δ ψ_mIs one of the signs of energy level generated in the oxidative phosphorylation process, and the result shows that the membrane potential of the panax stipuleanatus n-butanol extract treated membrane is obviously reduced (in comparison with the untreated drug-resistant strain control) ((P＜0.001）。

Claims

1. A pharmaceutical composition with drug-resistant fungus effect is characterized in that the pharmaceutical composition consists of an n-butyl alcohol extract of panax notoginseng and an antifungal drug; the antifungal drug is fluconazole; the mass ratio of the panax stipuleanatus n-butyl alcohol extract to the antifungal medicine is 2: 1; the drug-resistant fungi is clinical candida albicans fluconazole drug-resistant strains; the panax stipuleanatus n-butyl alcohol extract is obtained by the following steps:

(2) adding ethanol into the coarse powder for refluxing for 2 times, wherein the dosage of the ethanol is 5-15 times of the mass volume of the panax stipuleanatus coarse powder each time, and the dosage of the ethanol is 1-3 hours each time, filtering after refluxing, merging filtrate, and concentrating the extracting solution under reduced pressure until no alcohol smell exists to obtain panax stipuleanatus ethanol extract;

2. The pharmaceutical composition according to claim 1, wherein the ethanol concentration in step (2) is 80-90% by mass; the amount of the suspension water in the step (3) is 1-3 times of that of the ethanol extract of the panax notoginseng, and the amount of the petroleum ether and the n-butanol is 1/6-1/2 of the volume of the water phase during each extraction.

3. The pharmaceutical composition of claim 1, wherein the n-butanol extract of Panax notoginseng is effective in reducing the efflux pump function of drug-resistant fungi.