CN113101284B

CN113101284B - Application of allicin in preparation of anti-yeast drugs

Info

Publication number: CN113101284B
Application number: CN202110376983.9A
Authority: CN
Inventors: 叶枫; 李征途; 李准; 李少强; 占扬清; 林烨; 李新霞; 史荣梅; 刘睿婷
Original assignee: Guangzhou Institute Of Respiratory Health; Xinjiang Garlic Research Institute Co ltd; First Affiliated Hospital of Guangzhou Medical University
Current assignee: Guangzhou Institute Of Respiratory Health; Xinjiang Garlic Research Institute Co ltd; First Affiliated Hospital of Guangzhou Medical University
Priority date: 2021-04-08
Filing date: 2021-04-08
Publication date: 2022-03-08
Anticipated expiration: 2041-04-08
Also published as: CN113101284A

Abstract

The invention provides an application of allicin in preparing an anti-yeast drug, relating to the technical field of medicines. Experiments show that the allicin has better antibacterial action on cryptococcus and candida, and the allicin and antifungal drug also have better antibacterial activity on cryptococcus and candida. Experiments also show that allicin achieves the bacteriostatic or bactericidal effect by destroying the capsule or cell wall or cell membrane of cryptococcus. Allicin has good activity against cryptococcus both in vivo and in vitro. The allicin preparation can be used as a safe and effective medicament for clinically treating fungal infectious diseases with small toxic and side effects and low price.

Description

Application of allicin in preparation of anti-yeast drugs

Technical Field

The invention relates to the technical field of medicines, in particular to application of allicin in preparation of an anti-yeast medicine.

Background

In recent years, with the increase of the incidence of fungi, about 3 hundred million people all over the world are affected by serious mycoses, and pathogenic fungi include common yeasts such as cryptococcus and candida. In fungal diseases, the incidence of candida is located on the second and the incidence of cryptococcus is located on the third, but the current clinical medicines for treating fungal diseases are limited and expensive, and only comprise polyene, flucytosine and azole, wherein the azole has the lowest bactericidal activity, and the serious side effect and the increasing drug resistance of the existing antifungal medicines still remain problems to be solved at home and abroad. Therefore, the research and development of a novel antifungal medicine with high efficiency and low toxicity are urgently needed, and the antifungal medicine has important significance for improving the survival and the life quality of patients.

It is reported that garlic and its preparation haveBroad-spectrum antimicrobial effect, one of the main active ingredients is Allicin (Allicin), the chemical name is diallyl thiosulfinate, the molecular formula is C₆H₁₀OS₂Molecular weight is 162.27, and is produced by catalytic cracking of alliin purified from Bulbus Allii and alliinase. 2mol alliin reacts with alliinase to generate 1mol allicin, and the reaction process and the structural formula are shown in figure 1.

At present, chemical synthesis and structural identification of allicin are reported, but the application of allicin in yeast such as cryptococcus and candida in vitro and in vivo is not reported.

Disclosure of Invention

Therefore, the need exists for providing an application of allicin in the preparation of anti-yeast drugs in order to solve the above problems. The allicin or allicin is combined with antifungal drugs, has good inhibition effect on yeasts such as cryptococcus and candida, and provides a safe and effective drug with small toxic and side effects and low price for clinical treatment of fungal infectious diseases.

In one embodiment, the yeast is cryptococcus.

In one embodiment, the cryptococcus includes, but is not limited to, cryptococcus neoformans, cryptococcus gatherensis, cryptococcus glubuli.

In one embodiment, the cryptococcus includes, but is not limited to, cryptococcus genolyticus of VNI, VGI, VGII, VNIV.

In one embodiment, the yeast is candida.

In one embodiment, the Candida includes, but is not limited to, Candida albicans, Candida krusei, Candida parapsilosis, Candida tropicalis, Candida viticola, Candida Simplicissima.

In one embodiment, the allicin is obtained by catalytic cracking reaction of alliin and alliinase as raw materials.

The invention also provides application of allicin in preparation of a medicine for destroying capsules and/or cell walls and/or cell membranes of yeast.

The invention also provides application of the allicin combined antifungal drug in preparation of an anti-yeast drug.

In one embodiment, the antifungal agent is selected from: one or more than two of amphotericin B, fluconazole and voriconazole. Wherein, the allicin and amphotericin B have better inhibiting/sterilizing effect on cryptococcus or candida.

In one embodiment, the yeast is cryptococcus or candida.

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

experiments show that the allicin has the effect of resisting cryptococcus in vivo and in vitro. Allicin has good inhibitory effect on cryptococcus in vitro and has bactericidal effect on cryptococcus. The in vitro experiment results show that: the Minimum Inhibitory Concentration (MICs) of allicin to cryptococcus in vitro is 1-8 μ g/ml, and allicin 4MIC and 8MIC (8, 16 μ g/ml) have bactericidal effect on cryptococcus neoformans H99. In a microscopic living cell culture system, allicin with 4MIC can achieve the bactericidal effect on cryptococcus in a short time, and allicin with 2MIC can achieve the bactericidal effect only after being acted for a long time. After the transmission electron microscope shows that allicin acts on the cryptococcus neoformans for 8 hours, capsules can be seen in the 2MIC group to disappear, cell membranes and organelles deform, and a few organelles dissolve and die; and a large number of electron compact bodies appear in the cells of the 4MIC group, organelles are widely dissolved and necrosed, and some somatic cells even disappear completely. The in vivo experiment of the mouse shows that: the allicin can effectively improve the symptoms of cryptococcus infection mice, wherein 8mg/kg of allicin can better reduce the lung index, the load of cryptococcus pulmonale and improve the pathological damage of the lung compared with 4mg/kg of allicin; moreover, the drug effect of the allicin with the concentration of 8mg/kg can be compared favorably with that of fluconazole, and the two drugs have no obvious difference on the drug effect of cryptococcus infected mice.

Experiments also find that the allicin body has the effect of resisting candida in vitro. The Minimum Inhibitory Concentration (MICs) of allicin against Candida in vitro is 2-4 μ g/ml. The time-sterilization curve of 0-12h shows that the allicin has bacteriostasis to candida.

Experiments also find that the allicin combined antifungal drug has better effects on resisting cryptococcus and candida. Allicin in vitro combined amphotericin B has additive or synergistic effect on cryptococcus; the combination of fluconazole was additive or non-related to cryptococcus. Allicin combined amphotericin B and fluconazole have additive effects on candida, while the combined voriconazole has no relevant effects on candida.

The above experimental results show that the allicin preparation can be an effective drug for treating cryptococcosis and candidiasis.

Drawings

FIG. 1 is a diagram of alliin production by reaction of alliin with alliinase;

FIG. 2 is an HPLC chromatogram of allicin in the reaction solution of alliin and alliinase.

FIG. 3 is a time-kill curve for allicin against Cryptococcus neoformans H99.

FIG. 4 is a time series image of allicin against Cryptococcus neoformans H99.

FIG. 5 shows the transmission electron microscopy results of allicin against Cryptococcus neoformans H99.

FIG. 6 is a time-sterilization curve of allicin against ATCC22019 Candida parapsilosis.

FIG. 7 shows the body weight change rate of mice on the eighth day.

FIG. 8 shows the mouse cryptococcus lung load results.

Figure 9 is the mouse lung index.

Figure 10 is the results of HE staining for lung pathology in mice.

Figure 11 shows the results of GMS staining of mouse lung pathology.

Detailed Description

To facilitate an understanding of the invention, a more complete description of the invention will be given below in terms of preferred embodiments. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

In the following examples or test examples, the starting materials were all commercially available unless otherwise specified. In addition, AMB represents amphotericin B, FLU represents fluconazole, and VRC represents voriconazole.

Example 1

And (3) synthesizing allicin and determining a product.

Weighing 290mg and 580mg of alliin according to the mass ratio of 1:2, respectively placing into a 10ml measuring flask, adding water to dissolve and dilute to a scale, transferring the alliin solution into a 50ml conical flask with a plug, quickly adding the alliin solution into the alliin solution, uniformly mixing, and reacting at room temperature for 30 minutes. Precisely transferring 0.5ml of reaction solution into a 25ml measuring flask, adding 15ml of methanol, adding 1.0ml of internal standard solution, diluting with 1% formic acid solution to scale, shaking up, and filtering to obtain the final product. Precisely sucking 20 μ l of the test solution, injecting into high performance liquid chromatograph, recording chromatogram (see figure 2), and calculating allicin production (see table 1).

TABLE 1 measurement results of allicin content

Note: a. the_xDenotes the peak area of the sample solution, A_sThe peak area of the internal standard solution is shown.

Test example 1

In vitro drug sensitivity test of allicin to cryptococcus.

1.1. Material

1.1.1 drug positive controls: amphotericin B (Dalian Meiren Biotechnology Ltd.); fluconazole (Dalian Melam Biotechnology Ltd.). Experimental drugs: alliin (Allin Xinjiang Enlexin pharmaceutical Co., Ltd., batch No.: AL160520, sealed at 4 ℃ in the absence of light); allinase (Alliinase Xinjiang Enlexin pharmaceutical Co., Ltd., batch No.: 201801001, sealed at-20 ℃ C.).

1.1.2 strains

All clinical and standard cryptococcus neoformans strains (candida parapsilosis ATCC22019, candida krusei ATCC6258, H99 cryptococcus neoformans) were provided by 6 hospitals of guangdong province (first hospital affiliated to zhongshan university, first hospital affiliated to guangzhou medical university, first people hospital in foshan city, eighth people hospital in guangzhou, general hospital in south war zone, people hospital in revenge) and laboratories of the fungal research institute of shanghai changzheng hospital. All clinical strains were identified as cryptococcus neoformans by ink staining and MALDI-TOF MS mass spectrometry. All strains were activated 2 times on SDA plates before use and cultured at 35 ℃ for 2-3 d.

1.1.3 culture Medium

RPMI-1640 liquid medium: 10.4g of RPMI-1640 powder and 34.5g of MOPS are taken, 900mL of distilled water is added, after the mixture is fully mixed, the pH is adjusted to 7.0 +/-0.1 (25 ℃) by using 1mol/L Na0H solution, the volume is kept to 1000mL, the mixture is filtered and sterilized by using a micro-membrane with the diameter of 0.22 mu m, and the mixture is placed into a refrigerator at 4 ℃ for standby. SDA (Sabouraud Dextrose agar) Saburg Weak plate Medium.

1.1.4 reagents and instruments

DMSO (MYM biotechnology limited); MOPS (Guangzhou Jiebai Biotech Co., Ltd.: 1808GB 007); NaOH (Guangzhou Jiebai Biotech limited, batch No. 180526); TWeen 20 (cantonese biotechnology limited, guangzhou); a table top high speed refrigerated centrifuge (eppendorf model: ST 40R); turbidimeter (Biomerieux); a constant temperature incubator (Sanyo model: MIR 254); an autoclave (Wooson model: Tan20 c 18); electronic analytical balance (sartorius model: BSA 224S-CW); PH meter (sartorius model: PB-10).

1.2. Experimental methods

1.2.1 drug formulation

Preparing an experimental medicine allicin: weighing 11.4mg of alliinase in a 10ml volumetric flask, adding about 6ml of distilled water, shaking uniformly, adding 5.7mg of alliin, fixing the volume to the scale, shaking uniformly, and placing in a 37 ℃ water bath box for reaction for 15 min. The reaction solution was centrifuged at 12000rpm for 10min at 4 ℃. Centrifuging the supernatant in 3KD ultrafilter tube at 4 deg.C and 10000rpm for 10min to obtain filtrate (0.256 mg/ml allicin). The allicin is required to be prepared at present, filtered, stored at 4 ℃ in dark place, and taken out for later use.

Preparing positive drugs: amphotericin B and fluconazole were dissolved in DMSO to prepare 16000 μ g/ml stock solutions, which were stored in a-80 ℃ refrigerator.

1.2.2 bacterial suspension preparation

The bacteria solution in the frozen tube is activated for 2 times in the SDA plate. The activated strains are subjected to zone streaking and inoculated on an SDA plate, and cultured for 72h at 35 ℃. Diameter of picking>3 colonies of 1mm were mixed in physiological saline and adjusted to 0.5 McLeod. The prepared bacterial suspension was diluted 50-fold by culturing in RPMI-1640, and then diluted 20-fold again (the final dilution was 1000-fold) to a concentration of 1X 10³-5×10³CFU/ml.

1.2.3 drug sensitive plate configuration and inoculation

A sterile 96-well U-bottom plate was prepared by adding 100. mu.l of RPMI-1640 culture medium to each row of wells 1-11 and adding 200. mu.l of RPMI-1640 culture medium to each row of wells 12 as a blank. The diluted liquid medicine is taken in the No. 1 hole, and is fully and evenly mixed, and then 100 mul of the diluted liquid medicine is sucked in the No. 2 hole. Diluting at multiple ratio until the number 10 hole is reached, mixing uniformly, sucking 100 μ l of liquid medicine, discarding, and making the concentration of amphotericin B and fluconazole after dilution at number 1-10 hole multiple ratio be 16-0.3125 μ g/ml, and the concentration of allicin after dilution be 64-0.125 μ g/ml. The DMSO content in each well of the 96-well plate is less than 1%. Well 11 is a positive control, without any drug. In reference to the CLSI-M27 protocol of the American Clinical Laboratory Standards Institute (CLSI), Candida parapsilosis ATCC22019 and Candida krusei ATCC6258 were used as quality control strains to perform in vitro antifungal susceptibility tests by the broth microdilution method.

1.2.3 inoculation and interpretation of MIC results

And adding 100 mul of prepared bacterial suspension into the No. 1-No. 11 experimental wells and the positive control wells of the drug sensitive plate, marking, and incubating at 35 ℃ for 72 hours for result interpretation.

The minimum inhibitory concentration MIC interpretation method comprises the following steps: amphotericin B was 100% growth inhibited, i.e. clarified by eye, compared to the positive control wells; fluconazole was a significant reduction in turbidity (about 50% reduction) compared to the positive control wells.

1.2.4 quality control

In order to ensure the accuracy of the experimental result, the errors of the used instruments, reagents and operators are eliminated. The Candida parapsilosis ATCC22019 is used for quality control of medicines, culture media and the like used in the same batch of experiments, and the results refer to CLSI M60 file. One replicate was performed for each drug of the same strain. On the basis, the experiment result of 2 times of experiments of the same strain and the same drug is identical and is recognized when the strain grows well.

1.2. Results

The MIC of allicin to 46 cryptococcus neoformans clinical isolate and standard strain H99 is 1-8 μ g/ml, MIC₅₀And

MIC

₉₀2 and 8. mu.g/ml, respectively. Furthermore, the MICs of allicin to the quality control strains ATCC22019 and ATCC6258 were 4. mu.g/ml, and the MICs to Cryptococcus standard strain H99 were 2. mu.g/ml. Compared with the common clinical antifungal drugs, the MICs 90 of amphotericin B and fluconazole to cryptococcus are respectively 0.5 and 4 mu g/ml, and the MICs of amphotericin B and fluconazole to the standard strain are both in a quality control range. In addition, 2 of the 46 clinical isolates were found to be resistant to amphotericin B, while the MICs of allicin against 2 resistant strains (YQJ18 and YQJ20) were 1. mu.g/ml and 2. mu.g/ml, respectively. Therefore, the allicin has good in-vitro drug effect on cryptococcus and can be compared favorably with fluconazole. The results are shown in Table 2.

TABLE 2MIC results for allicin, amphotericin B and fluconazole against Cryptococcus

In the table, a represents the MIC value of amphotericin B to yeast, and B represents the MIC value of fluconazole to yeast. ECV represents an epidemiological boundary value, and the MIC value of the strain is less than or equal to ECV, which indicates that the strain has no acquired resistance and/or mutation resistance; if the MIC value is > ECV, the strain is acquired and/or mutation resistant.

Test example 2

Allicin combined amphotericin B and allicin combined fluconazole in vitro cryptococcus combined susceptibility test.

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

2.1 drug sensitive plate configuration and inoculation

Taking a 96-well plate A1 as a positive control well, and taking H12 as a negative control well; diluting the clinically common antifungal medicines by multiple ratios in the first row except A1 holes, and diluting the allicin by multiple ratios in the first column except A1 holes; B2-H11 are combined drug sensitive holes. So that the final concentration of amphotericin B from 2 to 11 is 0.0078, 0.0156, 0.0312, 0.0625, 0.125, 0.25, 0.5, 1, 2, 4 mug/ml (if the drug is fluconazole from 2 to 11 is 0.0312, 0.0625, 0.125, 0.25, 0.5, 1, 2, 4, 8, 16 mug/ml), and the final concentration of B-H allicin is 0.125, 0.25, 0.5, 1, 2, 4, 8, 16 mug/ml. And (3) fully and uniformly mixing the prepared bacterial suspension, adding the bacterial suspension into No. 1-No. 11 wells of 100 mu l of drug sensitive plates, culturing at 35 ℃ for 72h, and observing the result, or reading the result when the positive control wells are full.

2.2 interpretation of results of Combined drug sensitivity

And evaluating the interaction effect of allicin combined amphotericin B and allicin combined fluconazole on cryptococcus by using a partial inhibitory concentration (FIC) index. The FIC index is (MIC for the combination of the drugs A/MIC for the single side of the drug A) + (MIC for the combination of the drugs B/MIC for the single side of the drug B).

Evaluation criteria: the smaller the FIC, the better the combined interaction of the two drugs, the better the inhibition effect on experimental bacteria. When FIC is less than 0.5, the interaction of the two drugs is synergistic; 0.5-1.0 is additive effect; 1.0-2.0 is irrelevant; >2.0 is antagonistic. Wherein, the synergistic effect is as follows: the combined action of the two medicines is obviously greater than the sum of the single-medicine actions; additive action: the activity of the two drugs when combined is equal to the sum of the antibacterial activity of the two drugs when used singly; unrelated effects: the combined action activity of the two drugs is equal to the single drug activity; antagonism: the combined action of the two medicines is obviously lower than that of the single medicine.

2.3 combination of drug sensitivity results

Allicin and amphotericin B have additive or synergistic effect on cryptococcus; allicin in combination with fluconazole has additive or no related effect on cryptococcus. The results are shown in tables 3 and 4.

TABLE 3 allicin in combination with amphotericin B drug sensitization results

TABLE 4 allicin in combination with fluconazole sensitization results

Test example 3

And (3) measuring the time-sterilization curve of the allicin to the cryptococcus in vitro.

The drugs and experimental materials used were as described above. The strain used was a standard strain of cryptococcus neoformans H99.

3.1 Experimental methods

According to the result of the MIC of allicin to H99, allicin is prepared into 10ml of liquid medicine with the concentrations of 0.5MIC, 1MIC, 2MIC, 4MIC, 6MIC and 8MIC, 10 mu l of 0.5 McLee bacterial suspension is added, and the liquid medicine is evenly mixed and cultured at 35 ℃. And taking out partial bacterial liquid for 0h, 2h, 4h, 6h, 8h, 10h and 12h respectively, washing, uniformly coating the bacterial liquid on an SDA plate, culturing at 35 ℃ for 48-72h, and calculating the colony number of each time point.

3.2 results of the experiment

The time-sterilization curve of 0-12H proves that the allicin has bacteriostasis to the cryptococcus neoformans H99 standard strain at 0.5MIC, 1MIC and 2MIC, and has bactericidal action to the cryptococcus neoformans H99 standard strain at 4MIC and 8MIC (8, 16 mu g/ml). See fig. 3.

As will be understood, bacteriostatic action means that the action of the drug does not cause the death of the fungus but rather inhibits the growth of the fungus which is capable of growing when the drug is removed or the drug is diluted; the bactericidal action means that the action of the drug causes the death of the fungus, and the fungus cannot grow even after the drug is removed or the concentration of the drug is diluted.

Test example 4

Allicin was imaged in vitro on a time series of cryptococcus growth states.

The medicines and strains are as above.

4.1 Experimental methods

Adding the prepared allicin into cryptococcus bacterium liquid in 384-hole plate to make the concentration of cryptococcus bacterium be 5X 10⁴CFU/ml, allicin concentrations of 1MIC, 2MIC, 4MIC, and a control group without allicin was set. The cryptococcus was placed in a microscope live cell culture system (model: Tokai Hit Thermal Plate) for observation at different time points, and an Olympus IX83 full-electric microscope and a Prime 95B SCMOS camera were used to take pictures and record the pictures intermittently and continuously to observe the change of the quantity of the cryptococcus after long-term action and the division of the cryptococcus after short-term action of allicin.

4.2 results of the experiment

In long-term culture, the cryptococcus in the normal group without the drug is slightly increased after 24h, and is obviously increased at 48h, 72h and 96 h. The allicin with 1MIC basically has no cryptococcus division growth after 24 hours of action, a small amount of cryptococcus can be seen after 48 hours of action, a large amount of cryptococcus can be seen after 72 hours of action, and the cryptococcus has grown to the whole visual field after 96 hours of action. 2MIC, the growth of the cryptococcus is almost in a proliferation stagnation state, and no obvious increase of the cryptococcus is observed after 96 h. The amount of cryptococcus for 96h and 24h of 4MIC effect was not significantly different, and the result was similar to 2 MIC. The results of the long-term culture indicate that 1MIC of allicin may be inhibitory to cryptococcus, and 2MIC and 4MIC may be bactericidal.

To further observe the effect of allicin on the proliferative division of cryptococcus over a short period of time, cryptococcus cultures were observed for approximately 6h in serial photographs. The group without drug split very rapidly within 6h, almost every 40 min. The growth rate of cryptococcus fissions after 1MIC effect was significantly reduced, but not completely inhibited. After 6 hours, some cryptococcus can still divide 1-2 times. After 2MIC, no obvious cryptococcus division growth is observed within 6 h. 4MIC, the division growth of the cryptococcus is completely inhibited, the phenomenon of cryptococcus division does not occur, and even large pieces of cryptococcus fragments can be seen. See fig. 4. The continuous photographing of the cryptococcus in a short period is consistent with the intermittent photographing result in a long-term culture. The results were consistent with those of the time-kill curve. In conclusion, 4MIC allicin achieves bactericidal effect in a short time, while 2MIC takes a long time to achieve bactericidal effect.

Test example 5

And (3) observing the allicin in vitro on cryptococcus by using a transmission electron microscope.

The medicines and strains are as above.

5.1 Experimental methods

H99 Cryptococcus neoformans is cultured for 72H at 37 ℃, a single colony is collected in 10ml YPD liquid medium, the single colony is placed in a constant-temperature shaking table at 37 ℃ for incubation for 36H at 200rpm, the bacterium suspension is adjusted to 0.5 McLee concentration, 2MIC and 4MIC allicin are added into each tube, and a drug-free group is used as a blank control. Washing at 37 deg.C for 8 hr, washing at 4500rcf for 5min for 1 time, slowly adding fixing solution, observing with scanning electron microscope, and taking picture.

5.2 results of the experiment

After allicin acts on the cryptococcus neoformans for 8 hours, capsules of the 2MIC group can disappear, cell membranes and organelles are obviously deformed, some of the cells even have cell wall rupture to cause cytoplasm overflow, and a few of the organelles are dissolved and necrotized and comprise cell nuclei and mitochondria. The 4MIC group shows obviously different results, most of thallus capsules are complete, however, a large number of electron compactors appear in cells, organelles are widely dissolved and necrotized, and some thallus cells even disappear completely. See fig. 5.

Test example 6

In vitro drug sensitivity test of allicin to candida.

The drugs and experimental materials used were as described above. The clinical strains used were isolated and identified by the first hospital affiliated to Guangzhou medical university, and the standard strains were provided from the Guangzhou medical university microorganism resource repository.

6.1 Experimental methods

The experimental method is the same as that of test example 1, the added drug sensitive plate is marked, and the plate is incubated at 35 ℃ for 24 hours to judge the result.

6.2 results of the experiment

The clinical MIC of allicin to 50 clinically isolated Candida is 2-4 μ g/ml, and the MIC is₅₀And MIC₉₀Both were 4. mu.g/ml, and the MICs for the quality control strains ATCC22019 and ATCC6258 were 4. mu.g/ml. And the MICs of amphotericin B and fluconazole to the standard strain are within the quality control range (Table 5). In addition, among 50 clinically isolated candida strains, 9 fluconazole-resistant strains were found, including 5 candida tropicalis, 3 candida krusei and 1 candida glabrata (MIC 8-64 μ g/ml), wherein candida krusei is naturally resistant to fluconazole, and allicin has a MIC of 2-4 μ g/ml against candida. Therefore, the allicin has obvious advantages in the candida which is resistant to the fluconazole in an in vitro experiment.

TABLE 5 results of MIC of allicin, amphotericin B and fluconazole against Candida

In the table, a represents the MIC value of amphotericin B to yeast, and B represents the MIC value of fluconazole to yeast. ECV represents an epidemiological boundary value, and the MIC value of the strain is less than or equal to ECV, which indicates that the strain has no acquired resistance and/or mutation resistance; if the MIC value is > ECV, the strain is acquired and/or mutation resistant. S represents that the result of drug sensitivity test of the strain with epidemiological break points is sensitive, and R represents that the result of drug sensitivity test of the strain with epidemiological break points is drug resistance.

Test example 7

Combined drug susceptibility experiments of allicin combined with amphotericin B, allicin combined with fluconazole and allicin combined with voriconazole on candida in vitro.

The drugs, strains and experimental materials used were as described above. Each experiment was repeated 3 times.

7.1 results of the experiment

Allicin in combination with amphotericin b (amb) and Fluconazole (FLU) showed additive effect on candida (FICI <1), with a maximum reduction of 4-fold and 8-fold respectively compared to single use (tables 3, 4). Whereas allicin in combination with Voriconazole (VRC) had no relevant effect on candida (table 6).

TABLE 6 allicin in combination with voriconazole drug sensitization results

Test example 8

And (3) measuring the time-sterilization curve of the allicin to the candida in vitro.

The drugs and experimental materials used were as described above. The strain used was ATCC22019 Candida parapsilosis standard strain.

8.1 results of the experiment

As can be seen from the time-sterilization curve of 0-12h, the Candida parapsilosis ATCC22019 grows slowly in 0-4h, and the sterilization effect is positively correlated with the drug concentration along with the extension of the action time of allicin. 8MIC significantly reduced colony counts at 12h, log0MIC/log8MIC <3(0MIC corresponds to Negative control in FIG. 6), indicating that allicin was bacteriostatic to Candida parapsilosis at 12h (see FIG. 6).

Test example 9

Experiments on the protective effect of allicin on cryptococcus infected mice.

9.1 Experimental materials

9.1.1 the allicin and fluconazole as the same medicine.

9.1.2 animals SPF-grade female C57BL/6 mice, 6-8 weeks old, weighing 18-20g, were randomly assigned.

9.1.3 infection with the Strain H99 Cryptococcus neoformans, supplied by the laboratory of the Hospital institute for fungi, Shanghai Yangcheng.

9.2 Experimental methods

9.2.1 establishment of Cryptococcus infection mouse model

60 mice are purchased in advance and fed in cages, the mice are normally fed for about one week, the health state of the mice is ensured, and healthy mice are selected for experiments. The breeding conditions of the mice are as follows: the room temperature is maintained at 23-27 ℃, the relative humidity is controlled at 40% -60%, the feed is sufficient, and water is freely drunk.

9.2.2 preparation of strains and preparation of suspensions

The recovered H99 cryptococcus neoformans strain is inoculated on an SDA culture medium, and the strain is in a high-activity state after 2 passages. Counting the amount of cryptococcus and adjusting the concentration of bacteria to 5 × 10⁶CFU/ml, i.e. 0.5 McLeod concentration, such that the cell density is 2.5X 10⁵cells/50. mu.l, confirmation was repeated.

9.2.3 animal grouping

Randomly grouping, and simultaneously establishing blank control group (intragastric with 0.2ml PBS), noninfected high-dose allicin group (intragastric with 0.2ml allicin), and cryptococcus infection group (2.5 × 10)⁵cells/50. mu.l nasal drop infection), Cryptococcus infection + allicin high and low dose groups (0.2ml allicin 4, 8mg/kg gavage), positive drug control group (0.2ml 20mg/kg fluconazole gavage). Each group of mice was numbered and the mice were observed for viability.

9.2.4 mice anesthesia and inoculation

Mice were anesthetized with isoflurane and observed for mobility and vital signs. 50 μ l of the H99 bacterial liquid was taken for nasal drip infection, and the bacterial liquid was slowly inhaled when the mice inhaled, and the mouse status was observed when the bacterial liquid was not inhaled when the mice exhaled.

9.3 detection index

9.3.1 mouse weight and survival rate

After 4 hours of infection with cryptococcus H99, the drug was added with antifungal agents and the treatment was continued for 7 days, and the normal control group (PBS), the high-dose allicin alone group (8mg/kg), the model group (PBS), the high-and low-dose allicin treatment group (4, 8mg/kg), and the fluconazole treatment group (20mg/kg) were each administered to the mice by gavage in an amount of 0.2 ml. Mice were monitored daily for body weight and their survival was recorded for a total of 8 days.

9.3.2 pulmonary fungus load assay

Killing 4 mice in each group at 8 days after infection, taking out the right lung and removing fascia, fully grinding with magnetic beads, ultrasonically homogenizing the tissue, fully shaking, mixing (without separation), and taking out with a pipette0.1ml of the suspension was diluted 10-fold in an EP tube containing 0.9ml of PBS, and the dilution was shaken well and then sequentially performed by 10²、10³Diluting by times and marking. And (3) taking out 20 mul of the diluent, uniformly coating the diluent on an SDA plate, repeating the plate for 3 times, culturing in a constant-temperature incubator at 35 ℃ for two days, taking out the plate, and counting the clone, wherein the clone is multiplied by the dilution times to obtain the fungus load count.

9.3.3 pulmonary index

Each mouse was dissected and the intact mouse lung was removed, the lungs were washed with PBS and blotted dry with absorbent paper, the total lung weight was weighed on an analytical balance, and the lung index (lung weight/mouse weight) x 100 was calculated.

9.3.4 pathological conditions of Lung tissue

4 mice in each group are sacrificed, left lung tissues are separated and fixed by 4 percent formaldehyde solution for more than 24 hours, and the left lung tissues are placed in a dehydration box for dehydration, transparence, wax penetration, embedding, slicing, dewaxing rehydration, HE staining, GMS staining, dehydration mounting and the like, and then observed and photographed under a microscope.

9.4 results of the experiment

9.4.1 mouse eighth day rate of change in body weight

The weight of mice in the blank control group, the uninfected allicin treatment group with 8mg/kg allicin and the fluconazole treatment group is increased more than before, the weight of the mice in the 8mg/kg allicin treatment group on the eighth day is equal to that before infection, and no obvious difference exists in comparison with the blank control group. The weight of the cryptococcus infection model group and the allicin treatment group with 4mg/kg is obviously reduced compared with that of a blank control group. Compared with the fluconazole treatment group, the change of the body weight of the allicin treatment group of 8mg/kg is not obviously different. None of the mice in each group died during the observation period. See fig. 7.

9.4.2 cryptococcus pulmonale load

The load of the cryptococcus pulmonale of all the drug treatment groups is obviously reduced compared with that of the model group, and the load of the cryptococcus pulmonale of the high and low dose allicin treatment groups is not obviously different from that of the fluconazole treatment group. See fig. 8.

9.4.3 pulmonary index

Compared with the model group, the lung index of mice in the model group and the low-dose allicin treatment group is obviously increased compared with the normal group, the lung index of mice in the fluconazole treatment group and the lung index of mice in the high-dose allicin treatment group can be obviously reduced, and the 8mg/kg allicin treatment group has no obvious difference compared with the fluconazole treatment group. See fig. 9.

9.4.4 pathological mechanism of lung tissue

The lung pathology HE staining result shows that lung bronchial epithelial cells of the allicin group without infection, which are 8mg/kg, are closely arranged, have normal shapes, are dry and clean in lumens, and have no obvious difference compared with the normal group. The lung pathology of the model group can show that a large amount of inflammatory cells infiltrate into an alveolar cavity and a trachea, the alveoli are damaged, and large-area lung consolidation changes occur. The pulmonary lesion of the fluconazole and allicin treatment group with 8mg/kg is obviously improved, a small amount of inflammatory cell infiltration can be seen, and the tracheal cavity is clean. The 4mg/kg allicin treatment group can also obviously improve lung consolidation, but the tracheal cavity still can be infiltrated by a small amount of inflammatory cells, namely, the efficacy of the allicin treatment group on cryptococcus infection mice is better with the increase of allicin dose. See FIG. 10 (in the HE staining, a is blank control, b is non-infection 8mg/kg allicin group, c is model group, d is fluconazole treatment group, e is 8mg/kg allicin treatment group, and f is 4mg/kg allicin treatment group).

Lung pathology GMS staining results show that the alveolar spaces of the model groups have extensive cryptococcus infiltration, while the cryptococcus in the lungs of the fluconazole and high-dose allicin treatment groups are obviously reduced, and the cryptococcus infiltration only occurs in local lung tissues. The low dose allicin treated group also reduced cryptococcus pulmonale infiltration, but cryptococcus sporans was still visible in the alveoli. See FIG. 11(GMS staining with a model group, b fluconazole treated group, c allicin treated group 8mg/kg, d allicin treated group 4 mg/kg).

The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims

1. The allicin and amphotericin B are used in preparing medicine for resisting H99 cryptococcus.