CN110613720B - Application of veratrine in preparation of medicine for preventing and treating candida albicans infection - Google Patents
Application of veratrine in preparation of medicine for preventing and treating candida albicans infection Download PDFInfo
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- CN110613720B CN110613720B CN201910907500.6A CN201910907500A CN110613720B CN 110613720 B CN110613720 B CN 110613720B CN 201910907500 A CN201910907500 A CN 201910907500A CN 110613720 B CN110613720 B CN 110613720B
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Abstract
The invention aims to disclose application of veratrine in preparation of a medicament for preventing and treating candida albicans infection. The detection results of various indexes of the compound alliveratrine on growth influence, hypha inhibition rate, cytotoxicity, animal models and the like of the candida albicans show that the alliveratrine has no obvious influence on the growth of the candida albicans, wherein the compound alliveratrine can still effectively inhibit the hypha formation of the candida albicans when the concentration is 50 mu M, and the hypha formation rate is lower than 50%. In addition, the alliveratrine has a good protective effect on candida albicans infected human cell A549, and the compound alliveratrine has no obvious toxic or side effect on cells. In a mouse oral cavity model, the veratrine has good inhibition effect on the invasion of candida albicans.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to an application of veratrine in preparation of a medicine for preventing and treating candida albicans infection.
Background
Candida albicans (Candida albicans), also known as Candida albicans and Candida albicans, is a opportunistic pathogen that is found in nature and in the oral, respiratory, digestive, and vaginal mucosa of the human body. Under normal conditions, candida albicans generally cannot cause diseases, but can cause deep infection of body surfaces and even internal tissues and organs when the immunity of a host is low, and the disease is usually difficult to cure. The data show that the mortality rate of Candida albicans infection in immunodeficiency patients reaches 40% -70%.
Previous studies have shown that Candida albicans pathogenesis is associated with its yeast-hyphal transition. First, free yeast adheres to host cells, at which time Candida albicans does not exhibit toxicity; subsequently, the attached yeast forms hyphae gradually, which promotes infection of host tissues, thereby producing biofilms. Biofilms are a significant cause of candida albicans disease. Thus, the transformation of Candida albicans morphology is a critical step in pathogenesis.
The existing candida infection treatment drugs comprise amphotericin B and azole antibiotics, and the antibiotics can cause the defects of DNA pollution, bacterial drug resistance, superbacteria, human hazard and the like, for example, nephrotoxicity is the most common and serious complication of deoxycholic acid amphotericin B, and 50 percent of patients have acute renal injury and renal tubular acidosis.
Based on the method, the novel bacteriostatic strategy is utilized to design the compound alliveratrine for resisting candida albicans, starting from the unique yeast-hypha diphasic property of the candida albicans, the novel compound alliveratrine which is efficient, low in toxicity and not easy to generate drug resistance is designed in a targeted manner, and the method has important scientific significance and application prospect. The rhizome of veratrum nigrum can be used as a medicine, and at present, people use veratrum nigrum to treat stroke and also can be used for dissipating blood stasis, relieving pain and the like. However, the antibacterial performance of alliveratrine extracted from veratrum is not reported. The invention discovers for the first time that the natural extract alliveratrine in veratrum has excellent resistance to candida albicans.
Disclosure of Invention
The invention provides an application of veratrine in preparation of a medicine for preventing and treating candida albicans infection.
In order to realize the invention, the invention provides the application of the alliveratrine in preparing the medicines for preventing and treating the candida albicans infection.
Further, the alliveratrine is extracted from veratrum, and the chemical structural formula of the alliveratrine is as follows:
furthermore, when the concentration of the allicin is not less than 50 mu M, the formation of hyphae of candida albicans can be effectively inhibited, and the hyphae formation rate is lower than 50%.
Compared with the prior art, the invention has the following advantages and effects:
previous studies by the present inventors have identified that Diffusible Signal Factor (DSF) quorum sensing signals and derivatives thereof can strongly interfere with candida albicans yeast-hyphal state transitions. The study also successfully synthesizes and screens the compound alliveratrine which can inhibit the adhesion of candida albicans, the hypha form conversion and the pathogenicity. Meanwhile, the compounds alliveratrine have low toxicity, do not influence the growth of candida albicans and human cells, and are expected to promote the development of novel antifungal drug treatment. However, when the concentration of the allicin is not less than 50 mu M, the formation of hypha of candida albicans can be effectively inhibited, and the hypha formation rate is lower than 50%.
Drawings
FIG. 1A is the cytotoxicity of alliveratrine to A549 cells at a final concentration of 100. mu.M;
FIG. 1B is a graph of the effect of veratrine at a final concentration of 100 μ M on the cytotoxicity of Candida albicans SC 5314;
FIG. 1C is a graph of the effect of veratrine on Candida albicans SC5314 cytotoxicity under different concentration gradients;
FIG. 2A is a graph showing the results of measurement of the inhibitory rate of veratrine against Candida albicans hyphae formation at 3 different concentrations ranging from 25. mu.M to 100. mu.M;
FIG. 2B is a microscopic image of hyphal inhibition of DMSO, FLC (fluconazole, a broad-spectrum antifungal drug that can be used to treat Candida albicans infection) alliveratrine at a final concentration of 100. mu.M;
FIG. 3 is a graph showing the effect of the compound alliveratrine on the growth rate of Candida albicans;
FIG. 4 is a graph showing the effect of the compound alliveratrine on Candida albicans infection of the oral cavity of mice, using PBS as control;
figure 5 is a mass spectrum of the compound alliveratrine.
Detailed Description
The present invention will be described in further detail below with reference to examples of implementation and the accompanying drawings, but the embodiments of the present invention are not limited thereto.
Example 1: effect detection test
The test method comprises the following steps:
(1) activation of candida albicans strains:
Candida albicans standard strain SC5314 was activated in LB medium (tryptone 10g/L, yeast extract 5g/L, NaCl 10g/L, agar 15g/L), and cultured overnight in an incubator at 30 ℃.
(2) Effect of compound alliveratrine on the virulence of candida albicans strain SC5314 cell:
(a) recovery and culture of A549 cells: freeze-thawed A549 cells were transferred to 10% FBS-containing DMEM medium (Gioco Corp.) at 37 ℃ and 5% CO2Cultured overnight under the conditions.
(b) Preparation of A549 cells: a549 cells in DMEM (high glucose Medium) containing 10% fetal bovine serum at 1.5X 104Cell concentration per well was cultured overnight in 96-well plates. When the cells were 80% full of the bottom of the 96-well plate, the culture medium was discarded, and the cells were washed 3 times with 1 × PBS.
(c) Preparation of candida albicans: selecting fresh SC5314, inoculating into GMM culture solution, and shake culturing at 30 deg.C and 200rpm overnight; adjustment to OD with DMEM cell maintenance solution containing 1% FBS6001.0, 10-fold dilution with DMEM (1% FBS) (10 ≈ 10)6CFU/mL) for use.
(d) And (3) determining the cytotoxicity: adding compound alliveratrine with final concentration of 100 μ M into the cell maintenance liquid containing bacteria, adding 100 μ L into the prepared A549 cells, standing at 37 deg.C and 5% CO 2The cells were incubated in an incubator for 8h, 3 replicates per sample, with DMSO, FLC only controls being set up. In addition, the toxicity of the compound alliveratrine on the cells is measured, namely the compound alliveratrine with the final concentration of 100 mu M is added into the bacteria-free cell maintenance liquid and treated in the same way. In order to determine whether the compound alliveratrine is a dose-dependent compound alliveratrine, the compounds alliveratrine with final concentrations of 50. mu.M, 100. mu.M and 200. mu.M, respectively, were added to the cell maintenance liquid containing bacteria, 100. mu.L of the compound alliveratrine was added to the prepared A549 cells, and the same procedure was followed. Reference is made to Promega corporation CytoTox
NonRadioactive Cythe totoxicity Assay protocol measures cellular LDH activity, followed by GraphPad Prism 5 treatment of the data.
(3) Effect of compound alliveratrine on hyphae of candida albicans strain SC 5314:
SC5314 strain on LB solid plate is selected, inoculated into GMM culture solution (6.7g/LYNB + 0.2% glucose), cultured overnight at 30 deg.C under shaking at 200rpm, and measured to determine bacterial liquid OD600Diluting the bacterial liquid to OD with GMM6000.1. 500 mul of bacterial liquid is taken and put into a 1.5mL EP tube, and a compound alliveratrine with the final concentration of 100 mul is respectively added, and DMSO and FLC are respectively set as controls. Shaking, mixing, incubating in water bath at 37 deg.C for 6 hr, centrifuging at 5000rpm for 10min, discarding supernatant, adding 40 μ L GMM culture solution, resuspending thallus, observing mycelium formation under Leica DMi8 microscope, and taking pictures with different visual fields.
(4) Determination of the effect of compound alliveratrine on the growth of candida albicans strain SC 5314:
selecting single colony of strain SC5314, inoculating to GMM culture solution, performing shake culture at 30 deg.C and 200rpm overnight, and determining OD of bacterial solution600Diluting the bacterial liquid to OD with GMM6000.5. And (3) putting 1mL of the bacterial liquid into a 1.5mL EP tube, sequentially adding a compound alliveratrine with the final concentration of 100 mu M, shaking and uniformly mixing, adding 300 mu L of the compound alliveratrine into a 100-pore plate, setting 4 times of each treatment, and setting that only DMSO is added for treatment. Placing in a growth curve tester, measuring OD every 2h at 30 deg.C and 200rpm600Values, observed after 2d experimental results, GraphPad Prism 5 processed data.
(5) Effect of compound alliveratrine on infection of candida albicans strain SC5314 in the mouse oral cavity:
(a) preparation of candida albicans: fresh SC5314 was picked and inoculated into GMM medium and cultured overnight at 30 ℃ under shaking at 200 rpm. The next day, the cells were collected (5000rpm,10 min). Washed twice with PBS and twice with HBSS solution. Then resuspended in PBS and the bacterial solution diluted to OD6000.1. And adding a compound alliveratrine into the bacterial liquid to make the final concentration of the compound alliveratrine 100 mu M. The control group was PBS without treatment.
(c) Mouse preparation: male BALB/c mice of 6-8 weeks old, purchased from Experimental animals center, Guangdong province, were housed in Experimental animals center, university of south China agricultural. 3/group were randomly assigned, weight recorded and each mouse was marked.
(b) Infection: the day before infection, mice were injected subcutaneously with hydrocortisone (225 mg/Kg). On the day of infestation, cotton swabs of the same size are soaked in each of the above groups of liquids for at least 5 min. Meanwhile, mice were anesthetized with 10% chloral hydrate and placed on an isothermal pad at 37 ℃. The corresponding cotton bud was placed under the tongue of the mouse and infected for 75 min. On the next day of infection, an equal dose of hydrocortisone was again injected to promote candida albicans infection.
(d) Sampling: on the fifth day of infection, after euthanizing the mice, tongue tissue from the mice was taken and placed in formalin solution for pathological section analysis. The test was conducted according to the care and use regulations (NIH publication) of experimental animals under the health guidelines of the national institutes of health.
(6) Mass spectrometric detection of the compound alliveratrine:
the compounds were dissolved in methanol and their molecular formulas were verified using A8 column with a mobile phase of a1 (water + 0.01% formic acid) and B1 (methanol + 0.01% formic acid).
(II) results of the experiment
(1) The compound alliveratrine can inhibit the virulence of Candida albicans SC5314 to cells
Using DMSO and FLC as controls and the final concentration of the compound alliveratrine as 100 μ M, we tested the virulence effect of the compound alliveratrine itself on cells in the absence of candida albicans SC5314, as shown in 1A. FIGS. 1B-1C are graphs showing the effect of veratrine on the pathogenicity of Candida albicans cells to A549 cells. The toxicity of cells is detected by detecting the release amount of LDH, and when the cytotoxicity of candida albicans is detected, the release amount of LDH of a control group added with DMSO is taken as 100%, and the LDH release ratio of a compound allicin group is regulated. Data shown are the average of 3 biological replicates, and error bars reflect standard deviations. As shown in figure 1A, the compound alliveratrine has less toxicity to cells.
Meanwhile, we also tested whether the compound alliveratrine can attenuate the virulence of candida albicans SC5314 on cells. The results are shown in figure 1B, and the compound alliveratrine has strong protective effect on cells, and can reduce the toxicity of Candida albicans below 20%. In addition, the compound alliveratrine has certain inhibition effect on candida albicans infection under different concentration gradients, and the result is shown in figure 1C.
(2) The compound alliveratrine can inhibit hypha formation of Candida albicans SC 5314.
Taking the hypha formation rate of DMSO and BDSF as a control, the final concentration of the compound alliveratrine is 50-200 mu M, and whether the compound alliveratrine has influence on the hypha formation of Candida albicans SC5314 or not. FIGS. 2A-2B are graphs showing the effect of veratrine on Candida albicans hyphae formation; where this data shows the average of 3 biological experiments, the error bars reflect the standard deviation. As shown in figure 2A, the compound alliveratrine has strong hypha inhibition effect on Candida albicans. In addition, FIG. 2B shows the results of the compound alliveratrine at 100. mu.M under an inverted microscope. The growth of hypha is almost completely inhibited when the compound alliveratrine is 200 mu M, the hypha formation rate is lower than 20% when the compound alliveratrine is 100 mu M, and the hypha formation rate is lower than 50% when the compound alliveratrine is 50 mu M.
(3) The compound alliveratrine has an effect on the growth of candida albicans SC 5314.
FIG. 3 is a graph showing the effect of the compound alliveratrine on the growth rate of Candida albicans; and (3) controlling DMSO and fluconazole, wherein the final concentration of the compound alliveratrine is 100 mu M, and whether the compound alliveratrine has influence on the growth of the Candida albicans SC5314 or not. Data shown are the average of 3 biological replicates, and error bars reflect standard deviations. The results are shown in figure 3, and the compound alliveratrine has an inhibitory effect on the growth of candida albicans SC 5314.
(4) The allicin can inhibit infection of the candida albicans strain SC5314 to the oral cavity of the mouse.
FIG. 4 is a graph of the effect of the compound alliveratrine on Candida albicans oral infection in mice, with PBS and untreated bacteria as controls, and the final concentration of the compound at 100. mu.M. Data shown are the average results of 3 biological replicates. The results are shown in fig. 4, where alliveratrine has a strong protective effect on candida albicans infected mice oral cavity, and the amount of bacteria infecting only candida albicans group in pathological section is significantly greater than that of the sample group.
(5) The compound used in the invention is veratrine.
Fig. 5 is a mass spectrum of the compound used, the molecular weight of which is consistent with that of veratrine, so that the compound detected by the invention can be judged to be veratrine (Jervine).
In summary, we screened a compound alliveratrine that can resist Candida albicans and tested its ability to resist fungal pathogens. Wherein, the compound alliveratrine shows strong characteristics of inhibiting the hypha formation and pathogenicity of candida albicans cells, has no great influence on the growth of the candida albicans cells, and has no toxicity to human cells; furthermore, the compounds have excellent protective effect on mice infected by Candida albicans at 100. mu.M. Therefore, some of the compounds alliveratrine can be developed into novel medicaments for resisting candida albicans infection.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (4)
1. The application of the allicin as the only active component in the preparation of the medicine for preventing and treating Candida albicans infection is characterized in that: when the concentration of the allicin is 50 mu M, the formation of hypha of candida albicans is effectively inhibited, and the hypha formation rate is lower than 50%.
2. The application of the allicin as the only active component in the preparation of the medicine for preventing and treating Candida albicans infection is characterized in that: when the concentration of the allicin is 100 mu M, the formation of hypha of candida albicans is effectively inhibited, and the hypha formation rate is lower than 20%.
3. The use according to claim 1 or 2, wherein the alliveratrine is effective in inhibiting the formation of hyphae of Candida albicans at a concentration of not less than 50 μ M, with a hyphae formation rate of less than 50%.
4. The use according to claim 1 or 2, wherein said alliveratrine is effective in inhibiting Candida albicans infection in a mouse oral cavity model at a concentration of 100 μ M.
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| Neoverataline A and B, two antifungal alkaloids with a novel carbon skeleton from Veratrum taliense;Chang-Xin Zhou 等;《Tetrahedron》;20031231;第59卷;摘要 * |
| 中药抗真菌的活性筛选及作用机制研究;吴晶;《中国优秀硕士学位论文全文数据库（电子期刊）》;20180115(第1期);第18-21页 * |
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