Quercetin QCT is a dietary flavonoid and has been demonstrated to be antifungal against C. albicans biofilm. Methods: 17 C. Conclusion: Together with these results, it could be demonstrated that QCT could be a favorable antifungal agent and a promising synergist with FCZ in the clinical management of VVC caused by C.

The clinical symptoms of VVC include vulva erythema, edema, excoriation, and fissure formation as well as introital and vaginal erythema [ 2 ].

Several lines of evidence indicated that the aetiology of VVC might not be attributed to an opportunistic infection or an immunodeficiency, but might be associated with a hypersensitivity response to a genetically defined commensal organism [ 3,4,5 ].

Although VVC is usually not a fatal factor for woman life, the re occurrence of VVC challenges the life quality of the infected women [ 6 ]. Multiple factors including long-term overuse of over-the-counter antifungal drugs, one-dose managements, low exposure to azoles together with the growing immunocompromised patients such as diabetics and HIV women contribute to the ever-rising incidence of VVC [ 7,8 ].

albicans is able to adhere to biotic e. epithelial or endothelial cells and abiotic e. central venous and types of catheters surfaces to form biofilm, a complex three-dimensional structure of fungal cell agglomerates. A biofilm is a special phenotype of C. albicans composed of extracellular matrix, the chemical constitute of which consists of proteins, polysaccharides as well as DNA [ 10 ], and encased fungal cells which are supposed to have altered susceptibility to antifungal agents and metabolism compared with its planktonic counterparts [ 11 ].

A biofilm phenotype provides a potent shield of fungal cells from the attacks of host immune system and extraneous antifungal chemicals. The biofilm formation of C.

During the three stages, the fungal cells are continuously dispersing from the settlement to the environment for searching new colonization [ 12 ]. albicans biofilms have been considered to be one of the critical factors accounting for fungal resistance to traditional antimycotic drugs, including azoles, polyenes, echinocandins, etc.

Fluconazole FCZ is the most commonly used first-line drug of the azole family in clinical prophylaxis and treatment of mucosal and invasive Candida infections including VVC.

The target of FCZ is believed to be the lanosterol α demethylase which is an essential enzyme encoded by ERG11 gene in the ergosterol biosynthesis process of C.

albican [ 14 ]. albican isolates [ 15 ]. Of note, the resistance rate to C. Therefore, it is necessary to seek novel medicines to use alone or in combination with FCZ in the treatment of FCZ-resistant C. albicans isolated from VVC. Quercetin QCT , a dietary flavonoid, has been demonstrated to possess the antifungal function to manage clinical C.

albicans biofilms and sensitize the susceptibility of FCZ-resistant C. albicans isolates to FCZ [ 18,19 ]. albicans isolated from VVC patients.

albicans isolates. albicans reference strains SC and ATCC as well as fifteen clinical isolates from VVC patients were obtained from the Department of Gynecology, the First People's Hospital of Shangqiu and stored in yeast extract-peptone-dextrose YPD, Oxoid Ltd.

These isolates were confirmed by API®32C biochemical testing panel bioMérieux UK Ltd. Then the isolates were propagated in YPD medium for 24 h at 37°C, washed by phosphate-buffered saline PBS Sigma-Aldrich, Shanghai, China , collected by centrifugation and then resuspended in RPMI medium Sigma-Aldrich, Shanghai, China for use.

The antifungal activities of QCT and FCZ against planktonic cells were represented by minimum inhibitory concentration MIC , which was defined as the lowest drug concentration inhibiting visible growth. The combination of QCT and FCZ against biofilm cells were also performed by the checkerboard assay and represented by FICI as described above [ 22 ].

The metabolic activity was performed as described before with a few modifications [ 23 ]. After incubations with antifungal drug, the supernatant was pipetted out and each well was washed by pH 7.

Subsequently, the newly-prepared XTT solution was pipetted into the pre-washed wells. The T-K test were performed according to a previous procedure with a small modifications [ 24 ].

albicans isolates in a well bottom-flat polystyrene microtiter plate. The corresponding MICs in combination were used in the ten isolates for 48 h of incubation at 37°C.

At 8, 16, 24, 32, 40, and 48 h, the strain broth was pipetted out and spread on sabouraud dextrose agar SDA, Gibco, Invitrogen, Carlsbad, CA, USA to evaluate colony forming unit CFU for another 48 h of incubation at 37°C. The control contained fungal cells and broth medium with no drug. The interpretations were based on the h of results as follows: the synergism was defined as an increase of no less than fold in killing for the combined agents compared with the most active agent used alone, antagonism was defined as a decrease of no less than fold in killing for the combined agents compared with the most active agent alone, and the indifference was defined as a less than fold in killing for the combined agents compared with any agents used [ 25 ].

The control included no drug. After incubations, the supernatant was discarded and the wells were washed three times by sterile PBS. The adhered cells were detected by XTT assay as described above and observed by SEM as described below. After critical point drying, the samples were sputter coated with gold in a vacuum evaporator, and the morphologies of C.

alcbians 04 were observed by a scanning electron microscope SEM, JSMF, Japan. The CSH of C. albicans 04 was surveyed by the water-hydrocarbon twophase assay followed by a previous report [ 26 ].

After 10 min g of centrifugation, the fungal cells were re-suspended with sterile PBS and adjusted OD to 1. After the mixed solution was separated into two definite phases by 3 min vortex, the OD of the aqueous phase was detected.

The control was set as the OD of the aqueous phase without the octane overlay. The flocculation procedures were followed by a previous description with minor modifications [ 27 ].

Briefly, the adjusted C. Then the suspension was vortexed for several seconds and photographed immediately. The sediments of cells were observed by inverted microscope OLYMPUS, Tokyo, Japan after 2 min. As for the test on solid plate, the strain broth of C. The colony shape on solid plate was photographed by a digital camera Olympus, Tokyo, Japan.

Before the test on semisolid plate, GM-BCP medium 10 g peptone, 5 g glucose, 0. Finally, the plate was dissected and the transection of colony was photographed laterally. Then the slides were placed in wells of a 6-well plate containing both the strain broth of C.

The working solution of FDA , µL freshly diluted in sterile PBS was added into each pre-rinsed well of well microtiter plate with µL fungal solution. The plate was incubated in the dark at 37 °C for 30 min on a rocking table, and observed by a fluorescent microscope Olympus IX81, Tokyo, Japan at the emission wavelength of nm.

The biofilm biomass was measured by crystal violet staining. Briefly, the C. After 10 min of dyeing, the staining solution was pipetted out gently and the loose cells were removed by sterile PBS. Finally, the OD value was recorded at the wavelength of nm by a microplate reader.

The CLSM procedure were undertaken as described before with a few modifications [ 28 ]. Then the supernatant was aspirated and the wells were rinsed by sterile PBS for several times. Subsequently, the freshly-prepared FUN1 solution was added into each well for 30 min of incubation at darkness.

The images were taken by confocal laser-scanning microscope Olympus Fluoview FV , and a detailed three-dimensional image of biofilm was performed using Z-stacks depending on the height of the biofilm.

After washing by PBS, the pieces were pre-incubated with fetal calf serum FCS at 37°C for 4 h, and then transferred to a clean well microtiter plate, co-incubated with C.

After incubation, the next process were divided into two steps: on one hand, the supernatant µL was aspirated into a new well microtiter plate, and the OD value was detected at the wavelength of nm; on the other hand, the pieces were fetched out by forceps, rinsed by PBS, put into glass tubes with 1 mL PBS, and vortexed rigorously to detach the biofilm completely.

The solutions containing detached biofilm cells were serially 10 × diluted and spread on SDA plate for CFU counting.

The qRT-PCR were performed as previously described with several modifications [ 29 ]. The total RNA was extracted from C. albicans 04 using MagExtractor-RNA kit ToyoBo, Tokyo, Japan. The absorption was measured at and nm to confirm the purity of RNA and to calculate the amount of RNA.

Approximately 1 μg of the extracted total RNA was used to synthesize cDNA by ReverTra Ace qPCR RT Master Mix with gDNA Remover kit ToyoBo, Tokyo, Japan.

The primers of ALS1 , ALS3 , HWP1 , SUN41 , UME6 , ECE1 , PDE2 , NRG1 , HSP90 and ACT1 were listed in Table 1. The procedures of qRT-PCR was performed on ABI fluorescent quantitative PCR system Applied Biosystem including 95°C for 5 min, 40 cycles of 95°C for 15 s, 48°C for 15 s, 72°C for 45 s.

All data were normalized to housekeeping gene ACT1 reference gene. The relative target-gene expression was calculated as a fold change of 2 -ΔΔCt value as previously described [ 30 ]. The murine VVC model was established according to the previous procedures described in [ 31 ].

The animal care and use committee of the First People's Hospital of Shangqiu approved the designs and procedures of the experiment. All mice were housed in the animal center, given standard food and water provided ad libitum at room temperature for at least a week prior to next experiments.

Three days prior to inoculation, mice were injected with µl of sesame oil containing 0. Estrogen injections were administered once every other day for three times in a week. A loopful of C. albicans 04 blastoconidia from Sabouraud-dextrose agar was added into 10 ml of YPD broth supplemented with 0.

An aliquot of 20 μL of the stationary-phase suspension, generating an inoculum size of 2 × 10 6 blastoconidia, was injected into the vaginal lumen of the estrogen-treated mice. The normal controls were inoculated with 20 µL of sterile PBS. The infected mice underwent vaginal lavage immediately with 0.

The normal control and model control were treated with PBS. At day 1 postinoculation, the vaginal lumens of mice were lavaged by PBS with repeated aspiration and agitation with a pipette tip. The lavage fluids were combined on ice during processing, were removed to perform fungal colony counting, morphology and HE staining.

All experiments were performed triplicate in three different occasions. The data were presented as means ± standard deviation and calculated by SPSS Differences between groups were determined using analysis of variance ANOVA.

The susceptibilities of 17 C. The susceptibility test showed synergism in FCZ-resistant isolates under planktonic state, and both FCZ-sensitive and -resistant isolates under biofilm states when QCT and FCZ were combined. Due to their susceptibilities to the combination of QCT and FCZ under both planktonic and biofilm states, the 10 FCZ-resistant isolates were used in T-K test.

It could be observed that the antifungal activities of QCT and FCZ alone were weaker than that of their combination Fig. The following experiments were performed in C. albicans 04 isolate due to its strong susceptibility to the combination of QCT and FCZ.

f I: indifference. g S: synergismc. Time-Kill curves of ten clinical FCZ-resistant C. The adherence is the first step of fungal biofilm formation and the initial reversible adherence is especially important for subsequent biofilm development.

The similar conclusions could be made from the SEM results Fig. The Candida CSH and flocculation were also of essential indicators of fungal adherence. A study was conducted in Taiwan, to study the synergistic effect of quercetin, irinotecan and its metabolite SN in the gastric cancer cell line of humans both in vitro and in vivo.

Results for in vitro analysis revealed that low-dose SN combined with quercetin showed comparable therapeutic effects as that of high-dose SN β -catenin protein expression level increased in the group managed with high-dose SN, while it was less in quercetin alone and as well in combined low doses of SN with quercetin.

In vivo results showed high levels of cyclooxygenase-2 and several markers of epithelial-mesenchymal such as Twist1 and ITGβ6 in rat models treated with irinotecan, while their levels were low in group treated with quercetin combined with a low dose of irinotecan. Several studies showed anticancer effects of quercetin by apoptosis and suppress cell proliferation of breast, lung, oral and prostate cancer.

Toru Hisaka et al. for the first time examined antitumor effects of quercetin on thirteen liver cancer cell lines 13 HCC in vitro.

Quercetin alone and in combination with 5-fluorouracil 5-FU was given, and cell viability was performed by MTT assay. The results showed synergistic activity of quercetin and 5-FU by cycle arrest via induction of apoptosis [ 11 ].

EMT6 a breast cancer cell line was used to induce tumor in mice and was subcutaneously injected. These findings suggest synergistic activity of quercetin and cisplatin on breast cancer cell lines along with decreased side effects of cisplatin in animal models [ 42 ].

Among the most prevalent cancers in females, ovarian cancer ranks seventh worldwide. It is a serious malignancy affecting the reproductive system in females. Guangya Xu et al. enhanced the effect of quercetin as an anticancer agent but improving its solubility profile.

For this, a quercetin-loaded thermo-sensitive injectable hydrogel system Qu-M-hydrogel was made based on nanotechnology. Quercetin was encapsulated using MPEG-PCL and later added into a thermosensitive hydrogel.

The formulated hydrogel system showed a slower release of quercetin in vivo. Qu-M—hydrogel composites showed enhanced apoptosis and inhibition of cell growth effects on the ovarian cancer mouse model SKOV-3 [ 43 ].

Investigating the role of quercetin as an anticancer agent against lung cancer, researchers in China conducted a study in which nanoparticles were made comprised of gefitinib and quercetin. Antitumor activity was conducted both in vitro and in vivo.

Nanoparticles formed showed good entrapment and release of both drugs up to 12 h. Results for both in vitro and in vivo studies confirmed the enhanced antitumor effect of gefitinib and quercetin [ 44 ].

Deantari karliana et al. formulated nanoparticles gel consisting of lecithin-quercetin injected into chitosan-tocopherol polyethylene glycol succinate TPGS to study the effects on osteoarthritis.

White male Sprague—Dawley rats 2—3 month-old weighed — g were used for this study and divided into five groups. The amount of quercetin administered through nanoparticle gel was 0.

Significant reduction in inflammation and edema was observed at dose 3. A study reported in Korea gave the anti-inflammatory mechanism of two flavanols, namely galangin and quercetin in lipopolysaccharide-stimulated RAW Atopic dermatitis induced by a 2,4-dinitrochlorobenzene mouse model was used.

Both the compounds were given alone and also in combination with study the synergistic effect. Results of the study revealed that both these compounds reduce nitric oxide production, interleukin-6 and nuclear factor NF-kB. From histological and ear thickness measurement, it was concluded that reduction in inflammation and IgE levels were greatly reduced when these flavonoids were used in combinations.

Thus, quercetin and galangin combination provided new ways for the prevention of AD [ 46 ]. Ex vivo COX-1 and lipoxygenase LOX assays using human platelets were applied for assessing anti-inflammatory potential.

The method was based on the inhibitory potential of compounds like eicosanoids and prostaglandins catalyzed by inflammatory enzymes response, COX-1 and LOX.

Severe inflammation and inflammatory pneumonia is the risk factor associated with SARS-Cov Cytokine storm is responsible for death in infected patients mainly due to acute lung injury, acute respiratory distress syndrome and multiple organ dysfunction syndromes.

The study revealed that NLRP3 is an inflammasome responsible for the activation of several inflammatory mediators like NRF2, SIRT1 and TXNIP. Quercetin by affecting this inflammasome successfully suppressed NLRP3 and thus acts as a potential treatment for severe inflammation and in life-threatening conditions like COVID [ 47 ].

Endothelial cell function is affected by cytokines and pro-inflammatory stimuli such as high blood glucose levels. Quercetin was investigated to reduce the harmful effects of hyperglycemia and inflammatory conditions on vascular endothelium. The metabolomics approach was used to identify and quantify 27 human umbilical veins endothelial cell HUVEC metabolites.

A significant increase in lactate and glutamate concentrations was observed with the treatment of high glucose concentrations in HUVECs. Lactate and adenosine triphosphate concentrations were inhibited by quercetin, while inosine levels were increased.

TNF- α and quercetin reduced pyruvate concentration. Quercetin altered HUVEC metabolites balance toward less inflamed phenotype, both alone and in the presence of pro-inflammatory stimuli [ 48 ]. Mousumi Patra et al. reported a novel complex of calcium phosphate-quercetin-nanoparticles CPQN which was synthesized by precipitation method.

For this, quercetin, calcium nitrate and ammonium hydrogen phosphate were used as precursors, and stabilization was done by sodium citrate. Nanoparticles formed had a unique property of color change at different pH which could be used as a pH indicator.

These nanoparticles were used as fluorophores to mark biological cells. Besides this, CPQN was investigated for antioxidant activity on mouse neuroblastoma cell N2A, by H 2 O 2 -induced oxidative stress. The nanoparticles exhibited remarked antioxidant properties and thus emerged as a unique beneficial moiety [ 49 ].

Hassanien et al. formulated a new complex for diabetes consisting of cobalt complexed with quercetin, cobalt-quercetin complex CQC by mixing quercetin solution and cobalt chloride solution in molar concentrations.

Several histopathological and biochemical parameters were assessed along with blood glucose levels, and it was found that CQC efficiently reversed diabetes-induced changes through its strong antioxidant activity. Antihyperglycemic effects of CQC were comparable with insulin [ 50 ]. Another study was conducted in Egypt to investigate metal complexed with quercetin for the management of diabetes.

Here, Zn NO3 2·6H2O was added in the presence of ammonia solution to formulate quercetin-zinc complex having formula ZnQNO 3 H 2 O·5H 2 O which was confirmed by 1 HNMR. For analyzing the antidiabetic profile of the complex formed, a streptozotocin STZ -induced diabetes rat model was used.

Several biochemical parameters along with glycosylated hemoglobin were analyzed. Quercetin possesses permeability glycoprotein P-gp inhibitory activity which could be enhanced by conjugating it with metals. Six different metal complexes of quercetin Cu, Zn, Co, Vd, Mo, Ni were synthesized and analyzed in vitro by everted sac intestinal model of rats.

The permeability of atorvastatin was observed in different control and experimental groups. Yuzhi Mu et al. reported a novel pH responsive nano-micelle based on quercetin, chitaconic anhydride and chitosan QT-CA-CS.

Anticancer drug doxorubicin was encapsulated in QT-CA-CS self-assembled nano-micelles by ultrasound method.

The advantage of this complex was that drug efflux from the cancerous cell was inhibited due to inhibition of the P-glycoprotein pump. At an acidic pH of 4. These nano-micelles escaped the lysosomes and released doxorubicin and quercetin faster in the cytoplasm which resulted in synergistic anticancer activity against MCF-7 breast cancer cells [ 52 ].

Rafael de Oliveira Pedro et al. reported a simple method in which chitosan self-assembled amphiphilic nanoparticles loaded with quercetin were made for treating MCF-7 breast cancer cell lines.

A larger release profile at pH 5 was observed. MTT assay revealed inhibitory effects of quercetin in nanoparticle form due to greater uptake by the cells.

Nanoparticles were hemocompatible, showing the emergence of a novel drug delivery system for cancer therapy [ 53 ]. Juan-Juan Ma et al.

developed Zein-chitosan nanoparticles for loading quercetin ZCPs-Q to increase its solubility in water. Results revealed that ZCPs-Q increases the solubility of quercetin and stability in water.

Thus, these nanoparticles are suitable for loading nutraceuticals for enhancing cellular uptake with an increased antioxidant profile [ 54 ]. Jalil Rashedi et al.

formulated chitosan and quercetin nanoparticles by inotropic gelation method for loading 5-FU. Anticancer effects were observed on colon cancer of Wistar rats. Firstly, the tumor was induced by using 1, 2-dimethylhydrazine DMH and dextran sulfate sodium DSS.

Rats were provided with NPs in the form of an enema. Results revealed high encapsulation efficiency of quercetin with an increased release profile up to 24 h. A decrease in microvascular density and mitosis rate was noticed in all the treatment groups as compared to the control group, confirming the promising nature of NPs in site-specific colorectal cancer [ 55 ].

Wenhao Nan et al. reported the protective effects of quercetin against ultraviolet-B radiations to protect skin damage, cancer and photoaging along with inflammation.

Due to low hydrophilicity and percutaneous absorption, quercetin use was limited in topical preparations. For this, chitosan nanoparticles were made using sodium tripolyphosphate and quercetin was entrapped in these nanoparticles.

Results revealed remarkable skin penetration of quercetin with better stability and low cytotoxicity in HaCat cells. It was concluded that quercetin loaded on chitosan TPP nanoparticles can be used as topical preparation against ultraviolet-b radiations [ 56 ].

Virginia Tzankova et al. Wistar rats were taken and paracetamol-induced liver injury was treated with these nanoparticles which showed significantly decreased levels of serum transaminases ALT and AST and restored gluthation levels.

In vitro cell viability of HepG2 cells decreased with the treatment of encapsulated quercetin in nanoparticles. Li Lv et al. reported a new therapeutic drug delivery system by incorporating doxorubicin and quercetin on biotin-decorated nanoparticles.

The cytotoxic study was done on MCF-7 breast cancer cell lines and ADR. By inhibition of P-glycoprotein, enhanced cytotoxic effects were observed in biotin decorated nanoparticles as compared to nanoparticles without biotin.

Thus it was concluded that MDR cancer cells can be treated with these novel nanoparticles [ 58 ]. This review examined that quercetin is a safe dietary supplement with a variety of biological functions in animals as well as in humans.

Majority of the literature showed its safety profile in animals as an antimicrobial, antidiabetic, anticancer, antioxidant and anti-inflammatory agent. However, further evaluation in this regard with accurate outcomes is much needed.

Poor solubility and oral bioavailability of quercetin were a major problem in its use which was managed by making its complexes with polymers and metal ions in sustained released microspheres, nanospheres and liposomal dosage forms.

Synergistic effects of quercetin with anticancer, antimicrobials, antidiabetics and anti-inflammatory agents make it an interesting compound for exploring new treatment modalities for acute and chronic human diseases with lesser side effects and improved efficacy.

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rubrum strains cultured in the presence of the MIC of quercetin when compared to untreated cells. This result is comparable to the reduction caused by MICs of fluconazole and cerulenin, which ranged from 40 to In contrast, the 0.

As can be seen in Table 4 , trans-chalcone was the best compound to inhibit protoplast regeneration in the two T. rubrum strains when tested at the MIC and 0. Similar results were obtained for cerulenin, a finding suggesting that trans-chalcone also acts by disrupting cell membrane homeostasis.

Interestingly, protoplast regeneration was higher in the mutant strain than in the wild-type strain in the presence of 0. Half the MIC of quercetin did not reduce the number of regenerated wild-type fungal colonies, but caused a Quantitative RT-PCR was used to evaluate the modulation of FAS1 and ERG6 gene expression in the presence of flavonoids.

The FAS1 gene of T. rubrum was repressed in the presence of MICs of quercetin, trans-chalcone, fluconazole, and cerulenin. The ERG6 gene was induced in the presence of MICs of fluconazole and cerulenin and was repressed in the presence of MICs of trans-chalcone and quercetin after 16 h of growth of T.

rubrum Figure 2. Relative fold change in the FAS1 and ERG6 genes determined by quantitative RT-PCR. The IC 50 value was Chalcones 1,3-diarylpropenone are open-chain flavonoids that are mainly synthesized by plants. These compounds have been shown to exert significant antifungal activity, especially against dermatophytes [ 18 ].

Most chalcones inhibit the biosynthesis of the yeast cell wall [ 19 ], but there is evidence that chalcones also block FAS in yeast 12 and fatty acid synthase II in mycobacteria [ 20 ]. In the present study, the MICs of quercetin, trans-chalcone, cerulenin and fluconazole were always lower for the mutant strain than for the wild-type strain MYA This higher susceptibility of the mutant strain might be explained by disruption of the TruMDR2 gene.

This gene encodes an ABC transporter that is involved in the resistance to various antifungal agents, such as terbinafine, 4NQO, and ethidium bromide [ 15 ]. To our knowledge, this is the first study reporting the involvement of the TruMDR2 transporter in flavonoid-mediated FAS inhibition.

A reduction of ergosterol content was observed in the two T. rubrum strains when the fungus was grown in the presence of the MIC of quercetin. However, a higher percent reduction of ergosterol content was found when the strains were grown in the presence of 0.

In fungi, sterols are found in the plasma membrane where they play a role in membrane permeability. The protoplast regeneration assay has been used to evaluate the level of plasma membrane damage caused by antifungal compounds [ 21 , 22 ].

Quercetin and trans-chalcone reduced the number of regenerated protoplasts in the two strains, suggesting an effect of these compounds on the cell membrane. Interestingly, in the presence of the 0. On the other hand, ergosterol content was lower in the mutant strain In yeast, osmotic stress NaCl and sorbitol has been shown to increase the level of resistance of cells treated with drugs that inhibit ergosterol synthesis [ 23 ].

This phenomenon observed for yeast may explain the better regeneration seen in the T. rubrum mutant strain. However, further studies are needed to clarify these results. The expression of T.

rubrum FAS1 fatty acid synthases and ERG 6 ergosterol synthesis genes is modulated by the synthetic FAS inhibitors PHS11A [ 13 ] and PHS11B [ 14 ].

In this study, we also observed that the inhibition of fatty acid and ergosterol synthesis by quercetin and trans-chalcone may involve transcriptional modulation of these genes since they are down-regulated by both flavonoids.

In addition to transcriptional modulation of the FAS1 gene, experiments using S. cerevisiae cells have shown that quercetin and trans-chalcone inhibit the enzymatic activity of FAS. Quercetin showed a better inhibitory activity, but trans-chalcone showed a better antifungal activity.

This result suggests that the antifungal activity of trans-chalcone may be related to targets other than FAS, such as ergosterol synthesis. Ergosterol is a vital component of the fungal cell, but little is known about the genetics and biochemistry of the ergosterol biosynthesis pathway and only 20 genes involved in the biosynthesis of ergosterol in T.

rubrum have been sequenced [ 24 ]. Zhang et al. In addition, the ERG5 , ERG6 and ERG25 genes have been shown to be up-regulated in Candida albicans strain SCAR, which is resistant to fluconazole and amphotericin B. The higher expression of these genes may increase the conversion of lanosterol to eburicol and methyl fecosterol.

By altering the pathway at this particular point, the cell would no longer be susceptible to the effects of fluconazole or amphotericin B. Analysis of sterol content in this strain confirmed the hypothesis that resistance to the two antifungal agents was due to the accumulation of sterol intermediates, which is consistent with the inactivation of lanosterol demethylase, and to the increased expression of several ergosterol biosynthesis genes [ 25 ].

According to Leber et al. Furthermore, ergosterol has been reported to regulate its own synthesis by negative feedback mechanisms [ 27 — 30 ]. The complete assessment of the mode of action of quercetin and trans-chalcone will require the analysis of additional genes and other assays that could demonstrate the interference of FAS inhibition with fatty acid homeostasis in T.

One possibility is supplementation of the growth medium with exogenous fatty acids in the presence of quercetin and trans-chalcone. Nevertheless, the present results indicate that these compounds can be explored for the development of new antifungal drugs.

Quercetin and trans-chalcone presented the best antifungal activity among the flavonoids tested. The results of the FAS enzymatic assay showed that quercetin is a better inhibitor of this enzyme than trans-chalcone. However, the lower MIC of trans-chalcone when compared to quercetin suggests the involvement of another cellular target in addition to FAS.

Therefore, trans-chalcone is a potential candidate for the development of new antifungal drugs against T. rubrum since it simultaneously inhibits the synthesis of fatty acids and ergosterol, a fact reducing the risk of resistance.

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