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Ethnopharmacological Relevance: The traditional Chinese medicine formula known as Pulsatilla decoction was utilized to treat conditions such as bacterial dysentery, ulcerative colitis, and fungal infections like vulvovaginal candidiasis (VVC) caused by Candida albicans (C. albicans). In our prior research, it was shown that the n-butanol extract from Pulsatilla Decoction (BEPD) exhibited effective inhibition of C. albicans. Nevertheless, the exact mechanism by which BEPD hinders hyphal growth, a critical virulence factor of C. albicans, remains unclear., Aim of the Study: In the present study, the inhibitory effect and mechanism of the BEPD on C. albicans hyphal growth was predicted by transcriptome analysis, and further verified by in vitro and in vivo experiments., Materials and Methods: The BEPD was prepared and C. albicans was cultured to induce the hyphal state. Transcriptome analysis was conducted to predict the significant difference in enrichment genes and signaling pathways in the inhibitory effect of BEPD on C. albicans hyphae. Various methods, such as spot assay, time-growth curve analysis, Confocal laser scanning microscope (CLSM), scanning electron microscope (SEM), transmission electron microscope (TEM), flow cytometry, and spectrophotometer, were used to assess the effect of BEPD on hyphal structure and growth activity, lipid peroxidation level, peroxidase (CAT) activity, superoxide dismutase (SOD) activity, and apoptosis of C. albicans. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) was employed to examine the expression levels of genes associated with endoplasmic reticulum and peroxisome function. The VVC model was employed to evaluate the influence of BEPD on the growth of C. albicans hyphae in vivo., Result: The growth of C. albicans hyphae on solid culture media was significantly inhibited by BEPD. CLSM showed that the length of C. albicans hyphae was decreased and their vitality was lowered. SEM indicated that the hyphae of C. albicans were fractured, while TEM revealed damage to the organelles within the cells. GO enrichment and KEGG pathways analysis from transcriptomic data demonstrated that BEPD effectively suppressed the functioning of the endoplasmic reticulum and peroxisomes in C. albicans hyphae. RT-qPCR verified the decreased expression of genes associated with endoplasmic reticulum and peroxisome function by BEPD. Investigation of the endoplasmic reticulum revealed that BEPD elevated levels of reactive oxygen species (ROS) and apoptosis, indicating endoplasmic reticulum stress, as well as malondialdehyde (MDA), a marker of oxidative stress. Additionally, BEPD was shown to lower the activities of catalase (CAT) and superoxide dismutase (SOD). In animal trials, BEPD effectively hindered the growth of C. albicans hyphae in the vaginas of mice with VVC, thus reducing immune inflammatory damage to the vaginal mucosa of these mice., Conclusion: This study demonstrates that BEPD has an inhibitory effect on hyphae, which are an important virulence factor of C. albicans. This effect may be related to BEPD's inhibitory effect on endoplasmic reticulum (ER) and peroxisome function. The findings suggest that BEPD could potentially play a therapeutic role in C. albicans infectious diseases by inhibiting hyphae., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The emergence of the "super fungus" Candida auris poses a significant threat to human health, given its multidrug resistance and high mortality rates. Therefore, developing a new antifungal strategy is necessary. Our previous research showed that Baicalein (BE), a key bioactive compound from the dried root of the perennial herb Scutellaria baicalensis Georgi, has strong fungistatic properties against C. auris. Nevertheless, the antifungal activity of BE against C. auris and its mechanism of action requires further investigation. In this study, we explored how BE affects this fungus using various techniques, including scanning electron microscopy (SEM), Annexin V-FITC apoptosis detection, CaspACE FITC-VAD-FMK In Situ Marker, reactive oxygen species (ROS) assay, singlet oxygen sensor green (SOSG) fluorescent probe, enhanced mitochondrial membrane potential (MMP) assay with JC-1, DAPI staining, TUNEL assay and reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Our findings revealed that BE induced several apoptotic features, including phosphatidylserine (PS) externalization, metacaspase activation, nuclear condensation and DNA fragmentation. BE also increased intracellular ROS levels and altered mitochondrial functions. Additionally, transcriptomic analysis and RT-qPCR validation indicated that BE may induce apoptosis in C. auris by affecting ribosome-related pathways, suggesting that ribosomes could be new targets for antifungal agents, in addition to cell walls, membranes, and DNA. This study emphasizes the antifungal activity and mechanism of BE against C. auris, offering a promising treatment strategy for C. auris infection., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Candida auris, a rapidly spreading multi-drug-resistant fungus, is causing lethal infections under certain conditions globally. Baicalin (BE), an active ingredient extracted from the dried root of Scutellaria baicalensis Georgi, exhibits antifungal activity. However, studies have shown the distinctive advantages of Traditional Chinese medicine in combating fungal infections, while the effect of BE, an active ingredient extracted from the dried roots of Scutellaria baicalensis Georgi, on C. auris, remains unknown. Therefore, this study aims to evaluate the potential of BE as an antifungal agent against the emerging multidrug-resistant C. auris. Various assays and models, including microbroth dilution, time growth curve analysis, spot assays, adhesion tests, flocculation test, cell surface hydrophobicity assay, hydrolase activity assays, XTT assay, violet crystal assay, scanning electron microscope (SEM), confocal laser scanning microscope (CLSM), flow cytometry, Live/dead fluorescent staining, reactive oxygen species (ROS), cell wall assay, aggregation assay, porcine skin model, Galleria mellonella larvae (G. mellonella larvae) infection model, and reverse transcription-quantitative polymerase chain reaction (RT-PCR) were utilized to investigate how baicalein suppresses C. auris through possible multifaceted mechanisms. The findings indicate that BE strongly inhibited C. auris growth, adhesion, and biofilm formation. It also effectively reduced drug resistance and aggregation by disrupting the cell membrane and cell wall while reducing colonization and invasion of the host. Transcriptome analysis showed significant modulation in gene expression related to different virulence factors post-BE treatment. In conclusion, BE exhibits significant effectiveness against C. auris, suggesting its potential as a viable treatment option due to its multifaceted suppression mechanisms., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Ethyl caffeate (EC) is a phenylpropanoid compound derived from Elephantopus scaber. In our previous work, EC was investigated to have a strong synergistic antifungal effect against azole-resistant strains of Candida albicans when combined with fluconazole (FLU). However, the protective effect and mechanism of EC + FLU on oropharyngeal candidiasis (OPC) caused by drug-resistant strains of C. albicans have not been investigated. This study aimed to investigate the protective effect and mechanism of EC combined with FLU against C. albicans-resistant strains that lead to OPC. An OPC mouse model revealed that EC + FLU treatment reduced fungal load and massive hyphal invasion of tongue tissues, and ameliorated the integrity of the tongue mucosa. Periodic acid-Schiff staining results showed more structural integrity of the tongue tissues and reduced inflammatory cell infiltration after EC + FLU treatment. Phosphorylation of EGFR (epidermal growth factor receptor) and other proteins in the EFGR/JNK (c-Jun N-terminal kinase)/c-JUN (transcription factor Jun) signaling pathway was significantly downregulated by EC + FLU. EGFR and S100A9 mRNA expression were also reduced. The above results were verified in FaDu cells. ELISA results showed that the concentration of inflammatory factors in the cell supernatant was significantly reduced after EC combined with FLU treatment. Molecular docking revealed that EC exhibited high binding energy to EGFR. In conclusion, EC enhances the susceptibility of azole-resistant C. albicans to FLU, and the underlying mechanism is related to the inhibition of the EGFR/JNK/c-JUN signaling pathway. This result suggests that EC has potential to be developed as an antifungal sensitizer to treat OPC caused by azole-resistant C. albicans., (© The Author(s) 2023. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology.)

Traditional herbal monomers (THMs) are widely distributed in many traditional Chinese formulas (TCFs) and decoctions (TCDs) and are frequently used for the prevention and treatment of fungal infections. The antifungal activities of five common THMs, including sodium houttuyfonate (SH), berberine (BER), palmatine (PAL), jatrorrhizine (JAT) and cinnamaldehyde (CIN), and their potential for inducing cell wall remodeling (CWR), were evaluated against Candida albicans SC5314 and Candida auris 12372. SH/CIN plus BER/PAL/JAT showed synergistic antifungal activity against both Candida isolates. Furthermore, SH-associated combinations (SH plus BER/PAL/JAT) induced stronger exposure of β-glucan and chitin than their counterparts, while CIN triggered more marked exposure compared with CIN-associated combinations (CIN plus BER/PAL/JAT). Collectively, this study demonstrated the anti- Candida effect and the CWR induction potential of the five THMs and their associated combinations, providing a possibility of their in vivo application against fungal-associated infections.

Context: Fluconazole resistance is an intractable problem of treating Candida albicans, calling for more antifungal agents to enhance the activity of fluconazole., Objective: This work investigates the anti-C. albicans activities of sodium houttuyfonate (SH) and/or fluconazole and the associated mechanism., Materials and Methods: The minimum inhibitory concentrations (MICs) of SH and fluconazole both ranging from 0.5 to 1024 μg/mL were determined by broth microdilution method in 19 C. albicans isolates, and their fractional inhibitory concentration index (FICI) was evaluated by checkerboard assay. After MIC SH and/or MIC fluconazole treatments, the expressions of IFD6, PHR1, ZAP1, ADH5, BGL2, XOG1 and FKS1 were analyzed by quantitative reverse transcription polymerase chain reaction (qRT-PCR) in C. albicans 1601., Results and Conclusion: The MICs of SH alone ranged from 32 to 256 μg/mL and decreased 2-16-fold in combination. SH showed strong synergism with fluconazole with FICI <0.13-0.5. In C. albicans 1601, we observed that (i) the expression of the seven genes increased notably in a range between 3.71- and 12.63-fold (p < 0.05) when SH was used alone, (ii) the combined use of SH and fluconazole slightly inhibited the expression of IFD6 and PHR1 by 1.23- and 1.35-fold (p > 0.05), but promoted evidently the expression of ZAP1, ADH5, XOG1 and FKS1 by 1.98-, 3.56-, 4.10- and 2.86-fold (p < 0.05). The results suggested SH to be a potential synergist to enhance the antifungal activity of fluconazole in C. albicans resistant isolates, and the underlying mechanism may be associated with β-1,3-glucan synthesis and transportation.

Background: Candida tropicalis was one of the most common non-albicans Candida (NACA) species creating relatively high morbidity and mortality. We previously demonstrated that the antifungal effect of BBR in combination with FLC might be attributed to reactive oxygen species production, ergosterol content and efflux pump levels in a clinical C. tropicalis isolate., Methods: we employed thirteen clinical C. tropicalis isolates as well as four standard reference strains to evaluate the antifungal activity of BBR in combination with FLC under planktonic and biofilm states by analyzing the mRNA expressions of ERG11, CDR1 and MDR1 by qRT-PCR., Results: The susceptibility tests showed that BBR in combination with FLC produced a FICI range of 0.13-1 under planktonic state and 0.25-1 under biofilm state in the seventeen strains. The mRNA expressions of ERG11, CDR1 and MDR1 were upregulated 1.43-2.10 and 1.47-4.42 fold in the case of BBR/FLC used alone compared with the control. The combined usage of BBR plus FLC resulted in a decrease of 2.84- (p<0.01) and 2.39-fold (p<0.05) in the expression of ERG11 mRNA, 2.98- (p<0.05) and 3.06-fold (p<0.05) in the expression of CDR1 mRNA, 1.41- and 4.33-fold in the expression of MDR1 mRNA compared with the BBR/FLC used alone., Conclusion: The synergism of BBR with FLC might be relevant with the inhibitions on the mRNA expressions of ERG11 and efflux pumps., (Copyright © 2017 Elsevier Masson SAS. All rights reserved.)

The aim of this study was to investigate the antifungal activity of baicalin and its potential mechanism of action against Candida albicans biofilms. The standard techniques including microdilution method and checkerboard assay were employed to evaluate the susceptibilities of baicalin alone and in combination with fluconazole against planktonic and biofilm cells of C. albicans. Transmission electron microscope (TEM), scanning electron microscope (SEM), fluorescent microscope and flow cytometry were used to assess the apoptotic incidences induced by baicalin in biofilm cells. The expressions of four genes (RAS1, CAP1, PDE2 and TPK1) related to Ras-cAMP-PKA pathway were also analyzed by quantitative reverse transcription polymerase chain reaction (qRT-PCR). The results showed that minimum inhibitory concentration (MIC) and sessile minimum inhibitory concentration (SMIC50) of baicalin were 500 and 2000 μg/mL with fractional inhibitory concentration indexs (FICIs) ranging from 0.28 to 0.75. A series of events related to apoptosis were observed in baicalin-treated C. albicans biofilms, including extensive chromatin condensation along the nuclear envelope, ROS accumulation, MMP reduction, PS externalization, nuclear fragmentation, chromatin condensation, metacaspase activation and Cyt C release. Additionally, the expressions of RAS1 and TPK1 were up-regulated by 3.2 and 2.9 folds respectively, while those of CAP1 and PDE2 were down-regulated by 3.3 and 6.6 folds respectively after exposure to baicalin in biofilm cells. In conclusion, baicalin can suppress the development of C. albicans biofilms most likely due to inducing cell death via apoptosis., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Vulvovaginal candidiasis (VVC) is an inflammatory disease of the vagina mainly caused by Candida albicans (C. albicans), which affects around three-quarters of all women during their reproductive age. Although some antifungal drugs such as azoles have been applied clinically for many years, their therapeutic value is very limited due to the emergence of drug-resistant strains. Previous studies have shown that the adhesion of C. albicans to vaginal epithelial cells is essential for the pathogenesis of VVC. Therefore, preventing the adhesion of C. albicans to vaginal epithelial cells may be one of the most effective strategies for the treatment of VVC. Berberine (BBR) is a biologically active herbal alkaloid that was used to treat VVC. However, so far, its mechanism has remained unclear. This study shows BBR significantly inhibits the adhesion of C. albicans to vaginal epithelial cells by reducing the expressions of ICAM-1, mucin1, and mucin4 in vaginal epithelial cells, which play the most important role in modulating the adhesion of C. albicans to host cells, and balancing IL-2 and IL-4 expressions, which play a key effect on regulating the inflammatory response caused by C. albicans infection. Hence, our findings demonstrate that BBR may be a potential therapeutic agent for VVC by interfering with the adhesion of C. albicans to vaginal epithelial cells and represents a new pathway for developing antifungal therapies agents from natural herbs. [ABSTRACT FROM AUTHOR]

This study aimed to evaluate the mono- and dual- antifungal activities of paeonol (PAE) and fluconazole (FLZ)/amphotericin B (AmB). To this end, the effects of PAE and FLZ/AmB on cell surface hydrophobicity, hydrolase activity, morphological transition were investigated in vitro and in a Galleria mellonella infection model. The results showed a relatively high minimum inhibitory concentration (MIC) and sessile MIC (SMIC) of PAE alone. However, compared with the single drug, the combined use of PAE and FLZ/AmB had a potent synergistic potential to inhibit the virulence factors for Candida. The concomitant use of two drugs was consistently more effective than either drug alone for increasing survival rate, decreasing the fungal burden, and alleviating the pathological features of G. mellonella infected by the fungus. Taken together, these findings demonstrate the anti-Candida effects of PAE plus FLZ/AmB and their potential to increase the sensitivity of C. albicans to FLZ/AmB of PAE. [ABSTRACT FROM AUTHOR]

Context: Fungal infections caused by fluconazole-resistantCandida albicansare an intractable clinical problem, calling for new efficient antifungal drugs. Kaempferol, an active flavonoid, has been considered a potential candidate againstCandidaspecies. Objective: This work investigates the resistance reversion of kaempferol in fluconazole-resistantC. albicansand the underlying mechanism. Materials and methods: The antifungal activities of fluconazole and/or kaempferol were assessed by a series of standard procedures including broth microdilution method, checkerboard assay and time-kill (T-K) test in nine clinical strains as well as a standard reference isolate ofC. albicans. Subsequently, the morphological changes, the efflux of rhodamine 6G, and the expressions ofCDR 1,CDR 2, andMDR 1were analysed by scanning electron microscope (SEM), inverted fluorescence microscope and quantitative reverse transcription polymerase chain reaction (qRT-PCR) inC. albicansz2003. Results: For all the testedC. albicansstrains, the minimum inhibitory concentrations (MICs) of fluconazole and kaempferol ranged 0.25–32 and 128–256 μg/mL with a range of fractional inhibitory concentration index of 0.257–0.531. InC. albicansz2003, the expression of bothCDR 1andCDR 2were decreased after exposure to kaempferol alone with negligible rhodamine 6G accumulation, while the expression ofCDR 1,CDR 2andMDR 1were all decreased when fluconazole and kaempferol were used concomitantly with notable fluorescence of rhodamine 6G observed. Discussion and conclusion: Kaempferol-induced reversion in fluconazole-resistantC. albicansmight be likely due to the suppression of the expression ofCDR1,CDR2andMDR1. [ABSTRACT FROM AUTHOR]

Fungal infection caused by Candida albicans is a serious health problem, and as drug resistance worsens, new sources for therapeutic compounds are needed. Traditional Chinese medicine represents a wealth of such sources, and can be designed as suppositories for the treatment of recurrent vulvovaginal candidiasis (RVVC). This study aimed to develop a Pulsatilla suppository containing the n-butanol extract of Pulsatilla decoction (BEPD) to treat RVVC. A Pulsatilla suppository containing BEPD was prepared, and its performance, weight, drug content, dissolution time and percentage, stability, toxicology, and pharmacodynamics were evaluated. Biological compatibility tests and clinical evaluations were performed in female Sprague–Dawley rats. The Pulsatilla suppository melted completely within 30 min. In vitro anti- C. albicans activity, stability changes, and toxicology tests indicated stability and safety in the rats. Compared with RVVC model rats, high-dose BEPD suppository (40, 60 mg/kg) can significantly reduce the vaginal fungal load of rats, relieve neutrophil infiltration, reduce the content of TLR/MyD88 pathway-related proteins, and reduce the expression of inflammatory factors such as NLRP3, demonstrating the efficacy of the Pulsatilla suppository in RVVC. Pulsatilla suppository plays a role in the treatment of RVVC by inhibiting the activation of NLRP3 inflammasome through TLR/MyD88 signaling pathway. [Display omitted] [ABSTRACT FROM AUTHOR]