Research Progress on the Antifungal Mechanisms of Natural Products Against Candida Species (Part I)

Candida infection is one of the most common opportunistic fungal infections in clinical practice, which often occurs in immunocompromised individuals such as the elderly, cancer patients undergoing chemotherapy, post-transplant patients, and HIV-infected individuals. Due to the widespread use of traditional antifungal agents such as azoles, polyenes, and echinocandins, the emergence of naturally resistant Candida strains and the increasing proportion of non-albicans Candida infections have led to increasingly severe clinical Candida resistance. Natural products are known for their unique chemical diversity and biological activities, and have been widely used to treat various diseases for centuries. According to statistics, first-line antifungal drugs in clinical practice are often natural products or their derivatives. In recent years, significant progress has been made in the development of antifungal natural products, and a considerable number of natural molecules have been discovered to have therapeutic effects against different types of Candida. For example, biologically active molecules with well-defined chemical structures extracted from plant tissues and their derivatives have significant inhibitory effects on various Candida species, and have many advantages such as wide sources, few adverse reactions, and suitability for long-term or preventive use, making them an important source of novel antifungal agents.

Among the natural products against Candida, fungal metabolites are the main source, followed by plants. These compounds mostly belong to alkaloids, polyketides, terpenes, phenols, macrolides, and peptides. Among them, terpenes have the highest content, followed by alkaloids and polyketides.

① Terpenes are primarily metabolites of higher plants and endophytic fungi. Terpenes with anti-Candida activity include monoterpenes, sesquiterpenes, diterpenes, and triterpenes. Studies have shown that 14 triterpenes isolated from Ganoderma lucidum increase the sensitivity of Candida albicans resistant to fluconazole to fluconazole, and some of them can significantly inhibit Candida albicans. Sesquiterpenes isolated from the essential oil of Pinus sylvestris have antibacterial effects on Candida albicans, Candida glabrata, and Candida krusei.

② Alkaloids are nitrogen-containing basic compounds found in organisms and possess significant biological activities. There are many types of anti-Candida alkaloids, mainly including indoles, pyridines, and quinolones. Indoles and quinolones have a relatively broad antifungal spectrum, but their antibacterial activity is usually moderate. In contrast, the reported pyridine alkaloids exhibit strong antifungal activity.

③ Most of the polyketide compounds that are anti-Candida are isolated from fungal metabolites. For example, some polyketide compounds isolated from the endophytic fungus Phoma of mangrove plants show strong antibacterial activity against Candida albicans, with an MIC50 value of 2.62 μg/mL.

The mechanisms of action of natural products against Candida albicans mainly include inhibiting virulence factors of Candida, such as inhibiting Candida adhesion and invasion, yeast-to-hyphae morphological transition, biofilm formation, and synthesis and secretion of hydrolases; affecting cellular structural changes in Candida's cell wall, cell membrane, and mitochondria; inducing apoptosis in Candida cells; inhibiting the expression of drug target enzymes and efflux pumps in Candida; in addition, some natural products also enhance the anti-Candida effect by regulating immunity.

1.Inhibition of Adhesion and Invasion

Adhesion of Candida albicans is a crucial first step for its invasion and biofilm formation. The anti-Candida adhesion effect of plant active ingredients can be mainly confirmed through cell experiments. This involves co-culturing Candida with oral mucosal epithelial cells and observing the adhesion of Candida on the surface of oral mucosal epithelial cells under the action of plant active ingredients. Studies have found that when 400μg/mL paeonol and 1μg/mL fluconazole are used alone, the adhesion rates of Candida albicans to human oral mucosal epithelial cells are (93.33±4.84)% and (91.67±3.45)% respectively, while when used together, the adhesion rate is only (38.33±1.51)%, indicating that paeonol can synergistically inhibit the adhesion of Candida albicans to human oral mucosal epithelial cells with fluconazole. In addition, studies have shown that cinnamaldehyde and berberine, when used alone or combined with fluconazole, can not only inhibit the formation of Candida albicans germ tubes but also significantly inhibit their adhesion to human oral mucosal epithelial cells. The mechanism is that cinnamaldehyde, berberine, and fluconazole synergistically interfere with the synthesis of Candida albicans germ tube cell wall proteins and the regulation of related genes, thus inhibiting the formation of Candida albicans germ tubes and reducing their adhesion ability to host cells. Other scholars have selected the human colon adenocarcinoma cell line (Caco-2) to co-culture with Candida albicans and used the cell membrane probe (DiI) staining method to evaluate the effect of rhizoxin B on the ability of Candida albicans to invade host cells. The results showed that when rhizoxin B and fluconazole were used alone, the invasion rates of intracellular hyphae were (68±34)% and (57±16)% respectively, but when the two drugs were combined, the invasion rate of intracellular hyphae could be significantly reduced to (25±16)%, confirming that the plant active ingredient rhizoxin B has an anti-Candida invasion effect.

2.Inhibition of Yeast-to-Hyphae Morphological Transition

The yeast-to-hyphae morphological transition is the basis for Candida albicans to exert its pathogenicity. Candida albicans can adhere, penetrate, and invade host cells by transforming from the yeast form to the hyphal form, thereby exerting its pathogenicity. The relevant signaling pathways involved mainly include the cAMP/PKA signaling pathway, MAPK signaling pathway, Rim101-mediated pH signaling pathway, and Tup1-mediated negative regulatory pathway. Studies have shown that when the concentration of sanguinarine is 0.8 μg/mL, all Candida albicans in Spider medium remain in the yeast form, and no hyphae are formed. However, when the concentration of sanguinarine reaches 1.6 μg/mL, the expression levels of Candida albicans cAMP signaling pathway-related genes ALS3, HWP1, ECE1, HGC1, and CYR1 are significantly downregulated.

Research has found that 4 μg/mL of magnolol and honokiol can significantly inhibit the formation of Candida albicans hyphae. When their concentration reaches 16 μg/mL, no hyphae are formed in Spider medium. The possible mechanism is that magnolol and honokiol inhibit the expression of Ras1-cAMP-Efg1 signaling pathway-related genes RAS1, EFG1, TEC1, and CDC35. The quorum-sensing molecule farnesol can inhibit the activity of adenylate cyclase, acting on the Ras1-Cyr1-cAMP-Efg1 signaling pathway, thereby inhibiting the yeast-to-hyphae morphological transition and biofilm formation of Candida albicans. In contrast, tyrosol can promote the formation of hyphae and biofilms.

Another study found that andrographolide can affect the secretion of Candida albicans quorum-sensing molecules farnesol and tyrosol, with a negative regulatory effect on farnesol and a positive regulatory effect on tyrosol. This component can significantly downregulate the expression of quorum-sensing molecule-encoding genes CHK1 and PBS2, ultimately resulting in significant inhibition of the yeast-to-hyphae morphological transition and biofilm formation of Candida albicans.

3.The Impact of Natural Products on Candida Biofilms

The formation process of Candida biofilms can be broadly divided into four stages: adhesion to the host surface; proliferation of Candida cells; hyphal formation and production of extracellular matrix; dispersal of the biofilm-matured fungal cells and colonization of other sites. Research has shown that the antifungal mechanisms of plant-derived active compounds against Candida species include inhibiting the formation process of Candida biofilms, disrupting the spatial structure of Candida biofilms, and affecting the dispersal of biofilm cells.Studies have found that berberine exhibits inhibitory effects on the initial formation to maturation stages (6h, 12h, 24h, 48h) of biofilms of Candida albicans, Candida krusei, Candida dubliniensis, and Candida glabrata, with the strongest inhibition observed against 48h biofilms. Observations using confocal laser scanning microscopy and 3D reconstruction revealed that berberine can significantly disrupt the spatial structure of the biofilms, resulting in loose cell arrangement, decreased density, and thinner biofilm thickness.

Huangqin flavonoid, the main active ingredient of skullcap, has been found to reduce the surface hydrophobicity of biological membranes by decreasing the expression of CSH1, thereby reducing adhesion and inhibiting the formation of Candida albicans biofilms. Riccardin D, a compound extracted from bryophytes with a macrocyclic dibenzyl structure, delays hyphal formation and leads to defects in biofilm maturation by downregulating the expression of hypha-specific genes ALS1, ALS3, ECE1, EFG1, HWP1, and CDC35, and inhibiting the Ras-cAMP-EFG pathway. Lotusine, an isoquinoline alkaloid isolated from the stems of Stephania cepharantha, exerts an antifungal biofilm effect through a similar pathway. Nepodin, extracted from the roots of Rumex plants, inhibits the formation of biofilms in Candida albicans, Candida glabrata, and Candida parapsilosis by suppressing the expression of hypha and biofilm-related genes (ECE1, HGT10, HWP1, and UME6) and increasing the expression of transport genes (CDR4, CDR11, and TPO2). Xanthochymol and garcinol, two benzophenone compounds isolated from Garcinia pedunculata, effectively inhibit germ tube germination and induce apoptosis in early and mature biofilm cells, thereby directly killing hyphae and biofilms.

4. Inhibition of Hydrolytic Enzyme Synthesis and Secretion

The virulence-related hydrolytic enzymes of Candida albicans primarily include secreted aspartyl proteinase (Sap), phospholipase (PL), and lipase (Lip). These enzymes mediate the adhesion and invasion of Candida albicans to host cells, while also cleaving host immune response-related molecules, assisting Candida albicans in evading the host's immune defense mechanisms. Various plant-derived active ingredients can inhibit the synthesis of hydrolytic enzymes by downregulating their encoding genes, thus reducing their secretion. Studies have found that shikonin can inhibit the secretion of phospholipase from Candida albicans in a concentration-dependent manner. 8 μg/mL of shikonin can reduce the relative expression levels of PLB1 and PLB2 genes by 56.4% and 61.4%, respectively, and 16 μg/mL of shikonin can decrease the phospholipase secretion of Candida albicans by 56.3%. Other studies have discovered that when the concentration of sodium houttuyfonate reaches 240 mg/L, it can significantly inhibit the expression of phospholipase-encoding genes PLB1 and PLB2. Conversely, some plant-derived active ingredients can also exert effects by upregulating the expression of hydrolytic enzyme-related genes.

References

[1] Yu Yu, She Xiaodong, Liu Weida. Research Progress on Anti-Candida Natural Products [J]. Chinese Journal of Mycology, 2023, 18(02): 183-187.

[2] Xie Yufei, Zhou Peiru, Hua Hong, et al. Research Progress on the Anti-Candida Mechanisms of Plant Active Ingredients [J]. Chinese Journal of Mycology, 2022, 17(04): 315-318+329.

About the Author

 Xiaomichong, a researcher in drug quality, has long been committed to drug quality research and drug analysis method validation. Currently, she works for a large domestic drug research and development company, engaged in drug inspection analysis and analysis method validation.