Plants' Natural Products as Alternative Promising Anti-Candida Drugs.

Candida is a serious life-threatening pathogen, particularly with immunocompromised patients. Candida infections are considered as a major cause of morbidity and mortality in a broad range of immunocompromised patients. Candida infections are common in hospitalized patients and elderly people. The difficulty to eradicate Candida infections is owing to its unique switch between yeast and hyphae forms and more likely to biofilm formations that render resistance to antifungal therapy. Plants are known sources of natural medicines. Several plants show significant anti-Candida activities and some of them have lower minimum inhibitory concentration, making them promising candidates for anti-Candida therapy. However, none of these plant products is marketed for anti-Candida therapy because of lack of sufficient information about their efficacy, toxicity, and kinetics. This review revises major plants that have been tested for anti-Candida activities with recommendations for further use of some of these plants for more investigation and in vivo testing including the use of nanostructure lipid system.

INTRODUCTION

Candida is a fungal pathogen[1] which is mostly known to cause high rate of mycotic infection to human worldwide.[2] Candida is known to cause mucosal and deep tissue infections. Candida infects mucosal tissues including mouth, esophagus, gut, and vagina.[3] Vaginal candidiasis continues to be a world health problem to women.[4] Candidal infections are common in hospitalized patients and elderly people, and are difficult to control.[5] About 50% of adults have Candida yeasts in their mouth and it is responsible for superficial easily treated infections. However, candidal infections can spread through the body and become life threatening, in particular with immunocompromised patients.[67] Candidiasis represents a major cause of death.[8] Candida can switch between two major forms, yeast and hyphae forms. The switch from yeast to hyphae is considered a major infectious agent of Candida.[9] In addition, Candida spp. produces biofilms on synthetic materials, which facilitates adhesion of the organisms to devices and renders the organism relatively resistant to antifungal therapy.[10] Catheter-associated Candida biofilms can lead to bloodstream infections.[11] Candida-infected catheters, in particular those associated with microbial biofilms, can represent 90% of infections among hospital-admitted patients and hence considered as a major cause of death.[11] Several synthetic drugs are established in the treatment regimens of candidal infections as indicated in Table 1, however drug resistance is developed.

Table 1

Candida resistance to synthetic drugs

MECHANISMS OF CANDIDAL RESISTANCE TO SYNTHETIC DRUGS

The formation of biofilms in Candida and the transition from planktonic to sessile form are mainly associated with highly resistant phenotype. Other mechanisms of resistance include the expression of resistance genes, particularly those encoding efflux pumps, and the presence of persister cells.[17] Major synthetic drugs that develop candidal resistance include 5-flucytosin, amphotericin B, azoles, and echinocandins [Table 1].

PLANTS AS NATURAL SOURCES OF ANTI-CANDIDAL DRUGS

Plants are known for decades as the only source of medicines by traditional people.[18] Moreover, plants are still used as major remedies by several countries, particularly in Africa and Asia.[19] Several plant species showed effective anti-candidal activities [Table 2]. However, promoting a medicinal plant as an antimicrobial agent is challenging and requires more assessment including safety and efficacy prior to clinical study. Table 2 summarizes most of the reported plants tested for anti-candidal activities. Several of these plants showed promising minimum inhibitory concentration (MIC) such as peppermint (0.08 μg/mL), Thymus villosus (0.64 μg/mL), eucalyptus (0.05 μg/mL), lemongrass oil (0.06 μg/mL), Cinnamomum zeylanicum (0.01 μg/mL), ginger grass oil (0.08 μg/mL), and coriander (0.2 μg/mL), however they have never been deeply studied as anti-Candida drugs for the market use.

Table 2

Natural anti-Candida products, their botanical sources, and minimum inhibitory concentration

This review article provides an overview of the reported natural anti-Candida products identified from plants and their mechanisms [Table 2]. Additionally, the current review article explores the possible biotechnological applications for the production of anti-Candida drugs and enhancing their activities.

MECHANISM OF ACTION OF ANTI-CANDIDA NATURAL PRODUCTS

The anti-Candida mechanisms of action initiated by plant natural products can involve inhibition of germination and biofilm formation, cell metabolism, cell wall integrity, cell membrane plasticity, or can involve induction of apoptosis [Figure 1].

Figure 1

Representative drawing of the active sites and mechanisms of most tested plant anti-Candida agents

Inhibition of Candida biofilm formation and transition to hyphal form

The switch of Candida from yeast to hyphae is mainly accompanied by resistant biofilm formation. Candida biofilms are difficult to eradicate and are associated with resistance against many existing antifungals. Thymol which is a major constituent of thyme oil can interfere with biofilm metabolic activity and thus inhibits early and mature biofilm formation.[86] Anthraquinones isolated from Heterophyllaea pustulata showed significant activity against Candida tropicalis biofilm formation by interfering with the pro-oxidant–antioxidant balance leading to biofilm injury.[149] They also showed synergistic activity with amphotericin B. Geranium oil and its nanoemulsion showed antibiofilm activity against Candida albicans, C. tropicalis, and Candida glabrata. The smaller particle size of geranium nanoemulsion efficiently penetrates biofilms and hence damages the organism's cell membrane.[79] Similarly, cinnamic acid derivatives showed great antibiofilm activity against C. albicans at lower MIC compared to fluconazole. The most active cinnamic acid derivative is a hybrid of cinnamic acid with miconazole that leads to inhibition of biofilm at 2 μg/mL and reduction in metabolic activity of preformed biofilm at 8 μg/mL.[150151] Furthermore, lemongrass oil and its major constituents exhibit strong inhibitory activity on Candida biofilm formation, germ tube formation (GTF), adherence, and candidal colonization.[130] Many terpenes including carvacrol, geraniol, and thymol showed strong activity in reducing the development of C. albicans biofilms. Carvacrol was able to inhibit Candida biofilm regardless of the tested species and of the biofilm maturation state.[152]

Inhibition of Candida germ tube formation

GTF is a transitional stage between yeast and hyphal cells which is an essential stage for Candida virulence activity.[153] GTF increases fungal adherence and penetration to infected tissues.[154] It has been shown that essential oil of oregano inhibits C. albicans GTF to a higher extent compared to other essential oils.[33] The inhibition of GTF is mainly related to the lipophilicity of the essential oils and their interaction with the Candida cell membrane, leading to changes and loss of the structural and enzymatic constituents of fungal cells including 1,3-β-D-glucan synthases, adenosine triphosphatase (ATPase), mannans, and chitin that are required in GTF.[155156]

Alteration in Candida cell membrane

It has been reported that terpenes can cause alteration in Candida cell permeability by getting embedded between the fatty acyl chain in the membrane lipid bilayers and hence interrupting the lipid packing and consequently disturbing membrane structure and functions.[154] Geraniol increases the membrane fluidity by affecting the central part of the lipid bilayers.[157] Tea tree oil increases cell permeability and inhibits medium acidification.[114] Salvia sclarea oil and its major constituents, linalyl acetate and linalool, induce a significant increase in plasma membrane fluidity, which in turn induces cell apoptosis. Thymol affects cell membrane electrostatics and can create deviated membrane tension.[84] Coriander oil showed an increase in cell membrane permeability, loss of membrane potential, leakage of intracellular DNA, and damage of cytoplasmic membrane, thus causing impaired cellular functions.[84] Raphanus sativus antifungal peptide 2 (RsAFP2) is a plant defensin that can interact with the sphingolipid glucosylceramide (GlcCer) of susceptible fungal membranes but not with the human GlcCer, and hence can exhibit selective antifungal activity.[158159] The RsAFP2–GlcCer interaction can lead to increase in the permeability, Ca2+ influx, and growth arrest.[160] Permeabilization due to RsAFP2 is mainly due to induction of many signaling pathways associated with the formation of reactive oxygen species (ROS), apoptosis, and caspase activation.[161] Geraniol oil derived from palmarosa oil, ninde oil, rose oil, and citronella oil can disturb the uniformity of cell membrane by interrupting sterol biosynthesis and inhibition of plasma membrane ATPase which is crucial for cell survival.[162] Taxodone is a diterpenoid compound isolated from Metasequoia glyptostroboides and Taxodium distichum, can cause loss of cell membrane integrity, and increases cell permeability, thus causing rapid loss of nucleic acid, ions, and some essential metabolites.[163]

Interference with Candida mitochondrial respiratory chain

Respiration takes place in mitochondria that produce ATP required by all cells. The process is accompanied with the production of large amount of ROS such as hydrogen peroxide and hydroxyl radicals as by-products. ROS can cause damage to cell proteins, lipids, and DNA.[164] HsAFP1 is a plant defensin derived from Heuchera sanguinea that shows apoptotic action against C. albicans mainly due to accumulation of ROS leading to the induction of mitochondrion-dependent apoptosis.[165] Dill seed essential oil (DSEO) can inhibit mitochondrial dehydrogenases mainly due to the disruption of the citric acid cycle and thus the inhibition of ATP synthesis.[166] Furthermore, DSEO causes intracellular accumulation of ROS in C. albicans and hence has an antifungal activity.[166] In addition, amentoflavone derived from Selaginella tamariscina has been associated with the induction of mitochondrion-dependent apoptosis in C. albicans.[167] Lycopene is a carotenoid pigment mainly found in tomato that can cause accumulation of intracellular Ca2+ and interference with mitochondrial functions, such as cytochrome C release and mitochondrial depolarization, leading to caspase activation and ROS production and hence leads to mitochondrial dysfunction and apoptosis.[168]

Inhibition of Candida adherence

Essential oil of Rosmarinus officinalis showed anti-adherent activity of C. albicans. The biological activity of R. officinalis is mainly associated with its main chemical components, including cineole, limonene, and cymene.[91] Schinus terebinthifolius and Croton urucurana have also showed strong anti-adherent activity of C. albicans that is associated with the presence of apigenin. Apigenin can modulate gene expression and reduce the formation of glucan, leading to biofilm inhibition activity.[169]

Induction of Candida apoptosis

Baicalein is a flavonoid isolated from the roots of Scutellaria baicalensis Georgi and shows potent activity against fluconazole-resistant C. albicans. Baicalein mainly inhibits C. albicans by inducing programmed cell death (apoptosis) and reduction of drug extrusion out of the yeast cells.[170] Silibinin, a natural product extracted from Silybum marianum (milk thistle), can cause Candida apoptosis through interference with mitochondrial Ca2+ signaling. Ca2+ signaling plays an important role in physiological processes and it is associated with stress responses in fungi.[171]

Interference with Candida cell metabolism

Allicin isolated from Allium sativum (garlic) shows a strong anti-Candida activity mainly by inhibition of thiol-containing amino acids and proteins, therefore interfering with cell metabolism.[172] Human cells contain glutathione which can bind to allicin preventing cell damage whereas glutathione is lacking in Candida that makes allicin as selective and effective candidate in anti-Candida therapy.[173]

Interference with Candida cell wall integrity

Cell wall integrity is very important during growth and morphogenesis of Candida cells and in the face of external challenges that cause cell wall stress. Several natural products have showed interference effects with Candida cell wall integrity. For example, RsAFP2 defensin interacts with Candida cell wall GlcCers and hence damages cell wall integrity. Furthermore, it can disrupt the localization of septins and blocks the switch from yeast to hypha. The black tea polyphenols including catechins and theaflavins can cause Candida cell wall damage.[144] Similarly, casuarinin isolated from Plinia cauliflora can target C. albicans cell wall, leading to significant changes in the cell wall architecture including the outer glycoprotein layer and cell wall porosity.[174]

RESISTANCE OF CANDIDA TO PLANT NATURAL PRODUCTS

Candida strains lacking GlcCer in their membranes, either because of nonfunctional synthase enzyme or its complete absence (as in Saccharomyces cerevisiae or C. glabrata), are resistant to RsAFP2 and hence protected from cell permeabilization.[160] C. tropicalis shows resistance against Uncaria tomentosa, mainly due to the enhanced ability of Candida to form biofilms.[175]

TOXICITY OF NATURAL ANTI-CANDIDA PRODUCTS

The cytotoxic activities of anti-Candida natural products are rarely investigated and only few products have been tested. For example, the toxicity of geraniol oil was measured by hemolytic assay on human erythrocytes. Geraniol oil caused only 1% cell lysis at 5 μg/mL MIC compared to 10% lysis by amphotericin B or fluconazole at same tested concentrations, suggesting the safety of geraniol.[162] The cytotoxicity of Morinda royoc L extract was also investigated on vero cells (African green monkey kidney cells). M. royoc L extract showed no toxic activities according to criteria established by the American National Cancer Institute (IC50 ≥200 mcg/mL).[176] Furthermore, oral administration of M. royoc in rats showed no toxic effects, suggesting that M. royoc is a good anti-Candida product.[177]

IN VIVO INVESTIGATION OF NATURAL ANTI-CANDIDA AGENTS

The anti-candidal activities of suppositories made from saponins derived from Solanum chrysotrichum were investigated in vulvovaginal candidiasis mice model. S. chrysotrichum treatment showed no significant difference in clinical effectiveness compared to ketoconazole.[178] On the other hand, garlic tablets (Garcin) showed similar activity to fluconazole on Candida vaginitis in women admitted to a health-care center in Iran, suggesting that garlic could be an alternative to fluconazole in the treatment of Candida infection.[179] U. tomentosa extract was clinically investigated in fifty patients with denture stomatitis. U. tomentosa is effective as miconazole on C. albicans, C. tropicalis, C. glabrata, and C. krusei; however, C. tropicalis showed resistance due to its ability to biofilm formation.[175] The anti-candidal activity of Cassia fistula seeds was tested in mice model. The seed extract showed 6-fold decrease in C. albicans in blood samples and kidneys of the tested animals.[73180]

FUTURE PROSPECTIVE AND BIOTECHNOLOGY ADVANCES IN THE PRODUCTION OF ANTI-CANDIDA-ACTIVE PLANTS

The need for new anti-Candida agents is increasing, especially with the emergence of resistant Candida strains. The effectiveness of natural agents against different strains of fungi, particularly Candida, is confirmed in several publications. It has been reported that many patencies are using natural products as anti-Candida. For example, Indigo naturalis or indigo-producing plant extract has been used in the topical treatment of candidiasis.[181] A patent made from oral herbal preparation developed by Piramal Life Sciences showed efficient activity against oral candidiasis.[182] Pharmalp developed an anti-candidal formula derived from Epilobium parviflorum for the use in the prevention and/or treatment of Candida infection.[183]

The screening for anti-Candida natural active products increased significantly during the past two decades. Several investigations have assessed the anti-Candida activities of natural products of plants from different geographical regions in the world. For example, Duarte et al. examined the anti-Candida activities of extracts of 258 Brazilian medicinal plant species.[184] However, other regions are still in the preliminary investigation stages such as the Arabian deserts. Desert plants of the arid/hyperarid climates of the Arab Gulf region are exposed to several environmental stresses, such as heat, drought, and salinity. Such stresses may provide new active compounds which might have effective and unique anti-Candida activities.

On the other hand, modern biotechnology techniques can improve the activity of plant extracts including anti-Candida; for example, the development of nanostructure lipid system. Nanostructure lipid system can improve the antimicrobial activity of plant extract, reduce the required doses, and reduce side effects. Nanostructure lipid system improves the anti-Candida activity of aqueous ethanol extract of stems and leaves of Astronium sp.[185] The nanostructure lipid system can reduce the MIC of the plant extract ~ 9 times. Nanostructure lipid system can efficiently compartmentalize specific active components and modify their properties and behavior of plant extracts in a biological environment.[125] Moreover, recent advances in metabolomics and engineering of target pathways may provide an optimized commercial production of the natural compounds and enhancement of their activity. Usually, metabolomics using various bioanalytical tools such as nuclear magnetic resonance, liquid chromatography-mass spectrometry (MS), and gas chromatography-MS can be done to identify the potential anti-Candida compounds. Once these compounds are identified and their biosynthetic pathways are assigned, candidate genes can be identified in silico [Figure 2]. Consequently, target pathways can be engineered with overexpression of the desired transcription factors and genes or silencing of the undesired competitive genes and pathways to enhance their production levels [Figure 2].

Figure 2

Simplified flow chart showing the utilization of different biotechnological approaches for identifying, developing, and enhancing the production levels of anti-Candida candidate compounds from a plant source

CONCLUSION

As concluding remarks, several plant natural products have been tested for anti-Candida activities. Several of these plant products can target critical processes in Candida biological activities including cell wall integrity, cell membrane plasticity, cell metabolism, respiratory chain, adherence to host cell, germination and biofilm formation, or induction of apoptosis. Despite these great anti-Candida activities of plant products compared to controls, only few have been tested in vivo and none of them have ever been clinically used as anti-Candida. On the other hand, although some of these products including garlic, probiotics, peppermint, cinnamon, ginger, and propolis are present in the pharmaceutical market for other medical purposes, they have never been used as anti-Candida. The need for new anti-Candida is urgent since Candida is known as a serious resistant microbe, and hence promotion of some of the selected plant products for clinical testing will be beneficial.

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Conflicts of interest

There are no conflicts of interest.