Extracts of Trichocline sinuata (Asteraceae) as natural sensitizers in the photodynamic inactivation of Candida albicans.

Despite significant progress in the development of phototherapy drugs, it is widely recognized that natural products remain the primary source of new photoactive compounds. Exploring uncharted flora in the east-central region of Argentina may offer a vast array of opportunities to isolate new photoactive molecules or plant extracts with high potential for use in antimicrobial photodynamic therapy (aPDT) against Candida albicans. To assess the photofungicidal potential of T. sinuata ("contrayerba") against C. albicans, the extracts underwent spectroscopic and chromatographic analysis, resulting in the identification of furanocoumarin metabolites with similar spectrophotometric properties in all extracts. The extract profiles were created using UHPLC-DAD, and seven furanocoumarins (FCs) were detected. The highest photoinactivation against C. albicans was observed for dicholormethanic extracts (MFC = 62.5 µg/mL), equal to xanthotoxin employed as a positive control. Furthermore, we determine that photochemical mechanisms dependent on oxygen (type I and type II processes) and mechanisms independent of oxygen (photoadduct formation) are involved in the death of these yeasts. These results support the use of native plants of the east-central region of Argentina as potent sensitizers for aPDT and suggest that they can replace xanthotoxin in treating superficial yeast infections of the skin.

Fungal Biofilms as a Valuable Target for the Discovery of Natural Products That Cope with the Resistance of Medically Important Fungi-Latest Findings.

The development of new antifungal agents that target biofilms is an urgent need. Natural products, mainly from the plant kingdom, represent an invaluable source of these entities. The present review provides an update (2017-May 2021) on the available information on essential oils, propolis, extracts from plants, algae, lichens and microorganisms, compounds from different natural sources and nanosystems containing natural products with the capacity to in vitro or in vivo modulate fungal biofilms. The search yielded 42 articles; seven involved essential oils, two Brazilian propolis, six plant extracts and one of each, extracts from lichens and algae/cyanobacteria. Twenty articles deal with the antibiofilm effect of pure natural compounds, with 10 of them including studies of the mechanism of action and five dealing with natural compounds included in nanosystems. Thirty-seven manuscripts evaluated Candida spp. biofilms and two tested Fusarium and Cryptococcus spp. Only one manuscript involved Aspergillus fumigatus. From the data presented here, it is clear that the search of natural products with activity against fungal biofilms has been a highly active area of research in recent years. However, it also reveals the necessity of deepening the studies by (i) evaluating the effect of natural products on biofilms formed by the newly emerged and worrisome health-care associated fungi, C. auris, as well as on other non-albicans Candida spp., Cryptococcus sp. and filamentous fungi; (ii) elucidating the mechanisms of action of the most active natural products; (iii) increasing the in vivo testing.

Evaluation of the antifungal photodynamic activity of Thymophylla pentachaeta extracts against Candida albicans and its virulence factors.

BACKGROUND: Candida albicans is one of the most common causative of opportunistic infections. Treatment of candidiasis is challenging considering the few antifungal drugs available and the increase in resistance. Antimicrobial photodynamic therapy (aPDT) is a recently developed therapeutic option that combines a non-toxic photosensitizer (PS) and light to kill the microbial pathogens. Targeting virulence, defined as the ability of a pathogen to cause overt disease, represents another attractive target for the development of novel antifungal agents. Thymophylla pentachaeta (DC.) Small var. belenidium (DC.) is an endemic plant from Argentina in which the presence of thiophenes, biologically active compounds whose antifungal activity is enhanced by irradiation with Ultraviolet A (UVA), have been already described. PURPOSE: The purpose of this study was to evaluate the photodynamic antifungal activity of hexane (Hex), dichloromethane (DCM), ethyl acetate (EtOAc) and methanol (MeOH) extracts from T. pentachaeta var. belenidium and their inhibitory effects on C. albicans virulence factors as well as biofilm formation and eradication. STUDY DESIGN/METHODS: Antifungal photodynamic activity of Hex, DCM, EtOAc and MeOH extracts from different parts of the plant were assessed with the microbroth dilution, bioautography and the time-kill assays, under light and darkness conditions. The capacities of the most active extracts of inhibiting Candida virulence factors (adherence to epithelial cells, germ tube and pseudomycelium formation and hydrolytic enzyme secretion) were assessed. In addition, the activity against biofilm formation and eradication has been investigated by reaction with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) that quantifies living cells in these structures. RESULTS: Hex and DCM extracts from T. pentachaeta roots exhibited high photodynamic antifungal activity against C. albicans [Minimal fungicide concentrations (MFCs)= 7.8 µg/ml] under UVA light irradiation. Chemical analysis of active extracts (Hex and DCM from roots) revealed the presence of photoactive thiophenes. Both extracts generate reactive oxygen species through type I and II mechanisms. These extracts, at sub-inhibitory concentrations, under light conditions decreased the adherence of C. albicans to Buccal Epithelial Cells (BEC), inhibited germ tube formation and reduced esterase production. Finally, they demonstrated activity against preformed biofilms submitted to irradiation (MFCs= 3.91 µg/ml and 15.63 µg/ml for Hex and DCM extracts, respectively). CONCLUSION: Taking together, results demonstrated the strong photodynamic effects of T. pentachaeta root extracts under UVA irradiation, making them valuable alternatives to the already established antifungal drugs against C. albicans.

Natural products targeting the synthesis of ß(1,3)-D-glucan and chitin of the fungal cell wall. Existing drugs and recent findings.

BACKGROUND: During the last three decades systemic fungal infections associated to immunosuppressive therapies have become a serious healthcare problem. Clinical development of new antifungals is an urgent requirement. Since fungal but not mammalian cells are encased in a carbohydrate-containing cell wall, which is required for the growth and viability of fungi, the inhibition of cell wall synthesizing machinery, such as ß(1,3)-D-glucan synthases (GS) and chitin synthases (CS) that catalyze the synthesis of ß(1-3)-D-glucan and chitin, respectively, represent an ideal mode of action of antifungal agents. Although the echinocandins anidulafungin, caspofungin and micafungin are clinically well-established GS inhibitors for the treatment of invasive fungal infections, much effort must still be made to identify inhibitors of other enzymes and processes involved in the synthesis of the fungal cell wall. PURPOSE: Since natural products (NPs) have been the source of several antifungals in clinical use and also have provided important scaffolds for the development of semisynthetic analogues, this review was devoted to investigate the advances made to date in the discovery of NPs from plants that showed capacity of inhibiting cell wall synthesis targets. The chemical characterization, specific target, discovery process, along with the stage of development are provided here. METHODS: An extensive systematic search for NPs against the cell wall was performed considering all the articles published until the end of 2020 through the following scientific databases: NCBI PubMed, Scopus and Google Scholar and using the combination of the terms "natural antifungals" and "plant extracts" with "fungal cell wall". RESULTS: The first part of this review introduces the state of the art of the structure and biosynthesis of the fungal cell wall and considers exclusively those naturally produced GS antifungals that have given rise to both existing semisynthetic approved drugs and those derivatives currently in clinical trials. According to their chemical structure, natural GS inhibitors can be classified as 1) cyclic lipopeptides, 2) glycolipids and 3) acidic terpenoids. We also included nikkomycins and polyoxins, NPs that inhibit the CS, which have traditionally been considered good candidates for antifungal drug development but have finally been discarded after enduring unsuccessful clinical trials. Finally, the review focuses in the most recent findings about the growing field of plant-derived molecules and extracts that exhibit activity against the fungal cell wall. Thus, this search yielded sixteen articles, nine of which deal with pure compounds and seven with plant extracts or fractions with proven activity against the fungal cell wall. Regarding the mechanism of action, seven (44%) produced GS inhibition while five (31%) inhibited CS. Some of them (56%) interfered with other components of the cell wall. Most of the analyzed articles refer to tests carried out in vitro and therefore are in early stages of development. CONCLUSION: This report delivers an overview about both existing natural antifungals targeting GS and CS activities and their mechanisms of action. It also presents recent discoveries on natural products that may be used as starting points for the development of potential selective and non-toxic antifungal drugs.

Evaluation of Argentinean medicinal plants and isolation of their bioactive compounds as an alternative for the control of postharvest fruits phytopathogenic fungi

Abstract One of the main problems that fruit health goes through in recent years is the difficult eradication of their fungal pathogens after harvesting. This concern to the whole world because it represents huge losses of production, fruit export restrictions and consumers distrust. One of the alternatives to solve this problem could be the exploration of plants and their active compounds, which have proven to be antifungal against human pathogens, but now applied to the treatment of fruits health. In this work, eighteen plant species that grow in Argentina were evaluated against four phytopathogenic fungi that greatly affect the postharvest stage of fruits commercially important to our country. All the species studied were at least active against one fungus of the panel, while three of them displayed high antifungal properties inhibiting the growth of selected pathogens. In addition, bio-guided fractionation of these most active extracts, led to the isolation of some compounds which proved to be responsible for their antifungal activity. Although they are known compounds and were previously isolated from other natural sources, this is the first time that they are evaluated for their phytopathogenic activities against this panel of fungi.

The antifungal activity and mechanisms of action of quantified extracts from berries, leaves and roots of Phytolacca tetramera.

BACKGROUND: Phytolacca tetramera is an endemic plant from Argentina that is currently at serious risk because its environment is subjected to a high anthropic impact. A previous study has shown that berry extracts obtained from this plant display antifungal activity against multiple human-pathogenic fungi when tested with a non-standardized method. Further evidences of the antifungal properties of other parts of the plant and studies of mechanism of antifungal action of the antifungal chemically characterized extracts are required. PURPOSE: This study aimed to gain further evidence of the antifungal activity of P. tetramera berry, leaf and root extracts in order to find the most active extract to be developed as an Herbal Medicinal Antifungal Product. The medicinal usefulness of P. tetramera extracts as antifungal agents will serve as an important support to create concience and carry out actions tending to the preservation of this threatened species and its environment. MATERIALS AND METHODS: Chemical analysis of all P. tetramera extracts, including quantitation of selected markers, was performed through UHPLC-ESI-MS/MS and UPLC-ESI-MS techniques according to the European Medicines Agency (EMA). The antifungal activity of the quantified extracts was tested with the standardized CLSI microbroth dilution method against Candida spp. Antifungal mechanisms of the most active extract were studied by examination of morphological changes by phase-contrast and fluorescence microscopies and both, cellular and enzymatic assays targeting either the fungal membrane or the cell wall. RESULTS: The antifungal activity of twelve P. tetramera extracts was tested against Candida albicans and Candida glabrata. The dichloromethane extract from berries (PtDEb) showed the best activity. Phytolaccagenin (PhytG) and phytolaccoside B (PhytB) were selected as the main active markers for the antifungal P. tetramera extracts. The quantitation of these active markers in all extracts showed that PtDEb possessed the highest amount of PhytG and PhytB. Finally, studies on the mechanism of antifungal action showed that the most active PtDEb extract produces morphological changes compatible with a damage of the cell wall and/or the plasma membrane. Cellular and enzymatic assays showed that PtDEb would not damage the fungal cell wall by itself, but would alter the plasma membrane. In agreement, PtDEb was found to bind to ergosterol, the main sterol of the fungal plasma membrane. CONCLUSION: Studies of the anti-Candida activity of P. tetramera extracts led to the selection of PtDEb as the most suitable extract, confirming the antifungal properties of the threatened species P. tetramera. The new data give a valuable reason for the definitive protection of this sp. and its natural environment thus allowing further studies for the future development of an Herbal Medicinal Antifungal Product.

Approaches to the mechanism of antifungal activity of Zuccagnia punctata-Larrea nitida bi-herbal combination.

BACKGROUND: In our previous study the synergism of four combinations of Zuccagnia punctata (ZpE) and Larrea nitida (LnE) exudates with the reliable statistical-based MixLow method was assessed, and the markers of the most anti-C. albicans synergistic ZpE-LnE bi-herbal combination were quantified according to European Medicines Agency (EMA). PURPOSE: To study the mechanisms of action as well as the cytotoxic properties of the ZpE-LnE most synergistic combination found in the previous work. MATERIALS AND METHODS: Minimum Fungicidal Concentration (MFC) and rate of killing of ZpE-LnE were assessed with the microbroth dilution and the time-kill assays respectively. Morphological alterations were observed with both confocal and fluorescence microscopy on the yeast Schizosaccharomyces pombe. The ergosterol exogenous assay, the quantification of ergosterol, the sorbitol as well as glucan synthase (GS) and chitin synthase (ChS) assays were used to detect the effects on the fungal membrane and cell wall respectively. The capacity of ZpE-LnE of inhibiting Candida virulence factors was assessed with previously reported methods. The effect of ZpE-LnE and of ZpE or LnE alone on cell viability was determined on human hepatoma cells line Huh7. RESULTS: ZpE-Ln E was fungicidal killing C. albicans in a shorter time than amphotericin B and produced malformations in S. pombe cells. ZpE-LnE showed to bind to ergosterol but not to inhibit any step of the ergosterol biosynthesis. ZpE-LnE showed a low or moderate capacity of inhibiting GS and ChS. Regarding inhibition of virulence factors, ZpE-LnE significantly decreased the capacity of adhesion to eukaryotic buccal epithelial cells (BECs), did not inhibit the germ tube formation and inhibited the secretion of phospholipases and proteinases but not of haemolysins. ZpE-LnE demonstrated very low toxicity on Huh7 cells, much lower than that each extract alone. CONCLUSION: The fungicidal properties of ZpE-LnE against C. albicans, its dual mechanism of action targeting the fungal membrane's ergosterol as well as the cell wall, its capacity of inhibiting several important virulence factors added to its low toxicity, make ZpE-LnE a good candidate for the development of a new antifungal bi-Herbal Medicinal Product.

Hybrid combinations containing natural products and antimicrobial drugs that interfere with bacterial and fungal biofilms.

BACKGROUND: Biofilms contribute to the pathogenesis of many chronic and difficult-to eradicate infections whose treatment is complicated due to the intrinsic resistance to conventional antibiotics. As a consequence, there is an urgent need for strategies that can be used for the prevention and treatment of biofilm-associated infections. The combination therapy comprising an antimicrobial drug with a low molecular weight (MW) natural product and an antimicrobial drug (antifungal or antibacterial) appeared as a good alternative to eradicate biofilms. PURPOSE: The aims of this review were to perform a literature search on the different natural products that have showed the ability of potentiating the antibiofilm capacity of antimicrobial drugs, to analyze which are the antimicrobial drugs most used in combination, and to have a look on the microbial species most used to prepare biofilms. RESULTS: Seventeen papers, nine on combinations against antifungal biofilms and eight against antibacterial biofilms were collected. Within the text, the following topics have been developed: breaf history of the discovery of biofilms; stages in the development of a biofilm; the most used methodologies to assess antibiofilm-activity; the natural products with capacity of eradicating biofilms when acting alone; the combinations of low MW natural products with antibiotics or antifungal drugs as a strategy for eradicating microbial biofilms and a list of the low MW natural products that potentiate the inhibition capacity of antifungal and antibacterial drugs against biofilms. CONCLUSIONS AND PERSPECTIVES: Regarding combinations against antifungal biofilms, eight over the nine collected works were carried out with in vitro studies while only one was performed with in vivo assays by using Caenorhabditis elegans nematode. All studies use biofilms of the Candida genus. A 67% of the potentiators were monoterpenes and sesquiterpenes and six over the nine works used FCZ as the antifungal drug. The activity of AmpB and Caspo was enhanced in one and two works respectively. Regarding combinations against bacterial biofilms, in vitro studies were performed in all works by using several different methods of higher variety than the used against fungal biofilms. Biofilms of both the gram (+) and gram (-) bacteria were prepared, although biofilm of Staphylococcus spp. were the most used in the collected works. Among the discovered potentiators of antibacterial drugs, 75% were terpenes, including mono, di- and triterpenes, and, among the atibacterial drugs, several structurally diverse types were used in the combinations: aminoglycosides, ß-lactams, glucopeptides and fluoroquinolones. The potentiating capacity of natural products, mainly terpenes, on the antibiofilm effect of antimicrobial drugs opens a wide range of possibilities for the combination antimicrobial therapy. More in vivo studies on combinations of natural products with antimicrobial drugs acting against biofilms are highly required to cope the difficult to treat biofilm-associated infections.

Synergistic mutual potentiation of antifungal activity of Zuccagnia punctata Cav. and Larrea nitida Cav. extracts in clinical isolates of Candida albicans and Candida glabrata.

BACKGROUND: Zuccagnia punctata Cav. (Fabaceae) and Larrea nitida Cav. (Zygophyllaceae) are indistinctly or jointly used in traditional medicine for the treatment of fungal-related infections. Although their dichloromethane (DCM) extract have demonstrated moderate antifungal activities when tested on their own, antifungal properties of combinations of both plants have not been assessed previously. PURPOSE: The aim of this study was to establish with statistical rigor whether Z. punctata (ZpE) and L. nitida DCM extract (LnE) interact synergistically against the clinically important fungi Candida albicans and Candida glabrata and to characterize the most synergistic combinations. STUDY DESIGN: For synergism assessment, the statistical-based Boik's design was applied. Eight ZpE-LnE fixed-ratio mixtures were prepared from four different months of 1 year and tested against Candida strains. Lϕ (Loewe index) of each mixture at different fractions affected (ϕ) allowed for the finding of the most synergistic combinations, which were characterized by HPLC fingerprint and by the quantitation of the selected marker compounds. METHODS: Lϕ and confidence intervals were determined in vitro with the MixLow method, once the estimated parameters from the dose-response curves of independent extracts and mixtures, were obtained. Markers (four flavonoids for ZpE and three lignans for LnE) were quantified in each extract and their combinations, with a valid HPLC-UV method. The 3D-HPLC profiles of the most synergistic mixtures were obtained by HPLC-DAD. RESULTS: Three over four IC50ZpE/IC50LnE fixed-ratio mixtures displayed synergistic interactions at effect levels ϕ > 0.5 against C. albicans. The dosis of the most synergistic (Lϕ = 0.62) mixture was 65.96 µg/ml (ZpE = 28%; LnE = 72%) containing 8 and 36% of flavonoids and lignans respectively. On the other hand, one over four IC50ZpE/IC50LnE mixtures displays synergistic interactions at ϕ > 0.5 against C. glabrata. The dosis of the most synergistic (Lϕ = 0.67) mixture was 168.23 µg/ml (ZpE = 27%; LnE = 73%) with 9.7 and 31.6% of flavonoids and lignans respectively. CONCLUSIONS: Studies with the statistical-based MixLow method, allowed for the finding of the most ZpE-LnE synergistic mixtures, giving support to a proper joint use of both antifungal herbs in traditional medicine.

Argentinean Andean propolis associated with the medicinal plant Larrea nitida Cav. (Zygophyllaceae). HPLC-MS and GC-MS characterization and antifungal activity.

The chemical profile and botanical origin of Andean Argentinian propolis were studied by HPLC-ESI-MS/MS and GC-MS techniques as well as the antifungal activity according to CLSI protocols. Dermatophytes and yeasts tested were strongly inhibited by propolis extracts (MICs between 31.25 and 125 µg/mL). The main antifungal compounds were: 3'methyl-nordihydroguaiaretic acid (MNDGA) 1, nordihydroguaiaretic acid (NDGA) 2 and a NDGA derivative 3, showing strong activity against Trichophyton mentagrophytes, T. rubrum and Microsporum gypseum (MICs between 15.6 and 31.25 µg/mL). The lignans 1 and 2 showed activities against clinical isolates of Candidas spp., Cryptococcus spp., T. rubrum and T. mentagrophytes (MICs and MFCs between 31.25 and 62.5 µg/mL). The lignan and volatile organic compounds (VOCs) profiles from propolis matched with those of exudates of Larrea nitida providing strong evidences on its botanical origin. These results support that Argentinian Andean propolis are a valuable natural product with potential to improve human health. Six compounds (1-6) were isolated from propolis for the first time, while compounds 1 and 3-6 were reported for first time as constituents of L. nitida Cav.

Antifungal activity of saponin-rich extracts of Phytolacca dioica and of the sapogenins obtained through hydrolysis.

A saponin-rich extract of Phytolacca dioica L. berries, its acid hydrolysate, and its major aglycone, phytolaccagenin, were assayed for antifungal activity against ATCC standard cultures of Candida albicans and Cryptococcus neoformans, and against clinical isolates of these fungi. The activity of the extract was either low or negligible, but the hydrolysate, containing the sapogenins, including phytolaccagenin, and also pure phytolaccagenin, showed promising antifungal potency. Hydrolysis of a natural product extract is shown to be a useful modification leading to improved bioactivity.

Argentinean propolis from Zuccagnia punctata Cav. (Caesalpinieae) exudates: phytochemical characterization and antifungal activity.

This paper reports the in vitro antifungal activity of propolis extracts from the province of Tucuman (Argentina) as well as the identification of their main antifungal compounds and botanical origin. The antifungal activity was determined by the microdilution technique, using reference microorganisms and clinical isolates. All dermatophytes and yeasts tested were strongly inhibited by different propolis extracts (MICs between 16 and 125 microg mL(-1)). The most susceptible species were Microsporum gypseum, Trichophyton mentagrophytes, and Trichophyton rubrum. The main bioactive compounds were 2',4'-dihydroxy-3'-methoxychalcone 2 and 2',4'-dihydroxychalcone 3. Both displayed strong activity against clinical isolates of T. rubrum and T. mentagrophytes (MICs and MFCs between 1.9 and 2.9 microg mL(-1)). Additionally, galangin 5, pinocembrin 6, and 7-hydroxy-8-methoxyflavanone 9 were isolated from propolis samples and Zuccagnia punctata exudates, showing moderate antifungal activity. This is the first study matching the chemical profile of Z. punctata Cav. exudates with their corresponding propolis, giving strong evidence on the botanical origin of the studied propolis.

Value of the ethnomedical information for the discovery of plants with antifungal properties. A survey among seven Latin American countries.

ETHNOPHARMACOLOGICAL RELEVANCE: This study reports the antifungal evaluation of 327 plant species (92 families and 251 genera) from seven Latin American countries which were selected on the basis of their reported ethnomedical uses and compared them with plants selected at random. AIM OF THE STUDY: (a) The main aim of this study was to investigate whether the probability of detecting antifungal plants is higher when plants have reports of ethnopharmacological uses related to fungal infections (PAU group) than when they are selected at random (PNAU group). (b) The second objective was to determine, within the PAU group, whether the probability of obtaining a positive result will be higher when the plants are tested against dermatophytes, than against yeasts or Aspergillus spp. (c) The third goal was to investigate, within all MICs0.05). (c) Within the detected antifungal plants from both groups, plants of the PAU group displayed higher activities (lower MICs) than those of PNAU group against dermatophytes (p<0.05) but not against yeasts or Aspergillus spp. CONCLUSIONS: Considering that dermatophytes are the cause of superficial fungal infections, which can be easily detected and followed by traditional healers, our findings suggest that the ethnopharmacological approach is useful in guiding the detection of antifungal plants in Latin America mainly for infections in which the pathological expression is obvious and, therefore, the cure can be clearly observed.

Antifungal activity of Zuccagnia punctata Cav.: evidence for the mechanism of action.

Petroleum ether and dichloromethane extracts of fruits, aerial parts and exudate of Zuccagnia punctata Cav. (Fabaceae) showed moderate antifungal activities against the yeasts C. albicans, S. cerevisiae and C. neoformans (MICs: 62.5 - 250 microg/mL) and very strong antifungal activities against the dermatophytes M. gypseum, T. rubrum and T. mentagrophytes (MICs: 8 - 16 microg/mL) thus supporting the ethnopharmacological use of this plant. Antifungal activity-directed fractionation of active extracts by using bioautography led to the isolation of 2',4'-dihydroxy-3'-methoxychalcone (1) and 2',4'-dihydroxychalcone (2) as the compounds responsible for the antifungal activity. Second-order studies included MIC (80), MIC (50) and MFC of both chalcones in an extended panel of clinical isolates of the most sensitive fungi, and also comprised a series of targeted assays. They showed that the most active chalcone 2 is fungicidal rather than fungistatic, does not disrupt the fungal membranes up to 4 x MFC and does not act by inhibiting the fungal cell wall. So, 2',4'-dihydroxychalcone would act by a different mechanism of action than the antifungal drugs in current clinical use, such as amphotericin B, azoles or echinocandins, and thus appears to be very promising as a novel antifungal agent.