1. Introduction
Natural products (NPs) have greatly inspired the search for and development of new therapeutic agents. Over the last 40 years, almost half of the drugs approved by the U.S. Food and Drug Administration (FDA) have been based on NPs, either isolated, derived from them, or with modified molecules [
1,
2]. From this perspective, plants remain a great resource, since it is estimated that a large percentage of the world’s population still uses plants or traditional botanical preparations for the treatment of diseases [
2]. However, only 0.8% of the drugs approved by the FDA are considered botanical NPs or botanical drugs, probably because the category was newly introduced in 2012 [
1]. Botanical drugs are defined as complex mixtures that lack a primary active ingredient that has been documented for prior substantial human use. These mixtures can be composed of vegetable materials, parts of plants, algae, macroscopic fungi, or combinations of them [
3,
4]
Anacardium genus (Anacardiaceae) consists of approximately 20 species, and
Anacardium microcarpum,
Anacardium humile, and
Anacardium occidentale are the most widely studied, because of their medicinal and nutraceutical properties. Their common name is cashew, marañon, or cajú [
5,
6,
7]. The leaves and flowers of
A. occidentale have been used in traditional medicine to treat skin lesions and diarrhea and as anti-inflammatory agents. Some formal studies for this species report antibacterial and antifungal activity and strong antioxidant action, attributed to the presence of anacardic acids, cardanol, gallic acid, catechin, and quercetin derivatives [
8,
9,
10].
Regarding its antifungal potential, its activity has been briefly described by a few authors against
Candida albicans and
Candida tropicalis, both reference strains [
9,
11,
12]. Annually, invasive fungal diseases (IFDs) are responsible for the admission of more than one million patients to health services worldwide. In addition, they are associated with high mortality rates of ~40-60% [
13,
14,
15,
16,
17]. Among them, invasive candidiasis (IC) is the most frequent, and
C. albicans is still the predominant etiological agent, being isolated in 43.6% of cases [
18,
19]. In parallel,
C. auris has been described as a multidrug-resistant yeast that is difficult to diagnose. It was first described in 2009 in Japan and has spread worldwide. In 2016, an alert was issued by the Centers for Disease Control and Prevention (CDC) for the mandatory notification of cases attributed to
C. auris [
20,
21,
22]. It is estimated that to date between 3000 and 4000 cases have been reported [
23,
24]. In South America, the first cases were reported in Venezuela and Colombia between 2012 and 2013, according to prospective studies [
20,
24]. An increase in cases was detected in several hospitals around the world, the most recent outbreak being in a hospital in Brazil, forcing the closure of the medical center [
25]. Altogether, we are currently facing a pessimistic outlook, due to the increase in cases, the limited number of commercial antifungals, frequent cases of resistance, and the toxicity associated with them [
13,
26,
27].
The present study was aimed at evaluating the anti-candidal activity of the ethanolic leaf extract of A.occidentale and constructing an approach to determining the possible mechanism of action on a reference strain and a clinical isolate of C. albicans in order to provide a potential alternative natural antifungal product. A. occidentale treatment induced an accumulation of intracellular ROS and mitochondrial dysfunction and does not show cytotoxicity or hemolytic activity at the concentrations tested.
4. Conclusions
The emergence of resistance to conventional drugs and the toxicity of high doses highlights the importance of developing new alternatives for treating invasive fungal diseases. NPs, mainly those of botanical origin, belong to a field that has recently been approved by the FDA, although to date there is no botanical drug or derivative thereof for use as an antifungal. A. occidentale has been used in traditional preparations for the treatment of diseases. In the present study, an ethanolic extract of leaves from A. occidentale, containing several glycosylflavonoids and with the biflavone agathisflavone as the major compound, was found to inhibit the growth and proliferation of C. albicans and C. auris at 62.5 µg/ml and 125 µg/ml. These results were corroborated with confocal microscopy, SEM, and TEM. Additionally, the extract induced the accumulation of intracellular ROS and mitochondrial dysfunction and did not show cytotoxicity or hemolytic activity at the concentrations tested. This is the first time that the mechanism of action of the plant has been explored, and it was shown that the flavonoids present in it may be related to this activity. This investigation expands the current knowledge of botanical drugs as a potential alternative to combat invasive fungal disease.
Author Contributions
L.F.Q: conceptualization, methodology, investigation, visualization, data curation, original draft preparation and writing-review and editing. A.X.H.: methodology, data curation, writing—review, and editing. L.C.C.: methodology, data curation, writing—review and editing. Y.V-C.: methodology, data curation, writing—review and editing. C.P.B-C.: methodology, data curation, writing—review and editing. G.M.C.: investigation, supervision, data curation, resources, project administration, and original draft preparation. R.X.F.: investigation and supervision. C.M.P-G.: conceptualization, data curation, investigation, supervision, formal analysis, resources and original draft preparation. All authors have read and agreed to the published version of the manuscript.
Acknowledgments
The authors would like to acknowledge Pontificia Universidad Javeriana, MinCiencias, Ministerio de Educación Nacional, Ministerio de Industria, Comercio y Turismo e ICETEX, and Convocatoria Ecosistema Científico - Colombia Científico “Generación de alternativas terapéuticas en cáncer a partir de plantas a través de procesos de investigación y desarrollo transnacional, articulados en sistemas de valor sostenibles ambiental y económicamente” sponsor by World Bank (contract No. FP44842-221-2018). We also thank the Ministerio del Medio Ambiente de Colombia for allowing the use of genetic resources and products derived (Contract number 212/2018; Resolution 210/2020). Finally to Convocatoria del Fondo de Ciencia, Tecnología e Innovación del Sistema General de Regalías, en el marco del Programa de Becas de Excelencia Doctoral del Bicentenario, definido en el artículo 45 de la ley 1942 de 2018, Bogotá, Colombia.
Figure 1.
(a) Ultra-performance liquid chromatography (UPLC) chromatogram obtained from A. occidentale leaf extract at 350 nm. (b) Mass spectrum of Agathisflavone.
Figure 1.
(a) Ultra-performance liquid chromatography (UPLC) chromatogram obtained from A. occidentale leaf extract at 350 nm. (b) Mass spectrum of Agathisflavone.
Figure 2.
Minimal Inhibitory Concentration (MIC) and Maximal Fungicide Concentration (MFC) of A. occidentale against Candida albicans strains. (a) MIC by broth microdilution based on CLSI M27-A3. (b) MCF against ATCC SC5314 by subculturing on agar plate after MIC method.
Figure 2.
Minimal Inhibitory Concentration (MIC) and Maximal Fungicide Concentration (MFC) of A. occidentale against Candida albicans strains. (a) MIC by broth microdilution based on CLSI M27-A3. (b) MCF against ATCC SC5314 by subculturing on agar plate after MIC method.
Figure 3.
Effect of A. occidentale extract on growth kinetic of C. albicans strains. (a) C. albicans ATCC SC5314; (b) C. albicans PUJ/HUSI 256;. The figure was constructed with information from three independent 48-hour curves performed under the same protocol.
Figure 3.
Effect of A. occidentale extract on growth kinetic of C. albicans strains. (a) C. albicans ATCC SC5314; (b) C. albicans PUJ/HUSI 256;. The figure was constructed with information from three independent 48-hour curves performed under the same protocol.
Figure 4.
Confocal scanning fluorescence images of C. albicans ATCC SC5314 cells after exposure to 0.5 MIC (31.25 μg/mL), MIC (62.5 μg/mL), and 2MIC (125 μg/mL) of A. occidentale. Live or dead intact cells stained with Calcofluor White M2R dye (blue fluorescence), live or dead cells stained with FUN1 488 nm (green fluorescence), active metabolic cells stained with FUN1 532 nm (red fluorescence), and a merged image, respectively. Untreated cells in PBS were used as a negative control.
Figure 4.
Confocal scanning fluorescence images of C. albicans ATCC SC5314 cells after exposure to 0.5 MIC (31.25 μg/mL), MIC (62.5 μg/mL), and 2MIC (125 μg/mL) of A. occidentale. Live or dead intact cells stained with Calcofluor White M2R dye (blue fluorescence), live or dead cells stained with FUN1 488 nm (green fluorescence), active metabolic cells stained with FUN1 532 nm (red fluorescence), and a merged image, respectively. Untreated cells in PBS were used as a negative control.
Figure 5.
Scanning electron microscopy of C. albicans ATCC SC5314 cells treated with A. occidentale leaf extract. Approximately 1 x 108 cells were incubated without (a-c, controls), with 62.5 μg/ml (d-f) and 125 μg/ml of extract for 24 h. Masses of debris and cell leakage were observed on the cell surface after treatment. Image size: a-e-g: 1µm; b-c: 2 µm; f: 5 µm; d,h,i: 500 nm.
Figure 5.
Scanning electron microscopy of C. albicans ATCC SC5314 cells treated with A. occidentale leaf extract. Approximately 1 x 108 cells were incubated without (a-c, controls), with 62.5 μg/ml (d-f) and 125 μg/ml of extract for 24 h. Masses of debris and cell leakage were observed on the cell surface after treatment. Image size: a-e-g: 1µm; b-c: 2 µm; f: 5 µm; d,h,i: 500 nm.
Figure 6.
Transmission electron microscopy of Candida albicans ATCC SC5314 cells treated with A. occidentale leaf extract. Approximately 1 x 108 cells were incubated without (panels a–d) or with 125 μg/ml extract (panels e–h) for 4 h. Still intact cells treated presented cytoplasmic changes with the presence of several microvacuoles (panels e, g, h), membrane retraction (e, g), irregular cell wall (panels e-h) and disintegrated nucleus envelope (f). Bar: ~2μm.
Figure 6.
Transmission electron microscopy of Candida albicans ATCC SC5314 cells treated with A. occidentale leaf extract. Approximately 1 x 108 cells were incubated without (panels a–d) or with 125 μg/ml extract (panels e–h) for 4 h. Still intact cells treated presented cytoplasmic changes with the presence of several microvacuoles (panels e, g, h), membrane retraction (e, g), irregular cell wall (panels e-h) and disintegrated nucleus envelope (f). Bar: ~2μm.
Figure 7.
Measurement of ROS in C. albicans. Two concentrations were used (62.5-125 μg/mL) of A. occidentale; AmB 4 μg/mL was used as a positive control, and NAC 60mM as ROS a scavenger. All data are represented with the mean ± SD. Fluorescence was detected with a DCF-DA. This figure was constructed with information from three independent 2-hour experiments performed under the same protocol. Statistical analysis was done through ANOVA. (***) with 125 μg/mL produced more ROS than untreated control p <0.001. (**) with 62.5 μg/mL produced more ROS than untreated control p <0.005. (*) A. occidentale 125 μg/mL more ROS than A. occidentale 125 μg/ml + NAC p <0.005 (#) AmB 4 μg/mL more ROS than A. occidentale 125 μg/mL p <0.01.
Figure 7.
Measurement of ROS in C. albicans. Two concentrations were used (62.5-125 μg/mL) of A. occidentale; AmB 4 μg/mL was used as a positive control, and NAC 60mM as ROS a scavenger. All data are represented with the mean ± SD. Fluorescence was detected with a DCF-DA. This figure was constructed with information from three independent 2-hour experiments performed under the same protocol. Statistical analysis was done through ANOVA. (***) with 125 μg/mL produced more ROS than untreated control p <0.001. (**) with 62.5 μg/mL produced more ROS than untreated control p <0.005. (*) A. occidentale 125 μg/mL more ROS than A. occidentale 125 μg/ml + NAC p <0.005 (#) AmB 4 μg/mL more ROS than A. occidentale 125 μg/mL p <0.01.
Figure 8.
(a) Measurement of ROS in UPR mutants strain. We employed A. occidentale 62.5 μg/mL; AmB 4 μg/mL was used as a positive control. All data are represented with the mean ± SD. Fluorescence was detected with a DCF-DA. This figure was constructed with information from three independent 2 h experiments performed under the same protocol. (b) Phenotypic analysis of C. albicans UPR mutants.
Figure 8.
(a) Measurement of ROS in UPR mutants strain. We employed A. occidentale 62.5 μg/mL; AmB 4 μg/mL was used as a positive control. All data are represented with the mean ± SD. Fluorescence was detected with a DCF-DA. This figure was constructed with information from three independent 2 h experiments performed under the same protocol. (b) Phenotypic analysis of C. albicans UPR mutants.
Figure 9.
Possible mechanism of action associated with endoplasmic reticulum stress. Box 1: Ire1p is activated by ER stress. Upon activation, Ire1p undergoes autophosphorylation and dimerization. HAC1 mRNA is spliced by activated Ire1p, then, transcription factor Hac1 up-regulates UPR target genes, mainly Kar2p to restore homoeostasis. Box 2: After treatment with the extract on Δhac1 and Δkar2 suggest Ire1p is activated and spliced HAC1 mRNA, but the Hac1p transcription factor is in low quantity and/or its translocation to the nucleus is affected, preventing efficient expression of the UPR target genes and consequently Kar2p cannot alleviate stress, contributing to the increase of ROS in the ER leading to cell death by inefficient UPR.
Figure 9.
Possible mechanism of action associated with endoplasmic reticulum stress. Box 1: Ire1p is activated by ER stress. Upon activation, Ire1p undergoes autophosphorylation and dimerization. HAC1 mRNA is spliced by activated Ire1p, then, transcription factor Hac1 up-regulates UPR target genes, mainly Kar2p to restore homoeostasis. Box 2: After treatment with the extract on Δhac1 and Δkar2 suggest Ire1p is activated and spliced HAC1 mRNA, but the Hac1p transcription factor is in low quantity and/or its translocation to the nucleus is affected, preventing efficient expression of the UPR target genes and consequently Kar2p cannot alleviate stress, contributing to the increase of ROS in the ER leading to cell death by inefficient UPR.
Figure 10.
Effect on the mitochondrial function in C. albicans. Three concentrations of A. occidentale were used (31.25-125 μg/mL); sodium azide 10mM was used as a positive control and NAC 60mM as a ROS scavenger. All data are represented with the mean ± SD. Fluorescence was detected with rhodamine 123. The figure was constructed with information from three independent 2-hour experiments performed under the same protocol. Statistical analysis was done through ANOVA. (*) with 125 μg/mL produced more fluorescence than the positive control, p <0.002. (**); with 62.5 μg/mL, more ROS was produced than with the positive control p <0.007. (***).
Figure 10.
Effect on the mitochondrial function in C. albicans. Three concentrations of A. occidentale were used (31.25-125 μg/mL); sodium azide 10mM was used as a positive control and NAC 60mM as a ROS scavenger. All data are represented with the mean ± SD. Fluorescence was detected with rhodamine 123. The figure was constructed with information from three independent 2-hour experiments performed under the same protocol. Statistical analysis was done through ANOVA. (*) with 125 μg/mL produced more fluorescence than the positive control, p <0.002. (**); with 62.5 μg/mL, more ROS was produced than with the positive control p <0.007. (***).
Figure 11.
Confocal scanning fluorescence images of A. occidentale-induced inhibited growth in Candida albicans ATCC SC5314. Cells treated with 62.5 and 125 μg/mL of ethanolic extract after 4 hours were stained with Calcofluor-white (CW)/propidium iodide (PI). CW, excitation at 375 nm; PI, excitation at 555 nm. Scale bars, 10 μm.
Figure 11.
Confocal scanning fluorescence images of A. occidentale-induced inhibited growth in Candida albicans ATCC SC5314. Cells treated with 62.5 and 125 μg/mL of ethanolic extract after 4 hours were stained with Calcofluor-white (CW)/propidium iodide (PI). CW, excitation at 375 nm; PI, excitation at 555 nm. Scale bars, 10 μm.
Figure 12.
(a) Hemolytic profiles; tween 20 was used as positive control; and (b) Cell viability of fibroblasts L929 in the presence of A. occidentale extract.
Figure 12.
(a) Hemolytic profiles; tween 20 was used as positive control; and (b) Cell viability of fibroblasts L929 in the presence of A. occidentale extract.
Figure 13.
Effect of A. occidentale extract on the growth kinetic of (a) C. auris PUJ/HUSI 435 and (b) C. auris PUJ/HUSI 537. This figure was constructed with information from three independent 48-hour curves performed under the same protocol; (c) Images of C. auris PUJ/HUSI 435 after exposure to MIC and 2MIC from the LIVE/DEAD™ Yeast Viability kit from Thermo Fisher via confocal laser scanning microscopy; (d) SEM of C. auris PUJ/HUSI 435. The first photo is a cell without treatment, the second and third photos are cells treated with MIC, and then in (e) the permeability of the membrane was evaluated by means of PI staining after exposing C. auris PUJ/HUSI 435 for 4 hours to MIC.
Figure 13.
Effect of A. occidentale extract on the growth kinetic of (a) C. auris PUJ/HUSI 435 and (b) C. auris PUJ/HUSI 537. This figure was constructed with information from three independent 48-hour curves performed under the same protocol; (c) Images of C. auris PUJ/HUSI 435 after exposure to MIC and 2MIC from the LIVE/DEAD™ Yeast Viability kit from Thermo Fisher via confocal laser scanning microscopy; (d) SEM of C. auris PUJ/HUSI 435. The first photo is a cell without treatment, the second and third photos are cells treated with MIC, and then in (e) the permeability of the membrane was evaluated by means of PI staining after exposing C. auris PUJ/HUSI 435 for 4 hours to MIC.
Table 1.
List of heterozygous haploid deletion strains used in this study.
Table 1.
List of heterozygous haploid deletion strains used in this study.
Strains* |
Gene ontology (GO)** |
C. albicans Δhog1/HOG1 |
MAP kinase of osmotic-, heavy metal-, and core stress response; role in regulation of response to stress |
C. albicans Δmkc1/MKC1 |
MAP kinase; role in membrane perturbation, or cell wall stress |
C. albicans Δirei1/IREI1 |
Protein kinase involved in regulation of unfolded protein response |
C. albicans Δkar2/KAR2 |
Chaperone with role in translocation of proteins into the endoplasmic reticulum |
C. albicans Δhac1/HAC1 |
bZIP transcription factor with role in unfolded protein response |
C. albicans Δero1/ERO1 |
Role in formation of disulfide bonds in the endoplasmic reticulum |
Table 2.
Peak assignments of the A. occidentale extract using UPLC-PDA/qTOF-MS in negative mode.
Table 2.
Peak assignments of the A. occidentale extract using UPLC-PDA/qTOF-MS in negative mode.
Peak no. |
Compounds |
Rt (min) |
[M–H]- (m/z)
|
λmax (nm) |
References |
1 |
5-Methylcyanidin-3-O-hexoside |
10.7 |
463.0762 |
282.1-514.2 |
[44] |
2 |
Quercetin 3-O-α-L-rhamnoside |
11.5 |
447.0922 |
255.9-349.5 |
[45] |
3 |
Quercetin galloyl-O-deoxy-hexoside |
12.0 |
599.1026 |
257.1-349.5 |
[46] |
4 |
Quercetin 3-O-xylopyranoside |
13.5 |
431.0954 |
257.1-349.5 |
[41] |
5 |
Unknown flavonoid* |
14.3 |
Nd |
263.0-349.5 |
|
6 |
Unknown flavonoid* |
16.4 |
Nd |
258.2-347.2 |
|
7 |
Kaempferol 3-O-α-glucoside |
18.5 |
433.0925 |
266.6-348.3 |
[41] |
10 |
Agathisflavone |
30.8 |
537.0823 |
271.3-334.5 |
[40,46] |
Table 3.
Doubling times of A. occidentale against Candida albicans.
Table 3.
Doubling times of A. occidentale against Candida albicans.
Treatment (μg/mL)
|
Doubling time (hours) |
C. albicans ATCC SC5314 |
C. albicans PUJ/HUSI 256 |
0 |
2.78 84.05 22.58 7.61 3.16 |
3.43 153.4 55.61 17.40 4.31 |
250 |
125 |
62.5 |
31.25 |