Exploring the Biomedical Potential of Endophytic Fungi Isolated from Panamanian Mangroves

Abstract

Objective: The objective of this study was to assess the biomedical potential of endophytic fungi isolated from Panamanian mangrove plants. This was achieved through the biological evaluation of organic extracts to determine their antiparasitic, anticancer, toxicity activity, and inhibition of the enzyme α-glucosidase. Methods: Leaves from various mangrove plant species in five different zones along the Pacific coast of Panama were collected using standard procedures. Endophytic fungi present on the leaves of each plant mangrove species collected were isolated using standard microbiological protocols. Organic extracts of each fungal isolate were prepared through maceration. The antiparasitic activity against Plasmodium falciparum, Trypanosoma cruzi, and Leishmania donovani, toxicity against Artemia salina, and anticancer activity in the MCF-7 cell line were investigated following protocols established by our institution. Finally, the ability to inhibit the enzymatic activity of α-glucosidase was evaluated by monitoring the hydrolysis of PNP-G. Results: Forty-three fungal isolates were isolated from the mangrove leaves analyzed. Organic extracts were prepared from each isolate for subsequent biological evaluation. It was found that twenty-one of the organic extracts showed activity in at least one of the performed bioassays. In the brine shrimp test, most samples were nontoxic or exhibited weak toxicity. Two fungal extracts showed anticancer activity, three showed antileishmanial activity, and one exhibited weak activity against P. falciparum. The most significant biological activity shown by the evaluated extracts was the inhibition of the α-glucosidase enzyme, where 44% of the fungal extracts were active. Conclusion: The most promising biomedical activity detected in the fungal isolates associated with mangrove plants is the inhibition of the α-glucosidase enzyme. Among the active samples, a high percentage belonged to fungi identified within the class Dothidiomycetes, revealing that this class is a potential new source of compounds with hypoglycemic activity.

Introduction

Currently, the mangrove ecosystem faces several challenges that threaten its existence and are leading to its decline and potential extinction in several parts of the world, including Panama. One of the most important threats to mangroves is the loss and degradation of their habitat, which in most cases is associated with human pressures. This fact shows that many people are not aware of the ecological importance and the real benefits that the mangrove ecosystem brings us. Taking this into consideration and the fact that in Panama the number of scientific research on the mangrove ecosystem is very low, there is a need for more scientific studies to prevent the loss of knowledge about this crucial ecosystem, as large areas of mangroves continue to disappear rapidly. ¹

On the other hand, one of the most interesting aspects to investigate within the mangrove ecosystem is the presence of endophytic fungi and their bioactivities from the bioprospecting perspective. These microorganisms live inside plants without causing harm and have been isolated from all major groups of plants, ² including those found in extreme or unusual habitats.

The number of extant fungal endophytes is a topic of extensive debate, and current estimates suggest that the number of fungal species ranges between 1.5 and 5.1 million.^3,4 Mangrove endophytic fungi constitute the second ecological group of fungi of marine origin, ⁵ since estimates of the number of species of marine fungi are within the range of 10,000 to 12,500, although a recent report suggests that there may be up to 1 million undiscovered species. ⁶ This last estimate is highly credible because the specific composition of fungal endophytes can vary depending on factors like the type of mangrove species, geographic location, and prevailing environmental conditions.

Extensive studies of a total of 60 different mangrove plant species from different countries around the world have led to the isolation of numerous endophytic fungi. Among these species, Avicennia marina stands out with the highest number of identified taxa, totaling 142. It is closely followed by Rhizophora mangle with 125 taxa and Rhizophora apiculata with 109 taxa. ⁷ Interestingly, several mangrove plant species have not been systematically investigated for the presence of mangrove fungi, including Rhizophora harrisonii, Tabebuia palustris, Mora oleifera, and Pelliciera rhizophorae. These species are exclusively found in the American continent, and in the present study, we report the presence of endophytic fungi in the latter two species.

Many investigations on the fungus living in mangrove plants reveal that several fungal genera have recurrently emerged as commonly reported endophytes within mangroves. Among these genera are Alternaria, Aspergillus, Cladosporium, Colletotrichum, Fusarium, Penicillium, Pestalotiopsis, Phoma, Phomopsis, Phyllosticta, and Trichoderma, among others.^4,7,8

In recent times, there has been a notable rise in research publications concerning mangrove fungi. However, numerous mangrove regions worldwide remain largely unexplored, particularly in Africa, Australia, New Zealand, Central America, and South America. The mangrove ecosystems of Bangladesh, China, Indonesia, Myanmar, Pakistan, and Sri Lanka also suffer from a lack of comprehensive studies focused on mangrove fungi. ⁷ While Brazil and the United States have contributed a significant portion of the research conducted on the American continent, the countries boasting the highest diversity of mangrove plants, such as Costa Rica, Panama, and Colombia, have comparatively few reports available.

Endophytic fungi from mangrove plants have diverse biotechnological applications, including biodiesel production, bioremediation, and biomedical potential against various diseases.^9–12 In 2022, Chen et al published an insightful review that provides a comprehensive summary of publications up until 2020 on secondary metabolites derived from mangrove endophytic fungi and their associated bioactivities. This extensive work highlighted the isolation of 1387 compounds from this source, showcasing the remarkable diversity of metabolites. These compounds exhibit a broad spectrum of pharmacological activities, encompassing cytotoxic, antibacterial, antifungal, antiviral, antioxidant, lethality-toxicity, anti-inflammatory, enzyme inhibitory, insecticidal properties, and more. ¹³ This review constitutes a valuable resource for understanding the global relevance of the study of endophytic fungi associated with mangroves.

Most scientific reports related to the biomedical potential of endophytic fungi from mangrove plants come from studies carried out in Asia and Africa, while reports from the Americas are somewhat limited. Thus, it is crucial to generate more scientific information on this type of microorganism in our continent. In America, some studies on mangrove endophytic fungi have been conducted^14,15; however, more systematic studies are necessary, including most mangrove plant species reported in this continent, and such a study is feasible in Panama due to the presence of about 92% of the continental mangrove plant species reported in the literature. ¹⁶

This research aimed to study the diversity of endophytic fungi from Panamanian mangrove plants and evaluate their organic extracts in different bioassays to detect their biomedical potential in terms of their antiparasitic, hypoglycemic, and anticancer activities. The information generated here provides an overview of the diversity and biological activities of mangrove endophytes in the Americas, and due to the mangrove species composition, it could be representative of the continent.

Materials and Methods

Microorganisms

Thirty-three different sets of healthy and mature leaf samples belonging to eight different mangrove plant species (Avicennia bicolor, Avicennia germinans, Conocarpus erectus, Laguncularia racemosa, Mora oleifera, Pelliciera rhizophorae, Rhizophora mangle, Rhizophora racemosa) were collected from different places along the Pacific coast of the Republic of Panama [Chame Bay (8°39'29.2"N, 79°48'10.8"W), Juan Diaz (9°01'04.1"N, 79°26'14.0"W), Veracruz (8°55'34.5"N, 79°33'56.0"W), Horconcito (8°19'17.8"N, 82°09'31.7"W), Hermosa Beach (8°13'15.8"N, 82°09'42.9"W), La Barqueta Beach (8°17'37.0"N, 82°30'32.9"W), Pedregal Port (8°21'34.4"N, 82°25'59.8"W), and El Salado Beach (8°12'00.4"N, 80°29'00.7"W)] for the isolation of their endophytic fungi.

Endophytic fungi were isolated following previously reported protocols. ¹⁷ Briefly, leaves were washed to remove soil, salt, or sand residues. Leaves were surface-sterilized using a disinfection battery (Ethanol 70%-Sodium Hypochlorite 1% -Ethanol 70%) and rinsed with sterile distilled water. Leaves were cut into several 1 mm² pieces and placed in individual Petri dishes with Bacto™ Malt Extract agar with 1% artificial sea salt (MEA) and antibiotic (Chloramphenicol). The Petri dishes were left at room temperature (25 °C). Petri dishes were checked daily to observe fungal growth. The growing endophytic fungi were isolated and passed on to new Petri dishes containing (MEA) without antibiotics.

Endophytic Fungi Culture

Mangrove endophytic fungi were cultured in Petri dishes containing malt extract agar (MEA). A portion of the mycelium from each endophytic fungus (0.5 cm²) was placed in 20 Petri dishes. The Petri dishes were incubated at room temperature (25 °C) under static conditions for 21 days. Afterward, the dishes were frozen and kept at −30 °C.

Organic Extracts Preparation

The frozen Petri dishes containing both the endophytic fungi and culture medium were freeze-dried for 48 h and the mycelium and MEA were subsequently ground into a coarse powder. To extract the secondary metabolites from the endophytic fungi, overnight maceration (12 h each) was performed using Ethyl Acetate. The resulting organic solvent was filtered and collected in round bottom flasks. The extracted solution was then concentrated under reduced pressure using a rotary evaporator.

Endophytic Fungi Identification

Endophytic fungal strains that showed at least one biological activity were identified based on DNA sequence identity of their entire nuclear ribosomal internal transcribed spacer region (ITS, including the ITS 1, the 5.8S gene, and the ITS2) with those of taxonomic type material deposited in the National Institutes of Health genetic sequence database (GenBank). We used the Basic Local Alignment Search Tool (BLAST) ¹⁸ to compare the sequences generated with those in GenBank. Taxonomic assignment to the fungal isolates were made based on the GenBank top hit BLAST searches. Isolates with relatively low sequence identity (91%–96%) were only assigned to the family rank. DNA sequence identity of 97% or more was considered sufficient to assign a genus rank and more than 98% to assign species name.

DNA Extraction, PCR, and sequencing were performed using a previously reported protocol. ¹⁹

Toxicity Assay

Artemia salina nauplii were collected after 48 h of incubation. A bioassay was performed in a ninety-six wells plate. Samples and controls were dissolved in a solution containing 50 μL of DMSO and 550 μL of Artificial Sea Water (ASW). Sample tests were diluted into four different concentrations (1000, 500, 250 and 125 ppm). Each concentration was assessed in triplicate. The potassium dichromate solution and the solvent for sample preparation were used as the positive and negative control, respectively. Each well was filled with 100 μL of ASW containing 10 to 15 nauplii and 100 μL of test solution. The plate was incubated at room temperature (25 °C) for 24 h. After the incubation period, dead nauplii were counted with an inverted microscope. For this purpose, 100 μL of methanol was added to the well, and dead nauplii were counted after 30 min.^20,21

Antiparasitic Assays

Leishmania donovani and Plasmodium falciparum

Samples and parasites were incubated for 48 h. The amount of parasite in the culture was determined using a DNA cross-linking agent. PicoGreen® solution (1%) was added to wells, in a dark condition, after 48 and 72 h of incubation. The plates were shaken and read in a microplate reader set up to 485/20 nm for the excitation step and to 528/20 nm for emission. Positive controls were amphotericin B for L. donovani and chloroquine for P. falciparum assay.^20,22

Tripanosoma cruzi

Trypomastigotes were exposed to test samples, in three different concentrations, for 120 h. In this assay, a colorimetric method is used to determine the inhibition of parasite growth as detected by the reduction of β-galactosidase (β-Gal) as a reporter gene, expressed by the Tulahuen clone C4 of T. cruzi. Assays are performed on trypomastigotes, the intracellular form of the parasite infecting African green monkey kidney (VERO) cells, exposed during 120 h to different concentrations (50, 10, and 2 µg/mL) of the test substance. The resulting color from the cleavage of chlorophenol red-β-d-galactoside (CPRG) by β-Gal expressed by the parasite was measured using a Benchmark BioRad microplate reader at 570 nm. Nifurtimox was used as a positive control (IC₅₀ 0.15-13.4 µM).^20,22

Anticancer Assay

In a 96-well plate, 100 μL of RPMI buffer containing MCF-7 cells was added to each well and incubated for 24 h at 37 °C. Samples were diluted in DMSO and analyzed by duplicate. After the incubation period, 100 μL of the sample was added to each well. Color control was RPMI without cells. The positive control was Adriamycin. The 96-wells plate was incubated for 48 h at 37 °C under an atmosphere of 5% CO₂/95% air mixture. After the incubation period, cells were fixed with 50% trichloroacetic acid for 1 h, air-dried, and stained with sulforhodamine B (SRB). Excess SRB was eliminated by washing the 96-well plate with 1% acetic acid. Bound SRB was dissolved with 10 mM Tris. Plates were shaken and read in a microplate reader set up to 515 nm. ²³

Inhibition of Alpha-Glucosidase Assay

A 96 wells plate was used to measure the α-glucosidase inhibitory properties of the extract. Test samples and positive control were dissolved in DMSO. Samples test (20 μL) were added in wells containing 150 μL of the enzyme (32 mU/mL) and incubated for 7 min at 37 °C. After incubation, 150 μL of PNPG (2 mM) was added to each well, and the plate was incubated for 20 min at 37 °C. Afterward, the absorbance of p-nitrophenol released from the reaction was measured at 400 nm.^20,24 All assays were performed in duplicate.

The activity of samples was calculated as a percentage in comparison to a control (DMSO or MeOH instead of sample solution) according to the following equation:

$$\text{\%}Inhibition=\left(\frac{(\mathrm{\Delta }{A}_{control}-\mathrm{\Delta }{A}_{sample})}{\mathrm{\Delta }{A}_{control}}\right)\times 100\text{\%}$$

ΔA_control: absorbance of the control – absorbance of the blank

ΔA_sample: absorbance of the sample – absorbance of the sample blank.

The concentration required to inhibit the activity of the enzyme by 50% (IC₅₀) was calculated by regression analysis.^1,20

Statistical Analysis

The data from biological assays were expressed as the mean ± SD of three replicates. The analysis was performed using Excel 2016. One-way analysis of variance (ANOVA) and Tukey posttest were used to evaluate the possible differences among the means. P values ≤ .05 were considered significant differences.

Results

Fungal Diversity

A total of 43 different fungal strains were obtained from the leaves processed belonging to eight different species of mangrove plants (Avicennia bicolor, Avicennia germinans, Conocarpus erectus, Laguncularia, racemosa, Mora oleifera, Pelliciera rhizophorae, Rhizophora mangle, Rhizophora racemosa).

Toxicity Assay

Only eight isolates showed an IC₅₀ below 1 mg/mL, ML-EM05-10 isolated from L. racemosa, collected in Juan Diaz; ML-EM10-01, isolated from A. germinans, collected in Horconcito; ML-EM12-01 and 05 isolated from R. mangle, collected in Hermosa Beach; ML-EM18-11 isolated from A. germinans, collected in El Salado beach, ML-EM19-05 isolated from Conocarpus erectus; ML-EM20-07 isolated from R. racemosa and ML-EM21-06 isolated from R. mangle, all collected in El Salado beach (IC₅₀ = 803, 400, 785, 724, 789, 634, 381 and 668 µg/mL, respectively).

Antiparasitic Activity

Three of the tested endophytic fungi were active against Leishmania donovani, and only one isolate was active against Plasmodium falciparum. None of the isolates were found to be active against Trypanosoma cruzi. Endophytic fungus ML-EM05-10, isolated from L. racemosa and collected in Juan Diaz, showed 62.0% growth inhibition (IC₅₀ 609.9 μg/mL) against P. falciparum. Endophyte ML-EM10-02, isolated from A. germinans and collected in Horconcito, showed 78.0% of growth inhibition (IC₅₀ 70.4 μg/mL) against L. donovani. ML-EM12-05, isolated from R. mangle and collected in Hermosa beach, resulted in 74.0% of growth inhibition (IC₅₀ 132.9 μg/mL) against L. donovani, and ML-EM14-04, isolated from P. rhizophorae and collected in La Barqueta beach, provided 78.7% of growth inhibition (IC₅₀ 64.6 μg) against L. donovani.

Anticancer Activity

The anticancer bioassays resulted in only two fungal extracts possessing activity against MCF-7 cell line: ML-EM14-04 isolated from P. rhizophorae and collected in La Barqueta beach as well as ML-EM20-01, isolated from R. racemosa and collected in El Salado beach, each with 8.8% (IC₅₀ 2.9 μg/mL) and 27.5% (IC₅₀ 117.3 μg/mL) of cell growth, respectively.

α-Glucosidase Inhibition

Of the 43 isolates tested, 44% of the fungal extracts possess α-glucosidase inhibitory properties (see Table 1). From the bioactive results, 14 samples showed moderate activity (60% to 80% of inhibition); meanwhile, five samples showed strong activity (above 80% of inhibition).

Table 1.

Percentage of Inhibition of the α-Glucosidase Enzyme by the Active Organic Extracts.

Isolate	Percentage of inhibition a	IC50 b
ML-EM02-01	95.5 ± 0.5	0.87
ML-EM05-10	70.8 ± 0.7	41.80
ML-EM10-01	88.2 ± 0.4	2.94
ML-EM12-01	69.8 ± 0.1	41.18
ML-EM12-05	90.7 ± 04	1.47
ML-EM17-03	88.1 ± 0.3	2.69
ML-EM18-11	69.8 ± 0.8	40.95
ML-EM19-05	79.7 ± 0.9	12.72
ML-EM20-01	61.3 ± 0.7	85.33
ML-EM20-02	73.6 ± 0.6	32.83
ML-EM20-07	71.7 ± 0.5	36.08
ML-EM21-03	84.2 ± 0.8	7.25
ML-EM21-06	78.8 ± 1.1	14.82
ML-EM21-10	64.8 ± 0.5	64.72
ML-EM22-02	63.8 ± 0.8	69.89
ML-EM22-08	66.0 ± 1.5	58.53
ML-EM26-01	80.0 ± 0.7	10.68
ML-EM26-02	71.2 ± 0.6	37.76
ML-EM28-01	66.7 ± 0.8	55.41

^a

at 6.25 mg/mL, ^b in μg/mL.

Molecular Identification of Fungal Isolates

According to the main objective of our research, all fungal isolates that were active in at least one assay were molecularly identified based on their ITS sequences (Table 2). A total of 16 isolates were identified to the species rank and one isolate was identified only to the genus rank. Four isolates were only identified to the family level due to their low DNA sequence identity (<97%) to known fungi in the NCBI GenBank database. This last suggest potential for taxonomic novelties to be further studied, for example, isolates ML-EM22-08 (Botryosphaeriaceae sp.), ML-EM10-01 (Elsinoaceae sp. 1) and ML-EM20-02 and ML-EM20-07 (Elsinoaceae sp. 2) (See Table 2). Lagungularia racemosa was the mangrove species with the highest diversity of bioactive fungi with a total of four species active in at least one assay followed by R. racemosa with three bioactive endophyte species isolated from this host. Phyllosticta capitalensis was the most commonly found and host generalist bioactive species isolated from R. mangle, P. rhizophorae and M. oleifera.

Table 2.

Taxonomic Identification of Endophytic Fungi with Bioactivity.

Fungal isolate	Taxonomy based on comparison with GenBank top hit	Isolate family, order and Class*	Host plant	Query cover (%)	Identity (%)	GenBank Top hit Accession No.
ML-EM21-10	Phyllosticta capitalensis	Phyllostictaceae, Botryosphaeriales, Dothideomycetes	R. mangle	99	100	OL957169.1
ML-EM28-01	Phyllosticta capitalensis	Phyllostictaceae, Botryosphaeriales, Dothideomycetes	M. oleifera	100	100	OL957169.1
ML-EM26-01	Phyllosticta capitalensis	Phyllostictaceae, Botryosphaeriales, Dothideomycetes	P. rhizophorae	100	99.85	OL957169.1
ML-EM26-02	Phyllosticta capitalensis	Phyllostictaceae, Botryosphaeriales, Dothideomycetes	P. rhizophorae	100	100	OL957169.1
ML-EM21-03	Phyllosticta capitalensis	Phyllostictaceae, Botryosphaeriales, Dothideomycetes	R. mangle	99	100	OL957169.1
ML-EM18-11	Phyllosticta capitalensis	Phyllostictaceae, Botryosphaeriales, Dothideomycetes	A. germinans	99	100	OL957169.1
ML-EM14-04	Phyllosticta sp.	Phyllostictaceae, Botryosphaeriales, Dothideomycetes	P. rhizophorae	42	96.78	NR_156631.1
ML-EM22-08	Phyllostictaceae sp.	Phyllostictaceae, Botryosphaeriales, Dothideomycetes	L. racemosa	93	93.42	NR_182625.11
ML-EM02-01	Phaeophleospora eucalypticola	Mycosphaerellaceae, Mycosphaerellales, Dothideomycetes	L. racemosa	98	100	NR_145123.1
ML-EM05-10	Zasmidium musae	Mycosphaerellaceae, Mycosphaerellales, Dothideomycetes	L. racemosa	94	100	NR_176687.1
ML-EM10-02	Pantospora guazumae	Mycosphaerellaceae, Mycosphaerellales, Dothideomycetes	A. germinans	100	99.63	JN190956.1
ML-EM22-02	Pseudocercospora indonesiana	Mycosphaerellaceae, Mycosphaerellales, Dothideomycetes	L. racemosa	99	99.45	MH863211.1
ML-EM20-01	Hortaea werneckii	Teratosphaeriaceae, Mycospherellales, Dothideomycetes	R. racemosa	88	100	DQ168665.1
ML-EM10-01	Elsinoaceae sp. 1	Elsinoaceae, Myriangiales, Dothideomycetes	A. germinans	94	91.11	NR_165561.1
ML-EM20-02	Elsinoaceae sp. 2	Elsinoaceae, Myriangiales, Dothideomycetes	R. racemosa	96	95.14	NR_159836.1
ML-EM20-07	Elsinoaceae sp. 2	Elsinoaceae, Myriangiales, Dothideomycetes	R. racemosa	97	95.14	NR_159836.1
ML-EM21-06	Elsinoe embeliae	Elsinoaceae, Myriangiales, Dothideomycetes	R. mangle	83	98.52	NR_148136.1
ML-EM12-05	Elsinoe embeliae	Elsinoaceae, Myriangiales, Dothideomycetes	R. mangle	84	98.52	NR_148136.1
ML-EM19-05	Allophoma minor	Didymellaceae, Pleosporales, Dothideomycetes	A. erectus	95	100	MH861501.1
ML-EM17-03	Penicillium paxilli	Aspergillaceae, Eurotiales, Eurotiomycetes	A. bicolor	99	100	MH856391.1
ML-EM12-01	Hypoxylon fendleri	Hypoxylaceae, Xylariales, Sordariomycetes	R. mangle	97	98.26	MK287533.1

*Except for Hypoxylon fendleri ML-EM12-01 and Penicillum paxilli ML-EM17-03, all other fungi found to have bioactivity in the assays performed in this study, belong to the Dothidiomycetes class of fungi.

The endophytic fungi isolated belong to three classes of Ascomycota (Dothideomycetes, Eurotiomycetes, and Sordariomycetes). Among these, the dominant group is the Dothideomycetes with isolates located in the orders Botryosphaeriales, Mycosphaerellales, Myriangiales, and Pleosporales. Only two other endophytic fungi were found outside Dothidiomycetes: Hypoxylon fendleri (Sordariomyectes) and Penicillium paxilli, (Eurotiomycetes) orders.

Discussion

Mangroves in Panama, as in many parts of the world, are among the most vulnerable and underappreciated ecosystems. It is estimated that since 1969, over 50% of the mangrove coverage area has been lost in the country, and the pressures caused by human economic activities could lead to the loss of even more areas. ²⁵ This highlights the urgent need for research to generate more information and increase the value of the mangrove ecosystem, particularly of the plants which are the basic organisms of this ecosystem, and their endophytic microorganisms.

Due to the high concentrations of salts in the internal organs of mangrove plants, we expected them to be highly selective environments for the proliferation of endophytic microorganisms. This fact was evident in our results, in which we obtained only 43 different fungi isolated from numerous leaf samples belonging to eight different species of mangrove plants. Moreover, our results agree well with those of other similar studies on mangrove plants.^26–28

The biomedical potential of the isolated microorganisms was determined through in vitro biological evaluations using specific bioassays available in our research group. To accomplish this, organic extracts were prepared from each fungus and tested for their anticancer effects on MCF-7 cells, antiparasitic effects against Leishmania donovani, Plasmodium falciparum, and Trypanosoma cruzi, as well as their effects on the inhibition of α-glucosidase enzyme (well-known anti-diabetic target).

In bioprospecting studies from natural sources, the brine shrimp bioassay has traditionally been used as a preliminary screening method to evaluate the toxicity of crude extracts inexpensively and easily. We also used this bioassay as an initial biological evaluation and found that none of the extracts from the evaluated isolates had marked toxicity against Artemia salina, which is important because it indicates that active extracts could be harmless to mammalian cells. ²¹ There are limited reports in the literature regarding the activity of extracts from mangrove endophytic fungi, making it challenging to compare comprehensively with our study's findings. However, Abraham et al ²⁹ describes the analysis of 110 extracts of endophytic fungi using the brine shrimp bioassay, where researchers discovered that five extracts exhibited high toxicity against this crustacean. Remarkably, only 25.5% of the extracts showed no activity at the highest concentration (1000 ppm). ²⁹ In contrast, our study found that only 18.6% of the extracts demonstrated activity against Artemia at concentrations ≤1000 ppm, while 81.4% of our extracts were inactive. Therefore, both studies present contrasting results.

Regarding the results of the biological evaluations conducted on the organic extracts of the endophytic fungi of mangrove plants, we found that very few samples produced positive results except for the test against α-glucosidase. This could indicate that the extracts are not very toxic, that there may be some selectivity in their bioactivity, or that their activity may be present in other biological models that were not available to us.

For example, in the assay, using MCF-7 cells only the samples of the isolates ML-EM14-4 and ML-EM20-01 (with 8.8% and 27.5% inhibition of cell growth, respectively) showed anticancer activity. The low activity in the anticancer bioassay previously detected was consistent with the results of the evaluation of the toxicity of the extracts on the crustacean Artemia salina, where no sample showed a relevant toxic effect. Based on the results of both tests, we determined that the fungal extracts evaluated do not have significant potential as anticancer agents. In a recent study conducted by Rahaman et al ³⁰ 80 fungal isolates from nine species of mangrove plants were isolated from the Sundarban Mangrove Forests, which is recognized as the largest mangrove forest in the world. The authors examined the carcinogenic potential of these isolates using the MCF-7 cell line. Interestingly, only 9 isolates demonstrated antibacterial activity, indicating that approximately 11.2% of the tested samples exhibited such properties. In contrast, our study revealed only two isolates out of 43 that demonstrated some anticancer potential. As a result, the percentage of bioactive samples was considerably lower at 4.6%. This implies that anticancer activity is not widespread among mangrove endophytic fungi. Nevertheless, numerous reports in the literature highlight the presence of metabolites from mangrove endophytic fungi with anticancer activity. This biological activity has been extensively studied and documented, making it one of the key areas of focus in research.^11,28,31,32

A similar situation was found in the antiparasitic evaluations’ results, where only a few samples showed activity. In this case, organic extracts from Pantospora guazumae ML-EM10-02, Elsinoe embeliae ML-EM12-05 and Phyllosticta sp. ML-EM14-04 were active with relatively good antileishmanial activity. Only one sample showed moderate activity (ML-EM05-10) against P. falciparum, and the other samples were inactive against the three parasites evaluated. In general, the results suggest that the organic extracts from endophytic fungus from mangrove plants tested do not possess significant antiparasitic activity.

In the case of the antiparasitic activity of extracts from mangrove endophytes, there are few reports in the literature related to the antiparasitic potential of endophytic fungi isolated from mangroves. For example, the isolate identified as Penicillium expansum isolated from the mangrove Excoecaria agallocha was shown to be effective against the malaria parasite. ³³ Another study carried out in 2013 detailed the search for endophytic mangrove fungi with antimalarial properties, being the isolates Diaporthe sp. and Verticillium sp. the species that showed antimalarial activity against P. falciparum (3D7). ³⁴ Finally, Lasiodiplodia theobromae yielded a series of 1,4-naphthoquinones, while Fusarium sp., both isolated from Avicennia lanata, produced a series of dihydroisocoumarins. Remarkably, both sets of compounds exhibited notable activity against the parasite Trypanosoma brucei brucei. ³⁵

Among all the bioassays performed in this research, the α-glucosidase evaluations had the highest number of active samples, with 43% of the analyzed samples showing good hypoglycemic potential. The results of this study suggest that endophytes obtained from Panamanian mangrove plants are a promising source of hypoglycemic molecules. α-glucosidase is an enzyme responsible for breaking down disaccharides and releasing glucose, leading to hyperglycemia. Inhibition of this enzyme can help control blood glucose levels. ³⁶ These findings encourage us to consider this area as an opportunity for future research, with a focus on performing bio-guided chemical studies on endophytes with high hypoglycemic activities to identify the molecules responsible for the activity and verify their efficacy and toxicity.

It is worth noting that the literature contains reports that support that mangrove endophytic fungi are prolific products of α-glucosidase inhibitory compounds, since mangrove endophyte species such as Xylaria sp. BL321, Penicillium chermesinum (ZH4-E2), fungus B60 (unidentified), Meyerozyma guilliermondii, Alternaria sp., Aspergillus sp., Nectria sp., and Talaromyces amestolkiae, all isolated from mangrove plants, demonstrated varying degrees of inhibition against the enzyme α-glucosidase^37–41 Another interesting point in our study, the percentage of fungal isolates demonstrating activity against the alpha-glucosidase enzyme was higher compared to other studies that evaluated the same target, as in the case of the study of Rahaman et al, ³⁰ where only 18.7% of the fungal isolates exhibited this activity. However, the active fungal isolates in the Rahaman et al study were more potent in terms of activity.

Based on the results obtained in the identification process and the biological evaluations, we found that 19 of the 21 isolates, that showed bioactivity in at least one of the assays performed, belonged to the Dothidiomycetes class of fungi. Among the bioactive endophytic fungi of the Dothidiomycetes class, we identified representatives of the families Mycosphaerellaceae, Teratosphaeriaceae, Didymellaceae, Elsinoaceae, and Phyllostictaceae. This shows that the bioactivity observed within the Dothidiomycetes class is not limited to a specific family but may be widespread in this class. It should be noted that some of these isolates had low DNA sequence identity, underscoring the need for more rigorous and systematic investigation to accurately identify and taxonomically classify these fungi. The identification of the molecules responsible for the widespread α-glucosidase inhibitory activity found in the Dothidiomycetes and two other distantly related classes of fungi may provide clues as to the evolution and adaptation of these fungi to the extreme environments of mangroves.

The Dothideomycetes class is known for its remarkable diversity, encompassing a wide range of fungi, including numerous endophytic species, and our study reveals that this class appears to be widely distributed in mangrove plants. Interestingly, there are several reports in the literature revealing that some fungal isolates of mangrove plants belonging to this class produce novel polyketide compounds with antioxidant and antibacterial biological activities.^42–45 Therefore, conducting future studies to identify the active components in the fungal isolates discovered in the present study becomes crucial to advance our understanding of the biomedical potential of the Dothideomycetes class of fungi obtained from mangrove plants.

Conclusions

In conclusion, endophytic fungi isolated from mangrove plants are a promising source of bioactive secondary metabolites, with 48.8% of the isolates evaluated showing at least one in vitro activity. Approximately 81% of the bioactive samples from fungal isolates displayed selectivity for one of the bioassays performed. Dothidiomycetes was the class of fungi that showed the highest potential in the biological evaluations carried out in this study, with 19 of the 21 isolates that showed bioactivity belonging to this group.

The most promising biological activity detected in the microbial isolates associated with mangrove plants is the inhibition of the α-glucosidase enzyme. Therefore, this source of natural compounds holds great potential for the development of oral hypoglycemic agents. Moreover, the presence of hypoglycemic, anticancer, and antiparasitic activity in the evaluated extracts suggests a wide range of biomedical applications derived from endophytic fungi associated with mangrove plants. Active fungal isolates obtained from the Panamanian mangrove plants are promising candidates for future research aimed at identifying the compounds responsible for each of the observed biological activities.