Introduction

Microorganisms are a potential source of new bioactive metabolites that can be used in human health. The endophytic fungi isolated from different plants, mainly medicinal plants with the capacity to produce a range of biologically active metabolites, shifted the focus of new drug sources from plants to fungi [1]. One advantage in producing functional fungal metabolites is the possibility of large-scale and short time cultivation, usually using substrates of low cost. Steroids, alkaloids, peptides, terpenoids, tannins, polyketones, flavonoids, and phenolics are some examples of compounds produced by fungi. Several secondary metabolites produced by endophytic fungi had their biological activities (e.g., antimicrobial, antiviral, cytotoxic, immunosuppressive, antiparasitic, antioxidant) and mode of action already reported [2,3,4,5,6,7,8]. Endophytic fungi from the order Xylariales constitute one of the sources of secondary metabolites with huge chemical diversity, which makes it possible to suggest a partnership between mycologists and chemists [9]. Some natural products from fungi have been used to develop commercial drugs, as cited by Helaly et al. [9] and Hyde et al. [10]. Besides producing bioactive compounds, the endophytes present beneficial functions to the host plant by modulating responses in biotic and abiotic stresses [11].

Recently, molecular phylogenetic studies using ITS, LSU, rpb2, and tub2 DNA sequence shows that Muscodor species have affinities to the genera Emarcea and Induratia. Thus, all Muscodor species were transferred to Induratia [12]. Endophytic strains of the genus Induratia are important mycofumigation agents. They produce a mixture of volatile organic compounds (VOCs) composed of organic acids, alcohols, esters, ketones, and aromatic hydrocarbons [12,13,14].

VOCs produced by Induratia spp. completely suppressed the growth of phytopathogenic fungi R. solani, C. coffeicola, and Phoma sp. and showed fungicidal effect against A. ochraceus on coffee beans and F. verticillioides in corn seeds [15]. The extrolites of Induratia species have also shown promising biological activity with possible applications in the agriculture and pharmaceutical industries, and many patents have been granted to various companies [16].

Induratia species were reported as enzymes producer, which includes extracellular amylase, cellulase, lipase, pectinase, phytase, protease, endo β-1,4 glucanase, and exo β-1,4 glucanase [17, 18]. Kapoor and Saxena [19] described a potent antioxidant activity in the extract obtained from Induratia indica (as Muscodor indica) and inhibitory action of xanthine oxidase in the extract of Induratia darjeelingensis (as Muscodor darjeelingensis). These actions help in the development of alternatives for the control of hyperuricemia and diseases related to oxidative stress.

The effect of VOCs of Induratia yucatanensis (as Muscodor yucatanensis) were lethal to the endophytes Colletotrichum sp., Phomopsis sp., and Guignardia mangiferae, and to the phytopathogens Phythophthora capsici, Phythophthora parasitica, Rhizoctonia sp., and Alternaria solani. It also inhibited root elongation in amaranth, tomato, and barnyard grass, particularly those produced during the first 15 days of fungal growth [20]. The same authors evaluated organic extracts from the culture medium and mycelium of I. yucatanensis on the same endophytes, phytopathogens, and plants. They observed that the mycelial extract inhibited plants more than endophytes or phytopathogens, and that both extracts share 12 allelochemicals including benzene derivatives, phenolic compounds, cyclopentadienes, esters, lactones, alkanes, aldehydes, and carboxylic acids.

Qadri et al. [21] characterized the soluble compounds produced by I. yucatanensis Ni30 (wild type) isolated from Elleanthus sp. and found brefeldin A as the major compound in the culture broth, whereas its mutant type EV1 presented ergosterol and xylaguaianol C. Induratia cinnamomi (as Muscodor cinnamomi) was reported to control Rhizoctonia solani AG-2 root rot in tomato plants, produce the phytohormone indole-3-acetic acid that increases seed germination and root elongation, solubilize metal minerals, and tolerate herbicides and insecticides [22].

Protease inhibitors can be found as one of the molecules present in fungal extracts. Proteases, especially serine proteases and metalloproteases, help in hemostasis by acting during the activation and at the end (dissolving thrombi, clots, and fibrin networks) of blood’s coagulation cascade [23]. Due to its regulatory activity in the formation and dissolution of clots and thrombi, substances that modulate proteases are often used as antithrombotic and anticoagulant drugs [24].

Phospholipase A2 inhibitors, which also may be present in fungal extracts, are molecules of great importance in hemostatic modulation. This is because phospholipases A2 are capable of promoting an intense toxic activity [25], resulting mainly from their catalytic activity on membrane phospholipids, which leads to structural breakdown, alteration in the flow of ions behind the membranes, activation of endogenous phospholipases of cells, or even cell death. In addition, the breakdown of phospholipids can result in the release of arachidonic acid, which is a precursor to eicosanoids via cyclooxygenase and lipoxygenase pathways and, therefore, causing changes in hemostasis and inflammatory responses [26].

Several natural inhibitors of proteases and phospholipases A2 have already been isolated from plants and animals to be used in the control of various pathological processes, such as thrombosis, hemorrhage, and cancer [27]. The extract produced by a fungus isolated from caves in Brazil, Lecanicillium aphanocladii, promoted an increase in plasma clotting time, induced by snake venom [28]. Most of the natural enzymatic modulators described in literature act reversibly through transient interactions, such as hydrogen and hydrophobic interactions [26]. Other inhibitors act as metal chelators, sequestering or forming complexes with ions, which are essential cofactors for the catalytic activity of enzymes. They may also interfere with other biological activities that depend on catalytic activity [27,28,29].

There is still little information about the soluble molecules produced by Induratia species. Thus, we investigated the presence of enzymatic modulators in ethyl acetate extracts (consisting of non-volatile compounds) obtained from four endophytic fungi belonging to the species I. coffeana and I. yucatanensis on parameters that assist in the maintenance of human hemostasis. Snake venom enzymes, mainly phospholipases A2 and proteases, have high structural and functional homology with human enzymes. Therefore, they were used to induce the necessary effects in this study, which allowed the evaluation of the bioactivity of compounds of the fungi on processes that occur in the human body.

Materials and Methods

Human Biological Material

Human blood was used for the in vitro evaluation of the action of the fungal extracts on the cytotoxicity on erythrocytes, plasma coagulation, and thrombolytic activities. Blood samples (10 mL) from 2 volunteers (aged over 18 and healthy)—for each test—were collected by venipuncture in tubes for vacuum collection in the median cubital vein. The tests were carried out after approval by the human research ethics committee of the Federal University of Lavras (COEP—UFLA), with a project registered under the number CAAE/80767417.0.0000.5148.

Obtaining the Fungal Extracts

Fungi of the Induratia genus were isolated from fresh and healthy leaves of organic coffee plantations (Coffea arabica) from Zona da Mata region, Viçosa municipality, Minas Gerais, Brazil. They were identified using DNA’s internal transcribed spacer (ITS) sequences. Single spore cultures were cryopreserved and deposited in the Mycological Collection of Lavras (CML) at the Department of Phytopathology at the Federal University of Lavras, Brazil, under the culture accession numbers (GenBank accessions number): I. coffeana CML4009 (MN658674), I. coffeana CML4010 (MN658675), I. coffeana CML4020 (MN658680) and I. yucatanensis CML4016 (MN658684). The fungi were grown in plates containing the medium PDA (Potato Dextrose Agar) for seven days at 25 ℃. After growth, six disks of approximately 5 mm diameter from the fungal colonies were transferred to 1 L of ME broth (Malt Extract), incubated at 25 ℃ in the dark on a shaker at 125 rpm for 12 days. The supernatants were separated from the mycelia by vacuum filtration. The extraction was carried out with the addition of ethyl acetate to the supernatants, in the proportion of 1: 0.5 (supernatant/ ethyl acetate), followed by removal of the solvent by rotary evaporation process. The extracts were then dissolved in Dimethyl sulfoxide p.a. 99.9% (DMSO) at a concentration of 10 mg mL−1.

Snake Venom

The Bothrops atrox venom, used as a tool to induce activities, was bought from the Bio-Agents serpentarium (Batatais, São Paulo, Brazil). The venom was weighed and dissolved in phosphate-buffered saline (PBS, pH 7.4) at a concentration of 10 mg mL−1, and stored at − 20 ºC. Screening tests were carried out to define the minimum inducing doses for each activity [27]. The use of venom in our research was registered at the National System for the Management of Genetic Heritage and Associated Traditional Knowledge (SisGen, Brazil) under the number ADF95EA.

Evaluation of Fungal Extracts Effects on the Coagulation of Citrated Human Plasma Induced by Enzymes Present in the Snake Venom (Previous Incubation of the Extracts with the Venom)

The extracts were previously incubated with the Bothrops atrox venom (6 µg) in the proportions of 1: 0.01, 1: 0.25, 1: 0.5, and 1: 1 (venom: extract; weight: weight) for 10 min at 37 °C. The samples were then added to tubes containing citrated plasma (200 µL) and then timed until a hard clot was formed. Controls containing only the venom were also performed. The minimum coagulant dose used in the tests was previously defined, which was the minimum amount of venom capable of inducing plasma coagulation (6 µg) in an interval between 50 and 70 s.

Evaluation of the Effects of Fungal Extracts on the Coagulation of Citrated Human Plasma Induced by Enzymes Present in the Venom (Previous Incubation of the Extracts with Plasma)

The effects exerted by the extracts of endophytic fungi on plasma coagulation were evaluated as in the previous test. However, the extracts were incubated with citrated plasma (200 µL) for 10 min at 37 °C, and then the B. atrox venom (6 µg) was added at the same proportions 1:0.01, 1:0.25, 1:0.5, and 1:1 (venom: extract; w: w). Controls containing only the venom (6 µg) or containing only the fungal extracts were also performed.

Phospholipase A2 Activity

The phospholipase activity was evaluated on agar gel according to the method described by Gutiérrez et al. [30]. The gel was made with 0.01 M CaCl2, egg yolk as a source of phospholipids (phosphatidylcholine, phosphatidylserine, and phosphatidylethanolamine), 1: 3 v/ v of PBS pH 7.2, 1% bacteriological agar, and 0.005% sodium azide. The medium was then poured into Petri dishes at 45 to 50 ºC. The samples were previously incubated for 30 min at 37 °C in the proportions of 1: 0.01, 1: 0.25, 1: 0.5, and 1: 1 (venom: extract; w: w), and then applied to holes made in the gel. After that, they were placed into a cell culture chamber for 12 h at 37 ºC. The formation of a translucent halo around the hole is indicative of phospholipase activity. The halos were measured in millimeters by a digital caliper to quantify the activity and its inhibition. The values were then converted into a percentage, considering the positive control (B. atrox venom at 10 µg) as 100% activity.

Cytotoxicity Test on Human Erythrocytes

This activity was evaluated using the methodology described by Gutiérrez et al. [30], with adaptations referring to the replacement of the phospholipid substrate by human erythrocytes. A human blood sample (5 mL) was collected and diluted in PBS (25 mL). The blood was then washed twice, using centrifugation at 1200 RPM for 10 min each and the supernatant removed and replaced with PBS at each centrifugation step. The red blood cell concentrate obtained was adjusted to a hematocrit of approximately 1% in the gel composition. The amount of venom (20 µg) considered as the minimum hemolytic dose was determined in a pilot test. It corresponded to the dose responsible for the formation of a halo in the gel with a diameter between 15 and 18 mm. The tests were performed with venom, fungal extracts, and both previously incubated in the proportions of 1: 0.01, 1: 0.25, 1: 0.5, and 1: 1 (venom: extract; w: w) for 30 min at 37 °C, and then applied to the holes in the gels. The gels remained in a cell culture chamber at 37 ºC for 24 h. The cytotoxic activity and its inhibition were quantified by measuring the translucent halos formed by the samples. The values obtained were converted into a percentage, considering the values of the venom alone as 100% activity.

Thrombolytic Test

The thrombolytic activity was evaluated on blood clots formed in vitro, in 96-well plates, according to the methodology described by Cintra et al. [31]. Samples of fungal extracts were evaluated separately. Its modulating action on enzymes present in venoms was also evaluated, after previous incubations of the extract with venom in different proportions (1: 0.01, 1: 0.25, 1: 0.5, and 1: 1—venom: extract; w: w) for 30 min at 37 °C. Controls containing only PBS or venom were also performed. The samples were applied, in triplicate, to the clots and the plates remained in a cell culture chamber for 48 h at 37 °C. The thrombolytic activity was quantified by the volume of liquid released by the thrombi, being subtracted from all treatments the average of the sample volumes (controls with PBS) and considered as 100% of lysis the average of the volumes released by the thrombi in the positive controls (B. atrox venom at 20 μg).

Proteolytic Test Using Casein as Substrate

This activity was evaluated using the methodology described by Gutiérrez et al. [30], with adaptations referring to the replacement of the phospholipid substrate by casein. Casein was used at a concentration previously described by Wang et al. [32] in a liquid caseinolytic test. A casein solution of 5 mg mL−1 in 50 mM Tris–HCl buffer (pH 8.0) was used for the preparation of the gel.

The B. atrox venom (20 μg) and the extracts were previously incubated in the proportions of 1: 0.01, 1: 0.25, 1: 0.5, and 1: 1 (venom: extract; w: w), for 30 min at 37 °C. Subsequently, they were applied to the holes made in the gel and incubated for 48 h at 37 ºC in a cell culture chamber. Controls containing only venom or extracts were also evaluated.

The gel was stained with 1% black starch solution and destained in 10% acetic acid solution. The quantification of the activity was performed by measuring the diameters of the translucent halos, formed around the holes. The results were expressed in percentage, in which the controls containing only venom corresponded to 100% of proteolytic activity.

Statistical Analysis

The results were presented as the mean of the triplicates ± standard deviation. The significance between the means was determined by analysis of variance, followed by the Tukey’s test when the treatments were compared with the control (P ˂ 0.05).

Results

Evaluation of Fungal Extracts Effects on the Coagulation of Citrated Human Plasma Induced by Enzymes Present in the Snake Venom (Previous Incubation of the Extracts with the Venom)

Non-volatile metabolites present in the extracts from the culture filtrate of the fungi Induratia coffeana (CML4009, CML4010, CML4020) and I. yucatanensis (CML4016) increased the clotting time of the citrated plasma when previously incubated with the venom, in most proportions evaluated (Table 1). However, the extract of I. coffeana (CML4020), in the proportion 1: 0.1, showed a pro-coagulant activity by reducing the observed clotting time by half when compared to the positive control (6 µg of snake venom). This same extract, however, was also the one that demonstrated the greatest inhibition effect of venom-induced clotting, prolonging the clotting time by 105 s when evaluated in the proportion of 1: 0.5.

Table 1 Coagulation activity of Induratia spp. extracts when first incubated with Bothrops atrox venom, or previously incubated with the citrated plasma
Full size table

Evaluation of the effects of Fungal Extracts on the Coagulation of Citrated Human Plasma Induced by Enzymes Present in the Venom (Previous Incubation of the Extracts with Plasma)

The previous incubation of the extracts with the plasma resulted in an increase in the clotting time, induced by the venom, in comparison to the data obtained after incubation of the extracts with the venom (Table 1).

Phospholipase A2 Activity

The highest percentage of inhibition observed was 7.2% for the extract of I. coffeana (CML4020) evaluated in the proportion 1: 0.5 (venom: extract; m: m). All the other fungal extracts, under the conditions evaluated, did not have a statistically significant inhibitory effect on phospholipases A2 activity (Fig. 1b).

Fig. 1
figure 1

Caseinolytic activity (a), Phospholipase activity (b), Hemolysis activity (c), and Thrombolysis activity (d) induced by Bothrops atrox venom, after the incubation with extracts from the endophytic fungi of the genus Induratia. Controls containing only venom (20 µg) were considered as 100% activity. The results correspond to the average of the data obtained for each proportion (venom: extract, w: w) in triplicate, and the upper vertical bars represent the standard deviation of the samples. *The means statistically differ from the respective control. Different letters indicate that the means of the different proportions differ from each other by the Tukey test (P < 0.05)

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Cytotoxicity Test on Human Erythrocytes

The fungal extracts did not induce cytotoxic action on human erythrocytes in the absence of the venom, under the conditions evaluated in this study (data not shown). After interaction venom-fungal extract, statistically significant inhibition on the cytotoxic activity exerted by the venom was observed in the extract of I. coffeana (CML4020) in the proportion of 1: 0.1, I coffeana (CML4010) in the proportion of 1: 1, and I. coffeana (CML4009) in the proportions 1: 0.5, 1: 0.25, and 1: 0.1. In contrast, potentiation of the venom-induced hemolytic activity was also observed, mainly, for the extracts of I. yucatanensis (CML4016) in the proportion of 1: 0.5 (62.6%) and I. coffeana (CML4009) in 1: 1 (57.6%) (Fig. 1c).

Thrombolytic Test

The extracts in all proportions showed a statistically significant reduction in the venom-induced thrombolytic activity, except the extract of I. coffeana (CML4020) at the 1: 0.25 ratio (Fig. 1d). The extract of I. coffeana (CML4009) presented the most promising result with a 73.6% inhibition in the proportion 1: 0.5. This same extract induced 25.4% of thrombolytic activity (data not shown) when evaluated in the absence of venom. Once again, since it was already observed in the coagulate test, it is demonstrated the interactions between compounds present in the extract and blood components, which results in the destabilization of thrombi.

Proteolytic Test Using Casein as Substrate

All extracts showed inhibitory activity on the breakdown of casein induced by proteases present in the venom, except for I. coffeana (CML4010, CML4009) in the ratio 1: 1, I. coffeana (CML4009) and I. yucatanensis (CML4016) and in the ratio 1: 0.1 (Fig. 1a).

Discussion

Considering the presence of several bioactive components in fungal extracts [1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23], the data obtained in our work suggest that the increase in plasma coagulation time, as well as the pro-coagulant action, may be resulting of weak and reversible interactions of compounds present in the extracts with the coagulant proteases present in the venom. In addition, at some point, they may have enhanced the action of hemorrhagic proteases. As these are mixtures containing varied molecular structures and in unknown quantities, the molar ratios that can interact and change the evaluated activity may be random between the doses evaluated. This is well observed in the effect variation (inhibition or potentiation) resulting from the evaluation of the I. coffeana (CML4020) extract.

Natural enzyme inhibitors have been widely described with inhibition mechanisms based on weak (hydrogen and/ or hydrophobic interactions) and transients interactions in different regions of the enzyme structures (e.g., active site and cofactor binding site) [26]. Considering that the fungi of the genus Induratia have also been reported as producers of compounds with bioactive action [12, 14], it is likely that metabolites produced by these fungi have mechanisms of action similar to those already described for some natural compounds. The extract produced by a fungus isolated from Brazilian caves, Lecanicillium aphanocladii, also promoted an increase in plasma clotting time, induced by snake venom [21]. Based on the literature, the authors of this work suggest that the action of the molecules present in the fungus extract is through the hydrophobic interactions with the aromatic rings that make up the structure of amino acid residues present in the active site of proteases, or other regions. Links in different regions of the active site can result in conformational changes in enzymes, with a consequent reduction in catalytic activity [33, 34].

The previous incubation of the extracts with the plasma resulted in an even greater increase in the clotting time. This result may have occurred because the bioactive molecules of the evaluated fungi have a greater number of interactions with plasma proteins than with proteases present in the venom, making it difficult for proteolytic enzymes to access the cascade factors and, thus, delaying their activation.

In addition to the possible interactions of fungal bioactive compounds with the components of the coagulation cascade, which are made up of enzymes and proteins, it is also necessary to consider interactions of fungal compounds with anticoagulation regulators present in the plasma, such as capture and/or formation of complexes with ions that are cofactors of the coagulation cascade and venom enzymes.

It should be noted that the possible effects of fungal extracts on the coagulation cascade depend not only on the proportion of chemical compounds in relation to the enzymes present in the venom, but also on the type and quantity of molecules present in the different extracts, on the composition of the plasma, the incubation time of the samples, and other reaction conditions, such as pH and temperature.

Thrombin inhibitors correspond to a class of drugs that are prominent in the treatment of bleeding, in which they act on the coagulation cascade. Such inhibitors may inhibit some metalloproteases, preventing the transformation of prothrombin (Factor II) into thrombin. Thus, they reduce the action of this enzyme in fibrinogen proteolysis for the formation of fibrin, and, consequently, decrease the formation of clots and thrombi [35]. To be an ideal thrombin inhibitor, it must be effective, selectively, and have an extended half-life [36].

Our results suggest modulation of enzymes exerted by the evaluated extracts and that the compounds present in the extracts act selectively on the different classes of enzymes present in the B. atrox venom. Although the venom-induced effects are attributed to several enzymes that act by different mechanisms, a class of enzyme can be highlighted in each test carried out. The coagulation of citrated plasma is mainly attributed to serine proteases, which have an action similar to thrombin [26, 27]. Thus, the inhibitory action of fungal extracts on these enzymes and the activation of prothrombin present in the plasma can be suggested, which results in the observed increases in clotting time.

I. coffeana (CML4020) extract evaluated in the proportion of 1: 0.5 showed a promising inhibitory effect on phospholipases A2 activity. Phospholipases A2 comprise one of the most common classes of toxins in the composition of snake venoms, along with the various classes of proteases. They are enzymes capable of promoting an intense catalytic activity on phospholipids, affecting the transport across biological membranes, blood clotting, inflammatory response, and hemostasis [25]. In addition, they may have genotoxic and mutagenic activities [26]. Thus, phospholipase A2 inhibitors are also promising molecules for the development of therapeutic agents, with possible use in the prevention, control, and treatment of pathologies associated with exacerbated inflammation. The present work highlights a potential future application for the extract of the fungus I. coffeana (CML4020).

Regarding cytotoxicity on human erythrocytes, the fungal extracts did not induce cytotoxic action in the absence of the venom and, after the incubation venom-fungal extract, some extracts reduced the cytotoxicity of the venom, while others increased the cytotoxicity. In the cytotoxicity test, there is a more complex reaction context. Cells can undergo the action of peptides that bind to membrane-carrying proteins and thus, changing the influx of ions and the membrane potential. It can also go through the action of proteases that degrade protein-membrane components, activating endogenous proteases, and phospholipase A2 proteins that degrade membrane phospholipids. In addition, other molecules present in lesser amounts in the venom composition may also exert activities, such as biogenic amines [37].

In this context, the potentiation of the cytotoxic activity can be attributed to a variety of molecular interactions whose mechanisms are still unknown. The importance of the results obtained is in the fact that the extracts have had modulating effects on the activities induced by the venom, and have not induced activity on the molecules and cells when evaluated in the absence of the venom.

Fungal extracts showed a reduction in the venom-induced thrombolytic and proteolytic activity. Thrombi dissolution mechanisms are related to fibrin degradation by the fibrinolytic system [23]. Thus, considering the results obtained, we can suggest the presence of plasminogen-activating molecules, for their conversion into plasmin, in some extracts. Fungal extracts also increased the dissolution of venom-induced thrombi. Therefore, they characterize the action of molecules that exert positive modulation on metalloproteases, especially fibinogenolytic, fibrinolytic, hemorrhagic, and hemolytic, in which contains disinterring domain in its structure, according to Gutiérrez et al. [38]. This demonstrates that the selected microorganisms have a wide range of bioactive compounds, capable of acting both in the coagulation cascade (probably as protease inhibitors or through interactions with cofactors) and in the clot and thrombus dissolution system (degrading the final product of the cascade).

Casein represents a random protein substrate since it is not related to the context of hemostasis. Thus, it was possible to observe that the fungal extracts are also able to inhibit proteases that act in the extracellular context, for example, in the digestive and defense processes of the organism.

It can be concluded by the results presented in this work that the extracts of the endophytic fungi studied of the genus Induratia have molecules that exert modulating activity on the blood coagulation process. In addition, there is a possible mechanism of interaction with the components of the coagulation cascade and with coagulant/fibrinogenolytic proteases present in the Bothrops atrox venom. Although the inhibitory effect of fungal extracts was less on the enzymatic activity of phospholipases A2, the data indicate the presence of inhibitory molecules, mainly in the extract of I. coffeana (CML4020). This is the first report on the modulation activity of the non-volatile metabolites from Induratia spp. extracts on the blood coagulation process.

It is possible to suggest that the protease modulators present in the fungal extracts can be widely explored for future pharmaceutical applications, once they were tested using snake venoms as controls and activity-inducing substances. The various classes of proteases in the B. atrox venom act in processes closely related to a variety of human pathologies. As for example, they induce or inhibit blood clotting and platelet aggregation, degrade intra and extracellular components (apoptosis, cell replacement, debridement, and tissue regeneration), and activate the inflammatory response [38, 39].

The extracts of the endophytic fungi I. coffeana (CML4009, CML4010, CML4020) and I. yucatanensis (CML4016) evaluated in this study demonstrated modulating activity on the blood coagulation process, acting as inhibitors or potentiators of the catalytic activity exerted by different classes of proteases (hemorrhagic or fibrinogenolytic serine proteases and metalloproteases—mainly from classes P-II and P-III).

The intrinsic variations in the production of metabolites, existing in each microorganism, are responsible for the various applications attributed to them [31]. However, the fungi whose extracts were evaluated in the present study correspond to species little explored in the pharmaceutical and clinical area. Thus, future studies are necessary to enable a greater understanding of the classes of molecules produced by them, as well as their identification and wide characterization (pharmacological and toxicological).