In Silico Study of The Potential of Naturally Caffeoylquinic Acids From Lentinus Crinitus Basidiocarp With Zika Virus Inhibition Targets

The methanolic extract of the fungus Lentinus crinitus was submitted to biological assays, identication of the chemical composition by LC-MS, and in silico study by molecular docking with all identied compounds. The test against Artemis salina reached LD 50 > 1000 μg/mL within 24h, and total mortality within 48 hours; the antioxidant test 62.4% inhibition in 1.0 mg/mL was obtained. Only 16 compounds were identied from the LC-MS analysis based on the comparison of reports already recorded in the literature. Most of the compounds identied here are described for the rst time in the genus Lentinus. These results showed that the fungus is a producer of different classes of secondary metabolites biologically active. The results of the molecular docking simulation of the identied phytochemicals presented 1,13,4-di-O-Caffeoylquinic, as the leading promising candidate in the inhibition of the Zika virus. versus the percentage of DPPH Inhibition (% Inhibition). The results of the antioxidant potential of ve mushroom species identied as Pleurotus sp., Hygrocybe sp., Hygrophorus sp., Schizophyllum commune, and Polyporus tenuiculust. From each mushroom, two extracts were obtained, one with petroleum ether and the other with methanol.


Introduction
The term biodiversity, or biological diversity, describes the richness and variety of the natural world, plants, animals, and microorganisms that provide food, medicine, and much of the raw material consumed by man for decades (Purvis;Hector 2000).
Bacteria and fungi are the most studied among the most diverse microorganisms found in the Monera, Protista, and Fungi kingdoms. Fungi are microorganisms of complex nature that since antiquity have some of their species used as foods, due to the pleasant taste and their nutritional and medicinal properties, being also essential agents in various industrial processes such as obtaining enzymes, vitamins, antibiotic synthesis, extraction of polysaccharides, pigments, lipids, and glycolipids (Chang;Buswell, 1996). Previous studies have shown that these organisms produce many secondary metabolic scans with antitumor, antiviral, anti-in ammatory, antithrombotic, cytostatic, hypoglycemia, and antimicrobial activities (Ramos et al. 2010). Due to their industrial potential, fungi were previously seen as toxic are also used today as decomposers of these substances. Several studies have emerged, due to technological advances, thus demonstrating the interest of researchers focused on mycology, which is, therefore, the area of Biology destined to study fungi.
Fungi have been explored in medicine and other biotechnological processes involved in the production of enzymes and have presented great importance in the agricultural and ecological sectors, acting as a decomposer of plant residues and signi cantly degrading toxic substances symbiosis with plants (Nepomucena, 2010).
About their nature, fungi are eukaryotes, heterotrophic, achlorophilate, aerobic, microaerophilic, uni or pluricellular, with a cell wall usually composed of chitin and cellulose, in addition to other complex carbohydrates, with glycogen as a reserve substance, absorption nutrition, and may live as saprobes, parasites or symbionts with other organisms (Putzke; Putzke, 2004). They have the body formed by a tangle of laments, called hyphae, and their set is called mycelium. Hyphae vary in diameter, wall thickness, and pigment location. They constitute an extensive and heterogeneous group found in any ecological niche. The number of fungal species spread worldwide is estimated at 1.5 million, of which only 74,000 species have been described. Excluding insects, fungi are the most numerous living microorganisms (Esposito;Azevedo, 2004). This research arose from the need to conduct a study in the area of natural products to make a phytochemical analysis of the species of fungus that are born in the trees of the Baturité massif (hoses and the Indian denim tree), which are known as wooden ear, besides providing some information about the presence of secondary metabolites. The fungus Lentinus crinitus is a widespread species throughout Brazil and is often associated with discomfort trunks. They present centrally stipited, infundibuliform basidoma and verrugous abhimenial surface to the strigose (Ballaminut, 2007). This work aims to set up assays to evaluate the functional capacity of the fungus L. crinitus regarding the antioxidant, ecotoxicological, and antimicrobial potential and make the phytochemical evaluation.

Fungus collection and washing
The fungus used was collected from a tree in the municipality of Redemption, in the Baturité massif in Ceará at the following coordinates: 4° 13′ 17" S, 38° 42′ 55" E and characterized and identi ed by image recognition in the Mushroom Identify -Automatic picture recognition application, and compared with the records available on the Virtual Herbarium of Flora and Fungi website with access allowed in the link: http://inct. orabrasil.net/herbario-virtual/.

Fungus extraction
After washing the fungus, 143.59 g of this material was weighed for extraction with methanol. The heavy sample was immersed in the solvent for 24 h. Then, the supernatant was vacuum ltered using a rotary evaporator to obtain the extract.

Ecotoxicity test
Ecotoxicity tests were designed to assess or predict toxic effects on biological systems and readjust the relative toxicity of substances (Forbes;Forbes, 2008). Toxicity assays can be used as the lethality assay with cotyledons microcrustacean developed to detect bioactive compounds in plant extracts. Artemia salina is a microcrustacean species of the order Anostraca, used as a bioindicator of toxicity (Meyer et al. 1982).

Bioassay with Artemia salina
A sodium chloride (NaCl) solution at a concentration of 30 g/L was prepared with pH 8. After the solution's preparation, a system was mounted inside a beaker of 1.75 L. The cup contained a screen with a diameter of 0.02 cm thick. Then, 1.50 L of the prepared saline solution was placed in the system. Later, about 50 mg of Artemia salina cysts were added on one side, with the care that they did not go to the other side of the container that contained only saline solution. For this, a 10 W LED lamp was positioned on the other side with just the saline solution to attract the organisms to the other side of the system, thus providing uniformity.
It is also boiling point that the temperature remained controlled around 28 to 30ºC using a digital thermometer. The entire container was wrapped with aluminum foil to aid in the outbreak of cysts. The study's cysts for later use showed greater strength, uniformity of size, and attraction by light. This choice aims at homogenizing the physical conditions of test organisms. The incubation period for these organisms was around 48 hours. However, the presence of cysts was already evidenced in the rst 24 hours.

Ecotoxicity test against Artemia salina
After the nauplius outbreak, those who overcame the dividing screen in the center of the beaker were removed from the system. These organisms overcame the dividing screen in the center of the cup opposite the side that was cultivated. They were captured with a Pasteur pipette and transferred to 12 test tubes to contain ten artemias in each box. The boxes contained 4 mL of the same prepared saline solution and 2 mL of the methanol extract in different concentrations (0.0 g/ml, 1.0 g/ml, 0.5 g/ml, 0.25 g/ml, 0.125 g/ml, and 0.0625 g/ml). The tests were performed in triplicate for each extract/compound concentration of this analysis. The counting of this biological assay was made by the rst 24 and 48 hours after pipetting the larvae in the test tubes. In this study, living nauplius was considered, all those who presented movements inside the tube and were attracted to light, and dead, those at the boxes' bottom without any activities. A magnifying glass was used for better visualization and counting.

Antioxidant assay
Antioxidant activity was determined by capturing free radicals with the DPPH test. For this procedure's performance, Hegazi e El Hady's (2002) studies with some adaptations were used as references.
The methodology for antioxidant activity was based on the extinction measure of radical adsorption. This is because the DPPH has the purpura staining that is reduced, forming diphenylpicrilhydrazine of yellowish coloration. For this experiment, the methanol extract was methanol in several different concentrations (0.0 mg/ml,1.0 mg/ml,0.5 mg/ml,0.25 mg/ml,0.125 mg/ml,0.0625 mg/ml). For this experiment, the fungus's methanol extract was diluted in 2 mL of methanol in several different concentrations, as represented in Table 2, except for the white sample, where only DPPH and Methanol were added.
Only 2 mL of a DPPH solution was added to each concentration of 2 mL methanol extract of a solution of DPPH, except for the sample called a blank, where only the solvent was placed. After adding the DPPH in the absence of light, it waited 30 minutes for the solution to manifest. After that, the PG Instruments Ltd spectrophotometer at 520 nm was read. The measured absorbance of extract concentrations was converted into a percentage of antioxidant activity using equation 1. (1) Where: A sample is the absorbance of the solution with DPPH.
A blank is the absorbance of the DPPH solution without the sample. The reduction of the DPPH radical is followed by monitoring the decrease of its absorbance to a characteristic wavelength during the reaction. The graphs were plotted using the Origin version 9.0 program. The value of IC 50

High-Resolution Mass Conditions -XEVO-QToF
The ESI-model was acquired in the range of 110 -1180 Da, xed source temperature at 120 ºC, desolvation temperature 350 ºC, desolation gas ow of 500 L/h, extraction cone of 0.5 V, the capillary voltage of 2.6 kV. The ESI+ mode was acquired in the range of 110 -1180 Da, xed source temperature of 120 ºC, desolation temperature 350 ºC, desolation gas ow of 500

Antioxidant activity
A statistical treatment with all the data collected about the antioxidant activity to verify the percentage of inhibition of DPPH and in which concentration was higher the sequestration of free radical DPPH. Table 1 is below, which contains all data on antioxidant activity. Chromatographic identi cation of chemical constituents in the methanol extract The high-resolution LC-MS signi cantly improves the possibility of obtaining valuable information from different chromatographic pro les in an extract (Zou et al. 2014).
Liquid chromatography coupled with mass spectrometry (LC-MS/MS) was successfully applied in this study to identify the chemical constituents and their secondary metabolites present in the methanol extract of the fungus Lentinus crinitus. It was possible to identify a total of 16 compounds in positive ion modes and negative ion modes. Tables 2 and 3 show the compounds identi ed by the attempt method, compared with the literature records based mainly on their molecular ions, retention time, and presented fragmentations.    Ecotoxicity test against Artemia salina The mortality results of Artemia saline larvae obtained with the methanol extract of the fungus Lentinus crinitus were examined after the count of 24 and 48 hours and are represented in Table 4. Depending on the toxicity analysis, it became possible to determine the percentage of dead Artemia saline nauplius and thus determine LD 50 , the concentration necessary to kill 50% of the sample population (Artemia salina) the stratum.  Depending on the tabled data, the construction of a graph that more clearly exposes the results obtained was performed, making it possible to graphically analyze all the data obtained after the calculations were performed. The Figure S1 explains the data mentioned above in Table 4 about 24 hours because the same extract presented total mortality during 48 hours.

Molecular Docking with Compounds Identi ed Against Zika Virus Transferase
This study phase performed docking calculations for the 16 compounds newly identi ed by LC-MS/MS. The biological target selected was 5M5B (Zika virus transferase). The active site in which the molecules were embedded was the same as the native SAM (S-Adenosilmethionine) alloy, as shown in Table 5. The Discovery Studio, was used to visualize the interactions of the receptor-ligand. The choice was made due to the ease of handling the software and its optimized graphics that facilitate understanding the results more straightforwardly, as shown in Figure S3a-S3b (Biovia, 2015). In Figure S3a, it is possible to observe the result obtained orange to SAM in the co-

Antioxidant activity
The extract presented plausible inhibition percentages. It gave antioxidant action of 62.3% at the concentration of 1000 µg/mL. At the concentrations analyzed, the same extract with the maximum absorbance value of 0.602 in the concentration was 32.5 µg/mL. According to the description of Fig. 1, it is perceived that the higher the concentration, the higher the percentage of inhibition. It agreed with a study, which states that the higher the DPPH intake by the sample, the higher its antioxidant activity (Nascimento et al. 2011). Therefore, it is veri ed similarly that the higher the sample's concentration, the lower the absorbance, and the higher the DPPH consumption.
Analysis of some edible wild mushrooms' nutritional and antioxidant potential capacity of the genus Lentinus crinitus using DPPH elimination assay methanolic extracts determined that the mushrooms presented signi cant antioxidant activities (Ao; Deb, 2019). Among the ten species studied, Lentinus tigrinus given about (47.5 µg/ml, IC50) exhibited the largest sequester of DPPH radicals, followed by Lentinus squarrosulus (82.6 µg / ml, IC50). The IC50 value was calculated to study the inhibition capacity of mushrooms against DPPH radicals.
The authors concluded that the mushroom species showed appreciable DPPH radical sequestration activity, the oil ether extract of Pleurotus sp., which showed the highest % inhibition (83.04 %) concentration of 20 mg/mL (20,000 µg/mL). This ability to kidnap the free radical DPPH was twice that of Hygrocybe sp (Chye; Wong; Lee, 2008).
When evaluating the antioxidant potential of the fungus Tyllopiillus balllloui, found that it proved to be a good source of compounds with antioxidant activity. Other studies have shown promising results when evaluating the antioxidant activity of compounds produced by fungi, such as Penicillium citrinum, the endophilic fungi Viscumalbum L. and Penicillium sp. However, few results were found in the reviewed literature on the antioxidant activity of the methanol extract of the fungus Lentinus crinitus (Lima, 2009).
The analysis of the methanol extracts of various mushroom species presented signi cant antioxidant activity in vitro. The authors suggest that mushrooms can be a natural source of antioxidants, as a food supplement or in the pharmaceutical industry, and phenolic compounds are the main ones responsible for the antioxidant activity of edible mushrooms (Elmastas et al. 2007).
Compared to more potent natural antioxidants, the results obtained show considerably high activity for the fraction analyzed.
The present study suggests that Lentinus crinitus fungi can aid oxidative damage in cells induced by oxygen radicals and effectively exploit antioxidant potential in the medical-pharmaceutical area.

Chromatographic identi cation of chemical constituents in the methanol extract
Chromatography is a fundamental technique used to quantify various fungal species' chemical constituents and has been used by several authors in several studies. Researchers used high-e ciency reverse-phase liquid chromatography with a diode arrangement detector to analyze phenolic acids present in aqueous and methanolic extracts of mushrooms (Puttaraju et al. 2006). While also analyzed phenolic compounds of mushrooms with HPLC system and diode arrangement detector. Some phenolic acids were identi ed in 19 mushrooms from Poland, using high-e ciency reverse-phase liquid chromatography and electrospray ionization mass spectrometry (LC-ESI-MS/MS) (Nowacka et al. 2014).
The chromatogram showed the equivalent of 21 peaks in positive mode and nine peaks in negative mode. Only ten peaks were identi ed in positive mode and six in negative mode, highlighting the existence of distinct chemical classes present in the extract, such as Alkaloids, avonoids, sesquiterpenes, phenolic acids, and other types of compounds. The presence of avonoid and phenolic acids justi es the high antioxidant content that the extract presented.
Our analysis results indicate that the extract's chemical pro le is satisfactory because unpublished compounds were identi ed using this technique, not yet described in the literature to date, as shown in Fig. 2 and 3 below, based on their peaks and retention times.

Description of peaks displayed on chromatogram in positive mode
Ecotoxicity test against Artemia salina According to Figure S1 behavior, it was possible to conclude that the mortality of Artemia salina in 24 hours increases linearly as the concentration increases. Similarly, it was also perceived that the lower the concentration of the fungus extract, the lower the mortality of larvae. Thus, the lethal dose (LD 50 ) was calculated based on the line equation obtained by linear regression, considering the correlation of the logarithm of concentrations and the corresponding percentage of mortality. At the value of y (coordinates) is attributed to half of the maximum possible deaths (n/2), the result of x obtained (abscissas) the antilogarithm is applied, resulting in the nal value of the DL 50 (Rajeh et al. 2012).
The calculated LD 50 was equal to 2,747.286 µg/mL by constructing the linear regression, classi ed as a non-toxic sample.
The absence of cytotoxicity against Artemia salina indicates that the part of the evaluated extract can be well tolerated given the biological system. The nding of potential toxicological signals the need for further studies such as in vitro tests to clarify the aspects of toxicity. Although cytotoxicity represents a concern regarding the safety of use, recent studies address cytotoxic plants' antitumor potential (Pereira et al. 2015).

Molecular Docking with Compounds Identi ed Against Zika Virus Transferase
Molecular docking was successfully applied in this study using the AutoDock Vina software (Trott;Oslon, 2009). The compounds used as binders went through the entire three-dimensional space of the grid until nding the most favorable positions; at the end of the calculation, RMSD values were obtained for the poses where the ligands were coupled that are described in Table S1, with their a nity energies and the inhibition constant that was determined by the docking energy. As the docking works in the validation of the a nity of the luminant present in a given active site found in the receiver of interest, to affect the con rmation of the anchoring in this study, a nity energies lower than the redocking value of the SAM, of -7.3 Kcal/mol and RMSD deviations more minor than two angstroms (Å) were taken into account. Table S1 shows the three ligands that presented lower dating energy and K i than the native ligand SAM, 3,4-di-O-Caffeoylquinic acid, Gossypetin, and Asperterone. Thus, it was necessary to draw Figure  Proposal of the structures of the compounds identi ed in the extract in the positive ion mode.