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.
Description of peaks displayed on chromatogram in positive mode
Peak 1, molecular ion, with mass/load ratio (m/z) equal to 330.1699 was found in the methanol extract Lentinus crinitus, with an Rt = 0.94 and presented the fragments (183; 198; 297; 223), which corresponded to the compound previously identified as Erythratidine (Amer; Shamma; Freyer, 1991; Feitosa et al. 2012). Peak 2 presented at m/z 314.1741 with an Rt = 1.00 and gave the fragments (313.37, 298.32, 283.28), corresponding to the previously identified compound Erysotrine. Both belong to the class of Alkaloids, unpublished in the genus Lentinus. Peak 3, molecular ion, with mass/load ratio equal to 325.1070. It was found in the methanol extract Lentinus crinitus, with an Rt = 2.5, and presented the fragments (138.0598, 204.0835, 325.0946, 124.0075, 223.0419), which corresponded to the compound previously identified and yet reported in the literature as Eucalyptin. Belonging to the flavonoid class, a phenolic compound unheard of in the genus Lentinus crinitus. Several authors have reported phenolic compounds in several edible fungal extracts and different areas in Portugal, Spain, and Finland (Kim et al. 2008; Ribeiro et al. 2008; Jayakumar; Thomas; Geraldine, 2009; Palacios et al. 2011). Peak 4, with m/z 457.1365, was found in the extract with an Rt = 3.92 and presented the fragments (204.0932, 187.1129, 136.0653), which corresponded to the compound previously as a new alkaloid monoterpene called 15-demethylplumeride. Peak 5 with molecule ion m/z 181.0482 was found in the methanolic extract Lentinus crinitus, with an Rt = 6.02, and presented the fragments (151.0436; 181.0499; 133.0827), which corresponded to caffeic acid, identified early (Guo et al. 2008). An unprecedented compound for species, as there are no reports of these compounds' presence in the literature. Caffeic acid is an acid derived from catechol and can be found generally in several plant species, such as teas, coffee, and elderflower (Meinhart et al. 2017). As for peak 6, the molecular ion with m/z = 317.0312 was found in the extract, with Rt = 7.32, with the fragments (319. 12; 219.17; 133.09, 220.17), which corresponded with gossypetin reported in the literature, is a hexahydroxyflavone with hydroxyl groups placed in positions 3-, 3'-, 4'-, 5-7 and 8- and it works like a vegetable metabolite. According to the literature, a flavonoid in the genus Lentinus crinitus has not yet been identified. However, it is known that phenolic compounds are generated as secondary metabolites in plants and fungi. They are considered one of the most influential groups associated with antioxidant power, having already described their ability to melt metals and inhibit lipoxygenase and free radicals (Decker, 2009). Peak 7, whose molecular ion obtained is m/z = 219.1740, was found in the extract with an Rt = 7.77, presenting the various fragmentations (121, 181, 147, 203, 200) that allowed identifying the compound belonging to the class of sesquiterpenoid named (+) - Nootkatone. The presence of sesquiterpenes in the genus Lentinus was first reported by literature, (Wu, 2009), which was able to identify a total of 19 sesquiterpene compounds in several Lentinus species. Basidiomycetes are a rich source of terpenoids forming mushrooms (Schmidt-Dannert, 2014). Sesquiterpenoids are important secondary metabolites and have several pharmaceutical and nutraceutical properties. In particular, the upper basidiomycetes have a versatile biosynthetic repertoire for these bioactive compounds (Lee et al. 2020). Peak 8, a molecular ion with an m/z ratio equal to 481.1358, was found in the methanol extract Lentinus crinitus, with an Rt = 8.72 and presented the fragments (293.0456, 151.0406, 133.0764), which corresponded to the compound previously identified and reported in the literature as Noidesol A, which is a dihydrophenol-C-glycosides, which was isolated from the bark of a woody plant of the genus Gnetum gnemonoides (Shimokawa et al. 2010). Peak 9, a molecular ion with mass/load ratio equal to 403.1411, was found in this study with Rt = 9.52, and presented the fragments (402.13, 403.13), the search in the literature corresponded to Nobiletin, [systematic name: 2-(3,4-dimethoxyphenyl) -5,6,7,8-tetramethoxy-4H-chromen-4-one (Wang et al. 2007). It is a flavonoid found in citrus peels and has shown a wide range of physiological properties (Noguchi et al. 2016). For example, citrus flavonoids such as nobiletin may exhibit pharmacological activities, including antioxidant, anti-inflammatory damage, and, notably, actions to improve memory impairment (Hwang; Shih; Yen, 2015). Peak 12, a molecular ion with mass/load ratio equal to 365.1371, methanol extract Lentinus crinitus, with an Rt = 13.66, presented the fragments (365.1406, 163.0775, 121.0993), which corresponded to the compound previously identified and also reported by the literature (Nuclear et al. 2010), Asperterone, obtained from the endophilic fungus Aspergillus terreus crops, isolated from the plant with flower Mammea siamensis.
Fragments presented in the peaks of the chromatograms in negative mode
Peak 3, a molecular ion with a mass/load ratio equal to 515.1248, was found in the methanol extract Lentinus crinitus, in negative mode with an Rt = 1.40 and presented the fragments (353.0876, 173.0453, 179.03445, 191.0559), which corresponded to the acid compound 3,4-di-O-Caffeoylquinic acid, isolated from the Elephantopus mollis plant and showing a high polyphenolic content (Ooi et al. 2011). Peak 4, a molecular ion with m/z equal to 325.1070, was found in the methanolic extract Lentinus crinitus, in negative ion mode with an Rt = 2.50 and presented the fragments (138.0598, 204.0835, 325.0946, 124.0075, 223.0419), which corresponded to the previously identified compound, already in positive mode, and reported in the literature as Eucalyptin, belonging to the flavonoid class. Peak 5, a molecular ion with m/z equal to 325.1070, was found in the methanol extract Lentinus crinitus, in negative ion mode with an Rt = 6.08 and presented the fragments (197.0398; 167.0303; 153.0456), which corresponded to Siringic Acid. Siringic acid plays a role in the communication between plants and soil microorganisms through changes in the microbial communities of the soil rhizosphere and inhibition of cucumber seedlings' growth. It has antioxidant, antimicrobial, anti-inflammatory, and anti-endotoxic activities (Srinivasulu et al. 2018). Vanillic acid is a phenolic compound used with an extended stay in the industry as a flavoring and sometimes as a food preservative, and can be found in cereals, whole grains, fruits, herbs, green tea, juices, beers and has antioxidant, hepatoprotective, cardioprotective and antiapoptotic activities (Marakov; Uchuskin; Trushkov, 2018). Peak 7, a molecular ion with a mass/load ratio equal to 167.0367, was found in the methanol extract Lentinus crinitus, in negative mode with an Rt = 7.35 and presented the fragments (131.0181; 167.0381; 116.9339), which corresponded to vanillic acid (Hoffmann; Linuma; Herrmann, 2007). Vanillic acid is a phenolic compound used with an extended stay in the industry as a flavoring and sometimes as a food preservative, and can be found in cereals, whole grains, fruits, herbs, green tea, juices, beers and has antioxidant, hepatoprotective, cardioprotective and antiapoptotic activities (Almeida; Cavalcante; Vicentini, 2016). Peak 8, a molecular ion with mass/load ratio equal to 315.2512, was found in the methanolic extract Lentinus crinitus, in negative mode with an Rt = 12.64 and presented the fragments (315.2529, 183.0113), which corresponded to a fatty acid developed in Malaysia named 9,10-Dihydroxystearate acid (Koay et al. 2011).
Figure 4 and 5 describe the possible structures of the identified compounds.
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 LD50, the concentration necessary to kill 50% of the sample population (Artemia salina) the stratum.
Table 4
Mortality of Artemia saline with methanol extract, with a time of 24 hours to 48 hours.
|
Artemia salina Dead
|
|
Artemia salina Dead
|
|
24 h
|
Average 24h
|
48 h
|
Average 48h
|
Concentration
|
Tube 1
|
Tube 2
|
|
Tube1
|
Tube 2
|
|
1.000 mg/mL
|
3.0
|
2.0
|
2.5
|
10.0
|
10.0
|
10.0
|
0.500 mg/mL
|
6.0
|
5.0
|
5.5
|
10.0
|
10.0
|
10.0
|
0.250 mg/mL
|
4.0
|
4.0
|
4.5
|
10.0
|
10.0
|
10.0
|
0.125 mg/mL
|
1.0
|
1.0
|
5.0
|
9.0
|
10.0
|
9.5
|
0.062 mg/mL
|
5.0
|
5.0
|
4.5
|
10.0
|
10.0
|
10.0
|
0.031 mg/mL
|
5.0
|
3.0
|
4.0
|
10.0
|
10.0
|
10.0
|
Blank
|
0.0
|
0.0
|
0.0
|
0.0
|
0.0
|
0.0
|
The criterion of classification of the methanol extract of the fungus extract against Artemia saline used in this work was based on the values of LD50 established by literature (Meyer et al. 1982) using the evaluation criterion in which the sample is considered toxic or active those with LD50 < 1000 µg/mL and nontoxic or inactive samples with LD50 > 1000µg/mL. 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 Identified Against Zika Virus Transferase
This study phase performed docking calculations for the 16 compounds newly identified 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.
Table 5
SAM (S-Adenosilmethionine) native alloy grid used for Docking.
center_x
|
8.607370
|
center_y
|
43.187481
|
center_z
|
86.285630
|
size_x
|
40.0
|
size_y
|
40.0
|
size_z
|
40.0
|
Visualization of Receptor-Ligand Complex Interactions
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-crystallized form and in blue the SAM obtained by redocking with affinity energy equal to -7.3 Kcal/mol and the deviation in RMSD in redocking by binding at the same site. The affinity energy linker was equal to - 7.3 Kcal/mol and more minor variations in RMSD with 1.856 Å. Furthermore, Figure S3b shows the Docking of the compound 13,4-di-O-Caffeoylquinic acid (red) with the protein anchoring in the same region of the SAM (orange), with energy value -8.8 Kcal/mol and the deviation in RMSD equal to 1.872 Å and its affinity Ki (µM) = 0.689. The result is satisfactory since the ligands were coupled at the same active site of the target protein.
From Figure S4a and S4b, it is possible to verify the compounds' interactions with the central amino acid residues, highlighting Asp152, Trp93, Gly92, Gly64, for 3,4-di-O-Caffeoylquinic acid (compound 1), and SAM. 3D visualization facilitates understanding and visibility of the amino acids present. Figure S5 more specifies the ratio by highlighting the distances separately for the compounds.
Figure S5 shows the main interactions between protein residues and compound 1,4-di-O-Caffeoylquinic acid. The outstanding approximations were Van Der Waals' interactions with his 116, Gly 112, Ser 65, Gly 91, Lys 188, Val 136, Phe 139, Thr 110, and Arg 90; Other interactions were observed, such as two pi-alkyl interactions, with Ile 153 (4.84 Å and 5.74 Å) residues, and two conventional hydrogen bonds between Asp 137 (3.64 Å) and Val 138 (3.62 Å).