Survey of Edible Amanita in Northern Thailand and Their Nutritional Value, Total Phenolic Content, Antioxidant and α-Glucosidase Inhibitory Activities

Edible wild mushrooms are extremely popular among consumers and are highly valued for their potential economic benefits in northern Thailand. In this present study, a total of 19 specimens of edible Amanita were collected during investigations of wild edible mushrooms in northern Thailand during the period from 2019 to 2022. Their morphological characteristics and the phylogenetic analyses of the internal transcribed spacer (ITS) and partial large subunit (nrLSU) of ribosomal RNA, RNA polymerase II second-largest subunit (rpb2) and partial translation elongation factor 1-alpha (tef-1) indicated that the collected specimens belonged to A. hemibapha, A. pseudoprinceps, A. rubromarginata, A. subhemibapha, and Amanita section Caesareae. This is the first report of A. pseudoprinceps and A. subhemibapha from Thailand. Full descriptions, illustrations and a phylogenetic placement of all specimens collected in this study are provided. Subsequently, the nutritional composition and total phenolic content, as well as the antioxidant and α-glucosidase inhibitory activities, of each species were investigated. The results indicate that the protein contents in both A. pseudoprinceps and A. subhemibapha were significantly higher than in A. hemibapha and A. rubromarginata. The highest total phenolic content was found in the extract of A. pseudoprinceps. In terms of antioxidant properties, the extract of A. pseudoprinceps also exhibited significantly high antioxidant activity by 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS), 2,2-diphenyl-1-picrylhydrazyl (DPPH) and ferric reducing antioxidant power (FRAP) assays. However, the extract of A. rubromarginata had the lowest total phenolic content and level of antioxidant activity. Additionally, α-glucosidase inhibitory activity varied for different Amanita species and the highest level of α-glucosidase inhibitory activity was found in the extract of A. pseudoprinceps. This study provides valuable information on the nutrient content, phenolic content and the antioxidant and α-glucosidase inhibitory potential of edible Amanita species found in northern Thailand.

Several edible wild mushrooms are known to be a good source of essential dietary minerals, nutrients, and vitamins, which makes them an important source of food for humans [17,20,21]. These mushrooms have also been recognized as a source of many bioactive compounds (e.g., immunomodulatory compounds, phenolic compounds, polysaccharides, terpenoids and tocopherols) that exhibit various beneficial biological activities including anticancer, antidiabetic, anti-inflammatory, antimicrobial, antioxidant, cholesterol-reducing, immunomodulatory and neuroprotective properties [22,23]. Additionally, ethnomycologists have recorded vital information on the relevant consumption patterns and applications of wild edible mushrooms for medicinal purposes [24,25]. Thailand, a Southeast Asian country, has many species of edible wild mushroom that are particularly abundant during the rainy season (mid-May to October) each year. Generally, wild edible mushrooms are collected by local farmers for consumption and sale in local, roadside or city markets (Figure 1).  Preliminary investigations of edible wild mushrooms in northern Thailand have revealed the existence of many genera, e.g., Amanita, Astraeus, Boletus, Cantharellus, Lactarius, Phlebopus, Russula, and Termitomyces [26][27][28]. Edible Amanita species are the most popular variety of edible wild mushrooms in northern Thailand because of their palatable texture and flavor. However, the number of lethal and edible Amanita species that have been found in Thailand has remained a controversial issue due to the absence of comprehensive herbarium reference material, accurate descriptions and available molecular data [29].
During our ongoing studies of edible wild mushrooms in northern Thailand, we have collected specimens of edible Amanita species from natural forests, roadsides and local markets. Therefore, the present study aimed to identify the collected specimens based on their morphological characteristics and multi-gene phylogeny using the sequence data of ITS, nrLSU, rpb2, and tef-1. A full description, color photographs, illustrations and a phylogenetic tree of the collected specimens are provided. Moreover, the nutritional composition, total phenolic content, and antioxidant and α-glucosidase inhibitory activities of collected edible Amanita were investigated.

Sample Collection
The edible Amanita were surveyed and collected from natural forests, roadsides and local markets in Chiang Mai and Lamphun Provinces in northern Thailand during the rainy seasons of the years 2019 to 2022. Basidiomata were kept in plastic boxes and taken to the laboratory. Specimens were dried in a hot air oven at 45 • C until they were completely dry. After that, the dried specimens were kept in a plastic Ziplock bag and deposited in the Herbarium of Sustainable Development of Biological Resources (SDBR-CMU), Faculty of Science, Chiang Mai University, Thailand.

Morphological Observations
Fresh specimens were used to describe macromorphological data. Color names and codes were followed by Kornerup and Wanscher [30]. The dried specimens were examined for micromorphological data. Dried specimens were mounted in 5% aqueous KOH, Melzer's reagent, or 1% aqueous Congo red solution. A light microscope (Nikon Eclipse Ni U, Tokyo, Japan) was used to examine micromorphological features. Each microscopic structure's size data were derived from at least 50 measurements using the Tarosoft (R) Imaging Frame Work program. The terminology for microscopic features followed Largent et al. [31] and Bas [32]. Basidiospore statistics are expressed as (a-) b-c (-d), where 'a' and 'd' are the extreme values and 'b-c' is the range comprising 90% of all values. The Q value represents ratio of the length divided by the width of each basidiospore and Qm is the average Q of all specimens ± standard deviation.

DNA Extraction, Amplification, Sequencing, and Phylogenetic Analyses
A Genomic DNA Extraction Mini-Kit (FAVORGEN, Ping-Tung, Taiwan) was used to extract DNA from fresh tissue of each specimen. The ITS, nrLSU, rpb2, and tef-1 regions were amplified by polymerase chain reaction (PCR) using ITS5/ITS4 [33], LR0R/LR5 [34], Am6F/Am7R [35], and EF1-983F/EF1-1567R [36] primers, respectively. The PCR for these four domains was performed in separate PCR reactions on a peqSTAR thermal cycler (PEQLAB Ltd., Fareham, UK). The PCR programs of ITS, nrLSU, rpb2, and tef-1 genes were established by following the methods employed by Liu et al. [15] and Cai et al. [36]. PCR products were directly sequenced by the Sanger sequencing method at 1st Base Company (Kembangan, Malaysia). Sequence analysis was performed by a similarity search using the BLAST program available at NCBI (http://blast.ncbi.nlm.nih.gov, accessed on 12 November 2022). Sequences from this study, previous studies, and the GenBank database were selected and listed in Table 1. The combined dataset of ITS, nrLSU, rpb2, and tef-1 was used for the phy-logenetic analysis. MUSCLE [37] was used to perform multiple sequence alignments, and BioEdit v. 6.0.7 [38] was used to make any necessary improvements. Maximum likelihood (ML) and Bayesian inference (BI) methods were used to construct phylogenetic trees. The best substitution models were GTR+I+G for ITS, nrLSU and tef -1 and HKY+I+G for rpb2 from the Akaike Information Criterion (AIC) in jModeltest 2.1.10 [39]. The GTRCAT model with 25 categories was subjected to ML analysis using RAxML v7.0.3 and 1000 bootstrap replications [40,41]. MrBayes v3.2.6 [42] was used for the BI analysis, which evaluated the posterior probabilities (PP) using Markov chain Monte Carlo sampling (MCMC). Six simultaneous Markov chains were run from random trees for one million generations and trees were sampled every 100th generation. The first 25% of trees were discarded and the remaining trees were used for calculating PP value in the majority rule consensus tree. FigTree v1.4.0 [43] was used to visualize the tree topologies. Superscript " T " represents type species. "-" represents the absence of sequence data in GenBank database.

Nutritional Analysis
A total of six samples of edible Amanita (SDBR-CMUNK0775, SDBR-CMUNK0776, SDBR-CMUNK0780, SDBR-CMUNK0853, SDBR-CMUNK0855, and SDBR-CMUNK0857) obtained in this study were used in the analyses of nutrition, antioxidant, and α-glucosidase inhibitory activities because their dry weights were sufficient for testing. A Waring blender (New Hartford, CT, USA) was used to grind each dried sample. The nutritional composition (including ash, carbohydrate, fat, fiber, and protein) of each dried sample was determined using a method developed by the Association of Official Analytical Chemists (AOAC) [44] at the Central Laboratory Company Limited (Chiang Mai, Thailand).

Preparation of Mushroom Extracts
Ten grams (10 g) of each ground mushroom sample was extracted with 100 mL of absolute ethanol at 25 • C and 150 rpm for 24 h, as described by Kaewnarin et al. [45]. After that, each extract was placed in an ultrasonic bath (Elma Transsonic Digital, Singen, Germany) at 60 • C for 3 h. Whatman's No. 1 filter paper was used to filter the samples. The residue was then re-extracted twice with absolute ethanol as mentioned above. The ethanolic extract was then dried using rotary evaporation at 40 • C. The extract was dissolved in 100 mL absolute ethanol and kept at 4 • C until further determination.

Determination of Total Phenolic Content
The method of Thitilertdecha et al. [46] was modified slightly to determine the total phenolic content. Folin-Ciocalteu reagent at 0.5 mL was mixed with 2.5 mL deionized water and 0.25 mL mushroom extract. After 5 min, 0.5 mL of Na 2 CO 3 (20% w/v) was added. The mixture was incubated for 1 h in the dark at 25 • C. Measurements of absorbance at 760 nm were used to investigate the total phenolic content. The total phenolic content of the samples was calculated using a standard curve of gallic acid. Results were expressed as milligrams of gallic acid equivalents per gram of dry weight (mg GAE/g dw). Each sample extract was analyzed in five replicates.
2.6. Antioxidant Assay 2.6.1. ABTS Scavenging Assay The procedure of Re et al. [47] with slight modifications was used to determine the 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical scavenging activity. The stock solution of ABTS cation chromophore was prepared by facilitating a reaction between 100 mL of 2.45 mM K 2 S 2 O 8 and 100 mL of 7.0 mM ABTS solution. The solution was kept for 16 h in a dark place at room temperature. The ABTS solution was diluted with phosphate buffer (50 mM, pH 7.4) before use to yield an absorbance value of 0.70 ± 0.2 at 734 nm. A quantity of 2.9 mL of ABTS solution was mixed with 0.1 mL of each sample extract. The mixtures were incubated in the dark for 30 min at room temperature. A mixture of absolute ethanol and ABTS solution was used as the control. After incubation, the absorbance of each mixture was measured spectrophotometrically at 734 nm. Trolox was used as a reference compound. The ABTS scavenging activity was expressed as the Trolox equivalent antioxidant capacity per gram of dry weight (TE/g dw). Each sample extract was subjected to five replications.

DPPH Scavenging Assay
The method developed by Gülçin et al. [48] was used to determine the 2,2-diphenyl-1picrylhydrazyl (DPPH) radical scavenging activity. Initially, 1.5 mL of the 0.1 mM DPPH solution in methanol was combined with 0.5 mL of the sample extract. The mixtures were incubated at room temperature in the dark for 30 min. Then, the absorbance of each mixture was determined using spectrophotometry at 517 nm. Trolox was used as a reference compound. The DPPH scavenging activity was expressed as the TE/g dw. Five replicates were performed for each sample extract.

FRAP Assay
The method developed by Li et al. [49] was used to determine the ferric reducing antioxidant power (FRAP) activity. The FRAP reagent was prepared using a mixture containing 20 mL of 20 mM ferric (III) chloride, 10 mM 2,4,6-tripyridyl-s-triazine solution in 20 mL of 40 mM HCl, and 5 mL of 300 mM acetate buffer (pH 3.6). A quantity of 1.5 mL of FRAP reagent and 1.4 mL of acetate buffer (300 mM, pH 3.6) were mixed with 0.1 mL of each sample extract. Then, the mixture was incubated in the dark for 30 min at room temperature. Trolox was used as a reference compound. A mixture of absolute ethanol and FRAP solution was used as the control. After incubation, the absorbance of each mixture was measured spectrophotometrically at 593 nm. Trolox was used as a reference compound and the FRAP value was expressed as the TE/g dw. Five replicates were performed for each sample extract.

Determination of α-Glucosidase Inhibitory Activity
The procedure of Oki et al. [50] was modified to prepare the α-glucosidase solution from rat intestinal acetone powder. A quantity of 3 mL of 0.9% NaCl solution was mixed with 100 mg of intestinal acetone powder (Sigma-Aldrich Chemical Co., Saint Louis, MO, USA), homogenized by sonication, and stored in an ice bath. The enzyme mixture was centrifuged at 4 • C for 30 min at 8000 rpm. The supernatant was maintained in an ice bath and directly subjected to inhibitory assay. The α-glucosidase inhibitory assay was followed the procedure of Tanruean et al. [51] with some modifications. Each extracted sample (10 µL) was mixed with α-glucosidase solution (30 µL) and incubated at 37 • C for 15 min. Later, 70 µL of 37 mM D-maltose was then added and incubated at 37 • C for 15 min. The reaction was stopped after 10 min in boiling water. A glucose oxidase assay was used to determine the released glucose concentration of the reaction mixture. The peroxidase-glucose oxidase (PGO) reagent (900 µL) containing 1 capsule of PGO enzymes to 100 mL of water and 1.6 mL of o-dianisidine solution was added to the reaction mixture and it was then mixed for 30 min at 37 • C in a water bath. The absorbance of α-glucosidase activity was measured at 450 nm. The percentage of inhibition was calculated according to the formula: Percentage of inhibition = (Ao−As/Ao) × 100, where Ao is the absorbance of the control and As is the absorbance of the mixture containing the test compound. Acarbose (a standard synthetic inhibitor of α-glucosidase) was used for standard compound. Each sample extract was analyzed in five replicates.

Statistical Analysis
Statistical differences between treatments were assessed using one-way analysis of variance (ANOVA) with the SPSS program version 16.0 for Microsoft Windows. Significant differences at the p < 0.05 level were determined using Tukey's test. The Pearson correlation coefficients (r) of the total phenolic content with antioxidant and α-glucosidase inhibitory activities of extract samples were analyzed using the SPSS program at a significance level of p < 0.05.

Sample Collection and Morphological Observations
In this study, a total of 19 edible Amanita specimens were obtained ( Table 2). These specimens were initially classified into four Amanita species, namely A. hemibapha (4 specimens), A. pseudoprinceps (7 specimens), A. rubromarginata (4 specimens), and A. subhemibapha (4 specimens), based on their morphological characteristics. Subsequently, multi-gene phylogenetic analysis further confirmed their identification.

Phylogenetic Analyses
The aligned dataset of the combined ITS, nrLSU, rpb2, and tef-1 sequences consisted of 2831 characters including gaps (ITS: 1-903, nrLSU: 904-1676, rpb2: 1677-2316, and tef-1: 2317-2831). The matrix had 1192 different alignment patterns and 23.41% gaps or undetermined characters. A final ML Optimization Likelihood value of −15972.8506 was the best-scoring RAxML tree. For BI analysis, the final average standard deviation value of the split frequencies at the end of the total MCMC generations was calculated as 0.00723. The topology of the phylogenetic trees from the ML and BI analyses were similar. A phylogenetic tree obtained from the ML analysis is represented in Figure 2. Our phylogenetic tree was constructed with the aim of having similar outcomes to previous phylogenetic studies [4,[52][53][54]. The phylogenetic tree consisted of 62 specimens of Amanita sect. Caesareae and two specimens of Amanita sect. Vaginatae (the outgroup). The phylogenetic tree clearly separated the 19 specimens obtained in this study into four species clades, namely A. hemibapha (4 specimens), A. pseudoprinceps (7 specimens), A. rubromarginata (4 specimens), and A. subhemibapha (4 specimens) in Amanita sect. Caesareae with high supported values (BS = 100% and PP = 1.0).
Habitat: Solitary to scattered on soil in tropical deciduous forests dominated by Dipterocarpus and Shorea.
Traditionally, morphological characteristics have been the primary basis for the identification of Amanita species [7,8,11,32]. However, identification can be difficult due to the high phenotypic variability that is influenced by differing environmental conditions and geographic distributions. Therefore, it is crucial to identify the Amanita species using DNAbased methods. The current classification of the genus Amanita is based on combined data on their morphological characteristics and molecular data. Moreover, multi-gene molecular phylogeny has provided researchers with a powerful tool for the identification of the Amanita species [4,14,36,[52][53][54]58]. In this present study, specimens of the edible Amanita species collected in northern Thailand were identified as A. hemibapha, A. pseudoprinceps, A. rubromarginata, and A. subhemibapha based on morphological characteristics and multi-gene phylogenetic analyses. The results of morphological comparisons of four edible Amanita species in this study are presented in Table 3. Morphologically, the color of the pileus and the larger spore size found in A. pseudoprinceps clearly differentiate it from those other three species. Additionally, the yellow annulus and narrow spores in A. hemibapha clearly distinguish it from A. rubromarginata and A. subhemibapha. Remarkably, A. rubromarginata has a redder and more of an orange-red-shaded pileus and annulus than A. subhemibapha. The multi-gene phylogenetic analysis also supports the determination that A. hemibapha, A. pseudoprinceps, A. rubromarginata, and A. subhemibapha are different species. Four Amanita species obtained from natural forests, roadsides, and local markets in this study belonged to the Amanita section Caesareae. This section is a highly regarded edible mushroom in the genus Amanita [4,[16][17][18][19]. Prior to this study, the toxicological analysis of A. hemibapha showed that no amatoxins and phallotoxins had been discovered and that it should be regarded as an edible species [63]. However, further research is required to fully understand the edibility and safety of A. pseudoprinceps, A. rubromarginata, and A. subhemibapha based on their toxicological studies. As a result, our study should be considerably important and highly valuable in terms of stimulating deeper investigations of edible macrofungi in Thailand. It will also help researchers in understanding the distribution and ecology of Amanita. Orange to yellow 40-50 × 9-12 7.0-11.0 × 6.5-9.0

Nutritional Analysis
A total of six samples of four edible Amanita species (namely A. hemibapha, A. pseudoprinceps, A. rubromarginata, and A. subhemibapha) obtained in this study have been included in the experiments. In this study, the fruiting bodies of edible Amanita were analyzed for their nutritional composition, which included ash, carbohydrate, protein, fat and fiber. The results are presented in Table 4. The results indicate that the protein contents in A. pseudoprinceps and A. subhemibapha were significantly higher than A. hemibapha and A. rubromarginata. The highest content of fiber was found in A. pseudoprinceps. It was determined that A. rubromarginata had the highest ash content. In addition, the carbohydrate content in A. hemibapha was significantly higher than the other Amanita species. The highest fat content was obtained in A. rubromarginata, but this value was not found to be significantly different from the fat content of A. hemibapha. These results were consistent with previous studies, which reported that edible wild mushrooms to be natural sources of nutrients for human diets (high-protein and low-fat contents), while the nutritional composition of each mushroom is dependent upon the mushroom species [20,22,64,65]. The amounts of ash, carbohydrate, protein, fat, and fiber of the four edible Amanita species in this study were within the ranges mentioned in previous reports of edible Amanita. Accordingly, the ash (0.11-11.82% dry weight), carbohydrate (22.16-61.70% dry weight), protein (10.11-45.65% dry weight), fat (0.17-17.52% dry weight) and fiber (1.18-30.30% dry weight) contents were found in various edible Amanita species, namely A. caesarea, A. calyptroderma, A. fulva, A. hemibapha, A. princeps, A. rubescens, and A. zambiana [66][67][68][69][70][71][72][73][74][75]. When compared to the findings of other previously published reports, the protein content of the Amanita species obtained in this study was relatively higher than those of A. calyptroderma [75] and A. loosei [69]. With regard to the outcomes of this study, this is the first comprehensive report on the nutritional composition of A. pseudoprinceps, A. rubromarginata, and A. subhemibapha.

Determination of Total Phenolic Content
The total phenolic content of each extract of Amanita in this study is presented in Table 5. It was found that the total phenolic contents ranged from 0.94-1.62 mg GAE/g dw. The highest value of total phenolic content was found in the extract of A. pseudoprinceps, followed by the extracts of A. subhemibapha and A. hemibapha. The lowest value of total phenolic content was found in the extract of A. rubromarginata. Previous findings support the results of this study in that the amount of phenolic contents of edible wild mushrooms varied within different ranges and was dependent upon the various mushroom species [45,[76][77][78]. According to our results, the amounts of total phenolic content obtained in this study were within the previously reported ranges of phenolic content found in edible wild mushrooms and varied from 0.39-38.44 mg GAE/g dw [76][77][78][79]. The total phenolic contents in the methanolic extracts of A. caesarea [79], A. fulva [74], A. hemibapha [80], A. javanica [81], A. ovoidea [82], A. princeps [80,81], and A. zambiana [73] were reported as 0. 64, 0.39, 8.5, 18.01, 0.50, 14.29-16.80 and 8.76 mg GAE/g dw, respectively. Additionally, the total phenolic contents in the ethanolic extracts of A. javanica and A. princeps were 12.79 and 16.52 mg GAE/g dw, respectively [81]. When compared to the results of previously published reports, the phenolic contents of the ethanolic extracts of A. hemibapha, A. pseudoprinceps, A. rubromarginata, and A. subhemibapha obtained in this study have been found to be relatively higher than those of methanolic extracts of A. caesarea, A. fulva and A. ovoidea [74,79,82], while they were relatively lower than extracts of A. javanica, A. princeps and A. zambiana [73,81]. However, the phenolic content of A. hemibapha obtained in this study was lower than that of the previous report of Butkhup et al. [80]. It can be concluded from our experiments that, similarly to the results of previous studies, the total content of phenolic can be influenced by different phenolic compounds found in mushroom extracts, along with the extractability of the different solvents used in the preparation process [45,81,83,84]. According to several previous studies, catechin, р-coumaric acid, gallic acid, hydroxycinnamic acid, quercetin, protocatechuic acid, rosmarinic acid, and syringic acid were found to be the major phenolic components in the ethanolic extracts of edible wild mushrooms [45,[85][86][87]. Some previous investigations revealed that the Folin-Ciocalteu assay, a method typically used for detection and quantification of total phenolic content, might be unsuited for total phenolic content measurement in complex biological samples due to high interference from various reducing compounds contained in samples [88][89][90]. The effectiveness of the Folin-Ciocalteu assay is also hampered by its limited suitability for some phenolic compounds [89,90]. Therefore, the measurement of total phenolic content in this study will still be assessed using other techniques such as high-performance liquid chromatography (HPLC) or liquid chromatography-mass spectrometer mass spectrometry (LC-MS) for further studies to characterize and identify the phenolic compounds contained in mushroom extracts.

Antioxidant Assay
A single method cannot fully determine the antioxidant activity of mushroom extracts. Thus, in this study, three methods, namely ABTS, DPPH, and FRAP assays, were used to determine the antioxidant activity of the ethanolic extracts of different samples of edible Amanita species. The ABTS and DPPH values were determined by evaluating the scavenging abilities of ABTS and DPPH radicals, respectively (by measuring the decrease in ABTS and DPPH radical absorption after exposure to radical scavengers) [91,92]. The FRAP assay was used to measure the conversion of the ferric form (Fe 3+ ) to the ferrous form (Fe 2+ ) [92]. In this study, the highest values of DPPH activity were observed in the extract of the A. pseudoprinceps, followed by the extracts of A. hemibapha and A. subhemibapha ( Table 5). The lowest value of DPPH activity was observed in the extract of A. rubromarginata. Furthermore, the results indicated that all extracts exhibited positive results in terms of the ABTS and FRAP assays, while the ABTS values varied from 0.56 to 1.00 mg TE/g dw ( Table 5). The highest ABTS value was observed in the extract of A. pseudoprinceps, followed by the extracts of A. hemibapha, A. subhemibapha, and A. rubromarginata. In the FRAP system, the extract of A. pseudoprinceps had significantly higher FRAP values than the extracts from the other samples (Table 5). The results from the ABTS, DPPH, and FRAP assays were similar and demonstrated that the extract of A. pseudoprinceps exhibited significantly high antioxidant activity. The lowest level of antioxidant activity was found in the extract of A. rubromarginata. According to Pearson correlation (p < 0.05), the total phenolic content of mushroom extract samples showed a significant strong positive correlation with DPPH (r = 0.975) and FRAP (r = 0.948) activities (Table 6). However, the positive correlation between the total phenolic content and ABTS activity (r = 0.762) was not statistically significant. All extracts of the four edible Amanita species exhibited antioxidant activities. These results are consistent with those of previous studies which reported that the extracts of wild mushrooms (e.g., genera Amanita, Boletus, Cantharellus, Lactarius, and Russula) exhibited antioxidant activities that varied according to the mushroom species [45,66,78,[80][81][82][83]. Furthermore, recent research has indicated that wild mushrooms contain dietary ingredients that are alternative sources of natural antioxidants [45,77,93]. In this study, A. pseudoprinceps exhibited the highest level of antioxidant activity due to the fact that it possesses high total polyphenol content. This determination is supported by the results of previous studies, which reported that high phenolic content is responsible for the high antioxidant activity [45,83,94]. Prior to this present study, the antioxidant activities of A. caesarea, A. calyptroderma, A. hemibapha, A. javanica, A. loosei, A. ovidea, and A. princeps have been reported from a variety of assays employing different mechanisms including lipid peroxidation, metal chelation, reducing power and scavenging activity, among others [69,75,[79][80][81]. However, variations in the assays themselves, and the results they express, make it difficult to compare the outcomes obtained in this study with those of previous studies.

Determination of α-Glucosidase Inhibitory Activity
Importantly, α-glucosidase is one of the key enzymes related to hyperglycemia by leading to an increase in blood glucose levels [95,96]. Therefore, inhibition of the function of this enzyme can reduce and control the risk of hyperglycemia. In this study, the α-glucosidase inhibition activity of the extracts of each edible Amanita species was investigated in terms of the inhibition percentage. The results were then compared with those of acarbose (anti-diabetic drug). The results then revealed that all extract samples exhibited α-glucosidase inhibition activity, while the value of the inhibition percentage varied according to the differences in the extract samples ( Table 5). The value of α-glucosidase inhibition activity in the extract samples varied from 19.26% to 31.44% inhibition. However, all mushroom extracts were found to be less effective than acarbose, a synthetic standard Inhibitor of α-glucosidase (44.06% inhibition at concentration of 1 mg/mL). These results are supported by those of previous studies, which reported that the extracts of certain edible wild mushrooms (e.g., Amanita, Astraeus, Boletus, Lactarius, Phlebopus, Russula, Suillus, and Tylopilus) have potential as natural α-glucosidase inhibitors. Accordingly, the α-glucosidase inhibition activity varied from 9.72-78.75% for each different mushroom species [45,97,98]. In this study, the amounts of α-glucosidase inhibitory activity obtained in this study were within the ranges reported from previous studies. Compared with the outcomes of a report conducted by Pongkunakorn et al. [97], the α-glucosidase inhibitory activity of the methanolic extracts of A. hemibapha (19.26 and 20.37%) and A. rubromarginata (20.28%) obtained in this study were lower than the α-glucosidase inhibitory activity of the water extracts of A. hemibapha and A. princeps, which were reported at 22.66% and 25.54%, respectively. Interestingly, the α-glucosidase inhibitory activity of the methanolic extracts of A. pseudoprinceps obtained in this study was higher than the α-glucosidase inhibitory activity of the water extracts of both A. hemibapha and A. princeps [97]. Several previous studies have reported that the use of different solvents resulted in different patterns of active compounds in mushroom extracts, which were related to biological activities including α-glucosidase inhibitory activity [83,84,97,98]. Importantly, this study is the first report on the α-glucosidase inhibition activities of A. pseudoprinceps, A. rubromarginata, and A. subhemibapha. This study found that the extracts of A. pseudoprinceps displayed a high level of α-glucosidase inhibition activity over the other extracts, which could be related to their high total phenolic content. Additionally, the total phenolic content of all mushroom extracts and α-glucosidase inhibitory activity were shown to be significantly correlated by Pearson correlation (p < 0.05) ( Table 6). These results were similar to those of previous studies [45,99,100], which revealed that the α-glucosidase inhibitory activity of natural substances is strongly correlated with the phenolic compound content.

Conclusions
The edible Amanita specimens collected in northern Thailand were identified as A. hemibapha, A. pseudoprinceps, A. rubromarginata, and A. subhemibapha based on the relevant morphological characteristics and multi-gene phylogenetic analyses. These four Amanita species were selected for further experiments, wherein their nutritional composition, total phenolic content, antioxidant activities, and α-glucosidase inhibitory activities were evaluated. All Amanita species were high in protein and carbohydrate but low in fat content. Additionally, the methanolic extracts of these four Amanita species contained varied amounts of total phenolic content and exhibited varied results in terms of their antioxidant and α-glucosidase inhibitory activities. The highest levels of antioxidant and α-glucosidase inhibitory activities were found in the methanolic extract of A. pseudoprinceps. The findings of this investigation provide valuable information on the nutrient content, total phenolic content, and the antioxidant and α-glucosidase inhibitory potential of the edible Amanita species found in northern Thailand. Therefore, our results suggest that these four edible Amanita species can be representative of an alternative food source. These species are also a good source of natural antioxidants and exhibit potential to naturally inhibit α-glucosidase for human health benefits. However, future studies should be implemented to conduct a comprehensive mineral analysis and to identify the phenolic profiles present in each edible Amanita species.