Evaluation of bioactivities and phenolic contents of wild edible mushrooms from northeastern Thailand

Abstract

Twenty-five wild edible mushrooms from Northeastern Thailand were analyzed for their antioxidant activities, proteins, sugars, β-glucan, and phenolic profiles. Results showed that T. clypeatus and V. volvacea exhibited the greatest scavenging activity (83.07 and 86.60%) and reductive power (9.79 and 8.42 g Fe(II)/kg, respectively). T. clypeatus recorded the highest content of (+)-catechin and naringenin (13.40 and 0.74 g/kg dw), with V. volvacea the highest amount of quercetin and quercetin-3-O-rutinoside (1.82 and 1.16 g/kg dw, respectively). Both T. clypeatus and V. volvacea also exhibited the greatest amounts of β-glucan (125.23 and 344.43 g/kg dw) and protein (343.30 and 452.20 g/kg dw, respectively) among the mushroom species evaluated. Results suggested that both T. clypeatus and V. volvacea were a good source of healthy compounds, namely β-glucan and flavonoids, and could be used to mitigate diseases involving free radicals.

Introduction

Wild edible mushrooms have long been exploited by many Asian countries as food and medicine [1] and are now becoming more important in the human diet with their nutritional and culinary values [2]. Mushrooms have high protein, low fat and cholesterol contents, and are considered by many as an ideal source of nutrition, rich in minerals and vitamins [3]. Edible mushrooms accumulate a variety of secondary metabolites in their fruiting bodies including phenolic compounds, polysaccharides, polyketides, terpenes and steroids. Phenolic compounds are an excellent source of antioxidants and possess anti-tumor, cardiovascular, anti-viral, anti-microbial, anti-inflammatory, and anti-allergenic properties. They also help to balance blood sugar levels and support the body’s detoxification mechanisms [4–6]. Furthermore, numerous companies are developing capsules from mushroom combinations, and although expensive, these have proved beneficial to health by counteracting cancer [7].

Northeastern Thailand has the highest wild edible mushroom diversity in Asia, and some mushrooms have high gastronomic relevance and/or medicinal properties. The Phu Phan National Park, located in Northeastern Thailand, is well known for its diversified natural resources which offers favorable environmental conditions for the growth of wild edible mushrooms. In addition to the health benefits, these mushrooms are a food source with potential economic value for the local people. A recent study by Seephonkai et al. [8], reported that mushrooms in the genus Phellinus collected from Northeast Thailand exhibited excellent antioxidant activity.

In view of the mushroom species biodiversity in Northeast Thailand and their interesting pharmacological activities, the antioxidant properties and phenolic constituents of twenty-five species of wild edible mushrooms were investigated. This is the first study on the phenolic composition and antioxidative activities of wild edible mushrooms collected from native forests in Northeastern Thailand.

Materials and methods

Reagents and chemicals

Deionized water was prepared by a Milli-Q Water Purification system (Millipore, MA, USA). Acetonitrile, methanol, and phosphoric acid were of HPLC grade from Merck (Darmstadt, Germany). Folin-Ciocalteu reagent and TPTZ (2,4,6-tripyridyl-s-triazine) were purchased from Sigma-Aldrich Chemical Company (St. Louis, MO, USA). DPPH (2,2-diphenyl-1-picryl hydrazyl hydrate) was obtained from Fluka (Buchs, Switzerland). Pure standard phenolics, including myricetin, quercetin, rutin, (+)-catechin, (−)-epicatechin, naringenin and kaempferol were purchased from Sigma-Aldrich Chemical Company (St. Louis, MO, USA). β-1,3-glucan was obtained from Sigma-Aldrich (Seelze, Germany).

Mushroom sample preparation

Twenty-five species of mushroom fruiting bodies were purchased from the indigenous people who collect from forest areas of the Phu Phan Mountains within Phu Phan National Park, Sakon Nakhon Province between June and August 2016. The area is 400–567 m above sea level. The mushrooms were identified by Dr. Sujitar Jorjong, Department of Plant Science, Faculty of Natural Resources, Rajamangala University, Thailand. The names of the species studied and their natural habitats are presented in Table 1. The mushrooms were classified into three groups as gilled mushrooms, puffball mushrooms and shelf mushrooms.

Table 1.

Natural habitats of twenty-five wild edible mushroom species studied for bioactive compound content and antioxidant potential

Local name	Scientific name	Family	Natural habitat
Gilled mushroom
Het Ra Ngok Kao	Amanita princeps	Amanitaceae	Saprophytic (grows on the ground under the shade of trees, Dipterocarpus alatus, Hopea odorata and Anisoptera costata)
Het Ra Ngok Luang	Amanita hamibapa	Amanitaceae	Saprophytic (on the ground under the shade of trees, Dipterocarpus alatus, Hopea odorata and Anisoptera costata)
Het Hat	Lactarius volemus	Russulaceae	Saprophytic (grows on the ground in open forests)
Het Nam Mag	Russula luteotacta	Russulaceae	Saprophytic (grows on the ground in open forests)
Het Nam Mag Yai	Russula emetica	Russulaceae	Saprophytic (grows on the ground in open forests)
Het Na Kao	Russula alboareolata	Russulaceae	Saprophytic (grows on the ground in open forests)
Het Kow Kao	Russula galochroides	Russulaceae	Saprophytic (grows on the ground under Lithocarpus cantleyanus)
Het Kow	Russula cyanoxantha	Russulaceae	Saprophytic (grows on the ground under Lithocarpus cantleyanus)
Het Tan Yai	Russula nigricans	Russulaceae	Saprophytic (grows on the ground under Dipterocarpus alatus)
Het Tan Noi	Russula densfolia	Russulaceae	Saprophytic (grows on the ground under Dipterocarpus alatus)
Het Kai	Russula delica	Russulaceae	Saprophytic (grows on the ground under Shorea obtuse and Shorea siamensis)
Het Na Lae	Russula violeipes	Russulaceae	Saprophytic (grows on the ground in open forests)
Het Pluak Tab	Termitomyces fuliginosus	Amanitaceae	Symbiotic (in association with termite nests)
Het Pluak Jig	Termitomyces clypeatus	Amnitacca	Symbiotic (in association with termite nests)
Het Teen Rat	Tricholoma crassum	Tricholomataceae	Saprophytic (grows naturally in various habitats such as meadows, rice fields, forests and soil containing high organic matter)
Het Fang	Volvariella volvacea	Pluteaceae	Saprophytic (grown on rice straw)
Puffball mushroom
Het Pow Nung	Astraeus hygrometricus	Astraeaceae	Saprophytic (grows on the ground under the shade of trees, Dipterocarpus alatus)
Het Klum Pra	Alpova trappei	Sclerodertraceae	Saprophytic (grows on the ground in open forests)
Shelf mushroom
Het Hoo Noo	Auricularia auricula	Auriculariaceae	Saprophytic (grows on both dead and living wood)
Het Man Poo	Cantharellus cibarius	Cantharellaceae	Saprophytic (grows on the ground in woods especially Mangifera indica)
Hed Ma Het Man Poo Yai	Cra Craterellus aureus	Cantharellaceae	Saprophytic (grows on the ground in woods especially Mangifera indica)
Het Kon Kao	Lentinus squarrosulas	Polyporaceae	Saprophytic (grows on decaying plants of Shorea obtuse, Shorea siamensis and Mangifera indica)
Het Bod	Lentinus polychrous	Polyporaceae	Saprophytic (grows on decaying plants of Hopea odorata and Anisoptera costata)
Het Hom	Lentinus edodes	Tricholomataceae	Saprophytic (grows on decaying plants of Lithocarpus cantleyanus)
Het Tong Fon	Lentinus giganteus	Polyporaceae	Saprophytic (grows on the ground in woods especially Mangifera indica)

Two hundred and fifty samples of the twenty-five wild edible species collected (10 subsamples from each species) were analyzed for their phenolic composition and antioxidant activity. The mushroom samples were washed, air dried, and then dried in an oven at 60 °C. The dried mushrooms were ground to a fine powder (ca. 1 mm size) and stored in airtight plastic bags in desiccators at room temperature prior to analysis. A 1 g sample of each dried mushroom was mixed with 10 ml of 60% methanol and subjected to extraction by an ultrasonic cleaner (KUDOS Ultrasonic Equipment Co., Shanghai, China) for 1 h at room temperature. The samples were then centrifuged at 2,000 g for 15 min, the solvent was decanted and the residue was re-extracted with 10 ml of fresh the solvent. The two extracts were combined and the volume was adjusted to 20 ml. Appropriate aliquots of extracts were filtered through a 0.45 μm membrane filter and stored at −18 °C until required for analysis.

Protein analysis

Total nitrogen concentration was measured by elemental analysis (LECO TruSpec Micro CHNS Determinator, LECO Corporation, St. Joseph, MI, USA). Samples (2 mg) were placed in tin capsules in an oven for combustion at 1100 °C using pure oxygen (20 cm³) as the combustion gas and pure helium as the carrier gas. Carbon, hydrogen and sulfur were determined by infrared absorption while nitrogen was measured as N₂ using a thermal conductivity detection system.

HPLC analysis for β-glucan, sugars, and phenolic compounds

The concentrations of β-glucan and sugars were determined using HPLC according to Kelebek et al. [9] (Shimadzu Cooperation Analytical & Measuring Instruments Division Kyoto, Japan liquid chromatograph equipped with LC-20AD series pumping system, SIL-20A series auto injector system, RID-10A series refractive index detector, and Bio-Rad (Richmond, CA) Aminex HPX-87P (lead-based), 0.78 × 30 cm, 5 µm chromatograph column). Twenty microliters samples were injected into the column and separation was conducted at 85 °C with the mobile phase being deionized water at a flow rate of 0.6 ml/min. The identification of β-glucan and sugars in the samples was achieved by comparing retention times and spectra with those of authentic standards, and quantified by the external standard method.

HPLC was also used for determining the concentrations of specific phenolic compounds. The liquid chromatographic apparatus (Shimadzu LC-20AD) consisted of a pump (Shimadzu LC-20AD), an automatic injector (Shimadzu SIL-10AD) (20 μl injection volume) and controller coupled to a photodiode array detector (SPD-M20A) (set at 254 nm) interfaced to LC Solution software (Shimadzu Cooperation Analytical & Measuring Instruments Division Kyoto, Japan)). Separations were performed on an Apollo C₁₈ column (Alltech Associates, Deerfield, IL, USA) (4.6 mm × 250 mm, 5 µm) preceded by separation on an Inertsil ODS-3 guard column (4.0 mm × 10 mm, 5 µm; GL Science Inc., Tokyo, Japan). Both columns were placed in a column oven set at 40°C. Solvent A was acetonitrile in deionized water (2% v/v), acidified with phosphoric acid (0.2% v/v), while solvent B was acetonitrile in deionized water (97.8% v/v), acidified with phosphoric acid (0.2% v/v). The elution profile started with 20% solvent B, 50% solvent B at 30 min, 60% solvent B at 35 min, 20% solvent B at 40 min and re-equilibration of the column for 15 min at a flow rate of 0.6 ml/min. Components were identified by comparing their retention times and spectra with those of authentic standards and quantified by the external standard method.

Determination of total phenolic content (TPC)

The TPC in the mushroom samples was estimated using a Folin-Ciocalteu assay, following the method of Singleton and Rossi [10] with some modifications. Briefly, 12.5 µl of the sample was mixed with 12.5 µl of Folin-Ciocalteu reagent (1:9; Folin-Ciocalteu reagent: distilled water) and 12.5 µl of distilled water. After 6 min, 125 µl of 7% sodium carbonate (Na₂CO₃) solution and 100 µl of distilled water were added to the mixture and allowed to react for 90 min. Absorbance was then read at 760 nm using a microplate reader (Synergy HT, BiotTek Instruments, USA). Gallic acid at different concentrations was used as a standard. Samples were measured in three replicates.

Determination of total flavonoid content (TFC)

The aluminum chloride (AlCl₃) method was used to determine the TFC of the sample extracts according to Kim et al. [11] with some modifications. Briefly, 25 µl of the diluted sample or (+)-catechin was mixed with 125 µl of deionized distilled water (ddH₂O) and 7.5 µl of 5% sodium nitrite (NaNO₂) solution. After 5 min, incubation at room temperature, 15 µl of 10% AlCl₃ solution was added. After 5 min 50 µl of 1 M sodium hydroxide (NaOH) solution was added. The mixture was immediately diluted by the addition of 27.5 µl of ddH₂O, thoroughly mixed, and its absorbance read at 510 nm against a blank prepared with ddH₂O, using a microplate reader. (+)-Catechin at different concentrations was used as a standard. Samples were measured in three replicates. The results were expressed as g (+)-catechin equivalent per kg dry weight (g CE/kg dw).

DPPH radical scavenging activity

The scavenging activity of the mushroom samples was estimated according to the procedure described by Brand-Williams et al. [12] with some modifications. Firstly, 100 μl of the sample (1 g/l) was added to 100 µl of 0.2 mM DPPH methanolic solution. The mixture was mixed thoroughly, kept in the dark at room temperature for 1 h and then absorbance was measured at 520 nm using a microplate reader. Extraction solvent was used as a blank while the control was prepared by mixing 100 µl of DPPH and 100 µl of methanol. The scavenging effect was calculated based on the following equation:

$$\text{DPPH}\text{scavenging}\text{effect}(\%)=\left(\left(A_0-A_1\right)/A_0\right)\times 100,$$

where A₀ is the absorbance of the control and A₁ is the absorbance in the presence of the sample.

Ferric reducing antioxidant power (FRAP)

The FRAP assay was performed following the method of Benzie and Strain with some modifications [13]. Briefly, 30 μl samples (1 g/l) or the standard were added to 270 μl of freshly prepared FRAP reagent and mixed thoroughly. The absorbance was measured at 595 nm after 30 min. Ferrous sulfate (FeSO₄) was used as the standard. The antioxidant potential of the sample was estimated against a standard curve of ferrous sulfate and the FRAP value was expressed as mg Fe(II) equivalents per gram of extract.

Results and discussion

Chemical composition

β-Glucan is a polysaccharide which is typically found in various species of mushrooms including reishi, shiitake and maitake [14]. β-Glucans have many functional and bioactive properties including anticancer abilities, antioxidant effects and activities against infectious diseases [14, 15]. The wild edible mushrooms studied were a rich source of fungal β-glucan with values ranging between 6.17 and 344.39 g/kg dw (Table 2). Shelf mushrooms were the richest sources of β-glucan followed by gilled mushrooms and puffball mushrooms. Park et al. [16] and Toledo et al. [17] found that the concentration of β-glucan in mushrooms varied from 76.0–101.0 and 27.0–89.2 g/kg dw, respectively. These values were considerably lower than results obtained in this study, with highest β-glucan concentration in V. volvacea (344.39 g/kg dw), followed by L. squarrosulas (191.85 g/kg dw), R. violeipes (136.61 g/kg dw) and C. cibarius (132.91 g/kg dw). Among the wild edible mushrooms studied, A. trappei had the lowest β-glucan content (6.17 g/kg dw) while β-glucan was not detected in A. auricula. V. volvacea and L. squarrosulas showed the greatest potential for commercial exploitation.

Table 2.

β-glucan, protein and sugar concentration in twenty-five species of wild edible mushrooms in Thailand

Mushroom	β-Glucan (g kg−1 dw)	Protein (g kg−1 dw)	Sugar (g kg−1 dw)			Sugar alcohol (g kg−1 dw)
			Sucrose	Glucose	Fructose	Myo-inositol	Mannitol	Arabitol	Xylitol	Sorbitol
Gilled mushrooms
A. princeps	90.10 ± 5.31	372.60 ± 12.50	50.48 ± 1.83	7.23 ± 0.68	4.35 ± 0.60	ND	12.99 ± 1.61	9.34 ± 1.27	0.95 ± 0.02	0.84 ± 0.07
A. hamibapa	84.92 ± 6.51	359.81 ± 10.46	83.11 ± 5.23	21.57 ± 1.57	13.19 ± 0.63	ND	7.18 ± 0.53	1.18 ± 0.80	0.88 ± 0.03	ND
L. volemus	80.39 ± 6.13	289.24 ± 17.19	0.15 ± 0.02	10.34 ± 0.22	19.63 ± 1.21	ND	164.58 ± 13.16	ND	ND	ND
R. luteotacta	121.74 ± 5.49	306.37 ± 24.50	2.77 ± 0.07	ND	6.08 ± 0.11	ND	126.13 ± 7.53	50.44 ± 5.52	2.66 ± 0.20	1.32 ± 0.10
R. emetica	36.90 ± 6.62	301.23 ± 13.16	20.48 ± 1.02	1.72 ± 0.07	21.56 ± 5.02	4.49 ± 0.28	107.36 ± 12.35	ND	ND	ND
R. alboareolata	62.69 ± 5.49	320.84 ± 10.23	0.56 ± 0.01	ND	18.39 ± 0.43	ND	116.28 ± 1.25	ND	ND	ND
R. galochroides	81.60 ± 9.55	222.30 ± 18.75	2.73 ± 0.06	ND	9.11 ± 0.35	ND	132.87 ± 9.40	53.75 ± 5.18	0.90 ± 0.04	1.14 ± 0.03
R. cyanoxantha	59.57 ± 0.53	349.06 ± 15.14	8.48 ± 0.62	2.40 ± 0.14	13.61 ± 0.70	5.99 ± 0.14	131.42 ± 13.44	ND	0.72 ± 0.03	ND
R. nigricans	76.93 ± 6.69	285.51 ± 13.22	4.58 ± 0.18	23.86 ± 7.04	1.52 ± 0.02	ND	98.03 ± 7.78	38.12 ± 4.64	0.89 ± 0.10	1.06 ± 0.04
R. densfolia	88.18 ± 5.09	254.72 ± 19.21	1.67 ± 0.04	7.71 ± 0.05	43.23 ± 2.95	ND	115.26 ± 1.30	ND	ND	ND
R. delica	86.67 ± 8.44	348.05 ± 16.17	0.92 ± 0.10	0.18 ± 0.01	2.54 ± 0.18	ND	106.32 ± 8.78	39.92 ± 5.11	3.05 ± 0.16	1.60 ± 0.08
R. violeipes	136.61 ± 7.93	397.32 ± 21.20	6.35 ± 0.14	0.07 ± 0.01	17.68 ± 1.82	0.53 ± 0.10	43.52 ± 4.61	ND	ND	ND
T. fuliginosus	115.21 ± 5.10	444.31 ± 20.35	54.29 ± 3.41	0.23 ± 0.03	5.83 ± 0.16	ND	2.56 ± 0.14	ND	1.05 ± 0.02	1.73 ± 0.03
T. clypeatus	125.23 ± 4.22	343.37 ± 15.33	31.63 ± 2.10	0.06 ± 0.01	8.56 ± 0.20	ND	10.24 ± 1.10	8.20 ± 0.16	3.54 ± 0.11	1.39 ± 0.05
T. crassum	98.26 ± 8.72	487.10 ± 17.41	90.27 ± 7.53	8.19 ± 0.40	6.60 ± 023	ND	1.26 ± 0.12	1.76 ± 0.19	0.95 ± 0.05	11.34 ± 0.05
V. volvacea	344.39 ± 11.33	452.23 ± 25.67	27.15 ± 2.10	5.77 ± 0.30	9.68 ± 0.46	ND	10.66 ± 1.19	ND	2.48 ± 0.30	3.28 ± 0.10
Puffball mushrooms
A. hygrometricus	30.07 ± 7.24	264.81 ± 10.39	2.14 ± 0.09	2.40 ± 0.14	4.12 ± 0.20	ND	9.66 ± 0.89	2.17 ± 0.14	ND	ND
A. trappei	6.17 ± 1.13	241.34 ± 12.43	1.14 ± 0.05	ND	24.05 ± 1.63	ND	ND	ND	ND	ND
Shelf mushrooms
A. auricula	ND	73.42 ± 6.12	ND	ND	ND	ND	ND	ND	ND	ND
C. cibarius	132.91 ± 4.27	193.91 ± 11.28	1.41 ± 0.07	ND	9.89 ± 0.53	ND	0.87 ± 0.05	ND	5.51 ± 0.15	ND
Cra C. aureus	23.13 ± 3.41	223.84 ± 17.18	12.70 ± 1.33	ND	23.85 ± 7.41	ND	37.91 ± 1.28	ND	ND	ND
L. squarrosulas	191.85 ± 8.25	404.31 ± 20.37	8.54 ± 0.36	0.64 ± 0.04	20.71 ± 0.50	ND	6.99 ± 0.33	0.67 ± 0.01	2.88 ± 0.02	1.15 ± 0.04
L. polychrous	102.93 ± 6.07	355.15 ± 24.50	31.95 ± 2.79	7.49 ± 0.68	13.23 ± 0.68	2.33 ± 0.05	13.41 ± 1.71	2.97 ± 0.30	1.39 ± 0.03	2.58 ± 0.01
L. edodes	119.31 ± 7.75	376.53 ± 16.44	155.61 ± 16.37	5.60 ± 0.18	57.11 ± 2.54	ND	118.20 ± 6.33	ND	ND	ND
L. giganteus	88.40 ± 3.13	322.17 ± 13.15	3.75 ± 0.08	9.19 ± 1.64	18.35 ± 1.81	ND	8.16 ± 0.41	ND	ND	ND

Results are expressed as mean values ± SD (n = 10). ND not detected

Mushrooms have high concentrations of carbohydrates and proteins. However, protein concentrations are affected by a number of factors including mushroom species, stage of development, the part sampled, level of nitrogen available, location, and the particular characteristics of the natural habitat [18]. This report studied the chemical composition of the twenty-five most popular wild edible mushroom species among the local Isan population. Chemical compositions (expressed on dry weight basis) for the selected mushroom species are shown in Table 2. Protein was present at high concentrations and varied between 73.42 g/kg dw (A. auricula) and 487.10 g/kg dw (T. crassum). C. aureus, A. hygrometricus, R. nigricans, and R. alboareolata all recorded high protein contents at 223.84, 264.81, 285.51 and 320.84 g/kg dw, respectively; twice the values reported by Sanmee et al. [2]. Differences in protein concentration may result from location and ecological conditions. In general, wild mushrooms are richer sources of protein than commercial mushrooms. The protein content of sixteen gilled fungi (222.30–487.10 g/kg dw) was higher than that reported in oyster mushrooms (177.01–235.00 g/kg dw) [19].

L. edodes had the highest contents of sucrose and fructose (155.61 and 57.11 g/kg dw, respectively), while the highest glucose content was found in R. nigricans (23.86 g/kg dw) (Table 2). Fructose was present in all mushroom species except in A. auricula where it was not detected. Fructose and glucose concentration of R. alboareolata and A. hygrometricus were 17–20 times higher than those reported by Sanmee et al. [2]. However, here, glucose concentrations (0.07–23.90 g/kg dw) were similar to Croatian wild edible mushrooms (2.80–15.20 g/kg dw) [20] and Korean edible mushrooms (1.50–36.30 g/kg dw) [21].

The sugar alcohols mannitol, arabitol, xylitol and sorbitol were all found in the wild edible mushrooms studied (Table 2). Myo-inositol was only detected in R. galochroides, R. luteotacta, L. polychrous and R. violeipes. Mannitol, one of the phytochemicals of edible mushrooms, has the capacity to reduce blood pressure [22]. It also has diuretic effects [23]. With the exception of A. trappei and A. auricula, mannitol was present as the main polyol in all the wild edible mushrooms studied, with values ranging from 0.87 to 164.58 g/kg dw. Highest concentrations of mannitol were found in L. volemus (164.58 g/kg dw), R. galochroides (132.87 g/kg dw), R. cyanoxantha (131.42 g/kg dw), and R. luteotacta (126.13 g/kg dw), while the lowest was C. cibarius at 0.87 g/kg dw. Gilled mushrooms provided have the richest sources of mannitol. Arabitol was predominant in R. galochroides, R. luteotacta, R. delica and R. nigricans while the highest amount of sorbitol was detected in T. crassum. Arabitol has not previously been measured in A. hygrometricus [2] but was detected in this study.

Phenolic compounds profile

Limited investigations have been carried out regarding the individual profiles of phenolic compounds in wild edible mushrooms. Cultivated mushroom species are better known in terms of content; however, wild edible mushrooms are scarcely investigated and, to the best of our knowledge, the phenolic content of these twenty-five species has not been previously described. The phenolics, (+)-catechin and (−)-epicatechin were present in all twenty-five mushrooms (Table 3). Other phenolic compounds such as quercetin, quercetin-3-O-rutinoside, myricetin, naringenin and kaempferol were also detected. Quercetin and quercetin-3-O-rutinoside were the major flavonols ranging from 0.04–1.84 and 0.04–1.17 g/kg dw, respectively. The highest concentrations of quercetin were seen in R. luteotacta (1.84 g/kg dw) and V. volvacea (1.83 g/kg dw) while V. volvacea also had the highest concentrations of quercetin-3-O-rutinoside (1.17 g/kg dw) and myricetin (0.68 g/kg dw). Myricetin was detected in low concentrations in R. delica, T. fuliginosus, C. aureus, and L. giganteus ranging from 0.02 to 0.03 g/kg dw. However, these results were within the range of eight other edible mushrooms, A. bisporus, B. edulis, C. gambosa, C. cibarius, C. cornucopioides, H. marzuolus, L. deliciosus and P. ostreatus where values varied from 0.02 to 0.04 g/kg dw [24]. The most abundant flavonoids were (+)-catechin and (−)-epicatechin (flavan-3-ols) ranging from 0.12 to 13.40 g/kg dw and 0.20 to 6.09 g/kg dw, respectively. Total concentrations of the flavan-3-ols varied from 0.32 to 14.69 g/kg dw with the lowest concentration occurring in A. trappei and the highest in T. clypeatus. Naringenin is a flavanone that is also present in wild edible mushrooms, although less abundant than other flavonoids. It was the main component of the flavanone groups in the studied mushrooms (0.02–0.74 g/kg dw). The studied edible mushrooms had low quantities of kaempferol which occurs in some mushroom species in similar concentration (varying from 0.01 to 0.04 g/kg dw), except in L. polychrous where the kaempferol content was considerably higher (0.06 g/kg dw) than other mushrooms.

Table 3.

Concentrations of individual phenolic compounds in twenty-five wild edible mushroom species located in Thailand

Sample	Flavonols					Flavan-3-ols			Flavanone	Total HPLC polyphenols
	Quercetin	Quercetin-3-O-rutinoside	Myricetin	Kaempferol	Total flavonols	(+)-catechin	(−)-epicatechin	Total flavan-3-ols	Naringenin
Gilled mushrooms
A. princeps	0.51 ± 0.02	0.62 ± 0.04	0.07 ± 0.02	0.03 ± 0.01	1.24 ± 0.11	3.01 ± 0.12	4.70 ± 0.24	7.71 ± 0.51	0.11 ± 0.00	9.06 ± 0.89
A. hamibapa	0.31 ± 0.01	0.30 ± 0.02	0.06 ± 0.00	0.01 ± 0.00	0.68 ± 0.05	2.67 ± 0.14	0.91 ± 0.14	3.58 ± 0.40	0.17 ± 0.03	4.43 ± 0.65
L. volemus	0.12 ± 0.01	0.24 ± 0.01	ND	ND	0.37 ± 0.03	1.91 ± 0.17	0.20 ± 0.03	2.10 ± 0.28	ND	2.47 ± 0.45
R. luteotacta	1.84 ± 0.08	0.70 ± 0.07	0.26 ± 0.03	0.01 ± 0.00	2.81 ± 0.23	3.11 ± 0.20	6.09 ± 0.42	9.20 ± 0.88	0.59 ± 0.02	12.61 ± 1.61
R. emetica	0.10 ± 0.01	0.14 ± 0.01	0.15 ± 0.01	0.02 ± 0.00	0.42 ± 0.04	1.70 ± 0.02	1.32 ± 0.21	3.03 ± 0.31	0.31 ± 0.04	3.75 ± 0.59
R. alboareolata	0.52 ± 0.02	0.13 ± 0.00	ND	ND	0.65 ± 0.03	0.41 ± 0.02	0.46 ± 0.04	0.86 ± 0.09	0.34 ± 0.03	1.85 ± 0.22
R. galochroides	0.42 ± 0.01	0.13 ± 0.02	ND	ND	0.54 ± 0.03	0.82 ± 0.04	0.55 ± 0.03	1.37 ± 0.11	ND	1.92 ± 0.20
R. cyanoxantha	0.43 ± 0.03	0.10 ± 0.01	ND	ND	0.53 ± 0.05	0.66 ± 0.08	0.66 ± 0.06	1.32 ± 0.20	ND	1.86 ± 0.37
R. nigricans	0.07 ± 0.01	0.26 ± 0.03	0.09 ± 0.02	0.02 ± 0.00	0.44 ± 0.04	1.55 ± 0.11	0.43 ± 0.03	1.99 ± 0.21	0.23 ± 0.01	2.66 ± 0.38
R. densfolia	0.22 ± 0.02	0.04 ± 0.00	0.17 ± 0.02	0.03 ± 0.00	0.45 ± 0.06	0.21 ± 0.02	1.01 ± 0.06	1.23 ± 0.12	0.59 ± 0.02	2.27 ± 0.30
R. delica	0.14 ± 0.01	0.26 ± 0.06	0.02 ± 0.00	0.04 ± 0.01	0.47 ± 0.08	2.83 ± 0.18	1.67 ± 0.09	4.50 ± 0.39	0.05 ± 0.00	5.02 ± 0.67
R. violeipes	0.09 ± 0.01	0.72 ± 0.04	0.09 ± 0.01	0.02 ± 0.00	0.93 ± 0.07	6.48 ± 0.34	2.04 ± 0.14	8.52 ± 0.69	0.21 ± 0.01	9.66 ± 1.09
T. fuliginosus	0.73 ± 0.02	0.12 ± 0.01	0.02 ± 0.00	0.01 ± 0.00	0.88 ± 0.03	8.43 ± 0.50	2.81 ± 0.10	11.24 ± 0.85	0.19 ± 0.01	12.31 ± 1.27
T. clypeatus	0.92 ± 0.04	0.27 ± 0.03	0.07 ± 0.02	ND	1.28 ± 0.11	13.40 ± 0.74	1.29 ± 0.24	14.69 ± 0.98	0.74 ± 0.06	16.71 ± 1.63
T. crassum	0.11 ± 0.01	0.14 ± 0.01	0.05 ± 0.00	ND	0.31 ± 0.04	2.09 ± 0.12	0.31 ± 0.05	2.40 ± 0.24	0.02 ± 0.00	2.73 ± 0.40
V. volvacea	1.83 ± 0.01	1.17 ± 0.02	0.68 ± 0.06	0.04 ± 0.01	3.72 ± 0.44	6.73 ± 0.69	5.87 ± 0.38	12.61 ± 0.52	0.72 ± 0.02	17.04 ± 1.40
Puffball mushrooms
A. hygrometricus	0.04 ± 0.61	0.09 ± 0.04	0.04 ± 0.01	ND	0.18 ± 0.02	2.61 ± 0.11	3.78 ± 0.17	6.38 ± 0.39	0.28 ± 0.01	6.84 ± 0.61
A. trappei	ND	ND	ND	ND	ND	0.12 ± 0.01	0.20 ± 0.01	0.32 ± 0.01	ND	0.33 ± 0.02
Shelf mushrooms
A. auricula	0.05 ± 0.50	0.08 ± 0.03	0.08 ± 0.01	ND	0.23 ± 0.02	1.52 ± 0.16	0.40 ± 0.04	1.91 ± 0.28	0.05 ± 0.13	2.19 ± 0.44
C. cibarius	0.24 ± 0.01	0.37 ± 0.02	0.03 ± 0.00	ND	0.88 ± 0.07	0.51 ± 0.04	0.39 ± 0.03	0.90 ± 0.10	0.54 ± 3.19	2.32 ± 0.30
Cra C. aureus	0.10 ± 0.00	0.38 ± 0.01	0.03 ± 0.00	ND	0.52 ± 0.03	0.68 ± 0.06	0.37 ± 0.06	1.05 ± 0.17	0.40 ± 3.07	1.97 ± 0.33
L. squarrosulas	0.10 ± 0.00	0.46 ± 0.03	0.17 ± 0.02	0.02 ± 0.00	0.76 ± 0.10	4.86 ± 0.16	1.57 ± 0.10	6.42 ± 0.37	0.09 ± 0.58	7.28 ± 0.68
L. polychrous	0.40 ± 0.02	0.77 ± 0.05	0.46 ± 0.05	0.06 ± 0.01	1.68 ± 0.23	5.61 ± 0.23	2.09 ± 0.12	7.70 ± 0.50	0.09 ± 1.13	9.48 ± 1.06
L. edodes	0.39 ± 0.01	0.49 ± 0.04	0.16 ± 0.01	0.02 ± 0.00	1.05 ± 0.09	3.05 ± 0.19	1.90 ± 0.16	4.96 ± 0.49	0.04 ± 0.62	6.05 ± 0.85
L. giganteus	ND	0.12 ± 0.00	0.02 ± 0.00	ND	0.14 ± 0.01	0.66 ± 0.06	0.98 ± 0.08	1.64 ± 0.20	ND	1.78 ± 0.30

Data were expressed g kg⁻¹dw (mean ± SD) (n = 10). ND, not detected

Total flavonols, qurercetin, quercetin-3-O-rutinoside, myricetin and kaempferol; total flavan-3-ols, (+)-catechin and (−)-epicatechin; total HPLC polyphenols, qurercetin, quercetin-3-O-rutinoside, myricetin, kaempferol, (+)-catechin, (−)-epicatechin, and naringenin

Total phenolic and flavonoid contents

As the antioxidation properties of extracts were often linked to their concentrations of phenolic and flavonoid compounds, the mushroom extracts were tested for their TPC and TFC. The amount of TPC was calculated as gallic acid equivalents, while catechin was used as the reference for the TFC assay. T. clypeatus had the highest TPC (8.84 g GAE/kg dw) among the wild edible mushroom species evaluated (Table 4), followed by A. hamibapa and V. volvacea with values of 8.53 and 8.49 g GAE/kg dw, respectively. The TFC in T. clypeatus was the highest (5.10 g CE/kg dw), while the lowest TFC was exhibited in C. aureus (0.21 g CE/kg dw). These results were lower than those reported in Portuguese wild edible mushrooms which varied from 4.58–58.14 g GAE/kg dw and 1.78–33.00 g CE/kg dw for TPC and TFC, respectively [25]. However, they were within the range of edible mushrooms in Malaysia where H. tessulatus, P. eryngii, P. florida, A. polytricha and F. velutipes varied from 0.90–6.03 g GAE/kg dw and 0.20–6.95 g QE/kg dw for TPC and TFC, respectively [26]. The American Cancer Society has established 0.10 g per day of flavonoids as adequate for the mitigation of cancer and deteriorating illnesses [27]. Results for the wild edible mushrooms studied revealed that they all contained high flavonoids. The TFC values for T. clypeatus, V. volvacea and L. polychrous were 5.10, 3.45 and 2.27 g CE/kg dw, respectively, above the value suggested for daily intake by the American Cancer Society.

Table 4.

Total phenolic concentration, total flavonoid concentration and antioxidant activity of twenty-five wild edible mushrooms species in Thailand

Sample	Total phenolics (g GAE kg−1 dw)a	Total flavonoids (g CE kg−1 dw)b	DPPH (% scavenging activity)	FRAP (g Fe(II) kg−1 dw)
Gilled mushrooms
A. princeps	1.68 ± 0.38	0.62 ± 0.02	59.40 ± 1.19	3.01 ± 0.26
A. hamibapa	8.53 ± 0.91	0.81 ± 0.03	71.75 ± 8.94	7.54 ± 0.14
L. volemus	3.61 ± 0.36	0.52 ± 0.01	66.75 ± 2.18	0.92 ± 0.08
R. luteotacta	4.63 ± 0.20	2.09 ± 0.05	81.24 ± 5.10	7.53 ± 0.45
R. emetica	1.71 ± 0.46	0.75 ± 0.03	46.31 ± 2.16	0.20 ± 0.04
R. alboareolata	4.68 ± 0.61	1.06 ± 0.02	62.71 ± 1.35	2.66 ± 0.09
R. galochroides	2.38 ± 0.35	1.38 ± 0.01	69.76 ± 0.94	3.86 ± 0.23
R. cyanoxantha	2.91 ± 0.48	1.64 ± 0.02	78.74 ± 1.14	3.03 ± 0.36
R. nigricans	2.31 ± 0.26	1.03 ± 0.03	51.90 ± 2.06	0.32 ± 0.01
R. densfolia	5.62 ± 0.42	1.47 ± 0.05	55.43 ± 1.17	2.14 ± 0.02
R. delica	4.83 ± 0.42	1.75 ± 0.01	75.72 ± 1.18	3.74 ± 0.47
R. violeipes	2.26 ± 0.31	1.37 ± 0.06	63.59 ± 1.59	3.34 ± 0.11
T. fuliginosus	6.31 ± 0.50	2.19 ± 0.08	72.34 ± 8.11	4.47 ± 0.51
T. clypeatus	8.84 ± 0.53	5.10 ± 0.02	83.07 ± 2.41	9.79 ± 0.38
T. crassum	2.56 ± 0.54	1.53 ± 0.01	64.18 ± 1.77	0.37 ± 0.06
V. volvacea	8.49 ± 0.44	3.45 ± 0.08	86.60 ± 1.25	8.42 ± 0.67
Puffball mushrooms
A. hygrometricus	2.33 ± 0.68	1.49 ± 0.03	45.72 ± 1.18	0.41 ± 0.04
A. trappei	0.72 ± 0.02	0.78 ± 0.03	36.75 ± 3.74	0.24 ± 0.04
Shelf mushrooms
A. auricula	0.95 ± 0.25	0.15 ± 0.00	40.94 ± 0.73	0.10 ± 0.02
C. cibarius	1.41 ± 0.16	0.27 ± 0.01	64.10 ± 5.20	1.94 ± 0.39
Cra C. aureus	2.34 ± 0.55	0.21 ± 0.03	59.91 ± 1.35	4.12 ± 0.62
L. squarrosulas	5.42 ± 0.49	1.19 ± 0.02	71.68 ± 0.52	2.64 ± 0.18
L. polychrous	5.35 ± 0.14	2.27 ± 0.04	85.43 ± 1.04	3.91 ± 0.27
L. edodes	2.21 ± 0.46	0.24 ± 0.01	63.44 ± 3.64	2.50 ± 0.64
L. giganteus	1.46 ± 0.15	0.21 ± 0.00	56.90 ± 7.07	3.65 ± 0.50

^aTotal phenolic content (TPC) quantified by the Folin-Ciocalteu assay [g gallic acid equivalents (GAE) kg⁻¹ dw]

^bTotal flavonoid content (TFC) quantified by the AlCl₃ method [g catechin equivalent (CE) kg⁻¹ dw]. Results are expressed as mean values ± SD, n = 10. Antioxidant capacity measured by DPPH (% scavenging activity) and FRAP [g ferrous ion equivalent (g Fe kg⁻¹ dw] assay

DPPH radical-scavenging activity

Oxidative stress is considered as being one of the most serious causes of various diseases; therefore, searching for natural scavengers of reactive oxygen species is highly desirable. Phenolic compounds may contribute directly to antioxidation activity [28] and it has been reported that the antioxidant activity of mushroom extracts correlated well with the content of their phenolic compounds [25]. Highly effective free radical scavenging activity of more than 80% was seen in the extracts of V. volvacea (86.6%), L. polychrous (85.4%), T. clypeatus (83.1%), and R. luteotacta (81.2%) (Table 4). These results were lower than those reported in Tanzania for wild edible mushrooms with values of 94.1% and 93.5% for Cantharellus congolensis and Cantharellus cyanoxanthus, respectively [29]. However, the methanolic extract of L. volemus showed stronger scavenging activities than those reported by Keleş et al. [30]. Khatua et al. [31] mentioned that the ethanolic extract from R. delica scavenged DPPH radicals by 60% at 1.4 mg/mL, lower than values reported here. The least activity was observed in puffball mushrooms A. hygrometricus (45.7%) and A. trappei (36.8%). These results revealed that mushroom extracts with higher phenolic compounds also had higher scavenging activity. This finding has also been observed in other studies including Palacios et al. [24], Pereira et al. [25] and Tibuhwa [29]. Here, V. volvacea was observed to have high TPC and TFC values of 8.49 g GAE/kg dw and 3.45 g CE/kg dw, respectively, corresponding to a high scavenging capacity of 86.6%. In contrast, A. trappei had the lowest TPC and TFC values (0.72 g GAE/kg dw and 0.78 g CE/kg dw, respectively) and exhibited the lowest scavenging ability (36.8%).

Ferric reducing antioxidant power (FRAP)

Antioxidant capacity was estimated by the FRAP method, which determines the capacity of antioxidant components to reduce a Fe³⁺-TPTZ complex to Fe²⁺-TPTZ. Hence, a higher Fe³⁺-TPTZ reduction means a higher antioxidant capacity. The reducing power of antioxidant components may serve as an indicator of potential antioxidant capacity [32]. Reducing powers of T. clypeatus, V. volvacea, A. hamibapa and R. luteotacta were very high (9.79, 8.42, 7.54 and 7.53 g Fe(II)/kg dw, respectively). Reducing powers of T. clypeatus and V. volvacea were higher than those of the other mushrooms as they contained higher TPC and TFC (Table 4). Reducing powers of gilled mushrooms were generally higher than shelf mushrooms and puffball mushrooms. High antioxidant capacities in mushrooms can suppress reactive oxygen species which are related to aging and deteriorating illnesses [33]. They might also serve as a good resource for the future development of functional foods or drugs.

This is the first research into the phenolic composition and antioxidant activity of native wild edible mushroom species in Thailand. The study focused particularly on the antioxidant potential of twenty-five wild edible mushroom species from Northeastern Thailand and determined their chemical composition in terms of proteins, sugars, β-glucan, and phenolic compounds. Phenolic compounds were the main components responsible for the antioxidant capacity of all the wild mushrooms studied. T. clypeatus and V. volvacea demonstrated the strongest reducing powers and radical scavenging activities, as well as having the highest concentrations of phenolic compounds. Additionally, T. clypeatus and V. volvacea exhibited the highest amounts of β-glucan and protein. These findings suggest T. clypeatus and V. volvacea as highly recommended for consumption. Further research is required to establish the intracellular antioxidant activity of wild edible mushroom extracts prior to application as food supplements or in the development of nutraceuticals.