β -glucan and antioxidant activities of four edible mushroom extracts from Thailand

The commercial mushroom extracts from Thailand , Lentinus squarrosulus , Pleurotus sajor-caju , Pleurotus ostreatus, and Volvariella volvacea , could be sources of β-glucan and antioxidant. The objective of this research was to evaluate the β-glucan content and antioxidant activities of the mushroom extracts prepared from different extraction conditions. Various solvents, including water, ethanol, acid, and alkali were used for the mushroom extraction. The mushroom extracts were evaluated for their β-glucan content, total phenolic compounds, and antioxidant properties. Among all extraction conditions, the alkaline extracted (BE) P . ostreatus and P. sajor-caju showed high level of β-glucan content with 25.82 ± 3.87% w/w and 23.08 ± 0.56% w/w, respectively. Large amounts of total phenolic compounds were obtained from V. volvacea and L . squarrosulus extracted with water at 60 o C (W60) as 38.07 ± 2.53 and 34.78 ± 5.69 mg gallic acid equivalent/g extract, respectively. Excellent scavenging of ABTS radicals and FRAP assay were also observed in W60 of V. volvacea as 67.12 ± 4.41 and 36.46 ± 3.44 mg trolox equivalent/g extract, respectively. The metal chelating effect of P. ostreatus was the highest at 66.13 ± 1.63 mg EDTA equivalent/g extract but was not significantly different from V. volvacea (63.76 ± 0.51 mg EDTA equivalent/g extract) ( p > 0.05). In conclusion, alkaline extraction was the most suitable for β -glucan and metal chelating activity extracts whereas water extraction was suitable for antioxidant extracts. These simple extraction conditions could be applied in producing high bioactive compounds and antioxidant active ingredients from local mushrooms for further addition in food and health products.


Introduction
Mushrooms are consumed worldwide because they are good sources of vitamins, minerals, proteins, carbohydrates and bioactive compounds with low level of lipids and caloric contents (Kosanić et al., 2011;Ruthes et al., 2015).Different functional compounds, including terpenes, sterols, ceramides and phenolic compounds have been found in mushrooms.The major compound groups in mushrooms are polysaccharides which are responsible for health properties including enhancing the immune system.Bioactive polysaccharides in mushroom are ˗ and glucan, hemicelluloses, chitin, xylans, galactans, and mannans (Synytsya et al., 2009;Villares et al., 2012).-glucans are polysaccharides of D-glucose monomers linked by -glycosidic bonds.Most of glucans are derived from the fruiting bodies of mushrooms (Zhu et al., 2015).This compound has bioactive carbohydrates which usually function as biological response modifiers and have been used to decrease cholesterol absorption (Villares et al., 2012;Bak et al., 2014).β-glucan content in mushroom ranges from 0.21 to 0.53 g/100 g (dry weight basis) and the composition depends on cultivation conditions and origin of cultivated mushroom (Khan et al., 2018).The importance of biological activities of crude natural extract is anti-oxidation.Oxidation is the biological process that produce energy in living organisms using biological processes as fuel.However, an unregulated production of oxygenderived free radicals is involved in many diseases (Mau et al., 2002).Antioxidants can be effective in reducing oxidative damage by oxygen-derived free radicals.A past study showed that the intensity of antioxidant activity in mushroom depended on the species and the extraction solvents (Kosanić et al., 2011;Khan et al., 2018;Uddin et al., 2019;Barbosa et al., 2020a).

Commercial mushrooms including
Lentinus, Pleurotus and Volvariella genus are widely cultivated in many parts of Thailand.In Thailand, Lentinus squarrosulus (Hed Khon Kao), Pleurotus sajor-caju (grey oyster mushroom), Pleurotus ostreatus (oyster mushroom) and Volvariella volvacea (straw mushroom) are widely cultivated because they require higher temperatures for rapid growth (Kupradit et al., 2017).Moreover, large amounts of these mushroom fruiting bodies are available year-round in Thailand.Therefore, the potential of these commercial mushrooms for use as bioactive compound sources should be evaluated.However, bioactive compounds in mushroom vary according to cultivation regions, mushroom species, and extraction conditions.More recently, the extraction methods, structure, and bioactivities of polysaccharides from several mushroom species have been reviewed and reported in many publications (Hwang et al., 2018;Uddin et al., 2019;Barbosa et al., 2020a;Gong et al., 2020).Various extraction techniques, such as ultrasonic or microwave method, alkali or hot water extraction, have been applied for mushroom bioactive polysaccharide extraction including β-glucan (Xu et al., 2019).However, simple extraction conditions with safety and cost-effectiveness should be considered to obtain extracts with high bioactive compounds and antioxidant activities.For further application, the commercial mushrooms will be value-added and applied as βglucan and antioxidant sources for food and health products.Thus, the effects of extraction process on βglucan and antioxidant activities of these 4 different commercial mushroom species were evaluated.The objectives of the present study were to evaluate the antioxidative properties including ABTS radical cation scavenging activity (ABTS), Ferric reducing antioxidant power (FRAP) and metal chelating activity of the extracted commercially cultivated fruiting bodies of mushrooms using different extraction conditions.The contents of bioactive compounds including β-glucan and the potential antioxidant components such as total phenolic compounds of these mushroom extracts were also determined.

Sample preparations
The fruiting bodies of L. squarrosulus, P. sajor-caju, P. ostreatus and V. volvacea were collected from local mushroom farms in Nakhon Ratchasima, Thailand.The fruiting bodies of all mushrooms were cleaned and dried at 60 o C for 72 hr.The dried fruiting bodies were then ground by blender and stored at 4 o C for further extraction and analysis.

Extractions
Five different conditions were used to extract soluble compounds from the ground fruiting bodies of the mushrooms.The mushroom powder were weighed and transferred to Erlenmeyer flasks.The solvents, including water, ethanol, acid, and alkali were added to the mushroom powder and extracted using different conditions as follows: Water and ethanol extractions For water and ethanol extractions, water or 70% ethanol solution were added into the mushroom powder at the ratio of 1:20 (w/v).For the first batch of water extraction at 100 and 60 o C, 50 g of each mushroom powder were separately added to 475 mL of water.Then, the soluble fractions were extracted at 100 o C for 20 min using autoclave (W100) and 60 o C for 90 min using water bath (W60).A soluble fraction of the first batch were collected by filtration using Whatman No.1 filter paper then the mushroom residue were extracted again at 100 o C for 20 min (W100) and 60 o C for 90 min (W60) with 475 mL of water as the second batch.A soluble fraction of the second batch were collected by filtration through Whatman No.1 filter paper and separately pooled with the first batch of W100 and W60.For ethanolic extraction method, ethanol (70% v/v) (Et) were used as the extraction solvent.Fifty gram of each mushroom powder were added to 475 mL of 70% ethanol solution.The first extracted batch were incubated at 60 o C for 90 min using water bath.A soluble fraction of the first batch were collected by filtration through Whatman No.1 filter paper and the mushroom residue were extracted again at 60 o C for 90 min with 475 mL 70% ethanol solution.A soluble fraction of the second batch were collected by filtration through Whatman No.1 filter paper and pooled with the first batch of Et60.All filtrate of the total soluble fraction obtained from W100, W60 and Et60 were separately concentrated at 60 o C using a rotary evaporator (Büchi Rotavapor R-144, Switzerland) to remove the solvents.The concentrated extracts were dried at 60 o C to remove the residue solvent until a constant weight of crude extract was achieved.The dry weight of the mushroom crude extracts of W100, W60 and Et60 were obtained using a digital balance.

Acid extraction
For acid extraction (AE), 3.8% HCl were used as the extraction solution with some modifications from the procedure of Szwengiel and Stachowiak (2016).Briefly, 180 mL absolute methanol were added into the dry powdered fruiting bodies (20 g) of each mushroom and incubated at 80 o C in water bath for 8 hr to remove undesired soluble materials such as vitamins, polyphenols, monosaccharides, disaccharides, and others.After methanol incubation, the residue of mushroom was separated by centrifugation for 10 min at 3,000 rpm.The soluble fraction of methanolic extract were discarded and the mushroom residue was further extracted using 3.8% HCl.The acid solutions were added into the mushroom residue at the ratio of 1:10 (w/v) and incubated for 300 min at 30 o C in water bath.Then, the extracts were neutralized with 4M NaOH and filtrated through Whatman No.1 filter paper.The crude extracts were precipitated using 95% ethanol at the ratio of 1:5 (v/v) and incubated at 4 o C for 18 hr.The precipitate was harvested by centrifugation at 5,000 rpm for 15 min and dried at 60 o C, to remove the residue ethanol, until a constant weight was achieved.The dry weight of the mushroom crude extracts of AE were obtained using a digital balance.

Alkaline extraction
For alkaline extraction (BE), the extraction method was performed following Maity et al. ( 2013) with some modifications.The mushroom powder (20 g) were added to 180 mL of 4% NaOH.The extractions were performed at 80 o C in water bath for 60 min and the extracts kept overnight at 4 o C.Then, the extracts were neutralized using 35% HCl and the soluble fractions were filtrated through Whatman No.1 filter paper.The crude extracts from alkaline extraction were precipitated using 95% ethanol at a ratio of 1:5 (v/v) at 4 o C for 18 hr.The precipitate was harvested using centrifugation for 15 min at 5,000 rpm and dried at 60 o C, to remove the residue ethanol, until a constant weight was achieved.The dry weight of the mushroom crude extracts of BE were obtained using a digital balance.All extraction conditions are summarized in Table 1.The mushroom crude extracts were kept at 4 o C for further analysis.Each extract was analyzed in four replicates to measure the β-glucan content, total phenolic compound content and antioxidant activity.The data results were reported as mean values ± standard deviation.

Determination of β-glucan content
The concentration of β-glucan in all crude extracts of each mushroom were measured using the Mushroom and Yeast β-glucan Assay kit (Megazyme International, Wicklow, Ireland).The total glucan content in crude extracts were measured using acid hydrolysis with ice-cold 12 M sulfuric acid for 1 hr.Then, samples were hydrolyzed with 2 M sulfuric acid for 2 hr at 100˚C.After neutralization, a mixture of enzymes exo-1,3-β-glucanase and β-glucosidase were added in the reactions and incubated for 1 hr at 40˚C for glucose hydrolysis.The content of total glucan was measured after GOPOD reagents were added and incubated for 20 min at 40 o C. Finally, the absorbance of all reactions was determined by spectrophotometer at 510 nm.The contents of α-glucan were measured by alkaline hydrolysis with 2 M KOH and incubated on ice for 20 min.Then, a mixture of amyloglucosidase and invertase were added in the reactions.The glucose released in enzymatic hydrolysis reaction was determined by GOPOD reagent and incubated at the same condition as described above.The β-glucan concentration was calculated by subtracting the αglucan from the total glucan content.The β-glucan concentration in mushroom extracts were reported in unit of g β-glucan /100 g extract (or % w/w).

Determination of total phenolic content
The level of total phenolic compounds in the various mushroom extracts were measured according to the method of Matthaus (2002) with some modifications.
In brief, a 100 µL of the crude extract samples were added to 2 mL of Na2CO3 solution (2% w/v) and incubated for 2 min at room temperature.Then, 100 µL of Folin-Ciocalteau reagent (Merck, Darmstadt, Germany) was added and incubated for 30 min at room temperature under the dark condition.After a 30 min incubation, the absorbance was determined at 750 nm using a spectrophotometer.The concentration of total phenolic compounds was reported in unit of mg gallic acid equivalents per g extract (mg gallic acid eq./ g extract).

Antioxidant activity assay ABTS radical cation scavenging activity
The antioxidant activity was carried out using ABTS assay, with minor alteration, as described by Wiriyaphan et al. (2012).At first, the production of ABTS radicals (ABTS •+ ) were performed by mixing 7.4 mM ABTS solution and 2.6 mM potassium persulfate solution (in 10 mM phosphate buffer, pH 7.4).The ABTS radical (ABTS •+ ) were incubated under a dark condition at room temperature for 16 hr.
Then, the ABTS •+ stock solution was diluted with 10 mM phosphate buffer (pH 7.4) to adjust the absorbance value to 0.700±0.02at 734 nm.For sample analysis, antioxidant activity of the mushroom crude extracts was performed by mixing 20 μL of crude extracts with 1.980 mL of ABTS working solution and incubating at room temperature for 5 min in dark condition.The decrease in absorbance was determined at 734 nm.Results were reported as mg trolox equivalent per g extract (mg trolox eq./g extract).

Ferric reducing antioxidant power (FRAP)
FRAP assay was measured as described by Benzie and Strain (1996), with some modifications.The FRAP reagent was freshly prepared from mixing 300 mM acetate buffer (pH 3.6), 10 mM TPTZ solution in 40 mM HCl, and 20 mM FeCl3•6H2O solution at ratio of 10 : 1 : 1 (v/v).For mushroom crude extract analysis, 1 mL of FRAP reagent was added to 100 μL of crude extract sample.The reactions were incubated at 37 o C for 15 min.Then, all samples were measured for the absorbance at 593 nm.Results were reported as mg trolox equivalents per g extract (mg trolox eq./ g extract).

Metal chelating activity
Metal chelating activity assay were performed, with slight modifications, as described by Decker and Welch (1990).Fifty μL of 2 mM FeCl2 and 100 μL of 5 mM ferrozine were added into 100 μL of crude extract samples.The reactions were incubated at room temperature for 20 min.Then, the absorbance were determined at 562 nm.The metal chelating activity of the analyzed samples were reported in unit of mg EDTA equivalents per g extract.(mg EDTA eq./g extract).

Statistical analysis
All experiments were performed in four replicates.The data were reported as mean ± standard deviations and shown as error bars.Statistical analysis was performed by using PASW Statistics 18 Release 18.0.0software.Data were determined to be significantly different (p < 0.05) by one-way analysis of variance (one-way ANOVA).

Extraction yield
The yield of crude extracts from each mushroom using different extraction conditions is shown in Table 2.
The yield of the extracts varied from 45.39 ± 0.50 to 446.35 ± 11.62 mg/g dry weight according to mushroom species and extraction conditions.The crude extract yield from water extraction (W) of all 4 edible mushrooms showed significantly higher values than those from ethanol (Et), acid (AE), and alkaline (BE) extraction.The results indicated that major component (20% -45%) in the 4 mushrooms could be solubilized in water (Table 2).This might indicate that the large amount of soluble component in mushroom has high polarity as similar to previous reports (Cheung et al., 2003;Cheung and Cheung, 2005;Sudha et al., 2012).Most functional polysaccharides from mushrooms are polar molecules and highly soluble in water or alkaline solution.Thus, it can be solubilized by water (Gong et al., 2020;Leong et al., 2021).Large amount of water crude extracts were found in V. volvacea at 100 o C (446.35 ± 11.62 mg/g dry weight) and 60 o C (428.51 ± 69.51 mg/g dry weight) or 43% -45% dry weight of mushroom which were slightly higher than the report from previous works (17% -21% dry weight of mushroom) (Cheung et al., 2003;Cheung and Cheung, 2005).These results indicated that the cultivation regions and cultivation processes might have had an effect on the mushroom compositions resulting in obtaining different extraction yields.

β-glucan content
The β-glucan content of the various crude extracts from different extraction conditions of the 4 edible mushrooms were measured as shown in Figure 1.In P. ostreatus and P. sajor-caju, the β-glucan content from BE was significantly higher than those of other extraction conditions with 25.82 ± 3.87% and 23.08 ± 0.56% w/w, respectively (p<0.05).However, the βglucan content of Et60 from V. volvacea (20.69 ± 2.50% w/w) and L. squarrosulus (13.34 ± 1.40% w/w) were not significantly (p>0.05)different from BE with values at 16.63 ± 8.50% and 12.13 ± 1.38% w/w, respectively (Figure 1).Although the extraction yield of mushroom fruiting bodies was high in W100, large amounts of β-glucan content was observed in BE of P. ostreatus and P. sajor-caju and in Et60 of V. volvacea and L. squarrosulus.In our investigation, only 8-11% w/w β-glucan were obtained from W100 and W60 of P. ostreatus, P. sajor-caju and V. volvacea.These results indicated that the most suitable extraction condition for β-glucan preparation of P. ostreatus and P. sajor-caju was alkaline extraction and ethanolic extraction for V. volvacea and L. squarrosulus.The βglucan contents from various mushroom extracts have been investigated and reported.In 2008, the amount of crude β-glucan extracted from Chaga using alkaline extraction method was 13.7 % w/w.It was suggested that large amounts of soluble β-glucan may have been the cause of high dietary fiber content in Chaga (Rhee et al., 2008).In 2013, an alkaline extraction yield of 1.5 g of crude polysaccharide / 1 kg of mushroom fruiting body were obtained from somatic hybrid mushroom, Pleurotus florida and V. volvacea strains.
After the fractionation of crude polysaccharide extract through Sepharose 6B column, the water-soluble polysaccharide was identified as a β-glucan.The polysaccharide extracts from the alkaline extraction showed the activities of immunoactivation of macrophages, splenocytes, and thymocytes (Maity et al., 2013).These results indicated that the concentration of β-glucan content and biological activities varied according to mushroom species and extraction condition.Moreover, a number of research studies have reported some biological properties of mushroom β-glucans including their antitumor, antioxidative, immunomodulatory, anti-inflammatory and recurrent respiratory tract infections (Khan et al., 2018;Barbosa et al., 2020b).

Total phenolic content
In this research, the total phenolic content from the various mushroom extracts were determined and results shown in Figure 2. Total phenolic compounds in the crude extract of W100 and W60 were higher than those of other extraction conditions in all 4 edible mushrooms.The total phenolic content from W60 of V. volvacea (38.07 ± 2.53 mg gallic acid eq./g extract) were significantly higher than those from P. ostreatus and P. sajor-caju but not significantly different from L. squarrosulus (34.78 ± 5.69 mg gallic acid eq./g extract) at the same extraction condition.These results indicated that the total phenolic content in mushroom extracts contained high polarity compounds.Thus, the samples gave the highest amount of these compounds when water was used as the extraction solvent.Similar results were reported by Boonsong et al. (2016).Their results showed that a large amount of total phenolic compounds were obtained from Lentinus edodes, Pleurotus eous, P. sajor-caju and Auricularia auricular extracts when water was used as the extraction solvent (Boonsong et al., 2016).
A correlation between higher antioxidant activity and a large amount of total phenolics was found in mushroom water extract (Puttaraju et al., 2006).Thus, water-extracted samples were found to possess better antioxidant activities.

Antioxidant activity assay ABTS radical cation scavenging activity
The ABTS assay is widely used to determine the antioxidant activity of hydrophilic and lipophilic compounds (Sudha et al., 2012).In Figure 3, the ABTS activity revealed that the W100 and W60 of all 4 edible mushrooms were significantly higher than those from other extraction conditions when compared in the same mushroom species.This result indicated that the extraction temperature (100 and 60 o C) showed only minor effect on the ABTS assay of crude extracts when water was used as the extraction solvent.The high ABTS radical scavenging of mushroom extracts from water extraction might have been due to large amount of total phenolic content (Boonsong et al., 2016).The W60 of V. volvacea showed excellent scavenging of ABTS radicals at 67.12 ± 4.41 mg trolox eq./g extract which was significantly higher than those of other mushrooms at the same extraction condition (p<0.05).These results indicated that the ABTS scavenging activity varied depending on the mushroom species and extraction condition.

Ferric reducing antioxidant power, FRAP
In the FRAP assay, on reduction of Fe III-TPTZ complex by antioxidant, Fe II-TPTZ is formed which can be measured by spectrophotometer at 595 nm (Lakshmi et al., 2004).The results of antioxidant from the 4 edible mushrooms using FRAP assays are shown in Figure 4.The results showed that the values from water-extracted V. volvacea and L. squarrosulus were significantly higher than those from ethanolic, acid and alkaline extractions.The scavenging effects of V. volvacea showed its highest value at 36.46 ± 3.44 mg trolox eq./g extract in the FRAP assay obtained from crude extract using water as the extraction solvent at 60 o C. In L. squarrosulus, the excellent antioxidant activity by FRAP assay in W100 (30.27 ± 1.44 mg trolox eq./g extract) was not significantly different from W60 (28.14 ± 4.83 mg trolox eq./g extract) (p>0.05).The result correlated with the total phenolic compounds content.Similar results were reported by (Attarat and Phermthai, 2015).Their results showed that the scavenging effects of L. squarrosulus extract correlated with the total phenolic compounds (Attarat and Phermthai, 2015).The antioxidant activity of mushroom extracts highly depends on the mushroom species, cultivation regions and extraction process.Bioactive compounds of cultivated mushrooms can be different from each other because of the differences in growth conditions, compost types, chemical types and quantities used for hygiene during the mushroom cultivations (Yildiz et al., 2017).The strong antioxidant properties in mushroom include the components of ascorbic acid, tocopherols, polysaccharides, flavonoids, ergothioneine, carotenoids, phenolics and glycosides (Chun et al., 2021).Among the 4 commercial mushrooms in this research, the highest antioxidant activity using FRAP assay were found in W60 of V. volvacea.

Metal chelating activity
In metal chelating activity assay, mushroom extracts can interfere with the formation of Fe 2+ and the ferrozine complex.The chelating activity of mushroom extract can then capture Fe 2+ before ferrozine resulting in the decrease of red color observed in the reaction (Boonsong et al., 2016).The results of metal chelating activity from various mushroom extracts are shown in Figure 5.The metal chelating activity of all the 4 edible mushroom extracts obtained from BE were significantly higher than those from the other extraction conditions (p<0.05) (Figure 5).The highest chelating effect of 66.13 ± 1.63 mg EDTA eq./g extract were observed in BE of P. ostreatus.These results indicated that the metal chelating activity of BE from P. ostreatus was related to β-glucan contents in the crude extract.However, the chelating activity of BE from P. ostreatus was not significantly different from V. volvacea (63.76 ± 0.51 mg EDTA eq./g extract) but significantly higher than those from P. sajor-caju and L. squarrosulus when the same extraction method was compared.These results indicated that alkaline extraction was the most suitable extraction method for β-glucan and metal chelating activity from P. ostreatus.The antioxidant activity of polysaccharide extracted from P. ostreatus was similar to recent research (Uddin et al., 2019;Barbosa et al., 2020a).The antioxidant activity of polysaccharide is mainly associated with monosaccharides components, chain conformation, molecular weight, and associations of proteins (Barbosa et al., 2020a).

Conclusions
All results in our research indicated that the extraction conditions and mushroom species clearly influenced β-glucan content, total phenolic compounds and antioxidant properties which led to different antioxidant effects.All of the 4 commercial mushrooms-P.ostreatus, P. sajor-caju, V. volvacea and L. squarrosulus -could be used as an alternative source of bioactive compounds and antioxidant from fungi.Among the 4 commercial mushrooms in this research, alkaline extraction was the most suitable extraction condition for β-glucan preparation from P. ostreatus (25.82 ± 3.87% w/w) and P. sajor-caju (23.08 ± 0.56% w/w).The highest antioxidant activity using ABTS and FRAP assay were found in the water extract of V. volvacea.The simple and easy extraction conditions with cost-effectiveness in this research could be applied to produce β-glucan and antioxidant additives for food and health products safe for consumers.
Among the extraction conditions, alkaline extraction was the most suitable for β-glucan extraction in all of the 4 edible mushrooms in this research.The mushroom extraction using alkaline treatment caused a degradation of the fiber structure and destruction of cell walls.The linkage of cell wall protein and glucan are hydrolyzed.This resulted in intracellular polysaccharides and the alkali-soluble fraction being released (Leong et al., Chanida Kupradit et al. 6/10 Asian J Agric & Biol.2023(1).
2021).However, the biological activity of β-glucan also varied with different molecular weight which was related to the extraction conditions and mushroom species.

Figure- 1
Figure-1.β-glucan concentration of the various crude extracts from different extraction conditions of the 4 edible mushrooms.Standard deviations are shown by the error bars.Bars with different capital letters determine significant difference between mushrooms in each extraction condition while different lower cases indicate significant difference between extraction conditions for each mushroom (p<0.05)

Figure- 2 .
Figure-2.Total phenolic concentration of the various crude extracts from different extraction conditions of the 4 edible mushroom.Standard deviations are shown by the error bars.Bars with different capital letters determine significant difference between mushrooms in each extraction condition while different lower cases indicate significant difference between extraction conditions for each mushroom (p<0.05)

Figure
Figure-3.ABTS assay of the various crude extracts from different extraction conditions of the 4 edible mushrooms.Standard deviations are shown by the error bars.Bars with different capital letters determine significant difference between mushrooms in each extraction condition while different lower cases indicate significant difference between extraction conditions for each mushroom (p<0.05)

Figure- 4 .
Figure-4.Ferric reducing antioxidant power (FRAP) of the various crude extracts from different extraction conditions of the 4 edible mushrooms.Standard deviations are shown by the error bars.Bars with different capital letters determine significant difference between mushrooms in each extraction condition while different lower cases indicate significant difference between extraction conditions for each mushroom (p<0.05)

Figure- 5 .
Figure-5.Metal chelating activity of the various crude extracts from different extraction conditions of the 4 edible mushrooms.Standard deviations are shown in the error bars.Bars with different capital letters determine significant difference between mushrooms in each extraction condition while different lower cases indicate significant difference between extraction conditions for each mushroom (p<0.05)

Table - 2. Yield of mushroom crude extracts by different extraction conditions. Extraction condition Yield of crude extract ( mg/g dry weight)* P. ostreatus P. sajor-caju V. volvacea L. squarrosulus
*Values are reported as means ± S.D. of duplicate measurements.Data with different letters in the column were significantly different between the same mushroom species (p<0.05).