Phytochemical, Free Radical Scavenging Activity and Thin Layer Chromatography Analysis of Methanolic Extracts of Six Wild Mushroom Species Collected From the Shai Hills Reserve of Ghana

Owusu, Schwinger, Dzomeku, Obodai, and Asante: Phytochemical, Free Radical Scavenging Activity and Thin Layer Chromatography Analysis of Methanolic Extracts of Six Wild Mushroom Species Collected From the Shai Hills Reserve of Ghana

Authors

INTRODUCTION

Mushrooms are low in calories and high in proteins, vitamins and minerals and they therefore serve as valuable, healthy foods.1 Currently, the consumption of wild edible mushroom is increasing as a result of their good source of protein and micronutrients.2 Apart from their healthy food source, mushrooms serve as therapeutic foods and are also useful in the management of such diseases as hypertension, hypercholesterolemia and cancer, diabetes, neurodegenerative disorders and cardiovascular disorders. Mushrooms are also good sources of antioxidants and are therefore possible protective agents for the reduction of oxidative damage in humans without any side effects.3 In recent times edible mushrooms have attracted interest as functional foods as well as source of bioactive metabolites for the development of drugs and nutraceuticals.4,5 Some have also been found to be a source of phenolic compounds,6 flavonoids, terpenoids, sterols, ascorbic acid, ergothioneine and carotenoids.3,4,5,6,7

Various methods are available for the screening of pharmacologically active substances in extracts. One such method is the thin layer chromatography (TLC), a simple, quick reliable and inexpensive procedure that can be used for screening of plant extracts.8 The TLC is also very useful for preliminary study before other instrumental techniques are applied.9,10 The use of TLC for separation and purification of plant constituents depends on their size, shape and charge. 11 It serves as a quick way for monitoring the identity and purity of drugs; it is also used for detecting adulterations and substitutions, drug combination analysis and phytochemical preparations.12,13

The aim of this study was to characterize mushroom species collected from Shai Hills Reserve in Ghana on the basis of their nutrient content, phytochemical constituents and antioxidant potentials.

MATERIALS AND METHODS

Sample collection

Mushroom samples were collected from the Shai Hills Reserve in Ghana in 2014. These were collected in brown large envelopes and identified using cultural and morphological characteristics.14,15 The samples were later sun-dried and kept in the freezer until ready for use.

Preparation of sample

Mushroom samples were prepared by following a modified approach.16 The mushrooms were lyophilized (Labconco, Missouri) and pulverized into fine powder. Ten grams of the pulverized samples was extracted with 100 ml of methanol at 25°C at 20 g for 24 hours and filtered through Whatman No. 1 filter paper. The residue was extracted with two additional 100 ml portions of methanol as described above and combined ethanolic extracts were concentrated under reduced pressure below 40°C to obtain the crude extract. The crude extracts were re-dissolved in methanol at concentration 20 mg/ml and stored at 4°C for further analyses.

Antioxidant Activity (DPPH Free Radical Scavenging Activity) of Methanolic Extract

The diluted working solutions of the test extracts were prepared in methanol. Accurately 100 μl of test samples (0.6-20.0 mg/ml) in methanol was added with 5 μl DPPH solution in 96-well microtitter plates. An amount of 0.002% DPPH was prepared in methanol. One microliter of this solution was mixed with 1 ml of sample solution and the standard solution to be tested separately. These solution mixtures were kept in the dark for 20 min and optical density was measured at 517 nm using a spectrophotometer against methanol. The blank was used as 1 ml of methanol with 1 ml of DPPH solution (0.002%). The optical density was recorded and percent of inhibition was calculated using the formula given below.

Percent inhibition of DPPH activity = (A−B/A) × 100 where A is optical density of the blank and B is optical density of the sample.

Statistics and IC50

Decolorization was plotted against the sample extract concentration and a linear regression curve was established in order to calculate IC50 (μg/ml), which is the amount of sample required to decrease the absorbance of the DPPH free radical by 50%. All the analyses were carried out in triplicate and the results expressed as mean ± SD. Statistical analyses were performed using SAS computer software

Determination of total phenol content

Total phenolic content in the methanolic extracts was determined by using Folin-Ciocalteau reagent based on modified version.17 Each sample (150 μl, 10 mg/ml) was added with 1200 μl distilled water and 450 μl aqueous sodium carbonate solution. One hundred microliters of Folin-Ciocalteu reagent was added to the mixture and agitated. The mixture was allowed to stand for 90 minutes and the absorbance was measured at 760 nm by using UV/visible spectrophotometer (SpectraMax Plus384, United States). The concentration of total phenolic compounds was calculated based on standard curve of gallic acid (0.2-1.0 mg/ml) with the linear equation, y = 0.624x − 0.939, where R2 = 0.995. The results were expressed as μg of gallic acid equivalent (GAE/ μg) per gram of the extracts.

Determination of total flavonoid content

The modified aluminum chloride colorimetric method18 was used to determine flavonoid content. Mushroom extract (100 μl, 10 mg/ml) was mixed with distilled 500 μ water and sodium nitrite, NaNO2 (5%, 30 μl). The mixture was allowed to stand for 5 minutes. Aluminium chloride solution, AlCl3.H2O (10%, 60 μl) was added to the mixture and left for 6 minutes. Sodium hydroxide, NaOH (1M, 200 μl) and 110 μl distilled water were added to the solution and mixed well. Absorbance of the solution was measured at 510 nm (SpectraMax Plus384, United States) and the concentration of total flavonoids content was calculated based on standard curve of rutin (0.2-1.0 mg/ml) with the linear equation y = 0.0101x + 0.2238, where R2 = 0.9563. The results were expressed as μg of rutin equivalent (RE/ μg) per gram of the extracts.

Determination of microelements and microelements

The micro and macronutrients were determined by an atomic absorption spectrophotometric (AAS) method. The samples were digested in nitric acid solution and passed through the AAS system using different lamps. Calibration was done with related minerals in different concentrations for different mineral elements.

Determination of crude protein

An amount of 0.1 grams of the pulverized mushroom sample was weighed into labelled conical flasks and 5 ml of concentrated H2SO4 was added and shaken gently. The solution was placed on a preheated sand digester and after heating for a while, drops of H2O2 was added. The solution was made to cool after which it was topped with distilled water to 100 ml mark of the volumetric flask. An amount of five milliliters of the solution was transferred into a round bottom flask, after which five milliliters of 2% boric acid solution was added. The solution was distilled in the presence of 5 ml of 40% NaOH. The distillate was against 0.1M HCl until a colour change was observed. The titre value was recorded. The procedure was repeated three times for each sample to minimize error. Percent nitrogen was calculated by using the following formula:

Nitrogen(%)=(ab)×0.01×14×V×1001000×W×aliquot pipetted

Where a represents titre value of sample, b, the titre value of the blank, V represents the extraction volume (100 ml) and W represents the weight of the sample (0.1 g). The crude protein content was estimated by multiplying the percent nitrogen value by the factor 6.25.

Thin layer chromatography (TLC)

Methanolic extracts of mushroom samples were separated on silica gel thin layer aluminium plates of 15x5 cm with 3 mm thickness. Extracts were spotted manually using capillary tube. Solvent systems used for the separation of the following phytochemical compounds alkaloids, flavonoids, saponins and terpenes were a mixture of chloroform and diethylamine (9 : 1), a mixture of chloroform and ethylacetate (6 : 4), a mixture of chloroform : methanol : distilled water : toluene (8 : 1 : 0.5 : 0.5) and toluene, respectively. After separation of the phytochemicals compounds, reagents such as iodine and Dragendorff were used to identify the compounds. Colour of the spots was noted and retention factor (Rf) values calculated by using the following formula:

Rf=Distance travelled by soluteDistance travelled by solvent×100

RESULTS AND DISCUSSION

Antioxidant potential

The extracts of Ganoderma sp showed the strongest activities for DPPH radicals and its IC50 value was 1.56 x x 10-4 μg/ml (Table 1). The extract for the unknown species gave the lowest activity. Moderate activity was recorded by the extracts of Mycena sp, Marasmus sp, Termitomyces sp and Amauroderma sp.

Radical scavenging activity of mushroom samples studied by Shirmila and Radhamany3 was 450 μg/ml. The antioxidant potential of the methanolic extracts of the mushroom samples in this study recorded higher values.

Total phenol and flavonoid content

The highest total phenol content of 17.53 mg gallic acid equivalent g was recorded for Mycena sp. While the lowest amount was realised from the extract of Amauroderma sp (Table 1). Total phenol content as reported in this work is higher than those reported by Shirmila and Radhamany3 who reported a total mean value of 1.901+0.011 mg GAE /g of extract.

The highest flavonoid content of 23.75 mg/g RUE was recorded by Termitomyces sp. whilst Amauroderma sp. gave the lowest amount (Table 1). Total flavonoid content reported in the current study is higher than the value reported by Shirmila and Radhamany3. They reported a value of 0.39 mg quercitin equivalent/g of extract. However the values for the current work were lower than those other researchers who reported 248+7.63 mg/g and 42.063 mg/g for total phenol and total flavonoid, respectively.19

Micronutrient contents of mushrooms

Amauroderma sp had the lowest content of cadmium (2.30 mg/L) while the highest content of 8.40 mg/L was recorded by the unknown sp. (Table 2). The most abundant micronutrient was found to be iron ranging from 1613.67 mg/L to 3731.00 mg/L. Iron values in mushroom samples as reported20 ranged from 568–3904 lg/g. The iron values of the present study fall within this range.

Lead was highest in extract of Amauroderma sp. while the lowest was recorded for Marasmus sp. Manganese was highest in Marasmus (222.00 mg/L) and lowest in Ganoderma sp (26.00 mg/L). Zinc was highest in the unknown sp. (81.90 mg/L) and lowest in Amauroderma sp. (36.97 mg/L). Zinc content of mushroom samples ranged from 33.5–89.5 lg/g.23 Others21,22 also reported values that ranged from 29.3–158 lg/g and 45–188 lg/g, respectively. There were significant differences (P < 0.001) among the six different mushroom species with respect to heavy metal content. The manganese content of the mushrooms studied in the present work ranged from 26 mg/L to 222.00 mg/L. However, others also reported values that ranged from 7.1-91.3 lg/g,22 and values that ranged from 21.7–74.3 lg/g.23

Macronutrients and percentage crude protein content of mushrooms

Marasmus sp. recorded the highest amount of calcium (900.00 mg/L) while Termitomyces sp. scored the lowest (127.33 mg/L) (Table 3), however other researchers recorded lower values of 0.17 to 8.80 mg/g dw1. Potassium content was highest in the unknown sp. (5633.33 mg/L) and lowest in Amauroderma sp. (1000.00 mg). These values were higher than those recorded by Huseyin, et al. (2009) which ranged from 12.6-29.1 mg/g dw. The highest amount of magnesium was recorded by the unknown sp. (754.33 mg/L) while the lowest was recorded by Termitomyces sp. (214.67 mg/L). The level of magnesium content reported in this work was higher than those of other researchers who reported values that ranged from 0.90 mg/g to 4.54 mg/g dw. Sodium content was highest in the unknown sp. (9600.00 mg/L).1

Table 1

Free radical scavenging activity, total phenolic and total flavonoid mean content of the six mushroom species.

Mushroom speciesDPPH scavenging activity IC50 (μg/mL) (x 10-4)Total Phenol (mg/g extract) in GAETotal flavonoid (mg/g extract) in RUE
Marasmus5.65339.3410011.23500
Unknown21.06679.0520012.81286
Amauroderma sp.7.52002.541335.46498
Mycena sp.4.686717.528009.33410
Termitomyces sp.5.600016.8080023.75048
Ganoderma sp.1.560012.3423317.87333
Mean7.6811.2687813.41179
Lsd(0.05)1.000.19590.0503
Table 2

Mean content of micronutrients in six different mushroom species from the Shai Hills

 CadmiumIronLeadManganeseZinc
Marasmus2.303731.0099.00222.0062.30
Unknown8.401816.67155.3361.3381.90
Amauroderma sp.8.003040.00422.33129.0036.97
Mycena sp.5.302207.33120.0070.0070.17
Termitomyces sp.7.331624.67141.6759.0038.87
Ganoderma sp.7.731613.67411.3326.0065.33
Mean6.512338.89224.9494.5659.26
Lsd(0.05)0.3621.9335.697.223.44
Table 3

Mean content of macronutrient and percentage crude protein in six different mushroom species from the Shai Hills

 CalciumPotassiumMagnesiumSodiumProtein
Marasmus900.332466.67612.676966.6720.54
Unknown813.005633.33754.339600.0021.24
Amauroderma sp.829.671000.00440.677433.3311.09
Mycena sp.389.674133.33632.679333.3328.24
Termitomyces sp.127.334900.00214.679133.3321.88
Ganoderma sp.134.333166.67439.008066.6726.89
Mean532.393550.00515.678422.2221.64
Lsd(0.05)79.4483.863.458139.071.01
Table 4

TLC fingerprint for alkaloids in methanolic extracts of six different mushroom species from the Shai Hills

FractionsRf valuesMarasmusUnknownAmauroderma sp.Mycena sp.Termitomyces sp. (nkankuma)Ganoderma sp.
 0.49---+-+
Table 5

TLC fingerprint for flavonoids in methanolic extracts of six different mushroom species from the Shai Hills

FractionsRf valuesMarasmusUnknownAmauroderma sp.Mycena sp.Termitomyces sp. (nkankuma)Ganoderma sp.
A0.20+-----
B0.21-----+
C0.22-+----
D0.23---+--
EF0.25----+-
G0.40--+---
H0.43-+----
I0.51----+-
J0.63-+--++
K0.70-----+
L0.72+-----
M0.73---+--
N0.76-----+
O0.87----+-
P0.88-+----
Q0.90-----+
R0.96----+-
S0.97-+----
Total number of bands251255

The highest percentage of crude protein was recorded by Mycena sp. (28.24 %) and the lowest was recorded by Amauroderma sp. (11.09 %). There were significant differences (P < 0.001) among the six mushroom species with reference to the major elements and protein contents (Table 3). In a proximate and mineral composition analysis of four edible mushrooms in Nigeria’ researchers reported 37% crude protein in Termitomyces mammiformis.24

TLC profile for alkaloid

Alkaloid was present in only Mycena sp and Ganoderma sp with a band each of Rf value of 0.49 (Table 4).

TLC profile for flavonoid

A total of 18 different bands were recorded with Rf values that ranged between 0.20 and 0.97 (Table 5). Two different bands were recorded by the Marasmus sp. with Rf values of 0.2 and 0.72, respectively. Five different bands were recorded by the unknown sp. with Rf values of 0.22, 0.43, 0.63 and 0.88, respectively. Amauroderma sp. recorded only one band with Rf value of 0.40. Mycena sp. recorded a total of two different bands of Rf values of 0.23 and 0.73, respectively. A total of five different bands were recorded by Termitomyces sp. with Rf values of 0.25, 0.51, 0.63, 0.97 and 0.97, respectively. The Ganoderma sp. also recorded a total of five different bands of Rf values of 0.21, 0.63, 0.70, 0.76 and 0.90, respectively. The band with Rf value of 0.63 was common to the unknown species, Termitomyces sp. and the Ganoderma sp.

Table 6

TLC fingerprint for saponins in methanolic extracts of six different mushroom species from the Shai Hills

FractionsRf valuesMarasmusUnknownAmauroderma sp.Mycena sp.Termitomyces sp. (nkankuma)Ganoderma sp.
A0.16-+----
B0.41-+----
C0.44----+-
D0.59+-----
E0.62-----+
F0.66+-----
G0.68---+-+
H0.69---+--
I0.71--+---
J0.73-+----
K0.78--+---
L0.79-+----
M0.88----+-
N0.94+--+-+
O0.99+----+
Total number of bands442324
Table 7

TLC fingerprint for terpenes in methanolic extracts of six different mushroom species from the Shai Hills

FractionsRf valuesMarasmusUnknownAmauroderma sp.Mycena sp.Termitomyces sp.Ganoderma sp.
A0.07-++--
B0.10+-----
C0.11---+--
D0.13-----+
E0.16----+-
F0.27-+----
G0.28----+-
H0.31-----+
I0.37--+---
J0.43-----+
K0.55----+-
L0.56-++---
M0.61+--+-+
Total number of bands233234

TLC profile for saponin

A total of 15 different bands were recorded with Rf values ranging from 0.16 to 0.99 (Table 6). Extracts of Marasmus sp. recorded a total of four different bands with Rf values of 0.59, 0.66, 0.94 and 0.99, respectively. A total of four different bands were recorded by extracts from the unknown sp. at Rf values of 0.16, 0.41, 0.73 and 0.79, respectively. Extracts of the Amauroderma sp. gave two bands with Rf values of 0.71 and 0.78, respectively. A total of three different bands were recorded by extracts from the Mycena sp. with Rf values of 0.68, 0.69 and 0.94, respectively. Extracts of the Termitomyces sp. gave a total of two different bands with Rf values of 0.44 and 0.88, respectively. The Ganoderma sp. extracts gave a total of four different bands with Rf values of 0.62, 0.68, 0.94 and 0.99, respectively. The band with Rf value of 0.68 was common to the Mycena and Ganoderma spp. The band with the Rf value of 0.84 was common to the Marasmus, Mycena and Ganoderma spp. Similarly the band with the Rf value of 0.99 was common to the Marasmus and Ganoderma spp.

TLC profile for terpene

A total of 13 different bands were scored with Rf values ranging between 0.07 and 0.61 (Table 7). Extracts from the Marasmus sp. recorded a total of 2 different bands with Rf values of 0.10 and 0.61, respectively. Extracts from the unknown sp. recorded a total of three different bands of Rf values of 0.07, 0.27 and 0.56, respectively. The Amauroderma sp. extracts gave a total of three different bands with Rf values of 0.07, 0.37 and 0.56, respectively. Extracts from the Mycena sp. gave a total of 2 bands with Rf values of 0.11 and 0.61, respectively. The extracts from Termitomyces sp recorded a total of three different bands with Rf values of 0.16, 0.28 and 0.55, respectively. The Ganoderma sp. extracts gave a total of four different bands with Rf values of 0.13, 0.31, 0.43 and 0.61, respectively. The bands with Rf value of 0.07 and 0.56 was common to the unknown and Amauroderma spp. Similarly, the band with Rf value 0.61 was common to the Mycena and Ganoderma sp.

TLC profiles of mushrooms is a new area of research in Ghana. The TLC revealed the presence of single alkaloid compound which was present in only Mycena. The highest number of flavonoid compounds was detected in extracts of Ganoderma sp, Termitomyces and the unknown sp. concerning saponin profile the highest number of compounds were present in extracts of Marasmus sp. Ganoderma and the unknown sp. The highest number of terpene compounds was detected in extracts of Ganoderma sp.

CONCLUSION

The antioxidant potential of the methanolic extracts of the mushroom samples in this study recorded higher values. This indicates that mushrooms have high antioxidant properties and rationalizes further investigation in the potential discovery of new natural bioactive principles from these mushrooms.

GRAPHICAL ABSTRACT

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SUMMARY

  • Phytochemical analyses of the methanolic extract of the mushrooms revealed the presence of antioxidants, phenols and flavonoids.

  • The highest number of flavonoid compounds was detected in extracts of Ganoderma sp, Termitomyces and the unknown sp.

  • There is the need for further investigation with the view of discovering new natural bioactive principles from these mushrooms.

  • TLC profiles of mushrooms is a new area of research in Ghana.

ACKNOWLEDGEMENT

The authors are grateful to the office of research and innovative division of the University of Ghana for providing funds for the research.

ABOUT AUTHORS

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Dr. Ebenezer Owusu is a Senior Lecturer at the Department of Plant and Environmental Biology at the University of Ghana. He is a member of many scientific associations and has publications in many peer-reviewed journals.

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Mrs. Gladys Schwinger was an Assistant Lecturer at the Department of Plant and Environmental Biology at the University of Ghana. She is currently pursuing a PhD degree at the Institute for Environmental and Sanitation Studies (IESS), University of Ghana.

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Matilda Dzomeku is a Research Scientist and the head of the mushroom laboratory of CSIR-Food Research Institute.

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Professor Mary Obodai is a Principal Research Scientist and the Director of CSIR-Food Research Institute. She is an Associate Professor at the CSIR college of Science and Technology.

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Professor I. K. Asante is professor and Head of the Department of Plant and Environmental Biology at the University of Ghana. He has published extensively in many peer-reviewed journals.

Notes

[1] Conflicts of interest CONFLICT OF INTEREST None

ABBREVIATIONS USED

AAS

Atomic absorption spectrophotometric

DPPH

1, 1-diphenyl-2-picrylhydrazyl

IC50

half maximal inhibitory concentration

GAE

Gallic acid equivalent

Rf

Representative Fraction

RUE

Rutin equivalent

TLC

Thin layer chromatography

REFERENCES

1. 

Huseyin G, Yusuf U, Yusuf T, Kenan D , authors. Determination of mineral contents of wild-grown edible mushrooms. Food Chemistry. 2009;113(4):1033–6

2. 

Racz L, Papp L, Prokai B, Kovacz Z , authors. Trace element determination in cultivated mushrooms: an investigation of manganese, nickel, and cadmium intake in cultivated mushrooms using ICP atomic emission. Microchemical Journal. 1996;54(4):444–51

3. 

Shirmila Jose G, Radhamany PM , authors. Identification and determination of antioxidant constituents of bioluminescent mushroom. Asian Pacific Journal of Tropical Biomedicine. 2012;386–S391

4. 

Terpinc P, Abramovic H , authors. A kinetic approach for evaluation of the antioxidant activity of selected phenolic acids. Food Chemistry. 2010;121(2):366–71

5. 

Orhan I, Üstün O , authors. Determination of total phenol content, antioxidant activity and acetylcholinesterase inhibition in selected mushrooms from Turkey. Journal of Food Composition and Analysis. 2011;24(3):386–90

6. 

Vaz JA, Barros L, Martins A, Santos-Buelga C, Vasconcelos, Ferreira ICFR , authors. Chemical composition of wild edible mushrooms and antioxidant properties of their water soluble polysaccharidic and ethanolic fractions. Food Chem. 2011;126(2):610–6

7. 

García-Lafuente A, Moro C, Villares A, Guillamón E, Rostagno MA, D’Arrigo M, Martínez JA , authors. Mushrooms as a source of anti-inflammatory agents. American Journal of Community Psychology. 2011;48(2):125–41

8. 

Nikolova M, Valyovska-Popova N, Dimitrova M, Peev D , authors. High-mountain Bulgarian plants – free radical scavenging activity and flavonoid composition Nikolova et al. J. BioSci. Biotech. 2014;(SE/ONLINE). 29–33

9. 

Mohammad A, Bhawani SA, Sharma S , authors. Analysis of herbal products by thin-layer chromatography: a review. Int. J. Pharma Bio Sci. 2010;1(2):1–50

10. 

Braz R, Wolf LG, Lopes GC, deMello JCP , authors. Quality control and TLC profile data on selected plant species commonly found in the Brazilian market. Rev. Bras. Farmacogn. 2012;22(5):1111–8

11. 

Helftmann F , author. Chromatography:- Fundamental and applications of chromatographic and Electrophoretic Techniques. 5th. Elsevier; Amsterdam: 1992. p. 520–2

12. 

Hahn-Deinstrop E , author. Applied thin layer chromatography best practice and avoidance of mistakes. Wilkey.VCH; Weinheim, Germany: 2000

13. 

Wagner H, Bladt S, Zgainski EM , authors. Plant drug analysis. Athin layer chromatography altlas. Springer-Verlag; Berlin Heidelberg New York Tokyo: 1984. 1. p. 309

14. 

Roger P , author. Mushrooms and other Fungi of Great Britain and Europe. Irish Book Center. (1st Ed). 1989;ISBN-10: 0330264419.

15. 

Pegler D, Spooner B , authors. The Mushroom Identifier. 1992. Published by The Apple Press. ISBN 1-85075-361-5.

16. 

Tsai SY, Huang SJ, Lo SH, Wu TP, Lian PY, Mau JL , authors. Flavour components and antioxidant properties of several cultivated mushrooms. Food Chemistry. 2009;13(2):578–84

17. 

Harborne JB , author. General procedures and measurement of total phenolics. Methods in plant biochemistry. 1989;1:1–28

18. 

Barros L, Ferreira MJ, Queiros B, Ferreira I, Baptista P , authors. Total phenols, ascorbic acid, betacarotene and lycopene in Portuguese wild edible mushrooms and their antioxidant activities. Food Chemistry. 2007;103(2):413–9

19. 

Hamzah RU, Jigam AA, Makun HM, Egwim EC , authors. Phytochemical screening and antioxidant activity of methanolic extract of selected wild edible Nigerian mushrooms. Asian Pac J Trop Dis. 2014;4:153–7

20. 

Turkekul I, Elmastas M, Tuzen M , authors. Determination of iron, copper, manganese, zinc, lead, and cadmium in mushroom samples from Tokat, Turkey. Food Chemistry. 2004;84(3):389–92

21. 

Isılog˘lu M, Yilmaz F, Merdivan M , authors. Concentrations of trace elements in wild edible mushrooms. Food Chemistry. 2001;73(2):163–75

22. 

Tuzen M , author. Determination of heavy metals in soil, mushroom and plant samples by atomic absorption spectrometry. Micro chemical Journal. 2000;74(3):289–97

23. 

Soylak M, Saracoglu S, Tuzen M, Mendil D , authors. Determination of trace metals in mushroom samples from Kayseri, Turkey. Food Chemistry. 2005;92(4):649–52

24. 

Adejumo TO, Awosanya OB , authors. Proximate and mineral composition of four edible mushroom species from South Western Nigeria African. Journal of Biotechnology. 2005;10(10):1084–8