Bulletin of the Chemists and Technologists 2015

of Bosnia and Herzegovina

44
Print ISSN: 0367-4444 UDC: __________________________
Original Scientific Article
5-8
Online ISSN: 2232-7266

Phenolic content and antioxidant activity of mushroom extracts

from Bosnian market

Alispahić, A., Šapčanin, A.*, Salihović, M., Ramić, E., Dedić, A., Pazalja, M.
Faculty of Pharmacy, University of Sarajevo, Zmaja od Bosne 8 - Kampus, Sarajevo, Bosnia and Herzegovina

Article info Abstract: Mushrooms are well balanced food that provides definite nutrition and health benefits
Received: 6/5/2015
Accepted: 25/5/2015 for humans. Mushrooms are known to produce different kinds of bioactive compounds, generally
linked with mycelial cell wall, that help in enhancing the capacity of immune system to fight
Keywords: against carcinogens. To consider the importance of polyphenolic compounds and its presence in
mushrooms, antioxidant activity,
monomeric anthocyanine content, many varieties of mushrooms, the total antioxidant activity of dry boletus mushroom, white and
total phenolic content brown champignon, oyster mushroom and shiitake from bosnian markets was determined. Total
phenolic content was estimated as Galic acid equivalents /g spectrophotometrically according to
the Folin-Ciocalteus method. Total anthocyanine content was analysed by pH differential
spectrophotometric method at 525 and 700 nm. The radical scavenging activity (RSA) of
mushroom extracts was determined by 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay. The analysis
revealed that the total phenolic contents ranged from 4.94 mg GAEg-1 in oyster mushroom to
35.56 mg GAEg-1 in dry boletus mushroom. DPPH scavenging activity was the highest for brown
*Corresponding author:
E-mail: ida@bih.net.ba champignon with value of 88.33 % and the lowest one was for oyster mushrooms with value of
Phone: 00-387-33-586-187
Fax: 00-000-00-0000000 43.88 %. The mushrooms examined in the present study could represent easily accessible sources
of natural antioxidants.

INTRODUCTION

Mushrooms have been a part of human diet in many Phenolic acids were the major phenolic compounds
regions of the world for centuries due to organoleptic reported in mushrooms.
characteristic as well as the nutritional values (Wang and The antioxidant activity of anthocyanins including the
Xu, 2014). In nature, there are over 150 000 different protection of low density lipoproteins (LDL) against
types of mushrooms but only 10% is known and oxidation, has been demonstrated in a number of different
designated (Wasser, 2010). However, only about 2 000 in vitro systems. Phenols are important plant constituents
species are grown and cultivated for nutritional purposes. because of their scavenging ability due to their hydroxyl
The consumption of the mushrooms has even increased groups (Hatano et al., 1989). In this study the radical
remarkably over the past few decades (Gan et al., 2013). scavenging activity (RSA) as well as the polyphenolic
Mushrooms are tasteful food, full of proteins, rich in content and anthocyanins content of five edible
vitamin B, rich in different minerals and have almost all mushrooms (Boletus edulis, Agaricus bisporus, Agaricus
essential amino acids (Mujić et al., 2011). Examination of bisporus var. Avellaneous, Pleurotus ostreatus, Lentinula
antioxidant activity in mushroom extracts and content of edodes) commercially available at Bosnian market was
antioxidant compounds is currently very interesting aim investigated. The amount of total phenol content was
of research.. Mushrooms are found to be rich source of determined by Folin-Ciocalteu reagent method and total
these antioxidants with immense antiradical activity monomeric anthocyanin content was determined by pH
(Valentão et al., 2005). differential spectrophotometric method.
6 Alispahić et al.

MATERIAL AND METHODS temperature using distilled water as blank. A Spektronic

Genesiys TM2 UV-Vis spectrophotometer was used for
Plant material determination. The content of total monomeric
Mushroom samples of boletus mushroom (Boletus anthocyanins was expressed in mg of cyanidin-3-
edulis), champignon white (Agaricus bisporus), glucoside equivalents (CGE) per gram of mushrooms. A
champignon brown (Agaricus bisporus var. Avellaneus), molar extinction coefficient of cyanidin-3-glucoside of
oyster mushroom (Pleurotus ostreatus), shiitake 26900 Lmol-1cm-1 and molar weight (MW) (449.2 gmol-1)
(Lentinula edodes) were collected in Bosnian market. were used for calculations. The anthocyanin
Identification was done by comparing their concentration was calculated according to the following
morphological, anatomical and physiological equation:
characteristics and monographs with descriptions given in A x MW x Df x 1000
mg CGE/g =
the manual (Moser, 1983; Bessette et al., 2000; Uzelac, 𝜀𝜀 x 𝑙𝑙
2009). The investigated mushrooms are five of the most where:
commercially cultivated ones available in Bosnia. 𝐴𝐴 = (𝐴𝐴525 – 𝐴𝐴700 )𝑝𝑝𝑝𝑝 1.0– (A525–A700)𝑝𝑝𝑝𝑝 4.5

Chemicals and reagents MW- molecular weight of cyaniding-3-glucoside

Folin-Ciocalteu reagent (Kemika, Zagreb, Croatia), gallic Df- dilution factor,
acid (Fluka Chemica, Switzerland), anhydrous sodium ε - molar absorbance
carbonate (Kemika, Zagreb, Croatia) and methanol l - pathlength
(Merck, Darmstadt, Germany), 1,1-diphenyl-2-
picrylhydrazyl (DPPH) (Sigma Aldrich, St. Louis, USA) DPPH Radical Scavenging Activity Assay
and (Trolox) 6-hydroxy-2,5,7,8-tetramethylchroman-2- The mushroom extracts were mixed with methanol (96
carboxilic acid (Sigma Aldrich, St. Louis, USA), %) and 63 μmol/L solution of DPPH. After 30 min. at
hydrohloric acid (Sigma Aldrich, St. Louis, USA), room temperature, the apsorbance was measured at 517
potassium chloride (Fluka Chemica, Switzerland), acetic nm and converted into percentage of radical scavenging
acid (Fluka Chemica, Switzerland), sodium acetate activity (Zeković et al., 2010). The comparative analysis
(Sigma Aldrich, St. Louis, USA) cyanidine-3-galactoside of samples was made by calculating DPPH scavenging
(Fluka Chemica, Switzerland) were used in this study. All activity which stands for the relative decrease of
the chemicals were of analytical grade purity. absorbance in the samples analysed. DPPH scavenging
activity was calculated by using the following equation:
Sample preparation
The fresh mushrooms 0.5 g were cut into small pieces, DPPH 𝑠𝑠cavenging activity = 100 × (𝐴𝐴𝑐𝑐 – 𝐴𝐴𝑠𝑠)/𝐴𝐴𝑐𝑐
crushed in a mortar with pestle and consecutively
extracted with 10 mL of 80 % ethanol. After maceration, where:
extracts were put in centrifuge (Tehnica Železniki LC- Ac - absorbance of the control
320) at 4000 rpm for 20 min. and then the supernatant As- absorbance of the sample.
was separated. These obtained extracts were used for
further investigations.Obtained extracts were stored in a RESULTS AND DISCUSSION
refrigerator at 4oC until analysis.
Results obtained for total phenolics, total monomeric
Determination of total phenolic content anthocyanins and DPPH scavenging activity are
Total phenols (TP) were determined presented in Table 1.
spectrophotometrically with Folin-Ciocalteu reagent
(Waterhouse, 2002). The sample (2 mL) was dissolved in Table 1: Total phenolic content, total monomeric anthocyanins
content and DPPH scavenging activity of dry boletus, champignon
ethanol and mixed with 10 mL Folin-Ciocalteau’s white, champignon brown, oyster and shiitake mushrooms
reagent diluted 1/10 with distilled water. After few TP TMA
minutes sodium carbonate (8 mL) was added to this Name %RSA
(mg GAE /g) (mg CGE/g)
solution. This solution was stored in dark place for two
hours and after that, the absorbance was measured at 765 dry boletus
nm. A standard curve was prepared using gallic acid as mushroom 35.56 - 87.74
(Boletus edulis)
standard with a concentration range from 100 to 500
μg/mL. Results are expressed in mg of gallic acid champignon white
equivalents per gram (mg GAE g-1) of mushrooms. 6.43 0.134 87.77
(Agaricus bisporus)

Determination of total monomeric anthocyanins champignon brown

(Agaricus bisporus 7.66 - 88.33
Total monomeric anthocyanins (TMA) content was
var. Avellaneus)
quantified using a pH differential method (Giusti and
Wrolstad, 2001). Samples were diluted in two buffer oyster mushroom
6.27 - 43.88
solutions: potassium chloride buffer 0.025 M (pH 1.0) (Pleurotus ostreatus)
and sodium acetate buffer 0.4 M (pH 4.5) and then the
shiitake
absorbance was measured simultaneously at 525 nm and 4.94 0.134 71.85
(Lentinula edodes)
700 nm, after 15 minutes of incubation at room TP- Total phenols; TMA - Total monomeric anthocyanins; RSA -
temperature. Absorbance readings were made at room Radical Scavenging Activity
Bulletin of the Chemists and Technologists of Bosnia and Herzegovina 2015, 44, 5-8 7

It can be seen that total phenolics content ranged from Total monomeric anthocyanins in investigated extracts
4.94 mg GAE g-1 to 7.66 mg GAE g-1 (fresh weight) and were detected in champignon white and shiitake in an
35.56 mg GAE g-1 (dry weight). According to review amount of 0.134 mg CGE g-1 of fresh sample. Research
given by Mujić et al. (2011), content of total phenolics in resaults related to anthocyanin content in mushrooms are
mushrooms obtained in the range 7.8-23.07 mg GAE g-1. scarces.
29.49 to 32.21 mg GAE g-1 was determined by different Antioxidant activity of mushroom extracts investigated
investigators (Yildirim et al. 2012). On the other hand, it by DPPH method are presented in Table 1. The
is difficult to compare our results with finding of other antioxidant activity of brown champignon extract
authors due to differences in extraction method applied, exhibited a significant inhibition of DPPH with 88.33
mode of expression of results (on dry or fresh basis of %RSA and the lowest value determinated for oyster
mushrooms), etc. For instance, Yildirim et al. (2012) used mushrooms was 43.88 %RSA. Dry boletus, white and
methanol to extract bioactive compounds from dry brown champignons showed a higher capacity of
mushrooms. Ejelonu et al. (2013) extracted bioactive scavenging the DPPH radical than the extracts of oyster
compounds from dry mushrooms with distilled water and mushrooms and shiitake. These results can be considered
obtained results in the range from 103.34 mg GAE g-1 to as high antioxidant capacity, in the comparison with
123.35 mg GAE g-1. Furthermore, concentration of total antioxidant capacity from various extracts of edible
phenols in medicinal mushrooms was between 4.45 mg mushroom (Pleurotus eous) (Sudha et al., 2012).
GAE g-1 to 14.44 mg GAE g-1 (Abugri and McElhenney, Correlation between specific classes of bioactive
2013). Our results showed that the highest value of 7.66 compounds and antioxidant activity was also investigated.
mg GAE g-1 was deteminated for fresh champignon Obtained results are presented in Figure 1.
brown and the highest value of 35.56 mg GAE g-1 for dry
boletus mushroom.

90
80
70
60 Total phenols (mg GAE/g)

50
Total monomeris anthocyanins (mg
%

40 CGE/g)
30 DPPH scavenging activity (%RSA)
20
10
0
dry boletus champignon champignon oyster shiitake
mushroom white brown mushroom

Figure 1. Correlation between DPPH scavenging activity, total phenolic content and total anthocyanin monomeric content

Maximum value of 88.33 %RSA was determined for rich antioxidant contents make the mushroom ideal
champignon brown, and minimum value of 43.88 %RSA nutritional supplement. Considering that mushrooms are
was determined for oyster mushroom, and was about of yellow, white, brown and dark hue, researchers have
50% lower than those of champignons brown, and it was found that they are a good source of anthocyanins.
not in correlation with total phenolic content of Further studies are needed to identify which phenolic
investigated mushrooms. compounds are responsible for the antioxidant activity of
the species, and assess the way in which the phenolic
substances contribute to this activity. Additional in vivo
CONCLUSIONS antioxidant assays are necessary to confirm the potential
use of these species in the treatment of different
The results of this study indicate that examined deseases. So we can conclude that those mushrooms
mushroom extracts possess good antioxidant activity. In represent a rich source of phenolic compounds and
all examined samples phenolic compounds have been thereby might serve as possible nutraceutical food in
detected, but monomeric anthocyanin compounds were human diet, and could help in the reducing the oxidative
detected only in champignon white and shiitake. Due to damage.
their high content of antioxidants, extracts of some
mushrooms, especially champignon white and boletus,
may be used as materials of dietary supplements. Their
8 Alispahić et al.

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Summary/Sažetak
Gljive su dobro izbalansirana hrana koja pruža određene prehrambene i zdravstvene pogodnosti za čovjeka. Gljive
proizvode mnoge vrste bioaktivnih spojeva, uglavnom povezanih sa micelama ćelijskog zida, koji pomažu u jačanju
sposobnosti imunološkog sistema da se bori protiv kancerogenih tvari. Da bi se razmotrila važnost polifenolnih supstanci i
njihovo prisustvo u različitim vrstama gljiva, određena je ukupna antioksidativna aktivnost suhog vrgnja, bijelih i smeđih
šampinjona i šitaki gljiva sa bosanskog tržišta. Sadržaj ukupnih fenola je izražen kao ekvivalent galne kiseline /g
spektrofotometrijski metodom po Folin-Ciocalteu-u. Sadržaj ukupnih antocijanina je analiziran spektrofotometrijski pH-
diferencijalnom metodom na valnim dužinama 525 i 700 nm. Antiradikalna aktivnost (RSA) ekstrakata gljiva je određena
DPPH metodom. Analize su pokazale da se sadržaj ukupnih fenola kreće u rasponu od 4.94 mg GAE/g u bukovačama do
35.56 mg GAE/g u uzorku suhog vrgnja. Procenat inhibicije je bio najveći za smeđe šampinjone 88.33 %, a najmanji za
bukovače 43.88 %. Gljive ispitane u ovoj studij predstavljaju lako pristupačan izvor antioksidanata.