http://informahealthcare.com/phb ISSN 1388-0209 print/ISSN 1744-5116 online Editor-in-Chief: John M. Pezzuto Pharm Biol, 2015; 53(6): 824–830 ! 2015 Informa Healthcare USA, Inc. DOI: 10.3109/13880209.2014.943245
ORIGINAL ARTICLE
Antioxidant and anticholinesterase activities of five wild mushroom species with total bioactive contents Gulsen Tel1, Mehmet Ozturk1, Mehmet E. Duru1, and Aziz Turkoglu2 Department of Chemistry, and 2Department of Biology, Faculty of Sciences, Mug˘la Sıtkı Koc¸man University, Mug˘la, Turkey
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1
Abstract
Keywords
Context: Recently, mushrooms are interesting natural products to be investigated due to exhibiting various bioactivities. Objective: This study determines the antioxidant and anticholinesterase activities of various extracts of five wild mushroom species. In addition, the total bioactive contents, namely, ascorbic acid, b-carotene, and lycopene along with phenolic and flavonoid contents were also determined spectrophotometrically. Materials and methods: Antioxidant activity was tested by using five complementary tests; namely, b-carotene-linoleic acid, DPPH scavenging, ABTS+ scavenging, cupric-reducing antioxidant capacity (CUPRAC), and metal chelating assays. The in vitro anticholinesterase activity was tested against acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) enzymes using the Ellman method. The spectrophotometric methods were used to determine the total phenolic, flavonoid, ascorbic acid, b-carotene, and lycopene contents. Results: The current study has shown that ethyl acetate extracts of Ganoderma lucidum (Curtis) P. Karst (IC50: 1.55 ± 0.05 mg/mL) and Funalia trogii (Berk.) Bondartsev & Singer (IC50: 4.31 ± 0.18 mg/mL) exhibited good lipid peroxidation inhibitory activity. The DPPH, ABTS, and CUPRAC assays supported this activity. The ethyl acetate and methanol extracts of Funalia trogii and Ganoderma lucidum indicated good anticholinesterase activity. Ganoderma lucidum had rich phenolic and flavonoid contents, indicating 98.67 ± 0.32 mg PEs/g extract and 160.38 ± 1.25 mg QEs/g extract, respectively. Discussion and conclusion: The results demonstrate that some of the mushroom species tested herein could be used in food and pharmaceutical industries as natural antioxidants.
Funalia trogii, Ganoderma lucidum, Gyromitra esculenta, Lyophyllum decastes, Pleurotus ostreotus
Introduction All over the world, mushrooms are used as valuable healthy foods since they are low in calories, fat, and essential fatty acids whereas they are rich in proteins, vitamins, and minerals (Pereira et al., 2012; Reis et al., 2012). Evaluation of studies in the literature on mushroom species indicate that polysaccharides, b-glucans, polysaccharide-protein complexes, lanostentype triterpenoids, cyathane-type diterpenoids, steroids, alkaloids, and phenolic compounds were isolated from mushroom species previously. These isolated compounds exhibited anticancer, antioxidant, antitumor, antiviral, antibacterial, antifungal, anti-inflammatory, and immunomodulator activities (Moradali et al., 2007; Tong et al., 2009; Tu¨rkog˘lu et al., 2007). Funalia trogii (Berk.) Bondartsev & Singer is a nonpoisonous inedible mushroom species that grows on trees.
Correspondence: Mehmet E. Duru, Department of Chemistry, Faculty of Sciences, Mug˘la Sıtkı Koc¸man University, 48121 Mug˘la, Turkey. Tel: +90 252 2111494. Fax: +90 252 2111472. E-mail: [email protected], [email protected]
History Received 1 April 2014 Revised 9 June 2014 Accepted 7 July 2014 Published online 27 February 2015
So far a few studies on F. trogii have been reported. The antioxidant activity of methanol and water extracts has been studied using DPPH radical scavenging, metal chelating, and lipid peroxidation inhibitory assays (Yegenoglu et al., 2011). Ganoderma lucidum (Curtis) P. Karst has been used as a medicinal mushroom in traditional Chinese medicine for more than 2000 years. There are many reports on G. lucidum. In previous studies, several biological activities such as antioxidant (Yegenoglu et al., 2011), anticancer (Trajkovic et al., 2009), antitumor (Joseph et al., 2011; Liu et al., 2012), immunomodulatory (Wang et al., 2013), and anti-inflammatory properties (Joseph et al., 2011) of G. lucidum were investigated. Although, potentially fatal if eaten raw, Gyromitra esculenta (Pers. ex Pers.) Fr. is a popular delicacy in Scandinavia, Eastern Europe, and some regions of North America. There are some studies performed on G. esculenta mushroom (Arshadi et al., 2006; Leal et al., 2013). Lyophyllum decastes (Fries: Fries) Singer, commonly known as the fried chicken mushroom, is an edible species in the Lyophyllaceae family. In previous studies, several biological activities of L. decastes such as antitumor (Ukawa et al., 2000), hypocholesterolemic
Bioactivities of five wild mushroom species
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DOI: 10.3109/13880209.2014.943245
(Ukawa et al., 2001), and antidiabetic (Miura et al., 2002) properties were investigated. Pleurotus ostreotus (Jacq. ex Fr.) P.Kumm., the oyster mushroom, is another common edible mushroom. Antioxidant (Yim et al., 2010) and antimicrobial activities (Vamanu, 2013) of P. ostreotus were studied. The antioxidant activity by using ABTS radical scavenging and cupric-reducing antioxidant capacity (CUPRAC) assays of F. trogii and antioxidant activity of G. esculenta were studied for the first time in this study. In addition, the anticholinesterase activity of all studied mushroom species was investigated for the first time. Some diseases, such as atherosclerosis, diabetes, cancer, and cirrhosis have been related with oxidative damage caused by oxygen-centered free radicals. In fact, oxidation is necessary for many living organisms (Halliwell & Gutteridge, 1984). Nowadays, especially in food and pharmaceutical industries, synthetic antioxidants have been used against free radicals due to their easy accessibility (Ding et al., 2010). To the best of our knowledge, since synthetic antioxidants are suspected of being responsible for liver damage and carcinogenesis, some of them were restricted to be used in the food industry by some countries (Grice, 1988). After that, some of the antioxidants occurring naturally have received much attention as safe antioxidants (Scalbert et al., 2005). In contrast, it is also reported that oxygencentered free radicals contribute to cellular ageing and neuronal damage (Sastre et al., 2000). According to Soholm (1998), an excess amount of free radical species causing oxidative stress is also associated with the pathology of many diseases including Alzheimer’s disease. Supportively, the lack of vitamin E, a natural antioxidant, enhanced Alzheimer’s disease on a mouse model (Nishida et al., 2006). Thus, the development and utilization of more effective antioxidants of natural origin as well as anticholinesterase compounds are desired. Regarding the consumption of G. lucidum, G. esculenta, L. decastes, and P. ostreotus, and the usage of F. trogii, collected from U¸sak-Turkey, we aimed to investigate the antioxidant and anticholinesterase activities, and the total bioactive compounds of these species. The main goal of the study is to compare the results with each other and those in the literature.
Materials and methods Mushroom materials The species names, collection localities and dates, family, and edibility of five mushroom species were listed in Table 1. All species were identified by Dr. Aziz Tu¨rkog˘lu. Voucher specimen have been deposited in the
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Fungarium of Department of Biology, Mug˘la Sıtkı Koc¸man University. Spectral measurements and chemicals Bioactivity measurements were carried out on a 96-well microplate reader, SpectraMax 340PC384 (Molecular Devices, Silicon Valley, CA). The measurements and calculations of the activity results were evaluated by using Softmax PRO v5.2 software (Molecular Devices, Silicon Valley, CA). Methanol, n-hexane, pyrocatechol, quercetin ethanol, ferrous chloride, metaphosphoric acid, copper (II) chloride, ascorbic acid, 2,6-dichlorophenolindophenol, and ethylenediaminetetraacetic acid (EDTA) were purchased from E. Merck (Darmstadt, Germany). Polyoxyethylene sorbitan monopalmitate (Tween-40), Folin–Ciocalteu’s reagent (FCR), butylatedhydroxyl anisole (BHA), neocuproine, b-carotene, linoleic acid, a-tocopherol, 1,1-diphenyl-2-picryl-hydrazyl (DPPH), 2,20 -azinobis(3-ethylbenzothiazoline-6-sulfonic acid) salt 3(2-pyridyl)-5,6-di(2-furyl)-1,2,4-triazine-50 ,500 -disulfonic acid disodium salt (Ferene), acetylcholinesterase (AChE) from electric eel (Type-VI-S, EC 3.1.1.7, 425.84 U/mg, Sigma, St. Louis, MO), butyrylcholinesterase (BChE) from horse serum (EC 3.1.1.8, 11.4 U/mg, Sigma, St. Louis, MO), 5,50 -dithiobis (2-nitrobenzoic) acid (DTNB), galantamine, acetylthiocholine iodide, and butyrylthiocholine chloride were purchased from Sigma Chemical Co. (Sigma-Aldrich GmbH, Sternheim, Germany). All other chemicals and solvents were of analytical grade. Extraction Each mushroom species was extracted separately with 2.5 L hexane, ethyl acetate, and methanol (4 24 h), successively, at room temperature (25 C), filtered, and evaporated to dryness in vacuo. The crude extracts were stored in a refrigerator until required. Determination of total bioactive compounds The phenolic content in methanolic extracts of five wild mushroom species was determined with FCR according to the method of Slinkard and Singleton (1977) as described in the literature and expressed as micrograms of pyrocatechol equivalents per g extract (PEs). The following standard curve which was obtained by pyrocatechol standard was used to calculate total phenolic contents: y ¼ 0:0073½PEs0:1665 r 2 : 0:9980 The aluminum nitrate method was used to determine the ¨ ztu¨rk et al., 2011) flavonoid content in methanolic extracts (O
Table 1. Collection localities and dates, family and edibility of the mushroom species studied. No. 1 2 3 4 5
Mushroom
Collection localities and dates
Family
Edibility
Funalia trogii (Berk.) Bondartsev & Singer Ganoderma lucidum (Curtis) P. Karst Gyromitra esculenta (Pers. ex Pers.) Fr. Lyophyllum decastes (Fries: Fries) Singer Pleurotus ostreotus (Jacq. ex Fr.) P.Kumm.
U¸sak-E¸sme, September 2009 U¸sak-E¸sme, September 2009 U¸sak-Banaz, May 2009 U¸sak-Banaz, November 2008 U¸sak-Banaz, May 2008
Polyporaceae Ganodermataceae Discinaceae Lyophyllaceae Pleurotaceae
Not edible Not edible/consumed as tea Edible after cooking Edible Edible
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G. Tel et al.
Pharm Biol, 2015; 53(6): 824–830
and the results were expressed as quercetin equivalents per g extract (QEs). The following standard curve obtained by quercetin standard was used to calculate total flavonoid contents: y ¼ 0:0074½QEs þ 0:0133 r 2 : 0:9990 Ascorbic acid content in methanolic extracts was determined as ascorbic acid equivalents per g extract (AAEs). The method was based on the extraction of the sample with metaphosphoric acid and then the reaction with 2,6dichlorophenolindophenol (Barros et al., 2008). The following standard curve, which was obtained by authentic ascorbic acid, was used to calculate the ascorbic acid content:
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y ¼ 0:0537½AAEs0:9581 r 2 : 0:9988 b-Carotene and lycopene contents in methanol extracts were determined according to the method of Barros et al. (2008). The method was based on the measurement of the absorbance of methanol extract, solved in acetone–hexane mixture (6:4, v/v), at 453 (A453), 505 (A505), and 663 (A663) nm wavelengths. Contents of b-carotene and lycopene were calculated according to the following equations: Lycopene ðmg=100mLÞ ¼ 0:0458 A663 þ 0:372 A505 0:0806 A453 ; b-Carotene ðmg=100mLÞ ¼ 0:216 A663 0:304 A505 þ 0:452 A453 :
¨ ztu¨rk et al., 2011). BHA and with slight modifications (O -tocopherol were used as antioxidant standards. Results were given as A0.50, (mg/mL) that corresponds to the concentration providing 0.500 absorbance. Metal-chelating activity The metal-chelating activity of the extracts on Fe2+ was measured spectrophotometrically (Decker & Welch, 1990). Ethanol was used as a control, and EDTA was used as a chelating standard for comparison of the activity. The results were given as IC50 mg/mL. Determination of anticholinesterase activity AChE and BChE inhibitory activities were measured by slightly modifying the spectrophotometric method developed by Ellman et al. (1961). AChE from electric eel (5.32 103 U in 20 mL phosphate buffer for each well) and BChE from horse serum (6.85 103 U in 20 mL phosphate buffer for each well) were used as enzymes, while acetylthiocholine iodide and butyrylthiocholine chloride were employed as substrates of the reaction. DTNB [5,50 -dithio-bis(2-nitrobenzoic) acid] was used for the measurement of the cholinesterase activity. All conditions ¨ ztu¨rk et al., 2011). were described in our earlier publication (O Galantamine which is a standard drug for the treatment of mild Alzheimer’s disease was used as a reference compound. The results were given as percentage inhibition against 25, 50, 100, and 200 mg/mL concentrations, the IC50 was also given.
Determination of antioxidant activity -Carotene/linoleic acid assay The lipid peroxidation inhibitory activity was evaluated using b-carotene/linoleic acid assay as previously described ¨ ztu¨rk et al., 2011). Ethanol was used as a (Marco, 1968; O control while BHA and -tocopherol were used as antioxidant standards. The results were given as 50% inhibition concentration, mg/mL (IC50).
Statistical analysis All data on antioxidant and anticholinesterase activities as well as bioactive contents were the averages of triplicate analyses. Data were recorded as mean ± S.E.M. Significant differences between means were determined by Student’s ttest, p values50.05 were regarded as significant.
Results and discussions
DPPH free radical scavenging assay
Total bioactive compounds
The free radical scavenging activity was determined spectrophotometrically by the DPPH assay described by ¨ ztu¨rk et al., 2011). Blois (1958) with a slight modification (O Ethanol was used as a control while BHA and -tocopherol were used as antioxidant standards. The results were given as IC50 mg/mL.
The ABTS+ scavenging activity was determined according to the method of Re et al. (1999) with slight modifications ¨ ztu¨rk et al., 2011). Ethanol was used as a control while (O BHA and -tocopherol were used as antioxidant standards. The results were given as IC50 mg/mL.
Table 2 presents total phenols and flavonoids, ascorbic acid, and carotenoids concentrations in the extracts obtained from the mushroom species. Total phenolic and flavonoid contents of the extracts of mushroom species determined as pyrocatechol equivalents (PEs) and quercetin equivalents (QEs), respectively. The G. lucidum afforded rich phenolic content, exhibiting 98.67 ± 0.32 mg PEs/g extract. The most flavonoid rich mushroom was found to be G. lucidum (160.38 ± 1.25 mg QEs/g extract), while G. esculanta (7.64 ± 0.14 mg QEs/g extract) was the poorest (Table 2). Ascorbic acid was found to be between 2.11 and 5.60 mg/g extract in the all mushroom species. b-Carotene and lycopene were found to be between 0.07–0.43 and 0.03–0.67 mg/g extract, respectively (Table 2).
Cupric reducing antioxidant capacity
Antioxidant activity
The cupric reducing antioxidant capacity (CUPRAC) was determined according to the method of Apak et al. (2004)
Table 3 shows the antioxidant activity of the extracts of mushroom species. b-Carotene-linoleic acid, DPPH scavenging,
ABTS cation radical decolorization assay
Bioactivities of five wild mushroom species
DOI: 10.3109/13880209.2014.943245
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Table 2. Total bioactive contents of methanol extract of five wild mushroom speciesa.
Mushroom species Funalia trogii Ganoderma lucidum Gyromitra esculenta Lyophyllum decastes Pleurotus ostreotus
Total phenolic content (mg/g)
Total flavonoid content (mg/g)
Ascorbic acid (mg/g)
b-Carotene (mg/g)
Lycopene (mg/g)
78.03 ± 1.02 98.67 ± 0.32 28.03 ± 0.25 7.76 ± 0.05 7.16 ± 0.08
33.14 ± 0.05 160.38 ± 1.25 7.64 ± 0.14 36.95 ± 0.74 14.18 ± 0.48
4.33 ± 0.01 5.60 ± 0.40 2.86 ± 0.38 4.77 ± 0.41 2.11 ± 0.48
0.43 ± 0.02 nd 0.15 ± 0.01 0.07 ± 0.03 0.11 ± 0.00
0.23 ± 0.04 0.67 ± 0.02 0.07 ± 0.01 0.03 ± 0.00 0.05 ± 0.00
a
Values expressed are means ± S.D. of three parallel measurements (p50.05). nd: not detected.
Table 3. Antioxidant activity of the extracts of five wild mushroom species by the b-carotene-linoleic acid, DPPH, ABTS+, CUPRAC, and metal chelating assaysa.
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Antioxidant activity
Mushroom species Funalia trogii Ganoderma lucidum Gyromitra esculenta Lyophyllum decastes Pleurotus ostreotus Standards
Extracts
b-Carotene-linoleic acid assay IC50 (mg mL1)
DPPH assay IC50 (mg mL1)
ABTS+ assay IC50 (mg mL1)
CUPRAC assay A0.50 (mg mL1)
Metal chelating assay IC50 (mg mL1)
Hexane (FTH) Ethyl acetate (FTEA) Methanol (FTM) Hexane (GLH) Ethyl acetate (GLEA) Methanol (GLM) Hexane (GEH) Ethyl acetate (GEEA) Methanol (GEM) Hexane (LDH) Ethyl acetate (LDEA) Methanol (LDM) Hexane (POH) Ethyl acetate (POEA) Methanol (POM) BHAb a-Tocopherolb EDTAb
482.6 ± 1.25 4.31 ± 0.18 32.2 ± 0.24 4200 1.55 ± 0.05 123.7 ± 1.69 4200 52.7 ± 0.12 40.2 ± 0.10 316.2 ± 1.78 80.9 ± 1.24 60.26 ± 1.45 4200 127.3±1.86 32.5 ± 1.01 1.34 ± 0.04 2.10 ± 0.08 NT
4200 132.9 ± 1.37 206.2 ± 1.23 4200 49.1 ± 1.06 47.6 ± 1.02 4200 4200 202.6 ± 1.18 4200 4200 4200 4200 4200 4200 45.37 ± 0.47 7.31 ± 0.17 NT
4200 43.4 ± 0.48 98.8 ± 0.65 4200 1.06 ± 0.05 1.27 ± 0.07 4200 197.3 ± 1.16 88.7 ± 1.09 4200 4200 53.1 ± 0.63 4200 142.1 ± 1.26 192.1 ± 1.63 4.10 ± 0.06 4.31 ± 0.10 NT
285.8 ± 2.15 59.3 ± 1.87 227.8 ± 2.01 309.1 ± 2.76 19.2 ± 1.11 20.7 ± 1.21 247.6 ± 2.18 76.1 ± 1.98 256.8 ± 2.14 405.6 ± 2.87 114.3 ± 1.16 440.1 ± 2.41 236.4 ± 2.56 102.3 ± 1.05 433.3 ± 2.56 24.4 ± 0.68 89.4 ± 0.79 NT
4200 47.7 ± 0.65 4200 4200 4200 101.7 ± 0.78 120.9 ± 0.89 4200 262.9 ± 1.18 376.5 ± 1.25 408.1 ± 1.36 4200 250.8 ± 1.17 4200 4200 NT NT 3.47 ± 0.35
a
IC50 values represent the means ± S.E.M. of three parallel measurements (p50.05). Reference compounds; NT, not tested; BHA, butylatedhydroxyl anisole; EDTA, ethylenediaminetetraacetic acid.
b
ABTS+ scavenging, CUPRAC, and metal-chelating assays were used to determine the antioxidant activity. BHA and a-tocopherol, and EDTA were the positive standards for comparison of the activity. The results were found to be statistically significant (p50.05) when compared with those of controls in each test. In general, the ethyl acetate extracts and the methanol extracts of the mushroom species exhibited good lipid peroxidation inhibitory activity. The highest activity was observed in ethyl acetate extract of G. lucidum (GLEA, IC50: 1.55 ± 0.05 mg/mL), followed by ethyl acetate extract of F. trogii (FTEA, IC50: 4.31 ± 0.18 mg/mL), the methanol extract of P. ostreotus (POM, IC50: 32.5 ± 1.01 mg/mL) and the methanol extract of G. esculenta (IC50: 40.2 ± 0.10 mg/mL) in b-carotene linoleic acid assay. The hexane extracts, however, showed weak activity. As shown in Table 3, the lipid peroxidation inhibition effect of the mushroom extracts and standards decreased in the following order: BHA4GLEA 4 a-tocopherol4FTEA4FTM4POM 4GEM4GEEA4LDM4LDEA4GLM4POEA4LDH4FTH. To the best of our knowledge, G. lucidum is known as a good antioxidant source (Yegenoglu et al., 2011). When the activity of all mushroom species compared with those
of antioxidant standards, none of the mushroom species except G. lucidum showed better activity than the standards (Table 3). The results of DPPH and ABTS+ assays were also given in Table 3. In DPPH assay, the methanol (GLM) and the ethyl acetate extracts of G. lucidum (GLEA) exhibited close activity to those of antioxidant standards. The methanol extract of G. lucidum exhibited the highest activity (GLM, IC50: 47.6 ± 1.02 mg/mL), followed by ethyl acetate extract of G. lucidum (GLEA, IC50: 49.1 ± 1.06 mg/mL), and the ethyl acetate extract of F. trogii (FTEA, IC50: 132.9 ± 1.37 mg/mL). In ABTS + assay, however, the ethyl acetate extract (GLEA, IC50: 1.06 ± 0.05 mg/mL) and the methanol extract (GLM, IC50: 1.27 ± 0.07 mg/mL) of G. lucidum also showed higher radical scavenging activity. For these extracts, the ABTS+ assay supported the DPPH assay almost in all extracts of mushroom species. The difference between the tested extracts and control was statistically significant (p50.05) in both antiradical assays. The scavenging effects of the mushroom extracts and standards on the DPPH radical decreased in the following order: a-tocopherol 4BHA4GLM4GLEA4FTEA4GEM4FTM. As shown
Hexane Ethyl acetate Methanol Hexane Ethyl acetate Methanol Hexane Ethyl acetate Methanol Hexane Ethyl acetate Methanol Hexane Ethyl acetate Methanol Galantaminb
Extract NA 25.14 ± 1.25 3.79 ± 0.32 NA 23.77 ± 1.22 1.77 ± 0.51 NA 15.66 ± 3.17 10.18 ± 2.27 NA 20.42 ± 1.04 17.81 ± 2.11 NA NA NA 75.99 ± 0.45
25 mg NA 26.41 ± 2.45 16.72 ± 1.36 NA 34.20 ± 2.14 3.97 ± 0.21 NA 19.85 ± 3.04 16.36 ± 2.09 NA 22.74 ± 0.45 18.29 ± 0.18 NA 0.89 ± 0.01 0.50 ± 0.03 78.08 ± 1.08
50 mg NA 52.84 ± 2.49 28.34 ± 2.70 NA 40.24 ± 1.65 7.95 ± 1.67 NA 20.98 ± 3.01 19.96 ± 2.71 NA 22.98 ± 1.77 26.00 ± 2.55 NA 2.60 ± 0.12 4.71 ± 1.71 78.76 ± 0.52
100 mg
Acetylcholinesterase activity
a
NA, not active. Values expressed are means ± S.E.M. of three parallel measurements (p50.05). b Reference compounds.
Standard
Pleurotus ostreotus
Lyophyllum decastes
Gyromitra esculenta
Ganoderma lucidum
Funalia trogii
Mushroom species NA 61.78 ± 0.42 52.87 ± 2.98 NA 53.37 ± 2.58 8.44 ± 2.32 NA 21.42 ± 1.83 24.52 ± 2.41 NA 24.46 ± 0.74 28.06 ± 0.73 4.79 ± 0.02 3.91 ± 0.15 13.09 ± 1.47 80.41 ± 0.98
200 mg NA 94.6 ± 1.03 188.5 ± 1.65 NA 174.3 ± 1.39 4200 NA 4200 4200 NA 4200 4200 4200 4200 4200 5.0 ± 0.1
IC50 (mg mL1)
Anticholinesterase activity
Table 4. Acetyl- and butyryl-cholinesterase inhibitory activities of the extracts of five wild mushroom speciesa.
NA NA NA 2.77 ± 0.94 NA 22.64 ± 2.02 4.72 ± 0.96 14.29 ± 1.73 NA 3.28 ± 0.64 8.50 ± 1.03 NA NA 5.03 ± 1.19 NA 62.48 ± 0.02
25 mg 5.93 ± 1.05 NA NA 4.33 ± 0.90 NA 34.46 ± 2.68 6.87 ± 2.25 20.09 ± 0.17 1.63 ± 0.42 5.60 ± 1.13 9.84 ± 1.13 9.86 ± 1.90 6.47 ± 1.75 6.85 ± 0.69 NA 66.47 ± 0.46
6.13 ± 1.41 NA NA 9.91 ± 1.49 3.97 ± 0.25 48.55 ± 1.91 11.36 ± 0.56 24.59 ± 0.94 11.82 ± 0.10 7.82 ± 0.51 11.39 ± 0.97 18.55 ± 0.61 11.08 ± 1.61 10.17 ± 0.35 NA 71.43 ± 0.06
100 mg
12.92 ± 1.04 9.67 ± 1.49 NA 13.59 ± 1.51 12.29 ± 0.66 55.18 ± 1.09 21.15 ± 0.03 38.20 ± 1.60 23.39 ± 0.79 15.63 ± 0.39 17.90 ± 0.61 30.77 ± 0.32 19.04 ± 0.77 17.55 ± 1.31 5.60 ± 0.93 76.90 ± 0.20
20 mg
Butyrylcholinesterase activity 50 mg
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4200 4200 NA 4200 4200 121.8 ± 1.54 4200 286.7 ± 1.73 4200 4200 4200 4200 4200 4200 4200 11.6 ± 0.9
IC50 (mg mL1)
828 G. Tel et al. Pharm Biol, 2015; 53(6): 824–830
Bioactivities of five wild mushroom species
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DOI: 10.3109/13880209.2014.943245
in Table 3, the scavenging effect on the ABTS cation radical, however, decreased in the following order: GLEA4GLM4BHA4a-tocopherol4FTEA4LDM4GEM 4FTM4POEA4POM4GEEA. The CUPRAC of the extracts of mushroom species was given in Table 3. The difference between the tested extracts and the control was statistically significant (p50.05) in the assay. The ethyl acetate extract of G. lucidum (GLEA, A0.50: 19.2 ± 1.11 mg/mL) was found to be the best reductant followed by its methanol extract (GLM, A0.50: 20.7 ± 1.21 mg/mL) and the ethyl acetate extract of F. trogii (FTEA, A0.50: 59.3 ± 1.87 mg/mL). Moreover, the methanol and ethyl acetate extract of G. lucidum exhibited higher activity than those of antioxidant standards in CUPRAC and ABTS+ assays, which supported each other. The cupric reducing antioxidant capacity of the mushroom extracts and standards decreased in the following order: BHA4 GLEA4GLM4FTEA4GEEA4a-tocopherol4POEA4 LDEA4FTM4POH4GEH4GEM4FTH4GLH4LDH4 POM4LDM. Ferrous ions are considered as one of the effective pro-oxidants because the ferrous state of iron accelerates lipid oxidation by breaking down hydrogen and lipid peroxides to reactive free radicals through the Fenton reaction (Halliwell & Gutteridge, 1984). In fact, the reaction is very slow, and peroxidation accelerates when catalyzed by ferrous state iron. Table 3 shows the chelating effects of the extracts of mushroom species compared with EDTA on ferrous ions. The ethyl acetate extract of F. trogii (FTEA, IC50: 47.7 ± 0.65 mg/mL) showed the highest metal-chelating activity among the other extracts studied. However, none of the extracts have comparable results with that of EDTA. As seen in Table 3, the metal-chelating effect of the extracts and EDTA decreased in the following order: EDTA4FTEA4 GLM4GEH4POH4GEM4LDH4LDEA. Anticholinesterase activity Table 4 shows the AChE and BChE inhibitory activities of the extracts compared with that of galantamine. Against AChE enzyme, the ethyl acetate extract of F. trogii (FTEA, IC50: 94.6 ± 1.03 mg/mL) exhibited significant activity, followed by ethyl acetate extract of G. lucidum (GLEA, IC50: 174.3 ± 1.39 mg/mL). Against BChE enzyme, however, the most active extract was the methanol extract of G. lucidum (GLM, IC50: 121.8 ± 1.54 mg/mL). At the same conditions, the IC50 values of galantamine were 5.0 ± 0.1 and 11.6 ± 0.9 against AChE and BChE, respectively. In general, the studied mushroom species exhibited mild inhibitory activity against both enzymes. However, F. trogii possessed interesting AChE inhibitory activity. Therefore, in order to obtain AChE inhibitory compounds, isolation studies by bioactivityguided fractionation are needed.
Conclusion The results showed the antioxidant and anticholinesterase importance of the mushroom species. Four of them are commonly consumed as edible mushrooms in Anatolia as well as in some parts of the world due to their delicious taste.
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Thus, consuming these species may help people to protect them against lipid peroxidation and free radical damage. In addition, their extracts will probably be used for the development of safe food products and additives. Moreover, the ethyl acetate extracts of F. trogii and G. lucidum, against acetylcholinesterase, and methanol extract of G. lucidum, and ethyl acetate extract of G. esculenta against butyrylcholinesterase, demonstrated mild cholinesterase inhibitory activity. In order to treat mild Alzheimer’s disease, these species may be useful as mild cholinesterase inhibitory agents, particularly against AChE. However, further studies, especially in vivo activity tests on extracts and isolated constituents, are needed.
Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.
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Trajkovic LMH, Mijatovic SA, Maksimovic-Ivanic DD, et al. (2009). Anticancer properties of Ganoderma lucidum methanol extracts in vitro and in vivo. Nutr Cancer 61:696–707. Tu¨rkog˘lu A, Duru ME, Mercan N, et al. (2007). Antioxidant and antimicrobial activity of Laetiporus sulphureus (Bull.) Murrill. Food Chem 101:267–73. Ukawa Y, Andou M, Furuichi Y, et al. (2001). Hypocholesterolemic activity of hatakeshimeji (Lyophyllum decastes Sing.) mushroom in rats. J Jpn Soc Food Sci 48:520–5. Ukawa Y, Ito H, Hisamatsu M. (2000). Antitumor effects of (1!3)-b-D-glucan and (1!6)-b-D-glucan purified from newly cultivated mushroom, hatakeshimeji (Lyophyllum decastes Sing.). J Biosci Bioeng 90:98–104. Vamanu E. (2013). In vitro antioxidant and antimicrobial activities of two edible mushroom mycelia obtained in the presence of different nitrogen sources. J Med Food 16:155–66. Wang J, Wang Y, Liu X, et al. (2013). Free radical scavenging and immunomodulatory activities of Ganoderma lucidum polysaccharides derivative. Carbohydr Polym 91:33–8. Yegenoglu H, Aslim B, Oke F. (2011). Comparison of antioxidant capacities of Ganoderma lucidum (Curtis) P. Karst and Funalia trogii (Berk.) Bondartsev & Singer by using different in vitro methods. J Med Food 14:512–16. Yim Jr HS, Chye FY, Tan CT, et al. (2010). Antioxidant activities and total phenolic content of aqueous extract of Pleurotus ostreatus (cultivated oyster mushroom). Malays J Nutr 16:281–91.
ORIGINAL ARTICLE
Antioxidant and anticholinesterase activities of five wild mushroom species with total bioactive contents Gulsen Tel1, Mehmet Ozturk1, Mehmet E. Duru1, and Aziz Turkoglu2 Department of Chemistry, and 2Department of Biology, Faculty of Sciences, Mug˘la Sıtkı Koc¸man University, Mug˘la, Turkey
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1
Abstract
Keywords
Context: Recently, mushrooms are interesting natural products to be investigated due to exhibiting various bioactivities. Objective: This study determines the antioxidant and anticholinesterase activities of various extracts of five wild mushroom species. In addition, the total bioactive contents, namely, ascorbic acid, b-carotene, and lycopene along with phenolic and flavonoid contents were also determined spectrophotometrically. Materials and methods: Antioxidant activity was tested by using five complementary tests; namely, b-carotene-linoleic acid, DPPH scavenging, ABTS+ scavenging, cupric-reducing antioxidant capacity (CUPRAC), and metal chelating assays. The in vitro anticholinesterase activity was tested against acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) enzymes using the Ellman method. The spectrophotometric methods were used to determine the total phenolic, flavonoid, ascorbic acid, b-carotene, and lycopene contents. Results: The current study has shown that ethyl acetate extracts of Ganoderma lucidum (Curtis) P. Karst (IC50: 1.55 ± 0.05 mg/mL) and Funalia trogii (Berk.) Bondartsev & Singer (IC50: 4.31 ± 0.18 mg/mL) exhibited good lipid peroxidation inhibitory activity. The DPPH, ABTS, and CUPRAC assays supported this activity. The ethyl acetate and methanol extracts of Funalia trogii and Ganoderma lucidum indicated good anticholinesterase activity. Ganoderma lucidum had rich phenolic and flavonoid contents, indicating 98.67 ± 0.32 mg PEs/g extract and 160.38 ± 1.25 mg QEs/g extract, respectively. Discussion and conclusion: The results demonstrate that some of the mushroom species tested herein could be used in food and pharmaceutical industries as natural antioxidants.
Funalia trogii, Ganoderma lucidum, Gyromitra esculenta, Lyophyllum decastes, Pleurotus ostreotus
Introduction All over the world, mushrooms are used as valuable healthy foods since they are low in calories, fat, and essential fatty acids whereas they are rich in proteins, vitamins, and minerals (Pereira et al., 2012; Reis et al., 2012). Evaluation of studies in the literature on mushroom species indicate that polysaccharides, b-glucans, polysaccharide-protein complexes, lanostentype triterpenoids, cyathane-type diterpenoids, steroids, alkaloids, and phenolic compounds were isolated from mushroom species previously. These isolated compounds exhibited anticancer, antioxidant, antitumor, antiviral, antibacterial, antifungal, anti-inflammatory, and immunomodulator activities (Moradali et al., 2007; Tong et al., 2009; Tu¨rkog˘lu et al., 2007). Funalia trogii (Berk.) Bondartsev & Singer is a nonpoisonous inedible mushroom species that grows on trees.
Correspondence: Mehmet E. Duru, Department of Chemistry, Faculty of Sciences, Mug˘la Sıtkı Koc¸man University, 48121 Mug˘la, Turkey. Tel: +90 252 2111494. Fax: +90 252 2111472. E-mail: [email protected], [email protected]
History Received 1 April 2014 Revised 9 June 2014 Accepted 7 July 2014 Published online 27 February 2015
So far a few studies on F. trogii have been reported. The antioxidant activity of methanol and water extracts has been studied using DPPH radical scavenging, metal chelating, and lipid peroxidation inhibitory assays (Yegenoglu et al., 2011). Ganoderma lucidum (Curtis) P. Karst has been used as a medicinal mushroom in traditional Chinese medicine for more than 2000 years. There are many reports on G. lucidum. In previous studies, several biological activities such as antioxidant (Yegenoglu et al., 2011), anticancer (Trajkovic et al., 2009), antitumor (Joseph et al., 2011; Liu et al., 2012), immunomodulatory (Wang et al., 2013), and anti-inflammatory properties (Joseph et al., 2011) of G. lucidum were investigated. Although, potentially fatal if eaten raw, Gyromitra esculenta (Pers. ex Pers.) Fr. is a popular delicacy in Scandinavia, Eastern Europe, and some regions of North America. There are some studies performed on G. esculenta mushroom (Arshadi et al., 2006; Leal et al., 2013). Lyophyllum decastes (Fries: Fries) Singer, commonly known as the fried chicken mushroom, is an edible species in the Lyophyllaceae family. In previous studies, several biological activities of L. decastes such as antitumor (Ukawa et al., 2000), hypocholesterolemic
Bioactivities of five wild mushroom species
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DOI: 10.3109/13880209.2014.943245
(Ukawa et al., 2001), and antidiabetic (Miura et al., 2002) properties were investigated. Pleurotus ostreotus (Jacq. ex Fr.) P.Kumm., the oyster mushroom, is another common edible mushroom. Antioxidant (Yim et al., 2010) and antimicrobial activities (Vamanu, 2013) of P. ostreotus were studied. The antioxidant activity by using ABTS radical scavenging and cupric-reducing antioxidant capacity (CUPRAC) assays of F. trogii and antioxidant activity of G. esculenta were studied for the first time in this study. In addition, the anticholinesterase activity of all studied mushroom species was investigated for the first time. Some diseases, such as atherosclerosis, diabetes, cancer, and cirrhosis have been related with oxidative damage caused by oxygen-centered free radicals. In fact, oxidation is necessary for many living organisms (Halliwell & Gutteridge, 1984). Nowadays, especially in food and pharmaceutical industries, synthetic antioxidants have been used against free radicals due to their easy accessibility (Ding et al., 2010). To the best of our knowledge, since synthetic antioxidants are suspected of being responsible for liver damage and carcinogenesis, some of them were restricted to be used in the food industry by some countries (Grice, 1988). After that, some of the antioxidants occurring naturally have received much attention as safe antioxidants (Scalbert et al., 2005). In contrast, it is also reported that oxygencentered free radicals contribute to cellular ageing and neuronal damage (Sastre et al., 2000). According to Soholm (1998), an excess amount of free radical species causing oxidative stress is also associated with the pathology of many diseases including Alzheimer’s disease. Supportively, the lack of vitamin E, a natural antioxidant, enhanced Alzheimer’s disease on a mouse model (Nishida et al., 2006). Thus, the development and utilization of more effective antioxidants of natural origin as well as anticholinesterase compounds are desired. Regarding the consumption of G. lucidum, G. esculenta, L. decastes, and P. ostreotus, and the usage of F. trogii, collected from U¸sak-Turkey, we aimed to investigate the antioxidant and anticholinesterase activities, and the total bioactive compounds of these species. The main goal of the study is to compare the results with each other and those in the literature.
Materials and methods Mushroom materials The species names, collection localities and dates, family, and edibility of five mushroom species were listed in Table 1. All species were identified by Dr. Aziz Tu¨rkog˘lu. Voucher specimen have been deposited in the
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Fungarium of Department of Biology, Mug˘la Sıtkı Koc¸man University. Spectral measurements and chemicals Bioactivity measurements were carried out on a 96-well microplate reader, SpectraMax 340PC384 (Molecular Devices, Silicon Valley, CA). The measurements and calculations of the activity results were evaluated by using Softmax PRO v5.2 software (Molecular Devices, Silicon Valley, CA). Methanol, n-hexane, pyrocatechol, quercetin ethanol, ferrous chloride, metaphosphoric acid, copper (II) chloride, ascorbic acid, 2,6-dichlorophenolindophenol, and ethylenediaminetetraacetic acid (EDTA) were purchased from E. Merck (Darmstadt, Germany). Polyoxyethylene sorbitan monopalmitate (Tween-40), Folin–Ciocalteu’s reagent (FCR), butylatedhydroxyl anisole (BHA), neocuproine, b-carotene, linoleic acid, a-tocopherol, 1,1-diphenyl-2-picryl-hydrazyl (DPPH), 2,20 -azinobis(3-ethylbenzothiazoline-6-sulfonic acid) salt 3(2-pyridyl)-5,6-di(2-furyl)-1,2,4-triazine-50 ,500 -disulfonic acid disodium salt (Ferene), acetylcholinesterase (AChE) from electric eel (Type-VI-S, EC 3.1.1.7, 425.84 U/mg, Sigma, St. Louis, MO), butyrylcholinesterase (BChE) from horse serum (EC 3.1.1.8, 11.4 U/mg, Sigma, St. Louis, MO), 5,50 -dithiobis (2-nitrobenzoic) acid (DTNB), galantamine, acetylthiocholine iodide, and butyrylthiocholine chloride were purchased from Sigma Chemical Co. (Sigma-Aldrich GmbH, Sternheim, Germany). All other chemicals and solvents were of analytical grade. Extraction Each mushroom species was extracted separately with 2.5 L hexane, ethyl acetate, and methanol (4 24 h), successively, at room temperature (25 C), filtered, and evaporated to dryness in vacuo. The crude extracts were stored in a refrigerator until required. Determination of total bioactive compounds The phenolic content in methanolic extracts of five wild mushroom species was determined with FCR according to the method of Slinkard and Singleton (1977) as described in the literature and expressed as micrograms of pyrocatechol equivalents per g extract (PEs). The following standard curve which was obtained by pyrocatechol standard was used to calculate total phenolic contents: y ¼ 0:0073½PEs0:1665 r 2 : 0:9980 The aluminum nitrate method was used to determine the ¨ ztu¨rk et al., 2011) flavonoid content in methanolic extracts (O
Table 1. Collection localities and dates, family and edibility of the mushroom species studied. No. 1 2 3 4 5
Mushroom
Collection localities and dates
Family
Edibility
Funalia trogii (Berk.) Bondartsev & Singer Ganoderma lucidum (Curtis) P. Karst Gyromitra esculenta (Pers. ex Pers.) Fr. Lyophyllum decastes (Fries: Fries) Singer Pleurotus ostreotus (Jacq. ex Fr.) P.Kumm.
U¸sak-E¸sme, September 2009 U¸sak-E¸sme, September 2009 U¸sak-Banaz, May 2009 U¸sak-Banaz, November 2008 U¸sak-Banaz, May 2008
Polyporaceae Ganodermataceae Discinaceae Lyophyllaceae Pleurotaceae
Not edible Not edible/consumed as tea Edible after cooking Edible Edible
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Pharm Biol, 2015; 53(6): 824–830
and the results were expressed as quercetin equivalents per g extract (QEs). The following standard curve obtained by quercetin standard was used to calculate total flavonoid contents: y ¼ 0:0074½QEs þ 0:0133 r 2 : 0:9990 Ascorbic acid content in methanolic extracts was determined as ascorbic acid equivalents per g extract (AAEs). The method was based on the extraction of the sample with metaphosphoric acid and then the reaction with 2,6dichlorophenolindophenol (Barros et al., 2008). The following standard curve, which was obtained by authentic ascorbic acid, was used to calculate the ascorbic acid content:
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y ¼ 0:0537½AAEs0:9581 r 2 : 0:9988 b-Carotene and lycopene contents in methanol extracts were determined according to the method of Barros et al. (2008). The method was based on the measurement of the absorbance of methanol extract, solved in acetone–hexane mixture (6:4, v/v), at 453 (A453), 505 (A505), and 663 (A663) nm wavelengths. Contents of b-carotene and lycopene were calculated according to the following equations: Lycopene ðmg=100mLÞ ¼ 0:0458 A663 þ 0:372 A505 0:0806 A453 ; b-Carotene ðmg=100mLÞ ¼ 0:216 A663 0:304 A505 þ 0:452 A453 :
¨ ztu¨rk et al., 2011). BHA and with slight modifications (O -tocopherol were used as antioxidant standards. Results were given as A0.50, (mg/mL) that corresponds to the concentration providing 0.500 absorbance. Metal-chelating activity The metal-chelating activity of the extracts on Fe2+ was measured spectrophotometrically (Decker & Welch, 1990). Ethanol was used as a control, and EDTA was used as a chelating standard for comparison of the activity. The results were given as IC50 mg/mL. Determination of anticholinesterase activity AChE and BChE inhibitory activities were measured by slightly modifying the spectrophotometric method developed by Ellman et al. (1961). AChE from electric eel (5.32 103 U in 20 mL phosphate buffer for each well) and BChE from horse serum (6.85 103 U in 20 mL phosphate buffer for each well) were used as enzymes, while acetylthiocholine iodide and butyrylthiocholine chloride were employed as substrates of the reaction. DTNB [5,50 -dithio-bis(2-nitrobenzoic) acid] was used for the measurement of the cholinesterase activity. All conditions ¨ ztu¨rk et al., 2011). were described in our earlier publication (O Galantamine which is a standard drug for the treatment of mild Alzheimer’s disease was used as a reference compound. The results were given as percentage inhibition against 25, 50, 100, and 200 mg/mL concentrations, the IC50 was also given.
Determination of antioxidant activity -Carotene/linoleic acid assay The lipid peroxidation inhibitory activity was evaluated using b-carotene/linoleic acid assay as previously described ¨ ztu¨rk et al., 2011). Ethanol was used as a (Marco, 1968; O control while BHA and -tocopherol were used as antioxidant standards. The results were given as 50% inhibition concentration, mg/mL (IC50).
Statistical analysis All data on antioxidant and anticholinesterase activities as well as bioactive contents were the averages of triplicate analyses. Data were recorded as mean ± S.E.M. Significant differences between means were determined by Student’s ttest, p values50.05 were regarded as significant.
Results and discussions
DPPH free radical scavenging assay
Total bioactive compounds
The free radical scavenging activity was determined spectrophotometrically by the DPPH assay described by ¨ ztu¨rk et al., 2011). Blois (1958) with a slight modification (O Ethanol was used as a control while BHA and -tocopherol were used as antioxidant standards. The results were given as IC50 mg/mL.
The ABTS+ scavenging activity was determined according to the method of Re et al. (1999) with slight modifications ¨ ztu¨rk et al., 2011). Ethanol was used as a control while (O BHA and -tocopherol were used as antioxidant standards. The results were given as IC50 mg/mL.
Table 2 presents total phenols and flavonoids, ascorbic acid, and carotenoids concentrations in the extracts obtained from the mushroom species. Total phenolic and flavonoid contents of the extracts of mushroom species determined as pyrocatechol equivalents (PEs) and quercetin equivalents (QEs), respectively. The G. lucidum afforded rich phenolic content, exhibiting 98.67 ± 0.32 mg PEs/g extract. The most flavonoid rich mushroom was found to be G. lucidum (160.38 ± 1.25 mg QEs/g extract), while G. esculanta (7.64 ± 0.14 mg QEs/g extract) was the poorest (Table 2). Ascorbic acid was found to be between 2.11 and 5.60 mg/g extract in the all mushroom species. b-Carotene and lycopene were found to be between 0.07–0.43 and 0.03–0.67 mg/g extract, respectively (Table 2).
Cupric reducing antioxidant capacity
Antioxidant activity
The cupric reducing antioxidant capacity (CUPRAC) was determined according to the method of Apak et al. (2004)
Table 3 shows the antioxidant activity of the extracts of mushroom species. b-Carotene-linoleic acid, DPPH scavenging,
ABTS cation radical decolorization assay
Bioactivities of five wild mushroom species
DOI: 10.3109/13880209.2014.943245
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Table 2. Total bioactive contents of methanol extract of five wild mushroom speciesa.
Mushroom species Funalia trogii Ganoderma lucidum Gyromitra esculenta Lyophyllum decastes Pleurotus ostreotus
Total phenolic content (mg/g)
Total flavonoid content (mg/g)
Ascorbic acid (mg/g)
b-Carotene (mg/g)
Lycopene (mg/g)
78.03 ± 1.02 98.67 ± 0.32 28.03 ± 0.25 7.76 ± 0.05 7.16 ± 0.08
33.14 ± 0.05 160.38 ± 1.25 7.64 ± 0.14 36.95 ± 0.74 14.18 ± 0.48
4.33 ± 0.01 5.60 ± 0.40 2.86 ± 0.38 4.77 ± 0.41 2.11 ± 0.48
0.43 ± 0.02 nd 0.15 ± 0.01 0.07 ± 0.03 0.11 ± 0.00
0.23 ± 0.04 0.67 ± 0.02 0.07 ± 0.01 0.03 ± 0.00 0.05 ± 0.00
a
Values expressed are means ± S.D. of three parallel measurements (p50.05). nd: not detected.
Table 3. Antioxidant activity of the extracts of five wild mushroom species by the b-carotene-linoleic acid, DPPH, ABTS+, CUPRAC, and metal chelating assaysa.
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Antioxidant activity
Mushroom species Funalia trogii Ganoderma lucidum Gyromitra esculenta Lyophyllum decastes Pleurotus ostreotus Standards
Extracts
b-Carotene-linoleic acid assay IC50 (mg mL1)
DPPH assay IC50 (mg mL1)
ABTS+ assay IC50 (mg mL1)
CUPRAC assay A0.50 (mg mL1)
Metal chelating assay IC50 (mg mL1)
Hexane (FTH) Ethyl acetate (FTEA) Methanol (FTM) Hexane (GLH) Ethyl acetate (GLEA) Methanol (GLM) Hexane (GEH) Ethyl acetate (GEEA) Methanol (GEM) Hexane (LDH) Ethyl acetate (LDEA) Methanol (LDM) Hexane (POH) Ethyl acetate (POEA) Methanol (POM) BHAb a-Tocopherolb EDTAb
482.6 ± 1.25 4.31 ± 0.18 32.2 ± 0.24 4200 1.55 ± 0.05 123.7 ± 1.69 4200 52.7 ± 0.12 40.2 ± 0.10 316.2 ± 1.78 80.9 ± 1.24 60.26 ± 1.45 4200 127.3±1.86 32.5 ± 1.01 1.34 ± 0.04 2.10 ± 0.08 NT
4200 132.9 ± 1.37 206.2 ± 1.23 4200 49.1 ± 1.06 47.6 ± 1.02 4200 4200 202.6 ± 1.18 4200 4200 4200 4200 4200 4200 45.37 ± 0.47 7.31 ± 0.17 NT
4200 43.4 ± 0.48 98.8 ± 0.65 4200 1.06 ± 0.05 1.27 ± 0.07 4200 197.3 ± 1.16 88.7 ± 1.09 4200 4200 53.1 ± 0.63 4200 142.1 ± 1.26 192.1 ± 1.63 4.10 ± 0.06 4.31 ± 0.10 NT
285.8 ± 2.15 59.3 ± 1.87 227.8 ± 2.01 309.1 ± 2.76 19.2 ± 1.11 20.7 ± 1.21 247.6 ± 2.18 76.1 ± 1.98 256.8 ± 2.14 405.6 ± 2.87 114.3 ± 1.16 440.1 ± 2.41 236.4 ± 2.56 102.3 ± 1.05 433.3 ± 2.56 24.4 ± 0.68 89.4 ± 0.79 NT
4200 47.7 ± 0.65 4200 4200 4200 101.7 ± 0.78 120.9 ± 0.89 4200 262.9 ± 1.18 376.5 ± 1.25 408.1 ± 1.36 4200 250.8 ± 1.17 4200 4200 NT NT 3.47 ± 0.35
a
IC50 values represent the means ± S.E.M. of three parallel measurements (p50.05). Reference compounds; NT, not tested; BHA, butylatedhydroxyl anisole; EDTA, ethylenediaminetetraacetic acid.
b
ABTS+ scavenging, CUPRAC, and metal-chelating assays were used to determine the antioxidant activity. BHA and a-tocopherol, and EDTA were the positive standards for comparison of the activity. The results were found to be statistically significant (p50.05) when compared with those of controls in each test. In general, the ethyl acetate extracts and the methanol extracts of the mushroom species exhibited good lipid peroxidation inhibitory activity. The highest activity was observed in ethyl acetate extract of G. lucidum (GLEA, IC50: 1.55 ± 0.05 mg/mL), followed by ethyl acetate extract of F. trogii (FTEA, IC50: 4.31 ± 0.18 mg/mL), the methanol extract of P. ostreotus (POM, IC50: 32.5 ± 1.01 mg/mL) and the methanol extract of G. esculenta (IC50: 40.2 ± 0.10 mg/mL) in b-carotene linoleic acid assay. The hexane extracts, however, showed weak activity. As shown in Table 3, the lipid peroxidation inhibition effect of the mushroom extracts and standards decreased in the following order: BHA4GLEA 4 a-tocopherol4FTEA4FTM4POM 4GEM4GEEA4LDM4LDEA4GLM4POEA4LDH4FTH. To the best of our knowledge, G. lucidum is known as a good antioxidant source (Yegenoglu et al., 2011). When the activity of all mushroom species compared with those
of antioxidant standards, none of the mushroom species except G. lucidum showed better activity than the standards (Table 3). The results of DPPH and ABTS+ assays were also given in Table 3. In DPPH assay, the methanol (GLM) and the ethyl acetate extracts of G. lucidum (GLEA) exhibited close activity to those of antioxidant standards. The methanol extract of G. lucidum exhibited the highest activity (GLM, IC50: 47.6 ± 1.02 mg/mL), followed by ethyl acetate extract of G. lucidum (GLEA, IC50: 49.1 ± 1.06 mg/mL), and the ethyl acetate extract of F. trogii (FTEA, IC50: 132.9 ± 1.37 mg/mL). In ABTS + assay, however, the ethyl acetate extract (GLEA, IC50: 1.06 ± 0.05 mg/mL) and the methanol extract (GLM, IC50: 1.27 ± 0.07 mg/mL) of G. lucidum also showed higher radical scavenging activity. For these extracts, the ABTS+ assay supported the DPPH assay almost in all extracts of mushroom species. The difference between the tested extracts and control was statistically significant (p50.05) in both antiradical assays. The scavenging effects of the mushroom extracts and standards on the DPPH radical decreased in the following order: a-tocopherol 4BHA4GLM4GLEA4FTEA4GEM4FTM. As shown
Hexane Ethyl acetate Methanol Hexane Ethyl acetate Methanol Hexane Ethyl acetate Methanol Hexane Ethyl acetate Methanol Hexane Ethyl acetate Methanol Galantaminb
Extract NA 25.14 ± 1.25 3.79 ± 0.32 NA 23.77 ± 1.22 1.77 ± 0.51 NA 15.66 ± 3.17 10.18 ± 2.27 NA 20.42 ± 1.04 17.81 ± 2.11 NA NA NA 75.99 ± 0.45
25 mg NA 26.41 ± 2.45 16.72 ± 1.36 NA 34.20 ± 2.14 3.97 ± 0.21 NA 19.85 ± 3.04 16.36 ± 2.09 NA 22.74 ± 0.45 18.29 ± 0.18 NA 0.89 ± 0.01 0.50 ± 0.03 78.08 ± 1.08
50 mg NA 52.84 ± 2.49 28.34 ± 2.70 NA 40.24 ± 1.65 7.95 ± 1.67 NA 20.98 ± 3.01 19.96 ± 2.71 NA 22.98 ± 1.77 26.00 ± 2.55 NA 2.60 ± 0.12 4.71 ± 1.71 78.76 ± 0.52
100 mg
Acetylcholinesterase activity
a
NA, not active. Values expressed are means ± S.E.M. of three parallel measurements (p50.05). b Reference compounds.
Standard
Pleurotus ostreotus
Lyophyllum decastes
Gyromitra esculenta
Ganoderma lucidum
Funalia trogii
Mushroom species NA 61.78 ± 0.42 52.87 ± 2.98 NA 53.37 ± 2.58 8.44 ± 2.32 NA 21.42 ± 1.83 24.52 ± 2.41 NA 24.46 ± 0.74 28.06 ± 0.73 4.79 ± 0.02 3.91 ± 0.15 13.09 ± 1.47 80.41 ± 0.98
200 mg NA 94.6 ± 1.03 188.5 ± 1.65 NA 174.3 ± 1.39 4200 NA 4200 4200 NA 4200 4200 4200 4200 4200 5.0 ± 0.1
IC50 (mg mL1)
Anticholinesterase activity
Table 4. Acetyl- and butyryl-cholinesterase inhibitory activities of the extracts of five wild mushroom speciesa.
NA NA NA 2.77 ± 0.94 NA 22.64 ± 2.02 4.72 ± 0.96 14.29 ± 1.73 NA 3.28 ± 0.64 8.50 ± 1.03 NA NA 5.03 ± 1.19 NA 62.48 ± 0.02
25 mg 5.93 ± 1.05 NA NA 4.33 ± 0.90 NA 34.46 ± 2.68 6.87 ± 2.25 20.09 ± 0.17 1.63 ± 0.42 5.60 ± 1.13 9.84 ± 1.13 9.86 ± 1.90 6.47 ± 1.75 6.85 ± 0.69 NA 66.47 ± 0.46
6.13 ± 1.41 NA NA 9.91 ± 1.49 3.97 ± 0.25 48.55 ± 1.91 11.36 ± 0.56 24.59 ± 0.94 11.82 ± 0.10 7.82 ± 0.51 11.39 ± 0.97 18.55 ± 0.61 11.08 ± 1.61 10.17 ± 0.35 NA 71.43 ± 0.06
100 mg
12.92 ± 1.04 9.67 ± 1.49 NA 13.59 ± 1.51 12.29 ± 0.66 55.18 ± 1.09 21.15 ± 0.03 38.20 ± 1.60 23.39 ± 0.79 15.63 ± 0.39 17.90 ± 0.61 30.77 ± 0.32 19.04 ± 0.77 17.55 ± 1.31 5.60 ± 0.93 76.90 ± 0.20
20 mg
Butyrylcholinesterase activity 50 mg
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4200 4200 NA 4200 4200 121.8 ± 1.54 4200 286.7 ± 1.73 4200 4200 4200 4200 4200 4200 4200 11.6 ± 0.9
IC50 (mg mL1)
828 G. Tel et al. Pharm Biol, 2015; 53(6): 824–830
Bioactivities of five wild mushroom species
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DOI: 10.3109/13880209.2014.943245
in Table 3, the scavenging effect on the ABTS cation radical, however, decreased in the following order: GLEA4GLM4BHA4a-tocopherol4FTEA4LDM4GEM 4FTM4POEA4POM4GEEA. The CUPRAC of the extracts of mushroom species was given in Table 3. The difference between the tested extracts and the control was statistically significant (p50.05) in the assay. The ethyl acetate extract of G. lucidum (GLEA, A0.50: 19.2 ± 1.11 mg/mL) was found to be the best reductant followed by its methanol extract (GLM, A0.50: 20.7 ± 1.21 mg/mL) and the ethyl acetate extract of F. trogii (FTEA, A0.50: 59.3 ± 1.87 mg/mL). Moreover, the methanol and ethyl acetate extract of G. lucidum exhibited higher activity than those of antioxidant standards in CUPRAC and ABTS+ assays, which supported each other. The cupric reducing antioxidant capacity of the mushroom extracts and standards decreased in the following order: BHA4 GLEA4GLM4FTEA4GEEA4a-tocopherol4POEA4 LDEA4FTM4POH4GEH4GEM4FTH4GLH4LDH4 POM4LDM. Ferrous ions are considered as one of the effective pro-oxidants because the ferrous state of iron accelerates lipid oxidation by breaking down hydrogen and lipid peroxides to reactive free radicals through the Fenton reaction (Halliwell & Gutteridge, 1984). In fact, the reaction is very slow, and peroxidation accelerates when catalyzed by ferrous state iron. Table 3 shows the chelating effects of the extracts of mushroom species compared with EDTA on ferrous ions. The ethyl acetate extract of F. trogii (FTEA, IC50: 47.7 ± 0.65 mg/mL) showed the highest metal-chelating activity among the other extracts studied. However, none of the extracts have comparable results with that of EDTA. As seen in Table 3, the metal-chelating effect of the extracts and EDTA decreased in the following order: EDTA4FTEA4 GLM4GEH4POH4GEM4LDH4LDEA. Anticholinesterase activity Table 4 shows the AChE and BChE inhibitory activities of the extracts compared with that of galantamine. Against AChE enzyme, the ethyl acetate extract of F. trogii (FTEA, IC50: 94.6 ± 1.03 mg/mL) exhibited significant activity, followed by ethyl acetate extract of G. lucidum (GLEA, IC50: 174.3 ± 1.39 mg/mL). Against BChE enzyme, however, the most active extract was the methanol extract of G. lucidum (GLM, IC50: 121.8 ± 1.54 mg/mL). At the same conditions, the IC50 values of galantamine were 5.0 ± 0.1 and 11.6 ± 0.9 against AChE and BChE, respectively. In general, the studied mushroom species exhibited mild inhibitory activity against both enzymes. However, F. trogii possessed interesting AChE inhibitory activity. Therefore, in order to obtain AChE inhibitory compounds, isolation studies by bioactivityguided fractionation are needed.
Conclusion The results showed the antioxidant and anticholinesterase importance of the mushroom species. Four of them are commonly consumed as edible mushrooms in Anatolia as well as in some parts of the world due to their delicious taste.
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Thus, consuming these species may help people to protect them against lipid peroxidation and free radical damage. In addition, their extracts will probably be used for the development of safe food products and additives. Moreover, the ethyl acetate extracts of F. trogii and G. lucidum, against acetylcholinesterase, and methanol extract of G. lucidum, and ethyl acetate extract of G. esculenta against butyrylcholinesterase, demonstrated mild cholinesterase inhibitory activity. In order to treat mild Alzheimer’s disease, these species may be useful as mild cholinesterase inhibitory agents, particularly against AChE. However, further studies, especially in vivo activity tests on extracts and isolated constituents, are needed.
Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.
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