Antioxidant activity of fruiting body extracts from pycnoporus sanguineus mushroom

Vietnam Journal of Science and Technology 58 (2) (2020) 143-151 doi:10.15625/2525-2518/58/2/14400 ANTIOXIDANT ACTIVITY OF FRUITING BODY EXTRACTS FROM PYCNOPORUS SANGUINEUS MUSHROOM Tran Duc Tuong 1, * , Duong Xuan Chu 2 , Bui Thi Minh Dieu 3 1 Department of Physics - Chemistry - Biology Teacher Education, Dong Thap University, 783 Pham Huu Lau street, ward 6, Cao Lanh city, Dong Thap province, Viet Nam 2 Department of Pharmacy, Can Tho University of Medicine and Pharmacy, 179 Nguyen Van Cu street, An Khanh ward, Ninh Kieu district, Can Tho city, Viet Nam 3 Biotechnology Research and Development Institute, Campus II - Can Tho University, 3/2 street, Xuan Khanh ward, Ninh Kieu district, Can Tho city, Viet Nam * Email: tdtuong@dthu.edu.vn Received: 6 September 2019; Accepted for publication: 9 March 2020 Abstract. The objective of the this study is to provide an in vitro evidence for the potential antioxidant activity of the ethanolic and aqueous extracts from fruiting bodies of Pycnoporus sanguineus (L.: Fr.) Murrill mushroom via DPPH free radical scavenging and lipid peroxidation inhibition assays. DPPH free radical and lipid peroxidation inhibitory activities of ethanolic and aqueous extracts from fruiting bodies of P. sanguineus were examined in a dose-response manner. Ascorbic acid and trolox were used as a positive control for these assays. The results showed that the ethanolic extract of P. sanguineus possessed strong inhibitory activity on DPPH free radical and lipid peroxidation with IC50 values of 196.68 and 975.84 μg/mL, respectively. The aqueous extract also exhibited moderate activity against DPPH free radical and lipid peroxidation with IC50 values of 322.03 and 1311.24 μg/mL, respectively, which was significantly lower than that of ascorbic acid and trolox with IC50 values of 55.00 and 886.68 μg/mL, respectively. The IC50 values of P. sanguineus aqueous extract against DPPH and lipid peroxidation (322.03 and 1311.24 μg/mL, respectively) to be much higher than corresponding positive controls and therefore lower antioxidant activity inversely. The degree of DPPH free radical and lipid peroxidation inhibition correlated with the dose of samples (inhibitors). Based on the obtained results, the P. sanguineus mushroom possesses high potential in antioxidant activity and could be a potential source of natural antioxidants in food and pharmaceutical applications. Keywords: antioxidant activity, DPPH free radical, lipid peroxidation, Pycnoporus sanguineus. Classification numbers: 1.2.1; 1.3.1. 1. INTRODUCTION Pycnoporus sanguineus (L.: Fr.) Murrill (Syn. Trametes sanguinea (L.) Lloyd) mushroom has been considered as one of the 25 major medicinal macrofungi worldwide [1]. This Tran Duc Tuong, Duong Xuan Chu, Bui Thi Minh Dieu 144 mushroom is known to be rich in various bioactive substances with anti-bacterial, anti-fungal, anti-viral, antiparasitic, anti-oxidant, anti-inflammatory, anti-proliferative, anti-cancer, anti- tumour, cytotoxic, anti-HIV, hypocholesterolemic, anti-diabetic, anti-coagulant, hepatoprotective, and more other activities [2-5]. Qualitative phytochemical analysis of the extracts from fruiting bodies of the P. sanguineus revealed the presence of flavonoids, saponins, tannins, and terpenoids [5]. P. sanguineus mushroom has also been successfully cultivated on various agricultural by-products such as corn cobs, melaleuca bark, rice hulls [6-8]. Oxidative stress and cellular metabolism generate reactive oxygen species (ROS) that contribute to the etiology, pathogenesis and progression of several diseases including inflammations, cancer, and cardiovascular diseases [9]. Dietary antioxidants can inactivate ROS and provide protection from oxidative damage, and are therefore considered important therapeutic and prophylactic agents against disease development [10]. Lipid peroxidation is a free radical chain reaction, which causes oxidative damage to membrane lipids in biological systems. It impairs the biological functions of membrane, inactivates membrane bound enzymes and receptors and may change nonspecific calcium ion permeability [11, 12]. The basic pre-requisite for the occurrence of lipid peroxidation is inadequate free radical scavengers. The interest in finding natural antioxidants for use in the food products and the pharmaceutical markets have grown notably since the early 1980s. The investigations aim at replacing the commonly used synthetic antioxidants such as butylhydroxytoluene and butylhydroxyanisole, which have been prohibited due to their carcinogenic potential as well as other health problems they may cause, including a gain in liver weight and the considerable proliferation of the endoplasmatic reticule [13, 14]. Many studies have highlighted the benefits of the consumption of antioxidant substances in daily diet. This can result in an effective action to protect the organism against the oxidative processes. It was discovered that several diseases, including cancer, arteriosclerosis, diabetes, arthritis, malaria, AIDS, and heart disease, may be linked to the damage caused by different forms of extremely ROS. These substances are also related to the human ageing process [15]. An endogenous antioxidant system of the body takes care of ROS that are generated under normal physiological conditions. However overproduction of ROS and inadequate presence of antioxidants have been related to pathogenesis of disease conditions like diabetes, cancer, atherosclerosis etc. [16, 17]. Our body’s self antioxidant system comprises of enzymes like catalase, superoxide dismutase and glutathione which react to reactive species and neutralize them thereby protecting the body from damaging effect of free radical species and preventing oxidative stress [18]. The objective of this study is to provide an in vitro evidence for the potential antioxidant activity of the ethanolic and aqueous extracts from fruiting bodies of Pycnoporus sanguineus mushroom via DPPH free radical scavenging and lipid peroxidation inhibition assays. 2. MATERIALS AND METHODS 2.1. Materials The fruiting bodies of Pycnoporus sanguineus MH225776.1 were collected in Viet Nam. This mushroom was identified by Tran Duc Tuong of Dong Thap University, a corresponding author of the paper, based on morphological and molecular characterisation of mycelia and the Antioxidant activity of fruiting body extracts from Pycnoporus sanguineus mushroom 145 fruiting bodies. The P. sanguineus MH225776.1 was planted on the formula of compost consisting of 50 % corn cobs and 50 % rubber sawdust at the Biotechnology Research and Development Institute, Can Tho University, Viet Nam. 2.2. Chemicals and reagents 1,1-diphenyl-2-picrylhydrazyl (BDH, England); Cyclophosphamide (Baxter Oncology GmbH, Germany); Methanol, phosphate buffer pH 7.4, DMSO buffer, and ascorbic acid (Prolabo, France); Trolox, thiobarbituric acid, and tricloacetic acid (Sigma-Adrich Co., USA). 2.3. Methods 2.3.1. Extraction After being crushed, the fruiting bodies of P. sanguineus were soaked in solvents (ethanol 96 % v/v at room temperature during 72 hours and water at 80 ºC during 7 hours) at the ratio of 1:15. Next, soaking solution was filtered through filter paper. Finally, the filtrate was concentrated to remove solvents with a vacuum rotary evaporator (IKA RV 05 Basic - Germany), yielding total ethanolic and aqueous extracts. 2.3.2. Determination of DPPH free radical scavenging activity This assay was carried out using a modified procedure of Blois [19]; Chi and Huong [20]; Phuong et al. [21]; National Institute of Medicinal Materials [22]. DPPH solution was prepared in methanol (MeOH) and stored in the dark at room temperature for about 30 minutes. The samples were prepared in DMSO into solution with a range of concentrations of 50, 100, 200, 300 and 400 (µg/mL). 1 mL each of the samples with various concentrations were placed in tubes containing 1 mL DPPH solution (0.2 mM). The reaction mixture was shaken for 15 seconds and stabilized in the dark at room temperature for 30 minutes. A control was prepared using the same procedure replacing the extracts with 1 mL of DMSO. Color reaction absorption was measured at 517 nm using a microplate reader (Thermo Spectronic Genesys 10 UV Vis Spectrophotometer). Ascorbic acid used as a positive control for this assay was investigated with ranges of concentrations of 5, 10, 20, 40 and 80 (µg/mL). The experiment was arranged with three replications. Percentage of DPPH free radical scavenging activity is calculated as: DPPH free radical scavenging activity (%) = [(Abscontrol – Absextracts/ascorbic acid)/Abscontrol] × 100 Notes: Abscontrol: The optical density values of negative control. Absextracts/ascorbic acid: The optical density values of the extracts/ascorbic acid samples. Concentrations of the extracts resulting in 50 % inhibition of DPPH free radical (IC50) were determined graphically. 2.3.3. Determination of lipid peroxidation inhibitory activity Lipid peroxidation inhibitory activity assay was performed as previously described by National Institute of Medicinal Materials [22], Robak et al. [23]; Stroev and Makarova [24]; and Huong and Nghia [25] with modification as follows. Mice (40 - 50 g) were induced to increase MDA by cyclophosphamide (single dose 400 mg/kg body weight) for 7 days before they were used for the preparation of brain homogenate. Brain homogenate was prepared with homogenizer at 0 - 5ºC with phosphate buffer pH 7.4 (50 mM) (1:10) for 30 min. The homogenate was centrifuged for 15 min, and clear cell free supernatant was used for the study of in vitro lipid peroxidation. 0.2 mL each of the samples with various concentrations (10, 50, 100, Tran Duc Tuong, Duong Xuan Chu, Bui Thi Minh Dieu 146 500, 1,000, 1,500 and 2,000 µg/mL) were placed in tubes containing 1 mL of added brain homogenate. Continue adding phosphate buffer to a sufficient 2 mL mixture. After being incubated at 37 ºC for 15 min, the reaction was stopped by addition of 1 mL tricloacetic acid (10 %). The reaction mixture was centrifuged at a rate of 10,000 rpm at 5 ºC for 10 min. Take 1 mL of clear solution after centrifugation to react with 1 mL of thiobarbituric acid reagent (0.8 %). The mixture was then heated at 100 ºC for 15 min. The samples were cooled and the absorbance was measured at 532 nm using a microplate reader (Thermo Spectronic Genesys 10 UV Vis Spectrophotometer). A control was prepared using the same procedure replacing the extracts with 0.2 mL of DMSO. Trolox was used as a positive control for this assay. The experiment was arranged with three replications. Percentage of lipid peroxidation inhibition is calculated as: Lipid peroxidation inhibition (%) = [(Abscontrol – Absextracts/trolox)/Abscontrol] × 100 Notes: Abscontrol: The optical density values of negative control. Absextracts/trolox: The optical density values of the extracts/trolox samples. Concentrations of the extracts resulting in 50% inhibition of lipid peroxidation (IC50) were determined graphically. 2.3.4. Statistical analysis The data were statistically analyzed by One-Way analysis of variance (ANOVA) and Independent-Samples T Test of the SPSS Statistics 22.0 software. Statistical differences at p-values under 0.05 were considered significant and subsequently compared using the Tukey's test with 95 % confidence intervals. 3. RESULTS AND DISCUSSION 3.1. DPPH free radical scavenging activity As shown in Figure 1 and Figure 2, ethanolic and aqueous extracts from fruiting bodies of the P. sanguineus and ascorbic acid showed DPPH free radical scavenging effects in a dose- dependent manner at the examined concentrations. Inhibition at various concentrations is significantly different (p < 0.05). The highest DPPH free radical scavenging activity (at the concentration of ascorbic acid 80 μg/mL and extracts 400 μg/mL) was observed from the ascorbic acid (72.40 %), ethanolic extract (89.25 %) and aqueous extract (59.12 %) from fruiting bodies of the P. sanguineus with IC50 values being 55.00 μg/mL, 196.68 μg/mL and 322.03 μg/mL, respectively (Tab. 1). The results showed that ethanolic extract of mushroom had good DPPH free radical scavenging activity at all the examined concentrations. However, this inhibitory activity of ethanolic and aqueous extracts was significantly lower than that of ascorbic acid (positive control) (Fig. 1 & Tab. 1). DPPH free radical scavenging activity of the ethanolic extract was significantly higher than aqueous extract in the concentrations range of 50 - 400 µg/mL (p < 0.05) (Fig. 2 & Tab. 1). DPPH free radical scavenging activity of extracts from fruiting bodies of the P. sanguineus in this study is higher than that of the ethanolic extract (IC50 value at 1,326 μg/mL) and aqueous extract (IC50 value at 1,708 μg/mL) from fruiting bodies of Ganoderma lucidum reported by Huong and Hang [26] and methanolic extract with IC50 value at 9,000 μg/mL [27]. The previous study of Borderes et al. [28] showed the methanolic extract from P. sanguineus mycelium against DPPH free radical with IC50 values being 1,620 μg/mL [28]. This Antioxidant activity of fruiting body extracts from Pycnoporus sanguineus mushroom 147 inhibition of extract from P. sanguineus mycelium was lower than that of extracts from fruiting bodies of the P. sanguineus mushroom. Figure 1. DPPH free radical scavenging activity of ascorbic acid (positive control) at various concentrations. The values are expressed as the mean ± standard deviation of three replicates (One-Way ANOVA followed by Tukey's test). Means not sharing a common letter were significantly different (p < 0.05). Figure 2. DPPH free radical scavenging activity of ethanolic and aqueous extracts from fruiting bodies of the P. sanguineus at various concentrations. The values are expressed as the mean ± standard deviation of three replicates (Independent-Samples T Test). Means not sharing a common letter at the same concentration were significantly different (p < 0.05). Tran Duc Tuong, Duong Xuan Chu, Bui Thi Minh Dieu 148 Table 1. IC50 values of samples (ascorbic acid, ethanolic and aqueous extracts from fruiting bodies of the P. sanguineus) against DPPH free radical with correlation coefficient. Samples IC50 values (µg/mL)* Correlation coefficient Ascorbic acid 55.00 R² = 0.9979 Ethanolic extract 196.68 R² = 0.9888 Aqueous extract 322.03 R² = 0.9914 Note: *Concentrations of the samples resulting in 50 % inhibition of DPPH free radical. 3.2. Lipid peroxidation inhibitory activity Similar to the results of DPPH free radical scavenging activity, ethanolic and aqueous extracts from fruiting bodies of the P. sanguineus and trolox showed lipid peroxidation inhibitory effects in a dose-dependent manner at the examined concentrations. Inhibition at various concentrations range of 100 - 2000 μg/mL is significantly different (p < 0.05) (Fig. 3). Figure 3. Lipid peroxidation inhibition of trolox (positive control), ethanolic and aqueous extracts from fruiting bodies of the P. sanguineus at various concentrations. The values are expressed as the mean ± standard deviation of three replicates (One-Way ANOVA followed by Tukey's test). Means not sharing a common letter at the same concentration were significantly different (p < 0.05). Table 2. IC50 values of samples (trolox, ethanolic and aqueous extracts from fruiting bodies of the P. sanguineus) against lipid peroxidation with correlation coefficient. Samples IC50 values (µg/mL)* Correlation coefficient Trolox 886.68 R² = 0.9941 Ethanolic extract 975.84 R² = 0.9906 Aqueous extract 1311.24 R² = 0.985 Note: *Concentrations of the samples resulting in 50% inhibition of lipid peroxidation. Antioxidant activity of fruiting body extracts from Pycnoporus sanguineus mushroom 149 The highest lipid peroxidation inhibitory activity at the concentration of 2000 μg/mL of each samples was observed from trolox (95.53 %), ethanolic extract (90.45 %) and aqueous extract (69.71 %) from fruiting bodies of the P. sanguineus with IC50 values being 886.68 μg/mL, 975.84 μg/mL and 1311.24 μg/mL, respectively (Tab. 2). The results showed that ethanolic extract of mushroom had good lipid peroxidation inhibitory activity equivalent to trolox in the concentrations range of 500 - 1500 µg/mL (p < 0.05). However, this inhibitory activity of aqueous extracts was significantly lower than that of trolox (positive control). Lipid peroxidation inhibitory activity of the ethanolic extract was significantly higher than aqueous extract in the concentrations range of 500 - 2000 µg/mL (p < 0.05) (Fig. 3 & Tab. 2). Lipid peroxidation inhibition of extracts from fruiting bodies of the P. sanguineus in this study is higher than that of the ethanolic extract (IC50 value at 1,368 μg/mL) and aqueous extract (IC50 value at 2,818 μg/mL) from fruiting bodies of Ganoderma lucidum reported by Huong and Hang [26]. The result showed potent free radical scavenging activity of extracts from fruiting bodies of the P. sanguineus with the superior inhibitory activity in ethanolic extract. These inhibitory actions might be due to both ethanolic and aqueous extracts from fruiting bodies of this mushroom containing several phytochemicals such as flavonoids, saponins, and terpenoids reported by Tuong et al. [5]. The high antioxidant activity of the ethanolic extract from fruiting bodies of the P. sanguineus may be attributed to the high content of flavonoids. The earlier studies proved that, the secondary metabolites such as flavonoids, terpenoids, saponins had strong free radical scavenging capacities [29-32]. 4. CONCLUSION Pycnoporus sanguineus (Trametes sanguinea) presented a satisfactory antioxidant potential when compared to other mushrooms. The best results indicated that this was proved by DPPH free radical scavenging and lipid peroxidation inhibitory activities of ethanolic and aqueous extracts from fruiting bodies of this mushroom with the predominant inhibitory activity in ethanolic extract. P. sanguineus showed potential to activity as a natural source of antioxidant compounds. 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