how to calculate ec50 value for antioxidant activity using

Results are expressed as micrograms of gallic acid equivalents per milligram of dry weight (g GAE/mg) of extract. [25] An aliquot of 0.1 ml of the fractions (50-800 g/ml) was combined with 1.0 ml of reagent (0.6 M sulfuric acid, 28 mM sodium phosphate and 4 mM ammonium molybdate). The ABTS+ solution was diluted with water to an absorbance of 0.70 (0.02) at 734 nm. You do this on the Parameters tab. IC50 Calculator | AAT Bioquest Figure 2 shows the degradation rates of EESTG in comparison with the negative and positive controls while a comparison of anti-oxidant activities is displayed in Figure 3. Front. Antioxidant properties of several medicinal mushrooms. Adebayo, E. A., Oloke, J. K., Ayandele, A. [19] At the end of the extraction, the extract was filtered through Whatman filter paper No. Metal ion chelating activity was expressed as mg GAE/L. ABTS radical scavenging activity was determined according to Re et al. doi: 10.1016/S2221-1691(12)60194-4. Mycosphere 4, 661673. The petroleum ether extracts of Pleurotus sp. Official Method of Analysis 17th Edn. Careers, Unable to load your collection due to an error. EC50 estimation of antioxidant activity in DPPH assay using several DPPH is a stable, nitrogen-centered free radical which produces violet/purple color in ethanol solution and fades to shades of yellow color in the presence of antioxidants. 43, 18131819. Nuran et al. Summed up together, the EC 50 and AEAC values obtained for EESTG imply that the extract contains phytochemicals with antioxidant properties. Nutr. How do you calculate the FRAP activity of any plant - ResearchGate The total polyphenol content of the fresh samples obtained at room temperature and boiling was higher in aqueous extract of mycelium and in the methanolic extract of the fruiting body. GraphPad Prism 9 Curve Fitting Guide - The EC50 J. Microbiol. doi: 10.1016/S0891-5849(98)00315-3, Shimada, K., Fujikawa, K., Yahara, K., and Nakamura, T. (1992). In general, the extracts of dried samples showed higher reducing power than the extracts of fresh samples and tend to show greater reducing power by aqueous than methanolic extracts. For example, a dose-response experiment studies how well a drug decreases the growth of tumors grown in cell culture. [28] The reaction mixture in a final volume of 1.0 ml contained 100 l of 2-deoxy-D-ribose (28 mM in 20 mM KH2PO4 buffer, pH 7.4), 500 l of the extract at various concentrations (50800 g/ml) in buffer, 200 l of [1.04 mM EDTA and 200 M FeCl3] (1:1v/v), 100 l of 1.0 mM hydrogen peroxide (H2O2) and 100 l of 1.0 mM ascorbic acid. Pleurotus ostreatus (32783) from the American Type Culture Collection (ATCC) grown on malt extract agar (MEA) at 28C for 7 days (d) was used. FIGURE 3. Copyright 2016 Gonzlez-Palma, Escalona-Buenda, Ponce-Alquicira, Tllez-Tllez, Gupta, Daz-Godnez and Soriano-Santos. Spanos GA, Wrolstad RE. Although all extracts showed chelating activity, some of them had higher values, for example EC50 = 1932.06 0.95 mg GAE/L for the methanol extract obtained by boiling of dried primordium. This work was carried out in collaboration between all authors. Accessibility In the case of extracts from dried samples, the highest value of reducing power it showed by aqueous extract obtained by boiling of fruiting body with a value of 0.701 0.003, and the methanol extract obtained at room temperature of mycelium showed 0.645 0.009, both at a concentration of 100 mg GAE/L (Figures 1 and 2). The values reported in previous works were lower than those obtained in this research, with the same treatment and reported in the same units. Molecules 16, 31973211. During the growth, different metabolites are produced, when the microorganism is in the exponential phase of growth, are produced those called primary metabolites and is known that secondary metabolites start their production by some sort of stress observed by the cells, this stage is when the microorganism not grow anymore but still metabolically active (Pichersky and Gang, 2000). Dig. In May 2023, Frontiers adopted a new reporting platform to be Counter 5 compliant, in line with industry standards. [4] IC 50 values can be used to compare the potency of two antagonists. Values are the average of three replicates DS. 6H2O), ethanol, hydrogen peroxide etc., All reagents used were of analytical grade and products of Sigma Chemical Co. (St. Louis, USA) or BDH Chemical Ltd, Poole, UK. The lower the EC50 value the higher the antioxidant activity of a sample. doi: 10.1016/j.foodchem.2008.03.078, Keywords: antioxidant activity, chelating activity, polyphenols compounds, growth stages, Pleurotus ostreatus, Citation: Gonzlez-Palma I, Escalona-Buenda HB, Ponce-Alquicira E, Tllez-Tllez M, Gupta VK, Daz-Godnez G and Soriano-Santos J (2016) Evaluation of the Antioxidant Activity of Aqueous and Methanol Extracts of Pleurotus ostreatus in Different Growth Stages. Fungi produce a considerable amount of metabolites, including vitamin C, vitamin E, and beta-carotene as well as phenolic compounds, known as excellent antioxidants (Barros et al., 2007; Chye et al., 2008; Yin et al., 2008; Fatih et al., 2010; Keles et al., 2011; Adebayo et al., 2012; Shirmila and Radhamany, 2012). The antioxidant activity measured by the FRAP method of extracts from fresh samples were higher with respect to the dried samples. Sci. Screening of radical scavenging activity of some medicinal and aromatic plant extracts. Evaluation of. Microbiol. (2011) reported the flavonoids content from ethanolic, cold water and hot water extracts of two strains of Grifola frondosa; values were 1.093.05 and 0.110.76 mg EQ/g of sample dry, respectively. In this study, the EC (50) estimation was performed using a comparative approach based on various regression models implemented in six specialized computer programs: GraphPad Prism version. In general, fruiting bodies showed the highest antioxidant activity and reducing power, while the mycelium showed the highest chelating activity. 299, 152178. The reaction mixture consisted of 0.5 mL of extract, 3 mL of methanol, and 0.3 mL of 0.5 mM 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical solution in methanol. The bioactivity of polyphenols can be related by their ability to chelate metals, capability of inhibiting or reducing different enzymes such as cyclooxygenase, lipoxygenase and telomerase and free radical scavenging (Yin et al., 2008; Korley et al., 2014). This tutorial shows how to determine an estimate for an EC50 value using a semi-logarithmic plot in Excel The percentage of the radical scavenging activity (RSA) was calculated based on the following equation: Acont and Asample are the absorbance values (at 518 nm) for the control and sample, respectively. Free Radicals in Biology and Medicine; pp. The results were expressed as mean SD (standard deviation) and the EC50 values were obtained from the linear regression plots. The total antioxidant capacity was expressed as g equivalents of BHT by using the standard BHT graph. 111, 370376. Non-site-specific hydroxyl radical-mediated 2-deoxy-D-ribose degradation: Hydroxyl radical scavenging activity was measured by the ability of the extract to scavenge the hydroxyl radicals generated by the Fe3+ -ascorbate-EDTA-H2O2 system (Fenton reaction). Identification and determination of antioxidant constituents of bioluminescent mushroom. ABTS radical scavenging activity of P. ostreatus. Halliwell B, Gutteridge JM. Phenolics have received much scientific attention because they are the most widely-spread secondary metabolites in the plant kingdom and aside this, they are also known as sources of potential natural antioxidants because of their abilities to act both as efficient radical scavengers and metal chelators.[30]. 3rd ed. It was freshly prepared and warmed at 37C. Reducing power assay of aqueous extracts of dry Pleurotus ostreatus. 2, S386S391. The -carotene-linoleate bleaching (BCB) method employs an emulsified lipid and therefore applicable especially to investigate lipophilic antioxidants such as the antioxidant activity of essential oils. (2013) quantified total content of polyphenols and flavonoids, as well as, antioxidant activity of the extracts obtained with 60% ethanol and water of Agaricus bisporus (white button mushroom) and A. brasiliensis (Brazilian mushroom). IC 50 values are very dependent on conditions under which they are measured. If polar compounds such as ascorbic acid, rosmarinic acid, etc., are tested by the BCB method, they would be considered as weak antioxidants;[37] as a result of this factor, the reference compounds that we used in this study were quercetin and BHT. 77, 229235. Agro. In general, extracts of the fruiting body had a high amount of polyphenols, followed by primordium and by the mycelium (Table 1). J. Funct. Data can be copied directly from Excel columns. 43, 15081515. Terashima S, Schimizu M, Nakayama H, Ishikura M, Ueda Y, Imai K, et al. EC50 estimation of antioxidant activity in DPPH assay using several Antioxidant properties of wild edible mushrooms. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. In addition, those organic acids may have a protective role against various diseases due to their antioxidant activity (Valentao et al., 2005). National Library of Medicine Asample = Absorbance of sample after 6 min. Hydroxyl radical (HO) is one of the most powerful free radicals directly implicated in the irreversible damage inflicted by oxidative stress. The extract that showed the highest chelating activity was the methanol extract of dried mycelium obtained by boiling (EC50 = 13.17 0.13 mg GAE/L), followed by the aqueous extract of the dried fruiting body obtained by boiling, with an EC50 = 25.53 0.80 mg GAE/L. How to calculate IC50 value of DPPH radical scavenging assay? Mustofa, Valentin A, Benoit-Vical F, Plissier Y, Kon-Bamba D, Malli M. Antiplasmodial activity of plant extracts used in west African traditional medicine. Sorry, I forgot to ask, also how do I calculate standard deviation for IC50 given the SE?? All values are reported as means SD (n = 3). This mixture was incubated at room temperature for 30 min in darkness. 800 g/ml), the activity values of EESTG, ascorbic acid and BHT were 0.4897 0.0190, 1.1447 0.0580, 1.2560 0.0362, respectively. **P < 0.001; significantly different from quercetin and extract. doi: 10.1021/jf040465z. The result we obtained based on this assay confirms the antioxidant potency of EESTG in comparison to BHT as a standard antioxidant. The integrated antioxidant system. (2012) reported high chelating activity of methanol extract of the mushroom P. eryngii collected from different regions of the Tunceli province of Turkey (EC50 = 470.23, 218.31, and 292.99 mg GAE/mL for Ovacik, Pulumur and City center, respectively). Antioxidant and antidiabetic activities of extracts from Cirsium japonicum roots. The EC50 values of the root, leaf, whole plant and stem of Sida rhombifolia have been reported by Kamlesh et al. Res. Technol. At the minimum concentration of extract/standards used in this study (i.e. The Review of Natural Products I, Facts and Comparison; p. 637. In general, the flavonoids content was reported with a low value, representing a very small percentage of the total polyphenol content, however, the majority of the extracts presented antioxidant activity. Food Sci. Comparing the previous results with those reported in our study, the value obtained from first region was higher than the reported for aqueous extract (obtained at room temperature and boiling) of fresh fruiting body, as well as of the methanolic extract (obtained at room temperature) of fresh fruiting body and the aqueous extract (obtained by boiling) of the dried fruiting body. Guzmn et al. Evaluating the effect of gamma radiation on the total phenolic content, flavonoids and antioxidant activity of dried Pleurotus ostreatus (Jacq. doi: 10.1016/j.foodchem.2009.04.016. It was observed that the DPPH and ABTS radical scavenging activities were positively correlated to the concentration of the extract. T1 and T2 as Figure 1. 156406. EC50 Calculator | AAT Bioquest Ascorbate is used as standard and positive control for total antioxidant activity (phosphomolybdic acid . After incubation for 6 min, absorbance was determined in spectrophotometer at 734 nm. J. Agric. Overall, the fruiting body of P. ostreatus showed the best results and the possibility of continuing to investigate its functional properties of this fungus is opened. This parameter is typically employed not only to express the antioxidant capacity but also to compare the activity of different compounds with each other. Inclusion in an NLM database does not imply endorsement of, or agreement with, Don't over interpret the EC50. Biomed. Values are the average of three replicates DS. Trichloroacetic acid (0.25 mL) was added to the mixture and was centrifuged at 1000 rpm for 10 min at room temperature. 111. The reducing power of all samples was concentration dependent (Figures 14). TABLE 4. Total phenols, ascorbic acid, -carotene and lycopene in Portuguese wild edible mushrooms and their antioxidant activities. The concentration that provokes a response of 50 in this experiment is not the EC50. For statistical analysis, data were analyzed using Sigma Plot (version 11.0). A., and Adegunlola, C. O. doi: 10.5943/mycosphere/4/4/2. The result is normally expressed using the EC 50 value, defined as the concentration of antioxidant that causes a 50% decrease in the DPPH absorbance. The method was based on Hue et al. Quercetin and BHT (50800 g/ml) were used as positive controls. Karachi: Ferozsons; 1978. The absorbance was measured at 700 nm. The higher the AEAC value, the greater is the antioxidant activity. Tecnol. Pract. Hence, this assay quantifies the ability of the extract to prevent, impede or reduce the formation of free radicals and thus the anti-oxidant activity of the extract is directly measured by the extent to which the bleaching of -carotene can be prevented. The reaction mixture consisted of 0.07 mL of extract and 3 mL of the ABTS radical. Gan, C. H., Nurul, A. Analysis of total phenols and other oxidation substrates and antioxidants by means of Folin-Ciocalteu reagent. J. This calculator generates an EC 50 value typically thought of as the relative EC 50. EC 50 represents the concentration at which a substance exerts half of its maximal response. 122. Double distilled water was used as a control. doi: 10.1007/s13197-015-1783-6, Pichersky, E., and Gang, D. R. (2000). Finding an EC50 value using a semi-logarithmic plot - YouTube T1 and T2 as Figure 1. Antioxidant properties in the oyster mushrooms (Pleurotus spp.) doi: 10.1016/S1360-1385(00)01741-6, Pornariya, C., and Kanok-Orn, I. The different results also suggested that antioxidant activity couldnt be by polyphenols. The reaction mixtures were incubated for 60 min in the dark at room temperature and then, the absorbances were measured at 725 nm. (2008) reported chelating activity in petroleum ether and methanol extracts (obtained at room temperature) of the mushrooms Pleurotus sp. The antioxidant activity is determined by three major parameters: (1) the anodic peak current (I a ), usually the y-axis on a cyclic voltammogram; (2) the peak oxidation potential (E pa ), usually the x-axis on a cyclic voltammogram; and (3) the area under anodic current wave (Q) ( Zhang et al., 2011 ). 26, 12311237. Samples were dried in an oven at 58C for 24 h. Aqueous and methanolic extracts in both room temperature and boiling were obtained. where A0 is the absorbance of the negative control (without sample) at 532 nm, and A is the absorbance at 532 nm of the reaction mixture containing sample. Koleva II, van Beek TA, Linssen JP, de Groot A, Evstatieva LN. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). the contents by NLM or the National Institutes of Health. Afterward, 0.1 mL of ferrozine solution (5 mM) was added and mixed vigorously. Author IG-P carried out trials and prepared the protocol. Our result confirms the presence of flavonoids in the extract based on quercetin as the reference compound and the TFC expressed in microgram quercetin equivalents per milligram (g QE/mg) of dry extract. Aruoma OI, Laughton MJ, Halliwell B. Carnosine, homocarnosine and anserine: Could they act as antioxidants. Invitro antioxidant activities, total phenolics and flavonoid of wild edible mushroom Macrolepiota mastoidea (FR.) ABTS radical scavenging activity was positively correlated to the concentration of the extract. The polyphenols content depends on the type of sample and solvent and temperature used for extraction. Methanolic extract obtained at room temperature of the fruiting body and primordium, both dried showed the higher radical scavenging activity of DPPH and ABTS. Yim et al. EC50 values of the extracts evaluated in this study are shown in Table 5. and P. ostreatus 9.01 and 7.23 mg GAE/g, respectively. J. Pharm. [9] -carotene (0.2 mg in 1 ml chloroform), linoleic acid (0.02 ml) and Tween 20 (0.2 ml) were transferred into a round-bottomed flask. In the case of aqueous extracts, the supernatant was recovered by centrifugation and for methanolic extraction, a rotary evaporator was used until the total recovery of the solvent and the residue was solubilized in 5% dimethylsulfoxide. In this study, the EC(50) estimation was performed using a comparative approach based on various regression models implemented in six specialized computer programs: GraphPad Prism version 5.01, BLeSq, OriginPro 8.5.1, SigmaPlot 12, BioDataFit 1.02, and IBM SPSS Statistics Desktop 19.0. doi: 10.1080/21501203.2010.511292. Food Chem. Quantitation of nine organic acids in wild mushrooms. In methanol extracts of Pleurotus sp. J. Microbiol. Othman A, Ismail A, Ghani NA, Adenan I. Antioxidant capacity and phenolic content of cocoa beans. As a library, NLM provides access to scientific literature. The total phenolic content was assayed using Folin-Ciocalteau reagent. (2013). The spawn was obtained by inoculation of wet and sterile wheat grains (500 g) with 10 mycelium plugs taken from the peripheral of a colony by using a sterile cork borer (4 mm diam). Flavonoids are reported as antifungal compounds, because plants produce them as protection against fungal infections, then, these compounds might negatively affect fungal growth. The flavonoid content was calculated in mg quercetin equivalents per g of dried sample (mg QE/g). The total phenolic content was measured according to the method of Singleton et al. The mixture was incubated in the water bath for 20 min at 50C. Pleurotus ostreatus is a fungus very important because is industrially produced as food (oyster mushroom), a ligninolytic enzyme producer and as bioremediation agents in decontamination processes of materials rich in phenolic compounds, and recently as a biocompounds source (da Luz et al., 2012).
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