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Optimization of extraction of antioxidant polysaccharide from Pleurotus ostreatus (Jacq.) P. Kumm and its cytotoxic activity against murine lymphoid cancer cell line

Optimization of extraction of antioxidant polysaccharide from Pleurotus ostreatus (Jacq.) P. Kumm... Citation: Uddin Pk M.M, Islam MS, Pervin R, Dutta S, Talukder RI, Rahman M (2019) Optimization of The purpose of this study was to optimize the extraction method for polysaccharide from the extraction of antioxidant polysaccharide from fruiting bodies of Pleurotus ostreatus (Jacq.) P. Kumm and to assess the antioxidant and Pleurotus ostreatus (Jacq.) P. Kumm and its cytotoxic activity against murine lymphoid cancer cytotoxic potentials of polysaccharide. In this investigation, polysaccharides from Pleurotus cell line. PLoS ONE 14(1): e0209371. https://doi. ostreatus (Jacq.) P. Kumm were extricated by utilizing the hot water. One-single factor and org/10.1371/journal.pone.0209371 response surface methodology was established to optimize the extraction conditions for Editor: David A. Lightfoot, College of Agricultural polysaccharide from Pleurotus ostreatus (Jacq.) P. Kumm. Examination of antioxidant activ- Sciences, UNITED STATES ity of Pleurotus ostreatus polysaccharide (POP) was directed by utilizing 2, 2-diphenyl-1- Received: July 24, 2018 picrylhydrazyl (DPPH) and 2, 2-azino-bis-3-ethyl-benzothiazoline-6-sulfonic acid (ABTS) Accepted: December 4, 2018 techniques. Cytotoxicity of POP was evaluated using an MTT assay. The experimental data were fitted to a quadratic equation utilizing multiple regression investigations, and the ideal Published: January 3, 2019 conditions were as per the following: water/crude material proportion, 26.04 mL/g; an extrac- Copyright:© 2019 Uddin Pk et al. This is an open tion time of 62.08 minutes; and an extraction temperature 70.5˚C. Under such conditions, access article distributed under the terms of the Creative Commons Attribution License, which the polysaccharide yield was 5.32± 0.12% with the anticipated yield. POP showed good permits unrestricted use, distribution, and 2 scavenging activity against DPPH radical (p<0.001, EC = 1036.38μg/mL, R = 0.8313) reproduction in any medium, provided the original and ABTS radicals (p<0.001, EC = 824.37μg/mL, R = 0.8223), with a dose (p<0.001)- author and source are credited. and-time (p<0.001) dependent cytotoxic potential on Ehrlich ascites carcinoma cell line in Data Availability Statement: All relevant data are vitro. This demonstrated that polysaccharides (POP) had certain cancer prevention agent within the paper and its Supporting Information exercises. In this manner, these examinations give reference to additionally research files: the S1 Dataset: Impact of three variables on the POP extraction from Pleurotus ostreatus; S2 and reasonable improvement of Pleurotus ostreatus (Jacq.) P. Kumm polysaccharide and Dataset: Antioxidant activities of the POP. POP may prove a useful therapeutic agent, due to its robust antioxidant and cytotoxic Funding: The authors received no specific funding activity. for this work. RIT is employed by Popular Diagnostic Centre Ltd. Popular Diagnostic Centre Ltd. provided support in the form of salary for author RIT, but did not have any additional role in PLOS ONE | https://doi.org/10.1371/journal.pone.0209371 January 3, 2019 1 / 17 Optimization of extraction of antioxidant polysaccharide the study design, data collection and analysis, Introduction decision to publish, or preparation of the P. ostreatus, the oyster mushroom, is a consumable mushroom, generally growing all over the manuscript. The specific role of this author is world. Currently, it serves as a rich source of nourishment[1]. Oyster mushrooms are a rich articulated in the ‘author contributions’ section. source of vitamins, amino acids, and minerals, as well as antioxidants[2]. In addition to their Competing interests: RIT is employed by Popular role in nutrition, they possess medicinal properties. Recent research has elucidated the essen- Diagnostic Centre Ltd. There are no patents, tial role of Oyster mushroom’s extracts in lowering blood cholesterol[3], scavenging free radi- products in development or marketed products to declare. This does not alter our adherence to all the cal[4] and inhibiting enzyme activity[5]. In recent years, there has been an increasing interest PLOS ONE policies on sharing data and materials. in the extraction and characterizations of polysaccharides from edible mushrooms. No previ- ous study has investigated on polysaccharides extraction and study of biological activities from Pleurotus ostreatus. Polysaccharides, are a kind of macromolecule sugar polymer, found in a large number of plants and fungus [6–8]. Specialists have found that polysaccharides extri- cated from various plants possess compelling properties [9–11]. These properties include anti- tumor, antiviral activity, immunomodulation, antilipidemic impact and oxidation protection [12–14]. In order to harbor the full potential of the polysaccharides, the polysaccharide must be proficiently extracted from Oyster mushrooms. The proficient extraction will enable the advancement and use of this fungus on a wider scale. In recent times, there have been a few investigations centered on the extraction of polysaccharide from Oyster mushrooms. Extract- ing polysaccharide with hot water is the most widely recognized method utilized in the agri- cultural industry, because of its easy accessibility and safety [15]. By and large, extraction proficiency of polysaccharide is influenced by several factors: extraction time, extraction tem- perature and water to material proportion. Their effects on the polysaccharide yield may be autonomous. The response surface methodology (RSM) determines the optimum conditions for extracting polysaccharides. The RSM procedure enables the interplay of several factors relating to polysaccharide yield to be investigated simultaneously. RSM creates a polynomial equation and an inclined model by multiple regression fitting of response surface examination. The RSM enables the extent of the polytomy factors in influencing polysaccharide yield and to be assessed, and their optimum level to maximize yield[16,17]. RSM has been effectively used to optimize different biochemical and biotechnological processes[18]. Box-Behnken Design, a kind of RSM, was used to find optimum extraction parameters of polysaccharides extraction from Pleurotus ostreatus. The objective of this investigation was to optimize the conditions for polysaccharide extraction from Pleurotus ostreatus (Jacq.) P. Kumm and to find the ideal extraction condition (water to crude material ratio, extraction time and extraction tempera- ture) utilizing response surface methodology. The in vitro antioxidant activities of POP were assessed on the base of 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity and 2,2-azino-bis-3-ethyl-benzothiazoline-6-sulfonic acid (ABTS) radical scavenging activity. Additionally, the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay was used to measure in vitro role of cytotoxicity in Murine Lymphoid cancer cell line (EAC cell line). Materials and methods Materials and reagents Fresh fruiting bodies of Pleurotus ostreatus (Jacq.) P. Kumm were collected and authenticated from the Mushroom Development Institute, which belongs to the Department of Agriculture Extension, Ministry of Agriculture, Savar, and Dhaka-1340 in Bangladesh. Monosaccharide (D-glucose), vitamin C, 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2-azino-bis-3-ethyl-ben- zothiazoline-6-sulfonic acid (ABTS), thiazolyl blue tetrazolium bromide (MTT), trichloro ace- tic acid (TCA), alfa-Amylase, bovine serum albumin (BSA), acetonitrile, 1-phenyl-3-methyl- PLOS ONE | https://doi.org/10.1371/journal.pone.0209371 January 3, 2019 2 / 17 Optimization of extraction of antioxidant polysaccharide 5-pyrazolone (PMP), RPMI-1640 were purchased from Sigma Co., St. Louis, MO, USA, while sephadex-G-100 and DEAE cellulose were acquired from Pharmacia, Co., Sweden. Murine Lymphoid cancer cell line or Ehrlich ascites carcinoma (EAC) cells were obtained with the courtesy of Indian Institute of Chemical Biology, Kolkata, India. Dimethyl sulfoxide, chloro- form, butanol, sodium chloride, phenol, sulfuric acid, uronic acid, glucuronic acid were pur- chased from local agents (Diatech, Alfa Scientific Co. and Bio-trade BD). Extraction of Pleurotus ostreatus (Jacq.) P. Kumm polysaccharide (POP) POP was extracted by hot distilled water based on the intended conditions. After centrifuga- tion (3000 rpm for 20 min), 1/3 of the original volume of the main supernatant was achieved by hot air concentration at 56˚C. The starch in the concentrated solution was removed by alfa- amylase at 60˚C. An adequate volume of ethanol was added to the concentrated extraction solution and mixed well to precipitate the POP at 4˚C for 12 h. Then, the precipitates were gathered by centrifugation (3000 rpm for 20 min) and washed consecutively with acetone, ether and ethanol. The POP extract was liquefied with distilled water and protein was removed with Sevag reagent (chloroform: normal butanol, 4:1, v/v). The protein fractions from POP extract were removed by dialysis (MWCO 1400 Da, Union Carbide) and finally POP was col- lected by freeze drying. The POP yield was calculated using the following formula: Wpops Yð%Þ ¼ � 100 Wsample Where, Wpops and Wsample are the weights of POP and Pleurotus ostreatus (Jacq.) P. Kumm powder used respectively. Single-factor experiment design In this experiment, the following three variables were investigated: water to sample ratio (mL/ g), extraction time (minutes), and extraction temperature ( C). Their variable effects on the POP extraction were assessed. Each sample was extracted according to the above mentioned procedure for polysaccharide extraction. The effects of three different conditions on POP yield were compared by one-way analysis of variance (ANOVA) using SPSS 21.0 (SPSS Inc., Chi- cago, IL). Design of Box-Behnken The Box-Behnken design (BBD) was used to obtain the experiment design, analysis of results and regression models. Through the single factors experiment, the appropriate ranges of water to raw material powder (X ), extraction time (X ) and extraction temperature (X ) were deter- 1 2 3 mined and then the BBD of POP yield was carried out. The entire design was made up of 17 experimental runs, which were carried out in a random sequence. The three variables at the three levels were coded as -1, 0 and +1. Here, a second-order—polynomial formula was used to optimize the extraction conditions of the POP yield (the response) as follows [17]: P P P P 3 3 2 3 Y ¼ A þ A X þ A X þ A X X o i¼1 i i i¼1 ii i i¼1 j¼iþ1 ij i j Where, Y is the dependent variable and X X are the independent variables and A , A , A , i j o i ii and A are the regression coefficients of the independent variables that were estimated by the ij model for intercept, linear, quadratic, and interaction terms, respectively. Design Expert 8.0.5.0 was utilized for statistical investigation of fluctuation for every response and anticipat- ing the ideal conditions for the extraction of POP [19,20]. PLOS ONE | https://doi.org/10.1371/journal.pone.0209371 January 3, 2019 3 / 17 Optimization of extraction of antioxidant polysaccharide Purification of polysaccharide from Pleurotus ostreatus (POP) The dried POP was also deproteinated using the Sevage method[21] and 1.0 g of POP was dis- solved in 10 mL dH 0 and applied to a DEAE-52 cellulose column (3.5 cm × 20 cm), and eluted with deionized water, followed by different gradient of NaCl solutions (0 to 0.5 M) at a flow rate of 0.8 mL/min. The eluates were collected in 5 mL fractions automatically and poly- saccharide content was measured using phenol-sulfuric acid method[22]. The main identical fractions containing polysaccharide were collected and concentrated and further fractionated by using gel filtration chromatography on Sephadex G-100 column (110 cm × 1.6 cm) at 0.1 mL/min and eluted with distilled water. 4 mL fractions were collected per tube and polysac- charide content of each tube was measured using phenol-sulfuric acid method[22]. The major fractions were combined and lyophilized to yield POP, which was used for further studies. Analytical method validation The polysaccharide content in POP extract was analyzed by phenol-sulfuric acid method using glucose as the standard. The regression equation was Y = 0.0231x – 0.046 and the correlation coefficient was 0.996. A linear relationship between the polysaccharide content and absorbance was detected within the range of 0–80 μg/mL, identified at 490 nm. The precision was appraised by analyzing the reproducibility and intermediate precision variations at three dif- ferent concentrations with five replicates. The accuracy was evaluated with the pointed rescue test. Chemical composition analysis The phenol-sulfuric acid method was used to determine the total polysaccharide content of POP extract, using D-glucose as the standard[22]. Protein content of POP extract was deter- mined by Lowery method, using BSA as the standard[23]. The m-hydroxybiphenyl assay was used to measure uronic acid content of POP extract, using glucuronic acid as reference mate- rial [24]. The moisture content of POP was measured, referring to the method outlined in Kong et al (22). The pH values of the POP at the 2 mg/mL of POP were determined using a pH meter and the relative viscosity of POP (10 mg/mL) was determined using viscometer (Thermo Scientific HAAKE, 388–0100) at 25˚C[25]. Monosaccharide composition The monosaccharide composition of the POP was determined using HPLC by methods deter- mined by Chai, Y et al[26] with minor modifications. POP samples (2 mg) were hydrolyzed with trifluoroacetic acid (0.5 mL of TFA, 2 mol/L) in a wrapped flask occupied with nitrogen at 120˚C and after 2 h of hydrolysis, the excess TFA from the flask was removed by evaporation with ethanol at 45˚C. Monosaccharide standards and POP hydrolysate were then added to 0.5 mL of PMP (1-phenyl-3-methyl-5-pyrazolone, 0.5 mol/L in methanol) and 0.5 mL NaOH (0.3 mol/L) for derivatization at 70˚C for 30 min. After centrifugation (10,000 rpm, for 5 min), the supernatant was assorted with 0.05 mL HCl (0.3 mol/L), and the mixture was mined with chlo- roform to eliminate surplus PMP. The aqueous layer was sieved using a 0.22 μm membrane and used for monosaccharide composition analysis of POP by HPLC system, which was fur- nished with a UV detector (245 nm) and an Amethyst C18 column (4.6 mm × 250 mm, 5 μm, Sepax, USA). The mobile phase of HPLC system was a mixture of PBS (Phosphate buffered saline, 0.1 mol/L, pH 7) and acetonitrile (80:20, v/v) with a flow rate of 1 mL/min. The column was reserved at 25˚C, and the injection volume was 10 μL. PLOS ONE | https://doi.org/10.1371/journal.pone.0209371 January 3, 2019 4 / 17 Optimization of extraction of antioxidant polysaccharide UV-Vis and FT-IR analysis The purified POP was re-dissolved in water to achieve a concentration of 0.25 mg/mL. The UV-Vis spectra of the solution were recorded at 190–600 nm, using spectrophotometer (Dou- ble beam UV-visible light spectrophotometer, China) and the functional groups of POP were -1 acknowledged using FT-IR spectrophotometer, within 4000–500 cm in KBr pellets. DPPH radical scavenging activity DPPH radical scavenging activity of POP was determined according to a modified method of Sharma et al 2016 [27]. Vitamin C was used as the positive control. Briefly, 50 μL of each POP sam- ples ranging in concentrations between 0.2 to 1.2 mg/mL were prepared by dissolving POP solid in distilled water and then adding a 5 mL (0.004% w/v in ethanol) solution of DPPH. The mixture was vortexed and shielded with Aluminum foil and incubated for 30 min in room temperature in the dark environment. The blank control was 80% ethanol and measurements were taken in tripli- cate. After 30 minutes of incubation, discoloration of reaction was measured using spectropho- tometer at 517 nm. DPPH scavenging effect was determined using the following equation; ½A ðA A Þ�� 100 o 1 2 DPPH scavenging effectð%Þ ¼ Where, Ao is the absorbance of the control; A is the absorbance of the POP sample; A is 1 2 the absorbance of the POP sample under similar condition to A excepting ethanol instead of the DPPH. The effective concentration for 50% of free radical scavenging activity of positive control (Vitamin C) and POP samples were determined using standard curve (r = 0.9994). ABST radical scavenging assay The ABST radical scavenging of POP was carried out using the method of P. Li et al 2011[28]. Vitamin C was used as the positive control. The ABTS radical cation was produced via the reaction between ABTS solution (5 mL, 7 mM) and K S O aqueous solution (1 mL, 15 mM) 2 2 8 following 12 h in the dark. The ABTS radical cation solution was then diluted with deionized water to yield an absorbance of 0.70 at 734 nm. The ABTS radical cation answer (3 mL) was added to 0.75 mL POP solution (dissolved in distilled water) at varying concentrations (0.1, 0.2, 0.4, 0.6, 0.8, 1.0 and 1.2 mg/mL). After 15 minutes of reaction, the absorbance was mea- sured at 734 nm. The scavenging endeavor of POP in opposition to ABTS radical was evalu- ated with the aid of the accompanied equation: ½A ðA A Þ�� 100 0 1 2 ABST radical scavenging activity ð%Þ ¼ Where, A is the absorbance of the control; A is the absorbance of the POP solutions; and o 1 A is the absorbance of all the reaction reagents (Except POP solutions). Determination of cytotoxic activity of POP using MTT assay Murine Lymphoid Cancer Cell Line or EAC (Ehrlich ascites carcinoma), which are murine lym- phoid cancer cell lines were cultivated in RPMI-1640 medium (10% foetal calf serum, and 1% (v/v) penicillin-streptomycin) in a humidified atmosphere of 5% CO at 37˚C. EAC cells were planted (5 ×10 cells/well in 200 μL RPMI 1640 media) in 96-wells, with various concentrations of purified POP (0.001–1000μg/mL) added and incubated for 24, 48, and 72 hrs at 37˚C in 5% CO atmosphere. After the end of incubation, the medium was removed and wells were washed with 10 mM PBS and MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide) (20 μL, PLOS ONE | https://doi.org/10.1371/journal.pone.0209371 January 3, 2019 5 / 17 Optimization of extraction of antioxidant polysaccharide 5 mg/mL MTT in PBS) was added and hatched for 4 h at 37˚C. 100 μL of acidic isopropanol was added into each well and incubation continued for 3 h more. The absorbance was measured at 570 nm and the proliferation rate (PR) was determined using the following formulae: Tabs PR ¼ � 100 Cabs Where, Tabs and Cabs are the absorbance of test and control. Cytotoxicity of POP on EAC cells was determined as cells growth inhibition rate (IR): IR ¼ 100 PR Statistical analysis GraphPad Prism 6 (Pad Software, Inc., USA) was used to analyze data, and results are expressed as mean±SD. Error bars were expressed as 95% CI. All experiments were performed at least three times. P<0.05 was considered to be statistically significant, and P<0.01 was regarded as highly statistically significant. Design Expert Software (Version 9.0.4.1, State-Ease Inc., USA) was used to design of Box-Behnken and single-factor experiment. Results and discussion Impact of water to raw material ratio on the POP yield The polysaccharide was extracted at different water to raw materials ratios, with a fixed extrac- tion time and extraction temperature at 60˚C and 70 min respectively. This allowed the influ- ence of water to raw materials ratio on the percent yield of POP to be evaluated (Fig 1A). Impact of extraction time on the POP yield Extraction time is a vital parameter, influencing the extraction yield of polysaccharide. To research the impact of extraction time on the extraction yield of POP (Fig 1B), the polysaccha- ride was removed at various extraction time (40 50, 60, and 70 min), and the water to crude material proportion and extraction temperature were set as 25 mL/g and 70˚C, separately. The extraction yield of POP essentially increased from 3.44% to 5.23% when the extraction time increased from 40 to 60 min (P < 0.05). However, when this extraction time was exceeded, the extraction yield of POP only decreased marginally. Consequently, the extraction time of 60 min was selected for the BBD analysis. Impact of extraction temperature on the POP yield. To assess the impact of extraction temperature on the yield of POP (Fig 1C), the water to crude material proportion and extrac- tion time were kept at 25 mL/g and 60 min, individually. The extraction yield of POP increased from 3.49% to 5.28% when the temperature increased from 50 to 80˚C, and the POP yield at 70˚C was essentially higher than those of 50, 60 and 80˚C (P < 0.05). In any case, the extrac- tion yield of POP at 80˚C was marginally lower than that of 70˚C (P > 0.05). For this reason, the extraction temperature of 70˚C was chosen selected for the BBD analysis. Optimization of polysaccharide extraction from Pleurotus ostreatus Based on the single-factor tests, 17 runs were performed to streamline the extraction states of POP. The POP yields (%) of the 17 runs designed to assess the 3 autonomous factors including water to crude material proportion (X1), extraction time (X2) and extraction temperature (X3) are exhibited in Table 1. The POP yields range from 3.44% to 5.28%. The percentage of POP PLOS ONE | https://doi.org/10.1371/journal.pone.0209371 January 3, 2019 6 / 17 Optimization of extraction of antioxidant polysaccharide Fig 1. Effect of water to raw material ratio (A), extraction time (B), and extraction temperature (C) on POP yield. Bar charts with different lowercase letters significantly differ (p<0.05). POP, Pleurotus ostreatus polysaccharide. The % yield of POP increased from 3.57% to 4.36% and increased to 5.21% with an increasing water to raw materials ratio, from 15 mL/g to 25 mL/g. However, when the water to crude material proportion increased to 30 mL/g there was a reduction in the extraction yield of POP. As the water to crude material proportion increased, the disintegration of polysaccharide from the materials essentially increased, and the extraction yield of POP increased. In this way, the water to crude material proportion of 25 mL/g was selected for the BBD analysis. https://doi.org/10.1371/journal.pone.0209371.g001 nd yield (% Y) was described by the 2 order polynomial Eq (1) as follow: Y ð%Þ ¼ 58:88þ 1:52725 X þ 0:677375 X þ 0:649125 X þ 0:000775 X X 0:001475 1 2 3 1 2 ð1Þ 2 2 2 X X þ 0:000262X X 0:028825 X 0:005881 X 0:004181 X 1 3 2 3 1 2 3 Where, Y is the POP yield; X1, X2 and X3 are the factors of water to crude material propor- tion, extraction time and extraction temperature, individually. The aftereffect of ANOVA investigation is displayed in Table 2. The importance of co- efficients can be verified by their respective P-values, and a smaller P-value signifies a more significant comparing coefficient. In Table 2, the model P-value was less than 0.0001, which PLOS ONE | https://doi.org/10.1371/journal.pone.0209371 January 3, 2019 7 / 17 Optimization of extraction of antioxidant polysaccharide Table 1. Box-Behnken experiment design and the results of the POP yield. Run Water to raw material ratio (X ) (mL/g) Extraction time (X ) (min) Extraction temperature (X ) ( C) POP yield (%) 1 2 3 Actual value Predicted value 1 25 60 70 5.41 5.39 2 30 50 70 4.105 4.09 3 20 70 70 3.98 3.99 4 30 70 70 4.365 4.36 5 25 60 70 5.4 5.39 6 25 60 70 5.395 5.39 7 20 50 70 3.875 3.88 8 30 60 80 4.735 4.75 9 25 60 70 5.425 5.39 10 25 70 80 4.94 4.93 11 25 50 80 4.695 4.69 12 30 60 60 4.045 4.04 13 25 50 60 3.88 3.89 14 25 60 70 5.32 5.39 15 20 60 60 3.62 3.60 16 25 70 60 4.02 4.02 17 20 60 80 4.605 4.60 POP, Pleurotus ostreatus polysaccharide. https://doi.org/10.1371/journal.pone.0209371.t001 demonstrates that the relapse display for POP yield was highly significant. Furthermore, the P- estimations of the straight coefficients (X , X and X ), and the quadratic term coefficients (X , 1 2 3 1 2 2 X and X ) were less than 0.01, which shows the significant effects of these three variables on 2 3 the extraction yield of POP. The P-estimation of the communication term coefficient (X X ) 1 3 was observed to be lower than 0.05, showing the critical impact of this coefficient on the extrac- tion yield of POP. The other term coefficients (X X , X X ) were not significant (P > 0.05). The 1 2 2 3 ANOVA shows that water to crude material proportion (X ), extraction time (X ) and extrac- 1 2 tion temperature (X ) were significant single factors influencing the extraction yield of POP. The assurance coefficient (R ) of the model was 0.9988, showing that 0.12% of aggregate varieties couldn’t be clarified by the model. However the balanced assurance (adj-R2) esteem (0.9974) showed that a large portion of the POP yield variety could be anticipated by the relapse demonstrate. The coefficient of variety (C.V.) was at low esteem (0.7199%), demon- strating the investigation information and predicated information were comparative. As per the exploratory information, the lack of fit P-value (0.8978) was more than 0.05, proposing that the lack of fit was unimportant in identifying the pure error. The Adeq Precision (70.6326) in the present model was a sufficient flag, which demonstrated that the model could be utilized to explore the plan space. Optimization of extraction conditions The impacts of three variables on POP yield (%Y) were analyzed with three-dimensional response surface plot and contour plot, as shown in Fig 2. In this experiment, two variables were presented by response surface plot and contour plot whereas a variable was fixed at the 0 level. For water to crude material proportion and extraction time (Fig 2B), the contour plot is circular, which dem- onstrates that the shared connection between water to crude material proportion and extraction time is not significant (P > 0.05). The comparative pattern (Fig 2F) was found for extraction time PLOS ONE | https://doi.org/10.1371/journal.pone.0209371 January 3, 2019 8 / 17 Optimization of extraction of antioxidant polysaccharide and extraction temperature (P > 0.05). The elliptical contour plot (Fig 2D) showed the common cooperation between water to crude material proportion and extraction temperature was note- worthy (P < 0.05). These outcomes were in concurrence with the qualities in Table 2. Diagnostics accuracy Diagnostics accuracy is crucial when determining the appropriateness of the model for the genuine framework. Four diagnostics graphs for model accuracy are displayed in Fig 3. Fig 3D demonstrates the anticipated and the genuine test esteems. The spots of the anticipated and real esteems indicates ordinary circulation and are near the 45˚ line, confirming that the model has a decent adjustment. Fig 3A demonstrates a normal % probability plot of residuals for the ordinariness presumption; the lingering plot that moves towards a straight line, demon- strating that the typicality supposition was appropriate. The inside studentized residuals versus anticipated esteems are shown in Fig 3B. The plots of the inside studentized residuals scattered arbitrarily demonstrate that the first fluctuation was steady for all esteems. The inside studen- tized residuals versus trial run numbers are displayed in Fig 3C, and every one of the focuses are situated inside a restricted range. All information shows that the reaction surface model is connected to the POP extraction, and that the model is critical and exact. Validation of the model The ideal extraction conditions for the extraction of POP comprised of a water/crude material proportion of 26.04 mL/g, an extraction time of 62.08 minutes, and an extraction temperature 70.5˚C, which were determined by utilizing the second-order quadratic equation of the model by fathoming the regression equation and dissecting the response surface. The greatest antici- pated estimation of POP yield was 5.34%. With a specific end goal to verify the anticipated Table 2. ANOVA analysis for the quadratic model of POP extraction. Source Sum of Squares df Mean Square F-value p-value � � Model 6.59 9 0.7319 674.13 < 0.0001 � � A-X1 0.1711 1 0.1711 157.60 < 0.0001 � � B-X2 0.0703 1 0.0703 64.76 < 0.0001 � � C-X3 1.45 1 1.45 1338.76 < 0.0001 AB 0.0060 1 0.0060 5.53 0.0509 � � AC 0.0218 1 0.0218 20.04 0.0029 BC 0.0028 1 0.0028 2.54 0.1551 2 � � A 2.19 1 2.19 2013.91 < 0.0001 � � B 1.46 1 1.46 1341.41 < 0.0001 2 � � C 0.7361 1 0.7361 678.01 < 0.0001 Residual 0.0076 7 0.0011 Lack of Fit 0.0010 3 0.0003 0.1905 0.8978 Pure Error 0.0066 4 0.0017 Cor Total 6.59 16 R 0.9988 Adjusted R 0.9974 Predicted R 0.9961 Adeq Precision 70.6326 C.V. % 0.7199 � � p<0.001 was measured to be highly significant. https://doi.org/10.1371/journal.pone.0209371.t002 PLOS ONE | https://doi.org/10.1371/journal.pone.0209371 January 3, 2019 9 / 17 Optimization of extraction of antioxidant polysaccharide Fig 2. Response surface and contour plots for % yield of POP from Pleurotus ostreatus (Jacq.) P. Kumm. (A-B) Water to raw materials and extraction time (C-D) Water to raw materials and extraction temperature and (E-F) Extraction time and extraction temperature. https://doi.org/10.1371/journal.pone.0209371.g002 PLOS ONE | https://doi.org/10.1371/journal.pone.0209371 January 3, 2019 10 / 17 Optimization of extraction of antioxidant polysaccharide Fig 3. Diagnostic plots for the Box-Behnken model adequacy. (A) Normal plot of residuals, (B) plot of internally studentized residuals vs. predicted response, (C) Residual vs. run and (D). Predicted vs. actual. https://doi.org/10.1371/journal.pone.0209371.g003 estimation of POP yield and the applicability of the model, three parallel tests were performed under the previously mentioned ideal extraction conditions. The yield of POP was 5.32 ± 0.12% (n = 3), which drew nearer the anticipated estimation of 5.34%. The outcomes demon- strated that the RSM was fitting for enhancing the conditions for POP extraction, and the regression model was precise and relevant for extricating POP. PLOS ONE | https://doi.org/10.1371/journal.pone.0209371 January 3, 2019 11 / 17 Optimization of extraction of antioxidant polysaccharide Fig 4. UV-spectrophotometric analysis of partially purified polysaccharides (POP). A) Before proteolysis and B) after removing of -1 protein and nucleic acids. The peaks in the range of 950–1200 cm indicate the presence of polysaccharides, indicating the presence of -1 glycosidic bonds by 1,150–1,160 cm . https://doi.org/10.1371/journal.pone.0209371.g004 UV and FT-IR analysis Fig 4A and 4B show the UV-visible absorption spectra of polysaccharide. Fig 4B exhibits that polysaccharide fraction have no absorption at 260 and 280 nm in the UV spectrum, signifying the absence of nucleic acid and protein. The IR spectra of POP are shown in Fig 5. Chromatographic analysis A portion of POP (10g) was separated through a DEAE-Cellulose column and three portions were collected according to the chromatography profile (Fig 6A). A portion of POP was Fig 5. FT-IR spectral analysis of the polysaccharide (POP). The spectra indicate a noticeable C-H stretching and -1 bending vibration absorption in the range of 2929–2939 cm , which are characteristic peaks of the polysaccharide[29]. -1 In the marked region, the peak in the region of 1230 cm is due to C-C stretching vibration and the peaks towards -1 1100 cm suggesting that the peak was related to the starching vibration of C-O. https://doi.org/10.1371/journal.pone.0209371.g005 PLOS ONE | https://doi.org/10.1371/journal.pone.0209371 January 3, 2019 12 / 17 Optimization of extraction of antioxidant polysaccharide Fig 6. Chromatographic profile of POP. A) The elution profile of POP on DEAE-cellulose column and B) gel filtration elution curve of POP. POP, Pleurotus ostreatus polysaccharide. https://doi.org/10.1371/journal.pone.0209371.g006 further purified on gel filtration chromatography and a polysaccharide (POP) was obtained (Fig 6B). Chemical and sugar composition The monosaccharide composition analysis indicates that POP was composed of only glucose, confirming that it was a glucan. Table 3 shows the chemical and sugar composition of POP. The low moisture (14.11%) and viscosity (1.13) contents of POP confirmed that it is suitable for large scale production. The gel filtration analysis results showed that POP was a single reg- ular peak (Fig 6B), indicating that it was an identical polysaccharide. The molecular weight of POP was calculated as 154.6 × 10 k. In vitro antioxidant activity of POP obtained at optimum temperature, time and water to raw material ratio The scavenging activity of POP on DPPH radical assay has been extensively used to determine the antioxidant capacity of POP due to its easy availability and the immovability of DPPH Table 3. Chemical and sugar composition of POP. POP Total sugar content (%) 97.6±1.33 Protein content (%) 0.5±0.03 Uronic acid content (%) nd pH 7.41±0.04 Moisture content (%) 14.11±0.27 Relative viscosity 1.13±0.02 MW (kDa) 154.6 Monosaccharide Glc 100.0 composition (%) a Ara nd Gal nd Man nd Rha nd Values are expressed as mean±standard deviation. nd , Not Detected. POP, Pleurotus ostreatus polysaccharide. Glc, Glucose; Ara, Arabinose; Gal, Galactose; Man, Manose; Rha, Rhamnose https://doi.org/10.1371/journal.pone.0209371.t003 PLOS ONE | https://doi.org/10.1371/journal.pone.0209371 January 3, 2019 13 / 17 Optimization of extraction of antioxidant polysaccharide Fig 7. Antioxidant activities of the POP. (A) DPPH radical scavenging activity (p<0.001, EC = 1038.38 μg/mL, R = 0.8313) and (B) ABTS radical scavenging activity (p<0.001, EC = 824.37 μg/mL, R = 0.8223). POP, Pleurotus ostreatus polysaccharide. https://doi.org/10.1371/journal.pone.0209371.g007 radicals [30]. DPPH is a steady radical that focuses on nitrogen and demonstrates a greatest consumption at 517 nm in methanol [31,32]. The scavenging ability of POP on DPPH radical and ABTS radical was shown in Fig 7A and 7B. The results show the DPPH radical scavenging activities of POP increase gradually as the concentration of polysaccharides increases from 0.2 to 5.0 mg/mL. The POP demonstrates a concentration dependent free radical scavenging activity, with a significant activity found at higher concentrations. The outcomes demonstrate that the scavenging activity of DPPH in POP is low compared to that of vitamin C below 1.4 mg/mL (P<0.05) and in case of ABTS that concentration is 1.6 mg/mL (P<0.05). DPPH radicals scavenging by POP and vitamin C is equal at 1.2 mg/mL (72.23%) while at 1.4 mg/mL (81.02%) POP exhibit the same ABTS radi- cals scavenging activity, in comparison to vitamin C. Polysaccharides from plants and fungus have been perceived to be a promising source of antioxidant molecules. Numerous investiga- tions have demonstrated that polysaccharides enhance the action of cell reinforcement pro- teins in the body, scavenge free radicals, and repress lipid oxidation [33]. In vitro cytotoxic measure of POP using MTT assay The cytotoxic properties of purified POP was assessed by using the EAC cell line. The POP −3 3 extract at 10 to 10 μg/mL exhibit dose-and-time dependent inhibitory effects on the prolif- eration of EAC cells, with greater than a 90% suppression at the highest concentration (72 h). Fig 8 shows the dose-and-time dependent cytotoxic effects of POP on EAC cells. Conclusion The ideal polysaccharide extraction states of water/crude material proportion, ultrasound time, and ultrasound control were determined utilizing the RSM of BBD in view of single fac- tor tests. The RSM of BBD could effectively improve the extraction states of POP, and the regression model was pertinent for removing POP. The antioxidant activity of DPPH and ABTS radicals’ in vitro measures demonstrate that POP has cancer prevention agent properties PLOS ONE | https://doi.org/10.1371/journal.pone.0209371 January 3, 2019 14 / 17 Optimization of extraction of antioxidant polysaccharide Fig 8. Cytotoxicity of purified POP on murine lymphoid cancer cell line (EAC cell line). POP, Pleurotus ostreatus polysaccharide. https://doi.org/10.1371/journal.pone.0209371.g008 that increase with the increasing convergence of the polysaccharide arrangement. Hence, these outcomes suggest that polysaccharide from Pleurotus ostreatus (Jacq.) P. Kumm ought to be further investigated as a novel and potential natural antioxidant, which may prove a useful therapeutic drug. Additionally the purification and structure identification are improve the overall understanding of polysaccharide. Supporting information S1 Dataset. Impact of three variables on the POP extraction from Pleurotus ostreatus. (XLSX) S2 Dataset. Antioxidant activities of the POP. (XLSX) Acknowledgments The authors received no specific funding for this work. Funder provided support in the form of salaries for authors (MPK, MSI, RP, SD, RIT, and MR), but did not have any additional role in the study design, data collection & analysis, decision to publish, and preparation of the manuscript. PLOS ONE | https://doi.org/10.1371/journal.pone.0209371 January 3, 2019 15 / 17 Optimization of extraction of antioxidant polysaccharide Author Contributions Conceptualization: Md. Moyen Uddin Pk, Mohammad Sayful Islam. Data curation: Md. Moyen Uddin Pk, Mohammad Sayful Islam. Formal analysis: Md. Moyen Uddin Pk, Rumana Pervin. Investigation: Md. Moyen Uddin Pk, Mohammad Sayful Islam, Subhajit Dutta, Rabiul Islam Talukder. Methodology: Subhajit Dutta, Rabiul Islam Talukder. Resources: Md. Moyen Uddin Pk. Software: Md. Moyen Uddin Pk, Subhajit Dutta. Supervision: Matiar Rahman. Visualization: Md. Moyen Uddin Pk. Writing – original draft: Md. Moyen Uddin Pk. Writing – review & editing: Matiar Rahman. References 1. Fernandes A, Barros L, Martins A, Herbert P, Ferreira ICFR. Nutritional characterisation of Pleurotus ostreatus (Jacq. ex Fr.) P. Kumm. produced using paper scraps as substrate. Food Chem. England; 2015; 169: 396–400. 2. Jin Z, Li Y, Ren J, Qin N. 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Free Radic Biol Med. 2006; 41: 1187–1190. https://doi.org/10.1016/j. freeradbiomed.2006.07.017 PMID: 17015164 PLOS ONE | https://doi.org/10.1371/journal.pone.0209371 January 3, 2019 17 / 17 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png PLoS ONE Public Library of Science (PLoS) Journal

Optimization of extraction of antioxidant polysaccharide from Pleurotus ostreatus (Jacq.) P. Kumm and its cytotoxic activity against murine lymphoid cancer cell line

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Copyright: © 2019 Uddin Pk et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: All relevant data are within the paper and its Supporting Information files: the S1 Dataset: Impact of three variables on the POP extraction from Pleurotus ostreatus; S2 Dataset: Antioxidant activities of the POP. Funding: The authors received no specific funding for this work. RIT is employed by Popular Diagnostic Centre Ltd. Popular Diagnostic Centre Ltd. provided support in the form of salary for author RIT, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific role of this author is articulated in the ‘author contributions’ section. Competing interests: RIT is employed by Popular Diagnostic Centre Ltd. There are no patents, products in development or marketed products to declare. This does not alter our adherence to all the PLOS ONE policies on sharing data and materials.
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Abstract

Citation: Uddin Pk M.M, Islam MS, Pervin R, Dutta S, Talukder RI, Rahman M (2019) Optimization of The purpose of this study was to optimize the extraction method for polysaccharide from the extraction of antioxidant polysaccharide from fruiting bodies of Pleurotus ostreatus (Jacq.) P. Kumm and to assess the antioxidant and Pleurotus ostreatus (Jacq.) P. Kumm and its cytotoxic activity against murine lymphoid cancer cytotoxic potentials of polysaccharide. In this investigation, polysaccharides from Pleurotus cell line. PLoS ONE 14(1): e0209371. https://doi. ostreatus (Jacq.) P. Kumm were extricated by utilizing the hot water. One-single factor and org/10.1371/journal.pone.0209371 response surface methodology was established to optimize the extraction conditions for Editor: David A. Lightfoot, College of Agricultural polysaccharide from Pleurotus ostreatus (Jacq.) P. Kumm. Examination of antioxidant activ- Sciences, UNITED STATES ity of Pleurotus ostreatus polysaccharide (POP) was directed by utilizing 2, 2-diphenyl-1- Received: July 24, 2018 picrylhydrazyl (DPPH) and 2, 2-azino-bis-3-ethyl-benzothiazoline-6-sulfonic acid (ABTS) Accepted: December 4, 2018 techniques. Cytotoxicity of POP was evaluated using an MTT assay. The experimental data were fitted to a quadratic equation utilizing multiple regression investigations, and the ideal Published: January 3, 2019 conditions were as per the following: water/crude material proportion, 26.04 mL/g; an extrac- Copyright:© 2019 Uddin Pk et al. This is an open tion time of 62.08 minutes; and an extraction temperature 70.5˚C. Under such conditions, access article distributed under the terms of the Creative Commons Attribution License, which the polysaccharide yield was 5.32± 0.12% with the anticipated yield. POP showed good permits unrestricted use, distribution, and 2 scavenging activity against DPPH radical (p<0.001, EC = 1036.38μg/mL, R = 0.8313) reproduction in any medium, provided the original and ABTS radicals (p<0.001, EC = 824.37μg/mL, R = 0.8223), with a dose (p<0.001)- author and source are credited. and-time (p<0.001) dependent cytotoxic potential on Ehrlich ascites carcinoma cell line in Data Availability Statement: All relevant data are vitro. This demonstrated that polysaccharides (POP) had certain cancer prevention agent within the paper and its Supporting Information exercises. In this manner, these examinations give reference to additionally research files: the S1 Dataset: Impact of three variables on the POP extraction from Pleurotus ostreatus; S2 and reasonable improvement of Pleurotus ostreatus (Jacq.) P. Kumm polysaccharide and Dataset: Antioxidant activities of the POP. POP may prove a useful therapeutic agent, due to its robust antioxidant and cytotoxic Funding: The authors received no specific funding activity. for this work. RIT is employed by Popular Diagnostic Centre Ltd. Popular Diagnostic Centre Ltd. provided support in the form of salary for author RIT, but did not have any additional role in PLOS ONE | https://doi.org/10.1371/journal.pone.0209371 January 3, 2019 1 / 17 Optimization of extraction of antioxidant polysaccharide the study design, data collection and analysis, Introduction decision to publish, or preparation of the P. ostreatus, the oyster mushroom, is a consumable mushroom, generally growing all over the manuscript. The specific role of this author is world. Currently, it serves as a rich source of nourishment[1]. Oyster mushrooms are a rich articulated in the ‘author contributions’ section. source of vitamins, amino acids, and minerals, as well as antioxidants[2]. In addition to their Competing interests: RIT is employed by Popular role in nutrition, they possess medicinal properties. Recent research has elucidated the essen- Diagnostic Centre Ltd. There are no patents, tial role of Oyster mushroom’s extracts in lowering blood cholesterol[3], scavenging free radi- products in development or marketed products to declare. This does not alter our adherence to all the cal[4] and inhibiting enzyme activity[5]. In recent years, there has been an increasing interest PLOS ONE policies on sharing data and materials. in the extraction and characterizations of polysaccharides from edible mushrooms. No previ- ous study has investigated on polysaccharides extraction and study of biological activities from Pleurotus ostreatus. Polysaccharides, are a kind of macromolecule sugar polymer, found in a large number of plants and fungus [6–8]. Specialists have found that polysaccharides extri- cated from various plants possess compelling properties [9–11]. These properties include anti- tumor, antiviral activity, immunomodulation, antilipidemic impact and oxidation protection [12–14]. In order to harbor the full potential of the polysaccharides, the polysaccharide must be proficiently extracted from Oyster mushrooms. The proficient extraction will enable the advancement and use of this fungus on a wider scale. In recent times, there have been a few investigations centered on the extraction of polysaccharide from Oyster mushrooms. Extract- ing polysaccharide with hot water is the most widely recognized method utilized in the agri- cultural industry, because of its easy accessibility and safety [15]. By and large, extraction proficiency of polysaccharide is influenced by several factors: extraction time, extraction tem- perature and water to material proportion. Their effects on the polysaccharide yield may be autonomous. The response surface methodology (RSM) determines the optimum conditions for extracting polysaccharides. The RSM procedure enables the interplay of several factors relating to polysaccharide yield to be investigated simultaneously. RSM creates a polynomial equation and an inclined model by multiple regression fitting of response surface examination. The RSM enables the extent of the polytomy factors in influencing polysaccharide yield and to be assessed, and their optimum level to maximize yield[16,17]. RSM has been effectively used to optimize different biochemical and biotechnological processes[18]. Box-Behnken Design, a kind of RSM, was used to find optimum extraction parameters of polysaccharides extraction from Pleurotus ostreatus. The objective of this investigation was to optimize the conditions for polysaccharide extraction from Pleurotus ostreatus (Jacq.) P. Kumm and to find the ideal extraction condition (water to crude material ratio, extraction time and extraction tempera- ture) utilizing response surface methodology. The in vitro antioxidant activities of POP were assessed on the base of 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity and 2,2-azino-bis-3-ethyl-benzothiazoline-6-sulfonic acid (ABTS) radical scavenging activity. Additionally, the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay was used to measure in vitro role of cytotoxicity in Murine Lymphoid cancer cell line (EAC cell line). Materials and methods Materials and reagents Fresh fruiting bodies of Pleurotus ostreatus (Jacq.) P. Kumm were collected and authenticated from the Mushroom Development Institute, which belongs to the Department of Agriculture Extension, Ministry of Agriculture, Savar, and Dhaka-1340 in Bangladesh. Monosaccharide (D-glucose), vitamin C, 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2-azino-bis-3-ethyl-ben- zothiazoline-6-sulfonic acid (ABTS), thiazolyl blue tetrazolium bromide (MTT), trichloro ace- tic acid (TCA), alfa-Amylase, bovine serum albumin (BSA), acetonitrile, 1-phenyl-3-methyl- PLOS ONE | https://doi.org/10.1371/journal.pone.0209371 January 3, 2019 2 / 17 Optimization of extraction of antioxidant polysaccharide 5-pyrazolone (PMP), RPMI-1640 were purchased from Sigma Co., St. Louis, MO, USA, while sephadex-G-100 and DEAE cellulose were acquired from Pharmacia, Co., Sweden. Murine Lymphoid cancer cell line or Ehrlich ascites carcinoma (EAC) cells were obtained with the courtesy of Indian Institute of Chemical Biology, Kolkata, India. Dimethyl sulfoxide, chloro- form, butanol, sodium chloride, phenol, sulfuric acid, uronic acid, glucuronic acid were pur- chased from local agents (Diatech, Alfa Scientific Co. and Bio-trade BD). Extraction of Pleurotus ostreatus (Jacq.) P. Kumm polysaccharide (POP) POP was extracted by hot distilled water based on the intended conditions. After centrifuga- tion (3000 rpm for 20 min), 1/3 of the original volume of the main supernatant was achieved by hot air concentration at 56˚C. The starch in the concentrated solution was removed by alfa- amylase at 60˚C. An adequate volume of ethanol was added to the concentrated extraction solution and mixed well to precipitate the POP at 4˚C for 12 h. Then, the precipitates were gathered by centrifugation (3000 rpm for 20 min) and washed consecutively with acetone, ether and ethanol. The POP extract was liquefied with distilled water and protein was removed with Sevag reagent (chloroform: normal butanol, 4:1, v/v). The protein fractions from POP extract were removed by dialysis (MWCO 1400 Da, Union Carbide) and finally POP was col- lected by freeze drying. The POP yield was calculated using the following formula: Wpops Yð%Þ ¼ � 100 Wsample Where, Wpops and Wsample are the weights of POP and Pleurotus ostreatus (Jacq.) P. Kumm powder used respectively. Single-factor experiment design In this experiment, the following three variables were investigated: water to sample ratio (mL/ g), extraction time (minutes), and extraction temperature ( C). Their variable effects on the POP extraction were assessed. Each sample was extracted according to the above mentioned procedure for polysaccharide extraction. The effects of three different conditions on POP yield were compared by one-way analysis of variance (ANOVA) using SPSS 21.0 (SPSS Inc., Chi- cago, IL). Design of Box-Behnken The Box-Behnken design (BBD) was used to obtain the experiment design, analysis of results and regression models. Through the single factors experiment, the appropriate ranges of water to raw material powder (X ), extraction time (X ) and extraction temperature (X ) were deter- 1 2 3 mined and then the BBD of POP yield was carried out. The entire design was made up of 17 experimental runs, which were carried out in a random sequence. The three variables at the three levels were coded as -1, 0 and +1. Here, a second-order—polynomial formula was used to optimize the extraction conditions of the POP yield (the response) as follows [17]: P P P P 3 3 2 3 Y ¼ A þ A X þ A X þ A X X o i¼1 i i i¼1 ii i i¼1 j¼iþ1 ij i j Where, Y is the dependent variable and X X are the independent variables and A , A , A , i j o i ii and A are the regression coefficients of the independent variables that were estimated by the ij model for intercept, linear, quadratic, and interaction terms, respectively. Design Expert 8.0.5.0 was utilized for statistical investigation of fluctuation for every response and anticipat- ing the ideal conditions for the extraction of POP [19,20]. PLOS ONE | https://doi.org/10.1371/journal.pone.0209371 January 3, 2019 3 / 17 Optimization of extraction of antioxidant polysaccharide Purification of polysaccharide from Pleurotus ostreatus (POP) The dried POP was also deproteinated using the Sevage method[21] and 1.0 g of POP was dis- solved in 10 mL dH 0 and applied to a DEAE-52 cellulose column (3.5 cm × 20 cm), and eluted with deionized water, followed by different gradient of NaCl solutions (0 to 0.5 M) at a flow rate of 0.8 mL/min. The eluates were collected in 5 mL fractions automatically and poly- saccharide content was measured using phenol-sulfuric acid method[22]. The main identical fractions containing polysaccharide were collected and concentrated and further fractionated by using gel filtration chromatography on Sephadex G-100 column (110 cm × 1.6 cm) at 0.1 mL/min and eluted with distilled water. 4 mL fractions were collected per tube and polysac- charide content of each tube was measured using phenol-sulfuric acid method[22]. The major fractions were combined and lyophilized to yield POP, which was used for further studies. Analytical method validation The polysaccharide content in POP extract was analyzed by phenol-sulfuric acid method using glucose as the standard. The regression equation was Y = 0.0231x – 0.046 and the correlation coefficient was 0.996. A linear relationship between the polysaccharide content and absorbance was detected within the range of 0–80 μg/mL, identified at 490 nm. The precision was appraised by analyzing the reproducibility and intermediate precision variations at three dif- ferent concentrations with five replicates. The accuracy was evaluated with the pointed rescue test. Chemical composition analysis The phenol-sulfuric acid method was used to determine the total polysaccharide content of POP extract, using D-glucose as the standard[22]. Protein content of POP extract was deter- mined by Lowery method, using BSA as the standard[23]. The m-hydroxybiphenyl assay was used to measure uronic acid content of POP extract, using glucuronic acid as reference mate- rial [24]. The moisture content of POP was measured, referring to the method outlined in Kong et al (22). The pH values of the POP at the 2 mg/mL of POP were determined using a pH meter and the relative viscosity of POP (10 mg/mL) was determined using viscometer (Thermo Scientific HAAKE, 388–0100) at 25˚C[25]. Monosaccharide composition The monosaccharide composition of the POP was determined using HPLC by methods deter- mined by Chai, Y et al[26] with minor modifications. POP samples (2 mg) were hydrolyzed with trifluoroacetic acid (0.5 mL of TFA, 2 mol/L) in a wrapped flask occupied with nitrogen at 120˚C and after 2 h of hydrolysis, the excess TFA from the flask was removed by evaporation with ethanol at 45˚C. Monosaccharide standards and POP hydrolysate were then added to 0.5 mL of PMP (1-phenyl-3-methyl-5-pyrazolone, 0.5 mol/L in methanol) and 0.5 mL NaOH (0.3 mol/L) for derivatization at 70˚C for 30 min. After centrifugation (10,000 rpm, for 5 min), the supernatant was assorted with 0.05 mL HCl (0.3 mol/L), and the mixture was mined with chlo- roform to eliminate surplus PMP. The aqueous layer was sieved using a 0.22 μm membrane and used for monosaccharide composition analysis of POP by HPLC system, which was fur- nished with a UV detector (245 nm) and an Amethyst C18 column (4.6 mm × 250 mm, 5 μm, Sepax, USA). The mobile phase of HPLC system was a mixture of PBS (Phosphate buffered saline, 0.1 mol/L, pH 7) and acetonitrile (80:20, v/v) with a flow rate of 1 mL/min. The column was reserved at 25˚C, and the injection volume was 10 μL. PLOS ONE | https://doi.org/10.1371/journal.pone.0209371 January 3, 2019 4 / 17 Optimization of extraction of antioxidant polysaccharide UV-Vis and FT-IR analysis The purified POP was re-dissolved in water to achieve a concentration of 0.25 mg/mL. The UV-Vis spectra of the solution were recorded at 190–600 nm, using spectrophotometer (Dou- ble beam UV-visible light spectrophotometer, China) and the functional groups of POP were -1 acknowledged using FT-IR spectrophotometer, within 4000–500 cm in KBr pellets. DPPH radical scavenging activity DPPH radical scavenging activity of POP was determined according to a modified method of Sharma et al 2016 [27]. Vitamin C was used as the positive control. Briefly, 50 μL of each POP sam- ples ranging in concentrations between 0.2 to 1.2 mg/mL were prepared by dissolving POP solid in distilled water and then adding a 5 mL (0.004% w/v in ethanol) solution of DPPH. The mixture was vortexed and shielded with Aluminum foil and incubated for 30 min in room temperature in the dark environment. The blank control was 80% ethanol and measurements were taken in tripli- cate. After 30 minutes of incubation, discoloration of reaction was measured using spectropho- tometer at 517 nm. DPPH scavenging effect was determined using the following equation; ½A ðA A Þ�� 100 o 1 2 DPPH scavenging effectð%Þ ¼ Where, Ao is the absorbance of the control; A is the absorbance of the POP sample; A is 1 2 the absorbance of the POP sample under similar condition to A excepting ethanol instead of the DPPH. The effective concentration for 50% of free radical scavenging activity of positive control (Vitamin C) and POP samples were determined using standard curve (r = 0.9994). ABST radical scavenging assay The ABST radical scavenging of POP was carried out using the method of P. Li et al 2011[28]. Vitamin C was used as the positive control. The ABTS radical cation was produced via the reaction between ABTS solution (5 mL, 7 mM) and K S O aqueous solution (1 mL, 15 mM) 2 2 8 following 12 h in the dark. The ABTS radical cation solution was then diluted with deionized water to yield an absorbance of 0.70 at 734 nm. The ABTS radical cation answer (3 mL) was added to 0.75 mL POP solution (dissolved in distilled water) at varying concentrations (0.1, 0.2, 0.4, 0.6, 0.8, 1.0 and 1.2 mg/mL). After 15 minutes of reaction, the absorbance was mea- sured at 734 nm. The scavenging endeavor of POP in opposition to ABTS radical was evalu- ated with the aid of the accompanied equation: ½A ðA A Þ�� 100 0 1 2 ABST radical scavenging activity ð%Þ ¼ Where, A is the absorbance of the control; A is the absorbance of the POP solutions; and o 1 A is the absorbance of all the reaction reagents (Except POP solutions). Determination of cytotoxic activity of POP using MTT assay Murine Lymphoid Cancer Cell Line or EAC (Ehrlich ascites carcinoma), which are murine lym- phoid cancer cell lines were cultivated in RPMI-1640 medium (10% foetal calf serum, and 1% (v/v) penicillin-streptomycin) in a humidified atmosphere of 5% CO at 37˚C. EAC cells were planted (5 ×10 cells/well in 200 μL RPMI 1640 media) in 96-wells, with various concentrations of purified POP (0.001–1000μg/mL) added and incubated for 24, 48, and 72 hrs at 37˚C in 5% CO atmosphere. After the end of incubation, the medium was removed and wells were washed with 10 mM PBS and MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide) (20 μL, PLOS ONE | https://doi.org/10.1371/journal.pone.0209371 January 3, 2019 5 / 17 Optimization of extraction of antioxidant polysaccharide 5 mg/mL MTT in PBS) was added and hatched for 4 h at 37˚C. 100 μL of acidic isopropanol was added into each well and incubation continued for 3 h more. The absorbance was measured at 570 nm and the proliferation rate (PR) was determined using the following formulae: Tabs PR ¼ � 100 Cabs Where, Tabs and Cabs are the absorbance of test and control. Cytotoxicity of POP on EAC cells was determined as cells growth inhibition rate (IR): IR ¼ 100 PR Statistical analysis GraphPad Prism 6 (Pad Software, Inc., USA) was used to analyze data, and results are expressed as mean±SD. Error bars were expressed as 95% CI. All experiments were performed at least three times. P<0.05 was considered to be statistically significant, and P<0.01 was regarded as highly statistically significant. Design Expert Software (Version 9.0.4.1, State-Ease Inc., USA) was used to design of Box-Behnken and single-factor experiment. Results and discussion Impact of water to raw material ratio on the POP yield The polysaccharide was extracted at different water to raw materials ratios, with a fixed extrac- tion time and extraction temperature at 60˚C and 70 min respectively. This allowed the influ- ence of water to raw materials ratio on the percent yield of POP to be evaluated (Fig 1A). Impact of extraction time on the POP yield Extraction time is a vital parameter, influencing the extraction yield of polysaccharide. To research the impact of extraction time on the extraction yield of POP (Fig 1B), the polysaccha- ride was removed at various extraction time (40 50, 60, and 70 min), and the water to crude material proportion and extraction temperature were set as 25 mL/g and 70˚C, separately. The extraction yield of POP essentially increased from 3.44% to 5.23% when the extraction time increased from 40 to 60 min (P < 0.05). However, when this extraction time was exceeded, the extraction yield of POP only decreased marginally. Consequently, the extraction time of 60 min was selected for the BBD analysis. Impact of extraction temperature on the POP yield. To assess the impact of extraction temperature on the yield of POP (Fig 1C), the water to crude material proportion and extrac- tion time were kept at 25 mL/g and 60 min, individually. The extraction yield of POP increased from 3.49% to 5.28% when the temperature increased from 50 to 80˚C, and the POP yield at 70˚C was essentially higher than those of 50, 60 and 80˚C (P < 0.05). In any case, the extrac- tion yield of POP at 80˚C was marginally lower than that of 70˚C (P > 0.05). For this reason, the extraction temperature of 70˚C was chosen selected for the BBD analysis. Optimization of polysaccharide extraction from Pleurotus ostreatus Based on the single-factor tests, 17 runs were performed to streamline the extraction states of POP. The POP yields (%) of the 17 runs designed to assess the 3 autonomous factors including water to crude material proportion (X1), extraction time (X2) and extraction temperature (X3) are exhibited in Table 1. The POP yields range from 3.44% to 5.28%. The percentage of POP PLOS ONE | https://doi.org/10.1371/journal.pone.0209371 January 3, 2019 6 / 17 Optimization of extraction of antioxidant polysaccharide Fig 1. Effect of water to raw material ratio (A), extraction time (B), and extraction temperature (C) on POP yield. Bar charts with different lowercase letters significantly differ (p<0.05). POP, Pleurotus ostreatus polysaccharide. The % yield of POP increased from 3.57% to 4.36% and increased to 5.21% with an increasing water to raw materials ratio, from 15 mL/g to 25 mL/g. However, when the water to crude material proportion increased to 30 mL/g there was a reduction in the extraction yield of POP. As the water to crude material proportion increased, the disintegration of polysaccharide from the materials essentially increased, and the extraction yield of POP increased. In this way, the water to crude material proportion of 25 mL/g was selected for the BBD analysis. https://doi.org/10.1371/journal.pone.0209371.g001 nd yield (% Y) was described by the 2 order polynomial Eq (1) as follow: Y ð%Þ ¼ 58:88þ 1:52725 X þ 0:677375 X þ 0:649125 X þ 0:000775 X X 0:001475 1 2 3 1 2 ð1Þ 2 2 2 X X þ 0:000262X X 0:028825 X 0:005881 X 0:004181 X 1 3 2 3 1 2 3 Where, Y is the POP yield; X1, X2 and X3 are the factors of water to crude material propor- tion, extraction time and extraction temperature, individually. The aftereffect of ANOVA investigation is displayed in Table 2. The importance of co- efficients can be verified by their respective P-values, and a smaller P-value signifies a more significant comparing coefficient. In Table 2, the model P-value was less than 0.0001, which PLOS ONE | https://doi.org/10.1371/journal.pone.0209371 January 3, 2019 7 / 17 Optimization of extraction of antioxidant polysaccharide Table 1. Box-Behnken experiment design and the results of the POP yield. Run Water to raw material ratio (X ) (mL/g) Extraction time (X ) (min) Extraction temperature (X ) ( C) POP yield (%) 1 2 3 Actual value Predicted value 1 25 60 70 5.41 5.39 2 30 50 70 4.105 4.09 3 20 70 70 3.98 3.99 4 30 70 70 4.365 4.36 5 25 60 70 5.4 5.39 6 25 60 70 5.395 5.39 7 20 50 70 3.875 3.88 8 30 60 80 4.735 4.75 9 25 60 70 5.425 5.39 10 25 70 80 4.94 4.93 11 25 50 80 4.695 4.69 12 30 60 60 4.045 4.04 13 25 50 60 3.88 3.89 14 25 60 70 5.32 5.39 15 20 60 60 3.62 3.60 16 25 70 60 4.02 4.02 17 20 60 80 4.605 4.60 POP, Pleurotus ostreatus polysaccharide. https://doi.org/10.1371/journal.pone.0209371.t001 demonstrates that the relapse display for POP yield was highly significant. Furthermore, the P- estimations of the straight coefficients (X , X and X ), and the quadratic term coefficients (X , 1 2 3 1 2 2 X and X ) were less than 0.01, which shows the significant effects of these three variables on 2 3 the extraction yield of POP. The P-estimation of the communication term coefficient (X X ) 1 3 was observed to be lower than 0.05, showing the critical impact of this coefficient on the extrac- tion yield of POP. The other term coefficients (X X , X X ) were not significant (P > 0.05). The 1 2 2 3 ANOVA shows that water to crude material proportion (X ), extraction time (X ) and extrac- 1 2 tion temperature (X ) were significant single factors influencing the extraction yield of POP. The assurance coefficient (R ) of the model was 0.9988, showing that 0.12% of aggregate varieties couldn’t be clarified by the model. However the balanced assurance (adj-R2) esteem (0.9974) showed that a large portion of the POP yield variety could be anticipated by the relapse demonstrate. The coefficient of variety (C.V.) was at low esteem (0.7199%), demon- strating the investigation information and predicated information were comparative. As per the exploratory information, the lack of fit P-value (0.8978) was more than 0.05, proposing that the lack of fit was unimportant in identifying the pure error. The Adeq Precision (70.6326) in the present model was a sufficient flag, which demonstrated that the model could be utilized to explore the plan space. Optimization of extraction conditions The impacts of three variables on POP yield (%Y) were analyzed with three-dimensional response surface plot and contour plot, as shown in Fig 2. In this experiment, two variables were presented by response surface plot and contour plot whereas a variable was fixed at the 0 level. For water to crude material proportion and extraction time (Fig 2B), the contour plot is circular, which dem- onstrates that the shared connection between water to crude material proportion and extraction time is not significant (P > 0.05). The comparative pattern (Fig 2F) was found for extraction time PLOS ONE | https://doi.org/10.1371/journal.pone.0209371 January 3, 2019 8 / 17 Optimization of extraction of antioxidant polysaccharide and extraction temperature (P > 0.05). The elliptical contour plot (Fig 2D) showed the common cooperation between water to crude material proportion and extraction temperature was note- worthy (P < 0.05). These outcomes were in concurrence with the qualities in Table 2. Diagnostics accuracy Diagnostics accuracy is crucial when determining the appropriateness of the model for the genuine framework. Four diagnostics graphs for model accuracy are displayed in Fig 3. Fig 3D demonstrates the anticipated and the genuine test esteems. The spots of the anticipated and real esteems indicates ordinary circulation and are near the 45˚ line, confirming that the model has a decent adjustment. Fig 3A demonstrates a normal % probability plot of residuals for the ordinariness presumption; the lingering plot that moves towards a straight line, demon- strating that the typicality supposition was appropriate. The inside studentized residuals versus anticipated esteems are shown in Fig 3B. The plots of the inside studentized residuals scattered arbitrarily demonstrate that the first fluctuation was steady for all esteems. The inside studen- tized residuals versus trial run numbers are displayed in Fig 3C, and every one of the focuses are situated inside a restricted range. All information shows that the reaction surface model is connected to the POP extraction, and that the model is critical and exact. Validation of the model The ideal extraction conditions for the extraction of POP comprised of a water/crude material proportion of 26.04 mL/g, an extraction time of 62.08 minutes, and an extraction temperature 70.5˚C, which were determined by utilizing the second-order quadratic equation of the model by fathoming the regression equation and dissecting the response surface. The greatest antici- pated estimation of POP yield was 5.34%. With a specific end goal to verify the anticipated Table 2. ANOVA analysis for the quadratic model of POP extraction. Source Sum of Squares df Mean Square F-value p-value � � Model 6.59 9 0.7319 674.13 < 0.0001 � � A-X1 0.1711 1 0.1711 157.60 < 0.0001 � � B-X2 0.0703 1 0.0703 64.76 < 0.0001 � � C-X3 1.45 1 1.45 1338.76 < 0.0001 AB 0.0060 1 0.0060 5.53 0.0509 � � AC 0.0218 1 0.0218 20.04 0.0029 BC 0.0028 1 0.0028 2.54 0.1551 2 � � A 2.19 1 2.19 2013.91 < 0.0001 � � B 1.46 1 1.46 1341.41 < 0.0001 2 � � C 0.7361 1 0.7361 678.01 < 0.0001 Residual 0.0076 7 0.0011 Lack of Fit 0.0010 3 0.0003 0.1905 0.8978 Pure Error 0.0066 4 0.0017 Cor Total 6.59 16 R 0.9988 Adjusted R 0.9974 Predicted R 0.9961 Adeq Precision 70.6326 C.V. % 0.7199 � � p<0.001 was measured to be highly significant. https://doi.org/10.1371/journal.pone.0209371.t002 PLOS ONE | https://doi.org/10.1371/journal.pone.0209371 January 3, 2019 9 / 17 Optimization of extraction of antioxidant polysaccharide Fig 2. Response surface and contour plots for % yield of POP from Pleurotus ostreatus (Jacq.) P. Kumm. (A-B) Water to raw materials and extraction time (C-D) Water to raw materials and extraction temperature and (E-F) Extraction time and extraction temperature. https://doi.org/10.1371/journal.pone.0209371.g002 PLOS ONE | https://doi.org/10.1371/journal.pone.0209371 January 3, 2019 10 / 17 Optimization of extraction of antioxidant polysaccharide Fig 3. Diagnostic plots for the Box-Behnken model adequacy. (A) Normal plot of residuals, (B) plot of internally studentized residuals vs. predicted response, (C) Residual vs. run and (D). Predicted vs. actual. https://doi.org/10.1371/journal.pone.0209371.g003 estimation of POP yield and the applicability of the model, three parallel tests were performed under the previously mentioned ideal extraction conditions. The yield of POP was 5.32 ± 0.12% (n = 3), which drew nearer the anticipated estimation of 5.34%. The outcomes demon- strated that the RSM was fitting for enhancing the conditions for POP extraction, and the regression model was precise and relevant for extricating POP. PLOS ONE | https://doi.org/10.1371/journal.pone.0209371 January 3, 2019 11 / 17 Optimization of extraction of antioxidant polysaccharide Fig 4. UV-spectrophotometric analysis of partially purified polysaccharides (POP). A) Before proteolysis and B) after removing of -1 protein and nucleic acids. The peaks in the range of 950–1200 cm indicate the presence of polysaccharides, indicating the presence of -1 glycosidic bonds by 1,150–1,160 cm . https://doi.org/10.1371/journal.pone.0209371.g004 UV and FT-IR analysis Fig 4A and 4B show the UV-visible absorption spectra of polysaccharide. Fig 4B exhibits that polysaccharide fraction have no absorption at 260 and 280 nm in the UV spectrum, signifying the absence of nucleic acid and protein. The IR spectra of POP are shown in Fig 5. Chromatographic analysis A portion of POP (10g) was separated through a DEAE-Cellulose column and three portions were collected according to the chromatography profile (Fig 6A). A portion of POP was Fig 5. FT-IR spectral analysis of the polysaccharide (POP). The spectra indicate a noticeable C-H stretching and -1 bending vibration absorption in the range of 2929–2939 cm , which are characteristic peaks of the polysaccharide[29]. -1 In the marked region, the peak in the region of 1230 cm is due to C-C stretching vibration and the peaks towards -1 1100 cm suggesting that the peak was related to the starching vibration of C-O. https://doi.org/10.1371/journal.pone.0209371.g005 PLOS ONE | https://doi.org/10.1371/journal.pone.0209371 January 3, 2019 12 / 17 Optimization of extraction of antioxidant polysaccharide Fig 6. Chromatographic profile of POP. A) The elution profile of POP on DEAE-cellulose column and B) gel filtration elution curve of POP. POP, Pleurotus ostreatus polysaccharide. https://doi.org/10.1371/journal.pone.0209371.g006 further purified on gel filtration chromatography and a polysaccharide (POP) was obtained (Fig 6B). Chemical and sugar composition The monosaccharide composition analysis indicates that POP was composed of only glucose, confirming that it was a glucan. Table 3 shows the chemical and sugar composition of POP. The low moisture (14.11%) and viscosity (1.13) contents of POP confirmed that it is suitable for large scale production. The gel filtration analysis results showed that POP was a single reg- ular peak (Fig 6B), indicating that it was an identical polysaccharide. The molecular weight of POP was calculated as 154.6 × 10 k. In vitro antioxidant activity of POP obtained at optimum temperature, time and water to raw material ratio The scavenging activity of POP on DPPH radical assay has been extensively used to determine the antioxidant capacity of POP due to its easy availability and the immovability of DPPH Table 3. Chemical and sugar composition of POP. POP Total sugar content (%) 97.6±1.33 Protein content (%) 0.5±0.03 Uronic acid content (%) nd pH 7.41±0.04 Moisture content (%) 14.11±0.27 Relative viscosity 1.13±0.02 MW (kDa) 154.6 Monosaccharide Glc 100.0 composition (%) a Ara nd Gal nd Man nd Rha nd Values are expressed as mean±standard deviation. nd , Not Detected. POP, Pleurotus ostreatus polysaccharide. Glc, Glucose; Ara, Arabinose; Gal, Galactose; Man, Manose; Rha, Rhamnose https://doi.org/10.1371/journal.pone.0209371.t003 PLOS ONE | https://doi.org/10.1371/journal.pone.0209371 January 3, 2019 13 / 17 Optimization of extraction of antioxidant polysaccharide Fig 7. Antioxidant activities of the POP. (A) DPPH radical scavenging activity (p<0.001, EC = 1038.38 μg/mL, R = 0.8313) and (B) ABTS radical scavenging activity (p<0.001, EC = 824.37 μg/mL, R = 0.8223). POP, Pleurotus ostreatus polysaccharide. https://doi.org/10.1371/journal.pone.0209371.g007 radicals [30]. DPPH is a steady radical that focuses on nitrogen and demonstrates a greatest consumption at 517 nm in methanol [31,32]. The scavenging ability of POP on DPPH radical and ABTS radical was shown in Fig 7A and 7B. The results show the DPPH radical scavenging activities of POP increase gradually as the concentration of polysaccharides increases from 0.2 to 5.0 mg/mL. The POP demonstrates a concentration dependent free radical scavenging activity, with a significant activity found at higher concentrations. The outcomes demonstrate that the scavenging activity of DPPH in POP is low compared to that of vitamin C below 1.4 mg/mL (P<0.05) and in case of ABTS that concentration is 1.6 mg/mL (P<0.05). DPPH radicals scavenging by POP and vitamin C is equal at 1.2 mg/mL (72.23%) while at 1.4 mg/mL (81.02%) POP exhibit the same ABTS radi- cals scavenging activity, in comparison to vitamin C. Polysaccharides from plants and fungus have been perceived to be a promising source of antioxidant molecules. Numerous investiga- tions have demonstrated that polysaccharides enhance the action of cell reinforcement pro- teins in the body, scavenge free radicals, and repress lipid oxidation [33]. In vitro cytotoxic measure of POP using MTT assay The cytotoxic properties of purified POP was assessed by using the EAC cell line. The POP −3 3 extract at 10 to 10 μg/mL exhibit dose-and-time dependent inhibitory effects on the prolif- eration of EAC cells, with greater than a 90% suppression at the highest concentration (72 h). Fig 8 shows the dose-and-time dependent cytotoxic effects of POP on EAC cells. Conclusion The ideal polysaccharide extraction states of water/crude material proportion, ultrasound time, and ultrasound control were determined utilizing the RSM of BBD in view of single fac- tor tests. The RSM of BBD could effectively improve the extraction states of POP, and the regression model was pertinent for removing POP. The antioxidant activity of DPPH and ABTS radicals’ in vitro measures demonstrate that POP has cancer prevention agent properties PLOS ONE | https://doi.org/10.1371/journal.pone.0209371 January 3, 2019 14 / 17 Optimization of extraction of antioxidant polysaccharide Fig 8. Cytotoxicity of purified POP on murine lymphoid cancer cell line (EAC cell line). POP, Pleurotus ostreatus polysaccharide. https://doi.org/10.1371/journal.pone.0209371.g008 that increase with the increasing convergence of the polysaccharide arrangement. Hence, these outcomes suggest that polysaccharide from Pleurotus ostreatus (Jacq.) P. Kumm ought to be further investigated as a novel and potential natural antioxidant, which may prove a useful therapeutic drug. Additionally the purification and structure identification are improve the overall understanding of polysaccharide. Supporting information S1 Dataset. Impact of three variables on the POP extraction from Pleurotus ostreatus. (XLSX) S2 Dataset. Antioxidant activities of the POP. (XLSX) Acknowledgments The authors received no specific funding for this work. Funder provided support in the form of salaries for authors (MPK, MSI, RP, SD, RIT, and MR), but did not have any additional role in the study design, data collection & analysis, decision to publish, and preparation of the manuscript. PLOS ONE | https://doi.org/10.1371/journal.pone.0209371 January 3, 2019 15 / 17 Optimization of extraction of antioxidant polysaccharide Author Contributions Conceptualization: Md. Moyen Uddin Pk, Mohammad Sayful Islam. Data curation: Md. Moyen Uddin Pk, Mohammad Sayful Islam. Formal analysis: Md. Moyen Uddin Pk, Rumana Pervin. Investigation: Md. Moyen Uddin Pk, Mohammad Sayful Islam, Subhajit Dutta, Rabiul Islam Talukder. Methodology: Subhajit Dutta, Rabiul Islam Talukder. Resources: Md. Moyen Uddin Pk. Software: Md. Moyen Uddin Pk, Subhajit Dutta. Supervision: Matiar Rahman. Visualization: Md. Moyen Uddin Pk. Writing – original draft: Md. Moyen Uddin Pk. Writing – review & editing: Matiar Rahman. References 1. Fernandes A, Barros L, Martins A, Herbert P, Ferreira ICFR. Nutritional characterisation of Pleurotus ostreatus (Jacq. ex Fr.) P. Kumm. produced using paper scraps as substrate. Food Chem. England; 2015; 169: 396–400. 2. Jin Z, Li Y, Ren J, Qin N. 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