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Optimizing the Extraction of Polysaccharides from Bletilla Ochracea Schltr. Using Response Surface Methodology (RSM) and Evaluating Their Antioxidant Activity

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Optimizing the Extraction of Polysaccharides from Bletilla Ochracea Schltr. Using Response Surface Methodology (RSM) and Evaluating Their Antioxidant Activity processes Article Optimizing the Extraction of Polysaccharides from Bletilla ochracea Schltr. Using Response Surface Methodology (RSM) and Evaluating their Antioxidant Activity Bulei Wang 1, Yan Xu 1, Lijun Chen 1, Guangming Zhao 1, Zeyuan Mi 1, Dinghao Lv 2 and Junfeng Niu 1,* 1 National Engineering Laboratory for Resource Development of Endangered Crude Drugs in Northwest China, The Key Laboratory of Medicinal Resources and Natural Pharmaceutical Chemistry, The Ministry of Education, College of Life Sciences, Shaanxi Normal University, Xi’an 710119, China; [email protected] (B.W.); [email protected] (Y.X.); [email protected] (L.C.); [email protected] (G.Z.); [email protected] (Z.M.) 2 Shanxi Institute of Medicine and Life Sciences, Taiyuan 030006, China; [email protected] * Correspondence: [email protected]; Tel.: +86-29-85310680 Received: 24 February 2020; Accepted: 10 March 2020; Published: 16 March 2020 Abstract: Bletilla ochracea Schltr. polysaccharides (BOP) have a similar structure to Bletilla striata (Thunb.) Reichb.f. (Orchidaceae) polysaccharides (BSP). Therefore, BOP can be considered as a substitute for BSP in the food, pharmaceuticals and cosmetics fields. To the best of our knowledge, little information is available regarding the optimization of extraction and antioxidant activity of BOP. In this study, response surface methodology (RSM) was firstly used for optimizing the extraction parameters of BOP. The results suggested that the optimal conditions included a temperature of 82 ◦C, a duration of 85 min and a liquid/material ratio of 30 mL/g. In these conditions, we received 26.45% 0.18% as the experimental yield. In addition, BOP exhibited strong concentration-dependent ± antioxidant abilities in vitro. The half-maximal effective concentration (EC50) values of BOP against 1,1-diphenyl-2-picrylhydrazyl (DPPH ), 2,2 -azino-bis (3-ethylbenzothiazoline-6-sulphonate) · 0 (ABTS+ ), hydroxyl ( OH) and superoxide anion ( O ) radicals and ferrous ions (Fe2+) were · · · 2− determined as 692.16, 224.09, 542.22, 600.53 and 515.70 µg/mL, respectively. In conclusion, our results indicate that BOP can be a potential natural antioxidant, deserving further investigation. Keywords: Bletilla ochracea Schltr. polysaccharide; response surface methodology (RSM); antioxidant activities 1. Introduction The reactive oxygen species (ROS), including hydrogen peroxide (H2O2), superoxide anion ( O ), hydroxyl ( OH) and peroxyl radicals (ROO ), are generated in organismal metabolic processes · 2− · · and play important roles in cell signaling, homeostasis and growth regulation at the physiological level [1]. However, excessive ROS can oxidize almost every biomacromolecule (membrane lipid, nucleic acid, and protein), exacerbating many serious diseases, such as diabetes [2], atherosclerosis [3], Alzheimer’s disease [4], Parkinson’s disease [5] and cancer [6]. Currently, synthetic antioxidants (butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT)) are widely used in food, cosmetic and pharmaceutical industries to scavenge the excess ROS, though they might cause many undesirable adverse effects [7]. Thus, efficient and safe antioxidants should be developed imminently to address this problem. Processes 2020, 8, 341; doi:10.3390/pr8030341 www.mdpi.com/journal/processes Processes 2020, 8, x FOR PEER REVIEW 2 of 13 undesirable adverse effects [7]. Thus, efficient and safe antioxidants should be developed imminently to address this problem. In recent years, polysaccharides have been applied in the biological fields due to their relatively low toxicity and diverse bioactivities. Especially, the polysaccharide (BSP) from Bletilla striata (Thunb.) Reichb.f. (Orchidaceae) has demonstrated antioxidant [8], immune-regulatory [9] and wound-healing activities [10]. Because of the limited supply of B. striata, researchers should explore a new source of similar polysaccharides [11]. Bletilla ochracea Schltr., also named “HuangHuaBaiJi” (family: Orchidaceae), has been used as an alternative of B. striata in traditional Chinese medicine (TCM). In a previous study, we have reported that the polysaccharide (BOP) from B. ochracea contains a similar structure to BSP, which is [→(4-β-D-Man-1)4→(4-β-D-Glc-1)2→4-2-O-acetyl-β-D-Man-1→4- β-D-Man-1→4-3-O-acetyl-β-D-Man-1→4-2-O-acetyl-β-D-Man-1→] (Figure 1). Meanwhile, BOP also displayed anticancer activity, probably due to its immunostimulatory property [12]. Accordingly, Processes 2020, 8, 341 2 of 12 polysaccharides from B. ochracea should be further explored. Response surface methodology (RSM), a statistical model for predicting the optimal extraction parametersIn recent with years, high polysaccharides reliability and havereproducibility, been applied has in been the biological widely applied fields due for togaining their relativelymaximal lowyield toxicity in the extraction and diverse of polysacch bioactivities.arides [13]. Especially, For example, the polysaccharide Hu et al. optimized (BSP) the from extractionBletilla process striata (Thunb.)of the polysaccharide Reichb.f. (Orchidaceae) (CAPs) from has Camptotheca demonstrated acuminata antioxidant fruits [8 ],(microwave immune-regulatory power, 600 [9] andW; wound-healingliquid/solid ratio, activities 40:1 mL/g; [10 ].time, Because 14 min; of theand limited temperature, supply 70 of °C).B. striata Under, researchers those conditions, should the explore yield a(8.53%) new source was very of similar close to polysaccharides the predicted value [11]. (8.61%)Bletilla [14]. ochracea Schltr., also named “HuangHuaBaiJi” (family:In this Orchidaceae), study, BOP has was been isolated used by as anextracting alternative with of hotB. striata water.in Box–Behnken traditional Chinese design medicine(BBD), a (TCM).typical InRSM, a previous was firstly study, applied we have for reportedoptimizing that th thee independent polysaccharide variables, (BOP) from includingB. ochracea temperature,contains atime similar and structure liquid/material. to BSP, which Moreover, is [ (4- theβ-D-Man-1)4 in vitro antioxidant(4-β-D-Glc-1)2 activity4-2-O-acetyl- was qualitativelyβ-D-Man-1 and ! ! ! ! 4-quantitativelyβ-D-Man-1 4-3-O-acetyl-evaluated byβ-D-Man-1 electron 4-2-O-acetyl-paramagneticβ -D-Man-1resonance ](EPR) (Figure 1technique). Meanwhile, and BOP radical- also ! ! ! displayedscavenging anticancer experiments activity, (DPPH·, probably ABTS+·, due ·OH, to ·O its2− immunostimulatoryand Fe2+). In summary, property the results [12 ].are Accordingly, expected to polysaccharidesprovide the theoretical from B. and ochracea technicalshould suppo be furtherrts for the explored. further utilization of BOP. Figure 1. The chemical structures of Bletilla ochracea Schltr. polysaccharide (BOP). Figure 1. The chemical structures of Bletilla ochracea Schltr. polysaccharide (BOP). Response surface methodology (RSM), a statistical model for predicting the optimal extraction 2. Materials and Methods parameters with high reliability and reproducibility, has been widely applied for gaining maximal yield in the extraction of polysaccharides [13]. For example, Hu et al. optimized the extraction process 2.1. Experimental Materials and Chemicals of the polysaccharide (CAPs) from Camptotheca acuminata fruits (microwave power, 600 W; liquid/solid ratio,The 40:1 pseudobulbs mL/g; time, 14 of min; B. ochracea and temperature, were procured 70 ◦C). from Under thethose Nation conditions,al Engineering the yield Laboratory (8.53%) was for veryResource close Development to the predicted of Endangered value (8.61%) Chinese [14]. Crude Drugs in Northwest of China (Xi’an, Shaanxi Province).In this study, BOP was isolated by extracting with hot water. Box–Behnken design (BBD), a typical RSM,The was firstly2,2-diphenyl-1-picryl-hydrazyl applied for optimizing the (DPPH), independent 2,2 variables,′-azino-bis including(3-ethylbenzothiazoline-6- temperature, time andsulphonate) liquid/material. (ABTS), Moreover,dihydro-nicotinamide the in vitro antioxidant adenine dinucleotide activity was (NADH), qualitatively nitroblue and quantitatively tetrazolium evaluated(NBT), 1-methoxy-5-methylphenazinium by electron paramagnetic resonance methyl (EPR) sulfate technique (PMS), and 5,5-dimethyl-1-pyrroline radical-scavenging experiments N-oxide (DPPH(DMPO), ABTSand ferrozine+ , OH, Owereand purchased Fe2+). In from summary, Sigma-Aldrich the results (St. are Louis, expected MO, to USA). provide Petroleum the theoretical ether · · · · 2− and(60–90 technical °C), ethanol, supports sulfuric for the acid, further phenol, utilization ferrous of sulfate BOP. and other chemicals were obtained from a local chemical factory (Xi’an, China). All reagents and chemicals used were of analytical reagent (AR) 2.grade. Materials and Methods 2.1.2.2. Material Experimental Pretreatment Materials and and Polysaccharides Chemicals Extraction TheAccording pseudobulbs to previous of B. ochracea reportswere [15], procured the fromtubes the of National B. ochracea Engineering were washed, Laboratory air-dried, for Resource and Developmentpowdered. The of powders Endangered were Chinese successively Crude Drugspreproce in Northwestssed with ofpetroleum China (Xi’an, ether Shaanxi (60–90 Province).°C) and 80% ethanolThe for 2,2-diphenyl-1-picryl-hydrazyl depigmenting and delipidating. (DPPH), Then, 2,2 the0-azino-bis polysaccharides (3-ethylbenzothiazoline-6-sulphonate) were extracted from B. ochracea (ABTS), dihydro-nicotinamide adenine dinucleotide (NADH), nitroblue tetrazolium (NBT), 1-methoxy- 5-methylphenazinium
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