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Research Article Enzymatic Extraction, Purification, and Characterization of Polysaccharides from Penthorum Chinense Pursh: Natural Antioxidant and Anti-Inflammatory

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Research Article Enzymatic Extraction, Purification, and Characterization of Polysaccharides from Penthorum Chinense Pursh: Natural Antioxidant and Anti-Inflammatory Hindawi BioMed Research International Volume 2018, Article ID 3486864, 13 pages https://doi.org/10.1155/2018/3486864 Research Article Enzymatic Extraction, Purification, and Characterization of Polysaccharides from Penthorum chinense Pursh: Natural Antioxidant and Anti-Inflammatory Li-mei Lin ,1,2 Ling-jia Zhao ,1,2 Jing Deng,1,2 Su-hui Xiong ,1,2 Jie Tang,1,2 Ya-mei Li,1,2 Bo-hou Xia ,1,2 and Duan-fang Liao1,2 College of Pharmacy, Hunan University of Chinese Medicine, Changsha , China Collaborative Innovation Center for the Protection and Utilization of Chinese Herbal Medicine Resources in Hunan Province, Hunan University of Chinese Medicine, Changsha , China Correspondence should be addressed to Bo-hou Xia; [email protected] Received 26 August 2018; Accepted 8 November 2018; Published 27 November 2018 Academic Editor: Jane Hanrahan Copyright © 2018 Li-mei Lin et al. Tis is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Penthorum chinense Pursh (PCP) is a kind of functional food or medicine for liver protection. In the present work, Plackett-Burman design, steepest ascent method, and response surface methodology (RSM) were employed to obtain maximum total sugar yield. Te experimental yield of 6.91% indicated a close agreement with the predicted yield of 7.00% of the model under optimized conditions. Te major polysaccharide fraction (PCPP-1a) from PCPP was purifed and identifed as acidic polysaccharides with a high content of uronic acid (FT-IR, UV, HPGPC). PCPP had similar monosaccharide profle with PCPP-1a but was rich in galacturonic acid 2+ (HPLC). Both of PCPP and PCPP-1a possessed strong hydroxyl radical scavenging, DPPH radical scavenging, and Fe chelating activities. Moreover, they were revealed to show strong anti-infammatory activities by inhibiting NO, TNF-�,andIL-1� release compared to LPS treatment in RAW264.7 cells. Tese data suggest that the polysaccharides from PCP could be potential natural products for treating ROS and infammatory-related diseases. 1. Introduction that PCP exhibited antioxidant, hypoglycemic, antiprolif- erative, and anticomplement activities [19–22]. According Polysaccharide, a crucial biomacromolecule in living organ- to the preparation process of previous studies, the water- isms, is one of the most abundant storage carbohydrates solubleconstituentswerethemainfocus[23].Amongthem, of plants [1]. Botanical polysaccharides were involved in favonoids, favonoid glycosides, and other phenolic contents multiple bioactivities such as antioxidation, immunomodula- accounted for the major constituents [19, 22, 24]. Unfor- tion, antidiabetic, and anticancer [2–5]. Moreover, botanical tunately, studies on the extraction, characterization, and polysaccharides have attracted increasing attention for their bioactivities of Penthorum chinense Pursh polysaccharides health-related benefts and therapeutic use with relatively high security [6, 7]. Terefore, they have immense potential (PCPP) are limited. industrial applications in pharmaceuticals [8], cosmetics [9], Accordingly, this study was frst designed to optimize foods [10], and materials [11]. the extraction process of PCPP using design of experiment Penthorum chinense Pursh (PCP, Saxifragaceae) mostly (DOE). Te crude PCPP was then purifed by DEAE-52 distributed in China (Miao ethnomedicine), eastern Russia, cellulose and Sephadex G-150. Moreover, chemical analysis Japan, and Korea [12–16]. It has been used traditionally as (HPLC, HPGPC, and FT-IR), antioxidant activities, and anti- functional food or traditional Chinese medicine for treating infammatory efects of PCPP and its purifed fractions were liver disease [17, 18]. Recently, several studies indicated further characterized. 2 BioMed Research International Table 1: Experimental feld defnition for the Plackett-Burman design. Experimental values Symbol code Factors Low level (-) High level (+) ∘ X1 ( C) Extraction temperature 30 60 X2 (min) Extraction Time 10 30 X3 (ml/g) Liquid-to-solid ratio 10 30 X4 (W) Ultrasonic power 200 400 X5 (%) Enzyme concentration 2 4 X6 pH 4 5 2. Materials and Methods the signifcant factors from a multivariable system and considerably diminishes the number of experiments [26]. .. Materials. PCP was acquired from Gaoqiao natural Tis technique has been widely used to screen the signifcant herbal special market (Changsha, China). Te dried PCP factors prior to optimization [27]. A two-level (high and low) sample was pulverized by a disintegrator (HX-200A, China) PBD was tested for 6 selected factors (Table 1). Minitab 16.0 and passed through an 80-mesh screen (Harbin Ouerfu Filter (Minitab Inc., USA) was applied to create the design matrix Material Co., Ltd., China) to obtain the fnal powder. shown in Table 2. Te yield of PCPP was analyzed in triplicate 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro- for each test and the averages were regarded as the responses. mide (MTT), Dextrans of diferent molecular weights, 2,2- diphenyl-1-picrylhydrazyl (DPPH), and lipopolysaccharide .. Steepest Ascent Method. Steepest ascent method based (LPS) were obtained from Sigma Chemical Co. (St. Louis, on steepest ascent lines was applied to get rapidly to the MO, USA). Total antioxidant capacity assay kit (ABTS, neighborhood of the optimum. Tis way established an FRAP) was obtained from Beyotime Biotechnology (Jiangsu, efective path through the center of the design based on the China). Dulbecco’s Modifed Eagle medium (DMEM) and results from the PBD [28]. fetal bovine serum (FBS) were acquired from Gibco (Grand In the present work, tests with defned intervals for each Island,NY,USA).DEAE-52,SephadexG-150,monosaccha- response were conducted by the path of steepest ascent. Te ride standards (glucose (Glc), galactose (Gal), ribose (Rib), obtained design and results are displayed in Table 3. Once mannose (Man), glucuronic acid (GlcUA), rhamnose (Rha), there is no further achieved improvement in the response, xylose (Xyl), galacturonic acid (GalUA), arabinose (Ara), and thepointwouldbethegeneralvicinityoftheoptimumand fucose (Fuc)), and cellulase were acquired from Sinopharm could be taken as the center point of central composite design Chemical Reagent Co., Ltd. (CCD). .. Preparation of PCP and Determination of the Yield. .. Response Surface Methodology (RSM). In order to under- Afer drying to a constant weight and homogenization, PCP ∘ stand the interactions among the factors and optimize the powder was extracted three times with 80% ethanol at 60 C, extraction conditions for PCPP, RSM via central composite each for 5 h, to remove lipids, amino acids, monosaccharide, design (CCD), which includes various processes such as oligosaccharides, and some colored materials [25]. Pretreated regression analysis and factorial design was applied [29]. In samples were centrifuged (4000 rpm, 20 min), and the pow- this study, a CCD experimental plan with two variables and der was vacuum-dried to a constant weight. Each dried and three levels was utilized to get the optimal conditions for pretreated sample (5 g) was extracted by ultrasound-assisted PCPP. Te three levels of the signifcant factors (enzyme enzymatic (cellulase) extraction method at the designated concentration and liquid-to-solid ratio) were set as -1, 0, extraction conditions. Extraction conditions were controlled and 1 for high, intermediate, and low values, respectively by varying the ratio of liquid-to-solid, enzyme concentration, (�=1.414). Te experiments were executed based on the pH value, ultrasonic power, time, and temperature. Extracts matrix established by the Design-Expert sof (Version 10). were centrifuged at 5000 rpm for 10 min. Te supernatant Te design matrix with coded and actual values of the was merged and precipitated by overnight incubation with factor for 13 experiments which were constructed with 5 ethanol (80%; v/v; fnal concentration). Te precipitation central point replications is manifested in Table 4. Once the was harvested by centrifugation (5000 rpm, 20 min) and ∘ experiments had been conducted, the PCPP yield (Response lyophilized at -50 C to acquire crude polysaccharides. Te variable) was ftted with a second-order model. Te basic percentage of the yield of polysaccharides (%) was estimated form of the equation appears as follows: using the formula �(%)=�1/�0 × 100%, where Y was the � � PCPP extraction yield (%), �1 was the polysaccharides of �=� + ∑ � � + ∑ � � 2 +∑ ∑ � � � +� extraction (g), and �0 represented dried sample weight (g). 0 � � �� � �� � � (1) �=1 �=1 �=1 �=�+1 .. Plackett-Burman Screening Designs. Plackett-Burman where Y is the predicted response (PCPP yield), �0 is design (PBD) is a highly fractional design which identifes the constant term, ��, ���,and��� are the regression term BioMed Research International 3 Table 2: Te Placket-Burman design variables (in coded levels) with PCPP yield as response. Variable levels Run X1 X2 X3 X4 X5 X6 Yield (%) ∘ ( C) (min) (mL/g) (W) (%) 1-1-1-1-1-1-10.97 2-111-11-12.81 3-1111-111.55 4 1 1 -1 1 -1 -1 1.35 51-11-1-1-11.91 611-111-13.02 7111-1114.23 8-1-1-11112.61 9 -1 1 -1 -1 -1 1 0.64 10 1-1-1-1112.75 11 -1 -1 1 1 1 -1 4.23 12 1 -1 1 1 -1 1 2.17 Efect 0.4353 -0.1729 0.9265 0.2737 1.8454 -0.0567 Coefcient 0.2177 -0.0865 0.4633 0.1368 0.9227 -0.0283 t-Value 1.70 -0.68 3.62 1.07 7.22 -0.22 p-value 0.149 0.529 0.015∗ 0.333 0.001∗ 0.833 2 2 Press = 5.65, R = 0.9332, and adj-R =0.8530.∗Identifed
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