Structural characterization and antioxidant activity of a new polysaccharide from Bletilla striata fibrous roots

Carbohydrate Polymers 227 (2020) 115362

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Structural characterization and antioxidant activity of a new polysaccharide from Bletilla striata fibrous roots

T

Ziyan Chena, Yan Zhaoa, Mengke Zhanga, Xiufang Yangb, Pengxiang Yuec,d, Dikang Tange, ⁎ Xinlin Weia, a

School of Agriculture and biology, Shanghai Jiao Tong University, Minghang200240, Shanghai, People’s Republic of China Hangzhou Tea Research Institute, China COOP, Hangzhou City, 310012 Zhejiang, People’s Republic of China c Damin Foodstuff (Zhangzhou) Co. Ltd, Zhangzhou City, 363000, Fujian, People’s Republic of China d Nanjing Rongdian Food Technology Co., Ltd., Nanjing City, 211300 Jiangsu, People’s Republic of China e Lusheng Kangyuan Science and Technology Development Co., Ltd., Zunyi City, 564100 Guizhou, People’s Republic of China b

A R T I C LE I N FO

A B S T R A C T

Keywords: Bletilla striata polysaccharide Fibrous root Antioxidant activity Galactomannan

A new polysaccharide (pFSP) was first isolated from the originally discarded fibrous roots part of Chinese traditional herb, Bletilla striata. pFSP was composed of D-glucose, D-galactose and D-mannose in a molar ratio of 1: 2.03: 3.45 with molecular weight of 9.1 × 104 Da. It could effectively scavenge DPPH and superoxide radicals with inhibition rate of 64.47% and 72.27% at 5.0 mg/ml, higher than that of polysaccharide from Bletilla striata tuber. Structural investigations of the periodate oxidation studies and Smith-degradation as well as the FT-IR spectroscopy were performed, and combined with 1D and 2D NMR spectroscopy, the repeating unit of pFSP contained (1→4)-linked-α-D-Glcp, (1→4)-linked-β-D-Manp and (1→3,6)-linked-β-D-Manp units, together with the branches of (1→6)-linked-β-D-Galp and terminated with (1→)-linked-β-D-Manp residue.

1. Introduction Bletilla striata (Thunb.) Reichb. f. (Orchidaceae), an important hemostatic herb native to East Asia, was composed of aboveground ornamental flowers and underground pseudobulb tuber that was generally regarded as the medicinal part (He et al., 2017). Dating back to ancient China, Bletilla striata was applied to treat chapped skin, accelerate ulcerative carbuncle recovery, and prevent skin chilblain and wrinkles, due to its excellent anti-inflammation and tissue regeneration functions (He et al., 2017; Liao, Zeng, Hu, Maffucci, & Qu, 2019). Bletilla striata tuber polysaccharide (BSP) has been considered as the most essential components responsible for the exhibition of biological functions of Bletilla striata (Chen, Cheng, He, & Wei, 2018), widely used in cosmetic and chemical industries and served as a promising natural antioxidant or an excellent moisturizer (Kong et al., 2015; Wang et al., 2014). However, resulting from the lack of wide Bletilla striata resource and increasing market demands, the price of Bletilla striata has been increasing rapidly (Ren et al., 2016). For the aim of protection and the entire utilization of this precious plant, the originally discarded fibrous roots part has been considered as a probable material for extracting compounds of similar functions. Fibrous roots were actually grown on the tuber with an estimated

⁎

25% of the mass weight of total tuber (Jiang et al., 2013), but during processing were generally crushed into powder and subsequently discarded, for they were regarded as worthless products or even toxic part for skin use. However, several studies showed that the extracts from fibrous roots exerted comparable activity on fibroblast cells, more extraordinary DPPH scavenging activity and tyrosinase inhibition ability compared with the extracts from tuber (Jiang et al., 2013; Lee, Kim, Lee, & Leem, 2013). Although it is known that different parts of the plant may contain similar components, currently no research investigating polysaccharide from fibrous roots is available. In this study, water extraction and alcohol precipitation were applied for extraction and ion chromatography was employed for purification, after which a new polysaccharide (pFSP) was obtained. Structural characterization including composition of monosaccharides, type of glycosidic bonds and order of glycosyl chains were investigated to elucidated the preliminary structure of pFSP. The antioxidant effect of pFSP was evaluated by DPPH radical, superoxide radical and ABTS radical scavenging assays. Through the bioactivity comparison with BSP, this study strived to verify the commercial value of fibrous roots, accomplish the achievement of the full exploitation of Bletilla striata plant, and meanwhile provide insight investigation of pFSP, paving the way for its further utilization.

Corresponding author. E-mail address: [email protected] (X. Wei).

https://doi.org/10.1016/j.carbpol.2019.115362 Received 21 May 2019; Received in revised form 19 September 2019; Accepted 20 September 2019 Available online 22 September 2019 0144-8617/ © 2019 Elsevier Ltd. All rights reserved.

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2. Materials and methods 2.1. Chemical reagents DEAE-Sepharose Fast Flow gel was purchased from GE Co. (USA). Coomassie brilliant blue G-250, bovine serum albumin (BSA) and monosaccharide standards that included fucose, rhamnose, arabinose, glucosamine, galactose, glucose, xylose, mannose, fructose, galacturonic acid (GalA) and glucuronic acid (GlcA) were obtained from Solarbio Co. (Beijing, China). 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), Vitamin C (Vc), absolute ethyl alcohol, trichloroacetic acid (TCA), phenol (C6H5OH), ethylene glycol, methanol, deuterium oxide (D2O), petroleum ether, dimethyl sulfoxide, formic acid, phenolphthalein and acetic acid were purchased from Aladdin Co. (Shanghai, China). Concentrated sulfuric acid (H2SO4), hydrochloric acid (HCl) and sodium phosphate dibasic (Na2HPO4) were purchased from Sinopharm Chemical Reagent Co. (Shanghai, China). Sodium chloride (NaCl), sodium hydroxide (NaOH), sodium phosphate monobasic (NaH2PO4), trifluoroacetic acid (TFA), sodium periodate (NaIO4) and sodium iodate (NaIO3) were purchased from Macklin Biochemical Co. (Shanghai, China). All other chemicals were of analytical grade and all solutions were freshly prepared in distilled water. 2.2. Preparation and purification of polysaccharide samples The whole fresh Bletilla striata collected in November 2018 was provided by Hubei Shennong Herbal Medicine Decoction Pieces Co., Ltd., P.R. China. The fibrous roots were cut separated from the tuber body. Both of these two parts were dried at 60 °C using an electric blast drying oven for 48 h. Then the dried samples were crushed into powders by a micronizer and selected through a 60-mesh sieve. The dried fibrous root powders were pretreated with petroleum ether at 25 °C for 5 h to remove fats. After filtration, the powder residues were collected and suspended in distilled water for 2 h at 80 °C (the water to solid ratio was 50 ml/g). After pouring the aqueous phase, the extraction was similarly repeated. All extraction solutions were collected and concentrated to 1/10 vol (about 300 ml) by rotary evaporation. A 4-fold volume of 95% ethanol was added and left to stand overnight (about 12 h). The precipitate was collected by centrifugation (5000 r/min, 5 min) and then dissolved in distilled water. The solution was treated with 15% trichloroacetic acid to remove proteins until no peak of UV scanning at 250 nm. The aqueous phase was dialyzed against distilled water for 48 h (MWCO 3500 Da) and then lyophilized to obtain crude polysaccharide from fibrous roots. Then the crude polysaccharide was dissolved in distilled water at 50 mg/ml and loaded onto a DEAE-Sepharose fast flow gel column (2.5 cm × 60 cm) (Wang, Li, Liu, Chen, & Wei, 2015); The column was first washed with distilled water and then eluted with a step gradient (0.10, 0.15 and 0.20 M NaCl) at a flow rate of 2 ml/min. The sugar elute was gathered using automatic collector (5 min/tube) and then combined under the monitor through phenol-sulfuric acid method. The separated fractions were concentrated and dialyzed against distilled water for 72 h (MWCO 3500 Da), and then lyophilized to obtain purified polysaccharide. Finally, sample eluted with distilled water from fibrous roots was named as pFSP. The polysaccharide from tuber was obtained using the same extraction and purification methods (Fig. 1), namely pBSP as a positive control in follow-up bioactivity studies.

Fig. 1. Vital procedures involved in extraction and purification of polysaccharide from fibrous roots.

standard. The uronic acid content was determined based on a published method (Bitter & Muir, 1962; Blumenkrantz & Asboe-Hansen, 1973) and D-glucuronic acid was served as standard. 2.3.2. Determination of homogeneity and molecular weight of pFSP The molecular weight of pFSP was determined by high performance gel permeation chromatography (HPGPC). pFSP was dissolved in 0.02 M phosphate buffer (pH 6.8) to prepare sample solution of 2 mg/ ml concentration. After centrifugation at 10,000 r/min for 10 min, 20 μl of sample solution was injected into a HPGPC system through a 0.22 μm filter. The column (Shodex SB-804, 300 × 8 mm) was eluted with phosphate buffer at a flow rate of 0.3 ml/min, the column temperature was maintained at 25 °C. Dextran standards with different molecular weights (T3, T6, T10, T40, T100, T500, and T1000) were used to establish the standard curve for calibration (Zhu et al., 2019). 2.3.3. Monosaccharide composition analysis The identification and quantification of monosaccharide were determined by a published method (Wang, Mao, & Wei, 2012) with some modification. pFSP (2 mg) was dissolved into 2 ml of 2 M trifluoroacetic acid solution (TFA) and hydrolyzed at 110 °C for 3 h. The hydrolysate was evaporated with rotary evaporator, then followed by the addition of methanol. The mixture was similarly evaporated to dryness under reduced pressure and the process was repeated until removing TFA completely. Then the residue was dissolved in 1 ml distilled water and subsequently diluted to 50 ml for Ion chromatography (IC) measurement. 25 μl of treated sample solution was injected into a Dionex ICS5000+ system (CA, USA) with a Dionex CarboPac PA20 anion exchange column (150 mm × 3 mm) with temperature maintained at 30 °C. Eleven kinds of monosaccharide standards fucose, rhamnose, arabinose, glucosamine, galactose, glucose, xylose, mannose, fructose, GalA and GlcA were determined as references.

2.3. Structure characterization of pFSP 2.3.1. Chemical analysis The total sugar content was estimated by the phenol-sulfuric acid method (Dubois, Gilles, Hamilton, Rebers, & Smith, 1956), using dextran to establish standard curve. The content of soluble proteins was determined by Bradford method (Bradford, 1976), using BSA as

2.3.4. Sodium periodate oxidation and Smith degradation reaction 20 mg of sample was weighed and placed in a dark reaction flask. 2

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2.4.2. Superoxide radical scavenging assay Superoxide radical scavenging assay was determined based on previous method (Qu, Li, Zhang, Zeng, & Fu, 2016) with some modifications. Briefly, 2 ml of Tris−HCl buffer (50 mM, pH 8.2) was mixed with 1 ml of sample solution at various concentrations (0, 0.25, 0.5, 0.75, 1.0, 2.0, 3.0, 4.0 and 5.0 mg/ml). After the addition of 0.2 ml of 6 mM pyrogallol, the mixture was shaken vigorously and reacted at 25 °C for 5 min. Finally, 0.5 ml of 0.1 M HCl was added to terminate the reaction. The absorbance of samples was determined at 320 nm using Vc as a positive control. The superoxide radical scavenging ability was calculated by the following Formula (2):

0.015 M NaIO4 was added to calculate the amount of formic acid per mol of the glycosyl group by standard curve, and the amount of formic acid produced was titrated with 0.0086 M NaOH solution. After the OD value of the reaction system was stabilized through periodic acid oxidation, ethylene glycol was added to terminate the reaction. The reaction solution was dialyzed (MWCO 3500 Da) against tap water for 24 h, subsequently against deionized water for another 24 h. After concentration, 30 mg of NaBH4 was added to react overnight (about 12 h with stirring). The solution was neutralized to pH 5.5 with 50% acetic acid solution, then dialyzed, concentrated and lyophilized. The product was hydrolyzed with 2 M TFA at 110 °C for 3 h. The hydrolysate was evaporated to dryness with rotary evaporator, then followed by the addition of methanol. After removing TFA completely, the residue was dissolved in 1 ml distilled water and diluted to 20 ml for IC measurement.

As− Asb ⎞ Scavenging rate % = ⎛ 1 − *100% Ac− Acb ⎠ ⎝

2.3.5. FT-IR analysis The FT-IR spectrum of samples were collected on a Thermo Scientific Nicolet iS10 FT-IR spectrometer in the range of 4000–500 cm−1. 10 mg dried polysaccharide samples were ground together with spectroscopic grade KBr powders, and pressed into 1 mm pellets for measurement.

2.4.3. ABTS radical scavenging assay The capacity of polysaccharide samples to scavenge ABTS radicals was measured according to reported method (Xiao et al., 2019). Briefly, the ABTS solution (7 mM) was mixed with potassium persulfate solution (2.45 mM) for 12–16 h in the dark. The prepared ABTS solution was diluted 40–50 times with phosphate buffer (pH 7.4) to an absorbance of 0.70 ± 0.02 at 734 nm. Then 0.4 ml of sample solution at various concentrations (0, 0.25, 0.5, 0.75, 1.0, 2.0, 3.0, 4.0 and 5.0 mg/ ml) was added into 3 ml of ABTS•+ solution. The mixture was oscillated completely and the absorbance was measured at 734 nm until the absorbance was stabilized within 10 min at room temperature. Vc was adopted as positive control. The ABTS radical scavenging ability was calculated by the following Formula (3):

2.3.6. NMR spectroscopy analysis 20 mg of dried pFSP was first dissolved using D2O and then lyophilized. After repeating the same treatment twice, the H atom in sample was replaced completely by D atom. The treated pFSP was subsequently dissolved in 0.5 ml of D2O for detection (Liu et al., 2018). 1D and 2D NMR spectra including 1H, 13C, homonuclear chemical shift (1H -1H COSY), heteronuclear single quantum coherence spectroscopy (HSQC), heteronuclear multiple bond correlation spectroscopy (HMBC) were collected on an AVANCE III 600 MHz spectrometer (Bruker, Germany) with a 5 mm CPTCI 1H- probe. Here, the exact 1H NMR scanning frequency was 600.13 MHz, and the 13C NMR scanning frequency was 150.92 MHz. COSY, HSQC, and HMBC were conducted using the Bruker standard pulse program (Jin et al., 2019). All inspections were performed at 298 K.

Scavenging rate % = ⎛ ⎝

Ac− As ⎞*100% Ac ⎠

(3)

where Ac represents the absorption of the negative control (ABTS system with sample solvent), and As represents the absorption of the ABTS system with sample addition.

2.3.7. SEM analysis pFSP was dispersed on a conductive holder using adhesive tapes and then coated with a thin gold layer. The SEM analysis was performed on a scanning electron microscope (HITACHSU8010, JEOL, Japan). The image magnifications were 250×and 10000×.

2.5. Statistical analysis All assays were performed in triplicate, and the data were presented as the means ± standard deviation (SD) using the software Prism 7.0. The NMR spectra was analyzed by the software MestReNova 10.0.

2.4. In vitro antioxidant activities of pFSP compared with pBSP 2.4.1. DPPH free radical scavenging assay The DPPH free radical scavenging activity of polysaccharide samples was examined according to previous method (Zhou et al., 2014) with some modifications. Briefly, 3 ml of freshly prepared DPPH (0.1 mM in 50% ethanol) as the source of free radicals was added into 1 ml of sample solution containing various concentrations (0, 0.25, 0.5, 0.75, 1.0, 2.0, 3.0, 4.0 and 5.0 mg/ml). The mixture was rapidly shaken for 5 min and incubated at 25 °C for 25 min. Vc was used as a positive control and the absorbance was determined at 517 nm. The DPPH radical scavenging activity was calculated by the following Formula (1):

As− Asb ⎞ Scavenging rate % = ⎛ 1 − *100% Ac− Acb ⎠ ⎝

(2)

where Ac represents the absorption of the negative control (superoxide system with sample solvent), Acb represents the absorption of the system background (Tris−HCl buffer with sample solvent), As represents the absorption of the superoxide system with sample addition, and Asb represents the absorption of the sample background (Tris−HCl buffer together with samples).

3. Results and discussion 3.1. Structure characterization of pFSP 3.1.1. Compositions and molecular weight of polysaccharide samples After a series of separation and purification procedures, both pFSP and pBSP were mainly composed of carbohydrates (97.82% and 107.7%, respectively) with little proteins (0.48% and 0.10%, respectively), which could meet the standard of subsequent analysis in structure and bioactivity (Table 1). The yield of crude FSP and BSP was 1.33% and 11.02%, respectively, calculated as the percentage of dried materials weight. Though the amount of polysaccharide in fibrous roots was lower than that in tuber, the easy acquirement of high purity polysaccharide from fibrous roots indicated the roots were valuable to be potentially used together with the tuber. The uronic acid contents of BSP and FSP exhibited great diversity (0.12% and 7.74%, respectively), which was possibly related to the differences in raw materials. However, pFSP had lower uronic acid (1.95%), suggesting that other fractions containing acidic sugar existed in FSP.

(1)

where Ac represents the absorption of the negative control (DPPH with sample solvent), Acb represents the absorption of the system background (50% ethanol with sample solvent), As represents the absorption of the DPPH system with sample addition, and Asb represents the absorption of the sample background (50% ethanol together with samples). 3

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Table 1 Chemical composition of polysaccharide samples. (n = 3). Sample

Total sugar/%

Protein/%

Uronic acid/%

Yield/%

BSP pBSP FSP pFSP

69.70 107.7 73.87 97.82

1.23 0.10 3.60 0.48

0.12 0.28 7.74 1.95

11.02

± ± ± ±

2.93 7.50 5.00 0.16

± ± ± ±

0.12 1.00 0.25 0.88

± ± ± ±

0.41 0.14 0.14 1.10

1.33

As illustrated in Fig. 2a, pFSP was eluted in the HPGPC system as a single symmetrical peak. The molecular weight of pFSP was calculated to be 9.1 × 104 Da in reference to T-series dextran standard, lower than the 1∼5 × 105 da molecular weight scope of BSP as previously published (Chen et al., 2018). The polydispersity index Mw/Mn (PD) was calculated to be 1.22, which also indicated that a relatively homogeneous molecular weight in pFSP.

Fig. 3. FT-IR spectra of pFSP and FSP.

which indicated the existence of non-reducing terminal residues, (1→)linked or (1→6)-linked glycosidic bonds and the presence of residues containing trihydroxy groups with the glycosidic bonds type, such as (1→2)-linked, (1→2,6)-linked, (1→4)-linked or (1→4,6)-linked glycosidic bonds, respectively. Furthermore, we could infer that the portion of the bonds linked with the glycerol liberation and the bonds related to the erythritol production reached nearly to 1:1. Combined with the Smith degradation result shown in Fig. 2c, the liberations of glycerol and erythritol were 41.94% and 35.71% with a molar ratio of 1.17: 1, which indicated a consistence with the result of periodate oxidation assay. The molar concentration of glucose and galactose residues decreased to a minute quantity after degradation, whereas the mannose residues accounted for 15.83% of product. This result revealed that mannose might be the structural backbone of pFSP and at least partial mannose residues were linked by the manners of (1→3), (1→3,6), (1→2,4) or (1→3,4) (Jing et al., 2015).

3.1.2. Monosaccharide composition analysis Seen from Fig. 2b, FSP contained four kinds of monosaccharides: arabinose, galactose, glucose, and mannose with the molar ratio of 0.13:1.50:1.00:1.86. While pFSP only contained three kinds of monosaccharides: galactose, glucose, and mannose with the molar ratio of 2.03:1.00:3.45. In pFSP, mannose and galactose might be the residue constructing the backbone as they were the major monosaccharides with the content of 53.2% and 31.3%, respectively. BSP could be referred as a glucomannan based on the previous studies, for it was majorly consisted of mannose together with glucose (Wang et al., 2006, 2014). More interestingly, a new fraction extracted by Peng additionally contained galactose units with the content of 8.3% (Peng, Li, Xue, & Liu, 2014), which was exactly in corresponding to the types of monosaccharides in pFSP. It might be inferred that arabinose was a unique monosaccharide that only found in fibrous root polysaccharide. Moreover, there might exist a transformation relationship between the amount of galactose, glucose, and mannose in tuber and fibrous root polysaccharide.

3.1.4. FT-IR spectrum analysis The FT-IR spectra of FSP and pFSP were basically indistinguishable with sight difference merely in the intensity of bands, as shown in Fig. 3 The band appeared in 3200–3500 cm−1 region arose from OeH stretching vibration and another band in 1000–1200 cm−1 region attributed to CeOeC glycosidic band vibrations, indicating the characteristic of polysaccharide (Li et al., 2017; Qu et al., 2016). The weak signals at approximately 2900 cm−1 represented the CeH asymmetric

3.1.3. Periodate oxidation and Smith degradation analysis In periodate oxidation assay, the absorbance of oxidation product at 223 nm was stable after 5 days. According to calculation, the ratio of consumed NaIO4 and produced HCOOH during oxidation was 3:1,

Fig. 2. (a) Elution profile of pFSP on high performance liquid gel permeation chromatography. (b) Ion chromatograms of monosaccharide compositions of pFSP and FSP. (c) Ion chromatograms of Smith degradation fractions of pFSP. 4

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Fig. 4. NMR spectra of pFSP in D2O. (a) 13C spectrum. (b) 1H spectrum.

indicating that the chemical shift of H2 was 3.70 ppm. The typical H3 (3.47 ppm), H4 (3.88 ppm), H5 (3.57 ppm) and H6 (3.21 ppm) showed strong intra-correlations in 1H-1H COSY spectra (Table 2). From HSQC spectrum (Fig. S2), H1 exhibited a close connectivity with C1 in corresponding with H1/C1 signal (4.99/101.26 ppm) in the anomeric region. The carbon peaks of other C2-C6 could also readily be found in HSQC, corresponded nearly to the previously reported values (Ren et al., 2019). Considering all chemical signals, residue A could attribute to a (1→6)-linked-β-D-Galp unit. Similarly, the assignments of residue B, C, D, E could be determined respectively and were roughly in agreement with the related reference values in Table 2. The sequence of glycosidic residues of pFSP was analyzed by 1H-13C HMBC examination (Fig. S3). One series of inter-residual correlations were found between residue C H1 and C C6, C C1 and E H4; between residue D H1 and C C6, D H1 and D C4, D C1 and C H6, D C1 and D H4; between residue E H1 and E C4, E H1 and D C4. Hence, the characterized polysaccharide showed a sequence of the main chain: →E→ D→D→D→C→C→E→. Another series of clear inter-residual correlations were summarized between residue A H1 and A C6, A H1 and C C3; between residue B H1 and A C6. In accordance to these apparent assignments above, the branch chain of pFSP was characterized as the sequence of B→A→C→. As there existed two branch chains, the exact number of residue A in each branch chain were not sure. However, combined with the monosaccharide composition results, the total number of galactose residue in one repeating motif could be concluded. Here, a1 and a2 were used as positive integers to represent the number of residue A. According to the clear inter-residual HMBC correlations, a

stretching vibration (Wang et al., 2012). The peaks at approximately 1652 cm−1 were assigned to CeO stretching vibration of carboxylic products (Chen et al., 2015). The absorptions at 1062.5, 1118.7 and 1138.4 cm−1 suggested the pyranose ring of sugar residues and the signal at 950.8 cm−1 was also ascribed to α-D-glucopyranose, which were further proved in NMR analysis (Li et al., 2017). The bands at approximately 811 cm−1 and 868 cm−1 might contribute to the mannose residues (Qu et al., 2016), corresponding to the monosaccharide composition results. 3.1.5. NMR spectrum of pFSP The 13C NMR spectrum of pFSP was shown in Fig. 4a. Four peaks were presented in the range δ 90–110, accurately occurred at 101.26, 100.04, 99.51 and 97.85 ppm. A chemical shift at 174.6 ppm might indicate the presence of uronic acid (Yang et al., 2009). Though pFSP purified from FSP (eluted by distilled water) ought to be considered as a purely neutral polysaccharide, we inferred the uronic acid in pFSP as residue or impurity after purification. The 1H NMR spectrum (Fig. 4b) showed five anomeric proton signals appeared at 5.32, 4.99, 4.98 and 4.91 ppm, together with a not shown peak at 4.66 ppm due to the water suppression at about 4.70 ppm, and labeled E, A, B, C and D, respectively. The anomeric proton signals at 4.66, 4.91, 4.98, and 4.99 ppm were attributed to β-pyranose forms, whereas other signals at 5.32 ppm were assigned to α-pyranose units. All the NMR signals of residues were assigned in Table 2. For residue A, a proton chemical shift at 4.99 ppm was detected and a cross peak at 4.99/3.70 was obtained from COSY spectra (Fig. S1), 5

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Table 2 13 C and 1H NMR chemical shifts (ppm, δ) for pFSP. Label

Units

C/H-1

C/H-2

C/H3

C/H-4

C/H-5

C/H-6

Reference

A B C D E

→6)-β-D-Galp-(1→ β-D-Manp-(1→ →3,6)-β-D-Manp-(1→ →4)-β-D-Manp-(1→ →4)-α-D-Glcp-(1→

101.26/4.99 97.85/4.98 97.85/4.91 100.04/4.66 99.51/5.32

73.44/3.70 72.19/3.71 72.54/3.74 71.54/4.03 78.48/3.52

76.77/3.47 74.98/3.47 76.46/4.01 72.51/3.50 74.71/3.86

69.63/3.88 73.22/3.87 71.51/3.59 76.49/3.73 72.41/3.60

76.89/3.57 67.09/3.55 71.41/3.73 73.22/3.89 71.64/4.12

67.37/3.21 60.42/3.67 69.46/3.59 66.46/3.58 60.37/3.30

Ren et al. (2019) Zhang et al. (2019) Hao et al. (2019) Niu et al. (2014) Niu et al. (2014) and Ren et al. (2019)

Fig. 5. Possible repeating unit of pFSP.

which accounted for the lower solubility of pFSP than FSP (data not shown). The variation on morphology and shape between FSP and pFSP mainly contributed to the changes of intramolecular hydrogen bonds after purification (Li et al., 2017). It could be predicted that the interaction of polysaccharide chain decreased in pFSP and thus the polysaccharide fragments were easier to be torn up.

possible preliminary structure of pFSP was established in Fig. 5. On this occasion, the characterized pFSP showed a main chain of (1→4)-linked α-D-Glcp, (1→4)-linked-β-D-Manp and (1→3,6)-linked-βD-Manp units, corresponded with the Smith degradation result. Meanwhile, 5 portions of (1→4)-linked bonds, 4 portions of (1→6)linked bonds, 2 portions of (1→3,6)-linked bonds and 2 portions of (1→)-linked bonds could be concluded. The ratio of related bonds for producing the glycerol and erythritol within periodate oxidation was calculated as 6:5, nearly close to the IC result of product molar ratio of 1.17: 1. Hence, this predicted structure was reliable, considering the comprehensive results of monosaccharide composition, FT-IR spectra, periodate oxidation and Smith degradation analysis, and NMR analysis. Compared with the reported structures of polysaccharide from tuber (Chen et al., 2018; Liao et al., 2019; Peng et al., 2014; Wang et al., 2006; Zhao, Qi, Ying, & Jihua, 2017), (1→4)-linked mannose residue majorly accounted for the main chain of polysaccharide, both in the root and tuber polysaccharide. Interestingly, BSPF2 extracted by Peng et al. (2014) owned the branches composed of (1→6)-linked galactose residue as well, similar to the branch structure of pFSP. These results further confirmed that there might exist a close relationship between tuber and fibrous root polysaccharide, as their monosaccharide compositions and glycosidic bond types possessed relatively high similarity.

3.2. In vitro antioxidant activities of pFSP compared with pBSP The model of DPPH radical-scavenging was commonly used to evaluate free radical scavenging capacity (Zhang et al., 2016). As shown in Fig. 7a, the scavenging activities of pFSP and pBSP both dosedependently increased with concentrations from 0.25 to 5 mg/ml, but were less than that of Vc. The DPPH radical scavenging effect of Vc increased rapidly up to 97.17% at the concentration of 1.0 mg/ml. At 5.0 mg/ml, the scavenging activity of pFSP and pBSP were 64.47% and 23.35%, respectively. Among diverse reactive oxygen species (ROS), superoxide anion acted as a precursor to hydroxyl free radicals that had potential of inducing tissue damage through reacting with biological molecules (Li et al., 2014). Both pFSP and pBSP exhibited concentration-dependent scavenging effects, with the capacity of pFSP being significantly (P < 0.05) higher than that of pBSP (Fig. 7b). The highest scavenging rate was observed at 72.27% for pFSP (5.0 mg/ml) that was more than 2-fold of that (32.82%) for pBSP, but still lower than that of Vc (99.73%) at the same dose. The means of scavenging a protonated radical ABTS was extensively applied to evaluate the total antioxidant ability potential of natural products (Wang et al., 2018). As shown in Fig. 7c, the highest scavenging activity of pFSP on ABTS radicals reached 29.49% at 5.0 mg/ml, which was slightly higher than that of pBSP (18.90%). The antioxidant activities of natural products were generally a combined function of several factors, such as molecular weight, configuration, degree of polysaccharide chain branching, and even correlated to the selected antioxidant evaluation system (Leung, Zhao, Ho, &

3.1.6. SEM analysis of FSP and pFSP The microstructure shown in Fig. 6 illustrated the difference between FSP and pFSP. Both in the scale bar of 200 μm, FSP had a purely flake structure, whereas pFSP exhibited a mixture of filaments, sheets and amorphous states. In Fig. 6b, a flake layer with unregular curls was observed in FSP, indicating the strong attractions between functional groups on the surface to create the polysaccharide chains aggregation (Li et al., 2017). However, in Fig. 6d, the surface of pFSP seemed to be more smooth, but still rougher than that of tuber polysaccharide obtained by vacuum freezing method (Kong et al., 2015). The smooth surface of polysaccharide probably had negative effect on the rehydration performance to reduce the solubility of polysaccharide itself,

Fig. 6. (a) SEM images in the scale bar of 200 μm of FSP. (b) SEM images in the scale bar of 50 μm of FSP. (c) SEM images in the scale bar of 200 μm of pFSP. (d) SEM images in the scale bar of 50 μm of pFSP. 6

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Fig. 7. Scavenging effects of pFSP compared with Vc and pBSP: (a) DPPH assay, (b) Superoxide radicals assay, (c) ABTS radicals assay.

online version, at doi:https://doi.org/10.1016/j.carbpol.2019.115362.

Wu, 2009). Remarkably, the antioxidant effects of pFSP and pBSP measured in ABTS system both showed large gap compared with the value performed in DPPH model and superoxide anion system. It could be inferred that pFSP and pBSP exhibited radical scavenging effect in a similar way of donating the hydrogen preferred rather than provided the electron (Nilsuwan, Benjakul, & Prodpran, 2018). The higher branching degree of pFSP might excite the hydrogen atom-donates in anomeric region more readily. Meanwhile, the lower molecular weight and looser polysaccharide chain structure left pFSP a further interaction with radicals (Li et al., 2017). Hence, pFSP exhibited generally better antioxidative capacity than pBSP through these in vitro assays. Since pBSP could be served as a promising natural antioxidant (Kong et al., 2015), pFSP was able to be considered as a potential substitute as well.

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4. Conclusion In conclusion, a novel polysaccharide pFSP with an average molecular weight of 9.1 × 104 Da was obtained from the fibrous roots of Bletilla striata (Thunb.) Reichb. f. (Orchidaceae). Chemical structure analysis demonstrated that pFSP consisted of D-glucose, D-galactose and D-mannose in a molar ratio of 1: 2.03: 3.45. The main linkages in pFSP were (1→4)-linked-α-D-Glcp, (1→4)-linked-β-D-Manp and (1→ 3,6)-linked-β-D-Manp units, together with the branches of (1→6)linked-β-D-Galp and terminated with (1→)-linked-β-D-Manp residue according to the FT-IR spectroscopy, Smith-degradation and 1D and 2D NMR spectroscopy. pFSP could effectively scavenge DPPH and superoxide radicals with inhibition rate of 64.47% and 72.27% at 5.0 mg/ml, higher than that of polysaccharide from Bletilla striata tuber. Therefore, we may conclude that polysaccharide extracted from fibrous roots that originally was regarded as useless part of Bletilla striata has potential value as a promising antioxidant agent. To protect Bletilla striata resource and achieve the full exploitation of this precious plant, more work on the bioactivities of pFSP should be carried out for the further utilization in other areas. Acknowledgement The authors are grateful for the financial sponsoring of National Key R&D Program of China (No. 2018YFC1604400), National Key R&D Program of China (No. 2017YFD0400803), Shanghai Agricultural Achievement Transformation Project (No. 143919N0502), Shanghai Agriculture Applied Technology Development Program, China (No. 2018-02-08-00-08-F01545), Shanghai Biomedical key Program (No. 17391901302), Shanghai Technique Standard Special Program (No. 17DZ2203300) and Shanghai Yuemu cosmetics Co., Ltd entrusted Program -R&D of Natural Plants, Shanghai Food Safety and Engineering Technology Research Center (No. 19DZ2284200). Appendix A. Supplementary data Supplementary material related to this article can be found, in the 7

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