Effects of extraction methods on the physicochemical characteristics and biological activities of polysaccharides from okra (Abelmoschus esculentus)

International Journal of Biological Macromolecules 127 (2019) 178–186

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International Journal of Biological Macromolecules journal homepage: http://www.elsevier.com/locate/ijbiomac

Effects of extraction methods on the physicochemical characteristics and biological activities of polysaccharides from okra (Abelmoschus esculentus) Qin Yuan 1, Shang Lin 1, Yuan Fu, Xi-Rui Nie, Wen Liu, Yan Su, Qiao-Hong Han, Li Zhao, Qing Zhang, De-Rong Lin, Wen Qin ⁎, Ding-Tao Wu ⁎ College of Food Science, Sichuan Agricultural University, Ya'an 625014, Sichuan, China

a r t i c l e

i n f o

Article history: Received 20 November 2018 Received in revised form 14 December 2018 Accepted 9 January 2019 Available online 11 January 2019 Keywords: Okra polysaccharides Extraction method Antioxidant activity Binding properties Enzyme inhibition

a b s t r a c t The impacts of three extraction techniques, including hot water extraction (HWE), pressurized water extraction (PWE), and microwave assisted extraction (MAE), on the physicochemical characteristics, antioxidant activities, in vitro binding properties, and in vitro inhibitory activities on α-amylase and α-glucosidase of okra polysaccharides (OPPs) were investigated and compared. The extraction yields, constituent monosaccharides, and FT-IR spectra of OPP-W, OPP-P, and OPP-M extracted by HWE, PWE, and MAE, respectively, were similar. However, their molecular weights, intrinsic viscosities, uronic acids, and degree of esterification were different. Furthermore, results showed that OPP-W, OPP-P, and OPP-M exhibited remarkable antioxidant activities, binding capacities, and inhibitory activities on α-amylase and α-glucosidase. Indeed, the antioxidant activities of OPP-W were significantly lower than those of OPP-M and OPP-P, which might be attributed to the low molecular weights and high contents of unmethylated galacturonic acid of OPP-P and OPP-M. However, the binding capacities and inhibitory activities on α-amylase and α-glucosidase of OPP-W and OPP-P were similar, but significantly higher than those of OPP-M, which might be attributed to the low molecular weights of OPP-M. Results suggested that the PWE method could be a good potential technique for the extraction of OPPs with high bioactivities for industrial applications. © 2019 Elsevier B.V. All rights reserved.

1. Introduction The fruit of Okra (Abelmoschus esculentus L. Moench), known as lady's finger and gumbo, is an important vegetable crop and folk medicine [1,2]. It is an annual plant native to Africa, and has been grown in different countries around the world, mainly in tropical, sub-tropical, and warm temperate regions [2]. In recent years, okra has also been widely cultivated in the North and South China [3]. An increasing interest in okra fruit as functional food ingredients has been noticed due to its health benefits [4]. The health beneficial effects of okra fruit are owing to its various biological activities, such as cholesterol-binding capacity [4], anti-diabetic, and anti-hyperlipidemic effects [5]. Generally, polysaccharides are considered the main bioactive components in okra fruit, which possess various bioactivities, such as antioxidant [6,7], anti-hyperglycemic [7,8], immunomodulatory [9], and antifatigue activities [10]. Indeed, okra is rich in bioactive polysaccharides, and the contents of okra polysaccharides range from 10.35% to ⁎ Corresponding authors. E-mail addresses: [email protected] (W. Qin), [email protected] (D.-T. Wu). 1 These authors contributed equally to this work.

https://doi.org/10.1016/j.ijbiomac.2019.01.042 0141-8130/© 2019 Elsevier B.V. All rights reserved.

16.895% [6,11,12]. Therefore, okra polysaccharides could be explored further as functional food ingredients for industrial applications. Generally, extraction techniques exert significant influences on the extraction yields, physicochemical properties, and bioactivities of natural polysaccharides [13–15], which have great effects on the utilization of natural polysaccharides for functional food and pharmaceutical purposes. Currently, several extraction techniques are used to extract natural polysaccharides. Each technique has its own advantages and drawbacks when considering the extraction efficiency, convenience, cost, time consumption, and environmental impact. Hot water extraction (HWE) is a very common and easy method for the extraction of polysaccharides [13]. However, HWE has the disadvantages of long extraction time, high extraction temperature, and low extraction efficiency. Thus, some physical methods which could facilitate the extraction process have been taken into considerations. Microwave assisted extraction (MAE) and pressurized water extraction (PWE) have been indicated higher extraction efficiency than that of HWE [16,17]. MAE is based on the direct application of electromagnetic radiation, which has the ability to absorb electromagnetic energy and transform it into heat, to a material [14]. Compared with traditional methods, MAE has the advantages of less time consumption and high extraction

Q. Yuan et al. / International Journal of Biological Macromolecules 127 (2019) 178–186

efficiency [14,15,18]. Furthermore, PWE is based on the use of water at temperatures and pressures high enough to maintain the solvent in its liquid state throughout the extraction procedure [19]. The high temperature and pressure could increase the solubility of target compounds, and decrease the solvent viscosity and surface tension, allowing better penetration into the sample matrix [16]. PWE has been demonstrated to be useful in the extraction of natural polysaccharides [16,20,21]. Up to date, some extraction methods, such as HWE, MAE, and ultrasound assisted extraction [6,12,22], have been optimized for the improvement of extraction yields of polysaccharides from okra fruit. In addition, effects of different extraction solvents on the physicochemical properties and anti-adhesive activities of okra polysaccharides have been investigated [23–25]. However, to the best of our knowledge, effects of different extraction techniques on the physicochemical characteristics and biological activities of polysaccharides from okra have seldom been investigated. Therefore, in this study, effects of different extraction techniques (HWE, MAE, and PWE) on the physicochemical characteristics and biological activities of polysaccharides from okra were systemically investigated and compared. Results are helpful for providing a good potential technique for the extraction of okra polysaccharides with high quality for industrial applications.

179

2.2.2. Microwave assisted extraction The microwave assisted extraction (MAE) was performed according to a previously optimized method with minor modifications [15]. Briefly, 5.0 g of okra powders were firstly refluxed with 50 mL of 80% (v/v) ethanol at 80 °C for 1 h as described above (Section 2.2.1). Then, the polysaccharides were extracted with 150 mL of 50 mM PBS (pH 6.0) by the microwave extraction device (MKJ-J1-3, Qingdao Makewave Microwave Applied Technology Co., Ltd., Shandong, China) at 480 W and 80 °C for 10 min. Finally, the crude okra polysaccharides (OPP-M) were obtained according to the same treatment processes as described in Section 2.2.1.

2. Material and methods

2.2.3. Pressurized water extraction The pressurized water extraction (PWE) was performed on a high pressure reactor according to a previously optimized method with minor modifications [16]. Briefly, 5.0 g of okra powders were firstly refluxed with 50 mL of 80% (v/v) ethanol at 80 °C for 1 h as described above (Section 2.2.1). Then, the polysaccharides were extracted with 150 mL of 50 mM PBS (pH 6.0) by the laboratory-scale high pressure reactor (LEC-300, Shanghai Laibei Scientific Instruments Co., Ltd., Shanghai, China) at 55 °C and 1.6 MPa for 40 min. Finally, the crude okra polysaccharides (OPP-P) were obtained according to the treatment processes as described in Section 2.2.1.

2.1. Material and chemicals

2.3. Characterization of polysaccharides from okra fruit

Okra fruits (Abelmoschus esculentus cv. Kalong) were harvested at a commercial orchard (30°46′ 18.50″ N, 104°02′ 20. 02″ E) located in Chengdu, Sichuan Province, China. The whole okra fruits were washed with distilled water. Samples were frozen and freezing dried. Subsequently, the samples were ground to pass through a 60 mesh sieve, and stored at −20 °C for further analysis. Trifluoroacetic acid, sodium cholate, sodium deoxycholate, sodium glycocholate, sodium taurocholate, cholesterol, oleic acid, bovine serum albumin, sodium nitroprusside, cellulose, soluble starch, αamylase, α-glucosidase, acarbose, rhamnose (Rha), mannose (Man), glucuronic acid (GlcA), galacturonic acid (GalA), glucose (Glc), galactose (Gal), xylose (Xyl), arabinose (Ara), 1-phenyl-3-methyl-5-pyrazolone (PMP), m-hydroxydiphenyl, 2,2′-azino-bis(3-ethylbenzothiazoline-6sulfonic acid) (ABTS), and 4-nitrophenyl β-D-glucopyranoside (PNPG) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Heat-stable α-amylase and a free cholesterol assay kit were purchased from Solarbio (Beijing, China). All other reagents and chemicals used were of analytical grade.

2.3.1. Chemical composition analysis The chemical compositions, such as the content of total polysaccharides, content of uronic acids, and content of proteins, were analyzed by colorimetric methods. The content of total polysaccharides in OPPs was determined by the phenol-sulfuric acid method with a mixture standard [27]. The mixture standard was prepared by 50% of GalA and 50% of Gal. The content of uronic acids in OPPs was determined by using the m-hydroxydiphenyl method with GalA as a standard [28]. The content of proteins in OPPs was determined by using Bradford's method with bovine serum albumin as a standard [29].

2.2. Extraction of polysaccharides from okra fruit 2.2.1. Hot water extraction The hot water extraction (HWE) was performed according to a previously optimized method with minor modifications [26]. Briefly, 5.0 g of okra powders were firstly refluxed with 50 mL of 80% (v/v) ethanol at 80 °C for 1 h. After centrifugation at 4000 ×g for 15 min (Heraeus Multifuge X3R Centrifuge, Thermo Fisher scientific, Waltham, MA, USA), the supernatant was discarded. Then, the okra polysaccharides (OPPs) were extracted twice with 100 mL of phosphate buffer solution (PBS, 50 mM, pH 6.0) at 95 °C for 1 h with continuous shaking. After centrifugation (4000 ×g for 15 min), the extracts were combined, and concentrated to 1/3 of the original volume by a rotary evaporator under a vacuum at 60 °C. Then, the heat-stable α-amylase (4 U/mL) was used to remove starch. Subsequently, the extracts were precipitated with three volumes of 95% (v/v) ethanol overnight at 4 °C. The precipitations were washed twice with 70% of ethanol, and dissolved in deionized water. After centrifugation, the supernatant was dialyzed against deionized water for 3 days (Dialysis membrane, molecular weight cutoff: 3.5 kDa, Solarbio, Beijing, China). Finally, the crude okra polysaccharides (OPP-W) were freeze dried, and stored at −20 °C for further analysis.

2.3.2. Determination of molecular weights The absolute molecular weights (Mw) and polydispersities (Mw/Mn) of OPP-W, OPP-P, and OPP-M were measured by high performance size exclusion chromatography coupled with multi angle laser light scattering and refractive index detector (HPSEC-MALLS-RID) according to a previously reported method with minor modifications [30]. Briefly, HPSEC-MALLS-RID measurements were carried out on a multi angle laser light scattering detector (MALLS, DAWN HELEOS, Wyatt Technology Co., Santa Barbara, CA, USA) with an Agilent 1260 series LC system (Agilent Technologies, Palo Alto, CA, USA). TSK-Gel G5000PWXL (300 mm × 7.8 mm, i.d.) and TSK-Gel G3000PWXL (300 mm × 7.8 mm, i.d.) were used in series at 30 °C. The MALLS instrument was equipped with a He-Ne laser (λ = 658 nm). An Optilab rEX refractometer (RID, DAWN EOS, Wyatt Technology Co., Santa Barbara, CA, USA) was simultaneously connected. The mobile phase was 0.9% NaCl aqueous solution at a flow rate of 0.5 mL/min. The sample concentration was about 1.0 mg/mL. An injection volume of 100 μL was used. The Mw was calculated by the Zimm method of static light scattering based on the basic light scattering equation as follows, Kc 1 ¼ Rθ M w

1þ

16π2 bS2 N z 3λ2

sin2


! θ þ 2A2 c þ … 2

where K is an optical constant equal to [4π2n2(dn/dc)2]/(NAλ4); c, the polysaccharide concentration in g/mL; Rθ, the Rayleigh ratio; Mw, the weight average molecular mass; bS2Nz1/2, the radius of gyration; λ, the wavelength; n, the refractive index of the solvent (0.9% NaCl aqueous solution); dn/dc, the refractive index increment of OPPs in 0.9% NaCl

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aqueous solution; NA, the Avogadro's number; A2, the second virial coefficient. The dn/dc value of OPPs was selected as 0.146 mL/g according to a previous study [11].

band areas at 1700–1750 cm−1 (esterified uronic acids) and 1600–1630 cm−1 (free uronic acids). DE was calculated according to the equation as follows,

2.3.3. Determination of intrinsic viscosities The intrinsic viscosities ([η]) of OPP-W, OPP-P, and OPP-M were measured by an Ubbelohde viscosity method according to a previously reported method [31]. In brief, the samples were dissolved in deionized water, and then the solutions were taken into an Ubbelohde capillary viscometer at 25 °C for 15 min in a constant temperature bath. The kinetic energy correlation was assumed to be negligible, and the Huggins and Kraemer equations were used to estimate the value of [η] as follows,

DEð%Þ ¼

ηsp =c ¼ ½η þ k½η2 c

ð ln ηr Þ=c ¼ ½η−β ½η2 c where k and β are constants for a given polymer under certain conditions in a given solvent; ηsp/c is the reduced specific viscosity, and (ln ηr)/c is the inherent viscosity; c is the polymer concentration. 2.3.4. Determination of constituent monosaccharides Constituent monosaccharides of OPP-W, OPP-P, and OPP-M were measured by high performance liquid chromatography (HPLC) analysis according to a previously reported method with some modifications [32]. Briefly, each sample (4.0 mg) was hydrolyzed with 2.0 M trifluoracetic acid (2.0 mL) at 95 °C for 10 h. After hydrolysis, the hydrolysates were evaporated to dryness by a rotary evaporator under a vacuum, and washed with methanol for three times to remove the residue of trifluoroacetic acid. Subsequently, the dried hydrolyzates were dissolved in 1 mL of water for subsequent derivatization. Then, 50 μL of hydrolyzates were mixed with 50 μL of 0.6 M sodium hydroxide and 100 μL of 0.5 M 1-phenyl-3-methyl-5-pyrazolone (PMP) methanol solution. The mixture was incubated at 70 °C for 100 min with continuous shaking. Then, 80 μL of 0.3 M hydrochloric acid solution was used to neutralize the mixture, and the mixture was diluted to 1 mL with pure water. Furthermore, 1 mL of chloroform was added. After vigorous shaking and layering, the organic phase was discarded. The operation was performed in triplicate, and finally the solution was passed through a 0.22 μm organic syringe filter for HPLC analysis. A standard solution, containing Rha, Man, GlcA, GalA, Glc, Gal, Xyl, and Ara, was derivatized as described above. The PMP derivatives were analyzed by a Dionex UltiMate 3000 HPLC system (ThermoFisher scientific, Waltham, MA, USA) with a ZORBAX Eclipse XDB-C18 column (4.6 × 250 mm i.d. 5 μm, Agilent Technologies Inc., CA, USA) and a diode array detector (DAD, ThermoFisher scientific, Waltham, MA, USA). A 20 μL of PMP derivatives was injected into the HPLC system at the operation temperature of 30 °C, and eluted with a mixture of 0.1 M phosphate buffer solution (pH = 6.7) and acetonitrile (83: 17, v/v) at a flow rate of 1.0 mL/min. The wavelength of DAD was set at 245 nm. 2.3.5. Fourier transform infrared spectroscopy analysis The Fourier transform infrared (FT-IR) spectroscopy analysis of OPPW, OPP-P, and OPP-M was performed according to a previously reported method with minor modifications [33]. Briefly, each sample (1.0 mg) was mixed with 100 mg of dried KBr, and pressed into disk for the analysis. The IR spectra were recorded in the frequency range of 4000–500 cm−1 with a Nicolet iS 10 FT-IR (ThermoFisher scientific, Waltham, MA, USA). Furthermore, the esterification degree (DE) of OPP-W, OPP-P, and OPP-M was determined from FT-IR spectra according to previously reported methods [34,35]. The determination of DE was based on the




A1726 A1726 þ A1618

  100

2.4. Evaluation of biological activities of polysaccharides from okra fruit 2.4.1. Antioxidant activities of OPPs The ABTS radical cation scavenging activity of OPP-W, OPP-P, and OPP-M was measured according to a previously reported method with minor modification [36]. Briefly, the ABTS radical cation solution was generated by the interaction of 7 mM ABTS solution and 2.45 mM aqueous potassium persulfate at room temperature for at least 16 h in dark. The ABTS radical cation solution was diluted with phosphate buffer (0.2 M, pH 7.4) to an absorbance of 0.750 ± 0.02 at 734 nm. Then, 200 μL of ABTS radical cation working solution was mixed with 20 μL of each sample at different concentrations (0.5, 1.5, 2.5, 3.5, and 4.5 mg/mL) or phosphate buffer as a negative control in a 96-well microplate to react at 30 °C for 20 min. The absorbance at 734 nm was measured. The ABTS radical cation scavenging capacity was calculated as follows, ABTS scavenging capacity ð%Þ ¼


 Asample −Acontrol  100% 1− Ablank

where Asample is the absorbance of the mixture of sample and ABTS work solution; Acontrol is the absorbance of the mixture of pure water and sample; Ablank is the absorbance of the mixture of pure water and ABTS work solution. Furthermore, the nitric oxide radical scavenging capacity of OPP-W, OPP-P, and OPP-M was measured according to a previously reported method with some modifications [37]. Briefly, 50 μL of 10 mM sodium nitroprusside (SNP, prepared in 200 mM PBS, pH = 6.6) was mixed with 450 μL of each sample at different concentrations (0.5, 1.5, 2.5, 3.5, and 4.5 mg/mL), and incubated at 25 °C for 3 h in front of a visible polychromatic light source. Then, 250 μL of Griess reagent (1% sulfanilamide and 0.1% naphthylethylenediamine dihydrochloride in 2% phosphoric acid) was added into the mixture. Finally, the absorbance was measured at 540 nm. The nitric oxide radical scavenging capacity was calculated as follows,
Scavenging capacity ð%Þ ¼

1−

Asample −Acontrol Ablank

  100%

where Asample is the absorbance of the mixture of sample, SNP solution, and Griess reagent; Acontrol is the absorbance of the mixture of pure water and sample; Ablank is the absorbance of the mixture of pure water, SNP solution, and Griess reagent. The ferric reducing antioxidant power (FRAP) of OPP-W, OPP-P, and OPP-M was measured according to a previously reported method with some modifications [37]. Briefly, the FRAP working solution contained 300 mM acetate buffer (pH 3.6), 10 mM TPTZ (2,4,6-Tris(2-pyridyl)-striazine) solution in 40 mM HCl, and 20 mM ferric chloride solution at a ratio of 10:1:1. The working solution was warmed at 37 °C before usage. An aliquot of 100 μL sample was mixed with 3 mL of FRAP solution at 1 min intervals. After 4 min of incubation at 37 °C, the absorbance was read at 593 nm. Afterwards, the FRAP was expressed as the absorbance at 593 nm. 2.4.2. In vitro binding properties of OPPs The in vitro fat-binding, cholesterol-binding, and bile acid-binding capacities of OPP-W, OPP-P, and OPP-M were measured according to previously reported methods [31]. In brief, in order to determine the

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fat-binding capacity, 20 mg of each sample was dissolved in 2.0 mL of pure water. Then 1 mL of peanut oil was added, and the mixture was incubated at 37 °C for 2 h with continuous shaking. After centrifugation (10,000 ×g for 20 min), the supernatant was carefully removed. The bound oil of OPPs was extracted with petroleum ether for four times. Cellulose was used as a positive control, and the pure water was used as a substrate blank. The fat-binding capacity of OPPs was expressed as gram of binding fat per gram of OPPs (g/g). In addition, in order to determine the cholesterol-binding capacity, 20 mL of cholesterol micellar solution were prepared by sonication at 480 W for 1 h, composed of 10 mM sodium taurocholate, 5 mM cholesterol, 5 mM oleic acid, 132 mM sodium chloride, and 15 mM sodium phosphate buffer (pH 7.4). Then, 10 mg of each sample was added into 3 mL of micellar solution, and the mixture was incubated at 37 °C for 2 h with continuous shaking. After centrifugation (13,000 ×g, 1 h), the supernatant was collected for the determination of cholesterol. The content of cholesterol in the supernatant was measured by the free cholesterol kit. In brief, 20 μL of supernatant were mixed with 980 μL of free cholesterol working solution. After incubation at 40 °C for 5 min, the absorbance was measured at 500 nm. The content of binding cholesterol was calculated as the amount of cholesterol in the supernatant of the substrate blank subtracted from the amount in the supernatant of the sample. Cellulose was used as a positive control, and pure water was used as a substrate blank control. The cholesterol-binding capacity of OPPs was expressed as milligrams of binding cholesterol per gram of OPPs (mg/g). Furthermore, in order to determine the bile acid-binding capacity, the bile acid mixture was prepared with sodium cholate, sodium deoxycholate, sodium glycocholate, and sodium taurocholate with proportions as 35%, 35%, 15%, and 15% (w/w) in 50 mM phosphate buffer (pH 6.9), respectively. Each sample (20 mg) was digested with 1 mL of 0.01 M HCl in a shaking water bath at 37 °C for 1 h, which simulated gastric digestion. Then, to each sample, 4 mL of 1.4 μM bile acid mixture and 5 mL of porcine pancreatin (10 U/mL) were added, and incubate at 37 °C for 1 h with continuous shaking. After centrifugation (13,000 ×g, 1 h), the supernatant was collected for the determination of unbound bile acid. The content of bile acid was measured by furfural-sulfuric acid method. The binding bile acid was calculated as the amount of bile acid in the supernatant of the substrate blank subtracted from the amount in the supernatant of the sample. The cholestyramine and cellulose were used as positive and negative controls, respectively. The bile acid-binding capacity of OPPs was expressed as a percent of blank control (%). 2.4.3. In vitro α-amylase and α-glucosidase inhibition of OPPs α-Amylase inhibitory effect of OPPs was measured according to a previously reported method with slight modifications [31]. In brief, 100 μL of each sample at different concentrations (50, 100, 150, 200 and 250 μg/mL, respectively) was mixed with 100 μL of α-amylase solution (30 U/mL, dissolved in 0.1 M, pH 6.8 phosphate buffer), and incubated at 37 °C for 30 min with continuous shaking. Then, 200 μL of soluble starch (0.5%, w/v) was added into the mixture, and incubated at 37 °C for 10 min. Subsequently, 100 μL of mixture was incubated with 400 μL of 3,5-dinitrosalicylic acid (DNS) reagent at a boiling water bath for 5 min. Finally, the absorbance of the mixture was measured at 540 nm. Acarbose standard was used as a positive control. The α-amylase inhibitory activity was calculated as follows, α‐Amylase inhibitionð%Þ ¼


 Asample −Acontrol  100% 1− Ablank −Acontrol

of each sample at different concentrations (50, 100, 150, 200 and 250 μg/mL, respectively) was mixed with 40 μL of α-glucosidase solution (0.5 U/mL, dissolved in 0.1 M pH 6.8 phosphate buffer), and incubated at 37 °C for 30 min with continuous shaking. Subsequently, 100 μL of PNPG (4 mM, dissolved in 0.1 M pH 6.8 phosphate buffer) was added into the mixture, and incubated at 37 °C for 10 min. Finally, the absorbance of the mixture was measured at 405 nm, and the α-glucosidase inhibitor (acarbose standard) was used as a positive control. The αglucosidase inhibitory activity was calculated as follows,
α‐Glucosidase inhibitionð%Þ ¼

1−

Asample −Acontrol Ablank −Acontrol

  100%

where Asample is the absorbance of the mixture of sample, PNPG, and αglucosidase; Acontrol is the absorbance of the mixture of phosphate buffer (instead of sample and α-glucosidase) and PNPG; Ablank is the absorbance of the mixture of phosphate buffer, PNPG, and α-glucosidase. 2.5. Statistical analysis All experiments were conducted in triplicate, and data were expressed in means ± standard deviations. Statistical analysis was performed using Origin 9.0 software (OriginLab Corporation, Northampton, MA, USA). Statistical significances were carried out by one-way analysis of variance (ANOVA), taking a level of p b 0.05 as significant to Duncan's multiple range test. 3. Results and discussions 3.1. Physicochemical characteristics of OPPs 3.1.1. Chemical composition of OPPs The extraction yields and chemical compositions of OPPs extracted by HWE, PWE, and MAE are summarized in Table 1. As shown in Table 1, results showed that the extraction yields of OPP-W, OPP-P and OPP-M were similar, which were determined to be 14.18%, 14.30%, and 13.98%, respectively. Results are in accordance with previous studies that the extraction yields of okra polysaccharides range from 10.35% to 16.895% [6,11,12,26]. Indeed, the HWE, PWE, and MAE had no significant effects on the extraction yields of OPPs. However, considering the extraction time and extraction temperature of different extraction methods, MAE and PWE could be better than HWE [16,17]. There was no significant difference in the total polysaccharide contents of OPP-W, OPP-P, and OPP-M, which were determined to be 83.21%, 83.12%, and 82.97%, respectively. There was also no significant Table 1 Chemical composition, molecular weight (Mw), polydispersity (Mw/Mn), and intrinsic viscosity ([η]) of OPP-W, OPP-P, and OPP-M.

Extraction yield (%) Total polysaccharides (%) Total uronic acids (%) Protein content (%) Degree of esterification (%) [η] (dL/g) Mw × 106 (Da) Fraction 1 Fraction 2

where Asample is the absorbance of the mixture of sample, starch solution, α-amylase, and DNS reagent; Acontrol is the absorbance of the mixture of phosphate buffer, starch solution, and DNS reagent; Ablank is the absorbance of the mixture of phosphate buffer, starch solution, αamylase, and DNS reagent. α-Glucosidase inhibitory effect was measured according to a previously reported method with minor modifications [38]. Briefly, 200 μL

181

Mw/Mn Fraction 1 Fraction 2

OPP-W

OPP-P

OPP-M

14.18 ± 0.22a 83.21 ± 0.36a 50.35 ± 0.12a 2.73 ± 0.11a 33.55 ± 0.08a 3.13 ± 0.06a

14.3 ± 0.14a 83.12 ± 0.18a 48.73 ± 0.09b 2.86 ± 0.16a 28.99 ± 0.06b 3.07 ± 0.08a

13.98 ± 0.27a 82.97 ± 0.52a 45.50 ± 0.11c 2.93 ± 0.21a 20.24 ± 0.11c 2.45 ± 0.08b

6.597 (±0.56%)a 1.736 (±0.35%)a

6.396 (±0.56%)a 1.707 (±0.34%)a

5.859 (±0.48%)b 1.391 (±0.65%)b

1.05 1.19

1.10 1.12

1.12 1.08

OPP-W, hot water extraction of okra polysaccharides; OPP-P, pressurized water extraction of okra polysaccharides; OPP-M, microwave assisted extraction of okra polysaccharides. Values represent mean ± standard deviation, and superscripts a–c differ significantly (p b 0.05) among OPPs; statistical significances were carried out by ANOVA and Duncan's test.

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difference in the protein contents of OPP-W, OPP-P, and OPP-M, which ranged from 2.73% to 2.93%. Furthermore, the uronic acid contents significantly (p b 0.05) varied by different extraction methods. The uronic acid contents of OPP-W, OPP-P, and OPP-M were determined to be 50.35%, 48.73%, and 45.50%, respectively, which is similar with the previous studies [11,26]. The lower content of uronic acids in OPP-M might be attributed to the degradation of OPPs under microwave treatment [39,40].

column and the co-elution of various different molecules from 38 min to 42 min. Therefore, the molecular weights of polysaccharide fractions (fraction 1 and fraction 2) in okra fruit are summarized in Table 1. As shown in Table 1, molecular weights of polysaccharide fraction 1 and fraction 2 in OPPs ranged from 5.859 × 106 Da to 6.597 × 106 Da, and from 1.391 × 106 Da to 1.736 × 106 Da, respectively. Results showed that molecular weights of OPP-P extracted by PWE were similar with those of OPP-W extracted by HWE. However, the molecular weights of OPP-M extracted by MAE were significantly (p b 0.05) lower than those of OPP-W extracted by HWE. A sharp degradation of fraction 1 was also detected in the HPSEC chromatogram of OPP-M (Fig. 1A). Results suggested that the MAE method could degrade the molecular weights of OPPs. Similar studies have shown that the molecular weights of polysaccharides extracted by microwave assisted extraction are lower than that of conventional hot water extraction [13,15,39,40,42]. Furthermore, the polydispersities of polysaccharide fraction 1 and fraction 2 in OPPs ranged from 1.05 to 1.12, and from 1.08 to 1.19, respectively. Moreover, bioactivities of natural polysaccharides, such as binding properties, antioxidant activity, and anti-inflammatory activity, are influenced by the intrinsic viscosity [31,41]. Thus, intrinsic viscosities of OPPs extracted by different extraction methods were compared.

3.1.2. Molecular weights and intrinsic viscosities of OPPs Generally, bioactivities of polysaccharides are closely correlated to their molecular weights and intrinsic viscosities [31,41]. Therefore, effects of different extraction methods on the molecular weights and intrinsic viscosities of OPPs were investigated. Fig. 1A showed the HPSEC-RID-UV chromatograms of OPP-W, OPP-P, and OPP-M. Results showed that HPSEC chromatograms of OPP-W, OPP-P, and OPP-M were similar, and three polysaccharide fractions (Fig. 1A, 1 to 3) were detected. UV absorbance at 280 nm was detected in OPP-W, OPP-P, and OPP-M, which further confirmed that minor protein existed in OPPs [11,26]. In addition, the molecular weight of fraction 3 could not be precisely determined due to the relatively poor resolution of the

A

B RID UV

1

4.0 ×10-6

2

0.15

3.0 ×10-6

0.10

3

2.0 ×10-6 1.0 ×10-6

0.0

10.0

20.0

30.0

200

Absorbance (mAU)

Detector voltage (V)

OPP-W

5.0 ×10-6

Differential refractive index (RIU)

0.20

100

OPP-W

PMP

GalA Man

40.0

5.0

10.0

20.0

2

5.0 ×10-6 4.0 ×10-6 3.0 ×10-6

0.20

3

2.0 ×10-6 1.0 ×10-6

0.10

0.0

10.0

20.0

30.0

200

Absorbance (mAU)

Detector voltage (V)

RID UV

1

100

GalA

5.0 10.0

20.0

RID UV

5.0 ×10-6 4.0

×10-6

2 3.0 ×10-6

0.30

2.0

0.20

×10-6

3 1.0 ×10-6 0.10 10.0

20.0

30.0

Time (min)

30.0

Ara

40.0

50.0

Time (min)

1

0.0

Gal

Rha

0

40.0

200

Absorbance (mAU)

Detector voltage (V)

0.40

50.0

PMP

Man

40.0

Differential refractive index (RIU)

OPP-M

40.0

OPP-P

Time (min) 0.50

30.0

Time (min) Differential refractive index (RIU)

0.30

Ara

0

Time (min) OPP-P

Gal

Rha

OPP-M

100

PMP

Rha GalA Gal

Man

Ara

0 5.0 10.0

20.0

30.0

40.0

50.0

Time (min)

Fig. 1. HPSEC chromatograms (A) and HPLC profiles (B) of OPP-W, OPP-P, and OPP-M. OPP-W, hot water extraction of okra polysaccharides; OPP-P, pressurized water extraction of okra polysaccharides; OPP-M, microwave assisted extraction of okra polysaccharides; PMP, 1-phenyl-3-methyl-5-pyrazolone; Rha, rhamnose; GalA, galacturonic acid; Gal, galactose; Ara, arabinose; Man, mannose.

Q. Yuan et al. / International Journal of Biological Macromolecules 127 (2019) 178–186 Table 2 Molar ratios of constituent monosaccharides of OPP-W, OPP-P, and OPP-M. Monosaccharides and molar ratios

OPP-W OPP-P OPP-M

Rha

GalA

Gal

Ara

Man

1.00 1.00 1.00

4.95 4.04 1.88

2.34 2.38 1.17

0.89 0.72 0.32

0.09 0.16 0.24

OPP-W, hot water extraction of okra polysaccharides; OPP-P, pressurized water extraction of okra polysaccharides; OPP-M, microwave assisted extraction of okra polysaccharides; Rha, rhamnose; GalA, galacturonic acid; Gal, galactose; Ara, arabinose; Man, mannose.

As shown in Table 1, the intrinsic viscosities of OPP-W, OPP-P, and OPPM were determined to be 3.13 dL/g, 3.07 dL/g, and 2.45 dL/g, respectively, which is similar with previous studies that the intrinsic viscosities of okra polysaccharides range from 2.91 dL/g to 5.1 dL/g [11,26]. The intrinsic viscosity of OPP-P extracted by PWE was similar with that of OPP-W extracted by HWE. However, the intrinsic viscosity of OPP-M extracted by MAE was significantly lower than that of OPP-W. Results indicated that the intrinsic viscosity of OPPs is positively correlated to the molecular weights, which is similar with previous studies [31,41]. Indeed, results suggested that the intrinsic viscosity of OPPs could be decreased by microwave treatment due to the decrease of molecular weights. 3.1.3. Constituent monosaccharides of OPPs Monosaccharides are the natural basic units that determine the unique structures and properties of polysaccharides. Analysis of constituent monosaccharides is necessary for structural characterization. Fig. 1B showed that the HPLC-UV profiles of OPP-W, OPP-P, and OPPM were similar. Results showed that the constituent monosaccharides of OPP-W, OPP-P, and OPP-M were measured as Rha, GalA, Gal, Ara, and Man, which is similar with previous studies [7,11,26]. The molar ratios of Rha, GalA, Gal, Ara, and Man in OPP-W, OPP-P, and OPP-M were determined to be about 1.00:4.95:2.34:0.89:0.09, 1.00:4.04:2.38:0.72:0.16, and 1.00:1.88:1.17:0.32:0.24, respectively (Table 2). Results showed that Rha, GalA and Gal were the dominant

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monosaccharides in OPPs, which indicated that rhamnogalacturonan I (RG I) and homogalacturonan (HG) existed in okra polysaccharides [11]. Furthermore, results showed that the molar ratios of constituent monosaccharides in OPP-W were similar with those of OPP-P, while different from those of OPP-M. Results suggested that the MAE method had significant effects on the molar ratios of constituent monosaccharides in OPPs, Similar studies have shown that different extraction techniques affect the monosaccharide compositions of polysaccharides [17,42].

3.1.4. FT-IR spectra and esterification degree of OPPs The FT-IR spectra were used for determination of structure features of OPPs. As shown in Fig. 2, the FT-IR spectra of OPP-W, OPP-P, and OPPM were similar, which indicated that OPPs extracted by different extraction methods had similar structures. Briefly, the intense and broad bands around 3200 cm−1 and 3600 cm−1 are the characteristic bands of hydroxyl groups, and the broad band at 3415 cm−1 is due to the stretching vibration of hydroxyl group [11]. Bands in the region of 3000–2800 cm−1 are assigned to C\\H absorption that includes CH, CH2, and CH3 stretching vibrations [43]. Absorption bands between 1730 cm−1 and 1720 cm−1 correspond to C_O stretching vibration of esterified groups [11]. Furthermore, the intense band appeared at 1618 cm−1 is the C_O asymmetric stretching of COO−, suggesting the existence of uronic acids [44]. The band at 1420 cm−1 is attributed to bending vibration of C\\H or O\\H [9], and the band at 1147 cm−1 is the asymmetric C\\O\\C stretching vibration, suggesting the presence of \\OCH3 [45]. Typical protein band at 1651 cm−1 and 1555 cm−1 were not detected, which indicated the low amount of protein in OPPs [11]. Furthermore, effects of different extraction methods on the degree of esterification (DE) of OPPs were also investigated by FT-IR spectroscopy analysis. The significantly (p b 0.05) highest DE was observed in OPP-W (33.55%), followed by lower DE in OPP-P (28.99%), and the lowest DE in OPP-M (20.24%). Previous studies have indicated that the low DE is observed in pectins extracted under harsh extraction conditions (such as high temperature, high microwave power, and long microwave irradiation time) [18,46], because these harsh conditions could increase de-esterification of polygalacturonic chains.

OPP-P OPP-M OPP-W

100

Transmittance (%)

80 60 40 20 0 -20

2937 1726

-40 -60 4000

1618

3415

3500

1420

3000

2500

2000

Wave number

1500

1147

1000

500

(cm-1)

Fig. 2. FT-IR spectra of OPP-W, OPP-P, and OPP-M. OPP-W, hot water extraction of okra polysaccharides; OPP-P, pressurized water extraction of okra polysaccharides; OPP-M, microwave assisted extraction of okra polysaccharides.

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A

3.2. Antioxidant activities of OPPs

100

ABTS radical scavenging capacity (%)

80 a b

60

a

c b c

a

b

c

40 20

a

a

a

a

a

a

0 OPP-W

Concentration (mg/mL) 100

Nitric oxide radical scavenging capacity (%)

B

OPP-M

OPP-P

a

80

b c

a

b

c

a

b

c

60

a

a a a

40

a a

20 0 OPP-W

Ferric reducing antioxidant power (Absorbance at 593 nm )

C

OPP-M

OPP-P

3.3. In vitro binding properties of OPPs

Concentration (mg/mL) 0.25 0.20

a b

a

b

0.15

c

c

c

a

b

0.10 c

a

b

a a

b

0.05 0 OPP-W

OPP-P

Previous studies have shown that okra polysaccharides exert remarkable antioxidant activity [6,7]. So, in the present study, effect of different extraction methods on the antioxidant activity of OPPs was investigated. Fig. 3 showed the ABTS radical scavenging activities, nitric oxide radical scavenging activities, and ferric reducing antioxidant power (FRAP) of OPP-W, OPP-P, and OPP-M. As shown in Fig. 3, the ABTS radical scavenging activities, nitric oxide radical scavenging activities, and FRAP of OPPs exhibited a dose-dependent manner. Results showed that both ABTS radical and nitric oxide radical scavenging activities of OPP-W, OPP-P, and OPP-M were similar at low concentrations of 0.5 to 1.5 mg/mL. However, the significantly (p b 0.05) highest ABTS radical scavenging activities, nitric oxide radical scavenging activities, and FRAP were observed in OPPM at high concentrations of 2.5 to 4.5 mg/mL, followed by lower antioxidant activities in OPP-P, and the lowest antioxidant activities in OPP-W. Indeed, the IC50 values of ABTS radical scavenging activities of OPP-M, OPP-P, and OPP-W were determined as 2.50 mg/mL, 3.14 mg/mL, and 4.40 mg/mL, respectively. Additionally, the IC50 values of nitric oxide radical scavenging activities of OPP-M, OPP-P, and OPP-W were determined as 0.90 mg/mL, 1.28 mg/mL, and 1.75 mg/mL, respectively. Results further confirmed that OPP-M and OPP-P exhibited stronger antioxidant activities than that of OPP-W, which suggested that the MAE and PWE methods could be good potential techniques for the extraction of OPPs with relatively high antioxidant activity. Generally, the antioxidant activities of natural polysaccharides are correlated to their molecular weights and compositional monosaccharides (uronic acids) [47–49]. It is estimated that presence of electrophilic groups like keto or aldehyde in acidic polysaccharides facilitates the liberation of hydrogen from O\\H bond, and these groups can improve the radical scavenging activities [50]. The highest antioxidant activities observed in OPP-M might be partially attributed to its lowest molecular weights. Previous studies have shown that the low molecular weight polysaccharides exert high antioxidant activity [48,51]. In addition, the highest antioxidant activities observed in OPP-M might be also related to the relatively higher contents of galacturonic acid as well as the rate of unmethylated acid groups [51,52].

OPP-M

Concentration (mg/mL) Fig. 3. ABTS radical cation scavenging capacity (A), nitric oxide radical scavenging capacity (B), and ferric reducing antioxidant power (C) of OPP-W, OPP-P, and OPP-M. OPP-W, hot water extraction of okra polysaccharides; OPP-P, pressurized water extraction of okra polysaccharides; OPP-M, microwave assisted extraction of okra polysaccharides; the error bars are standard deviations; significant (p b 0.05) differences are shown by data bearing different letters (a–c); statistical significances were carried out by ANOVA and Ducan's test.

The over absorption of fat, cholesterol, and bile acid can lead some obesity issues, which are associated with cardiovascular disease, diabetes, and cancer [41]. Okra fruit exhibit strong oil-binding and cholesterol-binding capacities in vitro [4]. Indeed, some reports have revealed that okra fruits and okra polysaccharides can lower lipid and total serum cholesterol levels in high-fat diet-fed C57BL/6 mice [8]. Therefore, effects of different extraction methods on binding capacities of OPPs were estimated. In vitro fat, cholesterol, and bile acid binding capacities of OPPs are summarized in Table 3. Results showed that the capacities of fat-binding, cholesterol-binding, and bile acid-binding of OPP-W, OPP-P, and OPP-M ranged from 6.11 ± 0.08 g/g to 7.15 ± 0.11 g/g, from 116 ± 0.19 mg/g to 138.02 ± 0.22 mg/g, and from (31.06 ± 0.18)% to (44.23 ± 0.11)%, respectively. The fat-binding, cholesterol-binding, and bile acid-binding capacities of OPP-W and OPP-P were similar, but significantly (p b 0.05) higher than those of OPP-M. Previous studies have revealed that the binding properties of natural polysaccharides are influenced by their molecular weights and viscosities [31,41,53]. The lower binding capacities of OPP-M extracted by the MAE method might be attributed to its relatively low molecular weights and viscosities. Furthermore, compared with the positive control (cellulose), OPP-W, OPP-P, and OPP-M exerted significantly (p b 0.05) higher fat-binding and cholesterol-binding capacities. Indeed, the bile acid-binding capacities of OPP-W and OPP-P were also significantly higher than that of cholestyramine (a positive control). Results suggested that okra polysaccharides had great potential to be explored as functional food ingredients for the prevention of hypercholesterolemia and hyperlipidemia.

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Table 3 The fat-binding, cholesterol-binding, and bile acid-binding capacities of OPP-W, OPP-P, and OPP-M.

Fat-binding (g/g) Cholesterol-binding (mg/g) Bile acid-binding (%)

OPP-W

OPP-P

OPP-M

7.15 ± 0.11a 138.02 ± 0.22a 44.23 ± 0.11a

6.86 ± 0.17a 134.79 ± 0.25a 42.97 ± 0.19a

6.11 ± 0.08b 116.23 ± 0.19b 31.06 ± 0.18c

Positive control 1.01 ± 0.11c 19.23 ± 0.12c 40.74 ± 0.42b

OPP-W, hot water extraction of okra polysaccharides; OPP-P, pressurized water extraction of okra polysaccharides; OPP-M, microwave assisted extraction of okra polysaccharides. Cellulose was used as a positive control in fat-binding and cholesterol-binding capacity assay, respectively, and cholestyramine was used as a positive control in bile acid-binding capacity assay. Values represent mean ± standard deviation, and superscripts a–c differ significantly (p b 0.05) among OPPs; statistical significances were carried out by ANOVA and Duncan's test.

3.4. In vitro α-amylase and α-glucosidase inhibitory effects of OPPs

A

100

-Amylase inhibition (%)

Inhibition of α-glucosidase and α-amylase is one of the main strategies to counteract metabolic alterations related to hyperglycaemia and type 2 diabetes [54]. It is recognized that okra polysaccharides possess remarkable anti-hyperglycemic activity in vivo [7,8]. Therefore, effects of different extraction methods on the in vitro inhibitory effects of

80

a a a

60

b

a

a

b

a

a

b

a

40

b a

a

b

20 0 Acarbose

4. Conclusions

OPP-M

OPP-P

OPP-W

Concentration (µg/mL) -Glucosidase inhibition (%)

B

100 80

a

a

a

b

a b

60

a

a

a

b

a

40

b a

a

b

20

OPPs against target enzymes (α-amylase and α-glucosidase) for type 2 diabetes were investigated. As shown in Fig. 4, results showed that the α-amylase and α-glucosidase inhibitions of OPP-W and OPP-P were similar at concentrations of 50 to 250 μg/mL, but significantly (p b 0.05) higher than those of OPP-M. Indeed, the IC50 values of αamylase and α-glucosidase inhibitions of OPPs were determined to be 141.67 μg/mL and 125.15 μg/mL (OPP-W), 147.18 μg/mL and 131.18 μg/mL (OPP-P), and 179.74 μg/mL and 170.18 μg/mL (OPP-M), respectively. Results further confirmed that the inhibitory effects of OPP-W and OPP-P on α-amylase and α-glucosidase were higher than those of OPP-M. The inhibitory effect of polysaccharides on digestive enzymes is considered as a non-competitive interaction [55], and it is estimated that the reaction of enzymes and substrate would be inhibited by increasing the viscosity of surroundings [56]. Meanwhile, the inhibitory effects of polysaccharides on digestive enzymes decrease as the decrease of molecular weights [31,51]. Thus, the lower inhibitory effects of OPP-M on α-amylase and α-glucosidase might be attributed to its relatively low molecular weights. Furthermore, compared with the positive control (Acarbose), OPP-W, OPP-P, and OPP-M exerted much stronger inhibitory effects on α-glucosidase (Acarbose, IC50 = 393.76 μg/mL), as well as moderate inhibitory effects on α-amylase (Acarbose, IC50 = 36.85 μg/mL). Results suggested that the hypoglycemic activity of okra fruit was partially attributed to the strong α-glucosidase and α-amylase inhibition effect of OPPs, and OPPs could be explored further as functional food ingredients for the treatment of type 2 diabetes.

0

In this study, the effects of three different extraction methods (HWE, MAE, and PWE) on physicochemical structures, antioxidant activities, in vitro binding properties, and inhibitory activities on α-amylase and α-glucosidase of OPPs were investigated. The extraction yields, constituent monosaccharides, and FT-IR spectra of OPP-W, OPP-P, and OPP-M were similar. Additionally, results showed that the antioxidant activities of OPP-W were significantly lower than those of OPP-M and OPP-P, which might be attributed to their relatively low molecular weights and high contents of unmethylated galacturonic acid. However, the binding capacities and inhibitory effects on α-amylase and α-glucosidase of OPP-W and OPP-P were similar, but significantly higher than those of OPP-M, which might be attributed to the low molecular weight of OPPM. Results are helpful for the better understanding of the structurefunction relationships of okra polysaccharides, and the PWE method could a good potential technique for the extraction of okra polysaccharides with high bioactivities for industrial applications. Acknowledgments

Acarbose

OPP-W

OPP-P

OPP-M

Concentration (µg/mL) Fig. 4. In vitro inhibitory activities on α-amylase (A) and α-glucosidase (B) of OPP-W, OPPP, and OPP-M. OPP-W, hot water extraction of okra polysaccharides; OPP-P, pressurized water extraction of okra polysaccharides; OPP-M, microwave assisted extraction of okra polysaccharides; the error bars are standard deviations; significant (p b 0.05) differences are shown by data bearing different letters (a–c); statistical significances were carried out by ANOVA and Ducan's test.

This work was supported by the Scientific Research Foundation of Sichuan Agricultural University (grant number 03120321) and the Scientific Research Fund Project of Science and Technology Department of Sichuan Province (grant number 2018JY0149). Conflict of interest The authors declare that there are no conflicts of interest.

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References [1] C.H. Peng, C.C. Chyau, C.J. Wang, H.T. Lin, C.N. Huang, Y.B. Ker, Abelmoschus esculentus fractions potently inhibited the pathogenic targets associated with diabetic renal epithelial to mesenchymal transition, Food Funct. 7 (2016) 728–740. [2] F.B. Xia, Y. Zhong, M.Q. Li, Q. Chang, Y.H. Liao, X.M. Liu, R.L. Pan, Antioxidant and anti-fatigue constituents of okra, Nutrients 7 (2015) 8846–8858. [3] N. Jiang, Z. Zhang, D. Li, C. Liu, M. Zhang, C. Liu, D. Wang, L. Niu, Evaluation of freeze drying combined with microwave vacuum drying for functional okra snacks: antioxidant properties, sensory quality, and energy consumption, LWT Food Sci. Technol. 82 (2017) 216–226. [4] Y. Chen, B.-C. Zhang, Y.-H. Sun, J.-G. Zhang, H.-J. Sun, Z.-J. Wei, Physicochemical properties and adsorption of cholesterol by okra (Abelmoschus esculentus) powder, Food Funct. 6 (2015) 3728–3736. [5] V. Sabitha, S. Ramachandran, K.R. Naveen, K. Panneerselvam, Antidiabetic and antihyperlipidemic potential of Abelmoschus esculentus (L.) Moench. in streptozotocin-induced diabetic rats, J. Pharm. Bioallied Sci. 3 (2011) 397–402. [6] K. Wang, M. Li, X. Wen, X. Chen, Z. He, Y. Ni, Optimization of ultrasound-assisted extraction of okra (Abelmoschus esculentus (L.) Moench) polysaccharides based on response surface methodology and antioxidant activity, Int. J. Biol. Macromol. 114 (2018) 1056–1063. [7] T. Zhang, J. Xiang, G. Zheng, R. Yan, X. Min, Preliminary characterization and antihyperglycemic activity of a pectic polysaccharide from okra (Abelmoschus esculentus (L.) Moench), J. Funct. Foods 41 (2018) 19–24. [8] J. Liu, Y. Zhao, Q. Wu, A. John, Y. Jiang, J. Yang, H. Liu, B. Yang, Structure characterisation of polysaccharides in vegetable “okra” and evaluation of hypoglycemic activity, Food Chem. 242 (2018) 211. [9] W. Zheng, T. Zhao, W. Feng, W. Wang, Y. Zou, D. Zheng, M. Takase, Q. Li, H. Wu, L. Yang, Purification, characterization and immunomodulating activity of a polysaccharide from flowers of Abelmoschus esculentus, Carbohydr. Polym. 106 (2014) 335–342. [10] H. Gao, W. Zhang, B. Wang, A. Hui, B. Du, T. Wang, L. Meng, H. Bian, Z. Wu, Purification, characterization and anti-fatigue activity of polysaccharide fractions from okra (Abelmoschus esculentus (L.) Moench), Food Funct. 9 (2018). [11] F.M. Kpodo, J.K. Agbenorhevi, K. Alba, R.J. Bingham, I.N. Oduro, G.A. Morris, V. Kontogiorgos, Pectin isolation and characterization from six okra genotypes, Food Hydrocoll. 72 (2017) 323–330. [12] V. Samavati, Polysaccharide extraction from Abelmoschus esculentus: optimization by response surface methodology, Carbohydr. Polym. 95 (2013) 588–597. [13] L. He, X. Yan, J. Liang, S. Li, H. He, Q. Xiong, X. Lai, S. Hou, S. Huang, Comparison of different extraction methods for polysaccharides from Dendrobium officinale stem, Carbohydr. Polym. 198 (2018) 101–108. [14] H. Shang, S. Chen, R. Li, H. Zhou, H. Wu, H. Song, Influences of extraction methods on physicochemical characteristics and activities of Astragalus cicer L. polysaccharides, Process Biochem. 73 (2018) 220–227. [15] H. Dong, S. Lin, Q. Zhang, H. Chen, W. Lan, H. Li, J. He, W. Qin, Effect of extraction methods on the properties and antioxidant activities of Chuanminshen violaceum polysaccharides, Int. J. Biol. Macromol. 93 (2016) 179–185. [16] Y. Xu, F. Cai, Z. Yu, L. Zhang, X. Li, Y. Yang, G. Liu, Optimisation of pressurised water extraction of polysaccharides from blackcurrant and its antioxidant activity, Food Chem. 194 (2016) 650–658. [17] Y. Yan, X. Li, M. Wan, J. Chen, S. Li, M. Cao, D. Zhang, Effect of extraction methods on property and bioactivity of water-soluble polysaccharides from Amomum villosum, Carbohydr. Polym. 117 (2015) 632–635. [18] S.S. Hosseini, F. Khodaiyan, M.S. Yarmand, Optimization of microwave assisted extraction of pectin from sour orange peel and its physicochemical properties, Carbohydr. Polym. 140 (2016) 59–65. [19] M. Çam, Y. Hışıl, Pressurised water extraction of polyphenols from pomegranate peels, Food Chem. 123 (2010) 878–885. [20] P.S. Saravana, Y.J. Cho, Y.B. Park, H.C. Woo, B.S. Chun, Structural, antioxidant, and emulsifying activities of fucoidan from Saccharina japonica using pressurized liquid extraction, Carbohydr. Polym. 153 (2016) 518–525. [21] T.C.T. Lo, H.H. Tsao, A.Y. Wang, C.A. Chang, Pressurized water extraction of polysaccharides as secondary metabolites from Lentinula edodes, J. Agric. Food Chem. 55 (2007) 4196–4201. [22] M. Nagpal, G. Aggarwal, M. Jindal, A. Baldi, U.K. Jain, R. Chandra, J. Madan, Ultrasound, microwave and Box-Behnken Design amalgamation offered superior yield of gum from Abelmoschus esculentus: electrical, chemical and functional peculiarity, Comput. Electron. Agric. 145 (2018) 169–178. [23] N. Sengkhamparn, R. Verhoef, H.A. Schols, T. Sajjaanantakul, A.G. Voragen, Characterisation of cell wall polysaccharides from okra (Abelmoschus esculentus (L.) Moench), Carbohydr. Res. 344 (2009) 1824–1832. [24] N. Sengkhamparn, L.M.C. Sagis, R.D. Vries, H.A. Schols, T. Sajjaanantakul, A.G.J. Voragen, Physicochemical properties of pectins from okra (Abelmoschus esculentus (L.) Moench), Food Hydrocoll. 24 (2010) 35–41. [25] T.L. Christian, B. Simone, A. Niyaz, H. Andreas, Acetylated Rhamnogalacturonans from immature fruits of Abelmoschus esculentus inhibit the adhesion of Helicobacter pylori to human gastric cells by interaction with outer membrane proteins, Molecules 20 (2015) 16770–16787. [26] K. Alba, A.P. Laws, V. Kontogiorgos, Isolation and characterization of acetylated LMpectins extracted from okra pods, Food Hydrocoll. 43 (2015) 726–735. [27] M. Dubois, K.A. Gilles, J.K. Hamilton, P.A. Rebers, F. Smith, Colorimetric method for determination of sugars and related substances, Anal. Chem. 28 (1956) 350–356. [28] T.M. Filisetticozzi, N.C. Carpita, Measurement of uronic acids without interference from neutral sugars, Anal. Biochem. 197 (1991) 157–162. [29] M. Bradford, A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of proteindye binding, Anal. Biochem. 72 (1976) 248–254.

[30] D.T. Wu, S.C. Lam, K.L. Cheong, W. Feng, P.C. Lin, Z.R. Long, X.J. Lv, Z. Jing, S.C. Ma, S.P. Li, Simultaneous determination of molecular weights and contents of water-soluble polysaccharides and their fractions from Lycium barbarum collected in China, J. Pharm. Biomed. Anal. 129 (2016) 210–218. [31] S. Lin, H. Guo, M. Lu, M.Y. Lu, J. Gong, L. Wang, Q. Zhang, W. Qin, D.T. Wu, Correlations of molecular weights of β-glucans from Qingke (Tibetan hulless barley) to their multiple bioactivities, Molecules 23 (2018) 1710. [32] X. Sun, H. Wang, X. Han, S. Chen, S. Zhu, J. Dai, Fingerprint analysis of polysaccharides from different Ganoderma by HPLC combined with chemometrics methods, Carbohydr. Polym. 114 (2014) 432–439. [33] D.T. Wu, L.Z. Meng, L.Y. Wang, G.P. Lv, K.L. Cheong, D.J. Hu, J. Guan, J. Zhao, S.P. Li, Chain conformation and immunomodulatory activity of a hyperbranched polysaccharide from Cordyceps sinensis, Carbohydr. Polym. 110 (2014) 405–414. [34] C. Kyomugasho, S. Christiaens, A. Shpigelman, A.M.V. Loey, M.E. Hendrickx, FT-IR spectroscopy, a reliable method for routine analysis of the degree of methylesterification of pectin in different fruit- and vegetable-based matrices, Food Chem. 176 (2015) 82–90. [35] F. Abdenor, A. Padmesh, W. Markr, W. Martinak, Determining the degree of methylesterification of pectin by ATR/FT-IR: methodology optimisation and comparison with theoretical calculations, Carbohydr. Polym. 78 (2009) 847–853. [36] S. Lin, H. Guo, J.D.B. Gong, M. Lu, M.Y. Lu, L. Wang, Q. Zhang, D.T. Wu, W. Qin, Phenolic profiles, β-glucan contents, and antioxidant capacities of colored Qingke (Tibetan hulless barley) cultivars, J. Cereal Sci. 81 (2018) 69–75. [37] M. Yang, Q. Shen, L.Q. Li, Y.Q. Huang, H.Y. Cheung, Phytochemical profiles, antioxidant activities of functional herb Abrus cantoniensis and Abrus mollis, Food Chem. 177 (2015) 304–312. [38] M. Fan, X. Sun, Y. Qian, Y. Xu, D. Wang, Y. Cao, Effects of metal ions in tea polysaccharides on their in vitro antioxidant activity and hypoglycemic activity, Int. J. Biol. Macromol. 113 (2018) 418–426. [39] J.L. Wang, J. Zhang, X.F. Wang, B.T. Zhao, Y.Q. Wu, J. Yao, A comparison study on microwave-assisted extraction of Artemisia sphaerocephala polysaccharides with conventional method: molecule structure and antioxidant activities evaluation, Int. J. Biol. Macromol. 45 (2009) 483–492. [40] Y. Yuan, D. Macquarrie, Microwave assisted extraction of sulfated polysaccharides (fucoidan) from Ascophyllum nodosum and its antioxidant activity, Carbohydr. Polym. 129 (2015) 101–107. [41] H. Guo, S. Lin, M. Lu, J.D.B. Gong, L. Wang, Q. Zhang, D.-R. Lin, W. Qin, D.-T. Wu, Characterization, in vitro binding properties, and inhibitory activity on pancreatic lipase of β-glucans from different Qingke (Tibetan hulless barley) cultivars, Int. J. Biol. Macromol. 120 (2018) 2517–2522. [42] N. Wang, Y. Zhang, X. Wang, X. Huang, Y. Fei, Y. Yu, D. Shou, Antioxidant property of water-soluble polysaccharides from Poria cocos Wolf using different extraction methods, Int. J. Biol. Macromol. 83 (2016) 103–110. [43] R. Wang, P. Chen, F. Jia, J. Tang, F. Ma, Optimization of polysaccharides from Panax japonicus C.A. Meyer by RSM and its anti-oxidant activity, Int. J. Biol. Macromol. 50 (2012) 331–336. [44] L. Lin, J. Xie, S. Liu, M. Shen, W. Tang, M. Xie, Polysaccharide from Mesona chinensis: extraction optimization, physicochemical characterizations and antioxidant activities, Int. J. Biol. Macromol. 99 (2017) 665–673. [45] P.H.F. Pereira, T.Í.S. Oliveira, M.F. Rosa, F.L. Cavalcante, G.K. Moates, N. Wellner, K.W. Waldron, H.M.C. Azeredo, Pectin extraction from pomegranate peels with citric acid, Int. J. Biol. Macromol. 88 (2016) 373–379. [46] W. Wongweng, A.F.M. Alkarkhi, A.M. Easa, Effect of extraction conditions on yield and degree of esterification of durian rind pectin: an experimental design, Food Bioprod. Process. 88 (2010) 209–214. [47] J.W. Sheng, Y.L. Sun, Antioxidant properties of different molecular weight polysaccharides from Athyrium multidentatum (Doll.) Ching, Carbohydr. Polym. 108 (2014) 41–45. [48] Z.H. Kelishomi, B. Goliaei, H. Mandavi, A. Nikoofar, M. Rahimi, A.A. MoosaviMovahedi, F. Mamashli, B. Bigdeli, Antioxidant activity of low molecular weight alginate produced by thermal treatment, Food Chem. 196 (2016) 897–902. [49] M.Y. Duan, H.M. Shang, S.L. Chen, R. Li, H.X. Wu, Physicochemical properties and activities of comfrey polysaccharides extracted by different techniques, Int. J. Biol. Macromol. 115 (2018) 876–882. [50] J. Wang, S. Hu, S. Nie, Q. Yu, M. Xie, Reviews on mechanisms of in vitro antioxidant activity of polysaccharides, Oxidative Med. Cell. Longev. 2016 (2015) 5692852. [51] J.K. Yan, L.X. Wu, Z.R. Qiao, W.D. Cai, H.L. Ma, Effect of different drying methods on the product quality and bioactive polysaccharides of bitter gourd (Momordica charantia L.) slices, Food Chem. 271 (2019) 588–596. [52] Z. Mzoughi, A. Abdelhamid, C. Rihouey, C.D. Le, A. Bouraoui, H. Majdoub, Optimized extraction of pectin-like polysaccharide from Suaeda fruticosa leaves: characterization, antioxidant, anti-inflammatory and analgesic activities, Carbohydr. Polym. 185 (2018) 127–137. [53] S.H. Lee, G.Y. Jang, M.Y. Kim, I.G. Hwang, H.Y. Kim, K.S. Woo, M.J. Lee, T.J. Kim, J. Lee, H.S. Jeong, Physicochemical and in vitro binding properties of barley β-glucan treated with hydrogen peroxide, Food Chem. 192 (2016) 729–735. [54] A. Podsędek, I. Majewska, M. Redzynia, D. Sosnowska, M. Koziołkiewicz, In vitro inhibitory effect on digestive enzymes and antioxidant potential of commonly consumed fruits, J. Agric. Food Chem. 62 (2014) 4610–4617. [55] M. Espinal-Ruiz, F. Parada-Alfonso, L.P. Restrepo-Sánchez, C.E. Narváez-Cuenca, Inhibition of digestive enzyme activities by pectic polysaccharides in model solutions, Bioact. Carbohydr. Diet. Fibre 4 (2014) 27–38. [56] D.J. McClements, Y. Li, Review of in vitro digestion models for rapid screening of emulsion-based systems, Food Funct. 1 (2010) 32–59.