Preparation, characteristics, and antioxidant activities of carboxymethylated polysaccharides from blackcurrant fruits

Journal Pre-proof Preparation, characteristics, and antioxidant activities carboxymethylated polysaccharides from blackcurrant fruits

of

Suyang Duan, Meimei Zhao, Baoyu Wu, Shijie Wang, Yu Yang, Yaqin Xu, Libo Wang PII:

S0141-8130(19)35630-2

DOI:

https://doi.org/10.1016/j.ijbiomac.2019.11.078

Reference:

BIOMAC 13859

To appear in:

International Journal of Biological Macromolecules

Received date:

19 July 2019

Revised date:

10 October 2019

Accepted date:

7 November 2019

Please cite this article as: S. Duan, M. Zhao, B. Wu, et al., Preparation, characteristics, and antioxidant activities of carboxymethylated polysaccharides from blackcurrant fruits, International Journal of Biological Macromolecules(2019), https://doi.org/10.1016/ j.ijbiomac.2019.11.078

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© 2019 Published by Elsevier.

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Preparation, characteristics, and antioxidant activities of carboxymethylated polysaccharides from blackcurrant fruits

Suyang Duan, Meimei Zhao, Baoyu Wu, Shijie Wang, Yu Yang, Yaqin Xu*,1, Libo Wang*,1

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College of Science, Northeast Agricultural University, Harbin 150030, People’s

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Republic of China

* Corresponding author. Tel.: +86 451 55190732; fax: +86 451 55190243 [email protected] (Y. Xu), [email protected] (L. Wang).

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E-mail address:

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1 These authors contributed equally to this work.

Abstract In the present study, the native polysaccharide (RNP) extracted from blackcurrant fruits was carboxymethylated. Physicochemical characteristics and

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antioxidant activities in vitro of RNP and three carboxymethylated polysaccharides (CRNPs) were determined. GC analysis proved that RNP and CRNPs were composed of the same six monosaccharides (galacturonic acid, rhamnose, arabinose, mannose, glucose and galactose), but the molar ratios of monosaccharides were different. HPLC demonstrated that the molecular weights of CRNPs were improved. The assays of the

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antioxidant properties indicated that CRNPs possessed stronger scavenging activities

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on radicals (hydroxyl and superoxide radicals) and better anti-lipid peroxidation

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activities, as well as better protection effects on erythrocyte hemolyses in vitro

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compared with RNP. The activities of CRNPs were significantly enhanced with the

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increase of the degree of substitution (DS). These results proved that the carboxymethylation could effectively increase the antioxidant activities of the

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polysaccharide.

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Keywords: Blackcurrant polysaccharides; Carboxymethylation; Characteristics; Antioxidant activities.

1. Introduction Much work has revealed that the biological activities of polysaccharides can be improved by chemical modifications to introduce functional groups, such as sulfation,

Journal Pre-proof phosphorylation, acetylation, and carboxymethylation

[1-3]. Especially,

carboxyme-thylation is the most commonly used to modify the structure of polysaccharides,

and

carboxymethylated

polysaccharides

exhibited

stronger

immunomodula-ting, antioxidant, and antitumor activities than natural polysaccharides. Wang et al proved that the carboxymethylation could effectively increase water solubility and antioxidant activity of the polysaccharide from Tremella fuciformis [4]. Gao and Huang also reported that carboxymethylated

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garlic polysaccharide had the good scavenging effect on the superoxide anions and hydroxyl radicals [5]. Poria cocos sclerotium polysaccharide without anti-gastric

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adenocarci-noma activity showed strong anti-gastric adenocarcinoma activity after

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carboxymethylation [6]. The carboxymethylated polysaccharide had a stronger

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cholesterol-lowering activity than the polysaccharide from Morchella angusticepes Peck (PMEP), which indicated carboxymethylation not only affected the

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physicochemical properties of PMEP, but also increased its biological activities [3].

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Blackcurrant (Ribes nigrum L.) is a classical fruit that has long been used to

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make juice, jam, liqueur and some medicines [7]. A wide range of nutritional compounds, such as organic acids, unsaturated fatty acids, vitamins, polysaccharides, flavonoids, and anthocyanins, make blackcurrant one of the most notable species in the berry kingdom [8, 9]. In our recent researches, the polysaccharides obtained from blackcurrant fruits possessed many activities in vitro. For example, two new heteropolysaccharides (BCP-1 and BCP-2) were prepared after different purification steps. They both showed obvious inhibitory effects on the formation of dicarbonyl compounds and AGEs [10, 11]. Moreover, BCP-2 had good inhibitory activities on α-glucosidase and α-amylase. It is also found that blackcurrant polysaccharides after sulfation modification showed better inhibitory activities on the antioxidant and

Journal Pre-proof α-amylase [12]. However, to the best of our knowledge, the carboxymethylation modification of the polysaccharides from blackcurrant fruits has not been reported. For further research and utilization of the polysaccharides from blackcurrant fruits, carboxymethylated polysaccharides were synthesized and their antioxidant activities were investigated comparing with the native one. Based on the results, the effects of DS on antioxidant activities were also discussed.

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2. Materials and methods

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2.1. Materials and reagents

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Blackcurrant fruits (Heifeng) were purchased from the horticultural station in

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Northeast Agricultural University (Heilongjiang, China). 2-Thiobarbituric acid (TBA)

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were acquired from Huishi Biochemical Reagent Co., Ltd (Shanghai, China). T-series dextran standards were received from Baier Di Biotechnology Co. (Beijing, China).

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Soy lecithin and trichloroacetic acid (TCA) was obtained from Solarbio Life Sciences

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Co. (Shanghai, China). Trifluoroacetic acid (TFA) were acquired from Guangfu

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Chemistry Research Institute (Tianjin, China). 2,2′-azobis-2-methyl-propanimidamide dihydrochloride (AAPH) and Butylated hydroxytoluene (BHT) was purchased from Aladdin Reagent Co., Ltd (Shanghai, China). The 6% rabbit erythrocytes were purchased

from

Guangzhou

hongquan biological technology Co.

(Guangzhou,

China). D4006 macroporous resin was purchased from a chemical plant at Nankai University (Tianjin, China). Malondialdehyde (MDA) and glutathione (GSH) assay kits were acquired from Jiancheng Bioengineering Institute (Nanjing, China). All other chemicals were of analytical grade. 2.2. Preparation of RNP and its derivative CRNP

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2.2.1 Preparation of the crude polysaccharide from black currant The blackcurrant fruits (20.0 g) after homogenization were mixed with 400 mL water and 1.6% complex enzyme (papain/pectinase=2:1) in a beaker. Then ultrasonic-assisted enzymatic extraction was performed in the ultrasonic cell disintegrator (JY92-2D, Xinzhi Bio-Sciences Co. Ltd, Ningbo, China) at 600 W

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power for 30 min. Following centrifugation at speed of 3500 rpm for 15 min, the

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aqueous extracts were concentrated and precipitated with 80% (v/v) ethanol at 4 °C

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overnight. The precipitate was collected by membrane filtration (0.45 μm, Millipore,

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USA) and lyophilized after washing by anhydrous ethanol and acetone. D4006

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macroporous resin was used for decoloring the obtained polysaccharides. The polysaccharides solution (4.0 mg/mL) was loaded on the column (20 mm × 30 cm),

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and eluted with deionized water at 1.0 mL/min. The collected polysaccharides eluent

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was concentrated, lyophilized, and then the polysaccharides coded as RNP (Ribes

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nigrum L. polysaccharide) was obtained. 2.2.2. Preparation of carboxylmethylated polysaccharides The carboxymethylation of RNP was obtained by mixing 100.0 mg RNP with 5.0 mL isopropanol, then NaOH (20%, 2.0 mL) and monochloroacetic acid (MCA) was added to the mixture. The resulting solution was heated at different temperatures (60-100 °C) at various the ratio of MCA to polysaccharide (8:1-30:1) for various periods (20-60 min), respectively. The solutions were cooled to room temperature when the reactions were completed, then neutralized with 2.0 mol/L HCl, and dialyzed against deionized water for 96 h. The dialyzates were concentrated and

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precipitated with 4-fold volume of 95% ethanol. The precipitates were lyophilized to obtain CRNPs. 2.2.3. Measurement of DS Based on the amount of glycolic acid produced from the cleavage of carboxymethylated products, DS was determined using a colorimetric method [13].

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Briefly, CRNPs were added to 0.25 mol/L NaOH to prepare CRNPs solutions (1.0

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mg/mL). The chromotropic acid (0.1%, 5.0 mL) and concentrated H2SO4 (98%, 1.0

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mL) were added to 1.0 mL of CRNPs solution, and the mixture was incubated at

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100 °C for 30 min, then cooled to room temperature. After addition of ammonium

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acetate solution to a final volume of 25.0 mL, the absorbance was determined at 570 nm. DS was calculated by the equation: 162 B 76 - 80 B

(1)

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DS 

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where B is the amount of glycolic acid (mg/mg of sample), 76 is the molecular weight

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of glycolic acid, 162 is the average molecular weight of anhydro-monosaccharide unit, and 80 is the net increase in weight of each unit of sodium carboxymethylate group substituted.

2.3. Characterization of polysaccharides 2.3.1. Physicochemical properties of RNP and CRNPs Ninhydrin test, iodine test, Fehling reagent reaction, and ferric chloride were used to determine the chemical properties of RNP and CRNPs. The contents of total sugar and uronic acid were measured according to phenol-sulfuric acid method [14] and carbazole-sulfuric acid method using D-galacturonic acid as a standard [15],

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respectively. The solubilities of the samples in water were determined according to the reported method [16]. 2.3.2. Monosaccharide composition analysis The polysaccharides samples (20.0 mg) were hydrolyzed for 3 h with TFA (2.0 mol/L, 2.0 mL) at 120 °C. Then methanol was used to remove TFA completely under

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reduced pressure. The hydrolyzed products and standard monosaccharides were

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acetylated by the addition of hydroxylamine hydrochloride, pyridine and acetic

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anhydride on the basis of the method of Xu et al. [17]. The acetylated polysaccharides

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were then analyzed by GC (GC-2010, Shimadzu Corporation, Japan) with flame

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ionization detector and a RTX-1701 silica capillary column. The monosaccharide composition of RNP and CRNPs was determined comparing with the retention time

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against standards. The monosaccharide compositions were calculated based on the

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internal standard method using inositol as internal standard.

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2.3.3. Determination of molecular weights The polysaccharide samples and standard dextrans (T-10, T-40, T-70, T-110 and T-2000) (2.0 mg/mL) were prepared and filtered by 0.45 μm membranes. Then 10 µL solutions of samples were injected to high performance liquid chromatography (LC-600E-2487, Waters Corporation, America) equipped with a Waters Ultrahydrogel Linear column (7.8 mm × 30 cm) and a model 2414 refractive index detector (RID), and eluted with deionized water at 1.0 mL/min. According to the retention time of RNP and CRNPs, their molecular weights were calculated based on the standard curve. 2.3.4. IR spectra analyses

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The IR spectra of RNP and CRNPs were measured by Fourier-transform infrared spectrometer (ALPHA-T, Bruker Instruments, Germany) with KBr pellets within wave number of 500-4000 cm-1. 2.3.5. Congo red experiment The configurations of RNP and CRNPs in solution were determined by the

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Congo red experiment. Simply, Congo red solution (160 µmol/L, 1.0 mL) was added

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in the polysaccharide solutions (1.0 mg/mL, 1.0 mL), and then the mixed solution

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were diluted by NaOH solutions (1.0 mol/L, 0-4.0 mL) to final concentrations of

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NaOH (0.0-0.5 mol/L). The absorbance (λmax) was determined by a double beam

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UV-visible spectrophotometer (TU-1901, Beijing Purkinje General Instrument Co., Ltd, Beijing, China) ranging from 350 to 700 nm. The mixed solution without the

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2.3.6. SEM analyses

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polysaccharides was used as a control.

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RNP and CRNPs were adhered onto the copper metal stub, and sputtered 100 nm gold film by a sputter coater, respectively. Then, the polysaccharides were tested by JSM-6480A scanning electron microscope (SEM, JEOL, Ltd, Tokyo, Japan). 2.4. Assays of radical scavenging activities The hydroxyl and superoxide anion radicals scavenging activities were measured according to the previously reported method [18]. 2.5. Lipid peroxidation inhibition capacity Lecithin solution (10.0 mg/mL) was prepared by solving lecithin in 0.1 mol/L phosphate buffer (pH 7.4). The polysaccharide solutions (1.0 mL, 0.2-1.2 mg/mL) and

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FeSO4 (1.0 mL, 0.4 mmol/L) were added to lecithin solution (1.0 mL). After incubating at 37 °C for 60 min in the dark, TCA-TBA-HCl solution (2.0 mL) was added and incubated at 95 °C for 15 min. TCA-TBA-HCl solution was obtained by mixing TCA (7.50 g), TBA (0.19 g) and HCl (0.01 mol/L, 1.0 mL) with deionized water to a final volume of 50.0 mL. Then the reaction mixture was centrifuged at

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2000 rpm for 15 min. The supernatant was determined at 532 nm with BHT as the

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positive control. The lipid peroxidation inhibition capacity was calculated as

Ax  Ad 100% Ax

(2)

e-

Inhibition rate 

pr

following Eq. (2).

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where Ad is the absorbance of the solution with different concentrations of polysaccharides; Ax is the absorbance of the solution without the polysaccharides.

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2.6. Erythrocyte hemolysis assay

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2.6.1. Assay for erythrocyte hemolysis mediated by AAPH and H2O2

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The 6% rabbit erythrocytes were diluted into 3% erythrocytes suspension with aldrin solution. The erythrocyte suspension (1.0 mL) was mixed with 2.5 mL AAPH (12.5, 25, 50, 100, and 200 mmol/L) and 2.5 mL H2O2 (0.25, 0.5, 1.0, 2.0, and 4.0 mmol/L), respectively. The mixture was incubated at 37 °C for 3.0 h with gentle shaking, and 200 μL reaction mixture was taken out at every 0.5 h. After dilution with NaCl solution (0.9%, 4.0 mL) and centrifugation at 2000 r/min for 15 min, the absorbance (A) of the supernatant was tested at 415 nm (mediated by AAPH) and 540 nm (mediated by H2O2 ), respectively. 4.0 mL of distilled water was added to the reaction mixture to achieve complete hemolysis, which was then centrifuged and the

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absorbance (B) of the supernatant was determined. The hemolysis rate was calculated using the following formula: A Hemolysis rate  ( )  100% B

(3)

2.6.2. Protective effects of the polysaccharides on erythrocyte hemolysis Inhibitory rates of RNP and CRNPs against AAPH and H2O2-induced hemolysis

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were evaluated. Firstly, 1.0 mL of erythrocyte suspension (3%) was mixed with 0.5

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mL of the samples (0.2-4.0 mol/L) and incubated for 10 min at 37 °C, respectively.

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AAPH (100 mmol/L, 2.5 mL) or H2O2 (1.0 mmol/L, 1.5 mL) were added in the

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mixture, and the incubation continued at the same temperature for another 2 h. Then,

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200 μL reaction mixture was taken out and treated as described in the above Section 2.6.1. The erythrocytes were incubated with polysaccharides (4.0 mg/mL, 0.5 mL) in

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the absence of AAPH and H2O2 were tested as control groups. Finally, the percentage

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of hemolysis inhibition was calculated as follows:

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Hemolysis inhibition  (C 

A' ) 100% B'

(4)

where A′ is the absorbance of the supernatant diluted by NaCl solution (0.9%, 4.0 mL)； B′ is the absorbance of the supernatant diluted by 4.0 mL of distilled water; C is the hemilysis rate induced by AAPH (100 mmol/L) or H2O2 (1.0 mmol/L) after 2 h incubation without the polysaccharides. 2.6.3. Measurement of MDA A microscale MDA kit was used to monitor MDA content according to the manufacturer’s protocol. The principle of this method is based on color reaction. MDA can be colored with TBA, and the absorbance was measured at 532 nm. MDA

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concentrations in erythrocytes incubated with polysaccharides (4.0 mg/mL, 0.5 mL) without AAPH or H2O2 induction were determined as control groups. In present study, the content of MDA was expressed as mol/mL. 2.6.4. Determination of GSH GSH level was determined using GSH assay kit according to the instructions.

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Dithiodidinitrobenzoic acid reacts with sulfhydryl compounds to form yellow

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compounds, which can be detected at 420 nm. The contents of reduced GSH in

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erythrocytes by addition of polysaccharides (4.0 mg/mL, 0.5 mL) without AAPH or

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H2O2 induction were tested as control groups. GSH level was expressed as gGSH/L.

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2.7. Statistical analysis

All data were illustrated in mean ± standard deviation (SD). The statistical

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analyses were operated by SPSS (version 18.0, Chicago, USA). Statistical

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significance was tested at p < 0.05.

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3. Results and discussion

3.1. Preparation of carboxymethylated polysaccharides CRNPs were synthesised by reaction of RNP and MCA in the presence of sodium hydroxide. The procedure of carboxymethylation reaction is based on Williamson’s ether synthesis, belonging to mechanism of bimolecular nucleophilic substitution (SN2) [4]. First, hydroxyl groups of RNP react with sodium hydroxide to form alkoxides groups (nucleophilic reagent). Second, carboxymethyl groups are formed through SN2 reaction between RNP alkoxide and MCA. However, a side reaction may take place simultaneously producing sodium glycolate from sodium

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monochloroacetate and sodium hydroxide [19, 20]. Thirteen carboxymethylated derivatives of RNP were obtained by varying the reaction conditions (Fig. 1). The DS of the samples varied from 0.40 ± 0.03 to 1.10 ± 0.03. It was clear that the DS of CRNPs increased as the dosage of MCA (from 0.5 g to 1.5 g). However, further increasing in MCA dosage beyond 1.5 g did not lead to

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substantial increments in DS. The increasing of MCA dosage in a certain range might

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provided more opportunities to make sufficient reaction with RNP, resulting the

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raising of the DS. Further increasing in MCA dosage accelerated MCA reacted with

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sodium hydroxid to form by-product sodium glycolate [21].

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As was evident, the DS increased from 0.57 ± 0.02 to 1.10 ± 0.03 prominently as the reaction temperature enhanced from 50 to 70 °C. It might be due to enhancing

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solubility of RNP alkoxide and inducing better contacts between RNP alkoxides and

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MCA. Thus reaction of chloroacetate sodium with the hydroxyl groups was

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accelerated to form carboxymethyl groups. Further increase at reaction temperature above 70 °C resulted in decrement of the DS, indicating that glycolate formation (side reaction) prevailed the carboxymethylation reaction [4, 19]. It was seen that the DS increased by increasing reaction time from 1 to 4 h, and then started declining. The above results might be explained: Prolonging the duration of reaction might be helpful of swelling of RNP, facilitating the entry of MCA into RNP granules. Nevertheless, overlong reaction time (beyond 4 h) with a comparatively high temperature (70 °C) was in favor of the side reaction, such as formation of glycolate or degradation of RNP [16].

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Based on the reported studies, the value of DS had a great impact on the biological activities of the polysaccharides [21]. Thus, it is worth to evaluate the effect of DS on antioxidant activities. In this study, among these CRNPs, three carboxymethylated polysaccharides named as CRNP-1, CRNP-2, and CRNP-3 were selected for determination of antioxidant activities and further characterization. The

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(moderate), and 1.10 ± 0.03 (high), respectively.

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DS values of CRNP-1, CRNP-2, and CRNP-3 were 0.40 ± 0.03 (low), 0.77 ± 0.01

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3.2. Physicochemical properties of RNP and CRNPs

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As shown in Table 1, qualitative tests proved that RNP and CRNPs did not

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contain protein, starch, reducing sugars and polyphenols. The total sugar contents of CRNPs were not much different to that of RNP, and the uronic acid contents of

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CRNPs were higher than that of RNP. Water solubility of CRNPs was enhanced

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compared with RNP, and the solubility was improved with the increase of DS. This

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might be attributed to the increase in hydrophilic carboxyl groups [22]. 3.3. Characterization of RNP and CRNPs 3.3.1. Molecular weight and monosaccharide composition The elution peaks of RNP and CRNPs were single and narrow in HPLC chromatograms, indicating a relatively narrow molecular weight distribution (Fig. 2A). According to the retention time, Mw of RNP and CRNPs were calculated (Table 1). Compared with RNP, Mw of CRNPs was increased, and the increase in Mw were 55.05% for CRNP-1 (DS, 0.40 ± 0.03), 43.87% for CRNP-2 (DS, 0.77 ± 0.01), and 36.36% for CRNP-3 (DS, 1.10 ± 0.03), respectively. The results suggested carboxymethylated

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polysaccharides were obtained successfully without degradation in the present study. Similarly, Wang et al. reported that the Mw of three carboxymethylated polysaccharides derived from Cyclocarya paliurus polysaccharides were increased compared with native polysaccharides [21]. The monosaccharide composition was shown in Fig. 2B and Table 1. It could be

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found that CRNPs and RNP were composed of the same six monosaccharides

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(galacturonic acid, rhamnose, arabinose, mannose, glucose and galactose). However,

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molar ratios among these polysaccharides were different. Both Wang et al. [21] and

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Chen et al. [2] also reported that the carboxymethylated polysaccharides from

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Cyclocarya paliurus and corn silk were consisted of the same monosaccharides compared with native polysaccharides, respectively, but the molar ratios of different

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monosaccharides were changed. These results showed that carboxymethylation

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polysaccharides.

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modification had low impact on the monosaccharide composition of the

3.3.2. FT-IR analysis

The general profile of the spectra for RNP and CRNPs were similar, the typical absorption peaks of the polysaccharides were displayed (Fig. 2C). However, some differences in a specific spectra range could be observed. The band at 3406 cm-1 belonged to O-H stretching vibration and the peak at 2933 cm-1 was assigned to C-H stretching of the CH2 groups [3]. The absorption peak at 1738 cm-1 in RNP spectrum was assigned to carboxylic acid groups of galacturonic acid, the disappearance of the peak in CRNPs spectra showed that CRNPs were predominantly in salt form [4]. In

Journal Pre-proof addition, three new strong peaks at 1608 cm-1 , 1414 cm-1 and 1340 cm-1 were observed after carboxymethylation modification, and they were assigned to asymmetrical and symmetrical COO- stretching vibrations, respectively [3, 23]. These data fully indicated that the native polysaccharide was carboxymethylated successfully. The absorption at 1200-1000 cm-1 confirmed that RNP and CRNPs

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contained pyranoid ring [24].

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3.3.3. Congo red test and SEM analyses

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The maximum absorption wavelength (λmax) of Congo red-polysaccharide in

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NaOH solution will decrease remarkably with the increasing concentrations of

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alkaline, when the polysaccharides with triple-helical conformations interact with Congo red and undergo ahelix-coil transition [25, 26]. Therefore, Congo red test can

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be applied for determination of triple helix conformation of the polysaccharides. As

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shown in Fig. 3A, there were no red shifts of λmax of the samples at all concentrations,

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indicating both RNP and CRNPs had non triple-helix conformations. The analysis results of GC had revealed that RNP and CRNPs were all heteropolysaccharides, so the triple-helix structure was not easily formed [26]. The surface morphology changes of the carboxymethylated polysaccharides were characterized by SEM. As shown in Fig. 3B, RNP and CRNPs were of similar surface morphology, exhibiting flake-like structures with uneven surfaces. These results also suggested that there were no new features generated in the polysaccharides after carboxymethylation modification. 3.4. Free radical scavenging capacities

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Hydroxyl radical (OH∙) is one of the most reactive free radicals. It can easily cross cell membranes and react with most biomolecules, which leads to tissue damage or cell death [13]. Superoxide radical (O2-∙) can indirectly to cause cellular damage [27]. Therefore, it was necessary to evaluate these radicals scavenging activities for the protection of living systems. Comparing with the native polysaccharide and VC, O2-∙)

scavenging abilities of three carboxymethylated

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the radicals (OH∙,

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polysaccharides were evaluated. As shown in Fig. 4A and Fig. 4B, the scavenging

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activities of all the test samples were ascended rapidly with the increase of the

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polysaccharide concentration (p < 0.05, 0.2-1.2 mg/mL), although lower than that of

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VC under the same concentrations. The scavenging rates on both hydroxyl radical and superoxide radical increased in the order of: RNP < CRNP-1 < CRNP-2 < CRNP-3 <

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VC in 0.2-1.2 mg/mL. CRNP-1, CRNP-2 and CRNP-3 exhibited excellent scavenging

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capacities (70.69 ± 2.33%, 76.69 ± 1.69%, and 78.57 ± 0.38%) on hydroxyl radicals

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compared with RNP (69.09 ± 2.51%), and the maximum scavenging activities of RNP, CRNP-1, CRNP-2 and CRNP-3 toward superoxide radicals were 52.58 ± 2.44%, 63.63 ± 1.34%, 66.39 ± 1.09%, and 76.03 ± 1.12%, respectively. These results proved that the polysaccharides after carboxymethylation treatment showed stronger scavenging activity, and the inhibitory effects of carboxymethylated polysaccharides were improved with the increase of DS. These data were agreed closely with other reported results [28]. 3.5. Anti-lipid peroxidation capacities Lipid peroxidation can be initiated when fatty acids or fatty acyl side chains are

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attacked by free radicals [29]. This oxidation process not only affects the quality of foods containing lipids but also leads to structural and functional damage of the biomolecule [2]. For this reason, it is necessary to determine the function of these polysaccharides on lipid peroxidation phenomenon. As shown in Fig. 4C, RNP and CRNPs all exhibited anti-lipid peroxidation activities in a dose dependent manner (p

f

< 0.05) and increased in the following order: RNP < CRNP-1 < CRNP-2 < CRNP-3 <

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BHT. At 1.2 mg/mL, the inhibitory activities of RNP, CRNP-1, CRNP-2, and CRNP-3

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were 9.91 ± 1.37%, 36.92 ± 0.71%, 45.46 ± 1.75%, and 48.03 ± 0.26%, respectively.

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At the same concentrations, they were all lower than that of BHT, but BHT, as a kind

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of synthetic antioxidants, might be responsible for liver damage and carcinogenesis [30]. In contrast, the polysaccharides could be applied widely to foods or medicine for

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their natural and non-toxic advantages [31]. This result was in accordance with the

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report of Chen et al. [2], the corn silk polysaccharide after carboxymethylation

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modification had stronger inhibitory activity during the lipid peroxidation than the native polysaccharide.

3.6. Effect of the f polysaccharides on erythrocyte hemolysis Erythrocytes are the most plentiful cells of blood participating in numerous key biochemical and physiological functions. At the same time, erythrocytes are susceptible to attack by free radicals or reactive oxygen species (ROS), because they contain high levels of unsaturated lipids in the erythrocyte membrane [32-34]. Therefore, erythrocytes have been extensively used as a cellular model for evaluation

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of antioxidant activities of the compounds, such as polysaccharides, pigment and caffeic acid [32, 35, 36]. 3.6.1 Erythrocyte hemolyses induced by AAPH and H2O2 In erythrocytes models, oxidation damage can be induced by many reagents, such as AAPH, H2O2, and t-butyl hydroperoxide [37, 38]. In this present, AAPH and

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H2O2 were chosen as free radical initiators, because they could eventually cause

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hemolysis of erythrocytes. And the hemolysis inhibition rate is an indirect way to

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evaluate the intracellular antioxidant ability [39]. The erythrocyte hemolyses curves

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induced by AAPH and H2O2 at different concentrations were shown in Fig. 5A and 5B.

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Erythrocytes suspension without AAPH and H2O2 showed slight hemolysis (24.59 ± 0.55%) for 3 h. After addition of AAPH and H2O2, erythrocyte hemolyses were

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increased significantly in a concentration dependent manner (p < 0.05) in 2 h. Then

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the curves slowed down within the time range of 2-3 h. As shown in Fig. 5A, the

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hemolysis rate of erythrocytes was 81.71 ± 1.39% treated with 100 mmol/L of AAPH for 2 h, and then hemolysis rate changed gently. Likewise, when H2O2 (1.0 mmol/L) and erythrocytes were incubated for 2 h, the hemolysis rate of erythrocytes was 82.25 ± 1.27% (Fig. 5B), and there was no more significant increase with increasing concentrations and time. Therefore, AAPH and H2O2-induced oxidative damage model were established, namely, 1.0 mmol/L of H2O2, 100 mmol/L of AAPH and the incubation time of 2 h were selected for determination of erythrocytes hemolysis inhibition. 3.6.2. Protection effects of polysaccharides on erythrocyte hemolysis

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As shown in Fig. 5C and 5D, erythrocyte hemolyses induced by AAPH (100 mmol/L) and H2O2 (1.0 mmol/L) were both effectively reduced with addition of RNP and CRNPs in a dose-dependent manner. At 4.0 mg/mL, the inhibition rates of RNP, CRNP-1, CRNP-2, CRNP-3, and VC on hemolyses induced by AAPH were 48.07 ± 0.17%, 53.13 ± 0.41%, 57.99 ± 0.94%, 61.74 ± 0.94%, and 68.59 ± 0.06%,

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respectively. The inhibition effects of RNP and CRNPs on H2O2-induced hemolyses

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were similar to those of AAPH. When the concentration of polysaccharides was 4.0

pr

mg/mL, the inhibition rates of RNP, CRNP-1, CRNP-2, CRNP-3, and VC were 50.95

e-

± 0.76%, 54.75 ± 0.27%, 57.12 ± 0.79%, 63.69 ± 0.94%, and 67.05 ± 0.94%,

Pr

respectively. These data confirmed that CRNPs had stronger protection effects on erythrocyte hemolysis, the inhibition rates were slightly lower than those of VC under

al

the same concentrations. CRNP-3 with higher DS showed better activity, which was

rn

agree with the results of radicals scavenging activities. In addition, when erythrocytes

Jo u

were incubated with RNP and CRNPs in the absence of AAPH and H 2O2, the hemolysis rates (19.71 ± 0.61%-20.69 ± 0.22%) were nearly equal to the normal control (20.07 ± 0.19%), indicating that RNP and CRNPs had no induction damage on erythrocytes. 3.6.3. Effects of polysaccharides on MDA generation MDA, as one of the end products of lipid peroxidation, can alter the structure and function of cell membrane, eventually cause disruption of cellular metabolism [40]. As seen in Fig. 6A and 6B, RNP and CRNPs obviously inhibited the production of MDA with increasing concentration (p < 0.05). The MDA level were 82.94 ± 1.41

Journal Pre-proof

and 59.52 ± 0.59 nmol/mL after treatment with AAPH (100 mmol/L) and H2O2 (1.0 mmol/L) for 2 h, which indicated that AAPH and H2O2 caused the occurrence of lipid peroxidation in erythrocytes. However, when the cells were treated with different concentrations of polysaccharides, the levels of MDA were significantly decreased. At 4.0 mg/mL, MDA concentrations were reduced from 82.94 ± 1.41 to 56.73 ± 1.07,

f

49.33 ± 0.93, 47.79 ± 1.41, and 40.39 ± 2.14 nmol/L after incubation with RNP,

oo

CRNP-1, CRNP-2, and CRNP-3, respectively. Similarly, the levels of MDA in H2O2

pr

treated erythrocytes were decreased from 59.52 ± 0.59 to 35.17 ± 1.55, 26.71 ± 2.11,

e-

22.32 ± 1.76, and 20.63 ± 1.76 nmol/mL with treatment by 4.0 mg/mL of RNP,

Pr

CRNP-1, CRNP-2, and CRNP-3, respectively. But the protective effects of polysaccharides were weaker than VC under the same concentrations. It was also

al

found that CRNP-3 showed higher inhibitory activity in two oxidation systems

rn

compared with other polysaccharides.

Jo u

In addition, when the cells were incubated with RNP and CRNPs without AAPH or H2O2, the concentrations of MDA (6.43-6.76 nmol/mL) were close to that of the normal group (5.07 ± 1.01 nmol/mL), illustrating that RNP and CRNPs had no effects on MDA formation. Therefore, these results indicated that lipid peroxidation caused ,

by ROS generation could be efficiently inhibited by RNP and CRNPs. 3.6.4. Effect of polysacharides on GSH levels of erythrocytes GSH, which is composed of three amino acids as a reduced glutathione, plays a part in the regulation of cellular redox homeostasis by scavenging a variety of free radicals/ROS through its electrondonating property [40]. As shown in Fig. 6C and 6D,

Journal Pre-proof

erythrocyte GSH levels were determined as 2.10 ± 0.24 and 5.60 ± 0.76 mgGSH/L after AAPH (100 mmol/L) and H2O2 (1.0 mmol/L) treatment (2 h), respectively. Compared with GSH level (18.39 ± 0.84 mgGSH/L) in normal erythrocytes, the sharp reduction of GSH contents indicated that erythrocytes were under severe oxidative stress. The concentrations of GSH (16.32 ± 0.92-17.41 ± 0.56 mgGSH/L) incubated

f

by RNP and CRNPs without AAPH or H2O2 were close to that of the normal group,

oo

illustrating that all the polysaccharides had no obvious effects on GSH levels. It was

pr

obvious that treatment of erythrocytes with RNP and CRNPs before AAPH and H2O2

e-

addition increased the GSH concentrations. Moreover, CRNP-3 possessed stronger

Pr

ability for the restoration of GSH compared with other polysaccharides, GSH levels were 8.27 ± 0.64, 9.12 ± 0.24, 9.68 ± 1.11, and 13.32 ± 0.24 mgGSH/L with

al

incubation by 4.0 mg/mL of RNP, CRNP-1, CRNP-2, and CRNP-3 (Fig. 6C),

rn

respectively. Similarly, CRNP-3 appeared to be more effective than other

Jo u

polysaccharides for the restoration of GSH in erythrocytes treated by H 2O2 (Fig. 6D), but slightly lower than those of VC in the concentration range of 0.2-4.0 mg/mL. Based on the above results in Section 3.6, RNP and CRNPs had strong protection effects on erythrocyte hemolyses induced by AAPH and H2O2. Preincubation of erythrocytes with RNP and CRNPs reduced MDA levels in a dose-dependent manner. In addition, the restoration of GSH in erythrocytes treated by AAPH and H2O2 also implied that RNP and CRNPs can prevent the oxidative modification of membrane proteins and lipids. It is worth noting that CRNP-3 with higher DS possessed better ability to protect erythrocytes than other polysaccharides, which is almost similar to

Journal Pre-proof the regularity for the radicals (OH∙, O2-∙) scavenging and anti-lipid peroxidation. 4. Conclusion Three carboxymethylated polysaccharides CRNPs with different DS were prepared. RNP and CRNPs were all heteropolysaccharides and composed with six same monosaccharides in different molar ratios. RNP and CRNPs both had non

f

triple-helix conformations and exhibited irregular surface appearances. Compared

oo

with RNP, CRNPs had stronger antioxidant activities, and CRNP-3 with highest DS

pr

showed the best antioxidant activities suggesting that DS played an important role.

e-

The present results confirmed that carboxymethylated modification was helpful to

Pr

improve the antioxidant activities of the polysaccharides from blackcurrant fruits. In future, the detailed structure, bioactivities in vivo and safety for human health of

rn

Conflicts of interest

al

carboxymethylated polysaccharides need further study.

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There are no conflicts to declare. Acknowledgments

This work was financially supported by Postdoctoral Scientific Research Start-up Fund Program of Heilongjiang Province, China (LBH-Q17030) and Student Innovation Practical Training Project of Heilongjiang Province of China (NO. 201910224081). References [1] J.G. Wang, L.N. Zhang, Y.H. Yu and P.C.K. Cheung, Enhancement of antitumor activities in sulfated and carboxymethylated polysaccharides of Ganoderma

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f

Anal. 25 (2017) 898-907, https://doi.org/10.1016/j.jfda.2016.10.007.

Journal Pre-proof Table 1

Comparison of the properties of RNP and CRNPs Samples

RNP

CRNP-1

CRNP-2

CRNP-3

DS of the carboxymethylated derivatives from Blackcurrant polysaccharide DS

0.40 ± 0.03

0.77 ± 0.01

1.10 ± 0.03

15.65 ± 0.09

21.15 ± 0.08

23.95 ± 0.45

32.5 ± 1.03

total sugar (%)

51.95 ± 1.03

47.61 ± 0.55

49.35 ± 0.33

52.47 ± 1.23

Uronic acid (%)

19.01 ±1.04

20.01 ± 0.12

26.43 ± 0.63

29.88 ± 0.91

Ninhydrin reaction

–

–

–

–

Iodine test

–

–

–

–

Fehling reagent reaction

–

–

–

–

Ferric chloride reaction

–

–

–

–

Molecular weight (kDa)

8093

12548

11643

11036

f

Solubility (mg/mL)

1.00

1.00

Rhamnose

2.31

2.09

Arabinose

13.29

8.29

Mannose

0.95

galactose

5.13

Galacturonic acid

1.96

Jo u

rn

1.00

1.55

4.35

4.5

5.65

1.34

1.09

0.23

3.50

5.24

6.65

0.96

1.28

4.35

e-

Pr

al

– Means of negative color reaction

1.00

pr

Glucose

oo

Monosaccharide composition (molar ratio)

Journal Pre-proof 1.2

f cd

1.0 def

def

a cd

0.8 DS

ab 0.6 0.4

bc

ab de

ab

70 3 4

50 1.5 4

a

a

0.2 0.0 Temperature (ºC) 70 Chloroacetic acid (g) 0.5 Time (h) 4

70 1 4

70 1.5 4

70 2 4

60 1.5 4

80 1.5 4

90 1.5 4

70 1.5 1

70 1.5 2

70 1.5 3

70 1.5 5

Jo u

rn

al

Pr

e-

pr

oo

f

Fig. 1. The different preparation conditions and DS of CRNPs (Means with different letters within the same factors are significantly different, p < 0.05).

Journal Pre-proof

A 2.5

RNP CRNP-3 CRNP-2 CRNP-1

2.0

Voltage (V)

1.5

1.0

0.5

0.0 4

6

8

10

12

14

Time (min)

B

Rha Fuc Ara Xyl

f

Man GluGal Inositol

oo

Gal A Standard monosaccharides

pr

RNP

CRNP-2 CRNP-3 5

6

7

8

9

10

11

Retention time (min)

80

CRNP-3

60

CRNP-2 1738 CRNP-1

40

1623

20

0

1424

2933

3406 3500

13

al

RNP

rn

100

Jo u

Transmittance (%)

C

12

Pr

4

e-

CRNP-1

1019

1340

1414 1092 1608

3000

2500

2000

1500

1000

500

Wavenumber (cm-1)

Fig. 2. Characterization of RNP and CRNPs. (A) The HPLC profiles of molecular weight distribution of RNP and CRNPs; (B) Monosaccharide composition of RNP and CRNPs; (C) Fourier transform infrared spectra of RNP and CRNPs.

Journal Pre-proof

A

520

CR CR+RNP CR+CRNP-1 CR+CRNP-2 CR+CRNP-3

λmax (nm)

510

500

490

480

470 0.0

0.1

0.2

0.3

0.4

0.5

Pr

e-

pr

oo

f

NaOH concentration (mol/L)

Jo u

rn

al

Fig. 3. Structure analysis of RNP and CRNPs. (A) Effect of polysaccharide on the absorbance of Congo red. (B) Photomicrographs as recorded by SEM (magnification 200×, Scale bar 200 μm).

Journal Pre-proof

A 100

Scavenging effect (%)

80

60

40

VC RNP CRNP-1 CRNP-2 CRNP-3

20

0 0.0

0.2

0.4

0.6

0.8

1.0

1.2

Concentration (mg/mL)

B

100

f VC RNP CRNP-1 CRNP-2 CRNP-3

20

0.2

0.4

0.6

0.8

1.0

1.2

Concentration (mg/mL)

60

al

BHT RNP CRNP-1 CRNP-2 CRNP-3

40

rn

Inhibition effect (%)

80

Pr

100

pr

40

0

C

oo

60

e-

Scavenging effect (%)

80

0 0.2

Jo u

20

0.4

0.6

0.8

1.0

1.2

Concentration (mg/mL)

Fig. 4. (A) hydroxyl radical scavenging activity of RNP and CRNPs; (B) superoxide anion radical scavenging activity of RNP and CRNPs; (C) lipid peroxidation inhibition activity of RNP and CRNPs.

Journal Pre-proof

A

100

0 mmol/L 0.25 mmol/L 0.5 mmol/L 1.0 mmol/L 2.0 mmol/L 4.0 mmol/L

80

Hemolysis (%)

80

Hemolysis (%)

B

0 mmol/L 12.5 mmol/L 25 mmol/L 50 mmol/L 100 mmol/L 200 mmol/L

100

60

40

20

60

40

20

0

0 0.0

0.5

1.0

1.5

2.0

2.5

3.0

0.0

0.5

1.0

Incubation time (h)

1.5

2.0

2.5

3.0

Incubation time (h)

C

D

100

80

40

20

20

0

0.0

1.0

2.0

3.0

Concentration (mg/mL)

4.0

Pr

0

RNP CRNP-1 CRNP-2 CRNP-3 VC

40

e-

RNP CRNP-1 CRNP-2 CRNP-3 VC

60

pr

60

Hemolysis inhibition (%)

80

Hemolysis inhibition (%)

oo

f

100

0.0

1.0

2.0

3.0

4.0

Concentration (mg/mL)

Jo u

rn

al

Fig. 5. Determination of AAPH (A) and H2O2 (B) induced erythrocytes hemolysis and protective effects of RNP and CRNPs on AAPH-induced (C) and H2O2-induced (D) hemolysis

Journal Pre-proof

A

B 100

80

RNP CRNP-1 CRNP-2 CRNP-3 VC

80

MDA (nmol/mL)

MDA (nmol/mL)

60

60

40

RNP CRNP-1 CRNP-2 CRNP-3 VC

20

40

20

0

0 0.0

1.0

2.0

3.0

4.0

0.0

1.0

Concentration (mg/mL)

4.0

20

oo

f

RNP CRNP-1 CRNP-2 CRNP-3 VC

16

12

8

RNP CRNP-1 CRNP-2 CRNP-3 VC

8

4

0

e-

4

12

pr

GSH (mgGSH/L)

16

GSH (mgGSH/L)

3.0

D 20

0

0.0

1.0

2.0

3.0

Concentration (mg/mL)

4.0

0.0

Pr

C

2.0

Concentration (mg/mL)

1.0

2.0

3.0

4.0

Concentration (mg/mL)

Jo u

rn

al

Fig. 6. Effects of polysaccharides on MDA levels (in (A) AAPH and (B) H2O2-induced erythrocytes) and GSH levels (in (C) AAPH and (D) H2O2-induced erythrocytes)

Journal Pre-proof Highlights A native polysaccharide (RNP) and three carboxymethylated polysaccharides (CRNPs) were obtained. CRNPs possessed stronger scavenging activities on hydroxy radicals and superoxide radicals. CRNPs showed better protection effects on erythrocyte hemolyses than that of RNP.

Jo u

rn

al

Pr

e-

pr

oo

f

RNP and CRNPs could be potential antioxidants for functional food.

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6