Antioxidant activities of sulfated pumpkin polysaccharides

Accepted Manuscript Antioxidant activities of sulfated pumpkin polysaccharides

Ling Chen, Gangliang Huang PII: DOI: Reference:

S0141-8130(18)36522-X https://doi.org/10.1016/j.ijbiomac.2018.12.261 BIOMAC 11420

To appear in:

International Journal of Biological Macromolecules

Received date: Revised date: Accepted date:

28 November 2018 18 December 2018 28 December 2018

Please cite this article as: Ling Chen, Gangliang Huang , Antioxidant activities of sulfated pumpkin polysaccharides. Biomac (2018), https://doi.org/10.1016/j.ijbiomac.2018.12.261

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ACCEPTED MANUSCRIPT Antioxidant activities of sulfated pumpkin polysaccharides Ling Chen, Gangliang Huang* Active Carbohydrate Research Institute, Chongqing Normal University, Chongqing, 401331, China

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E-mail: [email protected]

Abstract: Pumpkin polysaccharide was extracted by hot water extraction method. It was modified

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with chlorosulfonic acid-pyridine to obtain sulfated pumpkin polysaccharides (SP1, SP2) with

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different degrees of substitution, which was 0.35 and 0.65, respectively. The total sugar contents of pumpkin polysaccharide and its sulfated derivatives were determined by phenol-sulfuric acid

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method, the ability of scavenging hydroxyl radicals and superoxide anions as well as the ability of reducing were determined. The results showed the scavenging effect of sulfated derivatives on

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hydroxyl radicals was not different after 0.8mg/mL and was lower than that of pumpkin polysaccharides. The sulfated pumpkin polysaccharides with different degrees of substitution had better scavenging effects on superoxide anions than pumpkin polysaccharide, but the reducing

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ability was lower than that of pumpkin polysaccharide.

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1. Introduction

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Key words: pumpkin polysaccharide; sulfated derivatives; antioxidant

Plant polysaccharides are polysaccharides with more than 10 degree of

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polymerization produced by plant cell metabolism. Nowadays, plant polysaccharide research is receiving more and more attention. The international scientific community even proposes that the 21st century is a century of polysaccharides [1]. Scientific experimental research shows that many plant polysaccharides have biological activity, and it has been proved that polysaccharides as free radical scavengers play an important role in preventing oxidative damage [2]. The biological activity of polysaccharides is related to its structure. The introduction of chemical groups can enhance the activity of polysaccharides or produce new activities. By molecular modification of polysaccharides, it is expected to obtain polysaccharide derivatives 1

ACCEPTED MANUSCRIPT with stronger activity and wider application range. Sulfation is a very effective modification method in structural modification. A certain hydroxyl group on the polysaccharide molecule is replaced by sulfate, which has anti-coagulation, anti-oxidation, anti-virus and other biological activities [3,4]. Pumpkin is the fruit of Cucurbitaceae plant. It is rich in carbonate, pectin,

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mineral salt, vitamin and pumpkin polysaccharide, which are beneficial to health. Pumpkin polysaccharide has hypoglycemic, hypolipidemic and anticancer effects [5].

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In addition, it can also remove various free radicals generated in the body during metabolism, including superoxide anions, hydroxyl radicals and other reactive oxygen

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species [6]. Herein, pumpkin polysaccharide was extracted by hot water method, and it was chemically modified by chlorosulfonic acid-pyridine method. The structures of

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pumpkin polysaccharide and its sulfated derivatives were identified by infrared spectroscopy and nuclear magnetic resonance (NMR) spectroscopy. The scavenging

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ability to hydroxy free radicals and superoxide anions as well as the reducing ability of pumpkin polysaccharide and its sulfated derivatives were determined by UV

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spectrophotometer. It provided an effective experimental basis for the antioxidation of

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pumpkin polysaccharide and its sulfated derivatives, and would lay a foundation for the further development and utilization of pumpkin polysaccharide.

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2. Experimental

2.1. Hot water extraction of pumpkin polysaccharide and protein removal

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The mixture was heated at 100 ℃ for 30 min with a ratio of pumpkin powder to water of 1:10 (V/m)，then heated at 60℃ for 2 h. After collecting supernatant by centrifugation and concentrating the supernatant by rotatory evaporator at 40 ℃, 4 volumes of ethanol were added for precipitation. The precipitate was washed three times with ethanol, and dissolved in a small amount of distilled water. After using Sevage method to get out of protein for three times, the supernatant was obtained by centrifugation. The supernatant was dialyzed against tap water for 3 d and dialyzed against distilled water for 2 d. The dialyzed pumpkin polysaccharide solution was 2

ACCEPTED MANUSCRIPT dried at 45℃ and ground into a powder. Finally, a relatively pure brown pumpkin polysaccharide powder was obtained.

2.2. Preparation of sulfated pumpkin polysaccharides 10 mL of pyridine was placed in a 250 mL three-necked flask equipped with a

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condenser and a stirring device, and cooled in an ice bath. Under stirring, 4 mL chlorosulfonic acid was slowly added. and the addition was completed in about 30

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min. The temperature was kept below normal temperature. When a large amount of pale yellow solid appeared in the flask, the ice water bath was removed. 0.5g

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polysaccharide was dissolved in 30 mL of DMF, and placed in a three-necked flask and stirred at a constant temperature in a boiling water for 1 h. After cooling down to

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room temperature, 1 mol/L NaOH solution was used to adjust the solution to neutral. 3-fold volume of absolute ethanol was added to stand overnight. The precipitate was

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collected and dissolved in an appropriate amount of water. It was dialyzed against tap water for 3 d, dialyzed against distilled water for 2 d, and the dialyzate was dried at to obtain sulfated pumpkin polysaccharide SP1. The amount of the

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45 ℃

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chlorosulfonic acid and the temperature were then changed to obtain a sulfated pumpkin polysaccharide SP2.

2.3. Antioxidant activity test of pumpkin polysaccharide and sulfated pumpkin

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polysaccharides

2.3.1. Scavenging of hydroxyl radicals

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Taking 1mL of pumpkin polysaccharide and its derivatives solution at a concentration of 0.1, 0.2, 0.4, 0.8, 1.6, and 3.2mg/mL in the test tube, respectively. Then, 9×10-3mol/L of 1 mL ferrous sulfate solution and 9×10-3 mol/L of 1 mL salicylic acid and ethanol solution (70% aqueous solution) were sequentially added to each test tube. The mixture was mixed, and then 9×10-3mol/L H2O2 solution (1 mL) was added to the mixture. After mixing, all the solutions were placed in a constant temperature water bath at 37 ℃ to react for 30 min. After cooling down to room temperature, the UV absorbance of the solution at 510nm [7] was measured. Using ascorbic acid (VC) as a positive control, each experiment was repeated for three times. 3

ACCEPTED MANUSCRIPT Calculation formula of hydroxyl radical scavenging rate: E(%) = (A0 - AS )/A0×100% A0 was the absorbance of the blank control solution. AS was the absorbance after adding the sample. 2.3.2. Superoxide anion scavenging capability

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First, 0.05 mol/L Tris-HCl buffer (pH=8.2, 3 mL) was added to the test tube. Then, 0.2 mL of pumpkin polysaccharide and its derivatives solution (using distilled

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water as control) at concentrations of 0.1, 0.2, 0.4, 0.8, 1.6, and 3.2 mg/mL were added, respectively. Then, the mixture was reacted in a 25 ℃ water bath for 10 min,

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and 12 μL of a 30 mmol/L pyrogallic acid solution was added at the same temperature. The mixture was mixed and reacted for 4 min, and the reaction was quenched with 0.5

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mL of concentrated hydrochloric acid, the absorbance of the mixture was measured at 320 nm [8]. The test was repeated for three times, using VC as a positive control. The

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calculation formula of superoxide anion removal rate: E (%)= [(Ai - A0 )/Ai ] ×100%

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Ai was the absorbance of the blank control solution.

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A0 was the absorbance after adding the sample. 2.3.3. Reduction capability

First, 2mL of the different concentrations (0.1，0.4，0.8，1.6，and 3.2mg/mL) of

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pumpkin polysaccharide and its derivatives solution was put in test tube, respectively. Then, 0.2 mol/L phosphate buffer (pH=6.6, 2 mL) and 2 mL potassium ferricyanide

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solution (1%, w/v) were added into each test tube, and then mixed in a water bath at 50℃ for 20 min. After the reaction, 2 mL trichloroacetic acid solution (10%, w/v) was added to the mixture, and then centrifuged for 10 min. 2 mL of supernatant were sucked through a straw, then 2 mL distilled water and 0.4 mL ferric chloride solution (1%, w/v) were added to the supernatant. These mixtures were fully mixed and reacted for 10 min at room temperature. After centrifugation, the UV absorbance of the supernatant was measured at 700nm [9]. Taking VC as a reference, each experiment was repeated for three times.

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ACCEPTED MANUSCRIPT 3. Results and discussion 3.1. Determination of total sugar content of pumpkin polysaccharide and its sulfated derivatives Using glucose as a standard, the total sugar content was measured by a phenol-sulfuric acid method, and the total sugar content of the pumpkin

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polysaccharide and its sulfated derivatives was calculated according to a linear

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regression equation. The results were shown in Fig. 1 and Table 1.

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Fig. 1. Standard curve with glucose as a standard.

3.2. Determination of the degree of substitution of sulfated pumpkin

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polysaccharides

The sulfate group concentration was determined by the BaSO4 turbidimetric method [10], and the formula for calculating the degree of substitution was as follows, DS = 1.62 × S%/(32 - 1.02 × S%) S% was the percentage of sulfate in the sample. The results were shown in Table 1.

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ACCEPTED MANUSCRIPT Table 1. Total sugar content and degree of substitution of pumpkin polysaccharide and sulfated derivatives. Polysaccharide

Total sugar

Degree of substitution

species

content (%)

（DS）

81.0

--

SP1

61.8

0.35

SP2

46.9

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P

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0.65

3.3. Infrared spectroscopy

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As could be seen from Fig. 2, the broad peak appearing at 3428 cm-1 was a characteristic absorption peak of the O-H stretching vibration. Since the

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polysaccharide molecule has many hydroxyl groups, the formation of intramolecular and intermolecular hydrogen bonds leads to a particularly broad peak. 2932 cm -1 was

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a C-H stretching vibration absorption peak, 1612 cm-1 was an absorption peak caused by bending vibration of O-H, 1333 cm-1 was an absorption peak caused by C-H

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variable angle vibration, a peak appearing at 962 cm-1 was the characteristic

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absorption peak of the sugar molecule, and the C-O absorption peak of the pyran ring structure was at 1033 cm-1. The absorption peak at 833 cm-1 was the characteristic absorption peak of variable angular vibration for α-CH type. The sulphated pumpkin

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polysaccharides had a strong absorption peak at 1261 cm-1, which was caused by asymmetric S=O stretching vibration absorption. In addition, the absorption peak at

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833 cm-1 was caused by the absorption of tensile vibration of C-O-S [11]. It could be seen that the sulfation reaction was successful.

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Fig. 2. Infrared spectra of pumpkin polysaccharide and its sulfated derivatives.

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3.4. 13Carbon nuclear magnetic resonance spectroscopy In Fig. 3, it could be seen that the carbonyl of pumpkin polysaccharide was at

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170ppm or so, the chemical shift of C1 was at 100ppm, the chemical shift of C2-C6

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was at 60-80ppm, and the methyl peak was at 52ppm. It indicated that the chemical shifts of C1 to C6 were changed after the sulfation modification, and the intensity of

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the peak was also decreased.

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P 13C NMR

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SP1 13C NMR

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SP2 13 C NMR

Fig. 3.

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carbon nuclear magnetic resonance spectra of pumpkin polysaccharide and

its sulfated derivatives.

3.5. The scavenging effect on hydroxyl radicals The scavenging effect of pumpkin polysaccharide and its sulfated derivatives on 9

ACCEPTED MANUSCRIPT hydroxyl radicals was shown in Fig. 4. It could be seen that the scavenging effect of VC on hydroxyl radicals was the best. Before 0.4mg/mL, the scavenging effect of sulfated pumpkin polysaccharides on hydroxyl radicals was better than that of unsulfated pumpkin polysaccharide. It was better but not much different. After 0.4mg/mL, with the increase of concentration, the scavenging effect of pumpkin

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polysaccharide on hydroxyl radicals was enhanced, and the effect of sulfated derivatives on scavenging hydroxyl radicals was also enhanced. Pumpkin

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polysaccharide showed a relatively flat trend. It indicated that the pumpkin polysaccharide without sulfation had better scavenging ability to hydroxyl radicals,

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and the introduction of sulfate group had little effect on the scavenging hydroxyl

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radicals.

radicals.

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Fig. 4. The ability of pumpkin polysaccharide and its sulfated derivatives scavenging hydroxyl

3.6. The removal effect on superoxide anions Fig. 5 showed the scavenging effect of pumpkin polysaccharide and its sulfated derivatives on superoxide anions. Using VC as a reference, the scavenging effect of pumpkin polysaccharide on superoxide anions was the worst, and the scavenging effect of SP2 was the best. It indicated that the introduction of sulfate group improved the ability of pumpkin polysaccharide to scavenge superoxide anions. The higher the 10

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degree of substitution, the better the scavenging effect.

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Fig. 5. Scavenging ability of pumpkin polysaccharide and its sulfated derivatives to superoxide

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anions.

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3.7. Determination of reducing power Fig. 6 showed the reducing ability of pumpkin polysaccharide and its sulphated derivatives. It could be seen that VC had the best reducing ability, and the reducing

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ability of polysaccharides had little change with the increase of concentration. After sulfation, the reducing ability of pumpkin polysaccharide showed a decreasing trend

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compared with underivatized pumpkin polysaccharide, but the overall difference was not significant. The reducing power of polysaccharide is related to its content. Since the content of polysaccharides was reduced after the sulfation, the reducing ability tended to decrease.

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Fig. 6. Reduction ability of pumpkin polysaccharide and its sulfated derivatives.

4. Summary

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Herein, pumpkin polysaccharide was extracted with hot water, and it was modified by chlorosulfonic acid-pyridine method to obtain sulfated derivatives with different

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degrees of substitution. The analysis of infrared spectroscopy and nuclear magnetic

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resonance spectroscopy indicated that the sulfation reaction was successful. The antioxidant activities of pumpkin polysaccharide and its sulfated derivatives were further studied. The results showed that the scavenging effect of sulfated derivatives

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on hydroxyl radicals decreased, indicating that the introduction of sulfate group had little effect on the scavenging of hydroxyl radicals with pumpkin polysaccharide. For

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the scavenging effect on superoxide anions, the introduction of sulfate group enhanced the scavenging of superoxide anions. It has shown that the effect of certain polysaccharide on scavenging superoxide anions is related to its rich hydroxyl group [12]. Pumpkin polysaccharide did not show a good scavenging effect, which might be related to its non-stretched steric structure. After sulfation, the originally included hydroxyl groups were released, thereby enhancing the scavenging ability to superoxide anions. The higher the degree of substitution of the sulfate group, the better the scavenging ability. The reducing ability of the polysaccharide is related to the polysaccharide content. The reducing ability of the sulfated pumpkin 12

ACCEPTED MANUSCRIPT polysaccharides was lowered because the polysaccharide content was lowered after sulfation. In general, the antioxidant activity of the sulfated pumpkin polysaccharide in some aspects has been improved. This provides an effective experimental basis for studying the antioxidant activity of other pumpkin polysaccharide derivatives, and it

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also lays a foundation for the application of pumpkin polysaccharide in organisms.

Acknowledgements

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The Project Sponsored by the Scientific Research Foundation for the Returned

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Overseas Chinese Scholars, State Education Ministry (No. 2015-1098). The work was also supported by Chongqing Key Research Project of Basic Science & Frontier

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Technology (No. cstc2017jcyjBX0012), Foundation Project of Chongqing Normal University (No. 14XYY020), Chongqing General Research Program of Basic

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Research and Frontier Technology (No. cstc2015jcyjA10054), and Chongqing Normal University Postgraduate's Research and Innovation Project (No. YKC17004),

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China.

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