Antioxidant activity of Momordica charantia polysaccharide and its derivatives

Antioxidant activity of Momordica charantia polysaccharide and its derivatives

Accepted Manuscript Antioxidant activity of Momordica charantia polysaccharide and its derivatives Fang Chen, Gangliang Huang, Zhiyuan Yang, Yupeng H...

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Accepted Manuscript Antioxidant activity of Momordica charantia polysaccharide and its derivatives

Fang Chen, Gangliang Huang, Zhiyuan Yang, Yupeng Hou PII: DOI: Reference:

S0141-8130(19)34616-1 https://doi.org/10.1016/j.ijbiomac.2019.07.129 BIOMAC 12881

To appear in:

International Journal of Biological Macromolecules

Received date: Revised date: Accepted date:

20 June 2019 5 July 2019 21 July 2019

Please cite this article as: F. Chen, G. Huang, Z. Yang, et al., Antioxidant activity of Momordica charantia polysaccharide and its derivatives, International Journal of Biological Macromolecules, https://doi.org/10.1016/j.ijbiomac.2019.07.129

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ACCEPTED MANUSCRIPT Antioxidant activity of Momordica charantia polysaccharide and its derivatives Fang Chen, Gangliang Huang*, Zhiyuan Yang, Yupeng Hou Active Carbohydrate Research Institute, Chongqing Key Laboratory of Inorganic Functional

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Materials, College of Chemisry, Chongqing Normal University, Chongqing 401331, China

Abstract: Momordica charantia polysaccharide (MCP) was extracted by hot water and

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chemically modified to obtain phosphorylated Momordica charantia polysaccharide (P-MCP) with degree of substitution 0.12 and sulfated Momordica charantia polysaccharide (S-MCP) with

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degree of substitution 0.45. The sugar content of the three polysaccharides was determined by phenol sulfuric acid method, 74.0%, 68.1% and 59.8% respectively. The scavenging ability of

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three polysaccharides to superoxide anion, hydroxyl radical and DPPH radical, as well as their anti-lipid peroxidation and reduction ability were determined. The results showed that the antioxidant activity of polysaccharides varied with different chemical modifications.

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Keywords: Momordica charantia polysaccharide, derivatives, preparation, antioxidant activity

1. Introduction

Polysaccharide isa natural mactomolecular compound composed of many monosaccharide molecules bound by glycoside bonds[1]. It is widely found in plants, animals and microorganisms.

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A large number of experiments have proved that polysaccharides isolated from natural products have remarkable effects on anti-virus, anti-oxidation, anti-cancer and improving human immunity[2].These polysaccharides have no cytotoxicity and have become one of the development directions of new drugs. The biological activity of polysaccharides is related to its structure. The introduction of chemical groups can enhance the activity of polysaccharides or make them produce new activitiesn[3].It is hopeful to obtain more active polysaccharide derivatives. Momordica charantia is thefruitof cucurbitaceae plant. It has special bitterness, not only has good edible value, but also has obvious medicinal value[4]. Modern scientific research has proved 1

ACCEPTED MANUSCRIPT that Momordica charantia contains active polysaccharides, which can improve immunity, anti-bacterial and anti-inflammatory effects[5]. In recent years, the research on active ingredients of

momordicacharantia

mainly

focused

on

momordicacharantia

protein

and

momordicacharantiasaponins [6].However, with the rapid development of polysaccharide research, Momordica charantia polysaccharide (MCP) has gradually attracted the attention of researchers.

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In this study, Momordica charantia polysaccharides were extracted by hot waterand modified by phosphorus oxychloride and chlorosulfonic acid. The structures of Momordica charanti polysaccharide and its phosphorylated and sulfated derivatives were identified by infrared (IR) spectroscopyand nuclear magnetic resonance (NMR) spectroscopy. And the antioxidant activity of

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Momordica charantia polysaccharide and itsderivatives were identified by ultraviolet (UV)

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spectrophotometry.

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2. Experimental

2.1. Extraction of MCP and proteinremoval

Momordica charantia polysaccharides were extracted by hot-water[7]. The fresh Momordica

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charantiawas weighed at 1500g and dried at 50 ℃ for 12 hours. The dried Momordica charantiawas crushed into Momordica charantia powder. The mixture of 50g Momordica

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charantia powder and 1000 mL distilled water was heated at 90℃ for 2 hours. Cool to room temperature. The solution was collected by centrifugation and concentrated to 100 mL at 60℃. Then 500 mL ethanol was added and a large amount of white sediment was obtained. White

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sediment was collected by centrifugation and dissolved in 200 mL distilled water. The protein was removed twice by Sevagemethod, and supernatant obtained by centrifugation. The supernatant was dialyzed in running water for 3 days, dialyzed in distilled water for 2 days, evaporated and concentrated, dried at 50℃ for 24 hours, and ground into powder to obtain Momordica charantia polysaccharide.

2.2. Preparation of phosphorylated Momordica charantia polysaccharide Take 0.5 g Momordica charantia polysaccharide in 10 mL DMF, and stir at room temperature for 8 hours. Take 7.5 mL pyridine and cool it in a three-necked flask for 30 minutes, add 2 mL phosphorus oxychloride slowly at low temperature(0℃), until light yellow solid appears. The

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ACCEPTED MANUSCRIPT DMF solution of Momordica charantia polysaccharide was slowly added into the three-neck flask, and the reaction temperature was raised to 35℃ for one hour. After the reaction, 1 mol/L NaOHsolution was added and the pH value was adjusted to 7. The mixture was dialyzed in running water for 3 days, dialyzed in distilled water for 2 days, evaporated and concentrated, dried at 50℃ for 24 hours, and ground into powder to obtain phosphorylated Momordica charantia

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Polysaccharide(P-MCP) [8].

2.3. Preparation of sulfated Momordica charantia polysaccharide

Take 0.5 g Momordicacharantia polysaccharide in 30 mL DMF, and stir at room temperature for 8 hours. Take 10 mL pyridine and cool it in a three-necked flask for 30 minutes, add 4 mL

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chlorosulfonic acid slowly at low temperature(0℃), until light yellow solid appears. The DMF solution of Momordicacharantia polysaccharide was slowly added into the three-neck flask, and

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the reaction temperature was raised to 80℃ for one hour. After the reaction, 1 mol/L NaOH solution was added and the pH value was adjusted to 7. The mixture was dialyzed in running

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water for 3 days, dialyzed in distilled water for 2 days, evaporated and concentrated, dried at 50℃ for 24 hours, and ground into powder to obtain

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polysaccharide(S-MCP) [9].

sulfated Momordica charantia

Antioxidant activity test

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2.4.1 Determination of scavenging capacity of hydroxyl radicals Momordica charantia polysaccharide and its sulfated andphosphorylated derivatives with 1 mL concentration of 0.1, 0.2, 0.4, 0.8, 1.6 and 3.2 mg/mL were added to the test tube. 1 mL 9 ×10-3mol/L FeSO4 solution and 1 mL 9 ×10-3mol/L salicylic acid-ethanol solution(70% ethanol

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solution) were added quickly. Finally, 1 mL 9×10 -3 mol/L H2O2solution was added to mix evenly. The mixture was reacted in water bath at 37℃ for 30 minutes and cooled to room temperature. The ultraviolet absorbance at 510 nm of the mixed solution was determined and recorded as AX, and Vc was used as a positive control [10]. The scavenging rate of hydroxyl radicals was calculated according to the following equation,E=[A0-(AX-AX0)] /A0×100%(Formula1). A0:Absorption of polysaccharides replaced by distilled water AX:Absorption of sample solution AX0:Absorption of Momordica charantia Polysaccharide and its derivatives 2.4.2 Determination of superoxide anion scavenging ability 3

ACCEPTED MANUSCRIPT Momordica charantia polysaccharide and its derivatives with 1 mL concentration of 0.1, 0.2, 0.4, 0.8, 1.6 and 3.2 mg/mL were added to the test tube(use isovolumetric distilled water as contrast). 3 mL Tris.HCl buffer solution(0.05 mol/L, pH=8.2) was added respectively, and the mixture was kept in a water bath at 37 ℃ for 10 minutes. Finally, 12 µL 30 mmol/L pyrogallolsolution was added, and the reaction time was 4 min. The ultraviolet absorbance of the

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mixture at 320 nm was determined immediately. Vc was used as a positive control [11]. The scavenging rate of superoxide anions was calculated according to the following equation, E=[(A0-AX)] /A0×100% (Formula 2) A0:Absorption of polysaccharides replaced by distilled water

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AX:Absorption of sample solution 2.4.3 Determination of reduction capacity

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Momordica charantia polysaccharide and its derivatives with 1 mL concentration of 0.1, 0.2, 0.4, 0.8, 1.6 and 3.2 mg/mL were added to the test tube. 0.2 mL phosphate buffer solution(0.2

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mol/L, pH=6.6) and 0.5 mL 1% potassium ferricyanide solution were added, respectively. The mixture was evenly mixed and reacted in a constant temperature water bath at 50℃ for 20

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minutes. The mixture was cooled to room temperature and 1 mL 10% trichloroacetic acid solution was added. After centrifugation, 1.5 mL supernatant was taken, 0.2 mL 1% FeCl 3 solution and 3

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mL distilled water were added, and the mixture was placed for 5 minutes. The ultraviolet absorbance of the mixture at 700 nm was determined. Vc was used as a positive control. The sample concentration is taken as abscissa and the absorbance as ordinate to draw a polyline map [12].

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2.4.4 Determination of anti-lipid peroxidation ability 100 mg soybean lecithin was dissolved in 100 mL phosphate buffer solution (0.1 mol/L, pH=7.4). Momordica charantia polysaccharides and its derivatives with 1 mL concentration of 0.1, 0.2, 0.4, 0.8, 1.6 and 3.2 mg/mL were added to the test tube (use isovolumetric distilled water as contrast). 1.8 mL soybean lecithin solution was added, respectively. The mixture was bathed in constant temperature water at 37℃ for 15 min. 0.5 mL 20% trichloroaceticsolution was added and the mixture was placed for 10 minutes. After centrifugation, 4 mL supernatant was taken and 0.5 mL 0.8% thiobarbituricacid (TBA) solution was added. The reaction was carried out in boiling water bath for 15 minutes and cooled to room temperature. VC was used as a positive control. The 4

ACCEPTED MANUSCRIPT ultraviolet absorbance of the mixture at 532 nm was determined and recorded as A [13]. The inhibitory rate of lipid peroxidation was calculated according to the following equation, E=[(A0-A)] /A0×100%(Formula 3) A0:Absorption of polysaccharides replaced by distilled water A:Absorption of sample solution

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2.4.5 Determination of DPPH free radical scavenging ability Weighted 4 mg DPPH accurately, dissolved in anhydrous ethanol and fixed volume in 100 mL volumetric bottle (concentration of 0.004% DPPH), preserved in dark conditions(0~4 ℃ ). Momordica charantia polysaccharides and their derivatives with 1 mL concentration of 0.1, 0.2,

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0.4, 0.8, 1.6 and 3.2 mg/mL were added to the test tube (use isovolumetric absolute ethanol as contrast). 4 mL DPPH solution were added respectively. The mixture was kept in darkness for 30

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min. Vc was used as a positive control. The ultraviolet absorbance of the mixture at 517 nm was determined and recorded as A.The scavenging rate of DPPH radical was calculated according to

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the following equation, E=[(A0-A)] /A0×100%(Formula 4)

A0:Absorption of polysaccharides replaced by absolute ethanol

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A:Absorption of sample solution

3. Results and discussion

3.1Deproteinizatioand yield of Momordica charantia polysaccharide

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Sevage method [14] is based on the denaturation of proteins in organic solvents. Free proteins are denatured into water-insoluble substances and removed by centrifugation. Sevage method has little effect on the structure of polysaccharides, but it needs repeated many times to achieve the desired effect. However, the number of repetitions had a significant effect on the yield of polysaccharides. The relationship between the number of deproteinization and the yield can be seen in Fig.1 and Table. 1.It can be seen that the protein content in polysaccharide can be greatly reduced by increasing the number of deproteinization. When the protein is removaled twice, there is almost no absorption peak at 260 nm, indicating that most of the proteins have been removed. When the number of deproteinization was increased again, the absorbance did not decrease

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Sevage method was enough.

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Fig. 1 The number of deproteinization. Table 1 The relation between the number of deproteinization and the yield.

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Number of deproteinization

1

2

3

5.3 %

4.8%

3.1%

1.5%

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Yield

0

3.2 Sugar content and degree of substitution

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The total sugar content was determined by phenol-sulfuric acid method[15]. Glucose was used as standard and standard linear graph was drawn. The total sugar content of samples was calculated by standard curve. The degree of substitution of phosphate group was determined by ammonia phosphomolybdate method, and The degree of substitution of sulfate group was determined by barium chloride-gelatin turbidimetry. The results were shown in Fig. 2 and Table 2. It can be seen that the degree of substitution of phosphate group is lower than that of sulfate group. The reason is that POCl3 is easy to hydrolyze into hydrogen chloride and phosphoric acid in air,

which

makes

the

reaction environment

become

strong acidic,

and some

momordicacharantia polysaccharides may hydrolyze. Chlorosulfonic acid is more stable than

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ACCEPTED MANUSCRIPT POCl3. During the reaction, only a few polysaccharides are hydrolyzed and the degree of substitution of the derivatives is higher. However, the sugar content of sulfated derivative decreased more, which may be the reason why chlorosulfonic acid reacts more intensely than

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phosphorus oxychloride and the reaction temperature is higher.

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Fig. 2 Standard curve of glucose.

Polysaccharide

Total sugar

Degree of

species

content

substitution

(%)

(DS)

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Table. 2 The total sugar content and substitution degree.

MCP

74.0



P-MCP

68.1

0.12

S-MCP

59.8

0.45

3.3 Infrared spectrum analysis 3.3.1 Infrared spectra of Momordicacharantiapolysaccharide Infrared spectra of Momordica charantia polysaccharide and its derivatives were shown in Fig. 3. The strong and wide absorption band at 3288cm-1 was caused by the stretching vibration of a 7

ACCEPTED MANUSCRIPT large number of hydroxyl groups[16], which was also the characteristic peak of hydroxyl groups on the sugar ring. 2941cm-1 was the C-H stretching vibration absorption peak[17], 1611cm-1 was the C-O asymmetric stretching vibration peak and C-H stretching vibration peak at 1413cm-1[18]. In sulfated Momordica charantia polysaccharide, a sharp absorption peak appeared at 1241cm-1, which was caused by S=O in sulfate radical[19].It can be seen that sulfation modification was successful. In phosphorylated Momordica charantia polysaccharide, the absorption peak appeared

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at 1271cm-1, but the absorption was weaker than S=O in sulfate radical, which indicated that the

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introduction of phosphate was successful, but its degree of substitution was low.

Fig. 3 The infrared spectrum.

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3.3.2 NMR atlas analysis

In Fig. 4, we can see that the chemical shifts of C1, C2-C5, carbonyl carbon (C6) and methyl peak are 104.36 ppm, 60-77 ppm, 170.79 ppm and 52.87 ppm respectively. As can be seen from Fig. 5, new resonance peaks such as 174.24 ppm, 169.12 ppm and 93.81 ppm appeared in sulfated Momordica charantia polysaccharides. This may be due to the influence of sulfuric acid group on the carbon and hydrogen atoms of polysaccharide ring.Fig. 6 is the

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C NMR of phosphorylated

Momordica charantia polysaccharide. New resonance peaks such as 60.61 ppm and 99.80 ppm can be seen. Phosphoric acid group is an electron-withdrawinggroup. Therefore, the chemical shifts of carbon atoms directly linked to phosphoric acid group move toward high field, while 8

ACCEPTED MANUSCRIPT those indirectly linked to phosphoric acid group move toward low field.In the 31P NMR of Fig. 7, three strong absorption peaks were observed at -0.18 ppm, -9.97 ppm and -23.05 ppm, indicating that three positions of the sugar ring were substituted by phosphoric groups. The highest strength

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was at -23.05 ppm, indicating that the position was most easily substituted.

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Fig. 4 13C NMR of Momordica charantiapolysaccharide.

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Fig. 5 13C NMR of sulfated Momordica charantia polysaccharide.

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Fig. 6 13C NMR of phosphorylated Momordica charantia polysaccharide.

Fig. 7 31P NMR of phosphorylated Momordica charantia polysaccharide.

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3.4 Analysis of antioxidant activity results 3.4.1 Scavenging capacity of hydroxyl radicals The scavenging principle of is that hydroxyl radicals are produced by Fenton reaction (Fe2++H2O2=•OH+OH-+Fe3+) [20]. The hydroxyl radicals react with salicylic acid to form

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2,3-dihydroxybenzoic acid. This substance has a special absorption peak at 510 nm, and then adds a substance which can scavenge hydroxyl radicals to reduce the peak at 510 nm. The hydroxyl radical scavenging activity of Momordica charantia polysaccharide and its derivatives was shown

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in Fig.8. It can be seen that with the increase of concentration, the scavenging effect of Momordicacharantia polysaccharide and its derivatives was increased. The scavenging effect of

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Vc was the best. When the concentration of Vc was more than 0.4 mg/mL, the scavenging effect of Vc was more than 90%. Generally speaking, the scavenging effect of Momordica charantia

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polysaccharide was lower than phosphorylated polysaccharide, but higher than sulfated polysaccharide. Semiacetal hydroxyl group with reductive property on polysaccharide molecule

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can reduce free radicals and prevent chain reaction of free radicals. The scavenging effect of derivatizedpolysaccharides on hydroxyl radicals is enhanced or weakened, which is related to the

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type, position and degree of substitution of substituents. These need further study.

Fig. 8 Hydroxyl radical scavenging capacity.

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ACCEPTED MANUSCRIPT 3.4.2 Scavenging capacity of superoxide anion It can be seen that the scavenging rate of Momordica charantia polysaccharide and its derivatives to superoxide anion is not high within the experimental concentration range, the maximum is not more than 60%. In the concentration of0 ~1.6 mg/mL, the scavenging rates of sulfated and phosphorylated Momordica charantiapolysaccharides were significantly higher than

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Momordica charantia polysaccharide. After the concentration exceeded 1.6 mg/mL, the clearance rate of sulfated Momordica charantia polysaccharide increased slightly, while that of phosphorylated Momordica charantia polysaccharide and Momordica charantia polysaccharide continued to increase at a higher rate. When the concentration reached 3.2 mg/mL, the clearance

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rate of phosphorylated polysaccharide was significantly higher than that of sulfated polysaccharide. In general, the scavenging rate of Momordica charantia polysaccharide is the

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lowest. It can be seen that the introduction of sulfate and phosphate groups can enhance the scavenging ability of Momordica charantia polysaccharide to superoxide anions. Chen [21] also

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proved that the introduction of sulfate and phosphoric acid can enhance the antioxidant activity of pumpkin polysaccharide. Compared with Momordica charantia polysaccharides, the scavenging

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effect of polysaccharides on O2 - was enhanced after chemical modification, which may be related

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to the water solubility and electronegativity of polysaccharides [22].

Fig. 9 Superoxide anion radical scavenging ability.

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ACCEPTED MANUSCRIPT 3.4.3 Reducing power It can be seen that the reducing power of Momordica charantia polysaccharide and its derivatives was much lower than Vc. The reducing power of phosphorylated Momordica charantia polysaccharide was similar to Momordica charantia polysaccharide. The reducing power of sulfated Momordica charantia polysaccharide was lower than Momordica charantia

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polysaccharide. With the increase of concentration, the reduction ability of polysaccharide and its derivatives of Momordica charantia was not significantly enhanced, and the maximum absorbance was not more than 0.2. This result may be related to the sugar content of polysaccharide. Sulfated Momordica charantia polysaccharide had the lowest sugar content, so its

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reducing power is also the weakest [23]. It can be seen that Momordica charantia polysaccharidehas the strongest antioxidant activity, while sulfated polysaccharidehas weaker

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antioxidant activity. This may be related to the position where sulfated groups replace. It may be that the position where they replace is the active site of polysaccharide, or that they connect to the

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active site of polysaccharide, so that the activity of sulfated polysaccharide can not be well

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displayed, thus reducing oxidative activity [24].

Fig. 10 Reduction capability.

3.4.4 Anti-lipid peroxidation ability

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ACCEPTED MANUSCRIPT Lipid peroxidation is a branch of free radical biology. It refers to the reaction of polyunsaturated fatty acids (PUFA) with free radicals to form intermediate free radicals, and then with molecular oxygen to form lipid peroxidation free radicals, causing rancidity and deformation[25]. For anti-lipid peroxidation, as shown in Fig. 11, the clearance rate of Vc was the highest. When the concentration was 3.2 mg/mL, the clearance rate reached more than 75%. However, the

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scavenging rate of Momordica charantia polysaccharide was the lowest, and the highest was not more than 35%. When the concentration of sulfated and phosphorylated Momordica charantia polysaccharides reached 0.8 mg/ml, their anti-lipid peroxidation ability exceeded 30%, which was much higher than that of Momordica charantia polysaccharide, and their changing trend was

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similar. The results showed that the successful introduction of sulfate and phosphate groups could

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improve the anti-lipid peroxidation ability of polysaccharide.

Fig. 11 Anti-lipid peroxidation capacity.

3.4.5 DPPH scavenging capacity The scavenging capacity of DPPH was determined by the strong absorption of DPPH radical at 517nm and the purple characteristic of its alcohol solution. When free radical scavengers exist, their absorption gradually disappears due to their single-electron pairing, and their fading degree is related to the number of electrons they receive. Therefore, rapid quantitative analysis can be carried out by spectrophotometer[26]. The scavenging effects of Momordica charantia 14

ACCEPTED MANUSCRIPT polysaccharides and its derivatives on DPPH free radicals are shown in Fig. 12. It can be seen that all three polysaccharides have scavenging effects on DPPH free radicals, but their scavenging abilities are quite different. As a control, the clearance rate ofVc was very high, reaching 83% at the concentration of 0.1 mg/mL. The removal rate of sulfated Momordica charantia polysaccharide was low, and the maximum was not more than 20%. The scavenging rate of

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phosphorylated Momordica charantia polysaccharide was significantly increased. At the concentration of 3.2 mg/mL, the scavenging rate reached 56%. The above results showed that the ability of DPPH free radical scavenging of Momordica charantia polysaccharide was enhanced after phosphorylation modification. It is reported that adding electron-withdrawing groups to

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pyrrole can enhance its antioxidant activity[27], so phosphate groups and sulfate groups of polysaccharide derivatives can improve the antioxidant activity of polysaccharide. The position

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and number of groups introduced can change the three-dimensional structure of polysaccharides, so that more hydroxyl groups in polysaccharides are exposed, thus affecting the scavenging ability

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of polysaccharides to DPPH free radicals.

Fig. 12 DPPH free radical scavenging capacity.

4. Conclusion Herein, hot-water extraction was used. Sevage method was used to remove protein. Momordica charantia

polysaccharide

was

successfully 15

prepared.

Sulfated

Momordica

charantia

ACCEPTED MANUSCRIPT polysaccharide was prepared by chlorosulfonic acid-pyridine method, and phosphorylated Momordica charantiapolysaccharide was prepared by phosphorus oxychloride-pyridine method. The sugar content of the three polysaccharides was measured by phenol-sulfuric acid method, 74.0%, 68.1% and 59.8% respectively. The degree of substitution of sulfated Momordica charantia polysaccharide was 0.45, and that of phosphorylated Momordica charantia

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polysaccharide was 0.12. The results of IR and NMR spectra showed that both chemical modifications were successful. The antioxidant activities of three polysaccharides were further studied. The results showed that sulfated Momordica charantia polysaccharide was stronger than Momordica charantiapolysaccharide in anti-lipid peroxidation and superoxide anion scavenging,

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which may be related to the degree of substitution and the position of substitution of sulfate group. As for phosphorylated Momordica charantiapolysaccharide, it showed good antioxidant activity

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in all aspects. It has been proved that phosphoric acid group has high nucleophilicity and can chelate metal ions. Therefore, phosphorylated Momordica charantia polysaccharide has better

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scavenging ability to •OH, O2- and other free radicals[28]. Therefore, surface modification without changing the bulk properties of polysaccharides is one of the effective ways to improve the

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activity of polysaccharides. There are few reports on chemical modification of Momordica charantia polysaccharide. The novelty of this study is to modify Momordica charantia

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polysaccharide by phosphorylation and sulfation, and to explore the changes of their antioxidant activity based on chemical modification. The results showed that the antioxidant activity of chemically modified Momordica charantia polysaccharides were significantly enhanced, which provided theoretical basis and practical support for the application of Momordica charantia

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polysaccharides as functional antioxidant food.

Acknowledgements The Project is sponsored by the Scientific Research Foundation for the Returned Overseas Chinese Scholars, State Education Ministry (No. 2015-1098). The work was also supported by Chongqing Key Research Project of Basic Science & Frontier Technology (No. cstc2017jcyjBX0012), Foundation Project of Chongqing Normal University (No. 14XYY020), Chongqing General Research Program of Basic 16

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

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