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Antioxidant activities of phosphorylated pumpkin polysaccharide
International Journal of Biological Macromolecules 125 (2019) 256–261
Contents lists available at ScienceDirect
International Journal of Biological Macromolecules journal homepage: http://www.elsevier.com/locate/ijbiomac
Antioxidant activities of phosphorylated pumpkin polysaccharide Ling Chen, Gangliang Huang ⁎ Active Carbohydrate Research Institute, Chongqing Normal University, Chongqing 401331, China
a r t i c l e
i n f o
Article history: Received 13 November 2018 Received in revised form 30 November 2018 Accepted 6 December 2018 Available online 07 December 2018 Keywords: Pumpkin polysaccharide Phosphorylation Antioxidant activities
a b s t r a c t Pumpkin polysaccharide was extracted with hot water, and it was chemically modified with phosphorus oxychloride-pyridine to obtain phosphorylated pumpkin polysaccharides (PP1, PP2) with different degrees of substitution. The degree of substitution of PP1 and PP2 was 0.01 and 0.02, respectively. The scavenging effect of pumpkin polysaccharide and its phosphorylated pumpkin polysaccharides on superoxide anions was determined by pyrogallol autooxidation. The scavenging effect of pumpkin polysaccharide and its phosphorylated pumpkin polysaccharide on hydroxyl radicals was determined by salicylic acid method. The reducing ability of pumpkin polysaccharide and its phosphorylated pumpkin polysaccharide was studied. It showed that the phosphorylated pumpkin polysaccharides with different degrees of substitution had higher scavenging ability to hydroxyl radicals and superoxide anions than the underivatized pumpkin polysaccharide. © 2018 Elsevier B.V. All rights reserved.
1. Introduction Pumpkin (Cucurbita moschata) is a short-day plant with a warm temperature and is drought tolerant. It is rich in starch, fat, reducing sugar, a variety of amino acids, vitamins and minerals, so it has a high therapeutic and health care function, known as “natural nutrition and health food.” Pumpkin polysaccharide is its main ingredient. Pumpkin polysaccharide is a brown powder, a non-specific immunopotentiator that enhances the body's immune function, promotes cytokine production, and produces multiple regulators by activating the immune system [1,2]. Free radical damage to the human body mainly has three aspects: the cell membrane is destroyed; the serum anti-protease is inactivated; and the damaged gene causes the occurrence and accumulation of cell mutation. The attack of the hydroxyl radicals on the human body begins with the cell membrane. The cell membrane is extremely elastic and flexible, so the cell membrane is extremely vulnerable for free radical attack. Once the electrons are taken away by free radicals, the cell membrane loses its elasticity and loses all its functions, leading to cardiovascular disease. It is more serious is that the attack of free radicals on genes can destroy the molecular structure of genes and lead to mutations in genes, causing systemic disorder throughout life. Superoxide anions are ubiquitous in living organisms and have strong oxidative properties and certain cytotoxicity. According to the study, with the
⁎ Corresponding author. E-mail address: [email protected] (G. Huang).
https://doi.org/10.1016/j.ijbiomac.2018.12.069 0141-8130/© 2018 Elsevier B.V. All rights reserved.
increase of age, the body's ability to remove superoxide anions gradually decreases, which causes the body to age. Therefore, the removal of free radicals is particularly important. A large number of experiments have shown that pumpkin polysaccharide has a good ability to scavenge free radicals and can be used as antioxidant within a certain concentration range. The extraction methods of pumpkin polysaccharide include alkali extraction [3], ultrasonic extraction and composite enzyme extraction [4]. From the experimental conditions and other aspects, the pumpkin polysaccharide was extracted with hot water and chemically modified. The structure of pumpkin polysaccharide and phosphorylated pumpkin polysaccharide was analyzed by infrared spectroscopy, and further confirmed by nuclear magnetic resonance (NMR) spectroscopy. The scavenging ability to superoxide anions and hydroxyl radicals, reducing ability of pumpkin polysaccharide and phosphorylated pumpkin polysaccharide were studied by UV spectrophotometer.
2. Experimental 2.1. Hot water extraction of pumpkin polysaccharide and protein removal Firstly, the mixture of pumpkin and water was mixed in a ratio of 10:1 (V/m). The mixture was heated for 30 min at 100 °C, and then heated for 2 h at 60 °C. The supernatant was centrifuged by a centrifuge and collected. The supernatant was concentrated at 40 °C by a rotary evaporator. After collecting the supernatant, 4 volumes of ethanol were added for precipitation. The precipitate was washed at least
L. Chen, G. Huang / International Journal of Biological Macromolecules 125 (2019) 256–261
three times with ethanol, then a small amount of distilled water was used for washing, then protein was removed at least three times by Sevage method [5,6], and the supernatant was obtained by centrifugation. The supernatant was dialyzed with tap water for 3 days and distilled water for 2 days. Pumpkin polysaccharide solution obtained by dialysis was dried and ground to powder at 45 °C. Finally, relatively pure brown pumpkin polysaccharide powder was obtained.
257
The calculation formula of superoxide anion removal rate: E ð%Þ ¼ ½ðAi −A0 Þ=Ai 100%
Ai was the absorbance of the blank control solution. A0 was the absorbance after adding the sample.
2.2. Preparation of the phosphorylated pumpkin polysaccharide 7.5 mL of pyridine was placed in a 250 mL three-necked flask, one of which was equipped with a condenser and the remaining neck was stoppered, a stirrer was placed in the bottle, and the connected device was placed in an ice bath for cooling. Under stirring, 1 mL chlorosulfonic acid was added drop by drop, and the addition was completed in about 10 min. When a large amount of pale yellow solid appeared in the flask, the ice water bath was removed. 0.5 g of the polysaccharide sample was first dissolved in 7.5 mL of DMF, stirred for at least 1 h, and then a mixture of the polysaccharide sample and DMF was added to the three-necked flask, and reacted at 80 °C for 3 h. After the reaction was completed and cooled to room temperature, the pH of the solution was adjusted to neutral using a 1 mol/L NaOH solution, then 3 volumes of absolute ethanol were added and allowed to stand overnight. The precipitate was collected by centrifugation and dissolved in an appropriate amount of water. Then, it was dialyzed against tap water for 3 days, dialyzed against distilled water for 2 days, and the dialyzate was dried at 45 °C to obtain the phosphorylated pumpkin polysaccharide (No. 1). Then, by changing the amount of phosphorus oxychloride and the reaction temperature, the phosphorylated pumpkin polysaccharide (No. 2) was obtained. 2.3. Antioxidant activity test of pumpkin polysaccharide and phosphorylated pumpkin polysaccharide 2.3.1. Determination of hydroxyl radical scavenging ability 1 mL of pumpkin polysaccharide and phosphorylated pumpkin polysaccharide solution were added to the prepared test tubes at concentrations of 0.1, 0.2, 0.4, 0.8, 1.6, and 3.2 mg/mL, respectively. Then, 1 mL of 9 × 10−3 mol/L ferrous sulfate solution and 1 mL of 9 × 10−3 mol/L salicylic acid-ethanol was sequentially added to each test tube. After the mixture was uniformly mixed, 1 mL of 9 × 10−3 mol/L H2O2 solution was further added to the mixture. After mixing again, all the solutions were placed in a constant temperature water bath at 37 °C for 30 min. After cooling to room temperature, the UV absorbance of all solutions at 510 nm was then measured. The clearance of hydroxyl radicals with ascorbic acid (VC) was used as a positive control and the entire experiment was repeated for three times [7]. The formula for calculating the hydroxyl radical scavenging rate: E ð%Þ ¼ ðA0 −AS Þ=A0 100%
A0 was the absorbance of the blank control solution. AS was the absorbance after adding the sample. 2.3.2. Determination of superoxide anion scavenging ability First, 3 mL of 0.05 mL/L Tris-HCl buffer (pH = 8.2) was added to the test tube. Then, 0.2 mL of pumpkin polysaccharide and phosphorylated pumpkin polysaccharide solution (using distilled water as control) at concentrations of 0.1, 0.2, 0.4, 0.8, 1.6, and 3.2 mg/mL were added, respectively, After mixing, the mixture was placed in a 25 °C water bath for 10 min, and 12 μL of 30 mmol/L pyro-gallic acid solution was quickly added at the same temperature. The mixture was allowed to react for 4 min and quenched with 0.5 mL of concentrated hydrochloric acid. The absorbance of the mixture was measured at 320 nm [8]. The test was repeated three times using VC as the positive control.
2.3.3. Determination of reducing power First, 2 mL of pumpkin polysaccharide and phosphorylated pumpkin polysaccharide solution with different concentrations (0.1, 0.4, 0.8, 1.6, and 3.2 mg/mL) were placed in separate tubes. Then, 2 mL of 0.2 mL/L phosphate buffer (pH = 6.6) and 2 mL of potassium ferricyanide solution (1%, w/v) were added to each tube, the mixture was uniformly mixed and then placed in a 50 °C water bath for 20 min. After the reaction, 2 mL of trichloroacetic acid solution (10%, w/v) was added to the mixture solution, followed by centrifugation for 10 min, and 2 mL of the supernatant after centrifugation was aspirated. Then, 2 mL of distilled water and 0.4 mL of ferric chloride solution (1%, w/v) were added to the supernatant. Finally, these mixtures were thoroughly mixed and reacted at room temperature for 10 min. After centrifugation again, the supernatant was measured for UV absorbance at 700 nm. Taking VC as a reference, each experiment was repeated for three times [9]. 3. Results and discussion 3.1. Determination of total sugar content of pumpkin polysaccharide and phosphorylated pumpkin polysaccharide The total sugar content was measured by the phenol sulfuric method [10] using glucose as the standard. The results were shown in Fig. 1 and Table 1. 3.2. Determination of the degree of substitution of phosphorylated pumpkin polysaccharide The degree of substitution of phosphate group was determined by the ammonium phosphomolybdate method, and the degree of substitution was calculated as follows: DS = 5.23P / (100 − 3.32P). The results were shown in Table 1. 3.3. Infrared spectroscopy It could be seen from the Fig. 2 that the broad peak appearing at 3428 cm−1 was the characteristic absorption peak of the O\\H stretching vibration. Since the polysaccharide molecule has many hydroxyl groups, the formation of intramolecular and intermolecular hydrogen bonds causes the peak to be particularly broad. 2932 cm−1 was the C\\H stretching vibration absorption peak; 1416 cm−1 was the absorption peak caused by C\\O stretching vibration of carboxyl groups; 1612 cm−1 was the absorption peak induced by the flexural vibration of O\\H; 1333 cm−1 was the absorption peak caused by C\\H variable angle vibration. The peak appearing at 962 cm−1 was the βtype absorption peak of the furan ring, which was caused by the symmetric stretching vibration of the furan ring. The C\\O absorption peak of the pyran ring structure was at 1018 cm−1. The absorption peak at 833 cm−1 was the characteristic absorption peak of the αtype C\\H variable angle vibration. 763 cm−1 was the C\\O\\C vibration absorption peak of D-glucopyranose ring. It illustrated that the pumpkin polysaccharide sugar chain was composed of furanose and pyranose. The absorption occurring at about 1270 cm−1 was the absorption of P_O stretching vibration in the phosphate group, indicating that the phosphate group might have been introduced, but the absorption was not significant due to the lower degree of substitution of the phosphate group.
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Fig. 1. The standard curve of glucose.
Table 1 Total sugar content and degree of substitution of pumpkin polysaccharide and phosphorylated pumpkin polysaccharide. Polysaccharide species
Total sugar content (%)
Degree of substitution (DS) Phosphorylated polysaccharide
P PP1 PP2
81.0 69.0 75.0
– 0.01 0.02
of C1 was at about 100 ppm, the chemical shifts of C2–C6 were at about 60–80 ppm, and the methyl group was at 52 ppm. From Fig. 3 (3.2 and 3.4) we could see that there was a strong absorption peak, indicating that the phosphate group was successfully introduced in the polysaccharide. It could be seen from Fig. 3(3.3 and 3.5) that the chemical shifts of C1–C6 were obviously shifted due to the introduction of phosphate group. 3.5. The scavenging effect on hydroxyl radicals
3.4. NMR spectra analysis In the NMR spectrum 3.1, we could see that the carbonyl position of the pumpkin polysaccharide was at about 170 ppm, the chemical shift
From Fig. 4, we could see that VC had the best scavenging effect on hydroxyl radicals. The unmodified pumpkin polysaccharide had the worst scavenging effect, and the pumpkin had better scavenging effect on hydroxyl radicals after phosphorylation. The scavenging effect
Fig. 2. Infrared spectra of pumpkin polysaccharide and its phosphorylated product.
L. Chen, G. Huang / International Journal of Biological Macromolecules 125 (2019) 256–261
259
3.3 PP1 13C NMR 3.1 P 13C NMR
3.5 PP2 13C NMR 31
3.4 PP2 P NMR
3.2 PP1 31P NMR
Fig. 3. 13C NMR and 31P NMR spectra of pumpkin polysaccharide and its phosphorylated product.
increased as the concentration of the polysaccharide increased, and the EC50 of PP2 was only 2.4 mg/mL. It indicated that the introduction of phosphate group could enhance the scavenging effect of polysaccharide on hydroxyl radicals, and the higher the degree of substitution, the better the scavenging effect.
3.6. The removal effect on superoxide anions From Fig. 5, we could see that the scavenging effect of VC on superoxide anions was still the best. The scavenging effect of pumpkin polysaccharide was the worst, and the scavenging effect of pumpkin polysaccharide after 1.6 mg/mL was significantly reduced. The scavenging effect of PP1 maintained a steady growth with increasing of concentration, while the scavenging effect of PP2 also showed a gentle increase after 1.6 mg/mL. In general, the introduction of phosphate group had a good scavenging effect on superoxide anions, but the increase in the degree of substitution had a degrading effect on the scavenging effect.
3.7. Reduction ability The reducing ability is related to the sugar content. From Fig. 6, we could see that the reduction of pumpkin polysaccharide and its phosphorylated product was far less than that of VC, but the reduction of pumpkin polysaccharide and its phosphorylated product was not much different. We could also see that the polysaccharide was more reductive after 0.8 mg/mL than its phosphorylated product. 4. Summary Herein, the use of hot water to extract pumpkin polysaccharides took a long time and the yield was low, but the operation was simple and cost-effective. Then, the pumpkin polysaccharide was modified by the phosphorus oxychloride-pyridine method, which was simple and had a high success rate. The phosphorylation reaction was successful by the analysis of infrared spectroscopy and NMR spectroscopy. The
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L. Chen, G. Huang / International Journal of Biological Macromolecules 125 (2019) 256–261
Fig. 4. The ability of pumpkin polysaccharide and its phosphorylated product to scavenge hydroxyl radicals.
alcoholic hydroxyl groups in the polysaccharide can be complexed with metal ions (such as Fe2+, Cu2+, etc.), which are necessary for generating radicals such as (•OH). So, the generation of hydroxyl radicals is suppressed [11,12]. The pumpkin polysaccharide after phosphorylation may increase the complexation effect due to the introduction of phosphate group. By the in vitro antioxidant activity test of pumpkin polysaccharide and its phosphorylated product, it was found that the removal of hydroxyl radicals by pumpkin polysaccharide after phosphorylation was significantly increased with the increase of concentration. The scavenging effect on superoxide anions was also higher than that of unmodified pumpkin polysaccharide. However, phosphorylated pumpkin polysaccharide with high degree of substitution was not better than
that with low degree of substitution after 1.6 mg/mL, and its scavenging effect was not significantly increased. From Figs. 4–5, we could also find that the scavenging effect of pumpkin polysaccharide and its phosphorylated product on superoxide anions was worse than that of hydroxyl radicals. Since the reducing ability was related to the sugar content, the reducing ability of the phosphorylated pumpkin polysaccharide was decreased. In conclusion, the overall antioxidant activity of pumpkin polysaccharides was enhanced with the introduction of phosphate group, which provided an effective basis for the further study of pumpkin polysaccharides and their phosphorylation. It also provided an effective theoretical basis for the antioxidant activity of pumpkin polysaccharides and their phosphorylated products in vivo. However,
Fig. 5. Scavenging ability of pumpkin polysaccharide and its phosphorylated product to superoxide anions.
L. Chen, G. Huang / International Journal of Biological Macromolecules 125 (2019) 256–261
261
Fig. 6. Reduction ability of pumpkin polysaccharide and its phosphorylated product.
the specific antioxidant mechanism of polysaccharides is still unclear, so further research is needed. Acknowledgements The Project 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 Research and Frontier Technology (No. cstc2015jcyjA10054), and Chongqing Normal University Postgraduate's Research and Innovation Project (No. YKC17004), China. References [1] Y. Liu, Y. Liang, Q.L. Lin, Q. Lu, M.H. Tian, F.X. Zhu, Research progress on polysaccharide extracted and antioxidant activity of pumpkin, Food & Machinery 30 (3) (2014) 239–243. [2] L. Chen, G. Huang, Extraction, characterization and antioxidant activities of pumpkin polysaccharide, Int. J. Biol. Macromol. 118 (Pt A) (2018) 770–774.
[3] H. Wang, Q.L. Zhang, Optimized extraction conditions of Stropharia rugosoannulata polysaccharides by alkaline method, Chin. Agric. Sci. Bull. 32 (26) (2016) 71–74. [4] Y. Zhao, Y.Q. Xu, C.S. Cui, Study on the optimum technology of polysaccharide extracting from pumpkin by ultrasonic-enzyme synergistic method, Acad. Period. Farm Prod. Process. 7 (2009) 13–15. [5] L.L. Cai, X.H. Li, F.R. Yu, Determination of protein content in polysaccharides from Bauhinia championi (Benth.), J. Fujian Univ. TCM 22 (3) (2012) 37–39. [6] Y. Liu, Y. Sun, G. Huang, Preparation and antioxidant activities of important traditional plant polysaccharides, Int. J. Biol. Macromol. 111 (2018) 780–786. [7] Q. Tang, G. Huang, F. Zhao, L. Zhou, S. Huang, H. Li, The antioxidant activities of six (1 → 3)-β-D-glucan derivatives prepared from yeast cell wall, Int. J. Biol. Macromol. 98 (2017) 216–221. [8] R. Liu, C. Xu, C.Y. Shi, Q.J. Chen, M. Zhang, Antioxidant activities of Fructus lycii polysaccharides and their hydrolysates, China Fruit Veg. 34 (4) (2014) 1–7. [9] Y. Liu, G. Huang, The derivatization and antioxidant activities of yeast mannan, Int. J. Biol. Macromol. 107 (Pt A) (2018) 755–761. [10] J.Y. Huang, Z.H. Tang, D.P. Li, Determination of polysaccharide content in Millettia speciosa Champ, J. Anhui Agri. 41 (3) (2013) (1180,1195). [11] H.J. Chang, X.Y. Wang, Extraction and antioxidant activity on pumpkin polysaccharide, J. Gansu Agric. Univ. 51 (3) (2016) 135–139. [12] X. Mei, C. Yi, G. Huang, Preparation methods and antioxidant activities of polysaccharides and their derivatives, Mini-Rev. Med. Chem. 17 (10) (2017) 863–868.
Contents lists available at ScienceDirect
International Journal of Biological Macromolecules journal homepage: http://www.elsevier.com/locate/ijbiomac
Antioxidant activities of phosphorylated pumpkin polysaccharide Ling Chen, Gangliang Huang ⁎ Active Carbohydrate Research Institute, Chongqing Normal University, Chongqing 401331, China
a r t i c l e
i n f o
Article history: Received 13 November 2018 Received in revised form 30 November 2018 Accepted 6 December 2018 Available online 07 December 2018 Keywords: Pumpkin polysaccharide Phosphorylation Antioxidant activities
a b s t r a c t Pumpkin polysaccharide was extracted with hot water, and it was chemically modified with phosphorus oxychloride-pyridine to obtain phosphorylated pumpkin polysaccharides (PP1, PP2) with different degrees of substitution. The degree of substitution of PP1 and PP2 was 0.01 and 0.02, respectively. The scavenging effect of pumpkin polysaccharide and its phosphorylated pumpkin polysaccharides on superoxide anions was determined by pyrogallol autooxidation. The scavenging effect of pumpkin polysaccharide and its phosphorylated pumpkin polysaccharide on hydroxyl radicals was determined by salicylic acid method. The reducing ability of pumpkin polysaccharide and its phosphorylated pumpkin polysaccharide was studied. It showed that the phosphorylated pumpkin polysaccharides with different degrees of substitution had higher scavenging ability to hydroxyl radicals and superoxide anions than the underivatized pumpkin polysaccharide. © 2018 Elsevier B.V. All rights reserved.
1. Introduction Pumpkin (Cucurbita moschata) is a short-day plant with a warm temperature and is drought tolerant. It is rich in starch, fat, reducing sugar, a variety of amino acids, vitamins and minerals, so it has a high therapeutic and health care function, known as “natural nutrition and health food.” Pumpkin polysaccharide is its main ingredient. Pumpkin polysaccharide is a brown powder, a non-specific immunopotentiator that enhances the body's immune function, promotes cytokine production, and produces multiple regulators by activating the immune system [1,2]. Free radical damage to the human body mainly has three aspects: the cell membrane is destroyed; the serum anti-protease is inactivated; and the damaged gene causes the occurrence and accumulation of cell mutation. The attack of the hydroxyl radicals on the human body begins with the cell membrane. The cell membrane is extremely elastic and flexible, so the cell membrane is extremely vulnerable for free radical attack. Once the electrons are taken away by free radicals, the cell membrane loses its elasticity and loses all its functions, leading to cardiovascular disease. It is more serious is that the attack of free radicals on genes can destroy the molecular structure of genes and lead to mutations in genes, causing systemic disorder throughout life. Superoxide anions are ubiquitous in living organisms and have strong oxidative properties and certain cytotoxicity. According to the study, with the
⁎ Corresponding author. E-mail address: [email protected] (G. Huang).
https://doi.org/10.1016/j.ijbiomac.2018.12.069 0141-8130/© 2018 Elsevier B.V. All rights reserved.
increase of age, the body's ability to remove superoxide anions gradually decreases, which causes the body to age. Therefore, the removal of free radicals is particularly important. A large number of experiments have shown that pumpkin polysaccharide has a good ability to scavenge free radicals and can be used as antioxidant within a certain concentration range. The extraction methods of pumpkin polysaccharide include alkali extraction [3], ultrasonic extraction and composite enzyme extraction [4]. From the experimental conditions and other aspects, the pumpkin polysaccharide was extracted with hot water and chemically modified. The structure of pumpkin polysaccharide and phosphorylated pumpkin polysaccharide was analyzed by infrared spectroscopy, and further confirmed by nuclear magnetic resonance (NMR) spectroscopy. The scavenging ability to superoxide anions and hydroxyl radicals, reducing ability of pumpkin polysaccharide and phosphorylated pumpkin polysaccharide were studied by UV spectrophotometer.
2. Experimental 2.1. Hot water extraction of pumpkin polysaccharide and protein removal Firstly, the mixture of pumpkin and water was mixed in a ratio of 10:1 (V/m). The mixture was heated for 30 min at 100 °C, and then heated for 2 h at 60 °C. The supernatant was centrifuged by a centrifuge and collected. The supernatant was concentrated at 40 °C by a rotary evaporator. After collecting the supernatant, 4 volumes of ethanol were added for precipitation. The precipitate was washed at least
L. Chen, G. Huang / International Journal of Biological Macromolecules 125 (2019) 256–261
three times with ethanol, then a small amount of distilled water was used for washing, then protein was removed at least three times by Sevage method [5,6], and the supernatant was obtained by centrifugation. The supernatant was dialyzed with tap water for 3 days and distilled water for 2 days. Pumpkin polysaccharide solution obtained by dialysis was dried and ground to powder at 45 °C. Finally, relatively pure brown pumpkin polysaccharide powder was obtained.
257
The calculation formula of superoxide anion removal rate: E ð%Þ ¼ ½ðAi −A0 Þ=Ai 100%
Ai was the absorbance of the blank control solution. A0 was the absorbance after adding the sample.
2.2. Preparation of the phosphorylated pumpkin polysaccharide 7.5 mL of pyridine was placed in a 250 mL three-necked flask, one of which was equipped with a condenser and the remaining neck was stoppered, a stirrer was placed in the bottle, and the connected device was placed in an ice bath for cooling. Under stirring, 1 mL chlorosulfonic acid was added drop by drop, and the addition was completed in about 10 min. When a large amount of pale yellow solid appeared in the flask, the ice water bath was removed. 0.5 g of the polysaccharide sample was first dissolved in 7.5 mL of DMF, stirred for at least 1 h, and then a mixture of the polysaccharide sample and DMF was added to the three-necked flask, and reacted at 80 °C for 3 h. After the reaction was completed and cooled to room temperature, the pH of the solution was adjusted to neutral using a 1 mol/L NaOH solution, then 3 volumes of absolute ethanol were added and allowed to stand overnight. The precipitate was collected by centrifugation and dissolved in an appropriate amount of water. Then, it was dialyzed against tap water for 3 days, dialyzed against distilled water for 2 days, and the dialyzate was dried at 45 °C to obtain the phosphorylated pumpkin polysaccharide (No. 1). Then, by changing the amount of phosphorus oxychloride and the reaction temperature, the phosphorylated pumpkin polysaccharide (No. 2) was obtained. 2.3. Antioxidant activity test of pumpkin polysaccharide and phosphorylated pumpkin polysaccharide 2.3.1. Determination of hydroxyl radical scavenging ability 1 mL of pumpkin polysaccharide and phosphorylated pumpkin polysaccharide solution were added to the prepared test tubes at concentrations of 0.1, 0.2, 0.4, 0.8, 1.6, and 3.2 mg/mL, respectively. Then, 1 mL of 9 × 10−3 mol/L ferrous sulfate solution and 1 mL of 9 × 10−3 mol/L salicylic acid-ethanol was sequentially added to each test tube. After the mixture was uniformly mixed, 1 mL of 9 × 10−3 mol/L H2O2 solution was further added to the mixture. After mixing again, all the solutions were placed in a constant temperature water bath at 37 °C for 30 min. After cooling to room temperature, the UV absorbance of all solutions at 510 nm was then measured. The clearance of hydroxyl radicals with ascorbic acid (VC) was used as a positive control and the entire experiment was repeated for three times [7]. The formula for calculating the hydroxyl radical scavenging rate: E ð%Þ ¼ ðA0 −AS Þ=A0 100%
A0 was the absorbance of the blank control solution. AS was the absorbance after adding the sample. 2.3.2. Determination of superoxide anion scavenging ability First, 3 mL of 0.05 mL/L Tris-HCl buffer (pH = 8.2) was added to the test tube. Then, 0.2 mL of pumpkin polysaccharide and phosphorylated pumpkin polysaccharide solution (using distilled water as control) at concentrations of 0.1, 0.2, 0.4, 0.8, 1.6, and 3.2 mg/mL were added, respectively, After mixing, the mixture was placed in a 25 °C water bath for 10 min, and 12 μL of 30 mmol/L pyro-gallic acid solution was quickly added at the same temperature. The mixture was allowed to react for 4 min and quenched with 0.5 mL of concentrated hydrochloric acid. The absorbance of the mixture was measured at 320 nm [8]. The test was repeated three times using VC as the positive control.
2.3.3. Determination of reducing power First, 2 mL of pumpkin polysaccharide and phosphorylated pumpkin polysaccharide solution with different concentrations (0.1, 0.4, 0.8, 1.6, and 3.2 mg/mL) were placed in separate tubes. Then, 2 mL of 0.2 mL/L phosphate buffer (pH = 6.6) and 2 mL of potassium ferricyanide solution (1%, w/v) were added to each tube, the mixture was uniformly mixed and then placed in a 50 °C water bath for 20 min. After the reaction, 2 mL of trichloroacetic acid solution (10%, w/v) was added to the mixture solution, followed by centrifugation for 10 min, and 2 mL of the supernatant after centrifugation was aspirated. Then, 2 mL of distilled water and 0.4 mL of ferric chloride solution (1%, w/v) were added to the supernatant. Finally, these mixtures were thoroughly mixed and reacted at room temperature for 10 min. After centrifugation again, the supernatant was measured for UV absorbance at 700 nm. Taking VC as a reference, each experiment was repeated for three times [9]. 3. Results and discussion 3.1. Determination of total sugar content of pumpkin polysaccharide and phosphorylated pumpkin polysaccharide The total sugar content was measured by the phenol sulfuric method [10] using glucose as the standard. The results were shown in Fig. 1 and Table 1. 3.2. Determination of the degree of substitution of phosphorylated pumpkin polysaccharide The degree of substitution of phosphate group was determined by the ammonium phosphomolybdate method, and the degree of substitution was calculated as follows: DS = 5.23P / (100 − 3.32P). The results were shown in Table 1. 3.3. Infrared spectroscopy It could be seen from the Fig. 2 that the broad peak appearing at 3428 cm−1 was the characteristic absorption peak of the O\\H stretching vibration. Since the polysaccharide molecule has many hydroxyl groups, the formation of intramolecular and intermolecular hydrogen bonds causes the peak to be particularly broad. 2932 cm−1 was the C\\H stretching vibration absorption peak; 1416 cm−1 was the absorption peak caused by C\\O stretching vibration of carboxyl groups; 1612 cm−1 was the absorption peak induced by the flexural vibration of O\\H; 1333 cm−1 was the absorption peak caused by C\\H variable angle vibration. The peak appearing at 962 cm−1 was the βtype absorption peak of the furan ring, which was caused by the symmetric stretching vibration of the furan ring. The C\\O absorption peak of the pyran ring structure was at 1018 cm−1. The absorption peak at 833 cm−1 was the characteristic absorption peak of the αtype C\\H variable angle vibration. 763 cm−1 was the C\\O\\C vibration absorption peak of D-glucopyranose ring. It illustrated that the pumpkin polysaccharide sugar chain was composed of furanose and pyranose. The absorption occurring at about 1270 cm−1 was the absorption of P_O stretching vibration in the phosphate group, indicating that the phosphate group might have been introduced, but the absorption was not significant due to the lower degree of substitution of the phosphate group.
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Fig. 1. The standard curve of glucose.
Table 1 Total sugar content and degree of substitution of pumpkin polysaccharide and phosphorylated pumpkin polysaccharide. Polysaccharide species
Total sugar content (%)
Degree of substitution (DS) Phosphorylated polysaccharide
P PP1 PP2
81.0 69.0 75.0
– 0.01 0.02
of C1 was at about 100 ppm, the chemical shifts of C2–C6 were at about 60–80 ppm, and the methyl group was at 52 ppm. From Fig. 3 (3.2 and 3.4) we could see that there was a strong absorption peak, indicating that the phosphate group was successfully introduced in the polysaccharide. It could be seen from Fig. 3(3.3 and 3.5) that the chemical shifts of C1–C6 were obviously shifted due to the introduction of phosphate group. 3.5. The scavenging effect on hydroxyl radicals
3.4. NMR spectra analysis In the NMR spectrum 3.1, we could see that the carbonyl position of the pumpkin polysaccharide was at about 170 ppm, the chemical shift
From Fig. 4, we could see that VC had the best scavenging effect on hydroxyl radicals. The unmodified pumpkin polysaccharide had the worst scavenging effect, and the pumpkin had better scavenging effect on hydroxyl radicals after phosphorylation. The scavenging effect
Fig. 2. Infrared spectra of pumpkin polysaccharide and its phosphorylated product.
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259
3.3 PP1 13C NMR 3.1 P 13C NMR
3.5 PP2 13C NMR 31
3.4 PP2 P NMR
3.2 PP1 31P NMR
Fig. 3. 13C NMR and 31P NMR spectra of pumpkin polysaccharide and its phosphorylated product.
increased as the concentration of the polysaccharide increased, and the EC50 of PP2 was only 2.4 mg/mL. It indicated that the introduction of phosphate group could enhance the scavenging effect of polysaccharide on hydroxyl radicals, and the higher the degree of substitution, the better the scavenging effect.
3.6. The removal effect on superoxide anions From Fig. 5, we could see that the scavenging effect of VC on superoxide anions was still the best. The scavenging effect of pumpkin polysaccharide was the worst, and the scavenging effect of pumpkin polysaccharide after 1.6 mg/mL was significantly reduced. The scavenging effect of PP1 maintained a steady growth with increasing of concentration, while the scavenging effect of PP2 also showed a gentle increase after 1.6 mg/mL. In general, the introduction of phosphate group had a good scavenging effect on superoxide anions, but the increase in the degree of substitution had a degrading effect on the scavenging effect.
3.7. Reduction ability The reducing ability is related to the sugar content. From Fig. 6, we could see that the reduction of pumpkin polysaccharide and its phosphorylated product was far less than that of VC, but the reduction of pumpkin polysaccharide and its phosphorylated product was not much different. We could also see that the polysaccharide was more reductive after 0.8 mg/mL than its phosphorylated product. 4. Summary Herein, the use of hot water to extract pumpkin polysaccharides took a long time and the yield was low, but the operation was simple and cost-effective. Then, the pumpkin polysaccharide was modified by the phosphorus oxychloride-pyridine method, which was simple and had a high success rate. The phosphorylation reaction was successful by the analysis of infrared spectroscopy and NMR spectroscopy. The
260
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Fig. 4. The ability of pumpkin polysaccharide and its phosphorylated product to scavenge hydroxyl radicals.
alcoholic hydroxyl groups in the polysaccharide can be complexed with metal ions (such as Fe2+, Cu2+, etc.), which are necessary for generating radicals such as (•OH). So, the generation of hydroxyl radicals is suppressed [11,12]. The pumpkin polysaccharide after phosphorylation may increase the complexation effect due to the introduction of phosphate group. By the in vitro antioxidant activity test of pumpkin polysaccharide and its phosphorylated product, it was found that the removal of hydroxyl radicals by pumpkin polysaccharide after phosphorylation was significantly increased with the increase of concentration. The scavenging effect on superoxide anions was also higher than that of unmodified pumpkin polysaccharide. However, phosphorylated pumpkin polysaccharide with high degree of substitution was not better than
that with low degree of substitution after 1.6 mg/mL, and its scavenging effect was not significantly increased. From Figs. 4–5, we could also find that the scavenging effect of pumpkin polysaccharide and its phosphorylated product on superoxide anions was worse than that of hydroxyl radicals. Since the reducing ability was related to the sugar content, the reducing ability of the phosphorylated pumpkin polysaccharide was decreased. In conclusion, the overall antioxidant activity of pumpkin polysaccharides was enhanced with the introduction of phosphate group, which provided an effective basis for the further study of pumpkin polysaccharides and their phosphorylation. It also provided an effective theoretical basis for the antioxidant activity of pumpkin polysaccharides and their phosphorylated products in vivo. However,
Fig. 5. Scavenging ability of pumpkin polysaccharide and its phosphorylated product to superoxide anions.
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Fig. 6. Reduction ability of pumpkin polysaccharide and its phosphorylated product.
the specific antioxidant mechanism of polysaccharides is still unclear, so further research is needed. Acknowledgements The Project 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 Research and Frontier Technology (No. cstc2015jcyjA10054), and Chongqing Normal University Postgraduate's Research and Innovation Project (No. YKC17004), China. References [1] Y. Liu, Y. Liang, Q.L. Lin, Q. Lu, M.H. Tian, F.X. Zhu, Research progress on polysaccharide extracted and antioxidant activity of pumpkin, Food & Machinery 30 (3) (2014) 239–243. [2] L. Chen, G. Huang, Extraction, characterization and antioxidant activities of pumpkin polysaccharide, Int. J. Biol. Macromol. 118 (Pt A) (2018) 770–774.
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