A studies on the extraction, purification, structure identification of polysaccharides from seed watermelon and their biological activities

Abstract:【Objective】Seed watermelon (Citrullus lanatus var. megulasnemus Lin et Chao) is rich in the phytochemical compounds such as polysaccharides, pectin, vitamins, trace elements, amino acids and so on, and thus has high nutritional value and benefit to human health. But, at present, seed water- melon is mainly used to get seeds as a snack, while the peel and pulp are discarded creating huge waste of resources. It is well known that polysaccharides have many biological functions, and their biological activity is closely related to their chemical structures. However, to date there have been few studies on the monosaccharide components and antioxidant activity of polysaccharides in seed watermelon. The purpose of this study was to identify the monosaccharide components in seed watermelon and their primary structures and biological activities. The results will be helpful for utilization of seed watermelon polysaccharides as a potential natural antioxidant in the food and pharmaceutical industry.【Methods】‘Neimeng Heizhongpian’seed watermelon pulp was used as the material to extract crude polysaccha- rides using water extraction and alcohol precipitation method. Effects of extraction time, extraction tem- perature and material-liquid ratio on the yield of crude polysaccharide were studied through an orthogo- nal experiment of L9(33) to optimize the extraction protocol to improve polysaccharide extraction yield. Proteins and pigments were removed from the crude polysaccharides by Sevag and by activated carbon, respectively, and polysaccharides were purified by Sephadex-G75 chromatography. The purified poly- saccharides SI, SII and SIII were obtained through DEAE-Sepharose chromatography. The polysac- charide fraction defined as SI with the highest contents of polysaccharides and uronic acids was used for qualitative analysis of monosaccharide component, and the relative molecular weight was measured by HPLC. The structures of SI were studied by IR and NMR. Finally, antioxidant activity and antibac- terial activity of SI were measured in vitro. The antioxidant activity was quantified by hydroxyl radi- cal scavenging capacity, 2, 2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging capacity, and ferric ion reducing antioxidant power (FRAP). Antimicrobial activities of SI were studied through determin- ing bacteriostatic diameter. Staphylococcus aureus, Bcillus subtilis, Escherichia coli, Rhizopus nigricus, Aspergillus niger and yeast were selected as the test strains【. Results】SI, which accounted for 70.26% of polysaccharide and 7.09% of uronic acid, was chosen for subsequent research. The results showed the relative molecular weight of SI was 1 747 Da. It was composed of mannose, rhamnose, galacturon- ic acid, glucose, galactose, xylose and arabinose with a molar ratio of 2.4∶2.2∶6.6∶36.8∶32.1∶7.2∶12.7. S I had some characteristic absorption peaks in the range of 400- 4 000 cm- 1. The three absorption peaks within 950-1 250 cm- 1 indicated that SI contains pyran ring and furan ring. There were several vibration absorption peaks within 750-920 cm- 1, suggesting that SI probably contains xylose, rham- nose, arabinose and mannose and other monosaccharides. The hydrogen signal peak of the 1H-NMR spectra in 4.971 mg·L-1 and 5.124 mg·L-1 and the carbon signal peak of the 13C-NMR spectra in 98.11 mg·L-1 and 101.55 mg·L-1 showed that the SI contains ɑ-D-glucose and ɑ-D-galactose. Similarly, the hydrogen signal peak of the 1H- NMR spectra in 4.887 mg · L- 1 and 4.686 mg · L- 1 and the carbon signal peakofthe13C-NMRspectrain94.6mg·L-1 indicatedthattheSIcontainsβ-D-mannoseandβ-L-rham- nose. In vitro antioxidant experimental results showed that SI possesses high activities of scavenging hydroxyl free radical and DPPH radical as well as high FRAP, and the antioxidant capacity of SI had dose-dependent manner within certain concentration range, but its antioxidant activity was lower than VC. In vitro antimicrobial test results showed that SI possessed some antimicrobial activity and had certain specificity, as it had had no inhibitory effect on black Rhizopus nigricans and Aspergillus niger, a weak inhibitory effect against Staphylococcus aureus, Bacillus subtilis and Escherichia coli, and a strong inhibitory effect on yeast, and the inhibition ability increased with the increase in SI concentration.【Conclusion】The result showed that polysaccharides of seed watermelon pulp possess antioxidant and antimicrobial activity in vitro, which is closely related to their chemical structure. They can be po- tentially used as natural antioxidant for food and pharmaceuticals industries.