Extraction And Separation Of Polysaccharides From Bergamot And Their Antioxidant Activities In Vitro

Increasing developments and applications have been paid to the natural polysaccharides because of the numerous bioactivities, such as anti-tumor, antioxidant, anti-inflammatory, antiviral effect, its little toxic side-effects and extensive utilization of the resources. Among these, because of the great antioxidant activities, natural polysaccharides were widely used in food, cosmetics and health care products. Bergamot(Citri Sarcodactylis Fructus) was the dry fruit of citron of rutaceae plants(Citrus bergamot Citrus medico l. Var. Sarcodactylis Swingle), chemical components mainly contained volatile oil, coumarone, flavonoids, polysaccharides and so on, possessed versatile biological activities including anticancer, anti-tumor, anti-inflammatory and antioxidant, had high research value as a genuine Chinese medicine materials in Chongqing. Nowadays, the main research of bergamot mainly focus on the essential oil and flavonoids, the study of Bergamot polysaccharide mainly for crude polysaccharides and the measurement of antioxidant activity was in molecular level and the study of structure was in preliminary stage, with less sample productivity and lack of effective simulation environment of the cells in the body level of antioxidant analysis and structure-activity relationship study. Based on this, this article in full on the basis of summarizing the predecessors’ research, a series of Bergamot polysaccharide were purified, structure and antioxidant activity in cell level were determined and measured, the structure-activity relationship between oxidation activity and the preliminary structure. It provides foundational and valuable reference information for both the further investigation and efficient utilization of Bergamot resources. In this paper, the main research works and achievements included the following sections.① Preparation and antioxidant activity of crude Bergamot polysaccharideBy the method of traditional hot water extraction and alcohol precipitation step by step, the crude Bergamot polysaccharides were separated, physical and chemical properties were analyzed and phenol-sulfuric acid method was carried for the determination of polysaccharide content, hydroxy diphenyl between colorimetric was used for determination of uronic acid content, finally antioxidant activity were analyzed at the cellular level of CAA(cellular antioxidant activity) after the optimization of experimental parameters. The results showed that with water extract-alcohol precipitation, three crude Bergamot polysaccharide, BP1, BP2 and BP3 were got, physical and chemical properties analysis showed that three kinds of crude polysaccharides contain uronic acid, did not contain impurities such as protein and nucleic acid. The polysaccharide content of the three kinds of crude polysaccharide were 17.3%, 19.9% and 16.6%, the content of uronic acid were 18.5%, 21.1% and 22.8%, respectively. The CAA tests showed that the fluorescent values were certain inhibited by which were generated from DCFH into DCF has in Hep G2 cells by the three kinds of crude polysaccharide, it showed that three kinds of polysaccharides has certain antioxidant effect by comparing the EC50 values and CAA, it learned that antioxidant effect of BP3 was obvious.②Preparation and antioxidant activity of uniformity Bergamot polysaccharideBergamot polysaccharide BP3 was carried for further purification by DEAE-52 cellulose column chromatography and Sephadex G-100 gel column chromatography, the purified matericals were collected, and CAA method was used for analysis and comparison of antioxidant activity of the polysaccharides. Results showed that four kinds of polysaccharide(BP3-1, BP3-2, BP3-3 and BP3-4) were purified by DEAE-52 cellulose column chromatography and Sephadex G-100 gel column chromatography. Single peak was appeared after the four polysaccharides purificated through Sephadex G-100 column, which meaned that the four kinds were uniformity polysaccharides. CAA method was used for the measurement of antioxidant activity, it showed that the polysaccharides had obvious inhibitation of fluorescence signals which were generated from Hep G2 cell. After analysis and comparison of the four polysaccharide EC50 and CAA, the results showed that the antioxidant capacity of the four polysaccharides were in turn of BP3-2 > BP3-1> BP3-3 > BP3-4.③Exploration of structure and antioxitant activity relationship of Bergamot homogeneity polysaccharideThe polysaccharide content and uronic acid content of the four homogeneity polysaccharides were calculated, spectrum analysis was carried by UV and IR, high performance liquid chromatography(HPLC) method was used to analyze the monosaccharide composition of polysaccharides, finally the relationship was explored between antioxidant activities and uronic acid content, monosaccharide composition, chain configuration. As a result, the content of the four kinds of polysaccharides were 63.1%, 18.0%, 21.1% and 63.1%, respectively. Uronic acid content was of 16.4%, 22.8%, 20.4% and 22.8%. Ultraviolet spectrum analysis showed that four kinds of polysaccharides were not including proteins and nucleic acid. Infrared spectrum was performed to analysis the chain configuration of the four polysaccharides, BP3.1 was of α-type, the remaining three were of β-type. Monosaccharide analysis revealed that the four kinds of polysaccharide were mainly consists of rhamnose and glucose by different proportion, BP3-1 was primarily consisted of rhamnose and glucose with the ratio of 12.45:1.00, BP3-2 with the ratio of 18.42:1.00, BP3-3 with the ratio of 0.03:1.00 and BP3-4 with the ratio of 0.026:1.00. Comprehensive comparison of BP3-2 showed low content of uronic acid, with the configuration of α-type, and the monosaccharide composition of rhamnose and glucose with the ratio of 18.42:1.00 had the best antioxidant effect.