Extraction,Fractionation And Anti-Fatigue Mechanism Of Polysaccharides From Brassica Rapa L.

The Tibet Plateau is known as “the third pole of the earth” due to the high altitude and large temperature difference between day and night.Brassica rapa L.is a popular dietary plant among residents in the Tibetan Plateau region,mainly used to relieve hypoxia,fatigue,cough,asthma,etc.Polysaccharide is one of the main bioactive compounds in Brassica rapa L.,which has anti-fatigue activity and antioxidant activity in vivo and in vitro.Fatigue is a protective physiological phenomenon to keep body from tissue damage due to excessive exercise and overwork.Fatigue can lead to decrease in physical function,and excessive fatigue can even cause disease.The anti-fatigue activity of polysaccharides from Brassica rapa L.had been verified,however,the anti-fatigue mechanism is not yet clear.Therefore,Brassica rapa L.grown in Yushu,Qinghai province,were used as the raw material in this thesis.The extraction and fractionation,physicochemical characterization,and anti-fatigue mechanism of polysaccharides from Brassica rapa L.were investigated.The crude polysaccharides were separated from Brassica rapa L.by ultrasound-assisted hot water extraction process,which was optimized by response surface methodology.The optimal extraction parameters were: extraction time of 55 min,liquid-to-material ratio of 43 m L/g,extraction temperature of 60 ?,and ultrasonic power of 360 W.The yield under this extraction condition was 1.45±0.11%.Four polysaccharide fractions,BRP-20,BRP-40,BRP-60,and BRP-80,were isolated by gradient ethanol precipitation method.The results of chemical composition analysis showed that the total sugar content of the four fractions was78.41%?90.55%.The weight average molecular weight was 339.4,294.0,246.3,and 70.4k Da,respectively,descending with the increasing ethanol concentration.In addition,the monosaccharide composition,surface morphology,and microstructure of the four fractions were also different.The results of antioxidant activity experiments in vitro indicated that all the four fractions had antioxidant activity.What's more,BRP-80,the fraction with the smallest weight average molecular weight(Mw=70.4 k Da),showed the strongest antioxidant activity.The concentration of 5.0 mg/m L BRP-80 scavenged more than 80% of both DPPH and ABTS radicals,66.78% of hydroxyl radicals and showed ferric reducing power.Therefore,in this thesis,BRP-80 was selected as the test substance of animal experiments to investigate its anti-fatigue activity and mechanism.The results of the forced swimming tests showed that BRP-80 significantly prolonged the swimming time of mice with dose-dependent effect.The mice in BRP-80 high dose group(200 mg/kg/d)had a significantly higher(p<0.05)swimming time of 33.25±5.38 min,compared to the control group(10.5±2.64 min);the swimming time of BRP-80 middle dose group(100 mg/kg/d)was 21.5±3.10 min,closed to the positive control salidroside group(100mg/kg/d,26.25±4.35 min).Subsequently,the anti-fatigue mechanism of BRP-80 was investigated.The results suggested that strenuous exercise can lead to an imbalance of oxidative stress in the body,which could be reversed by up-regulating antioxidant enzymes activity and reduced malondialdehyde content in liver and muscle tissues.What's more,BRP-80 could protect muscle tissue of mice and reduced creatine kinase activity in serum after exercise significantly.BRP-80 also could up-regulated lactate dehydrogenase activity in muscle,reducing serum lactic acid levels after exercise,and relieved the accumulation of metabolites that caused fatigue.It also significantly elevated hemoglobin levels in the blood,improved the oxygen supply capacity,promoting aerobic respiratory energy supply.The results of biochemical indexes of energy metabolism showed that BRP-80 could significantly increase glycogen level,and enhance the vitality of some key enzymes in energy metabolism process,exerting anti-fatigue activity by regulating energy metabolism.The results of western blot indicated that BRP-80 increased Nrf2 and HO-1 protein expression in liver and activated PI3K/Akt/m TOR signaling pathway in skeletal muscle.The results of intestinal flora analysis showed that BRP-80 could influence the intestinal microbial composition of mice,which in turn affected the body metabolism and exerted anti-fatigue activity.