Comparative Studies On Chemical Structure And Anti-fatigue Effect Of Polysaccharides Both From Turnip (Brassica Rava L.) And Maca (Levidium Mevenii Walp.)

In this dissertation,the chosen materials Brassica rapa L.and Lepidium meyenii Walp.,were both from the Qinghai-Tibet Plateau.Maca(Lepidium meyenii Walp.)was initially transplanted from Peru of South America to Yunnan of China in 2003 and then to Tibetan regions approximately in 2010.Tibetan turnip("Yuan gen",Brassica rapa L.)is traditionally multipurpose used as medicine,food and forage with long plant history in Qinghai-Tibet Plateau.At present,a great many research had been carried out on its bioactive effects of Maca at home and abroad,which mainly on the enhancement of sexual ability,fertility and anti-fatigue ability.However,less attention had been given to Tibetan turnip(Brassica rapa L.).Polysaccharides,the major secondary metabolites in the two plateau plants,have been considered as the materials basis of multitudinous bioactive effects.In this study,crude turnip polysaccharide(TP)and maca polysaccharide(MP)were extracted by traditional method of hot water extraction and ethanol precipitation.Refined TP and MP were further purified and obtained.High performance gel permeation chromatography(HPGPC),scanning electron microscope(SEM)and atomic force microscope(AFM)were adopted to comparatively study their physical properties.Infrared spectroscopy(IR),monosaccharide composition,methylation,gas chromatography-mass spectrometer(GC-MS)and nuclear magnetic resonance(NMR)were further conducted to comparatively analyze their chemical structures.Then,the weight-loaded mice swimming model was used to comparatively evaluate their anti-fatigue effects.The hydrogen peroxide induced PC 12 cell model was constructed to compare their anti-oxidative activities.The Western Blot and immunofluorescence were adopted to investigate the effects on Bax and Bcl-2 expression levels.Major results were obtained as follows:(1)Pufied TP(purity of 94.61%in DW)and MP(purity of 99.20%in DW)were obtained by traditional extraction and purification method with the yield of 0.94%,1.06%in dry weight respectively.The mean weight average molecular weight(Mw)of TP and MP were 193.18 kDa,793.5 kDa respectively.MP also contained superlarge molecular weight polysaccharide(3143 kDa).(2)The monosaccharide composition analysis indicated that they were both acidic polysaccharides with high content of D-GalA.There were seven,six types of monosaccharides detected in TP and MP respectively.The monosaccharide molar percentage of TP was D-Ga1A:D-Man:L-Ara:D-Gal:D-Glc:L-Rha:D-GlcA=67.73:17.19:10.20:3.19:1.11:0.34:0.23(moL,%)and MP was D-GalA:D-Glc:L-Ara:D-Man:D-Gal:L-Rha = 35.07:29.98:16.98:13.01:4.21:0.75(moL,%).The D-GalA content of TP(67.73%)was nearly twice of that of MP(35.07%),but the D-Glc content of TP(67.73%)was much less than that of MP(29.98%).D-GlcA was not detected in MP.On the basis of monosaccharide composition,the methylation,GC-MS and NMR revealed that the TP backbone was composed of ?-1,3,6-GalpA and ?-1,3-Galp(A)were alternatively linked in 1,3 glycosidic linkage form.And MP backbone was composed of ?-1,3-Galp(A),?-1,3-Glcp and a-1,3-Manp glycosidic linkages.(3)Both TP and MP showed good anti-fatigue activity ranging the tested dosages,the data revealed that the mid dosage TP showed no significant improvement(p>0.05)comparing with control group(average swimming speed 10.31±4.54 cm/s,total swimming duration 46.56±29.67 min),however,the high dosage TP(100 mg/kg.bw/d)could not only significantly accelerate swimming speed(15.15±3.25 cm/s,p<0.05),but also significantly accelerate the total swimming duration(106.10±42.00 min,p<0.05);MP exhibited better performance which initially showed the anti-fatigue activity at the mid dosage by significantly accelerating swimming speed and elongating swimming duration.Moreover,the high dosage MP could significantly accelerate the average swimming speed(17.21 ±5.21 cm/s,p<0.01)and elongate the swimming duration to(171.67±69.68 min,p<0.01).Then,the biochemical analysis results of mice serum indicated that high dosage TP could significantly advance Glutathione Peroxidase(GSH-Px)activity(p<0.05)comparing with normal control and MP advanced the GSH-Px activity to with the mid(p<0.05)and high dosage(p<0.01)respectively.But they showed no improvement on superoxide dismutase(SOD)activities.Both TP and MP showed the ability of lowering the lactic dehydrogenase(LDH)activity.As compared with normal control,the high dosage TP significantly reduced LDH activity(p<0.01),and also the mid dosage MP(p<0.05);moreover the high dosage of MP had strong reducing effect on LDH activity(p<0.01).Besides,administration of TP and MP could lower the metabolic waster levels,including blood urea nitrogen(BUN),lactic acid(LA)and malondialdehyde(MDA).TP showed better LA lowering effect than MP and they showed nearly the same effect on MDA.But TP showed weaker effect on reducing BUN than MP.However,as for energy substances,there was no any remarkable divergence on muscle glycogen between normal control and drug administration groups;and the hepatic glycogen content inversely showed decreasing trend.(4)In hydrogen peroxide induced oxidative damage PC 12 cell model,high dosage(10?80 ?g/mL)of TP and MP showed cell prohibition activities.However,low dosage(0.001?1.0?g/mL)of TP and MP showed protective effect in dosage dependent manner.MP exhibited stronger protective effect to PC 12 than that of TP with the same concentration.In hydrogen peroxide induced oxidative damage cell model,both polysaccharide samples protected PC 12 cells from oxidative stress via upregulating Bcl-2 protein expression level.Although there were some divergences on monosaccharide composition and chemical between TP and MP,both of them showed good anti-fatigue effects in mouse weight-loaded swimming model.At the same time,TP and MP also exhibited good anti-oxidative activities in PC 12 cell model.In one word,MP showed better anti-fatigue effect than that of TP.