| Tibetan turnip(Brassica rapa L.)is an edible,medical and feeding plant grown under a unique natural condition with extremely cold and hypoxic environment in Tibet.Tibetan turnip possesses the effects of "heat-clearing and detoxification","tonic and anti-hypoxia","fatigue alleviating" and "wind disperseing" according to Tibetan medical books and "Sichuan Provincial Standard of Tibetan Medicinal Herbs".Moreover,data from our previous studies and other literatures showed that Tibetan turnip and its extracts could promote the mice’s ability of hypoxia-tolerance.However,few studies on separation and purification of anti-hypoxia compounds from Tibetan turnip have been reported,let alone research about its action mechanisms.In this study,we used a feeding-trial to affirm the effects of Tibetan turnip on promoting hypoxia-tolerance in humans.Then,physical-hypoxia and chemical-hypoxia cellular models were established respectively based on the dPC12 cell viability.The neuroprotective effective fraction of Tibetan turnip was selected after evaluating by the models in vitro.Next,the active compounds were isolated,purified and identified from this fraction,and the most effective one was screened by the same cellular models.At last,the anti-hypoxia efficacies and mechanisms of the active compound were investigated both in vitro and in vivo.The main research contents and results are listed as bellows:1.A 7-day and self-control human feeding trial was conducted to investigate the effects of Tibetan turnip micron powder(7.5 g x 2 times/d)on promoting hypoxia-tolerance in healthy humans.Subjects were required to undergo a hypoxia inhalation test(HIT)(7.1%O2)and a cardiopulmonary function evaluation(Bruce treadmill protocol)before(1st day)and after(9th day)the trial.Simultaneously,the serum anti-oxidative activities,blood biochemistry and routine indexes were evaluated.Results demonstrated that turnip powder could effectively increase the subjects’ SpO2 and alleviate their hypoxia symptoms during HIT.The anaerobic threshold,peak O2 pulse(male)and peak VO2/kg(female)were significantly improved after a 7-day consumption of turnip powder during the Bruce treadmill test(P<0.05).As for the blood analysis,anti-oxidative activities were boosted effectively after the 7-day treatment of turnip powder.Moreover,mean corpuscular hemoglobin concentration in the turnip-treated males increased significantly(P<0.05).Above all,those findings further confirmed that Tibetan turnip(micro powder)could effectively improve the hypoxia-tolerance in healthy humans.2.Physical-hypoxia and chemical-hypoxia cellular models were established using neuron-like dPC12 cells(differentiated PC 12 cell).The conditions of the physical-hypoxia model were:dPC 12 cells were cultured in a tri-gas incubator for 36 h with an oxygen concentration of 0.5%;the conditions of the chemical-hypoxia model were:dPC12 cells were co-incubated with 300 μmol/L of CoCl2 for 36 h.The ethanol extracts of Tibetan turnip were separated stepwise by organic solvents so as to obtain different polar fractions.And the n-butanol-ethyl acetate(medium polar)fraction was proved to have great neuroprotective effects on hypoxia-induced dPC12 cells,which was considered to be the effective component of Tibetan turnip.3.Ten compounds were isolated and purified from the above component using silica gel column chromatography,sephadex LH-20,solid-phase extraction,and prep-HPLC.And six phenolic acids(4-hydroxyl-benzoic acid-glucopyranoside,trans-p-coumaric acid-glucopyranoside,cis-p-coumaric acid-glucopyranoside,trans-ferulic acid-glucopyranoside,ferulic acid,sinapic acid)were identified using by LC/Q-TOF/MS,NMR and single-crystal X-ray diffraction.The results of activity evaluation in vitro showed that cis-and trans-p-coumaric acid-β-D-glucopyranoside exhibited the most remarkable neuroprotective effect,which exists mainly in the form of trans-structure.4.The mechanism of trans-p-coumaric acid-β-D-glucopyranoside(CG)on neuroprotective effect was determined using the physical-hypoxia cellular model.Results indicated that at the concentration of 5-50 μmol/L CG could dose-dependently increase the cell viability,block the lactic dehydrogenase(LDH)leakage and reverse the apoptosis of hypoxia-induced PC 12 cells.Moreover,50 μmol/L of CG could significantly inhibit the loss of mitochondrial membrane potential(MMP)and the release of Cytochrome C from mitochondria to the cytosol,increase the expression of anti-apoptosis proteins(Bcl-2/Bax and Bcl-XL),suppress the activities of pro-apoptosis proteins(Caspase-3 and Caspase-9)in the hypoxia-induced dPC12 cells(P<0.05).Similar trends from qRT-PCR analysis were observed among the mRNAs that encode the corresponding proteins after pretreating with CG in the hypoxia-induced dPC12 cells.5.The normobaric hypoxia-tolerance test was conducted according to the"Inspection and Assessment Standard for Health Food".We found that taking 50 mg/kg.bw/d n-butanol fraction of Tibetan turnip for 30 days could significantly prolong the survival time of male mice in the test.Then,an acute-hypoxia mice model was established to investigate the anti-hypoxia effects of n-butanol fraction and its bioactive compound CG’s aglycone,i.e.trans-p-coumaric acid(CA).The model conditions were:the oxygen concentration in hypoxic cabin was dropped from 21%to 9.5%within 15 min,and kept for 6 h.Results indicated that administration with n-butanol fraction(50 mg/kg.bw/d)and CA(25,100 mg/kg.bw/d)for 7 days could significantly reduce the pulmonary water content of acute-hypoxia-induced mice(P<0.05),which could be due to their abilities of improving the activity of Na+,K+-ATPase,enhancing antioxidant capacity(SOD↑,CAT↑ and MDA↓)and inhibiting proinflammatory factors(TNF-α,IL-1β and IL-6).Moreover,taking 50 mg/kg.bw/d n-butanol fraction of Tibetan turnip could promote the ATP level in the brain of acute-hypoxia-induced mice,which might be involved in increasing the activities of anaerobic metabolism-related enzymes including PK,PFK and LDH. |
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