The Anti-ageing Effect Of Active Ingredient α-Tocopherol From Acer Truncatum Bunge Oil And Related Potential Mechanism

Forest foods are widely popular for being green,environmentally friendly and rich in nutritional value.Acer truncatum bunge seed oil is a newly included forest food resource in China,which is abundant in natural α-tocopherol and has various biological effects such as antioxidant and atherosclerosis prevention.In this study,the anti-ageing effects and molecular mechanisms of α-tocopherol in Acer truncatum bunge seed oil were evaluated using the HepG2 cellular senescence model,the Cryptomeria hidrophila senescence model and the mouse natural senescence model to provide a theoretical basis for the development and utilization of Acer truncatum bunge seed oil as a forest food.1.Extraction and detection of α-tocopherol from Acer truncatum bunge seed oilThe oil was extracted from the seed kernels of Acer truncatum by cold pressing.The oil was light yellow to yellow in colour,with the inherent odour and taste of Acer truncatum bunge seed oil and no off-flavour.The clarity of the oil obtained was clarified and transparent.The natural α-tocopherol content of Acer truncatum bunge seed oil was measured to be 110.45 mg/kg.α-tocopherol was then purified from the deodorized distillate of Acer truncatum bunge seed oil,and the purified naturalα-tocopherol was a yellowish mucilage with almost no odour.The purity ofα-tocopherol in the purified sample was 96.8%by HPLC analysis.It can be used for subsequent biological function experiments.2.The inhibitory effect of α-tocopherol on cellular senescence and its mechanismA premature senescence model was established using HepG2 human embryonic liver cancer cells to observe the effects of α-tocopherol on senescent cells.Firstly,the MTS method and HE staining showed that 0-200 μg/mL of α-tocopherol had no significant effect on the cellular activity of HepG2 cells.25 μg/mL,50 μg/mL and 100μ/mL of α-tocopherol treatments significantly reduced D-gal-induced cellular damage and cellular senescence.Further observation of apoptosis and cell cycle by flow cytometry revealed that D-gal induced HepG2 cells into early apoptosis and late apoptosis with a total apoptosis rate of 11.9%.α-tocopherol intervention significantly decreased the rate of modulation.D-gal treatment caused significant cellular G0/G1 phase block and α-tocopherol significantly alleviated cellular G0/G1 phase block,suggesting that α-tocopherol may inhibit cellular senescence by inhibiting apoptosis and regulating the cell cycle.Western blot observed that the expression trends of the key cell cycle proteins p53 and p21 were generally consistent with the cell cycle results,indicating that α-tocopherol plays a regulatory role on the expression of key proteins for cellular senescence,thereby inhibiting premature cellular senescence.3.Inhibitory effect and mechanism of α-tocopherol on natural ageing of nematodesA natural senescence model was developed using Caenorhabditis elegans to observe the effect of α-tocopherol on natural senescence in Caenorhabditis elegans.Firstly,the survival rate of nematodes in the α-tocopherol-treated group was significantly increased compared to the control group,and both the mean and maximum life span were extended.The effects of α-tocopherol on the motility,swallowing ability and resistance to heat stress of Caenorhabditis elegans were then observed.The results showed that α-tocopherol treatment significantly improved nematode motility,and the wavelength and amplitude of nematode trajectories were significantly increased compared to the control group.α-tocopherol could enhance the swallowing ability and heat stress resistance of Caenorhabditis elegans to a certain extent,indicating that α-tocopherol treatment could enhance the vitality and resistance of nematodes.Under oxidative stress conditions,the reactive oxygen radicals ROS in nematodes increased,while the oxidative stress in Caenorhabditis elegans showed a decreasing trend in the α-tocopherol treated group.The changes in the MDA content of the oxidants in the nematodes were observed and a reduction in the MDA content of the nematodes at different concentrations of α-tocopherol compared to the control group was observed.In comparison,the antioxidant enzymes CAT and GSH-Px in nematodes showed a gradient increase in concentration,with the enzyme activity of CAT increasing by 79.23%in the 100 μg/mL treated group,126.37%in the 200μg/mL treated group and 168.26%in the 400μg/mL treated group compared to the blank group.The increase in enzyme activity was positively correlated with the increase in α-tocopherol concentration;the enzyme activity of GSH-Px enzyme increased by 14.51%in the 100 μg/mL treatment group,12.87%in the 200 μg/mL treatment group and 18.03%in the 400 μg/mL treatment group relative to the blank group.This indicates that the inhibitory effect of α-tocopherol on the natural aging of nematodes is related to the antioxidant function.4.The inhibitory effect and mechanism of α-tocopherol on natural aging in miceC57BL/6 male mice were used to establish a natural aging model to observe the effect of α-tocopherol on natural aging in mice.The 8-week-old C57BL/6 male mice were randomly divided into natural aging group and α-tocopherol-protected group.The naturally aged mice were gavaged with saline daily,while theα-tocopherol-protected mice were gavaged with α-tocopherol solution daily until 71 weeks of age,when 8-week-old C57BL/6 male mice of the same strain were set up as young controls.The results showed that the naturally aged mice grew some white hairs and showed skin shedding behavior,while they gained weight and moved slowly.The mice in the α-tocopherol-protected group had no white hair,were leaner and longer,and moved more quickly than the mice without α-tocopherol.The plasma,skin and liver SOD and CAT levels increased and MDA levels decreased in the protected group compared to the naturally aged group.α-Tocopherol improved the pathological structure of skin and liver tissues,resulting in a marked increase in appendages and a return of the nucleus to the centre of the cell.Gene microarray analysis showed that α-tocopherol up-regulated 49 genes and down-regulated 57 genes in skin tissues and up-regulated 49 genes and down-regulated 48 genes in liver tissues of naturally aged mice.KEGG analysis of the differentially expressed genes in skin tissues revealed that α-tocopherol treatment regulated signaling pathways related to carbon metabolism,amino acid biosynthesis,PPAR signaling pathway,insulin signaling pathway,pentose phosphorylation pathway and myocardial contraction.KEGG analysis of differentially expressed genes in liver tissues revealed that α-tocopherol treatment regulated signaling pathways related to sterol biosynthesis,PPAR signaling pathway,insulin signaling pathway,bile secretion,glycogen synthesis and catabolism,and AMPK signaling pathway.