Analysis Of 4'-O-Methylpyridoxin Analogues In Ginkgo Biloba Seeds And Its Biological Effects

Ginkgo biloba seeds are widely used as food and medicine in China,but the toxicity of G.biloba seeds has been restricting the utilization of G.biloba seeds resources.4'-O-methylpyridoxin?MPN?as the toxic substance in G.biloba seeds was investigated in this work.The contents and composition of MPN analogues in G.biloba seeds at different regions,growth stages and processing methods was detected.The apoptotic effect of MPN on human gastric mucosal epithelial cells?GES-1?was studied.The effect of MPN on rat growth and metabolize was investigated after 15 days feeding.Finally,the effects of MPN on vitamin B₆,neurotransmitter and amino acid metabolism in rats with different ages were explored.The results of this study provide an important theoretical reference for comprehensive evaluation of G.biloba seed safety and toxicity mechanism.1.The differences of MPN analogues in G.biloba seeds from China regions were investigated.The results showed that the contents of MPN,MPNG,PMP,PLP,PN,PL and PM in G.biloba seeds from nine regions of China were ranged from 112.59–158.81?g/g,64.02–172.83?g/g,3.34–12.73?g/g,21.47–111.30?g/g,205.00–316.72?g/g,44.66–109.17?g/g and47.82–138.19?g/g,respectively.The contents of in G.biloba seeds from Taizhou and Yuxi had the lowest total MPN?TMPN?contents with its values at 177.47?g/g and 187.17?g/g,respectivel.The highest total vitamin B₆?VB₆?contents were found in Suizhou and Chengdu with its values at577.38?g/g and 586.81?g/g,respectively.Principal component analysis?PCA?showed that the contents of MPN analogues in G.biloba seeds from Liupanshui,Guilin and Yuxi,Putian and Chengdu regions were similar.2.The content and composition changes of MPN ananlogues in G.biloba seeds were studied regarded as different cultivars?Fozhi and Maling?and tree ages?10-years-old and 15-years-old?.The results showed that the highest TMPN contents were found in G4 stages and its values was249.30?g/g,295.62?g/g and 267.85?g/g in Maling-15,Fozhi-10 and Maling-10,respectively.The content of VB₆ in three G.biloba seeds samples decreased gradually from 895.56?g/g to386.37?g/g,1079.60?g/g to 408.02?g/g and 846.74?g/g to 257.92?g/g during the whole growth period.PCA results showed that cultivars have greater impact on the content and composition of MPN analogues in G.biloba seeds compared to tree years.3.The effects of different drying and cooking methods on MPN analogues in G.biloba seeds were investigated.The results showed that sun-drying could significantly reduce the content of MPN analogues in G.biloba seeds,while freeze-drying have no effects on them.With the higher hot air drying temperature,the contents of MPN and TMPN gradually decreased,while the contents of MPNG increased.After hot air and vacuum drying,PLP content in G.biloba seeds increased significantly,while PMP,PM and PL content decreased.Vacuum drying?60??can keep the content of VB₆ better.The contents of MPN and MPNG in G.biloba seeds were significantly decreased after high temperature baking and frying treatment.The content of PM and PN can be well preserved by microwave treatment in a short time.PCA results showed that the effects of frying and boiling on MPN analogues in G.biloba seeds were different from those of microwaving and baking treatment.Toxic substances in G.biloba seeds were significantly reduced after sun-drying and frying treatment.4.The inhibitory effect of MPN on the growth of different human cells and the mechanism of apoptosis were studied.The results showed that the IC₅₀ values of MPN for different human cells were human neuroblastoma SHSY5Y cells?130.40?M?<human gastric mucosal epithelial GES-1cells?133.40?M?<human small intestinal epithelial Caco-2cells?139.80?M?<human normal hepatocytes L-02 cells?152.00?M?<human renal tubular epithelial HK2 cells?178.30?M?.High dose of MPN?100?M?had an effect on the nucleus of GES-1 cells,and the proportion of apoptotic and necrosis cells reached 43.80%.PI staining showed that MPN had an effect on DNA replication of GES-1 cells.The percentage of G1/G0 and G2/M cells increased significantly while that of S-phase cells decreased significantly.JC-1 staining showed that MPN could significantly reduce mitochondrial membrane potential in GES-1 cells.Westen blotting results showed that MPN significantly increased the expression of apoptotic proteins C-caspase-8and Bax in GES-1 cells.5.The effects of different concentration of MPN on the growth and metabolism of rats were studied.The results showed MPN had no effect on the body weight,organ weight and coefficient of rats.Histopathological observation showed that the dose of MPN of 50mg/kg.BW could induce nucleus contraction of brain and the vacuolation and hemorrhage of heart tissue in rats.MPN at low dose?10mg/kg.BW?and medium dose?30mg/kg.BW?had a greater effect on the content of amino acids in plasma of rats.MPN had significant effects on protein,lipid and metabolic products in plasma.High dose of MPN could decrease the anti-oxidative stress ability of rats.Low and medium doses of MPN could increase the levels of PLP,PL and VB₆ in rat plasma.6.The effects of MPN administration on plasma and brain MPN residues,VB₆,5-hydroxytryptamine,dopamine and amino acid analogues in young and adult rats were investigated.The levels of MPN in serum and brain were 38.11–10.03?M,25.39–6.29?M,48.99–7.98?g/g and 34.23–3.88?g/g in young and adult rats between 0-6h.At the same time,the content and trend of PM,PLP and PL in plasma and brain of young rats treated with MPN changed greatly.After MPN administration,plasma and brain levels of 5-hydroxytryptamine and dopamine decreased in both groups.There were more kinds of amino acid analogues in plasma of young rats than that of adult rats.PCA results showed that amino acid analogues in the brain of young rats had greater difference before and after MPN administration.