| Ginkgo biloba is the dried mature seed of Ginkgo(Ginkgo biloba L.).It can be used as a medicine or as a food for a long time.All previous dynasties have "ginkgo toxic" records,in long-term practice has proved that a large number of Ginkgo consumption will produce severe toxic reactions,and even life-threatening.However,the toxic substance base of ginkgo and the mechanism of toxicity is not clear,leading to its safety and food restriction.It also restricts the value embodiment and industrialization development of this precious edible and medicinal resource in china.In view of this,this thesis is based on the analysis and evaluation of Ginkgo multiple toxic substances,characterization and toxicity studies carried out in vivo toxicity component of ginkgolic acid and MPN.Furthermore,through the investigation and optimization of the processing technology of Ginkgo biloba,the methods and mechanisms of reducing the toxicity of Ginkgo biloba during processing were studied,which provided a scientific basis for the industrial development of ginkgo fruit for safe consumption.Literature researchBy combing and summarizing the related literature of Ginkgo biloba,the present research situation of Ginkgo toxicity and the research methods of toxic Chinese medicine were summarized.In order to provide scientific reference for the study of the toxic substances and the in vivo process of the fruit,with modern toxicity research methods.Experimental study1.Analysis and evaluation of multiple toxic substances in Ginkgo bilobaThrough the establishment of corresponding detection method,qualitative and quantitative analysis of toxic substances in ginkgo ginkgo acid,pyridoxine and cyanide compounds.The control method of ginkgolic acids in Ginkgo extracts were determined,the results show that GA(15:1)461.86?g/g?922.35?g/g,GA(17:2)86.68?g/g?227.75?g/g,GA(17:1)81.67?g/g?279.75?g/g,The MPN in Ginkgo biloba was determined.The result showed that the content of MPN was 256.38?g/g?373?g/g.The cyanide in ginkgo is analyzed using picric acid test strip.The results showed that the cyanide content is lower than the detection limit,negative reaction.Conclusion cyanide is not a toxic substance causing acute poisoning of ginkgo,and there is a big difference between GAs and MPN in different batches.The method is reliable and suitable for the evaluation and analysis of the toxic components in Ginkgo biloba.2.Study on the characteristics and compound toxicity of Ginkgo toxic substances based on in vivo process2.1 Toxicity characterization and in vivo process of ginkgolic acidsThe acute toxicity of GAs was evaluated by zebrafish toxicity test and LD50 acute toxicity test of mice.The toxic dose was determined and the toxic symptoms were described.The results showed that GAs induced zebrafish intoxication was dose dependent and time dependent.The toxicity was slower and there was no acute toxicity.After oral administration of GAs,no acute poisoning symptoms occurred in mice.The death concentration occurred at 3?4 days after administration,LD50 was 7.89 g/kg,and 95%confidence interval was(7.19?8.68)g/kg,which was in low toxicity level.Pharmacokinetics,tissue distribution and metabonomics of oral GAs in rats were studied:Pharmacokinetic parameters of GAs and its metabolites After intragastric administration,the C-T curves of high(900,mg/kg),medium(300 mg/kg)and low(100 mg/kg)dose groups appeared double peaks phenomenon.In pharmacokinetic studies of GAs metabolites,6 metabolites that are qualitatively reliable and have a peak area have been selected.The relative change curve was drawn for pharmacokinetic study.Hydroxylated products,carbonylation of hydroxylated products(double oxidation products)and glucuronidated products of GA(15:1)and GA(17:1)were studied.The amount of metabolite production in the high dose group was significantly less than that in the medium and low dose groups,and the time required for each metabolite reached a peak,and there was a lag phenomenon compared with the original compound.Tissue distribution study After oral administration of GAs,the rats were rapidly distributed in the main organs and mainly accumulated in the liver.At 0.5 h,the liver was the most widely distributed and subsequently decreased.The content of GAs in brain increased gradually to 4 h,and then decreased gradually.The tissue distribution of the 5 GAs is approximately the same.Excretion study After intragastric administration of GAs at 900 mg/kg,the rats were excreted mainly from feces,the fecal excretion rate was 29.6%,the urinary excretion rate was 0.004%,and the bile excretion rate was 3.54%.The excretion trend of the 5 GAs in each excreta was consistent.The excretion of urine was mainly concentrated in 6?12 h,the excretion of feces was mainly concentrated in 12?48 h,and the excretion of bile was relatively homogeneous in 24 h.The excretion rate of the compounds in excreta was similar to that in the administration of GAs,and there was no significant difference in the excretion of each ginkgolic acids in rats.Serum metabonomics After oral administration of GAs,the serum levels of BUN and CRE were significantly increased,and the levels of TP and ALB decreased significantly,suggesting that GAs has some damage to liver and kidney function.The activities of related enzymes ALT,AST,LDH and CK in serum were significantly decreased,which might be related to the inhibitory effect of GAs on enzyme activity in vivo.In principal component analysis(PCA)diagrams,there was a significant difference between the samples of different groups.After third days of oral administration of GAs,samples were the longest distance from the blank samples,suggesting a significant change in the endogenous components of the body at third days.On the seventh day,the endogenous substances gradually returned to normal levels,suggesting that the body has the ability to recover itself.This result was consistent with the result of LD50 test of oral administration of GAs in mice.The potential biomarkers for 14 key organisms mainly include different types of LysoPC and LysoPE.These biomarkers are mainly concentrated in the metabolic pathways of sphingolipid metabolism,glycerophospholipid metabolism and primary bile acids.These suggest that GAs toxicity may act on these metabolic pathways and cause toxic reactions.2.2 Study on material characterization and toxicity of MPN in vivoThe acute toxicity of MPN was evaluated by zebrafish toxicity test and LD50 acute toxicity test of mice.The toxic dose was determined and the toxic symptoms were described.The results showed that MPN could be neurotoxic to zebrafish larvae,and had influence on the morphology and hatching of zebrafish.Mice with oral MPN have convulsions.The toxic dose was close to the death dose.LD50 is 35.20 mg/kg,and 95%confidence interval is(33.18?37.33)mg/kg.The toxicity of MPN poisoning is consistent with the symptoms of Ginkgo poisoning,suggesting that MPN is the main toxic substance causing acute poisoning of ginkgo.Pharmacokinetics,tissue distribution and metabonomics of oral MPN in rats were studied:Pharmacokinetic parameters of MPN The rats treated with MPN,in the plasma of pyridoxic acid(4-PA),pyridoxine(PN)and pyridoxal(PL)blood concentration increased.These results suggest that 4-PA,PL and PL may be the metabolites after oral administration of MPN.On the other hand,it may also be due to the fact that MPN is more responsive to phosphate kinase PKH than vitamin B6,thereby inhibiting phosphorylation of PL,resulting in elevated levels of PL in the body.4-PA is an oxidation product of PL and is excreted as a terminal metabolite of vitamin B6.MPN,a derivative of vitamin B6,may also exist in this metabolic process.Tissue distribution After oral administration of MPN,the rats were distributed rapidly in the tissues and reached the maximum distribution at 5 min,then decreased gradually.All the main organs were distributed,and the amount of liver was larger.At 24 h,the MPN content in the tissue samples has been reduced to a lower level.4-PA was distributed in heart,liver,spleen,lung and kidney,and could not be detected in brain tissue.At each time point,the content of 4-PA in liver and kidney was higher than that in heart samples,and the content of 4-PA in heart samples was lower.The content of 4-PA in lung and spleen samples was lower in 15 min.4-PA mostly distributed in the liver and kidney tissues,and the content of 4-PA in the lung and spleen increased between 0.5 h?1 and h,and decreased greatly at 4h.At 24 h,4-PA was detected in all tissue samples,most of which were distributed in the liver and kidney.This may be related to metabolism in the liver and ultimately renal excretion.Pharmacokinetic parameters of MPN-5'-glucoside After oral administration of MPN-5'-glucoside,the content of MPN-5'-glucoside in the plasma increased rapidly and decreased rapidly within 4 h.MPN was rapidly generated in plasma and decreased rapidly within 4 h.The concentrations of 4-PA,PL and PN in plasma were increased synchronously,and the metabolic tendency was similar.It suggests that MPN-5-glucoside metabolism produces MPN,further causing changes in 4-PA,PL,and PN.Serum metabonomics After oral administration of MPN in rats,serum metabonomics study was performed within 24 h,and 13 biochemical parameters were measured.The results showed that the levels of TBIL,LDH,BUN,CRE,BUN and CRE in serum were significantly changed after oral administration of MPN.After 5 min,the serum levels of BUN and CRE were significantly decreased compared with the blank group,and there was a significant difference between the 24 h group and the blank group.The level of TBIL decreased significantly,and the activity of LDH increased significantly after administration,then decreased gradually in the whole experiment,and returned to the level similar to that of the blank group at 24 h.2.3 Compound toxicity characterization of the main toxic substances in Ginkgo biloba:GAs pharmacokinetic effects on MPN rats Rats given GAs and MPN,AUC(0-t)of MPN significantly decreased.AUC(0-t)of 4-PA in plasma was decreased similar to MPN.AUC(0-t)of PN has a small increase phenomenon,while the AUC(0-t)of PL is similar to that given MPN alone,indicating that GAs has a significant effect on the internal processes of MPN.After administration of GAs,the MPN,4-PA,PN,and PL Tmax were delayed in comparison with oral administration of MPN alone.This may be related to the absorption of MPN by GAs or the inhibition of enzyme activity in vivo,thus delaying the peak time of MPN and its metabolite and decreasing the Cmax of MPN.MPN effect on the parameters of GAs in rats When the administration of GAs was given alone,the C-T curve existed double-peak.After giving GAs and MPN at the same time,there was no obvious double-peak phenomenon in GAs.The AUC(0-t)of GA(13:0)increased compared with GAs alone,and the AUC(0-t)of the other four GAs decreased slightly.Cmax of GA(13:0)and GA(15:1)increases,while GA(17:2),GA(15:0)and GA(17:1)decrease slightly.The content of GAs metabolite in plasma changed obviously,the hydroxylation product decreased obviously,and the content of glucuronic acid increased.After the combination of MPN and GAs,oral administration has an effect on the in vivo process of GAs.This effect may be related to the length of the fatty acid chain of ginkgolic acids.Study of pyridoxine compounds combined toxicity At the same time,the pharmacokinetic parameters of MPN-5 '-glucoside were not significantly affected by MPN administration.But the increase of 4-PA,PL and PN in plasma is greater than that of MPN-5-glucoside alone.MPN-5-glucoside produces MPN in the body after oral administration and further produces metabolites such as 4-PA,PL and PN.3.Study on Optimization and mechanism of Ginkgo reduction methodIn the edible part,the germ is the most toxic part,and its weight accounts for 2.82%of the total weight of the edible part,but the GAs contains 62.1%of the GAs in the edible part,and the MPN contains 3.87%.It was suggested that the removal of the embryo from Ginkgo biloba could effectively reduce the toxicity of the edible parts of Ginkgo biloba.Processing temperature and drying speed are the main factors affecting the content of toxic substances in Ginkgo biloba.The content of GAs and MPN in Ginkgo biloba was changed less than 50?.50? and higher temperature(60?,70?,80?,90? and 100?),while maintaining the humidity conditions in ginkgo,the loss rate of MPN was above 90%.However,the content of MPN in ginkgo was almost unchanged during the rapid drying process.The content of GAs treated at high temperature(90?,100?)was reduced by about 55%.The results indicated that the content of MPN and the transformation may itself contain the enzyme,the enzyme activity under low temperature is not enough,the rapid loss of moisture in Ginkgo during rapid drying process,decreased enzyme activity in less water conditions,the reduction of MPN is limited.Compared with the chromatogram of Ginkgo samples before and after treatment,the MPN peaks in Ginkgo biloba samples were significantly reduced to hardly recognized when treated 12 h,and the other three peaks were obviously increased in the chromatogram.The higher the temperature,the faster the transformation rate of MPN in Ginkgo biloba,and there is a balance point.The three conversion products increase with the increase of processing time at the beginning of heating,and the higher the temperature is,the more the conversion product grows,the faster the rate is.It is suggested that there is a balance between the components of Ginkgo in the heating process,and finally reach a stable state.With the standard comparison,one product is determined as MPN-5'-glucoside.Presumably,this is the biotransformation product of MPN in the presence of Ginkgo nut glucosidase.Comparing the toxicity of MPN and MPN-5 '-glucoside with zebrafish,the results showed that the toxicity of MPN-5'-glucoside was less than MPN.On the basis of the above studies,a method based on enzymatic method to remove ginkgo toxicity was developed and the process was validated.Fresh ginkgo in moisture control and 60 degrees of temperature,so that the moisture content of more than 50%,maintain 7 hours,then heated to 90??100?,drying to the required content,and then continue to maintain the temperature for 1 hours.The low toxic ginkgo can be used as the raw material for the development of Ginkgo products. |
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