Absorption,Transportation,Residual Distribution Of Chiral ¹⁴C-Dufulin In Rhizome Plants And Metabolic Transformation Of Its Raceme

Dufulin is the first independently developed plant immune induced antiviral agent in China,which is also a biologically derived antiviral agent synthesized fromα-phosphoamides in sheeps.It is a effective pesticide against viral diseases in plants such as tobacco,rice,cucumber and tomato,and has a chiral structure.However,the transport behavior and metabolic rules of Dufulin and its chiral enantiomers in plants are still unknown,which is very important for environmental risk assessment of Dufulin.In this paper,using S/R-[thiazolyl-2-¹⁴C]-Dufulin as tracers,the absorption,transport,and accumulation of different enantiomers of Dufulin in different tissues and organs of rhizome crops（radish and garlic）were studied with isotope tracing methods.Emphasis was placed on whether the behavior of Dufulin enantiomers in plants has chiral selectivity.At the same time,using high specific activity[thiazolyl-2-¹⁴C]-Dufulin racemates and[benzyl-¹⁴C]-Dufulin racemates as tracers,combining advanced analytical techniques such as radioisotope tracing and HPLC-LSC/QTOF-MS,the residue,metabolic transformation,and mechanism of Dufulin racemates in the tested plants were systematically studied through parallel and combined experiments.The purpose is to provide theoretical basis and technical support for scientific evaluation of safety of Dufulin.The main conclusions were as follows:（1）The absorption,translocation,and directional accumulation of Dufulin enantiomers in root and stem crops were clarified when they were applied on the leaf surface.There was no chiral selectivity in the absorption,translocation,and directional accumulation of different Dufulin enantiomers.Rapid transport to other tissues and organs of the plant occurred after the Dufulin enantiomers were absorbed by the treated leaves.¹⁴C Dufulin began to be transferred after two hours of application,but most of the Dufulin remained in the treated leaves.In the root crop cherry radish,more than 90%of the ¹⁴C Dufulin enantiomers remained on the treated leaves 28 days after application,while only 6.47%of S-¹⁴C Dufulin and 6.32%of R-¹⁴C Dufulin were transferred from the leaves to other locations.In the succulent root of cherry radish（edible parts）,the final ratios of S-¹⁴C-Dufulin and R-¹⁴C-Dufulin were 3.00%and 2.93%,while the accumulation of S-¹⁴C-Dufulin and R-¹⁴C-Dufulin in the lateral roots of cherry radish was almost negligible,with only 0.11%of S-¹⁴C-Dufulin and 0.12%of R-¹⁴C-Dufulin remaining.In bulb crop garlic plants,after 28 days of application,the proportion of S-¹⁴C-Dufulin in labeled leaves decreased from 97.017%to 58.954%,while R-¹⁴C-Dufulin decreased from 96.273%to 59.592%.This indicated that nearly half of the Dufulin enantiomers and metabolites have been transferred to other sites,with stronger transport ability than in cherry radish.At the garlic bulb,the proportions of S-¹⁴C-Dufulin and R-¹⁴C-Dufulin were 15.378%and14.378%,making them the site with the highest content of Dufulin enantiomers except for labeled leaves.Only a small fraction of S-Dufulin and R-Dufulin were transported to the roots.The concentrations of S-¹⁴C-Dufulin and R-¹⁴C-Dufulin in different tissues of cherry radish decreased in the following order:labeled leaf（LL）>leaves below the labeled leaf（LBLL）>leaves above the labeled leaf（LALL）>lateral roots（LR）>succulent roots（SR）.The concentration in various tissues of garlic decreased in the following order:LL>LBLL>Bulb>LALL>Root.This indicated that Dufulin was more likely to accumulate in old leaves rather in new ones in rhizome crops.However,the concentration of Dufulin enantiomers in the root was very low,which was similar to the situation of Dufulin in cherry radish.The results of single factor analysis showed that when Dufulin enantiomers were applied through leaves,there was almost no chiral selectivity of Dufulin enantiomers in cherry radish and garlic plants（p>0.05）.（2）The rules of absorption,translocation,and directional accumulation of Dufulin enantiomers in root crops were clarified.No chiral selectivity was observed for the absorption,translocation,and directional accumulation of Dufulin enantiomers.The result of root absorption experiment indicated that Dufulin could be absorbed by plants from soil through their roots,but the absorbing ability for Dufulin enantiomers of roots was weak.In root crops,only 0.18%of S-Dufulin and 0.15%of R-Dufulin were absorbed by cherry radish,while more than 99%of parent compound and metabolites remained in the soil.The enrichment ability of the underground part of cherry radish was significantly stronger than that of its aboveground part.More than half of the Dufulin enantiomers remained in the lateral roots.The proportion of S-Dufulin and R-Dufulin in the succulent root gradually increased,especially after the succulent root expanded（42 days after application）,reaching a maximum of 46.13%and 41.67%,respectively.The absorption ability for Dufulin enantiomers of garlic plants from soil was also poor,with less than 1%of Dufulin enantiomers being absorbed by garlic 49 days after application.However,compared to the total absorption of cherry radish plants,garlic was more likely to absorb Dufulin enantiomers from soil.From 14 to 49 days after application,the proportion of S-Dufulin and R-Dufulin in the garlic root were not less than 70%,indicating that most of the Dufulin enantiomers accumulated in the root after entering the garlic,while only a small part being transported to the bulb and leaf tissues.The concentration of Dufulin enantiomers in various tissues of cherry radish decreased in the following order:LR>SL>SR,and Root>Leaves>Bulb in garlic plants.Both S-Dufulin and R-Dufulin had the highest concentrations at the root,indicating that Dufulin is not easily accumulated at the stem and leaves,but more likely accumulated at the root.There was almost no significant differences between the two Dufulin stereoisomers in the absorption,transportation,and accumulation processes in various plant tissues and organs（p>0.05）when Dufulin enantiomers were applied through the root.This only indicated that Dufulin enantiomers do not have chiral selectivity in these two plants,not for other plants.（3）Dufulin enantiomers were prone to transported to the vegetative organs of rhizomatous plants and remain in the root,making it difficult to transport from the roots to the upper ground.Based on the calculated transport concentration factors（TCFs）in the foliar application experiment,it was found that Dufulin had a stronger ability to move to the edible site in cherry radish than to move to other sites.The TCF of S-¹⁴C-Dufulin and R-¹⁴C-Dufulin decreased in the following order:SR>LBLL>LALL>LR 28 days after application.In garlic,the TCF of S-¹⁴C Dufulin and R-¹⁴C Dufulin in the bulb were the highest among all sampling times.The TCF of S-Dufulin and R-Dufulin in different tissues decreased in the following order:Bulb>LALL>LBLL>Root.This indicated that both S-Dufulin and R-Dufulin enantiomers had poor transport ability from labeled leaves to roots,and Dufulin enantiomers were more easily transported to plant organs that store nutrients,whether they were the succulent root of cherry radishes or the bulb of garlics.In the root application experiment,the ability of two crops to enrich Dufulin enantiomers was determined through bioconcentration factor（BCF）calculations.The BCF of cherry radish plants reached its maximum value on 21 days,with S-Dufulin being 0.3580±0.0558 and R-Dufulin being 0.3456±0.0558.In garlic,the BCF of both reached the maximum of 0.0699±0.0068 and 0.0664±0.0085,respectively,at the same time 35 days after application.This indicated that the ability of garlic to enrich Dufulin enantiomers in soil was worse than that of cherry radish in soil.At each sampling time,the BCF of S-Dufulin and R-Dufulin in the root（the succulent root and lateral roots）of cherry radish were significantly higher than that of the stem and leaf tissues.The BCF of the garlic root was also significantly higher than that of the garlics stem and leaf,which also confirmed that Dufulin enantiomers were more likely to accumulate in the root of rhizome crops,and the enrichment ability of the plant root was the strongest.The translocation factor（TF）describes the transportation capacity of Dufulin enantiomers in rhizome crops.In cherry radish,the TF of S-Dufulin and R-Dufulin reached their maximum of 0.0877±0.0142 and 0.0826±0.0095,respectively,at the last sampling time.In garlic,the TF of S-Dufulin and R-Dufulin increased after initial decrease.The TF of S-Dufulin ranged from 0.0706±0.0072 to 0.1692±0.0187,while the TF of R-Dufulin ranged from 0.0474±0.0073 to 0.1516±0.0056.This result indicated that the transportation capacity of Dufulin from the bottom to the top in rhizome plants was limited.Compared to cherry radishes,garlic had a stronger transportation capacity.（4）The residues and metabolites of Dufulin in rhizome crops were confirmed,and the metabolic pathway and mechanism of Dufulin in rhizome crops were clarified.In the metabolic experiment,the residual amount of Dufulin extracted from the stem and leaf of cherry radish was over 98%,and the residual rate of Dufulin extracted from the succulent root was over 87%.The extractable residues of garlic leaves was above 88%,while the extractable residues of bulb remains above 80%.Dufulin was more likely to form bound residues in garlic than in cherry radishes.In this paper,five metabolites of Dufulin were identified in rhizome crops.Among them,three metabolites were compared with reference substances by performing chromatography and their structures have been confirmed,including the conjugates of meta hydroxy Dufulin,glucose Dufulin,and conjugation of glucose malonic acid with hydroxy Dufulin.M1,M2,and M3,M4 were detected in the stem,leaves,and the succulent root of cherry radishes.In the leaves and the bulb of garlic plants,there were Ⅰ phases metabolite M2 and Ⅱ phases metabolites M3,M4,and M5.Unlike cherry radishes,there was no methyl hydroxylated hydroxy Dufulin M1 found in garlic,but there was an additional metabolite M5,which was the conjugate of disaccharide and hydroxy Dufulin.In the stem and leaf tissues and the succulent root of cherry radishes,only the proportion of the parent compound of Dufulin exceeded 10%and the concentration exceeded 0.01 mg/kg,meeting maximum residue amount setting.In garlic leaves,also only the parent compound met the setting.However,the proportion and concentration of the parent compound and four metabolites in garlic bulbs all exceeded 10%and 0.01mg/kg.Therefore,it is recommended to define the residue of Dufulin in cherry radish as the parent compound of Dufulin.Residues in garlic leaves are defined as the parent compound of Dufulin,while it is recommended that the four metabolites and the parent compound should all be included in the residue definition for garlic bulbs.Although it is recommended that the parent compound of Dufulin be included in the residue definition in cherry radish and garlic,its estimated daily intake（EDI）is far less than the acceptable daily intake（ADI）according to the dietary assessment risk assessment,and their hazard quotient（HQ）is also far less than 1.Therefore,in the edible parts of cherry radishes and garlics,the parent compound of Dufulin cannot pose a threat to humans.