A Preliminary Study On The Chemical Components, Identification And Toxicity Of Sulfur-fumigated Ophiopogon Japonicu

Sulfur-fumigation is a highly efficient and important traditional post-harvest handling process for foods,agriculture products,and Chinese herbal medicines(CHMs).However,sulfur-fumigated(SF)CHMs have recently emerged as a controversial topic because of the following issues:(1)the side effect of sulfur itself;(2)the superfluous residues in SO2 and detrimental heavy metals in CHMs;and(3)transformation of the bioactive compounds.Thus,it is of great importance to develop a rapid and sensitive approach to ascertain the sulfurfumigation state of a given medicinal herb for CHM quality control.Ophiopogonis Radix(Maidong in Chinese),is a widely used CHM.According to CHM theory,Ophiopogonis Radix nourishes the yin,promotes body fluid production,moistens the lung,eases the mind and clears away heart fire.Phytochemical studies have revealed various biologically active compounds,including steroidal saponins,homoisoflavonoids,and polysaccharides,which possess therapeutic effects against acute and chronic inflammation,diabetes,cardiovascular diseases,and other disorders.However,there was limited studies focused on SF Ophiopogonis Radix.Therefore,in the present study,a comprehensive study is investigated to clarify the influence of sulfur-fumigation on the secondary metabolites,discriminate SF from none-sulfur-fumigated(NSF)Ophiopogonis Radix,and the potential toxicity of SF Ophiopogonis Radix.The main contents and results were described as follows:1.A comprehensive qualitative study of SF Ophiopogonis RadixIn this part,SF Ophiopogonis Radix samples was prepared in our lab following the modified procedures similar to that performed by illicit wholesalers.An UHPLC-LTQ-Orbitrap mass spectrometer qualitative method was established for target identification of steroidal saponins,homoisoflavonoids,and their sulfur-containing derivatives.The diagnostic product ions(DPIs)and neutral loss fragments were speculated according to the summarized fragmentation behaviors of known compounds,which supplied tremendous help for the identification of secondary metabolites in Ophiopogonis Radix.Meanwhile,the sulfurcontaining derivative candidates were automatically assembled based on the potential reaction types using Metlab software,which were then screened according to the HR-MS1 data sets.And then,a novel strategy based on DPIs,NLFs,and HR-MS1 data sets were established to implement rapid identification of steroidal saponins.As a result,a total of 356 compounds were identified,including 39 homoisoflavonoids,31 homoisoflavonoids sulfur derivatives,191 steroidal saponins,and 95 steroidal saponins sulfur derivatives.This study clarified the transformation of secondary metabolites of Ophiopogonis Radix during sulfur fumigation process and the established strategy provided new insights for the fast identification of compounds in CHMs.2.Discrimination of SF and NSF Ophiopogonis RadixIn this part,a novel and efficient strategy to rapidly discriminate SF from NSF Ophiopogonis Radix based on the MOCA strategy was established.It was characterized by the multiple data-acquisition means including NIR,HPLC-DAD/ELSD,and LC-HR-MS coupled with various multivariate statistical analysis.Modified analytical conditions were employed to obtain satisfied data sets of 26 batches Ophiopogonis Radix(13 SF and 13 NSF).And then,PCA and PLS-DA were employed to construct satisfied model to discriminate SF samples from NSF samples.As a result,a rapid and accurate method was established based on the NIR metabonomics.The synergy interval partial least squares(SiPLS)with 3 intervals and twodimensional correlation spectrum(2D-COS)were employed to screen the potential wavebands(around 5 000 cm-1)which was in accordance with the reported S-H and S-OH perssad.The markers,screened based on LC-HR-MS metabonomics,fulfilled three different criteria:position in S plot(p>0.05 and p(corr)>0.3),VIP value(>1.5),and t test(p<0.05).And a total of 45 markers were identified,among which,38 were assigned to be sulfate/sulfite derivatives of the reported steroidal saponins,and 9 were steroidal saponins.In order to explore whether the 38 sulfate/sulfite containing markers was the contributing factor to the changes in the NIR spectrum,a reference standard of the main representative steroidal saponins chosen from the 45 identified markers,namely ophiopogonin D,and one batch Ophiopogonis Radix were sulfur-fumigated and analyzed using the same methods.The results indicated that the established MOCA strategy was reliable and credible in the discrimination of SF Ophiopogonis Radix which could further employed in other SF CHMs.3.The potential toxicity of SF Ophiopogonis RadixIn this part,in vitro and in vivo experiments were respectively conducted to explore the potential toxicity of SF Ophiopogonis Radix.First,high-content screening(HCS)technology based on HepG2 cells was carried out.Different groups,including negative drug,positive drug,Ophiopogonis Radix,and SF Ophiopogonis Radix,were set to obatain multi-parameters of cytotoxicity(cells number,nuclear intensity,GSH reduce level,ROS level,mitochondrial membrane potentia-MMP).As a result,all of the five targets were negative in Ophiopogonis Radix group,but the GSH reduce level and mitochondrial membrane potentia parameters were positive in SF Ophiopogonis Radix group.This result indicated that the Ophiopogonis Radix was at more risk after sulfured fumigated.In the UHPLC-Q-Exactive based metabonomics experiment,chemometrics was used to screen the potential discriminatory markers,and a total of 15 metabolites were identified.After MetPA analysis,Tryptophan metabolism,Valine,leucine and isoleucine degradation,Terpenoid backbone biosynthesis,Phenylalanine metabolism,and Phenylalanine,tyrosine and tryptophan biosynthesis were enriched and should be the possible metabolic pathways when compared the biological samples of Ophiopogonis Radix group and SF Ophiopogonis Radix group.On the same condition,Ophiopogonis Radix expressed no hepatotoxicity based on HCS analysis,whereas the SF Ophiopogonis Radix influenced the targets of MMP and GSH and expressed the hepatotoxicity on HepG2 cells,which were relating to energy metabolism.Similarly,amino acid metabolism caused the disorder of glycometabolism,lipid metabolism,which would lead to energy metabolism.