Effect Of Sulfur Fumigation On The Quality Of TCMs Containing α,β-Unsaturated Carbonyl Compounds

Sulfur fumigation,as a traditional processing method of Traditional Chinese Medicinal Materials,can make it beautiful in color and conducive to storage.However,the influence of sulfur fumigation on the quality of Traditional Chinese Medicine(TCM)has been controversial.On the one hand,sulfur dioxide produced in sulfuring process is generally considered to be toxic substance.Although it is allowed to be legally added in food,excessive sulfur dioxide residue may bring potential health risks.On the other hand,sulfur fumigation may lead to chemical changes,which may lead to changes in effectiveness and safety.Sulfur dioxide residue in TCM has been limited in Chinese Pharmacopoeia,but researches about chemical transformation mechanism caused by sulfur fumigation are rare.Compounds containing α,βunsaturated carbonyl,electrophilic alkenyl,benzyl alcohol hydroxyl,hemiacetal hydroxyl,aldehyde group and glycosidic bond can transform into other compounds in sulfuring process.Electrophilic alkenyl,benzyl alcohol hydroxyl,hemiacetal hydroxyl,aldehyde group and glycosidic bond exist in compounds as the form of substituents.And α,β-unsaturated carbonyls can also serve as a part of mother nucleus of coumarins,quinones and chromogen ketones.Nucleophilic addition reaction can happen between α,β-unsaturated carbonyls and sulfinic acid.Sulfur fumigation may lead to disconnection of double bond in mother nucleus,which may induce far-reaching impact on the effectiveness and safety of TCM.On the other hand,compounds containing α,β-unsaturated carbonyl involve the most kinds of compounds,such as coumarins,quinones,chromogen ketones,et al.It covers most TCM such as medicine of Umbelliferae and Labiatae.But there is no systematic study on the effect of sulfur fumigation on TCM containing compounds with α,β-unsaturated carbonyls.Therefore,this study focuses on these questions.In this study,Angelicae Dahuricae Radix rich in coumarin,Salviae Miltiorrhizae Radix rich in naphthoquinone,phenylacrylic acid and Saposhnikoviae Radix rich in chromogen ketones were selected as representatives to study the effects of sulfur fumigation on chemical composition,efficacy and toxicity of TCM.The main contents and conclusions are shown as follows:1.Effect of sulfur fumigation on coumarins based on metabolomics,histochemistry,separation and purification methodAngelicae Dahuricae Radix was treated by sulfur fumigation,chemical components of Angelicae Dahuricae Radix before and after sulfured were analyzed by UPLC-PDA-Q-TOF-MS.Based on this,a rapid screening and identification method for chemical components of Radix Angelicae dahuricae was established.183 compounds were identified,including 32 furocoumarins,56 bifuranocoumarins,8 trifuranocoumarins,34 sulfur-containing coumarins,and 53 other compounds.PCA and OPLS-DA were used to screen and characterize components only exist in sulfured medicinal materials.Based on this,the chemical transformation mechanism in the process in sulfuring process was speculated.The results showed that a large number of sulfurcontaining compounds produced during sulfuring process.There were two main types of sulfurcontaining compounds,one was dihydrocoumarin sulfonic acid compounds with carbon-carbon double bond(between C-3 and C-4)broken,and the other was ring-opening dihydrocoumarin sulfonic acid compounds with lactone bond broken.DESI-MS was used to locate the chemical components in Angelica dahurica before and after sulfur fumigation.The results showed that coumarins were mainly located in the phloem of unsulfured Angelicae Dahuricae Radix,and dihydrocoumarin sulfonic acid was located in the phloem of sulfured Angelicae Dahuricae Radix,which also reflected that coumarins could be transformed into sulfur-containing compounds.Two purified sulfur-containing compounds were obtained by separation and purification for the first time,and the structures were determined by NMR,Mosher and ECD method,which were 3,4-dihydrobyakangelicin-4-sulfonic acid,(4R,12S)-3,4-dihydrooxypeucedanin hydrate4-sulfonic acid.2.Effect of sulfur fumigation on chromogenic ketones and quinones based on metabolomics,synthesis,separation and purification methodPLC-PDA-Q-TOF-MS,PCA and OPLS-DA were used to analyze chemical difference between sulfured and unsulfured Salviae Miltiorrhizae Radix and Saposhnikoviae Radix.The results showed that chromogen ketones,miltirone and phenylacrylic acid could react with sulfinic acid to form sulfur-containing compounds.One sulfur-containing compound in sulfured Saposhnikoviae Radix was obtained by synthesis,separation and purification for the first time,and its structure was determined by NMR.It was named 2,3-dihydroprim-Oglucosylcimifugin-2-sulfonic acid.3.Effect of sulfur fumigation on compounds containing α,β-unsaturated carbonyl based on synthesis methodCompounds containing α,β-unsaturated carbonyl(including coumarins,chromogenic ketones,flavonoids,phthalides and phenylacrylic acids with different structures)selected to be added into supersaturated sodium pyrosulfite solution at room temperature to simulate the chemical reaction during sulfuring process.The results showed that in addition to flavonoids,other α,β-unsaturated carbonyl compounds can react with sulfinic acid to form sulfurcontaining derivatives.It is suggested that it is universal that α,β-unsaturated carbonyl compounds can react with sulfinic acid,but when there are large conjugated groups(such as flavonoids and most tanshinones)connected to carbon-carbon double bonds of α,β-unsaturated carbonyls,the addition reaction is not easy to occur due to the influence of steric hindrance and conjugation effect.4.Effect of sulfur fumigation on the quality of Angelicae Dahuricae Radix was evaluated based on study of anti-inflammatory and cytotoxicityTaking Angelicae Dahuricae Radix as the research object,anti-inflammatory activity of(4R,12S)-3,4-dihydrooxypeucedanin hydrate-4-sulfonic acid,oxypeucedanin hydrate,water extraction of sulfured and unsulfured Angelicae Dahuricae Radix was evaluated using the antiinflammatory model of zebrafish induced by lipopolysaccharide.It resulted that there was no significant difference in anti-inflammatory activity between water extraction of sulfured and unsulfured Angelicae Dahuricae Radix,and there was no significant difference in the antiinflammatory activity between(4R,12S)-3,4-dihydrooxypeucedanin hydrate-4-sulfonic acid and oxypeucedanin hydrate.The cytotoxicity of(4R,12S)-3,4-dihydrooxypeucedanin hydrate-4-sulfonic acid,oxypeucedanin hydrate,water extraction of sulfured and unsulfured Angelicae Dahuricae Radix was evaluated were also evaluated using human liver L02 cells,human renal proximal convoluted tubular HK2 cells and rat adrenal pheochromocytoma PC 12 cells.The results showed that the cytotoxicity of(4R,12S)-3,4-dihydrooxypeucedanin hydrate-4-sulfonic acid was significantly lower than that of oxypeucedanin hydrate at the same concentration.However,there was no significant difference in cytotoxicity between water extraction of sulfured and unsulfured Angelicae Dahuricae Radix at the same concentration.It is suggested that sulfur fumigation has little effect on the quality of Angelicae Dahuricae Radix.At the same time,it was found that(4R,12S)-3,4-dihydrooxypeucedanin hydrate-4-sulfonic acid could be transformed into its prototype compound during heating,and the sulfur dioxide residue was significantly reduced.Reasonable decocting can partially offset the effect of sulfur fumigation on the quality of Angelicae Dahuricae Radix.In conclusion,this is the first study to discuss the effect of sulfur fumigation on the chemical composition of TCM was discussed from the perspective of functional groups from different levels.LC-MS analysis,separation and purification,structure identification,effectiveness and safety evaluation were used to explore the impact of sulfur fumigation on the quality of TCM.Through comprehensive analysis and evaluation,it showed that addition reaction between α,β-unsaturated carbonyl compounds and sulfinic acid were universal in sulfuring process,in addition to compounds with strong conjugated group connected to carboncarbon double bond of α,β-unsaturated carbonyl.Attention should be paid to such components.In addition,although sulfur fumigation can lead to the chemical transformation,it has no significant effect on their anti-inflammatory activity and cytotoxicity,suggesting that sulfur fumigation has limited impact on the quality of Angelicae Dahuricae Radix.The effects of sulfur fumigation on the quality of medicinal materials may be different due to different types of chemical components.So,further relevant research should be carried out to scientifically evaluated effects of sulfur fumigation on quality of TCM.