| Liliaceae,as a traditional and commonly used Chinese medicine in China,has the characteristics of not easy to dry and easy to mildew during storage and transportation due to its high starch content.There are often reports of using sulfur fumigation.Moreover,Liliaceae is mostly used for both medicine and food,so people pay more attention to its quality and drug safety.A large number of studies have shown that sulfur fumigation often produces new compounds（hereinafter referred to as"sulfur fumigation markers"）,which affect the quality of traditional Chinese medicine and seriously threaten the safety of drug use.However,most of the current studies focus on the quality impact of a single medicinal material.At present,it is of great practical significance to carry out the quality evaluation of sulfur fumigated traditional Chinese medicine from the perspective of the same family or genus,explore the sulfur fumigated markers and their common transformation rules,and establish a stable and reliable quality evaluation system of sulfur fumigated traditional Chinese medicine to clarify how sulfur fumigation affects the quality of traditional Chinese medicine.Objective:In this study,9 common Liliaceae traditional Chinese medicines,Fritillaria thunbergii（Zhebeimu）,F.cirrhosa（Chuanbeimu）,F.ussuriensis（Pingbeimu）,F.hupehensis（Hubeibeimu）,F.pallidiflora（Yibeimu）,Lilium lancifolium（Baihe）,Ophiopogon japonicus（Maidong）,Anemarrhena asphodeloides（Zhimu）,and Smilax glabra（Tufuling）were used as the representatives of Liliaceae traditional Chinese medicine to analyze the changes of their chemical composition before and after sulfur fumigation,to identify the differential components,to explore the common or characteristic sulfur fumigation markers,and to investigate their safety in order to elucidate the mechanism of sulfur fumigation on the quality.It is expected to clarify the mechanism of sulfur fumigation affecting the quality and safety of Liliaceae traditional Chinese medicines,and provide basis for market supervision and quality control.Methods:1.UPLC-QTOF-MS/MS and data analysis software were used to analyze the different components of 9 Liliaceae traditional Chinese medicines before and after sulfur fumigation.Masslynx mass spectrometry software was used to mine common or characteristic sulfur fumigation markers.Through literature review and comparison with standard products,the decomposition and transformation rules of sulfur fumigation markers were summarized;2.Using extensive targeted metabolomics to determine the chemical components of 9 Liliaceae traditional Chinese medicines before and after sulfur fumigation,and analyzing the sulfur free difference components before and after sulfur fumigation by PCA,OPLS-DA and other multivariate statistical methods;3.Structural verification and identification of sulfur fumigation markers using standard reaction and nuclear magnetic resonance technology;4.In vitro cytotoxicity test and zebrafish model test were used to evaluate the safety of the decoction and common sulfur fumigation markers of Liliaceae traditional Chinese medicines;5.According to the pharmacopoeia method and UPLC-MS method,the sulfur dioxide residue of 123 batches of Liliaceae traditional Chinese medicines and the sulfur fumigation markers of Liliaceae were detected respectively on the market,to verify the feasibility of judging whether they were sulfur fumigated through the detection of the sulfur fumigation markers of Liliaceae,and to serve market supervision.Results:1.Based on UPLC-QTOF-MS/MS metabolomics data analysis,20 sulfur fumigation markers were mined from 5 Beimu,including 1 common sulfur fumigation marker,which was mainly divided into two categories:isosteroidal alkaloids and octadecenoic acids;10 phenylacrylic acids and furostanol saponins sulfur fumigation markers in Baihe,6 furostanol saponins sulfur fumigation markers in Maidong and Zhimu,and 2 flavonoid glycosides sulfur fumigation markers in Tufuling were analyzed.Based on this,the transformation rules of 9 Liliaceae traditional Chinese medicines during sulfur fumigation were summarized,that is,isosteroidal alkaloids and phenylacrylic acids can react with sulfite by addition,and octadecanoic acids,flavonoid glycosides and furostanol saponins can react with sulfite by esterification to produce corresponding sulfur-containing derivatives.2.Through extensive targeted metabolomics analysis,743 different compounds were selected from 1061 Liliaceae compounds,of which 312 were increased and 431were decreased after sulfur fumigation.The relatively large changes in relative content were mainly alkaloids,flavonoids,terpenes,and amino acids.Most of the compounds increased in content were primary metabolites such as amino acids,and most of the compounds decreased in content were secondary metabolites such as alkaloids and flavonoids.Further explore the sulfur-free common markers of Liliaceae traditional Chinese medicines,analyze and obtain 5 different compounds before and after sulfur fumigation of 5 Beimu,including Vasicine,4-methyl-5-thiazoleethanol,Deethylatrazine,Guanosine 3’,5’-cyclic monophosphate and diallyl disulfide,and 1 different compound before and after sulfur fumigation of 9 Liliaceae traditional Chinese medicines,which is4-methyl-5-thiazoleethanol.3.The Structural verification results show that some of the products from the reaction of Peimisine,Regaloside A,and Regaloside B with sodium pyrosulfite exist in their corresponding sulfur fumigated samples.The partial sulfonation products of Peimisine were mainly isolated and prepared,and a monomer of 100 mg was obtained.At the same time,its structure was identified by nuclear magnetic resonance technology and was named Pemisine sulfonic.4.The results of cytotoxicity showed that compared with the normal control group,the aqueous decoction of 9 Liliaceae traditional Chinese medicines before and after sulfur fumigation could inhibit the growth of L-02,HK-2 and PC-12 cells in varying degrees,but there was no significant difference at the same dose.The results of acute toxicity of zebrafish model showed that cardiovascular toxicity of Pemisine was greater than that of Pemisine sulfonic at doses of 10 and 20 ng/tail.At the concentrations of 625,1250 and2500 mg·L-1,the aqueous decoction of Zhebeimu and the sulfur fumigated Zhebeimu had certain toxicity to the liver,but there was no significant difference.Anti-inflammatory results showed that compared with the model control group,Pemisine,Pemisine sulfonic,Zhebeimu aqueous decoction,and sulfur fumigated Zhebeimu aqueous decoction all had anti-pneumonia effects.At the same concentration,the anti-pneumonia effect of Pemisine is better than that of Pemisine sulfonic.At the same low concentration,the anti-pneumonia effect of sulfur fumigated Zhebeimu aqueous decoction is better than that of Zhebeimu aqueous decoction.With the increase of concentration,the anti-pneumonia effect of the two is equivalent.5.The Pemisine sulfonic（m/z 508.27）was used as the discriminating ion of Beimu,and the Dihydrohydrate Regaloside A sulfonic（m/z 497.09）,Dihydrohydrate Regaloside B sulfonic（m/z 539.10）,Regaloside A disulfonic（m/z 561.06）,Regaloside B disulfonic（m/z 603.06）were used as the discriminating ion of Baihe,and the Ophiopogonin B（m/z 981.43）and Ophiofuroside K（m/z 1127.49）were used as the discriminating ions of Maidong,and the ion chromatograms was extracted.The results showed that among 89 batches of Fritillaria,8 batches of Zhebeimu,5batches of Pingbeimu,4 batches of Chuanbeimu,5 batches of Hubeibeimu,and 4 batches of Yibeimu detected common sulfur fumigation markers,the detection rate is 29.21%.Among 19 batches of Baihe,5 batches detected sulfur fumigation markers,the detection rate is 26.32%.And 15 batches of Maidong did not detect sulfur fumigation markers.At the same time,compared with the detection results of sulfur dioxide residue,it was found that sulfur fumigation markers as internal quality control indicators could reflect the sulfur fumigation situation of traditional Chinese medicines more quickly and accurately.Based on the Pemisine sulfonic,the rapid detection of sulfur-fumigated Beimu can be realized to achieve the goal of"one standard and multiple evaluation".Conclusion:In this study,the quality evaluation of sulfur fumigated traditional Chinese medicines was carried out for the first time from the perspective of the same family and genus.The common and characteristic sulfur fumigation markers of 9Liliaceae traditional Chinese medicines were excavated,and their decomposition and transformation rules were summarized.The composition and types of different compounds before and after sulfur fumigation of 9 Liliaceae traditional Chinese medicines were systematically characterized,filling the research gap of the influence of sulfur fumigation on isosteroidal alkaloids,phenylacrylic acids and octadecanoic acids.At the same time,a common sulfur fumigation marker in Beimu was obtained,which realized the rapid identification of sulfur fumigation of traditional Chinese medicines in the market.From the perspective of safety evaluation,combined with the actual drug use characteristics and habits,proper sulfur fumigation（referring to the sulfur dioxide residue limit in ISO 22590:2010）will not cause safety problems for Liliaceae traditional Chinese medicines.This study will provide demonstration and technical support for the study of sulfur fumigation of other traditional Chinese medicines with similar chemical components.At the same time,the quality and safety evaluation of traditional Chinese medicines based on the common sulfur fumigation markers can provide guarantee for the realization of controllable quality,safety and effectiveness of traditional Chinese medicines. |
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