| In the field of traditional Chinese medicine(TCM),sulfur-fumigation has been widely employed to protect herbs from insects and molds,maintain their moisture,and promote appearance during post-harvest handling and storage,therefore used in the primary processing and storage.At present,the abuse of sulfur fumigation for TCM is extremely common,especially in the processing and storage of raw materials.On one hand,sulfur dioxide is a food additive allowed to be used at home and abroad.Usually,the substance is added to food as sulfite to exert effects of anti-corrosion,bleaching and anti-oxidation.On the other hand,excessive sulfur-fumigation could induce chemical transformations of active components in herbs,generate sulfur-containing derivatives,and even lead to pharmacological risks.It is urgent to formulate a feasible sulfur fumigation process for TCM to control pest and mildew,meanwhile minimize the residual hazards.Although standards of sulfur dioxide have been formulated worldwide,there is no uniform evaluation standard of sulphur dioxide in TCM considering of problems caused by sulfur fumigation and sulfur dioxide residues.Excessive sulfur dioxide in herbs could be detected even without sulfur fumigation according to some sulfur dioxide standards,for air contains sulfur dioxide,plant itself contains sulfur,and some chemical compositions in herbs are sulfur-contained(such as those in Gastrodia Rhizoma).Therefore,the rationality of current sulfur dioxide standard of TCM needs further verification.In addition,it is necessary to study and formulate the sulfur dioxide limit standards for TCM,based on the characteristics and usage habits of TCM.In addition,there is no stable and reliable markers for assessing the extent of sulfur fumigation.Therefore,the risk of sulfur dioxide in TCM was assessed in this paper.And based on this,systematical study of sulfur fumigation process and sulfur fumigation markers were carried out with Gastrodia Rhizoma(GR)and Achyranthis bidentatae Radix(AB).The results are as follows:1.Risk assessment of sulfur dioxide in TCM considering medication habitsAccording to the THQ risk assessment model proposed by the United States Environmental Protection Agency and daily allowable intake of sulfur dioxide(ADI =0.7 mg/(kg·d))in JECFA,taking into account of the exposure frequency and exposure times,the maximum exposure risk of sulfur dioxide was evaluated to calculate a reasonable standard limit value of sulfur dioxide.And the maximum residue limit of sulfur dioxide for TCM is calculated to be 750 mg/kg,which is consistent with the limit value of sulfur dioxide(500-1000mg/kg)of vegetable and fruit regulated by Codex Standards for Food Additives.In particular,the reference value is in line with the characteristics and medication habits of TCM,considering that TCM is not eaten daily like foods.2.Discussion on the effect of sulfur fumigation on the quality of GR based on metabolomics and sulfur fumigation technologyIn order to find a stable and reliable intrinsic indicator of sulfur control,UPLC-Q-TOF-MS based metabolomics and multivariate statistical analysis was applied to analyze 'the dififerential metabolites of GR and AB before and after sulfur fumigation.The results showed that six sulfur-fumigated markers were identified in GR,including two sulfur-containing phenolic acids(p-hydroxybenzyl hydrogen sulfite and p-mercaptobenzyl hydrogen sulfate),one sulfur-containing disaccharides(3-0-(6-0-sulfo-?-d-galactopyranosyl)-p-d-glucopyranose),one glycolipid(Gingerglycolipid B),and two phospholipids(Phosphatidylinositol(18:2/0:0)and Phosphatidylinositol(0:0/18:2)).Among them,?-hydroxybenzyl hydrogen sulfite was chosen as an important quality control indicator,with high abundance and good specificity in sulfur fumigated GR samples,to determine whether GR samples were sulfur fumigated or not.The p-hydroxybenzyl hydrogen sulfite in sulfur fumigated GR was chemically synthesized(SO2 reacted with ?-hydroxybenzyl alcohol),isolated and identified by NMR.And the monomer purity of this sulfur fumigation marker was more than 99%.At the same time,based on UPLC-MS/MS,a quantitative analysis method of the sulfur fumigation marker was established to detect the sulfur fumigation degree of GR.In order to further to verify the stability of the marker,an experiment was conducted from aspects of storage time and heat treatment.The results showed that the marker had good stability under long-term storage(0 to 8 months)and heating(at 50? 60?,70?and 80 ?)for 2 h,4 h,8 h,12 h and 24 h.In summary,the marker could be used as an important index to evaluate whether GR sample was sulfur fumigated and the sulfur fumigation degree.To further study the effect extent of sulfur fumigation on the quality of GR,an UPLC method was established for the simultaneous determination of eight active components(adenosine,gastrodin,4-hydroxybenzyl alcohol,p-hydroxybenzaldehyde,parishin A,parishin B,parishin C,and parishin E)in GR.On this basis,the effects of different sulfur fumigation processes,different storage times and decoction steps on chemical composition and sulfur dioxide residues of sulfur funigated GR were discussed respectively.The results showed that sulfur fumigation could significantly inhibit mold mildew.The effects of different sulfur dosages(the weight ratio of the sulfur to herbal material,1:20,1:40,1:80)and fumigation time(0-24 h)on the quality of GR were studied.And results showed that sulfur funigation for 1 hour with ratio of 1:40 or for less than 2 h with ratio of 1:80 just fumigated the surface of GR,and could ensure the limited value of 750 mg/kg for sulfur dioxide.So the best sulfur funigation process that can guarantee the quality of medicinal materials with a satisfactory anti-mold effect,meanwhile control the residual amount of sulfur dioxide,is fumigation for 1 h with 1:40 the ratio of sulfur to medicinal material.While results of study on storage time showed that residual amount of sulfur dioxide in GR decreased gradually in storage,and decreased more than 50%after 4 months of storage.In addition,boiling of sulfur fumigated GR led large amount of sulfur dioxide to spread with the water vapor into the atmosphere,and only a small amount of chemical composition and sulfur dioxide would be absorbed with the decoction.In this study,in vitro cell safety evaluations were performed for sulfur markers and water extracts of sulfur fumigated GR.In order to clarify whether the p-hydroxybenzyl hydrogen sulfite had cytotoxicity on human body,its effects of different doses on HK-2,L-02 and PC-12 activity were evaluated.The results showed that the component showed a good promotion for the growth of liver cells and kidney cells.According to the United States Pharmacopoeia toxicity classification,the safe dose of this component on PC-12 cell was obtained in the range of 0-1000 ?mol/L.Moreover,effects of water extract of sulfur fumigated GR and AB under the best sulfur fumigation process on the viability of these three kinds of cells were analyzed.The results showed that in the normal concentration range,compared with un-sulfur fumigated group,sulfur fumigated GR and AB extracts had little effect on the viability and cytotoxicity of HK-2,L-02 and PC-12 cells.3.Effect of sulfur fumigation on the quality of AB based on metabolomics and sulfur fumigation technologyAn UPLC-Q-TOF-MS based metabolomics and multivariate statistical analysis was applied to analyze the differential metabolites of GR and AB before and after sulfur fumigation.The results showed that three triterpene saponins(Betavulgaroside ??Betavulgaroside ??Betavulgaroside ?)and two amides(Feruloyl-4-O-methyldopamine and Moupinamide)were identified as characteristic markers in AB samples.However,the trace amounts of sulfur-containing ingredients in sulfur-fumigated AB samples were not ruled out.An UPLC-MS/MS method of simultaneous determination of five active components(ecdysone,betaine,oleanic acid,ferulic acid and 5-hydroxymethyl furfural)in AB was established.On this basis,the effects of different sulfur dosages(the weight ratio of the sulfur to herbal material,1:20,1:40,1:80)and fumigation time(0-24 h)on the quality of AB were studied.And results showed that sulfur fumigation for 2 h with ratio of 1:80 could not influenced the quality of AB.And sulfur fumigation for 1 hour with ratio of 1:40 could minimized the quality of AB and just ensure the limited value of 750 mg/kg for sulfur dioxide.In addition,effects of water extract of sulfur fumigated AB under the best sulfur fumigation process(fumigation for 1 h with 1:40 the ratio of sulfur to medicinal material)on the viability of these three kinds of cells(HK-2,L-02 and PC-12 cells)were analyzed.The results showed that in the normal concentration range,compared with un-sulfur fumigated group,sulfur fumigated AB extracts had little effect on the viability and cytotoxicity of HK-2,L-02 and PC-12 cells.Therefore,the chosen sulfur fumigation process can ensure the quality and safety of AB.Through the above researches,the risk assessment model of sulfur dioxide in TCM was established and the maximum theoretical limit of sulfur dioxide was obtained.The optimal sulfur fumigation process based on GR and AB was proposed,which was the safest and most effective to ensure the quality of medicinal materials.Moreover,a set of reasonable and feasible research program for sulfur fumigation quality control for TCM was developed,which could be used to quickly find the characteristic sulfur fumigation markers.And a safe and reliable quality control index of sulfur fumigated GR was obtained.This provides new ideas about the quality control and safety evaluation of other sulfur-fumigated TCM,and provides reference for the formulation of limit standard of sulfur dioxide in TCM. |
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