A practical protocol for comprehensive evaluation of sulfur-fumigation of Gastrodia Rhizoma using metabolome and health risk assessment analysis.

Gastrodia Rhizoma is one of the most heavily sulfur-fumigated edible and medical herbs in the marketplace. We developed a practical protocol using an ultra-performance liquid chromatography coupled with quadrupole time-of-flight-MSE (UPLC/QTOF-MSE)-based metabolome and health risk assessment model to identify characteristic sulfur-fumigated markers, dissect chemical transformation mechanisms, and control the quality of sulfur-fumigated Gastrodia Rhizoma. Two sulfur-containing p-hydroxybenzyl products, one sulfur-containing disaccharide, one glycolipid, and two phospholipids were selected and identified as markers based on multivariate statistical analysis. In particular, the sulfur-containing markers p-hydroxybenzyl hydrogen sulfite and trace p-mercaptobenzyl hydrogen sulfate were positively correlated with the active major phenolics. Moreover, a practical index the time of the minimum content was useful for evaluating the extent of the sulfur-fumigation under different weight ratios of the sulfur to herbal materials (1:20, 1:40, and 1:80). Ultimately, the 1:40 ratio within 1h of sulfur-fumigation was considered as safe and efficient for herb quality preservation under the maximum residue limit of 750mg/kg. This study shows that the practical protocol-based discriminated markers and practical limits can be applied to quality assurance of sulfur-fumigation and non-fumigation Gastrodia Rhizoma and other edible or medical materials.

The promoter of an antifungal protein gene from Gastrodia elata confers tissue -specific and fungus-inducible expression patterns and responds to both salicylic acid and jasmonic acid.

Gastrodia antifungal proteins (GAFPs) are a group of mannose-binding lectins purified from Gastrodia elata that show strong resistance against a wide spectrum of fungi. The GAFP-2 promoter was analyzed for its ability to control the expression of the reporter gene, beta-glucuronidase (GUS) in transgenic tobacco plants. The GUS assays revealed that the GAFP-2 promoter is expressed in a tissue-specific manner, which mainly expressed in the vascular cells. The highest GUS activity was observed in roots, followed by stems. GAFP-2-GUS expression was strongly induced by the fungus Trichoderma viride and by the plant stress regulators, salicylic acid and jasmonic acid in the stably transformed tobacco plants. The -537 region of the GAFP-2 promoter was sufficient for its tissue-specific and inducible expression of the promoter.