Optimization of sulfation process conditions for dandelion root polysaccharides and their protective effect on oxidative damage of HepaRG cells
The activity of polysaccharides is usually related to the composition of sugar units, molecular weight, type of glycosidic bonds, main chain configuration, branch chains, and spatial configuration. Compared with the original polysaccharides, structurally modified polysaccharides have significantly improved water solubility, which is beneficial for enhancing polysaccharide activity, and some may even generate new biological activities. Phosphorylation, sulfation, carboxymethylation, and acetylation are commonly used methods for polysaccharide chemical modification. Among them, sulfated polysaccharides have attracted increasing attention from researchers due to their excellent biological activities, such as anticoagulant, anti-inflammatory, antioxidant, anti-tumor, and immune enhancement. The sulfation of polysaccharides refers to the reaction in which the hydroxyl groups on the polysaccharide molecular chain are replaced by sulfate groups, and the resulting product is also known as polysaccharide sulfate. The commonly used methods for polysaccharide sulfation include chlorosulfonic acid pyridine method, concentrated sulfuric acid method, Nagasawa method, etc. The chlorosulfonic acid pyridine method has the advantages of economical reaction reagents, simple reaction conditions, and high product recovery rate, and is therefore commonly used as a method for polysaccharide sulfation.
Dandelion root is the dried root of the Asteraceae plant dandelion, which has various pharmacological activities such as antioxidant, lipid-lowering, and liver protection. Research by Ma Hongmei et al. shows that polysaccharides are the most abundant active ingredient in dandelion roots, with a content of up to 42.75%. Our research group found that dandelion root polysaccharides have good in vitro antioxidant activity, which is reflected in their ability to scavenge DPPH radicals, hydroxyl radicals, and superoxide anion radicals, showing a certain dose effect relationship. However, we found in our research that the water solubility of dandelion root polysaccharides is not very good, which limits their ability to exert better pharmacological effects. Literature reports that sulfation of polysaccharides can improve their water solubility and enhance their biological activity. Therefore, the author considers sulfation modification of dandelion root polysaccharides. H2O2 is a common reactive oxygen species molecule that can induce cell apoptosis or necrosis by disrupting proteins and lipids, especially DNA in the genome and mitochondria. Due to its ability to effectively increase intracellular oxidative stress levels and simulate the damage caused by oxygen free radicals in vivo, H2O2 is often used as a model substance for establishing in vitro cellular oxidative stress damage. Therefore, this study intends to use H2O2 to establish a cellular oxidative stress damage model and investigate the protective effect of sulfated dandelion root polysaccharides on H2O2 induced cell damage.
In summary, this study used the chlorosulfonic acid pyridine method to modify dandelion root polysaccharides with sulfuric acid. The degree of substitution was used as an indicator to optimize the sulfuric acid esterification process using response surface methodology. The protective effects of dandelion root polysaccharides and their sulfates on H2O2 induced oxidative damage in HepaRG cells were preliminarily explored, with the aim of providing valuable references for the development and utilization of dandelion root polysaccharides.

This experiment used the chlorosulfonic acid pyridine method to modify dandelion root polysaccharides through sulfation, and optimized the sulfation process using response surface methodology. The optimal conditions for the sulfation reaction were obtained as a reaction temperature of 83 ℃, a pyridine to chlorosulfonic acid ratio of 8, and a reaction time of 3 hours. Under these conditions, the degree of substitution of sulfated dandelion root polysaccharides was 1.13. Dandelion root polysaccharides modified with sulfuric acid have a good protective effect on H2O2 induced oxidative damage cells. This study indicates that sulfation modification helps to enhance the antioxidant capacity of dandelion root polysaccharides, providing new ideas and directions for the further development and utilization of dandelion root polysaccharides.