变叶海棠叶总黄酮的分离纯化及体外抗炎活性研究

Separation and Purification of Flavonoids in Malus toringoides (Rehd.) Hughes Leaves with Macroporous Resins and Evaluation of Anti-Inflammation Activities in Vitro

摘要:

本研究通过吸附和解吸附实验，考察并优化了变叶海棠（Malus toringoides (Rehd.) Hughes）叶中总黄酮的纯化工艺及工艺参数。通过比较12种不同极性的大孔吸附树脂对变叶海棠叶总黄酮的吸附、解吸附能力，最终选取AB-8树脂进行实验。结果表明，最优静态吸附、解吸附工艺参数为：pH=4 条件下静态吸附3 h; 70%乙醇（pH=7）静态解吸附3 h；最优动态吸附、解吸附工艺参数：以2BV h-1的流速上样9BV（柱体积）样品溶液，随后在2BV h-1流速条件下，分别以6BV水、6BV 20%乙醇、6BV 40%乙醇、6BV 60%乙醇、6BV 80%乙醇对上样柱进行洗脱，在此条件下分离纯化，变叶海棠叶总黄酮纯度由32.79%提高到59.18%。经红外光谱扫描，光谱具备黄酮类物质特征官能团，证明黄酮类物质的存在。此外，对精制后的变叶海棠叶总黄酮进行体外抗炎作用研究，在50-400 μg mL-1浓度条件下，变叶海棠叶总黄酮对脂多糖（lipopolysaccharide, LPS）诱导巨噬细胞炎症反应有明显的抑制作用，可能是通过抑制与炎症相关的基因表达来抑制NO、IL-6、IL-1β、TNF-α的分泌。

Abstract:

The purpose of this research was to study the purification process of flavonoids in Malus toringoides (Rehd.) Hughes leaves (FMTs), and the purification parameters were optimised by adsorption and desorption tests. AB-8 resin was selected by comparing the adsorption and desorption ability of FMTs on twelve types of resins. The results showed that the optimal purification parameters of static adsorption and desorption were the sample at pH=4 with 3 h of adsorption; the desorption solvent was 70 % ethanol (pH = 7.0) with 3 h of desorption. The best dynamic parameters were a sample loading amount of 9 bed volumes (BV) at a feeding flow rate of 2 BV h-1 and washing the column with 6 BV of water, followed by subsequent flushing with 20 % (6 BV), 40 % (6 BV), 60 % (6 BV) and 80 % (6 BV) ethanol in water (v/v) at an elution flow rate of 2 BV h-1. Under these conditions, the purity of FMTs increased from 32.79% to 59.18%. The existence of flavonoids was confirmed by their characteristic functional groups that were identified by infrared spectroscopy. Meanwhile, FMTs in the dosage range of 50 to 400 ?g mL-1 showed a significant anti-inflammatory effect on lipopolysaccharide (LPS)-induced RAW 264.7 mouse macrophage cells, which could inhibit the secretion of NO, IL-6, IL-1β and TNF-α through the inhibition of inflammatory-related gene expression.