茯苓皮水溶性三萜的制备、结构鉴定及其抗炎活性研究

Preparation, structural identification and anti-inflammatory activity of water-soluble triterpenoids from Poriae cutis

中文关键词:  茯苓  茯苓皮  三萜  抗炎  成盐  水溶性  茯苓酸A钠

英文关键词:Poria cocos  Poriae cutis  triterpenoid  anti-inflammatory  salt formation  solubility enhancement  sodium poricoic acid A

中文摘要:

      目的 制备茯苓皮水溶性三萜, 进行结构鉴定并探究其体外抗炎活性。方法 采用成盐法制备茯苓皮水溶性三萜, 通过制备型高效液相色谱法(preparative high performance liquid chromatography, Prep-HPLC)对水溶性三萜进行分离纯化, 超高效液相色谱-四极杆飞行时间质谱法(ultra performance liquid chromatography-quadrupole-time-of-flight mass spectrometry, UPLC-QTOF-MS/MS)对三萜进行结构鉴定, 采用脂多糖(lipopolysaccharide, LPS)刺激小鼠巨噬细胞RAW 264.7建立炎症模型, 研究茯苓皮水溶性三萜体外抗炎活性。结果 将茯苓皮水溶性三萜制备成钠盐后, 其溶解度由(0.89±0.02) mg/mL提高到(18.12±0.05) mg/mL。通过Prep-HPLC对茯苓皮水溶性三萜进行分离纯化, 得到4个峰, 通过UPLC-QTOF-MS/MS对三萜进行鉴定, 鉴定出茯苓酸A钠和茯苓酸D钠。茯苓皮水溶性三萜和茯苓酸A钠可抑制LPS诱导的RAW 264.7细胞分泌一氧化氮(nitric oxide, NO), 同时可降低细胞因子如肿瘤坏死因子-α (tumor necrosis factor-alpha, TNF-α)、白细胞介素-1β (interleukin-1 beta, IL-1β)和白细胞介素-6 (interleukin-6, IL-6)的产生, 具有较高的抗炎活性, 而茯苓皮三萜和茯苓酸D钠没有抗炎活性。结论 茯苓皮水溶性三萜在水中的溶解度是茯苓皮三萜的20.36倍, 且具有良好的抗炎活性。本研究提供了一种通过成盐反应提高茯苓皮三萜水溶性和抗炎活性的方法。

英文摘要:

      Objective To prepare the water-soluble triterpenoids from Poriae cutis, conduct structural identification and explore their the anti-inflammatory activity in vitro. Methods The water-soluble triterpenoids from Poriae cutis were prepared by salt formation method, separation and purification of water-soluble triterpenoids from Poriae cutis were separated by preparative high performance liquid chromatography (Prep-HPLC), structural identification of the triterpenoids was performed by ultra performance liquid chromatography- quadrupole-time-of-flight mass spectrometry (UPLC-QTOF-MS/MS), and an inflammatory model was established by stimulating mouse macrophages RAW 264.7 with lipopolysaccharide (LPS) to investigate the in vitro anti-inflammatory activity of water-soluble triterpenoids from Poriae cutis. Results After preparing water-soluble triterpenoids from Poriae cutis into sodium salts, the solubility increased from (0.89±0.02) mg/mL to (18.12±0.05) mg/mL. Four peaks were obtained by separating and purifying water-soluble triterpenoids from Poriae cutis by Prep-HPLC. The triterpenoids were identified by UPLC-QTOF-MS/MS, and sodium poricoic acid A and sodium poricoic acid D were identified. The water-soluble triterpenoids from Poriae cutis and sodium poricoic acid A could inhibit LPS induced secretion of nitric oxide (NO) in RAW 264.7 cells, while reducing the production of cytokines such as tumor necrosis factor alpha (TNF-α), interleukin-1 beta (IL-1β), and interleukin-6 (IL-6), exhibiting high anti-inflammatory activity. However, Poriae cutis triterpenoids and sodium poricoic acid D had no anti-inflammatory activity. Conclusion The solubility of water-soluble triterpenoids is about 20.36 times higher than that of triterpenoids from Poriae cutis and has good anti-inflammatory activity. This study provides a method to improve the water solubility and anti-inflammatory activity of water-soluble triterpenoids from Poriae cutis through salt formation reaction.