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UPLC-PDA-QTOF/MS 分析虎杖根及根茎的抗炎成分及其对脂多糖激活的 RAW264.7 巨噬细胞的作用。

Anti-inflammatory constituents in the root and rhizome of Polygonum cuspidatum by UPLC-PDA-QTOF/MS and lipopolysaccharide-activated RAW264.7 macrophages.

机构信息

The Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, 100193, China.

Tianjin University of Traditional Chinese Medicine, Tianjin, 300193, China.

出版信息

J Pharm Biomed Anal. 2021 Feb 20;195:113839. doi: 10.1016/j.jpba.2020.113839. Epub 2020 Dec 26.

Abstract

The root and rhizome of Polygonum cuspidatum (Hu-Zhang) has been used for treatment of various inflammatory disorders in China. In our pervious study, we found that three fractions (HZE-30, HZE-60 and HZE-95) from the ethanol extract of Hu-Zhang (HZE) all could inhibit NO production, and HZE-60 shows the most potent anti-inflammatory activity. In order to understand the major contribution constituents of Hu-Zhang responsible for its anti-inflammatory effect, quantitative composition-activity relationship method was performed. Firstly, the constituents in HZE-60 were characterized using an ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS) approach. Second, quantitative analyzed five major constituents identified in HZE-60 and compare the difference of five major constituents in HZE and three anti-inflammatory activity fractions. Finally, evaluated the anti-inflammatory effects of major constituents in lipopolysaccharide (LPS)-activated RAW264.7 macrophages. The results showed that a total of 31 compounds were identified from HZE-60, including 12 anthraquinones, 7 diphenylethenes, 9 phenols and 3 others. The contents of five major constituents (polydatin (6), resveratrol (7), emodin-1-O-β-d-glucoside (15), emodin-8-O-β-d-glucoside (21) and emodin (31)) were simultaneously determined by UPLC-PDA with good linearity (correlation coefficients > 0.9990) and satisfactory repeatability (RSD < 0.99 %), precision (RSD < 0.01 %), stability (RSD < 0.67 %) and recoveries (99.52 %-101.23 %, RSD < 0.91 %). All five major constituents could be detected in HZE and HZE-60 fraction, but only 6 was detected in HZE-30, and 31 in HZE-95. Moreover, 7, 15 and 21 exhibited significant anti-inflammatory activity via suppressing supernatant pro-inflammatory mediators, such as NO, tumor Necrosis Factor-α (TNF-α), interleukin-6 (IL-6) and monocyte chemotactic protein-1 (MCP-1). Therefore, we conclude that the bioactivity of HZE is the syngeneic effect of its constituents, and 7, 15 and 21 should make great contributions for the anti-inflammatory effect of Hu-Zhang. The findings define the anti-inflammatory chemical constituents of Hu-Zhang, which will benefit further investigation on its quality control and the mechanism of action.

摘要

虎杖的根和根茎在中国一直被用于治疗各种炎症性疾病。在我们之前的研究中,我们发现虎杖乙醇提取物的三个馏分(HZE-30、HZE-60 和 HZE-95)都可以抑制 NO 的产生,其中 HZE-60 显示出最强的抗炎活性。为了了解虎杖中负责抗炎作用的主要成分,我们采用了定量构效关系方法。首先,采用超高效液相色谱-四级杆飞行时间质谱联用(UPLC-Q-TOF/MS)方法对 HZE-60 中的成分进行了表征。其次,对 HZE-60 中鉴定的 5 种主要成分进行定量分析,并比较了 HZE 和 3 种抗炎活性馏分中 5 种主要成分的差异。最后,评估了主要成分在脂多糖(LPS)激活的 RAW264.7 巨噬细胞中的抗炎作用。结果表明,从 HZE-60 中鉴定出 31 种化合物,包括 12 种蒽醌类、7 种二苯乙烯类、9 种酚类和 3 种其他类化合物。通过 UPLC-PDA 同时测定了 5 种主要成分(虎杖苷(6)、白藜芦醇(7)、大黄素-1-O-β-D-葡萄糖苷(15)、大黄素-8-O-β-D-葡萄糖苷(21)和大黄素(31))的含量,具有良好的线性(相关系数>0.9990)和令人满意的重复性(RSD<0.99%)、精密度(RSD<0.01%)、稳定性(RSD<0.67%)和回收率(99.52%-101.23%,RSD<0.91%)。所有 5 种主要成分均能在 HZE 和 HZE-60 馏分中检测到,但在 HZE-30 中仅能检测到 6,在 HZE-95 中仅能检测到 31。此外,7、15 和 21 通过抑制上清液中促炎介质(如 NO、肿瘤坏死因子-α(TNF-α)、白细胞介素-6(IL-6)和单核细胞趋化蛋白-1(MCP-1))的产生而表现出显著的抗炎活性。因此,我们得出结论,HZE 的生物活性是其成分的协同作用,7、15 和 21 应该对 HZE 的抗炎作用做出巨大贡献。这些发现确定了虎杖的抗炎化学成分,这将有利于其质量控制和作用机制的进一步研究。

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