Mu Huai-Xue, Lin Cheng-Yuan, Huang Lin-Fang, Yang Da-Jian, Lu Ai-Ping, Han Quan-Bin, Bian Zhao-Xiang
School of Chinese Medicine, Hong Kong Baptist University, Kowloon Tong, Hong Kong, China.
Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences, Beijing, 100193 China.
Chin Med. 2016 Feb 13;11:5. doi: 10.1186/s13020-016-0077-x. eCollection 2016.
This study aims to identify the major anti-inflammatory components in the petroleum ether extract of Bupleurum malconense (Chaihu), by bioassay-guided fractionation, and to investigate the anti-inflammatory mechanisms of active components in lipopolysaccharide (LPS)-stimulated murine macrophage RAW-Blue cells.
A QUANTI-Blue assay was used to guide fractionation of B. malconense root extract. The petroleum ether extract which exerted significant secreted embryonic alkaline phosphatase (SEAP) inhibition effect was purified by silica gel column chromatography and assisted with reverse phase HPLC. The major bioactive compound which significantly inhibited SEAP activity was obtained and its anti-inflammatory effects in LPS-induced RAW-Blue cells were measured by the overproduction of NO (Griess method), gene expression of Il-1β, Tnf-α and iNos (real-time PCR). In parallel, protein expressions of COX-2, iNOS and IκB-α were determined by western blot.
In bioassay-guided fractionation using LPS-stimulated mouse macrophage RAW-Blue cells, (+)-3'-angeloxyloxy-4'-keto-3',4'-dihydroseselin (Pd-Ib) was identified by MS and NMR spectral analyses. Pd-Ib (5, 10, 20 μg/mL) suppressed the gene expression of Il-1β (P < 0.0001, P < 0.0001, P < 0.0001 for three respective concentrations), Tnf-α (P = 0.006, P = 0.001, P < 0.0001 for three respective concentrations) and iNos (P = 0.009, P < 0.0001, P < 0.0001 for three respective concentrations) in LPS-stimulated macrophages. The production of cyclooxygenase-2 (P = 0.019, P = 0.002, P < 0.0001), iNOS (P < 0.0001, P < 0.0001, P < 0.0001 for three respective concentrations) and NO (P < 0.0001, P < 0.0001, P < 0.0001 for three respective concentrations) significantly decreased when macrophages were treated with Pd-Ib (5, 10, 20 μg/mL) in the presence of LPS. Pd-Ib (5, 10, 20 μg/mL) suppressed the nuclear activation of NF-κB while it up-regulated the IκB-α level (P = 0.028, P = 0.013, P = 0.005 for three respective concentrations) in LPS-stimulated macrophages.
Pd-Ib isolated from B. malconense suppressed LPS-induced inflammatory responses in macrophages by inhibiting NF-κB activity and reducing the expression of iNOS, COX-2 as well as pro-inflammatory cytokines.
本研究旨在通过生物活性追踪分离法鉴定滇柴胡石油醚提取物中的主要抗炎成分，并研究活性成分在脂多糖（LPS）刺激的小鼠巨噬细胞RAW-Blue细胞中的抗炎机制。
采用QUANTI-Blue检测法指导滇柴胡根提取物的分离。对具有显著分泌型胚胎碱性磷酸酶（SEAP）抑制作用的石油醚提取物进行硅胶柱色谱纯化，并辅以反相高效液相色谱。获得了显著抑制SEAP活性的主要生物活性化合物，并通过检测一氧化氮（NO）过量产生（Griess法）、白细胞介素-1β（Il-1β）、肿瘤坏死因子-α（Tnf-α）和诱导型一氧化氮合酶（iNos）基因表达（实时定量PCR）来测定其在LPS诱导的RAW-Blue细胞中的抗炎作用。同时，通过蛋白质免疫印迹法检测环氧化酶-2（COX-2）、诱导型一氧化氮合酶（iNOS）和核因子κB抑制蛋白α（IκB-α）的蛋白表达。
在使用LPS刺激的小鼠巨噬细胞RAW-Blue细胞进行生物活性追踪分离时，通过质谱和核磁共振光谱分析鉴定出（+）-3'-当归酰氧基-4'-酮基-3',4'-二氢邪蒿素（Pd-Ib）。Pd-Ib（5、10、20μg/mL）可抑制LPS刺激的巨噬细胞中Il-1β（三种浓度下P分别为<0.0001、<0.0001、<0.0001）、Tnf-α（三种浓度下P分别为0.006、0.001、<0.0001）和iNos（三种浓度下P分别为0.009、<0.0001、<0.0001）的基因表达。当巨噬细胞在LPS存在下用Pd-Ib（5、10、20μg/mL）处理时，环氧化酶-2（三种浓度下P分别为0.019、0.002、<0.0001）、iNOS（三种浓度下P均为<0.0001）和NO（三种浓度下P均为<0.0001）的产生显著降低。Pd-Ib（5、10、20μg/mL）可抑制LPS刺激的巨噬细胞中NF-κB的核激活，同时上调IκB-α水平（三种浓度下P分别为0.028、0.013、0.005）。
从滇柴胡中分离得到的Pd-Ib通过抑制NF-κB活性以及降低iNOS、COX-2和促炎细胞因子的表达，抑制LPS诱导的巨噬细胞炎症反应。
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