Identification of the effective constituents for anti-inflammatory activity of Ju-Zhi-Jiang-Tang, an ancient traditional Chinese medicine formula

• An approach was proposed to identify effective constituents in JZJT.
• Activity index was suggested to evaluate the contribution of every constituent.
• Combination of NMR with LC–MS was efficient to characterize TCM formulae.
• 108 constituents were identified, including eighteen potential novel compounds.
• Polymethoxy flavones were major active constituents for anti-inflammatory activity.

The anti-inflammatory constituents of Ju-Zhi-Jiang-Tang (JZJT), a formula used for thousands of years in China, were identified by LC–MS and pharmacological activity evaluation. In this study, the whole extract of formula was separated into multiple components to facilitate the analytical process. To characterize their contributions to pharmacological activity of formula, activity indexes of constituents were proposed and calculated for the first time, which integrated the chemical and pharmacological information of multiple components. Among the 151 constituents detected in JZJT by LC–Q-TOF-MS and LC–IT-MS, a total number of 108 constituents were identified unambiguously or tentatively, including eighteen potential novel compounds. And, the structures of some constituents were confirmed by NMR. According to their activity indexes, polymethoxy flavones were indicated as the major active constituents responsible for the anti-inflammatory activity of JZJT. To verify the feasibility of activity indexes in predicting the active constituents, nine compounds with positive and negative index values were selected to validate their anti-inflammatory activity in vitro. The results showed that two polymethoxy flavones with higher positive index values, i.e., nobiletin and tangeretin can significantly exert anti-inflammatory effects, while other compounds with negative values did not show any activity. In conclusion, our results indicated the proposed approach might be an efficient and rapid way to identify active constituents of TCM formulae.