Ganpu tea, an emerging pu-erh compound tea, which is cofermented with the peel of Citrus reticulata “Chachi,” has been widely favored by Chinese consumers due to its potential health effects and distinct flavor and taste. So far, the influence of this cofermentation procedure on the chemical profile of pu-erh tea has barely been addressed yet. In this work, an ultra-high-performance liquid chromatography-Q Exactive Orbitrap mass spectrometry (UHPLC-QE Orbitrap MS)-based qualitative and quantitative method combined with multivariate analysis was conducted to comprehensively investigate the chemical changes in pu-erh tea after cofermented with Citrus peel. A total of 171 compounds were identified based on a three-level strategy, among which seven phenolic acids, 11 flavan-3-ols, and 27 flavonoids and flavonoid glycosides were identified from pu-erh tea for the first time. Eighty-nine main constituents were selected for further quantitative analysis using a validated method. Both the principal component analysis (PCA) of untargeted metabolomics and orthogonal partial least squares discriminant analysis (OPLS-DA) models of targeted components revealed the significant chemical profile disparity between the raw pu-erh tea and Ganpu tea. It showed that Citrus tea cofermentation process significantly decreased the total contents of phenolic acids, flavan-3-ols, and flavonoid aglycones, while most of the quercetin glycosides and myricetin glycosides as well as the vitexin were significantly increased. In addition, hesperidin, a flavonoid glycoside only existed in Citrus, was first found in pu-erh tea after cofermented with Citrus. This study clearly profiled the chemical composition and content changes of pu-erh tea after cofermented with Citrus peel, which revealed that Citrus tea cofermentation process further accelerated the fermentation of pu-erh tea and improved the unique flavor of tea.

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Foam cell formation is the hallmark of the development and progression of atherosclerosis. The aim of this study was to investigate the regulatory effects of three polymethoxyflavones (PMFs), namely, tangeretin (TAN), 5,6,7,3′,4′,5′-hexamethoxyflavone (HxMF), and 3,5,6,7,8,3′,4′-heptamethoxyflavone (HpMF) on macrophage-derived foam cell formation and to further explore the molecular mechanisms. The RAW264.7 macrophage-derived foam cell model was successfully induced by oxidized low-density lipoprotein (ox-LDL) (80 μg/ml). It showed that TAN, HxMF, and HpMF alleviated ox-LDL-induced NO release while also inhibiting the expression of IL-1β, IL-6, and TNF-α in RAW264.7 cells. Uptake of excess ox-LDL was inhibited by TAN, HxMF, and HpMF, resulting in the reduction of its foam cell formation. Moreover, TAN, HxMF, and HpMF promoted HDL-mediated cholesterol efflux. Western blot experiment showed that TAN, HxMF, and HpMF inhibited the expression of scavenger receptor class A type I (SRA1) and cluster of differentiation 36 (CD36), while upregulating peroxisome proliferator-activated receptor γ (PPARγ), liver X receptor α (LXRα), phospholipid ATP-binding cassette transporter G1 (ABCG1), and scavenger receptor class B type I (SRB1) expression. Together, our findings suggested that PMFs inhibited foam cell formation might inhibit lipid uptake via downregulating SRA1/CD36 expression and promote cholesterol efflux from foam cells via upregulating PPARγ/LXRα/ABCG1/SRB1 expression. This antiatherosclerotic activity is expected to provide new insights into the development of healthcare uses for PMFs.