Multiomics Analyses of Citrus aurantium L. Var. Amara and Ginger Reveals Lipid Metabolism, Bile Acid Biosynthesis, and Gut Microbiome Rebalance Supporting Their Anti‐Obesity Effects

Both the flower of Citrus aurantium L. var. amara (CAVA) and rhizome of Zingiber officinale Roscoe (ginger) are food and medicinal homologous plants that have been used in China for aiding gastric digestion and preventing obesity. However, the combinatorial use of the two plants on obesity remains elusive. Our endeavor aimed to identify the optimal synergistic ratio between CAVA and ginger and to explore the underlying mechanism of their anti-obesity effects. Aqueous CAVA and ginger extracts were prepared separately and then combined into nine different ratios. The constituents of CAVA and ginger were unambiguously characterized by employing LC-MS. High-fat diet (HFD)-induced obese C57BL/6J mice were established and then administered with the nine combinations of CAVA-G extracts for 6 weeks. The trajectory of mice's body weights was analyzed. Besides, hematoxylin and eosin (HE) staining of the liver and oil red O staining of adipose tissue were performed. ELISA assay was employed to measure serum levels of total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C). Moreover, serum metabolic profiling was conducted through UPLC-Q-TOF/MS analysis. Gut microbiota analysis was performed via 16S rRNA gene sequencing. Pattern recognition and Pearson correlation analysis were used to pinpoint the key endogenous metabolites and microbiota. Two groups of CAVA-G combination treatment (C3 and A1) significantly prevented the increase of weight in mice. According to our analysis, the best anti-obesity effect was achieved when the ratio between CAVA and ginger was 37:63. The levels of TC and LDL-C were dramatically decreased in the C3 group, whereas the level of TG was significantly reduced in the A1 group. Interestingly, HDL-C level was increased dramatically in the C3 group. Compared with the model group, a total of 16 and 25 biomarkers were identified for groups C3 and A1, respectively. These biomarkers are mainly implicated in lipid metabolism and primary bile acid biosynthesis. Interestingly, the abnormal diversity of gut microbiota was induced by HFD feeding. Treatment with C3 or A1 significantly increased the relative abundance of Akkermansia and Novosphingobium, while reducing the relative abundance of Dorea, Bacteroides and Roseburia. Of note, this is the first report that Novosphingobium is involved in preventing obesity. These findings will layer a foundation for the usage of CAVA-G for preventing obesity.