Bioinformatics-Guided Identification of Ethyl Acetate Extract of Citri Reticulatae Pericarpium as a Functional Food Ingredient with Anti-Inflammatory Potential

Abstract

Citri Reticulatae Pericarpium (CRP) is one of the most commonly used food supplements and folk medicines worldwide, and possesses cardiovascular, digestive, and respiratory protective effects partially through its antioxidant and anti-inflammatory functions. The unique aromatic flavor and mild side effects make CRP a promising candidate for the development of anti-inflammatory functional food. However, recent studies show that the crude alcoholic extract and some isolated compounds of CRP show compromised anti-inflammatory activity, which became the main factor hindering its further development. To identify the bioactive compounds with anti-inflammatory potential, and improve the anti-inflammatory effects of the extract, a bioinformatics-guided extraction protocol was employed in this study. The potential bioactive candidates were identified by combing network pharmacology analysis, molecular docking, principal components analysis, k-means clustering, and in vitro testing of reference compounds. Our results demonstrated that 66 compounds in CRP could be grouped into four clusters according to their docking score profile against 24 receptors, while the cluster containing flavonoids and phenols might possess a more promising anti-inflammatory function. In addition, in vitro anti-inflammatory tests of the seven reference compounds demonstrated that hesperitin, naringenin, and gardenin B, which were grouped into a cluster containing flavonoids and phenols, significantly decreased LPS-induced NO, TNF-α, and IL-6 production of macrophages. While the compounds outside of that cluster, such as neohesperidin, naringin, hesperidin, and sinensetin showed little effect on alleviating LPS-induced NO and proinflammatory cytokine production. Based on the chemical properties of selected compounds, ethyl acetate (EtOAc) was selected as the solvent for extraction, because of its promising solubility of flavonoids and phenols. Furthermore, the ethanol alcoholic extract was used as a reference. The chemical profiling of EtOAc and crude alcoholic extract by HPLC/MS/MS also demonstrated the decreased abundance of flavonoid glycosides in EtOAc extract but increased abundance of phenols, phenolic acid, and aglycones. In accordance with the prediction, the EtOAc extract of CRP, but not the crude alcoholic extract, significantly decreased the NO, IL-6, and TNF-α production. Taken together, the results suggested selective extraction of phenols and flavonoids rich extract was able to increase the anti-inflammatory potential of CRP partially because of the synergistic effects between flavonoids, phenols, and enriched polymethoxyflavones. Our study might pave the road for the development of ethyl acetate extract of CRP as a novel functional food with anti-inflammatory function.

Sinensetin Attenuated Macrophagic NLRP3 Inflammasomes Formation via SIRT1-NRF2 Signaling

“…To date, the effect of SNS on mediating immunocytes activities remains largely elusive. It is reported that flavonoids, but not SNS, significantly decreased LPS-induced inflammatory cytokines in macrophages via bioinformatics and in vitro approach, such as TNF-α and IL-6 production . However, others reported that SNS performed as an immunity enhancer counters immunosuppression via elevating transcription and secretion of IFN-γ, IL-2, and IL-6 .…”

“…It is reported that flavonoids, but not SNS, significantly decreased LPS-induced inflammatory cytokines in macro- phages via bioinformatics and in vitro approach, such as TNF-α and IL-6 production. 29 However, others reported that SNS performed as an immunity enhancer counters immunosuppression via elevating transcription and secretion of IFN-γ, IL-2, and IL-6. 30 To further confirm the role of SNS in regulating macrophagic activities in inflammatory injury, we performed in vitro and in vivo studies and then found that macrophage indeed largely infiltrated in LPS-induced injury sites, which accompanied with releasing massive pro-inflammatory cytokines, including TNF-α, IL-1β, and IL-6.…”

Comprehensive identifying flavonoids in Citri Reticulatae Pericarpium using a novel strategy based on precursor ions locked and targeted MS/MS analysis

“…And except for its medicinal value with abundant resources, Citrus reticulata Blanco is also an edible material with highly exploitable value. Modern studies correlated to its pharmacological actions, biological activities, and clinical applications have revealed that treatment of cardiovascular disease 1 , anti-oxidant 2 , anti-inflammatory 3 , anti-tumor 4 as well as reducing blood lipid are its important effects 5 . However, with the development of evidence-based medicine and/or evidence-based pharmacy, clarification of the active secondary metabolites in Citri Reticulatae Pericarpium is the fundamental science, which needs to be systematically addressed.…”

Simultaneous determination of twenty-nine active compounds in fuzhengjiedu granules by HPLC-QQQ-MS/MS

“…It is well known that the most serious obstacle in the natural products development and research is to establish the best quality control method by identifying the representative active chemical constituents in the preparations, especially for complex herbal preparations [ 10 ]. The main active components of these eight herbs in Fuzhengjiedu Granules are as follows: chlorogenic acid (S 1 ), neochlorogenic acid (S 2 ), cryptochlorogenic acid (S 3 ), caffeic acid (S 5 ), isochlorogenic acid C (S 10 ), and isochlorogenic acid A (S 12 ) in JYH, protocatechuic aldehyde (S 4 ) and syringic acid (S 6 ) in ZJC, liquiritin (S 7 ), isoliquiritin (S 8 ), neoisoliquiritin (S 14 ), neoliquiritin (S 16 ), liquiritigenin (S 18 ), isoliquiritigenin (S 24 ), and licochalcone A (S 28 ) in GC, naringin (S 9 ), hesperidin (S 11 ), naringenin (S 22 ), and hesperetin (S 25 ) in CP, benzoylmesaconine (S 13 ), benzoylaconine (S 15 ), benzoylhypaconine (S 17 ), mesaconitine (S 19 ), aconitine (S 20 ), and hypaconitine (S 21 ) in DFP, psoralen (S 23 ) and bergapten (S 26 ) in WZMT, 6-gingerol (S 27 ) in GJ, and pogostone (S 29 ) in GHX [ 7 , [11] , [12] , [13] , [14] , [15] , [16] , [17] , [18] ], respectively. Related literature and studies have shown that these twenty-nine compounds are the foremost active ingredients in each individual.…”