Management of Citrus sinensis peels for protection and treatment against gastric ulcer induced by ethanol in rats.

Introduction: Stomach ulcer is one of the most prevalent disorders worldwide. The study was aimed to isolate and characterize the major polymethoxylated flavonoids in Citrus sinensis peels petroleum ether extract and investigate its protective and curative effect on gastric ulcer.Material and methods: Some spectral analyses were used for identification of the isolated compounds from the petroleum ether extract of Citrus sinensis peels. One oral dose (0.5 ml/100 g b.wt.) of absolute ethanol was orally given to rats after starvation for 24 h to induce gastric ulcer. To explore the protective and curative role of the plant extract, it was orally (250 mg/kg b.wt.) given for 1 week either before or post-ulcer induction. A reference drug, ranitidine (100 mg/kg b.wt.), was also evaluated. Stomach acidity, gastric volume, lesion counts, glutathione (GSH), superoxide dismutase (SOD), malondialdehyde (MDA), succinate dehydrogenase (SDH), lactate dehydrogenase (LDH), acid phosphatase (AP), interlukin-10 (IL-10) and prostaglandin E2 (PGE2) were estimated. Stomach histopathological features were monitored.Results: Nine polymethoxy flavonoids were identified from the extract. Treatment with C. sinensis peels extract recorded amelioration in all parameters.Conclusion: Citrus sinensis petroleum ether peels extract had protective and curative effects against gastric ulcer. Therefore, the extract recorded anti-secretory, anti-ulcerative, anti-inflammatory, and antioxidant effects. Its healing action exceeded its protective role due to its richness in polymethoxylated flavonoids.

Direct Analyses of Secondary Metabolites by Mass Spectrometry Imaging (MSI) from Sunflower (Helianthus annuus L.) Trichomes.

Helianthus annuus (sunflower) displays non-glandular trichomes (NGT), capitate glandular trichomes (CGT), and linear glandular trichomes (LGT), which reveal different chemical compositions and locations in different plant tissues. With matrix-assisted laser desorption/ionization (MALDI) and laser desorption/ionization (LDI) mass spectrometry imaging (MSI) techniques, efficient methods were developed to analyze the tissue distribution of secondary metabolites (flavonoids and sesquiterpenes) and proteins inside of trichomes. Herein, we analyzed sesquiterpene lactones, present in CGT, from leaf transversal sections using the matrix 2,5-dihydroxybenzoic acid (DHB) and α-cyano-4-hydroxycinnamic acid (CHCA) (mixture 1:1) with sodium ions added to increase the ionization in positive ion mode. The results observed for sesquiterpenes and polymethoxylated flavones from LGT were similar. However, upon desiccation, LGT changed their shape in the ionization source, complicating analyses by MSI mainly after matrix application. An alternative method could be applied to LGT regions by employing LDI (without matrix) in negative ion mode. The polymethoxylated flavones were easily ionized by LDI, producing images with higher resolution, but the sesquiterpenes were not observed in spectra. Thus, the application and viability of MALDI imaging for the analyses of protein and secondary metabolites inside trichomes were confirmed, highlighting the importance of optimization parameters.

Chrysosplenetin inhibits artemisinin efflux in P-gp-over-expressing Caco-2 cells and reverses P-gp/MDR1 mRNA up-regulated expression induced by artemisinin in mouse small intestine.

CONTEXT: CYP3A4 and P-gp together form a highly efficient barrier for orally absorbed drugs and always share the same substrates. Our previous work revealed that chrysosplenetin (CHR) significantly augmented the rat plasma level and anti-malarial efficacy of artemisinin (ART), partially due to the uncompetitive inhibition effect of CHR on rat CYP3A. But the impact of CHR on P-gp is still unknown. OBJECTIVE: The present study investigates whether CHR interferes with P-gp-mediated efflux of ART and elucidates the underlying mechanism. MATERIALS AND METHODS: P-gp-over-expressing Caco-2 cells were treated with ART (10 µM) or ART-CHR (1:2, 10:20 µM) in ART bidirectional transport experiment. ART concentration was determined by UHPLC-MS/MS method. Healthy male ICR mice were randomly divided into nine groups (n = 6) including negative control (0.5% CMC-Na solution, 13 mL/kg), ART alone (40 mg/kg), verapamil (positive control, 40 mg/kg), ART-verapamil (1:1, 40:40 mg/kg), CHR alone (80 mg/kg), ART-CHR (1:0.1, 40:4 mg/kg), ART-CHR (1:1, 40:40 mg/kg), ART-CHR (1:2, 40:80 mg/kg) and ART-CHR (1:4, 40:160 mg/kg). The drugs were administrated intragastrically for seven consecutive days. MDR1 and P-gp expression levels in mice small intestine were examined by performing RT-PCR and western blot analysis. ABC coupling ATPase activity was also determined. RESULTS: After combined with CHR (1:2), Papp (AP-BL) and Papp (BL-AP) of ART changed to 4.29 × 10 - 8 (increased 1.79-fold) and 2.85 × 10 - 8 cm/s (decreased 1.24-fold) from 2.40 × 10 - 8 and 3.54 × 10 - 8 cm/s, respectively. Efflux ratio (PBA/PAB) declined 2.21-fold (p < 0.01) versus to ART alone. ART significantly up-regulated both MDR1 mRNA and P-gp levels compared with vehicle, while CHR in combination ratio of 0:1, 0.1:1, 1:1, 2:1 and 4:1 with ART, reversed them to normal levels as well as negative control (p < 0.05). The ATPase activities in ART-CHR 1:4 and CHR alone groups achieved a slight increase (p < 0.05) when compared with ART alone. DISCUSSION AND CONCLUSION: These results confirm that CHR inhibited P-gp activity and reverse the up-regulated P-gp and MDR1 levels induced by ART. It suggested that CHR potentially can be used as a P-gp reversal agent to obstruct ART multidrug resistance.

Activation of human bitter taste receptors by polymethoxylated flavonoids.

Tangeretin and nobiletin are polymethoxylated flavonoids in citrus peel. Both tangeretin and nobiletin are bitter; however, their bitterness has not been evaluated using human bitter taste receptors (hTAS2Rs). We screened 25 kinds of hTAS2Rs and found that hTAS2R14 and hTAS2R46 received both compounds.

Polymethoxylated flavonoids in citrus fruits: absorption, metabolism, and anticancer mechanisms against breast cancer.

Polymethoxylated flavonoids (PMFs) are a subclass of flavonoids found in citrus fruits that have shown multifunctional biological activities and potential anticancer effects against breast cancer. We studied the absorption, metabolism, species source, toxicity, anti-cancer mechanisms, and molecular targets of PMFs to better utilize their anticancer activity against breast cancer. We discuss the absorption and metabolism of PMFs in the body, including the methylation, demethylation, and hydroxylation processes. The anticancer mechanisms of PMFs against breast cancer were also reviewed, including the estrogen activity, cytochrome P-450 enzyme system, and arylhydrocarbon receptor (AhR) inhibition, along with various molecular targets and potential anticancer effects. Although PMFs may be advantageous in the prevention and treatment for breast cancer, there is a lack of clinical evidence and data to support their efficacy. Despite their promise, there is still a long way to go before PMFs can be applied clinically.

Polymethoxylated flavonoids (PMFs)-loaded citral nanoemulsion controls green mold in citrus by damaging the cell membrane of Penicillium digitatum.

Citrus is susceptible to Penicillium digitatum (P. digitatum) infection in post-harvest storage, resulting in enormous economic losses. This study aimed to investigate the antifungal activity and potential mechanism of the combination of Polymethoxylated flavones (PMFs) and citral (two natural antifungal components derived from citrus) against P. digitatum in vitro and citrus fruit. The results show that PMFs can enhance the antifungal activity of citral nanoemulsion, and PMFs-loaded citral nanoemulsion (PCT) has significant antifungal activity in a concentration-dependent manner. PCT can evidently inhibit spore germination and mycelial growth in vitro, and effectively control the growth of green mold on postharvest citrus fruit. Furthermore, PCT treatment resulted in the alteration of mycelia morphology, accumulation of reactive oxygen species, and membrane lipid peroxidation. These changes can disrupt the normal structure and function of the cell membrane, as evidenced by the reduction of total lipid and ergosterol content in the mycelia and the stronger red fluorescence of the cells emitted after PI staining. Based on the above results, we infer that PCT has a strong inhibitory effect on P. digitatum, and its potential mechanism is related to the destruction of the cell membrane. Therefore, PCT can be considered as a botanical fungicide for citrus preservation.

Citrus Peel Extracts: Effective Inhibitors of Heterocyclic Amines and Advanced Glycation End Products in Grilled Pork Meat Patties.

In the present study, citrus peels were extracted using various conventional and deep eutectic solvents (DESs). Compared to other citrus peel extracts, the DES extract based on choline chloride showed notably higher total phenolic and flavonoid content levels, along with superior antioxidant activity, among these extracts. Consequently, this study aimed to further investigate the inhibitory effects of the choline chloride based DES extract on the production of both free and bound heterocyclic amines (HAs) and advanced glycation end products (AGEs) in roast pork meat patties. The results indicated that the addition of choline chloride-based DES extracts, particularly the choline chloride-carbamide based DES extract, can effectively reduce the oxidation of lipids and proteins by quenching free radicals. This approach proves to be the most efficient in reducing the formation of both HAs and AGEs, leading to a significant reduction of 19.1-68.3% and 11.5-66.5% in free and protein-bound HAs, respectively. Moreover, the levels of free and protein-bound AGEs were reduced by 50.8-50.8% and 30.5-39.8%, respectively, compared to the control group. Furthermore, the major phenolics of citrus peel extract identified by UHPLC-MS were polymethoxylated flavonoids (PMFs) including hesperidin, isosinensetin, sinensetin, tetramethoxyflavone, tangeretin, and hexamethoxyflavone, which inferring that these compounds may be the main active ingredients responsible for the antioxidant activity and inhibition effects on the formation of HAs and AGEs. Further research is needed to explore the inhibitory effects and mechanisms of PMFs with different chemical structures on the formation of HAs and AGEs.

Chemicalome and metabolome profiling of polymethoxylated flavonoids in Citri Reticulatae Pericarpium based on an integrated strategy combining background subtraction and modified mass defect filter in a Microsoft Excel Platform.

Detection of metabolites in complex biological matrixes is a great challenge because of the background noise and endogenous components. Herein, we proposed an integrated strategy that combined background subtraction program and modified mass defect filter (MMDF) data mining in a Microsoft Excel platform for chemicalome and metabolome profiling of the polymethoxylated flavonoids (PMFs) in Citri Reticulatae Pericarpium (CRP). The exogenously-sourced ions were firstly filtered out by the developed Visual Basic for Applications (VBA) program incorporated in the Microsoft Office. The novel MMDF strategy was proposed for detecting both target and untarget constituents and metabolites based on narrow, well-defined mass defect ranges. The approach was validated to be powerful, and potentially useful for the metabolite identification of both single compound and homologous compound mixture. We successfully identified 30 and 31 metabolites from rat biosamples after oral administration of nobiletin and tangeretin, respectively. A total of 56 PMFs compounds were chemically characterized and 125 metabolites were captured. This work demonstrated the feasibility of the integrated approach for reliable characterization of the constituents and metabolites in herbal medicines.

Structural Pharmacokinetics of Polymethoxylated Flavones in Rat Plasma Using HPLC-MS/MS.

The purpose of this study was to investigate the pharmacokinetics of the polymethoxylated flavonoids kumatakenin, pachypodol, and retusin, which contain two, three, or four methoxy substitutions, using a validated ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method in rats. The pharmacokinetic results demonstrated that the elimination half-lives for kumatakenin, pachypodol, and retusin were 30 ± 11.6, 39.4 ± 19.5, and 106.9 ± 26 min, respectively, for the low dose group and 54.5 ± 16.5, 33.8 ± 10, and 134.6 ± 34.7 min for the high dose group. The results suggested that the area under the curve values (AUC) for the analytes did not correlate with the number of methoxy groups. Pachypodol had the lowest AUC, which may have been correlated with lipophilicity, for both the low and high dose groups. In conclusion, the polymethoxylated flavonoid pachypodol is more hydrophilic than kumatakenin or retusin, which were correlated with the pharmacokinetic results.

Characterization of major metabolites of polymethoxylated flavonoids in Pericarpium Citri Reticulatae using liver microsomes immobilized on magnetic nanoparticles coupled with UPLC/MS-MS.

The peels of citrus fruits (Pericarpium Citri Reticulatae, PCR) have long been used in traditional Chinese medicines (TCMs). Polymethoxylated flavonoids (PMFs) were found to be the main components present in PCR extracts, but their metabolism remains unclear which restrain the utilization of this TCM. In the present work, rat liver microsomes were immobilized on magnetic nanoparticles (LMMNPs) for in vitro metabolic study on the whole PMFs of PCR. LMMNPs were characterized by transmission electron microscope and Fourier-transform infrared spectrum. The relative enzyme binding capacity of LMMNPs was estimated to be about 428 µg/mg from thermogravimetric analysis. Incubation of LMMNPs with PMFs produced demethylated metabolites of PMFs, six of which were identified by ultrahigh pressure liquid chromatography-mass spectrometry (UPLC-MS/MS). The 3'-hydroxylated tangeretin (T3) was detected from the metabolites of tangeretin for the first time, which suggested that 4'-demethylated and 3'-hydroxylated derivative of tangeretin (3'-hydroxy-5,6,7,8,4'-pentamethoxyflavone, T4) was not only derived from 4'-demethylated tangeretin (T2) as previously reported, but also from T3. This is the first investigation of the metabolism of the whole PMFs, which may shed light on the mechanism of action of PCR.

Antimicrobial activity of wax and hexane extracts from Citrus spp. peels

Antibacterial and antifungal properties of wax and hexane extracts of Citrus spp. peels were tested using bioautographic and microdilution techniques against three plant pathogenic fungi (Penicillium digitatum, Curvularia sp., and Colletotrichum sp.), two human pathogens (Trichophyton mentagrophytes and Microsporum canis), and two opportunistic bacteria (Escherichia coli and Staphylococcus aureus). Two polymethoxylated flavonoids and a coumarin derivative, were isolated and identified from peel extracts, which presented antimicrobial activity especially against M. canis and T. mentagrophytes: 4',5,6,7,8-pentamethoxyflavone (tangeritin) and 3',4',5,6,7,8-hexamethoxyflavone (nobiletin) from C. reticulata; and 6,7-dimethoxycoumarin (also known as escoparone, scoparone or scoparin) from C. limon.