Flavonoid extracts of Citrus aurantium L. var. amara Engl. Promote browning of white adipose tissue in high-fat diet-induced mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Obesity has become a public burden worldwide due to its booming incidence and various complications, and browning of white adipose tissue (WAT) is recognized as a hopeful strategy to combat it. Blossom of Citrus aurantium L. var. amara Engl. (CAVA) is a popular folk medicine and dietary supplement used for relieving dyspepsia, which is recorded in the Chinese Materia Medica. Our previous study showed that blossom of CAVA had anti-obesity potential, while its role in browning of WAT was still unclear. AIM OF THE STUDY: This study aimed to characterize the constituents in flavonoids from blossom of CAVA (CAVAF) and to clarify the anti-obesity capacities especially the effects on browning of WAT. MATERIALS AND METHODS: Gradient ethanol eluents from blossom of CAVA were obtained by AB-8 macroporous resin. 3T3-L1 cells and pancreatic lipase inhibition assay were employed to investigate the potential anti-obesity effects in vitro. HPLC and UPLC/MS assays were performed to characterize the chemical profiles of different eluents. Network pharmacology and molecular docking assays were used to reveal potential anti-obesity targets. Furthermore, high-fat diet (HFD)-induced mice were constructed to explore the anti-obesity actions and mechanisms in vivo. RESULTS: 30% ethanol eluents with high flavonoid content and great inhibition on proliferation of 3T3-L1 preadipocytes and pancreatic lipase activity were regarded as CAVAF. 19 compounds were identified in CAVAF. Network pharmacology analysis demonstrated that AMPK and PPARα were potential targets for CAVAF in alleviating obesity. Animal studies demonstrated that CAVAF intervention significantly decreased the body weight, WAT weight, serum TG, TC and LDL-C levels in HFD-fed obese mice. HFD-induced insulin resistance and morphological changes in WAT and brown adipose tissue were also markedly attenuated by CAVAF treatment. CAVAF supplementation potently inhibited iWAT inflammation by regulating IL-6, IL-1ß, TNF-α and IL-10 mRNA expression in iWAT of mice. Furthermore, the gene expression levels of thermogenic markers including Cyto C, ATP synthesis, Cidea, Cox8b and especially UCP1 in iWAT of mice were significantly up-regulated by CAVAF administration. CAVAF intervention also markedly increased the expression levels of PRDM16, PGC-1α, SIRT1, AMPK-α1, PPARα and PPARγ mRNA in iWAT of mice. CONCLUSION: CAVAF treatment significantly promoted browning of WAT in HFD-fed mice. These results suggested that flavonoid extracts from blossom of CAVA were probably promising candidates for the treatment of obesity.

Potential bio-functional properties of Citrus aurantium L. leaf: chemical composition, antiviral activity on herpes simplex virus type-1, antiproliferative effects on human lung and colon cancer cells and oxidative protection.

This study examined the antioxidant, anticancer and antiviral properties of the methanolic extracts from bigarade (Citrus aurantium L.) leaves at two development stages. Ferulic acid, naringin and naringenin were the principal phenolic components of young and old leaves. The highest total antioxidant capacity was obtained in young leaf extracts (YLE). These latter also exhibited the highest antiradical DPPH (1,1-diphenyl-2-picrylhydrazyl) and ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) activities, while the highest iron chelating and reducing power activities were observed in old leaf extracts (OLE). The potent anticancer activity was observed in YLE for human lung carcinoma (A-549) and in OLE for colon adenocarcinoma (DLD-1) cells. YLE showed the highest virucidal effects as compared to OLE and the positive control acyclovir against herpes simplex virus type-1 (HSV-1) propagation in Vero cells during the absorption and replication periods. The young and old leaves might be a source of natural antioxidants and protective agents against oxidative damage.

Effects of Citrus aurantium L. on Gastrointestinal Motility and Gastric Cancer Cell Proliferation.

Background: To research the impact of a Chinese traditional medicine (Citrus aurantium L.) on gastric cancer proliferation and mice gastrointestinal motility. Materials and Methods: The intestinal transit rates (ITRs) and gastric emptying (GE) values in mice with experimentally induced gastrointestinal motility dysfunction (GMD) and in normal mice were calculated to research the in vivo influences of C. aurantium L. on gastrointestinal motility. CCK-8 was used to examined the effect of C. aurantium L. on gastric cancer proliferation. Results: The GE and ITR values were dose-dependently and notably added by C. aurantium L. in normal ICR mice (with 1 g/kg C. aurantium L., ITR values: 53.3% ± 0.8% versus 64.3% ± 0.9% and 53.3% ± 0.8% versus 79.8% ± 2.0%, p < 0.01; GE values: 59.3% ± 0.8% versus 70.1% ± 1.9% and 59.9% ± 0.8% versus 69.9% ± 2.1%, p < 0.01). Compared with the normal mice, the GMD mice's ITRs were notably declined; however, C. aurantium L. could dose-dependently and significantly reverse it. In addition, in the model of delayed GE induced by loperamide and cisplatin, C. aurantium L. administration reversed the GE deficit. Furthermore, C. aurantium L. significantly reduced gastric cancer proliferation. Conclusion: The results indicate that C. aurantium L. could become a new drug for gastrointestinal prokinetic and gastric cancer therapy.

An approach of pectin from Citrus aurantium L. for superabsorbent resin with superior quality for hygiene products: Salt resistance, antibacterial, nonirritant and biodegradability.

In this paper, a kind of superabsorbent resin (SAR) with superior quality for hygiene products was developed using Fructus Aurantii Immaturus pectin (FAIP) from Citrus aurantium L.. FAIP-g-AM/AMPS SAR was established by free radical graft co-polymerization with FAIP as skeleton structure, N, N'-Methylene-bis (acrylamide) (MBA) as the cross-linker. Meanwhile, the functional monomers of acrylamide (AM) and 2-acrylamido-2-methylpropane sulfonic acid (AMPS) were introduced. The structure and morphology of FAIP-g-AM/AMPS were characterized by FTIR, 13C NMR, XRD, SEM and TG-DSC analysis. The results confirmed that the AFIP-g-AM/AMPS SAR was successfully prepared, which exhibited a three-dimensional (3D) network structure and an excellent thermal stability. The absorption and retention capacity of FAIP-g-AM/AMPS was comparable to or even better than commercial diapers and sanitary napkins. Significantly, FAIP-g-AM/AMPS itself exhibited excellent antibacterial and safety. FAIP-g-AM/AMPS has an inhibition ratio of 97.1 % for Escherichia coli (E. coli) and 98.5 % for Staphylococcus aureus (S. aureus), and was non-irritating and non-allergic to the skin. In addition, FAIP-g-AM/AMPS presented amazing biodegradability and a weight loss reached 37.1 % after 30 days by soil burial test. The research provides a safe and high-performance SAR, which expected to be used in hygiene products such as baby diapers, adult incontinence pads and sanitary napkins.

The Protective Effects of Sour Orange (Citrus aurantium L.) Polymethoxyflavones on Mice Irradiation-Induced Intestinal Injury.

Sour orange (Citrus aurantium L.) is one of the biological sources of polymethoxyflavones (PMFs), which are often used to deal with gastrointestinal diseases. The intestine is highly sensitive to irradiation damage. However, limited certain cures have been released for irradiation-induced gastrointestinal injury, and the potentials of sour orange PMFs as radio-resistance agents have not been fully discussed yet. The present study aims to (1) investigate the PMF components in 12 sour orange cultivars, (2) determine the protective effects of PMFs on irradiation-induced intestinal injury by treating mice that received 12 Gy abdominal irradiation with different doses of PMFs and observing the changes in organ indexes and pathological sections and (3) test cytotoxicity of PMFs by CCK-8 method. The results showed that sour orange PMFs appeared to have high intraspecies similarity. Besides, PMFs protected mice from irradiation-induced injury by alleviating body weight loss, reliving organ index changing and maintaining the intestinal structure. Finally, IC50 concentrations to cell line CCD 841 CoN of PMFs and nobiletin were calculated as 42.23 µg/mL and 51.58 µg/mL, respectively. Our study uncovered PMF contents in 12 sour orange materials and determined the protective effects on irradiation-induced intestinal injuries, providing guidance for the utilization of sour orange resources.

Comprehensive chemical profiling of the flowers of Citrus aurantium L. var. amara Engl. and uncovering the active ingredients of lipid lowering.

The flowers of Citrus aurantium L. var. amara Engl. (FCAVA) is popularly consumed as an edible tea for anti-hyperlipidemia. But the active ingredients are not fully clear. In this study, ultra-high performance liquid chromatography coupled to quadrupole time of flight tandem mass spectrometry (UHPLC-QTOF-MS/MS) with diagnostic product ions and neutral loss filtering strategy were successfully used for comprehensive characterization of chemical components in FCAVA. A total of 228 constituents, including 46 organic acids, 12 coumarins and 170 flavonoids, were tentatively characterized (30 confirmed with reference standards). Among them, nineteen flavonoids in 70 batches of FCAVA from different geographical origins were quantified by UHPLC tandem triple quadrupole mass spectrometry (QQQ-MS), which displayed satisfactory linearity, sensitivity, precision, accuracy, and stability. According to analytical results, the distribution of nineteen flavonoids in different geographical origins of FCAVA was clarified. In addition, the effect on LDL uptake of twenty-five flavonoids was investigated in HepG2 cell. It was found that the acacetin, diosmetin and rutin dose-dependently enhanced LDL uptake in HepG2 cells comparing to control. Furthermore, in a hyperlipidemia C57BL/6J mice model, administration of acacetin, diosmetin and rutin (30 mg/kg/d, intragastric, for three weeks) significantly decreased the levels of total cholesterol (TC), triglyceride (TG) and low density lipoprotein cholesterol (LDL-C) in plasma, respectively. Overall, these findings indicated the potential of FCAVA in the development of functional food or medicine for the prevention and treatment of hyperlipidemia, which could be considered for the improvement of quality standardization of FCAVA.

Inhibitory effect of chloroform extracts from Citrus aurantium L. var. amara Engl. on fat accumulation.

BACKGROUND: Excess lipid accumulation can accelerate the development of various metabolic diseases. Blossoms of Citrus aurantium L. var. amara Engl. (CAVA) have been reported to possess inhibitory capacities on lipid deposition. However, the constituents responsible for the observed bioactivity and the underlying mechanisms are still not clearly understood. PURPOSE: To screen constituents from blossoms of CAVA with inhibitory effects on lipid accumulation and to explore the action mechanism. METHODS: The chloroform (CHL) extracts are prepared from blossoms of CAVA by fractional extraction and are characterized using LC-MS assay. 3T3-L1 preadipocytes are induced with differentiation medium (DMI) and treated with CHL extracts. High fat diet (HFD)-induced obese mice are further established and administrated with CHL extracts for 12 weeks. Hematoxylin and eosin (HE) staining, Oil Red O staining, ELISA, RT-qPCR, western blot and 16S rRNA gene sequence methods are employed. RESULTS: 14 compounds are identified in CHL extracts and trigonelline hydrochloride, nobiletin and 7-demethylsuberosin are most abundant. CHL extracts treatment significantly inhibit differentiation of 3T3-L1 cells by regulating expression of preadipocyte factor-1 (Pref-1), fatty acid synthase (FAS) and CCAAT/enhancer binding protein α (C/EBPα). CHL extracts intervention also significantly attenuate features of obesity and improved plasma biochemical profiles in HFD-fed mice. HFD-triggered hepatic steatosis and epididymal adipose tissues (EATs) hypertrophy are also reversed by CHL extracts administration through enhancing antioxidant responses and modulating lipogenesis and energy expenditure-related genes and proteins. 16S rRNA gene sequence data further show that CHL extracts enhance the diversity of gut microbiota. CHL extracts at lower concentrations reduce the ratio of Firmicutes to Bacteroidetes and the abundance of Erysipelotrichaceae. CHL extracts at higher doses markedly increase the abundance of Lachnospiraceae. CONCLUSION: These findings suggest that CHL extracts probably suppress lipid accumulation through inhibiting differentiation of 3T3-L1 cells and attenuating metabolic syndromes in HFD-fed mice.

The levels of bioactive ingredients in Citrus aurantium L. at different harvest periods and antioxidant effects on H2 O2 -induced RIN-m5F cells.

BACKGROUND: Citrus aurantium L. (Aurantii fructus) is a multi-purpose citrus fruit with high medicinal and nutritional value, but currently there are no data that can be used to investigate the appropriate harvest time to obtain high-quality citrus bioactive ingredients from it. RESULTS: Phytochemicals and the levels of the main bioactive ingredients were investigated by ultra high performance liquid chromatography electrospray ionization quadrupole time-of-flight mass spectrometry (UHPLC-ESI-Q-TOF/MS). The flavanone, polymethoxyflavone, coumarin, synephrine, and limonin content in the citrus fruit was analyzed at different harvest periods, and significant differences, ranging from 0.03 ± 0.01 to 116.26 ± 40.20 g kg-1 (DW), were shown. These compounds were present in higher amounts in June and then decreased gradually, while the biomass accumulation of most of them showed an increasing tendency around harvest time. The H2 O2 -induced RIN-m5F cells model was employed to evaluate their antioxidant capacity. Citrus fruit harvested from June 11 to July 7 possessed an excellent antioxidant capacity by inhibiting the intensity of intracellular reactive oxygen species (ROS) (P < 0.01) and improving superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH) activity (P < 0.01). The chemical composition and antioxidant capacity of citrus leaves, stems, and roots were also evaluated, and these showed great variation compared with other citrus fruits. Multivariate statistical analysis indicated that harvesting time was related closely to the phytochemical contents and antioxidant capacity. CONCLUSION: Citrus fruit can be appropriately harvested from June to early July when the levels of bioactive ingredients and antioxidant activity reach higher values. This research provides practical information for producing high-quality citrus products. © 2020 Society of Chemical Industry.

Citrus aurantium L. Dry Extracts Ameliorate Adipocyte Differentiation of 3T3-L1 Cells Exposed to TNFα by Down-Regulating miR-155 Expression.

Citrus aurantium L. dry extracts (CAde) improve adipogenesis in vitro. These effects are dependent from an early modulation of CCAAT/enhancer-binding protein beta (C/Ebpß) expression and cyclic Adenosine Monophosphate (cAMP) response element-binding protein (CREB) activation. C/Ebpß and Creb are also targets of miR-155. This study investigated whether CAde regulates miR-155 expression in the early stages of adipogenesis and whether it ameliorates adipocyte differentiation of cells exposed to tumor necrosis factor-alpha (TNFα). Adipogenic stimuli (AS) were performed in 3T3-L1 pre-adipocytes treated with CAde, TNFα, or both. Gene and miRNA expression were determined by quantitative real-time PCR. Adipogenesis was evaluated by Oil-Red O staining. CAde treatment enhanced AS effects during the early adipogenesis phases by further down-regulating miR-155 expression and increasing both C/Ebpß and Creb mRNA and protein levels. At variance, TNFα inhibited 3T3-L1 adipogenesis and abolished AS effects on miR-155, C/Ebpß, and Creb expression. However, in cells exposed to TNFα, CAde improved adipocyte differentiation and restored the AS effects on miRNA and gene expression at early time points. In conclusion, this study identified miR-155 down-regulation as part of the mechanism through which CAde enhances adipogenesis of pre-adipocytes in vitro. Furthermore, it provides evidence of CAde efficacy against TNFα negative effects on adipogenesis.

Systematic Detection and Identification of Bioactive Ingredients from Citrus aurantium L. var. amara Using HPLC-Q-TOF-MS Combined with a Screening Method.

Bitter orange, Citrus aurantium L. var. amara (CAVA), is an important crop and its flowers and fruits are widely used in China as a food spice, as well as in traditional Chinese medicine, due to its health-promoting properties. The secondary metabolites that are present in plant-derived foods or medicines are, in part, responsible for the health benefits and desirable flavor profiles. Nevertheless, detailed information about the bioactive ingredients in CAVA is scarce. Therefore, this study was aimed at exploring the phytochemicals of CAVA by high performance liquid chromatography/quadrupole time-of-flight mass spectrometry (HPLC-Q-TOF-MS). Here, a systematic screening method combined with HPLC-Q-TOF-MS was presented. This technique was used to systematically screen metabolites, primarily from the complex matrix of CAVA, and to identify these compounds by their exact masses, characteristic fragment ions, and fragmentation behaviors. A total of 295 metabolites were screened by the screening method and 89 phytochemicals were identified in the flowers, fruits, roots, leaves, and branches of CAVA. For the first time, 69 phytochemicals (flavonoids, alkaloids, terpenoids, etc.) were reported from CAVA. The results highlight the importance of CAVA as a source of secondary metabolites in the food, medicine, and nutraceutical industries.

Relationship between volatile components, antimicrobial and antioxidant properties of the essential oil, hydrosol and extracts of Citrus aurantium L. flowers.

INTRODUCTION: In the Mediterranean Region, essential oil, hydrosol, and ethanol extract of C. aurantium flowers have a long history of usage in different products such as a flavoring agent and an ingredient of many traditional anti-infectious and skin care products. The present study was undertaken to compare the antimicrobial activity, antioxidant activity and phytochemical composition of essential oil (EO), hydrosol and ethanol extract of Cyprus Citrus aurantium L. flowers. METHOD: The chemical composition of samples was determined by GC/MS. The total phenolic and flavonoid contents were determined by Folin-Ciocalteu colorimetric method and aluminum chloride colorimetric assay, respectively. Antioxidant activity was evaluated by 1,1-diphenyl-2-picrylhydrazyl (DPPH) and hydrogen peroxide (H2O2). The antimicrobial activity of the samples was determined by disc diffusion and broth microdilution methods against five foodborne pathogenic bacteria. RESULT: The EO and ethanol extract showed significant antimicrobial activity against all tested pathogens which were attributed to the presence of important phytochemical classes such as polyphenols, flavonoids, alkaloids, and terpenes. The EO showed higher antimicrobial activity followed by ethanol extract with minimum inhibitory concentrations ≤1562mg/l and ≤6250mg/l, respectively, against Amoxycillin resistant Bacillus cereus and other test organisms. Significantly (p≤0.05) the higher total phenolic (81.37mg GAE/g) and flavonoid contents (20.34mg QE/g) were obtained in the ethanol extract with the higher antioxidant activity as compared to EO and hydrosol with half maximal inhibitory concentration (IC50) values of 96.07µg/ml in the DPPH and 66.50µg/ml in the H2O2 assay. A significant correlation (R2≥0.94) was found between phenolic content and IC50 values for DPPH and H2O2 assays. ß-Cholesta-5,22-dien-3-ol, herboxide isomer and isochiapin-B are reported for the first time for essential oil. CONCLUSION: Citrus aurantium L. flowers have strong potential for the isolation of antimicrobial and antioxidant agents for further use in food and medicine industries as natural preservatives.

Vascular Protection of TPE-CA on Hyperhomocysteinemia-induced Vascular Endothelial Dysfunction through AA Metabolism Modulated CYPs Pathway.

A high concentration of homocysteine (Hcy) in plasma induces vascular endothelial dysfunction, and it may ultimately accelerate the development of cardiovascular diseases (CVDs). Although several B vitamins have been clinically applied for hyperhomocysteinemia (HHcy) treatment, the outcomes are not satisfied due to their limited therapeutic mechanism. Hence, in order to improve the curative effect, development of new effective therapeutic strategies should be put on the agenda. Total phenolic extracts of Citrus aurantium L. (TPE-CA) is a naturally obtained phenolic mixture, mainly containing flavones, flavanones and their glycosyl derivatives, flavonols, polymethoxyflavones and coumarins. Previous reports indicated that bioactive phenolic compounds possessed potent vascular protective effects and regarded as a protective agent against CVDs. Intriguingly, the exact mechanism underlying the suppressed effects of TPE-CA on HHcy could assist in revealing their therapy on CVDs. Here, the multi-targeted synergistic mechanism of TPE-CA on HHcy-induced vascular endothelial dysfunction was uncovered in a deduced manner. TPE-CA treatment exhibited an obvious superiority than that of B vitamins treatment. Network pharmacology was employed to identify the interrelationships among compounds, potential targets and putative pathways. Further experimental validation suggested that the treatment of TPE-CA for HHcy could not only effectively reduce the Hcy level in plasma through up-regulating transsulfuration pathway in Hcy metabolism, but also restore the HHcy-induced vascular endothelial dysfunction by activating cytochrome P450 enzymes (CYPs) epoxygenase signal cascades and inhibiting CYPs hydroxylase signal cascades in arachidonic acid (AA) metabolism.

Citrus aurantium L. var. amara Engl. inhibited lipid accumulation in 3T3-L1 cells and Caenorhabditis elegans and prevented obesity in high-fat diet-fed mice.

Natural products with anti-obesity effects and few side effects have attracted great attention recently. Citrus aurantium L. var. amara Engl. (CAVA) is popularly consumed as an edible and medicinal resource in China. However, its anti-obesity effects were poorly understood. The anti-obesity effects of CAVA extracts were systematically evaluated using 3T3-L1 cells, Caenorhabditis elegans (C. elegans) and high fat diet (HFD)-fed mice. Flavonoid-rich (EA) extracts with neohesperidin, hesperidin and naringin comprising 32.15%, were isolated from CAVA. EA extracts treatment significantly inhibited differentiation of 3T3-L1 preadipocytes by modulating lipid metabolism-related mediators. EA extracts supplementation also inhibited antioxidant responses in C. elegans by decreasing reactive oxygen species generation and malonaldehyde value, and increasing superoxide dismutase content. EA extracts feeding markedly decreased triglyceride (TG) content, and affected expression of genes involved in lipid and glucose metabolism in wild type C. elegans. TG content in mdt-15 (XA7702) mutants was not decreased by EA extracts administration, suggesting that EA extracts treatment might inhibit lipid accumulation in C. elegans dependent on mdt-15. EA extracts intervention further reduced body weight gain and modulated plasma biochemical parameters in HFD-fed mice. EA extracts treatment prevented HFD-induced epididymal adipose hypertrophy, liver oxidative injuries and steatosis. EA extracts administration also strongly prevented HFD-induced reduction of gut microbial diversity, decreased the Firmicutes-to-Bacteroidetes ratio and the Erysipelotrichaceae abundance, and enhanced the Bifidobacteriace abundance in HFD-fed mice. EA extracts from blossoms of CAVA were excellent antiobesogenic candidates that acted through multiple mechanisms that acted simultaneously.

Optimization of Ultrasonic-Assisted Extraction of Total Phenolics from Citrus aurantium L. Blossoms and Evaluation of Free Radical Scavenging, Anti-HMG-CoA Reductase Activities.

The objective of this study was to develop an ultrasonic-assisted procedure for the extraction of total phenolics from Citrus aurantium L. blossoms (CAB) and evaluate the free radical scavenging activity and anti-HMG-CoA reductase activity of the total phenolics. In this work, a Box- Behnken design based on single-factor experiments was used to explore the optimum extraction process. Under the optimum conditions (extraction solvent 70.31% ethanol, extraction temperature 61.94 °C, extraction time 51.73 min, and liquid-to-solid ratio 35.63 mL/g), the extraction yield of total phenolics was 95.84 mg gallic acid equivalents (GAE)/g dry matter (DM), which was highly consistent with the theoretical value (96.12 mg GAE/g DM). The higher contents of total phenolics and five main phenolic compounds obtained from the optimized ultrasonic-assisted extraction (UAE) proved its efficiency when compared with conventional heat reflux extraction (HRE). The total phenolic extract showed excellent free radical scavenging properties against 1,1-diphenyl-2-picrylhydrazyl radical (DPPH·), 2,2'-azino-bis-(3-ethylbenzthiazoline-6-sulphonic acid) radical (ABTS+·), hydroxyl radical (·OH) and superoxide anion radical (·O2-), with IC50 values of 197.007, 83.878, 218.643, and 158.885 µg/mL, respectively; the extracts also showed good inhibition of ß-hydroxy-ß-methylglutaryl-CoA reductase (HMG-CoA reductase) activity, with an IC50 value of 117.165 µg/mL. Total phenolics from CAB could be a potential source of natural free radical scavenger and HMG-CoA reductase inhibitor.

Synergistic enhancement and hepatoprotective effect of combination of total phenolic extracts of Citrus aurantium L. and methotrexate for treatment of rheumatoid arthritis.

Rheumatoid arthritis (RA) is an autoimmune inflammatory disorder characterized by joint destruction and bone damage. Methotrexate (MTX) is recommended as the first-line disease-modifying agent for the treatment of RA. However, the clinical efficacy of MTX is limited due to its low response and side effects, especially hepatotoxicity. Total phenolic extracts of Citrus aurantium L. (TPE-CA) are rich in dietary bioactive flavonoids, which show beneficial effects on liver health and are regarded as therapeutic tools against inflammatory diseases. In this study, the efficacy of MTX, alone or in combination with TPE-CA, for the treatment of collagen-induced arthritis and protection against hepatic injury in rats was investigated. TPE-CA and MTX combination effectively reduced the inflammatory symptoms and joint damage by inhibiting the NF-κB pathway. Moreover, TPE-CA significantly ameliorated MTX-induced chronic hepatic injury by enhancing antioxidant enzymes activities, suppressing hepatic cytochrome P450 2E1 expression, and modulating the nuclear factor erythroid 2-related factor 2/heme oxygenase-1 pathway. This combination regimen not only provided synergistic enhancement but also exhibited hepatoprotective effect against chemically induced chronic hepatotoxicity. This could be an alternative strategy to improve the low response of MTX in RA treatment.

Bioactive constituents obtained from the fruits of Citrus aurantium.

Three new compounds, citrusauranosides A (1), B (2), and C (3), along with 22 known compounds (4-25) were obtained from the 70% ethanol-water extract of the fruits of Citrus aurantium L.. Their structures were identified by spectroscopic methods. Among the known compounds, 14, 15, 17 and 23 were firstly obtained from Citrus genus, and the NMR data of 17 is reported here for the first time. Moreover, inhibitory effects of all compounds on motility of mouse isolated intestine tissue were determined. As a result, 1, 4-8, and 11 displayed significant inhibitory effects on contraction tension.

Citrus aurantium L. and Its Flavonoids Regulate TNBS-Induced Inflammatory Bowel Disease through Anti-Inflammation and Suppressing Isolated Jejunum Contraction.

Inflammatory bowel disease (IBD) is a serious digestive system disease, for which the clinical therapeutic choices remain limited. Dried fruits of Citrus aurantium L. (CAL) are a traditional medicine used for regulation of the digestive system. The aim of this study was to identify the regulatory effects of CAL on IBD and to clarify the mechanism of the active compounds. In trinitrobenzene sulfonic acid-induced IBD rats, 125 to 500 mg/kg of oral CAL significantly alleviated weight loss and diarrhea, decreased colitis inflammatory cell infiltration, and inhibited pro-inflammatory cytokine production. The mechanisms of characteristic flavonoids in CAL were evaluated involving inflammation and intestine contraction aspects. Naringenin, nobiletin, and hesperetin showed anti-inflammatory effects on lipopolysaccharide-induced RAW cells. The mechanism may be related to the inhibition of the tumor necrosis factor-α (TNF-α)-induced nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway to suppress cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) expressions. Naringenin and nobiletin showed inhibitory effects on isolated jejunum contraction. The mechanism of naringenin is partly related to COX, NOS, inositol triphosphate (IP3), and finally, to decreased jejunum motility. This study demonstrated that CAL, and its flavonoids' regulatory effects on IBD through anti-inflammation and inhibition of intestine muscle contraction, can provide basic information on developing new drugs or supplements against IBD based on CAL.

Polyphenols from Blossoms of Citrus aurantium L. var. amara Engl. Show Significant Anti-Complement and Anti-Inflammatory Effects.

Citrus aurantium L. var. amara Engl. (CAVA) was traditionally used as a digestant or expectorant in China. Crude polyphenols (CAVAP-W) extracted from blossoms of CAVA were mainly composed of eriocitrin/neoeriocitrin, eriocitrin/neoeriocitrin, rhoifolin, hesperidin, naringin, rutin, veronicastroside, neohesperidin, and hesperetin by LC-MS analysis. CAVAP-W showed significant anticomplement and anti-inflammatory effects. Due to the close relationship between anticomplement and anti-inflammatory activity, the anti-inflammatory effect was further investigated and the results showed that CAVAP-W significantly suppressed production of interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), interleukin-1beta (IL-1ß), and mRNA expression of inducible nitric oxide synthase (iNOS), IL-6, TNF-α, IL-1ß, and cyclooxygenase-2 (COX-2) in lipopolysaccharides-stimulated RAW264.7 cells. Furthermore, CAVAP-W inhibited mitogen-activated protein kinase (MAPK) phosphorylation and NF-κB activation through suppressing nuclear translocation of nuclear factor-kappa B (NF-κB) P65, degradation and phosphorylation of IκBα, phosphorylation of IκKα/ß, c-Jun N-terminal kinase (JNK), and P38, and activation of COX-2, thereby exerting the anti-inflammatory effects.

Various Antioxidant Effects Were Attributed to Different Components in the Dried Blossoms of Citrus aurantium L. var. amara Engl.

Citrus aurantium L. var. amara Engl. (CAVA) was traditionally used as an edible and medicinal material in China. Total flavonoids (CAVAF), alkaloids (CAVAA), polysaccharides (CAVAP), coumarins (CAVAC), and neroli (CAVAO) were extracted from CAVA. Hesperidin, naringin, and neohesperidin composed 83.94% of CAVAF, and synephrine represented 50.56% of CAVAA. On the basis of 1,1-diphenyl-2-picrylhydrazyl radical (DPPH•), 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt radical cation (ABTS• +), hydroxyl radical (•OH), ferric-reducing antioxidant power (FRAP), and reducing power assays, the antioxidant activities of five components were comprehensively and comparatively investigated. CAVAF had a stronger DPPH• scavenging effect and FRAP and reducing power. CAVAP and CAVAA exhibited comparable •OH scavenging effects to vitamin C. CAVAA showed the highest ABTS• + scavenging activity. In conclusion, different constituents varied significantly toward different sources of free radicals and other oxidants. It is obvious that CAVA has various antioxidant effects, which are attributed to different components.

Two new phenolic glycosides isolated from the fruits of Citrus aurantium.

The present study was designed to investigate the chemical constituents of the fruit of Citrus aurantium L.. The compounds were isolated and purified by various chromatographic techniques, and their structures were elucidated on the basis of physicochemical properties and spectral data. Two new phenolic glycosides (compounds 1 and 2) were obtained and identified as 1-O-3, 5-dihydroxyphenyl-(6-O-4-hydroxybenzoyl)-ß-D-glucopyranoside (1) and 1-O-3, 5-dihydroxyphenyl-(6-O-3-methoxy-4-hydroxy benzoyl)-ß-D-glucopyranoside (2), respectively.