Dalia Hamdan

Inhibition of P-glycoprotein activity by limonin and other secondary metabolites from Citrus species in human colon and leukaemia cell lines.

P-glycoprotein (P-gp), a membrane transporter encoded by the MDR1 gene in human cells, mediates drug efflux from cells and plays a major role in causing multidrug resistance; which is one of the most accepted mechanisms for failure of chemotherapy in cancer treatment. In this study, we investigated the effects of nine naturally occurring compounds isolated from Citrus jambhiri Lush and Citrus pyriformis Hassk (Rutaceae) for their potential to modulate the activity of P-gp in the multidrug-resistant human leukaemia cell line CEM/ADR5000. Limonin, deacetylnomilin, hesperidin, neohesperidin, stigmasterol and ss-sitosterol-O-glucoside inhibited the efflux of the P-gp substrate rhodamine 123 in a concentration-dependent manner. Some of these compounds were more active than verapamil, which was used as a positive control. Treatment of drug-resistant Caco-2 cells with the most active C. jambhiri and C. pyriformis compounds increased their sensitivity to doxorubicin and completely reversed doxorubicin resistance, which agrees with a decreased P-gp activity. Limonin was the most potent P-glycoprotein inhibitor - when it was applied at a non-toxic concentration of 20 microM, it significantly enhanced doxorubicin cytotoxicity 2.98-fold (P<0.001) and 2.2-fold (P<0.001) in Caco2 and CEM/ADR5000 cells, respectively. These isolated Citrus compounds could be considered as good candidates for the development of novel P-gp/MDR1 reversal agents which may enhance the accumulation and efficacy of chemotherapy agents.

Effect of hesperidin and neohesperidin from bittersweet orange (Citrus aurantium var. bigaradia) peel on indomethacin-induced peptic ulcers in rats.

Hesperidin and neohesperidin are the major flavanones isolated from bittersweet orange. It was recently reported that they have potent anti-inflammatory effects in various inflammatory models. In the present study, the effects of hesperidin and neohesperidin on indomethacin-induced ulcers in rats and the underlying mechanisms were investigated. Gastric ulcers were induced in rats with a single dose of indomethacin. The effects of pretreatment with hesperidin and neohesperidin were assessed in comparison with omeprazole as reference standard. Ulcer index, gene expression of gastric cyclooxygenase-2 (COX-2), tumor necrosis factor alpha (TNF-α), lipid peroxidation product, malondialdhyde (MDA), and reduced glutathione (GSH) content in stomach were measured. Furthermore, gross and histopathological examination was performed. Our results indicated that both hesperidin and neohesperidin significantly aggravated gastric damage caused by indomethacin administration as evidenced by increased ulcer index and histopathological changes of stomach.

Secondary metabolites of ponderosa lemon (Citrus pyriformis) and their antioxidant, anti-inflammatory, and cytotoxic activities.

Column chromatography of the dichloromethane fraction from an aqueous methanolic extract of fruit peel of Citrus pyriformis Hassk. (Rutaceae) resulted in the isolation of seven compounds including one coumarin (citropten), two limonoids (limonin and deacetylnomilin), and four sterols (stigmasterol, ergosterol, sitosteryl-3-β-D-glucoside, and sitosteryl-6ʹ- O-acyl-3-β-D-glucoside). From the ethyl acetate fraction naringin, hesperidin, and neohesperidin were isolated. The dichloromethane extract of the defatted seeds contained three additional compounds, nomilin, ichangin, and cholesterol. The isolated compounds were identified by MS (EI, CI, and ESI), 1H, 13C, and 2D-NMR spectral data. The limonoids were determined qualitatively by LC-ESI/MS resulting in the identification of 11 limonoid aglycones. The total methanolic extract of the peel and the petroleum ether, dichloromethane, and ethyl acetate fractions were screened for their antioxidant and anti-inflammatory activities. The ethyl acetate fraction exhibited a significant scavenging activity for DPPH· free radicals (IC50 = 132.3 μg/mL). The petroleum ether fraction inhibited 5-lipoxygenase with IC50 = 30.6 μg/mL indicating potential anti-inflammatory properties. Limonin has a potent cytotoxic effect against COS7 cells [IC50 = (35.0 ± 6.1) μM] compared with acteoside as a positive control [IC50 = (144.5 ± 10.96) μM]

Chemical composition of the essential oils of variegated pink-fleshed lemon (Citrus x limon L. Burm. f.) and their anti-inflammatory and antimicrobial activities.

The volatile secondary metabolites of essential oils from fruit peel and leaves of variegated pink-fl eshed lemon (Citrus x limon) were investigated using GLC and GLC-MS (gas-liquid chromatography-mass spectroscopy). Altogether 141 compounds were identified and quantified, accounting for 99.59% and 96.33% of the total hydrodistilled peel and leaf oil, respectively. Limonene occurred in higher amounts in fruit peel (52.73%) than in leaf oil (29.13%). Neral (12.72%), neryl acetate (8.53%), ρ-menth-1-en-7-al (4.63%), β-pinene (6.35%), and nerol (4.42%) were the most abundant constituents in leaf oil, whereas γ-terpinene (9.88%), β-pinene (7.67%), geranial (4.44%), and neral (3.64%) dominated in the fruit peel oil. The antioxidant, anti-inflammatory, antitrypanosomal, and antimicrobial activities of the fruit peel essential oil were evaluated. The oil had a low antioxidant activity with an IC50 value of (26.66 ± 2.07) mg/ml as compared to the efficient antioxidant ascorbic acid [IC50 (16.32 ± 0.16) μg/ml]. The oil moderately inhibited soybean 5-lipoxygenase (5-LOX) with an IC50 value of (32.05 ± 3.91) μg/ml and had moderate antitrypanosomal activity [IC50 (60.90 ± 0.91) μg/ml]. In addition, moderate antimicrobial activities were detected against Gram-positive bacteria (Bacillus subtilis, Staphylococcus capitis, Micrococcus luteus), one Gram-negative bacterium (Pseudomonas fluorescens), and yeasts (Saccharomyces cerevisiae, Candida parapsilosis)

Secondary Metabolites Isolated from Dichloromethane Fraction of Rough Lemon Stem and Hepatoprotective Effect of Limonianin.

Aim: To investigate the dichloromethane fraction obtained from an aqueous methanol extract of green stems of Citrus jambhiri Lush. and evaluate the hepatoprotective effect of limonianin. Study Design: Isolation and identification of secondary metabolite and hepatoprotective activity of limonianin. Place and Duration of Study: Faculty of Pharmacy, Zagazig University and Institute of Pharmacy and Molecular Biotechnology, Heidelberg University, between February 2012 and January 2014. Methodology: Psyllic acid, sinensetin, nobiletin, 6-demethoxytangeretin, limonianin, hesperetin, quercetin were isolated from the green stem of Citrus jambhiri. Their identity was unambiguously confirmed via different spectroscopic methods (UV, MS, H NMR, C NMR, H-COSY, HSQC and HMBC). The liver enzymes alanine aminotransferase (ALT), Original Research Article British Journal of Pharmaceutical Research, 4(16): 1963-1975, 2014 1964 aspartate aminotransferase (AST), superoxide dismutase (SOD), malondialdehyde (MDA) and total antioxidant capacity (TAC) were used to evaluate the hepatoprotective effect of limonianin. Results: Limonianin exhibited an equal or even better hepatoprotective activity than the known and medicinally used flavonoid mixture silymarin on human hepatoma cells (HepG2) against D-galactosamine-induced hepatotoxicity. Limonianin at concentration 5 μM exhibited a reduction of ALT, AST and MDA with 46.50, 42.10 and 24.36%, respectively while SOD and TAC were elevated with 38.56 and 391.67%, respectively compared with D-galactosamine (D-GaIN) treated cells. At concentration of 10 μM, limonianin shows elevation of SOD and TAC with 74.56 and 791.67%, respectively compared with silymarin (63 and 766.67%, respectively). Conclusion: The findings of this study showed that limonianin could be used as ideal hepatoprotective agent.