Investigation of the interaction for three Citrus flavonoids and α-amylase by surface plasmon resonance.

Flavonoids, a class of natural drugs with broad biological activity, exhibit inhibitory effect on α-amylase. Citrus peel is a good source of flavonoids. The real-time interactions between three Citrus flavonoids (naringin, neohesperidin, hesperidin) and α-amylase were investigated by surface plasmon resonance biosensor, and were compared with the α-amylase inhibitors, acarbose. These results showed the binding affinities of naringin, neohesperidin and hesperidin with α-amylase reach the highest at pH6 with KD values of 2.27±0.18mM, 3.09±0.20mM and 3.51±0.09mM, and can be reinforced with 0.2M NaCl and 0.1M CaCl2, respectively. The results of 1, 1-diphenyl-2-picryl hydrazyl radical assay indicate that the antioxidant activities of naringin, neohesperidin and hesperidin are significantly inhibited by interacting with α-amylase, and the inhibition percentage are 47.61±0.034%, 22.81±0.037% and 21.01±0.051%, respectively. Additionally, it is found that both the number and the position of hydroxyl group play an important role in the interaction of three Citrus flavonoids and α-amylase. These results provide useful information for rapid screening inhibitors of α-amylase from plant-based food.

Inhibitory effects of flavonoids on biofilm formation by Staphylococcus aureus that overexpresses efflux protein genes.

This study evaluated the efficacy of glycone (myricitrin, hesperidin and phloridzin) and aglycone flavonoids (myricetin, hesperetin and phloretin) in inhibiting biofilm formation by Staphylococcus aureus RN4220 and S. aureus SA1199B that overexpress the msrA and norA efflux protein genes, respectively. The minimum inhibitory concentration (MIC) and minimum biofilm inhibitory concentration (MBIC50 - defined as the lowest concentration that resulted in ≥50% inhibition of biofilm formation) of flavonoids were determined using microdilution in broth procedures. The flavonoids showed MIC >1024 µg/mL against S. aureus RN4220 and S. aureus SA1199B; however, these compounds at lower concentrations (1-256 µg/mL) showed inhibitory effects on biofilm formation by these strains. Aglycone flavonoids showed lower MBIC50 values than their respective glycone forms. The lowest MBIC50 values (1 and 4 µg/mL) were observed against S. aureus RN4220. Myricetin, hesperetin and phloretin exhibited biofilm formation inhibition >70% for S. aureus RN4220, and lower biofilm formation inhibition against S. aureus SA1199B. These results indicate that sub-MICs of the tested flavonoids inhibit biofilm formation by S. aureus strains that overexpress efflux protein genes. These effects are more strongly established by aglycone flavonoids.

Hesperidin alleviates rat postoperative ileus through anti-inflammation and stimulation of Ca(2+)-dependent myosin phosphorylation.

AIM: Postoperative ileus (POI) is a postoperative dysmotility disorder of gastrointestinal tract, which remains one of the most perplexing problems in medicine. In the present study we investigated the effects of hesperidin, a major flavonoid in sweet oranges and lemons, on POI in rats. METHODS: SD rats were administered hesperidin (5, 20, and 80 mg·kg(-1)·d(-1), ig) for 3 consecutive days. POI operation (gently manipulating the cecum for 1 min) was performed on d 2. The gastrointestinal motility and isolated intestinal contraction were examined 1 d after the operation. Then the myosin phosphorylation and inflammatory responses in cecum tissue were assessed. Smooth muscle cells were isolated from rat small intestine for in vitro experiments. RESULTS: The gastric emptying and intestinal transit were significantly decreased in POI rats, which were reversed by administration of hesperidin. In ileum and cecum preparations of POI rats in vitro, hesperidin (2.5-160 µmol/L) dose-dependently increased the spontaneous contraction amplitudes without affecting the contractile frequency, which was blocked by the myosin light chain kinase (MLCK) inhibitor ML-7 or verapamil, but not by TTX. Furthermore, administration of hesperidin increased the phosphorylation of MLC20 in the cecum tissue of POI rats. Moreover, administration of hesperidin reversed the increased levels of inflammatory cytokines, iNOS and COX-2 in cecum tissue of POI rats. In freshly isolated intestinal smooth muscle cells, hesperidin (5-80 µmol/L) dose-dependently increased the intracellular Ca(2+) concentration as well as the phosphorylation of MLC20, which was abrogated by ML-7 or siRNA that knocked down MLCK. CONCLUSION: Oral administration of hesperidin effectively alleviates rat POI through inhibition of inflammatory responses and stimulation of Ca(2+)-dependent MLC phosphorylation.

Hesperetin stimulates differentiation of primary rat osteoblasts involving the BMP signalling pathway.

Hesperidin found in citrus fruits has been reported to be a promising bioactive compound for maintaining an optimal bone status in ovariectomized rodent models. In this study, we examined the capacity of hesperetin (Hp) to affect the proliferation, differentiation and mineralization of rodent primary osteoblasts. Then, the impact of Hp on signalling pathways known to be implicated in bone formation was explored. We exposed osteoblasts to physiological concentrations of 1 microM Hp (Hp1) and 10 microM Hp (Hp10). Neither proliferation nor mineralization was affected by Hp at either dose during 19 days of exposure. Hp at both doses enhanced differentiation by significantly increasing alkaline phosphatase (ALP) activity from Day 14 of exposure (Day 19: Hp1: +9%, Hp10: +14.8% vs. control; P<.05). However, Hp did not induce an obvious formation of calcium nodules. The effect of Hp10 on ALP was inhibited by addition of noggin protein, suggesting a possible action of this flavanone through the bone morphogenetic protein (BMP) pathway. Indeed, Hp10 significantly induced (1.2- to 1.4-fold) mRNA expression of genes involved in this signalling pathway (i.e., BMP2, BMP4, Runx2 and Osterix) after 48 h of exposure. This was strengthened by enhanced phosphorylation of the complex Smad1/5/8. Osteocalcin mRNA level was up-regulated by Hp only at 10 microM (2.2 fold vs. control). The same dose of Hp significantly decreased osteopontin (OPN) protein level (50% vs. control) after 14 days of culture. Our findings suggest that Hp may regulate osteoblast differentiation through BMP signalling and may influence the mineralization process by modulating OPN expression.

Pharmacological exploration of the sedative mechanism of hesperidin identified as the active principle of Citrus sinensis flowers.

The infusion of flowers of several species of Citrus genera is used as a sedative to treat insomnia in Mexican traditional medicine. The aims of this study were to investigate the sedative effect of different extracts of flowers of Citrus sinensis (L.) Osbeck (Rutaceae) and describe the pharmacological action mechanism of the sedative active compounds of this plant. The methanol and dichloromethane extracts, obtained from the flowers of Citrus sinensis (L.) Osbeck (Rutaceae), showed a dose-dependent sedative effect in the exploratory cylinder model in mice, with ED50 (ip) values of 47.04+/-12.03 mg/kg and 129.15+/-21.25 mg/kg, respectively. Hesperidin (ED50=11.34+/-2.48 mg/kg) was identified in the methanol extract as the sedative active principle of this plant. The pre-treatment with atropine (1 mg/kg I. P.), flumazenil (2 mg/kg I. P.), clonidine (0.01 mg/kg I. P.), isoproterenol (0.3 mg/kg I. P.), haloperidol (0.3 mg/kg I. P.), WAY 100 635 (3 mg/kg I. P.), P-chlorophenylalanine (250 mg/kg I. P., twice per day for 2 days), forskolin (3 mg/kg I. P.) and rolipram (0.173 mg/kg I. P.) did not modify the sedative effect of 30 mg/kg hesperidin. However, the sedative effect of this compound was potentiated by yohimbine (1.25 mg/kg I. P.) and buspirone (1 mg/kg I. P.), and reverted by pretreatment with aminophylline (30 mg/kg I. P.), caffeine (30 mg/kg I. P.) and several doses of 1,3-dimethyl-8-phenylxanthine (10, 30 and 54.7 mg/kg I. P.). These results suggest that adenosine receptors might be involved in the sedative action of hesperidin, identified as the active principle of the flowers of Citrus sinensis.