Fig. 22.1 Chemical structure of hesperidin isolated from Pericarpium Citri Reticulatae

venous ulcers and it is used for the treatment of chronic venous insufficiency, hemorrhoids and the prevention of postoperative thromboembolism (Kanaze et al. 2003).

We tested the hypothesis that hesperidin might affect development of DR through the regulation of NO or iNOS. To assess this, in this study, hesperidin was isolated from Pericarpium Citri Reticulatae of Newhall Citrus reticulata Blanco in order to explore the effect of hesperidin on expression of iNOS in cultured rabbit retinal pigment epithelial (RPE) cells under the condition of high glucose in vitro.

22.2 Materials and Methods

22.2.1Preparing Hesperidin Extract of Pericarpium Citri Reticulatae

Pericarpium Citri Reticulatae was cut into pieces, dried at 30–40◦C, and crushed into 1–2 mm particles by Chinese herbal medical pulverizer. The particles (100 mg) were weighted accurately and soaked by 500 ml distilled water for 30–45 min. After the particles absorbed water sufficiently, they were washed by distilled water to make them colorless and then filtered. They were carried out with 400 ml saturated calcareous water in the ultrasonic cleaning bath (Sonoc, PRC) which worked at

25 kHZ frequency at 25◦C for 30 min. After that, the liquid was adjusted to pH 13.0 by 5 mol/L NaOH and removed of dregs of Pericarpium Citri Reticulatae through the vacuum filtration. The filtrate was neutralized into pH 4.5 by 1 mol/L. When the crystals were separated from the filtrate, the crystals were obtained by the filtration and changed into the crude hesperidin at 70◦C by vacuum dehydration. The crude hesperidin was dissolved with 500 ml 50% ethanol containing 1% NaOH and filtered to remove the insoluble. The filtrate was shifted into pH 5.0 by 1 mol/L HCl in order to get the sediment. The sediment was washing by 50% ethanol and then by diluted water to approach the neutrality. Finally, the refined hesperidin was extracted after the sediment was arescent at 70◦C by vacuum dehydration.

22.2.2Identification of Hesperidin by High Performance Liquid Chromatogram (HPLC)

Identification of hesperidin was carried out on HPLC system (Agilent HP1100, USA), consisting of the G1311A pump and G1311A UV spectrophotometric detector. Data collection and integration were accomplished using a ChromatoSolution. The analytes were determined at room temperature on an analytical column (Hypersil C18, 4.6 × 250 mm, 5 μm). The mobile phase consisted of a mixture of acetonitrile, water, and methanol (20:79.6:0.4, v/v). The mobile phase was passed under vacuum through a 0.45 μm membrane filter and degassed before use. The analysis was carried out at a flow rate of 1.5 ml/min with the detection wavelength set at 283 nm.

22.2.3 Cell Culture

For the isolation of RPE cells, rabbit eyes were cut circumferentially through the sclera approximately 1 mm posterior to the ora serrata; after the vitreous was aspirated and the retina gently separated from the RPE cell layer, the eye cup was washed with Dulbecco s minimum essential medium (DMEM; Gibco, USA) and incubated at 37◦C with 0.05% trypsin and 0.02% ethylenediaminetetraacetic acid (EDTA; Sigma, USA). After 1 h the trypsin solution was aspirated from the eye cup and replaced with DMEM supplemented with 20% fetal bovine serum (FBS; Sijiqing, PRC). The RPE cells, released by gentle pipetting, were transferred to a 75 cm2 tissue culture flask containing DMEM supplemented with 20% FBS without antibiotics. The cells were cultured at 37◦C in a humidified atmosphere of 5% CO2 and 95% air; the medium was changed every 4 days thereafter. After 4–5 weeks, when the cells had reached confluence, they were passaged by trypsinization. The third to fifth passage cells were used for all experiments.

22.2.4 MTT Cell Viability Assay

Rabbit RPE cells were plated at a density of 5 × 104 cells/well in a 96 well plate to determine the protection of hesperidin. The cells were divided into 6 groups including the control group cultured in DMEM, the model group cultured in DMEM containing 33 mmol/L glucose without any drug and four experimental groups which were exposed to hesperidin at the concentration of 10, 20, 40 and 80 mg/L at 37◦C under 5% CO2 for 2 h and then cultured in DMEM containing 33 mmol/L glucose. After incubated cells in the presence of hesperidin for 48 h, viable cells were stained with MTT [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl-tetrazolium bromide] (0.5 mg/ml) for 4 h. The medium was then removed and the formazan crystals produced were dissolved by adding dimethylsulfoxide (200 μl). The optical density (A value) at 490 nm was measured by the microplate reader (Thermo, USA).

22.2.5 Assay of NO Production

The levels of NO produced were monitored by determining nitrite levels in culture medium. This was performed by mixing medium with Griess reagent (1% sulfanilamide, 0.1% N-1-naphthylenediamine dihydrochloride, and 2.5% phosphoric acid). 100 μl of medium was collected at the indicated time points for the determination of nitrite level. Absorbance was measured at 540 nm after incubation for 10 min using the 721 spectrophotometer (Tuopu, PRC).

22.2.6 Cellular Immunohistochemistry of iNOS

The glutaraldehyde-fixed RPE cells were reduced with sodium borohydride (0– 1%) for 10 min. They were incubated in 0.3% hydrogen peroxide (H2O2) in 90% methanol for 30 min to inactivate the endogenous peroxidase activity. After washing in 0.01 M PBS, sections were blocked for 4 h in 10% goat normal serum and then incubated in the primary antiserum for iNOS (1:500 dilution, rabbit polyclonal, Zhongshan Goldenbridge, PRC). The secondary antibody used was biotinylated goat anti-rabbit IgG (1:200 dilution, Zhongshan Goldenbridge, PRC) for 6 h at 4◦C. The antigen-antibody complex was localized employing an avidin-biotin-peroxidase system (ABC kit, Zhongshan Goldenbridge, PRC) according to the manufacturer’s instructions. Peroxidase staining was developed using 3,3 -diaminobenzidine tetrahydrochloride (0.06%) as chromogen, in 0.1 M acetate-imidazole buffer (pH 7.4) together with H2O2 (0.06%) and nickel sulphate (0.5%). To demonstrate the specificity of the antibody, negative control was incubated and processed similarly but with theomission of the primary antiserum. The results of iNOS expressed in RPE cells were present of integral optical density (IOD) using the semiquantitative analysis by computer image analytical system (Leica, Germany).