The leaves and twigs of O. schwerinae were collected from Kunming, Yunnan Province, China, in April 2011, and identified by Professor Joo Hwan Kim (Gachon University, Korea). A voucher specimen (no. DiAB-141) was deposited in the Herbarium of the Diabetic Complications Research Team, Korea Institute of Oriental Medicine, Korea. Air-dried twigs and leaves of O. schwerinae (4 kg) were extracted with 12 l ethanol (EtOH) three times by maceration. The combined extracts were filtered and concentrated using a vacuum evaporator, and 104.16 g of the EtOH extract was obtained. The EtOH extract was suspended in 500 ml H₂O. This H₂O suspension was fractionated by liquid-liquid partitioning using n-hexane (500 ml), EtOAc (500 ml) and n-BuOH (500 ml) to yield an n-hexane fraction of 6.30 g, an EtOAc fraction of 27.0 g and an n-BuOH fraction of 10.08 g. All of the factions were tested for AGE formation. The EtOAc fraction (27 g), which had the highest inhibitory effect on AGE formation, was loaded into a column (7×57 cm) that was packed with 70–230 mesh silica gel. Silica gel (Merck Millipore, Darmstadt, Germany) chromatography was performed using a mobile phase of CHCl₃ and MeOH (gradient of 40:1-0:1, v/v); ten fractions were obtained. The fifth fraction (700 mg) was loaded onto a column (60x2.5 cm) packed with Sephadex LH-20 gel (GE Healthcare Life Sciences, Chalfont, UK), and column chromatography was performed using a mobile phase of MeOH and distilled H₂O (gradient of 1:1-1:0, v/v). A total of OSSC1E-K19 (10 mg; yield, 0.04%) was isolated. The structure of OSSC1E-K19 was identified as 4-hydroxy-3′,5′-dimethoxy-(1,1′-biphenyl)-3-O-β-D-glucopyranoside by comparing its nuclear magnetic resonance (NMR) and high resolution electrospray ionization mass (HRESIMS) data with those in the literature (11); 1H and 13C-NMR spectra were obtained using a Bruker Avance³⁰⁰ NMR spectrometer (Bruker AXS, Karlsruhe, Germany) with tetramethylsilane as an internal standard, and HRESIMS was recorded on a Shimadzu LCMS-IT-TOF spectrometer (Shimadzu Corporation, Kyoto, Japan). The chemical structure of OSSC1E-K19 is presented in Fig. 1.

Ten milligrams per milliliter bovine serum albumin (BSA; Roche Diagnostics, Basel, Switzerland) in 50 mM phosphate buffer (pH 7.4) containing 0.01% sodium azide (to avoid microbial contamination; cat. no. S-8032, Sigma-Aldrich, St. Louis, MO, USA) was added to a 0.2 M glucose and fructose solution (Sigma-Aldrich); this solution was added to the samples (1–150 µM). Aminoguanidine (AG; cat. no. 396494; Sigma-Aldrich) was used as a positive inhibitor. After incubation for 14 days at 37°C, AGEs-specific fluorescence was determined using a spectrofluorometer (Synergy HT; BIO-TEK, Winooski, VT, USA; excitation at 370 nm and emission at 440 nm). The concentration leading to 50% inhibition of AGE formation (IC₅₀) was determined.

AGE-BSA (Wako Pure Chemical Industries, Ltd., Osaka, Japan) was conjugated with horseradish peroxidase (HRP) using a peroxidase labeling kit-NH2 (Dojindo Molecular Technologies, Inc., Kumamoto, Japan). HRP-labeled AGE-BSA was incubated with or without OSSC1E-K19 or AG in collagen-coated microtiter plates. HRP-labeled AGE-BSA was incubated to react with collagen at 37°C for 4 h. After washing with a solution of 0.05% Triton-X 100 in phosphate-buffered saline (PBS), 100 µl 3,3′,5,5′-tetramethylbenzidine chromogen (cat. no. T4444; Sigma-Aldrich) was added followed by incubation for 15 min. The absorbance at 450 nm was determined using an absorbance plate reader (Synergy HT) after the addition of 40 µl 1 N H₂SO₄.

AGE-RSA was synthesized as previously described (12). Non-modified RSA (fraction V, low-endotoxin; Sigma-Aldrich) was used as the control. The components of AGE-RSA were determined using a glucose-derived AGE ELISA kit (Cosmo Bio, Tokyo, Japan). The modified levels of AGE-RSA were nearly 200-fold higher than those in the non-modified control. No endotoxins were detected using the E-Toxate kit (cat. no. ET0200-1KT; Sigma-Aldrich).

Injection of AGE-RSA was performed as previously described (13). A total of 40 male eight week-old Sprague-Dawley rats (Orient Bio, Inc., Sungnam, Korea) were used in the present study. Rats were housed four per cage in a sterile humidified environment (50–60%) at 23±1°C, with a 12-h light/dark cycle. Food and water were provided ad libitum throughout the experiment. The rats were divided into five groups: 6 µg RSA-injected normal rats; rats injected with 6 µg AGE-RSA i.v.; and rats injected with AGE-RSA i.v. and treated with different concentrations of OSSC1E-K19 (50, 100 or 150 µM). For the negative control, 150 µM OSSC1E-K19 was injected. Three days after i.v. injection, the rats were anesthetized and sacrificed with an overdose of zolazepam (10 mg/kg; Virbac, Carros, France). The present study was approved by the Institutional Animal Care and Use Committee of the Korea Institute of Oriental Medicine (protocol no. 12–079; Daejeon, Korea).

The rats were deeply anesthetized by intraperitoneal injection of zolazepam (10 mg/kg; Virbac), after which PBS containing fluoresceindextran (FD40S; Sigma-Aldrich) was injected into the left ventricle of the heart. The retinal flat was mounted, and the fluorescent images were captured using an Olympus BX51 microscope with a DP71 digital camera (Olympus, Tokyo, Japan).

Retinal sections were de-waxed and re-hydrated by sequential immersion. The slides were boiled in 10 mM sodium citrate buffer (pH 6.0) at 121°C for 10 min. The slides were incubated with a mouse anti-VEGF antibody (1:500; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) for 2 h. After washing, the sections were labeled with fluorescein isothiocyanate-conjugated donkey anti-mouse immunoglobulin G (1:3,000; Santa Cruz Biotechnology, Inc.) for 1 h at room temperature. Finally, the sections were mounted in fluorescent mounting medium (Dako North America, Inc., Carpinteria, CA, USA) containing DAPI counterstain. The sections were observed and images were captured by fluorescence microscopy (BX51; Olympus, Tokyo, Japan).

Retinas were homogenized (Precellys 24; Bertin Technologies, Saint-Quentin en Yveline, France) in solutions containing 250 mM sucrose, 1 mM ethylenediaminetetraacetic acid, 0.1 mM phenylmethylsulfonyl fluoride (Sigma-Aldrich) and 20 mM potassium phosphate buffer (Sigma-Aldrich; pH 7.6). Retinal protein (20 µg) was resolved by 12% SDS-PAGE and transferred onto a polyvinylidene difluoride membrane (Bio-Rad Laboratories, Inc., Hercules, CA, USA) and the membranes were incubated with the following primary monoclonal antibodies overnight at 4°C: Antibodies against VEGF (1:1,000; cat. no. ab1316; Abcam, San Francisco, CA, USA) and occludin (1:1,000; cat. no. 711500; Invitrogen Life Technologies, Inc., Carlsbad, CA, USA) were used. Monoclonal anti-β-actin antibody (1:3,000; cat. no. A1978) was purchased from Sigma-Aldrich. Either goat anti-mouse IgG HRP (cat. no. sc-2005) or anti-rabbit IgG HRP-conjugated antibody (cat. no. sc-2004; 1:2,000; Santa Cruz Biotechnology, Inc.) was used as a secondary antibody. Immunoreactive bands were visualized using an enhanced chemiluminescence western blotting detection system (Amersham Bioscience, Amersham, NJ, USA). Images were captured and quantification was performed using a LAS-3000 (Fujifilm, Tokyo, Japan).

Values are expressed as the mean ± standard error of the mean. Statistical analysis was performed using one-way analysis of variance followed by Tukey's multiple comparisons test or an unpaired Student's t-test. GraphPad Prism 6.0 (GraphPad Inc., La Jolla, CA, USA) was used for all statistical analyses. P<0.05 was considered to indicate a statistically significant difference between values.

OSSC1E-K19 was examined for its ability to inhibit AGE formation. As shown in Table I, OSSC1E-K19 dose-dependently inhibited the formation of AGE-BSA (IC₅₀, 118.49±4.85 µM). The inhibitory activity of OSSC1E-K19 was greater than that of AG (IC₅₀, 1,040.70±44.17 µM). In addition, OSSC1E-K19 dose-dependently inhibited the cross-linking of AGE-BSA with collagen, and the IC₅₀ value of OSSC1E-K19 was 8.20±0.60 mM (Fig. 2). AG also reduced cross-linking between AGE-BSA and collagen (IC₅₀, 1.39±0.13 mM).

A major clinical hallmark of DR is enhanced vascular leakage culminating in overt BRB breakage (14). To test the ability of OSSC1E-K19 to inhibit AGE-induced vascular permeability, the present study performed fluorescein angiography in rats that were injected with AGE-RSA i.v.. As shown in Fig. 3, the fluorescence intensity was enhanced, with dye leakage occurring throughout the entire retina in the AGE-RSA-injected eyes; however, these leaks were restricted to the vasculature in the saline-injected normal rat eyes and in OSSC1E-K19-injected eyes.

The inhibitory effects of OSSC1E-K19 on retinal VEGF expression in AGE-RSA-injected eyes were assessed by determining retinal VEGF expression using immunofluorescence staining and western blot analysis. In AGE-RSA-injected eyes, immunofluorescence staining for VEGF revealed that VEGF (green) was present in the ganglion cell layer, while treatment with OSSC1E-K19 significantly decreased retinal VEGF expression in AGE-RSA-injected eyes (Fig. 4A). These immunofluorescence staining results were confirmed by western blot analysis. As shown in Fig. 4B, AGE-RSA treatment led to a significant increase in VEGF expression compared with that in the saline-injected normal group. However, treatment with OSSC1E-K19 markedly inhibited the expression of VEGF in AGE-RSA-injected eyes. Similarly, treatment with OSSC1E-K19 almost completely eliminated the increase in retinal vascular permeability that was observed in AGE-RSA-injected eyes (Fig. 3). These results strongly suggested that OSSC1E-K19 inhibits retinal vascular permeability via the suppression of retinal VEGF expression.

The loosening of TJs causes BRB breakdown (15). The present study evaluated the expression of occludin, a well-known TJ protein. AGE-RSA-injected eyes exhibited significantly lower levels of occludin than those of saline-injected normal mice. However, the decreased occludin expression in the AGE-RSA-injected eyes was restored by OSSC1E-K19 treatment (Fig. 5). These findings suggested that OSSC1E-K19 blocks vascular leakage, possibly by stabilizing junctional proteins.