Bioactive compounds isolated from apple, tea, and ...

Phytomedicine 23 (2016) 200–213

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Bioactive compounds isolated from apple, tea, and ginger protect against dicarbonyl induced stress in cultured human retinal epithelial cells Chethan Sampath a, Yingdong Zhu b, Shengmin Sang b, Mohamed Ahmedna a,∗ a b

Department of Health Sciences, College of Arts & Sciences, Qatar University, Doha 2713, Qatar Center for Excellence in Post-Harvest Technologies, North Carolina Agricultural and Technical State University, NC 28081, United States

a r t i c l e

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Article history: Received 20 June 2015 Revised 1 December 2015 Accepted 21 December 2015

Keywords: Advanced glycation end products Methylyglyoxal Bioactive food compounds HRPE cells Oxidative stress Diabetes

a b s t r a c t Background: Methylglyoxal (MGO) is known to be a major precursor of advanced glycation end products (AGEs) which are linked to diabetes and its related complications. Naturally occurring bioactive compounds could play an important role in countering AGEs thereby minimizing the risk associated with their formation. Methods: In this study, eight specific bioactive compounds isolated from apple, tea and ginger were evaluated for their AGEs scavenging activity using Human Retinal Pigment Epithelial (H-RPE) cells treated with MGO. Results: Among the eight specific compounds evaluated, (-)-epigallocatechin 3-gallate (EGCG) from tea, phloretin in apple, and [6]-shogaol and [6]-gingerol from ginger were found to be most effective in preventing MGO-induced cytotoxicity in the epithelial cells. Investigation of possible underlying mechanisms suggests that that these compounds could act by modulating key regulative detoxifying enzymes via modifying nuclear factor-erythroid 2-related factor 2 (Nrf2) function. MGO-induced cytotoxicity led to increased levels of AGEs causing increase in Nε -(Carboxymethyl) lysine (CML) and glutathione (GSH) levels and over expression of receptor for advanced glycation end products (RAGE). Data also showed that translocation of Nrf2 from cytosol to nucleus was inhibited, which decreased the expression of detoxifying enzyme like heme oxygenase-1 (HO-1). The most potent bioactive compounds scavenged dicarbonyl compounds, inhibited AGEs formation and significantly reduced carbonyl stress by Nrf2 related pathway and restoration of HO-1 expression. Conclusions: These findings demonstrated the protective effect of bioactive compounds derived from food sources against MGO-induced carbonyl stress through activation of the Nrf2 related defense pathway, which is of significant importance for therapeutic interventions in complementary treatment/management of diabetes-related complications. © 2016 Published by Elsevier GmbH.

Introduction Diabetes is one of the chronic diseases associated with obesity, which is an emerging global epidemic. The International Diabetes Federation reported an alarming increase of obesity in the Middle East and North Africa (MENA) region where Kuwait, Qatar and

Abbreviations: MGO, Methylglyoxal; AGEs, Advanced glycation end products; HRPE, Human Retinal Pigment Epithelial cells; CML, ε -Carboxymethyl lysine; GSH, Glutathione; RAGE, Receptor for advanced glycation end products; HO-1, Heme oxygenase-1; Nrf2, Nuclear factor-erythroid 2-related factor 2; AG, Aminoguanidine; EGCG, (-)-epigallocatechin 3-gallate; ECG, (-)-epicatechin 3-gallate; EC, (-)epicatechin; PH, Phloretin. ∗ Corresponding author. Tel.: +974 44034848; fax: +974 4403 4501. E-mail addresses: [email protected] (C. Sampath), [email protected] (M. Ahmedna). http://dx.doi.org/10.1016/j.phymed.2015.12.013 0944-7113/© 2016 Published by Elsevier GmbH.

Saudi Arabia, top the list with prevalence rates in the range 23– 24% (IDF 2013). During prolonged incubation and at high concentrations of glucose, it reacts with proteins/lipids non enzymatically generating reactive dicarbonyl intermediates like α -oxoaldehydes such as Methylglyoxal (MGO) and glyoxal (GO) which induce the formation of advanced glycation end products (AGEs) (Hegab et al. 2012). These intermediate compounds, once generated, react quickly with amino acids like lysine, arginine and cysteine on proteins exerting toxicity to cells and tissues (Nagaraj et al. 2002). It has been reported that accumulation of AGEs exacerbates diabetes-related complications affecting the eyes, kidneys, nervous system and blood vessels (Hegab et al. 2012). Hence, trapping of MGO and GO result in limiting the formation of AGEs and could serve as a basis for effective prevention/management of diabetic complications for a long term (Rahbar 2007). The mechanism

C. Sampath et al. / Phytomedicine 23 (2016) 200–213

reported in the literature suggests that AGEs bind to a receptor for advanced glycation end products (RAGE) which, in turn, impairs the vessel functions inducing a series of inflammatory processes, such as increasing the levels of oxidative stress, up-regulating the expression of adhesion molecules, enhancing intima proliferation and promoting angiogenesis (Goldin et al. 2006). In vitro and in vivo studies suggest that limiting the expressions of RAGE in vascular cells could modulate the levels of various proinflammatory mediators and prevent the development of vascular dysfunction (Figarola et al. 2007). Nε -(Carboxymethyl) lysine (CML) and Nε (Carboxyethyl) lysine (CEL) are the best-characterized AGEs generated from GO and MGO, respectively. The levels of CML and CEL in diabetic patients are about 2–3 folds higher than those in healthy people (Teerlink et al. 2004). The generation of CML has been associated with progression of inflammation, atherosclerotic calcification, and diabetes (Hofmann et al. 2002). Earlier studies have shown that Nrf2 phosphorylation activates antioxidant responsive element (ARE), glutamate cysteine ligase (GCL) and HO-1 which converts MGO into lactic acid by glyoxalase-1 (Desai & Wu 2007; Vander Jagt 2008; Keum, Owuor, Kim, Hu, & Kong 2003; Li & Kong 2009). Naturally occurring compounds, especially polyphenols, are of great interest considered as potential candidates to prevent the formation of AGEs, especially given their proven safety and efficacy (compared to synthetic compounds) in the prevention of cancer, hyperglycemia, heart disease and aging. Phenolic compounds like quercetin have been reported to possess a strong ability to attenuate oxidative damage by activating Nrf2 (Yeh & Yen 2006; Weng, Chen, Yeh & Yen 2011). For instance, higher intakes of quercetin have been found to reduce the risk of type 2 diabetes (Knekt et al. 2002). Apples being a rich source of polyphenols are considered to be beneficial in promoting good health. Qi et al. (2006), reported that consumption of whole apples and cereal bran decreased plasma glucose levels in non-insulin-dependent diabetic patients. It was also shown that women who had consumed an apple or more a day reduced their risk for type 2 diabetes by up to 28% (Song et al. 2005). Tea consumption regularly has been reported to reduce cancer, heart disease, obesity and diabetes in humans, animal models, and cell lines (Grove & Lambert 2010; Naito & Yoshikawa 2009). For example, the consumption of Oolong tea has been reported to decrease plasma glucose and fructosamaine levels in diabetic patients (Hosoda et al. 2003) while green tea consumption promoted glucose metabolism in healthy human volunteers and reduced blood glucose levels both in diabetic db+ /db+ mice and streptozotocininduced diabetic mice (Tsuneki et al. 2004). A study by Sabu et al. (2002), found that catechins from green tea interacts with glucose metabolism by decreasing serum glucose levels in alloxaninduced diabetic rats. Green, Oolong, and black tea intakes were all reported to reduce plasma and liver triglyceride levels and plasma cholesterol levels in type 2 diabetes in Zucker rats and SpragueDawley rats fed with high sucrose diet (Hasegawa, Yamda, & Mori 2003; Yang, Wang, & Chen 2001). It has also been reported that tea catechins exhibited antithrombogenic activity in streptozotocindiabetic rats by normalizing the thromboxane A2 (TXA2): prostacyclin I2 (PGI2) ratio and thereby improving kidney function (Yang, Choi, & Rhee 1999; Choi, Chang, & Rhee 2002; Rhee, Kim, & Kwag 2002). In another in vivo study, green tea extract was found to prevent AGEs formation and collagen crosslinking delaying collagen aging in C57BL/6 mice (Rutter et al. 2003). Ginger (Zingiber officinale Rosco) is derived from Zingiberaceae and has been used worldwide as spice, dietary supplement, and traditional medicine for centuries (Butt and Sultan 2011). Several in vivo studies have found that ginger extract possess antidiabetic properties (Bordia, Verma, & Srivastava 1997; Akhani, Vishwakarma, & Goyal 2005; Bhandari, Kanojia, & Pillai 2005;

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Kadnur & Goyal 2005; Al-Amin, Thomson, Al-Qattan, PeltonenShalaby, & Ali 2006; Ojewole 2006; Kato et al. 2006; Islam & Choi 2008; Nammi, Sreemantula, & Roufogalis 2009; Madkor, Mansour, & Ramadan 2011; Ramudu et al. 2011; Shanmugam, Mallikarjuna, Kesireddy, & Sathyavelu Reddy 2011). Saraswat et al. (2010) found that ginger was effective against the development of diabetic cataract in rats mainly through its antiglycation activity. However, the active components in ginger responsible for the observed antidiabetic effects are still unknown. This study was designed to investigate the cytoprotective properties of eight major bioactive compounds isolated from apple, tea and ginger against MGO-induced carbonyl stress and postulate a possible mode of action for the observed cytoprotective effects. Materials and methods Materials Methylglyoxal, quercetin, chlorogenic acid, phloretin, (-)-epicatechin 3-gallate (ECG), 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT), 4-vinylpyridine and Aminoguanidine (AG) were purchased from Sigma Chemicals (St. Louis, MO, USA). Anti-HO-1 (P 249) antibody was obtained from Cell Signaling Technology (Danvers, MA, USA). Anti-Nrf2 (SC 20) and anti-RAGE (SC 5563) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-GAPDH was purchased from Abcam (Cambridge, MA, USA). Dulbecco’s Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F-12), Fetal Bovine Serum (FBS), L- glutamine, Non-Essential Amino Acids (NEAA), Streptomycin/Penicillin at 10,000 μg/ml/10,000 U/ml) and phosphate- buffered saline (PBS) were procured from Hyclone (Belgium). Bioactive compounds (-)-epigallocatechin 3-gallate (EGCG), and (-)-epicatechin (EC) were purchased from Jiang Su Dehe Bio-Tech Co., LTD (Jiangsu, China). Compounds from Ginger ([6]-Gingerol and [6]-Shogaol), were isolated and characterized by members of the research team at North Carolina A&T State University, USA (Sang et al. 2009). All compounds were > 95% pure. Cell culture and treatments Human retinal pigmented epithelial (HRPE) cells were obtained from Lonza (Basel, Switzerland). Retinal pigment epithelial (RPE) cells form the outer blood retina barrier and play a key role in the pathological process of neovascularization. HRPE cells are well characterized cell lines expressing growth factors and receptors, which are used in the detoxification of AGEs (Spilsbury et al. 2000; Dunn et al. 1996; Tanihara et al. 1997). Cells were cultured in DMEM/F-12 medium (supplemented with 10% FBS, 3 mM glutamine, NEAA, and antibiotics (streptomycin/penicillin) and incubated at 37 °C in a humidified atmosphere of 5% CO2 . Viability assay MGO-induced cell toxicity was assessed using MTT colorimetric assay. For this assay, HRPE cells were seeded in 96-well plates (5 × 104 cells/well). The bioactive compounds (phloretin, ECG, EC, quercetin, [6]-shogaol, chlorogenic acid and [6]-gingerol) were dissolved in DMSO, whereas EGCG was dissolved in water and subsequent dilutions were made using cell growth media. Cells at 70– 80% confluence were placed in DMEM/F-12 media free of FBS. The cells in the media were treated with MGO at different concentrations, ranging from 0–100 mM, and incubated for 24–96 h. In a different set of experiment, cells were treated with compounds at different concentrations, ranging from 10–100 μM, and incubated for

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24 and 96 h, respectively. After exposure to MGO/compounds for 24 and 96 h, media was completely taken out and the cells were incubated with MTT at 5 mg/ml for 3 h. During this incubation time, mitochondrial dehydrogenases of living cells reduced MTT to purple formazan. This insoluble purple formazan product was dissolved with DMSO, forming a colored solution. The absorbance was read at 570 nm using a microplate reader (Tecan, USA). The results were expressed as the percentage of control treatment representing 100% viability. Protective role of bioactive compounds HRPE cells (5 × 104 cells/well) were plated in 96-well plates and allowed to attach for 48 h at 37 °C and 5% CO2 . The bioactive compounds (phloretin, ECG, EC, quercetin, [6]-shogaol, chlorogenic acid and [6]-gingerol) were dissolved in DMSO, whereas EGCG was dissolved in water and subsequent dilutions were made using cell growth media. At 70–80% confluence, cells were pre-incubated with bioactive compounds at concentrations ranging from 10– 50 μM for 3 h respectively, then co-treated with MGO (1 mM) and incubated at 37 °C for 24 and 96 h, respectively. After the incubation period, the media was aspirated and the cells were treated with MTT (5 mg/ml) and incubated at 37 °C for 3 h after which MTT was aspirated and 100 μl of DMSO added before the absorbance was read at 570 nm using a microplate reader (Tecan, USA). The experiment was repeated independently to confirm the results. Determination of AGEs formation The formation of AGEs was monitored using a fluorescence assay (Lv et al. 2011). HRPE cells were plated into 6-well plates and incubated at 37 °C in a humidified atmosphere of 5% CO2 . At 70– 80% cell confluence, media was replaced with DMEM/F-12 free of FBS and the cells were pre-treated with bioactive compounds/AG at 25 μM respectively for 3 h, and then co-treated with MGO and incubated at 37 °C for 24 and 96 h. The medium was replaced every 24 h during the 96 h incubation. At the end of the incubation period, cells were trypsinized (0.05%), collected into tubes and centrifuged at 130 X g for 5 min. The cell pellet was reconstituted with 200 μl PBS and sonicated 3 times for 5 s each then centrifuged at 10,000 X g for 15 min at 4 °C. An aliquot of the supernatant was taken for protein concentration using BCA method. The cell lysate was transferred to a black 96-well plate and the fluorescence intensity was recorded by a multi-mode microplate reader (Biotek, VT, USA) at an excitation of 360 nm and emission of 460 nm. PBS was used as a blank and the fluorescence intensity was normalized with untreated cells.

reader (Tecan, USA). The concentration of CML in samples was determined by plotting the optical density of the samples into a standard curve. Three compounds phloretin, 6-shogaol and 6-gingerol were selected based on their relatively low inherent cytotoxicity, high cytoprotection, inhibition of AGEs and CML generation data. All had the highest effect at the lowest concentration. PLS-DA described in the statistical analysis section below classification also placed these compounds together and closer to the control group. Hence, these three compounds were selected to move to the tests designed to better understand the mechanisms of action leading to the observed cytoprotection effect.

Evaluation of RAGE and HO 1 expressions by western blotting HRPE cells were plated into 150 × 15 mm culture plates and incubated at 37 °C in 5% CO2 . At 70–80% confluence, cells were pre-treated with 25 μM phloretin, [6]-shogaol and [6]-gingerol, respectively, then co-treated with MGO for 96 h. At the end of 96 h, cells were lysed using RIPA buffer (Thermo Scientific, USA). Proteins were separated by 10% SDS– polyacrylamide gel electrophoresis at 100 v followed by transfer to polyvinylidenedifluoride (PVDF) membranes for 1 h at 4 °C. The membranes were then blocked with 5% BSA in 1X PBS, for 1 h at room temperature followed by incubation with primary antibody against RAGE (1:500) and HO 1 (1:200) at 4 °C overnight. Next day, the membranes were incubated with a HRP-conjugated secondary antibody (1:1000) for 1 h at room temperature. The membranes were developed using electrochemiluminescence (ECL) kit (BioRad, USA) according to manufacturer’s protocol. The signal densities on the blots were measured with Image J software and normalized using anti-GADPH as an internal control.

Preparation of cytosol and nuclear extract for NrF2 expression HRPE cells were plated into 150 × 15 mm culture plates and incubated at 37 °C in 5% CO2 until 70–80% confluence after which cells were pre-treated with 25 μM of phloretin, [6]-shogaol and [6]-gingerol individually for 3 h and co-treated with MGO (1 mM) for 96 h. After the 96 h treatment, proteins from nuclear and cytosol were extracted using NE-PER Nuclear and Cytoplasmic Extraction Kit protocol (Thermo Fisher Scientific, USA) and the expression of cytosol and nuclear NrF2 expression was then assessed by western blot using primary antibody against NrF2 (1:200) following the procedure described in the previous western blot section.

Quantification of CML by Enzyme Linked Immune Assays (ELISA)

Measurement of the intracellular glutathione forms and their ratio

For quantification of CML, samples were prepared following the procedure described above under the AGEs assay section and CML determined using the CML ELISA Kit protocol (Cosmo Bio Co. Ltd., Japan). Microtiter well plates were pre-coated with an antibody specific to CML. Standards or samples were added to the appropriate wells with a biotin-conjugated polyclonal antibody preparation specific to CML and Avidin conjugated to Horseradish Peroxidase (HRP) was then added to each well and incubated at 37 °C for 1 h. A TMB (3,3 ,5,5 tetramethyl-benzidine) substrate solution was then added to each well for visualization where only those wells that contain CML, biotin-conjugated antibody and enzymeconjugated Avidin exhibit a change in color. The enzyme-substrate reaction was terminated by the addition of sulphuric acid solution and the color change was measured at 450 nm by a microplate

The reduced glutathione (GSH) content was measured using a HT Glutathione Assay kit (Trevigen, USA). Briefly, HRPE cells were plated in 150 × 15 mm culture plates and incubated at 37 °C in 5% CO2 until 70–80% confluence, then cells were pre-treated with 25 μM phloretin, [6]-shogaol and [6]- gingerol respectively for 3 h and then co-treated with MGO (1 mM) incubated for 96 h. At the end of 96 h incubation, cells were trypsinized (0.05%), collected into tubes and centrifuged at 130 X g for 5 min. The cell pellet was washed once with cold 1X PBS and the pellet was then reconstituted with 5% (w/v) metaphosphoric acid the suspension was sonicated 3 times, 5 s each, before centrifugation at 14,000 X g for 5 min at 4 °C. The clear supernatant was then used for glutathione assay using the Trevigen kit protocol. The Glutathione assay is based on an optimized enzymatic recycling method for the


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Fig. 1. Chemical structures of the bioactive compounds from apple (Quercetin, Phloretin, Chlorogenic Acid), ginger ([6]-Shogoal, [6]-Gingerol), and tea (-)-epigallocatechin 3-gallate, (-)-epicatechin 3-gallate, (-)-epicatechin.

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Day 1 Day 4

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100 90 80 70 60 50 40 30 20 10 0 0.0

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Fig. 2. Cytotoxic effects of MGO on HRPE cells. Data are presented as means ± SD (n = 5).

quantification of glutathione. The sulfhydryl group of the reduced form (GSH) reacts with DTNB (5,5 -dithiobis-2-nitrobenzoic acid, Ellman’s reagent) to produce a yellow-colored 5-thio-2nitrobenzoic acid (TNB) that absorbs at 405 nm. The rate of TNB production is directly proportional to this recycling reaction which is, in turn, directly proportional to the concentration of glutathione in the sample. The absorbance of TNB was recorded at 405 nm to quantify glutathione levels in each sample using a standard curve with correction for protein concentration. The protein concentration in cell lysates was determined using a Pierce BCA kit (Thermo Fisher Scientific, USA). For quantification of oxidized glutathione (GSSG), samples and GSSG standards were treated with 2 M 4-vinylpyridine (1 μl/ 50 μl sample) which is used to block free thiols present in the

reaction, then incubated at room temperature for one hour. Samples were subsequently treated with 150 μl of the reaction mixture (glutathione reductase mixture) and the absorbance was recorded at 405 nm using a microplate reader (Biotek, USA). Reduced cellular glutathione was quantified by subtracting the values measured in oxidized samples from the total glutathione. The ratios of reduced (GSH) to oxidized (GSSG) glutathione were calculated and used to represent cellular redox status after treatment with the compounds. Statistical analysis Data was analyzed using Analysis of Covariance (ANCOVA). Differences between treatment and control means were evaluated with Dunnett’s post hoc test using SPSS software (SPSS

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Fig. 3. Screening of cytotoxic effects of bioactive compounds on HRPE cells. (a). 24 h incubation (b). 96 h incubation. Data are presented as means ± SD (n = 5). Legends: Q = Quercetin, PH = Phloretin, 6-S = 6-Shogoal, 6-G = 6-Gingerol, CGA = Chlorogenic Acid, EGCG = (-)-epigallocatechin 3-gallate, ECG = (-)-epicatechin 3-gallate, EC = (-)-epicatechin, AG = Aminoguanidine.


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Fig. 4. Protective effects of the bioactive compounds against MGO-induced damage on HRPE cells (a). 24 h incubation (b). 96 h incubation. Data are presented as means ± SD (n = 5). Legends: PH = Phloretin, EGCG = (-)-epigallocatechin 3-gallate, ECG = (-)-epicatechin 3-gallate, EC = (-)-epicatechin, AG = Aminoguanidine.

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Fig. 5. Partial least square discriminate analysis (a). Grouping based inherent compound toxicity (b). Grouping based on inhibitory activity against MGO-induced cell death (Cytoprotection). Legends: Q = Quercetin, PH = Phloretin, 6-S = 6-Shogoal, 6-G = 6-Gingerol, CGA = Chlorogenic Acid, EGCG = (-)-epigallocatechin 3-gallate, ECG = (-)-epicatechin 3-gallate, EC = (-)-epicatechin, AG = Aminoguanidine.

Inc., USA). Means were judged different at the 5% significance level. Partial least squares regression discriminant analysis (PLSDA) was performed on the cytotoxicity and cytoprotection data to determine/group the best performing bioactive compounds taking into account all measured variables. Results The chemical structures of the major bioactive compounds isolated from tea, apple, and ginger are presented in Fig. 1. The common structure consists of diphenylpropanes (C6 –C3 –C6 ), which includes two aromatic rings linked through three carbons that form an oxygenated heterocycle. This structure is suspected of playing a major role in trapping dicarbonyl compounds. MGO-induced cell toxicity, presented in Fig. 2, shows that MGO at the low concentrations of 0.1 and 0.5 mM had a negligible effect on the viability of HRPE cells (99 ± 6% and 98 ± 4% of cell viability, respectively) following 24 h incubation. However, cell viability decreased from 80 ± 3% to 10 ± 5% when the concentration of MGO increased from 1 to 10 mM. MGO at 100 mM was found to be lethal to cell within 24 h of incubation. Following exposure to MGO for 96 h, cell viability decreased to 20%, 50% & 80 % at 0.1, 0.5, and 1 mM of MGO, respectively, while this long exposure made the 10 mM a lethal dose to cells. Overall, cytotoxicity of MGO increased both with increasing concentrations and incubation time.

Cytotoxic effects of bioactive compounds on HRPE cells To evaluate the potential inherent toxicity of the eight specific bioactive compounds (EGCG, ECG, EC, quercetin, [6]-shogaol, phloretin, chlorogenic acid and [6]-gingerol) against HRPE cells, cells were incubated with different concentrations of each bioactive compound for 24 and 96 h after which the cell viability was determined using MTT assay. The results of cell viability assays, presented in Fig. 3a, show that treatment with EGCG, ECG, EC, quercetin and chlorogenic acid at the concentrations of 10, 25 and 50 μM had no significant effect on the viability of HRPE cells compared to the untreated cells. However, EGCG, ECG, and EC at 100 μM significantly (p < 0.05) decreased cell viability to 78 ± 6%, 80 ± 1%, and 87 ± 3%, respectively. In contrast, treatment with phloretin and chlorogenic acid at concentrations up to 100 μM had no significant effect on the viability of HRPE cells (Fig. 3a). Hence, these compounds would be suitable for use in the full test range without concerns about inherent toxic side effect. It was also observed that the other compounds such as [6]-shogaol and [6]- gingerol had potential cytotoxic effects at 50 μM where cell viability was reduced to 80 ± 2% and 83 ± 2%, respectively when compared to the untreated cells. AG, used as a positive control, showed toxicity at 100 μM (78 ± 2%) whereas at 10, 25 and 50 μM it had no significant effect on cell viability with 96 ± 2%, 100 ± 4% and 93 ± 3%, respectively.


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Fig. 5. Continued

As observed for MGO exposure, the compounds showed increased effect with time (Fig. 3b). The results indicate that EGCG, ECG, EC, quercetin and chlorogenic acid at concentrations of 10, 25 and 50 μM did not significantly influence the viability of the cells after 96 h of incubation. The cytotoxic effect of EGCG and ECG, at 100 μM significantly reduced cell viability to 67 ± 6% and 78 ± 6% as shown in Fig. 3b compared with untreated cells. Compounds from ginger, [6]-shogaol and [6]-gingerol, at 50 μM reduced cell viability significantly after 24 h exposure to 78 ± 5% and 89 ± 7%, respectively. Interestingly, AG at higher concentration (50 & 100 μM) attained significant compound toxicity after 96 h incubation reducing cell viability to 85% & 63%. Protective effects of the bioactive compounds on MGO-induced damage on HRPE cells To determine the protective effects of the bioactive compounds on HRPE cells from MGO-induced cell death, cultures were pretreated with bioactive compounds at different concentrations for 3 h respectively prior to co-treatment with MGO at 1 mM for 24 and 96 h period. As shown in Fig. 4a, the percentage of viable HRPE cells was significantly decreased in cultures treated with MGO alone compared with the control cultures (80 ± 3% vs. 100 ± 2%). Pretreatment of HRPE cultures with EGCG, ECG, EC, quercetin and chlorogenic acid at the concentrations of 10, 25 and

50 μM prior to exposing them to MGO showed a dose-dependent increase in cell viability. However, EGCG, EC and ECG at 100 μM concentration did not fully suppress MGO-induced cytotoxicity as expected most likely due to their inherent cytotoxicity. Overall, pretreatment with EGCG, ECG and EC reduced the toxic effects of MGO on HRPE cells. Data show that AG, phloretin, [6]-shogaol and [6]-gingerol were more effective than the other compounds since they showed the highest protective effect at a lower concentration of 25 μM. At 96 h incubation, the effect of bioactive compounds on MGO-induced cell toxicity followed a trend similar to that observed at 24 h incubation. As shown in Fig. 4b, the viability of cultures treated with MGO was significantly decreased compared with the control cultures from 100 ± 3% to 30 ± 5%. Pretreatment of HRPE cultures with AG, phloretin, [6]-shogaol, chlorogenic acid and [6]-gingerol at the concentrations of 10 and 25 μM significantly increased cell viability over the levels of cells exposed to MGO only. However, EGCG, quercetin, and chlorogenic acid at 50 μM achieved about 89 ± 3 %, 85 ± 3% and 69 ± 3% cell viability respectively, against MGO-induced cell toxicity. The use of phloretin at 50 μM and higher did not significantly increase cell viability beyond the levels achieved at 25 μM. Whereas AG, [6]-shogaol and [6]-gingerol enhanced cell viability at lower concentrations, they exhibited cytotoxic effect at concentration at 50 μM and higher.

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Fig. 6. Total AGEs accumulation in HRPE cells by bioactive compounds. Data are presented as mean ± SD (n=5). ∗ P < 0.05, ∗∗∗∗ P < 0.0001 vs. untreated group; #### P < 0.0001 vs. group treated with MGO alone. Legends: Q=Quercetin, PH = Phloretin, 6-S = 6-Shogoal, 6-G = 6-Gingerol, CGA=Chlorogenic Acid, EGCG = (-)-epigallocatechin 3-gallate, ECG = (-)-epicatechin 3-gallate, EC = (-)-epicatechin, MG = Methylglyoxal, VC = vehicle control, CTR = Control.

Partial Least Square Discriminate Analysis (PLS-DA) was performed on both the cytotoxicity and cytoprotection data to determine the best performing bioactive compounds overall in terms of maintaining high cell viability. Fig. 5a, shows that bioactive compounds fell into 3 groups for cytotoxicity among which group # 3 was considered to be the most lethal while group # 1 was the least toxic to HRPE cells. The control treatment fell under group 1 with most bioactive compounds clustered close to the control group indicating reduced toxicity. In terms of cytoprotection, Fig. 5b, clustered compounds into 3 groups based on cytoprotection against MGO-induced cell death. The most cytoprotective compounds overall fall under group 1 along with the control indicating complete reversal of MGO-induced cytotoxicity by the bioactive compounds in group 1 Among the tested compounds, phloretin, [6]-gingerol, EGCG and [6]-shogoal were found to possess higher cytoprotective effect at lower concentration at which they exhibited relatively low inherent cytotoxicity. Data analysis also showed that cell viability levels for the 3 best performing bioactive compounds were similar to the level observed in untreated control but significantly higher the cell viability observed in cultures exposed to MGO.

nificantly increased in MGO treated group at both exposure times compared to the control (P < 0.0001) whereas, pre-treatment with the bioactive compounds significantly decreased AGEs formation within 24 h (P < 0.0001) with further decrease over time. Quantification of CML As expected, the control cells (which were not treated with the compounds or MGO) showed no CML generation while the cells exposed to MGO alone generated about 225 and 847 picogram of CML after 24 and 96 h of incubation, respectively. However, cells pre-treated with bioactive compounds exhibited inhibitory effect against CML generation. The compounds EC, ECG, quercetin and chlorogenic acid was significantly different from the untreated group. Among all the compounds screened, EGCG, phloretin, [6]shogoal and [6]-gingerol had the highest rates of inhibition, with 89%, 95%, 91% and 90% CML inhibition, respectively, after 96 h incubation (Fig. 7). Western blot analysis

Total AGEs measurement Incubation of HRPE cells with MGO led to an increase in fluorescence intensity due to the formation of AGEs which possess fluorescence properties (Fig. 6). However, cells exposed to bioactive compounds showed a reduction of fluorescence intensity suggesting that the formation of MGO-induced AGEs may have been inhibited. After 96 h of exposure to MGO, AGEs formation increased by 39% of which 83%, 89% and 77% was inhibited/reversed by phloretin, [6]-shogaol and [6]-gingerol, respectively. ECGC was also found to be a potent inhibitor of AGEs, especially within the first 24 h exposure. As shown in Fig. 6, the AGEs formation sig-

To postulate the mechanisms by which these bioactive compounds protect HRPE cells against MGO-induced cytotoxicity, western blot analyses were measured on potential therapeutic markers. Based on the results obtained for cytoprotective, AGEs formation and CML inhibition, the most potent compounds namely, phloretin, [6]-shogoal and [6]-gingerol, were selected for western blot analysis. The data show that RAGE expression significantly increased (P < 0.001) in the MGO-treated group compared to the control group (Fig. 8a). Phloretin exhibited a weaker effect while [6]-shogaol and [6]-gingerol significantly reduced MGO-induced RAGE expression (Fig. 8a).


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Fig. 7. Inhibition of CML generation by bioactive compounds at different time intervals. ∗∗∗∗ P < 0.0001 vs. untreated group Legends: EGCG = (-)-epigallocatechin 3-gallate, ECG = (-)-epicatechin 3-gallate, EC = (-)-epicatechin.

The role of Nrf2 expression was measured by its translocation activity from cytoplasm to the nucleus, triggering stress-induced defense mechanism. HRPE cells treated with MGO alone significantly increased Nrf2 expression in cytoplasm suppressing the defense mechanism (Fig. 8b). As shown in Fig. 8c, the translocation of Nrf2 in to the nucleus was significantly increased in cells pre-treated with phloretin, whereas [6]-shogaol and [6]-gingerol did not seem to trigger significant translocation. Interestingly, phloretin activated Nrf2 expression nearly two folds yet its effect on reducing the expression of RAGE was limited during MGO induced cytotoxicity. To further evaluate the underlying protective mechanisms of these bioactive compounds, expression of HO-1 was studied. Cells pre-treated with phloretin, [6]-shogaol and [6]-gingerol significantly enhanced (p < 0.001) the expression of HO-1 (Fig. 8d) compared to cells treated with MGO without prior exposure to bioactive compounds. These results could explain the protective role of phloretin, [6]-shogoal and [6]-gingerol which may be through activating the expression of Nrf2 in the nucleus which, in turn, up regulates the enzymes involved in the defense mechanism, particularly HO-1. Glutathione redox balance To understand glutathione redox status, it is necessary to measure both the reduced and oxidized forms of glutathione. Glutathione redox status was analyzed after 96 h treatment and data presented in Fig. 9. MGO alone decreased GSH levels much more than GSSG which resulted in lowering of GSH/GSSG ratio compared to untreated cells. However, pre-treatment of cells with phloretin,

[6]-shogaol and [6]-gingerol increases GSH substantially. While GSSG content remained relatively unchanged compared to cells that were not pre-treated with bioactive compounds (Fig. 9A), the sharp increase in GSH resulted in significantly higher GSH/GSSG ratio (Fig. 9B). This rise in GSH/GSSG ratio was attributed to the beneficial effect of bioactive compounds. Discussion The formation or accumulation of AGEs has shown to play a significant role in promoting diabetic complications. Naturally occurring bioactive compounds from food sources have been reported to counter the formation of AGEs. Recently, in vitro studies have reported that naturally occurring flavonoids can scavenge the reactive dicarbonyl species effectively thereby reducing the formation of AGEs. Hence, this study was designed to investigate for the antiAGEs activity of specific bioactive compounds from common food sources (tea, apple, and ginger) using HRPE cells which are the best characterized cell lines for evaluation of AGEs (Kim et al. 2007). Data obtained show that EGCG, EC, ECG, [6]-gingerol and [6]shogaol exhibited some level of toxicity at higher concentrations towards the HRPE cells. Although all the eight compounds showed protective activity, the most potent cytoprotective and least inherently cytotoxic compounds were EGCG, phloretin, [6]-gingerol and [6]-shogaol. The use of these compounds enabled near complete prevention of carbonyl stress in the retinal cells. Phloretin, [6]-gingerol and [6]-shogaol showed effectiveness at lower concentration than EGCG, which had its best effect at 50 μM. The findings of this study are consistent with those reported by Zhu et al. (2015). Earlier studies have reported that EGCG and phloretin are

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Fig. 8. Western blot analysis: (a). RAGE expression (b). accumulation of nrf2 in the cytosol (c). translocation of nrf2 from cytosol to nucleus, and (d). HO 1 expression. Quantitative data from FluorChem. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, ∗∗∗∗ P < 0.0001 vs. untreated group; # P < 0.05, ### P < 0.001, #### P < 0.0001 vs. group treated with MGO alone. Legends: PH=Phloretin, 6-S=6-Shogoal, 6-G=6-Gingerol.

capable of trapping GO and MGO which could explain the observed reduction in AGEs (Sang et al. 2007; Shao et al. 2008). Overall, results from this study demonstrated that bioactive compounds such as phloretin from apple, [6]- shogaol and [6]- gingerol from ginger and EGCG from tea can efficiently inhibit the carbonyl induced stress and revert its impact on cell viability and cellular markers when compared with a known therapeutic agent aminoguanidine (AG), which was used in this study as a positive control. It was reported that AG acts on α and β -dicarbonyl compounds by preventing the formation of AGEs (Thornalley 2003). Increase in AGEs formation such as CML was observed when cells treated with MGO alone, and subsequently pre-treated with the compounds inhibited the formation of AGEs. This protective effect exhibited by the bioactive compounds against carbonyl stress in HRPE cells is likely

due to quenching of MGO, especially since previous studies suggest that MGO is the main dicarbonyl compound responsible for albumin glycation (Sadowska-Bartosz et al. 2014). Earlier reports also verify that various classes of polyphenols such as flavanols, flavonols, isoflavones, chalcones, and oligomers of flavanols can act as inhibitory molecules against the formation of AGEs by trapping MGO (Lv, Shao, Chen, Ho, & Sang 2011; Peng et al. 2012). As for the mechanism of action, the compounds analyzed in this study seem to actively influence the key targets responsible for trapping of dicarbonyl species. The Western blot data clearly indicate RAGE mediated cellular activating events by blocking translocation of Nrf2 suppressing the activation of the defense system by MGO-induced carbonyl stress. Holik et al. (2013) reported that an increase in CML up-regulated the RAGE expression. Literature reports indicate


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Fig. 9. Change in Glutathione forms: (a). GSH level and GSSG level, (b). GSH/GSSG ratio as indicators of carbonyl stress in HRPE cells after 96 h incubation following pretreatment with the bioactive compounds. # P < 0.05, ### P < 0.001, vs. group treated with MGO alone. Legends: PH=Phloretin, 6-S=6-Shogoal, 6-G=6-Gingerol.

that natural compounds could activate Nrf2 and induce phase II detoxifying enzymes (Na & Surh, 2008; Zúñiga-Toalá et al., 2013; Kim, Kim, Shim, & Chang 2014). Similar observations were reported for Schisandra sphenanthera extracts which enhanced the accumulation of Nrf2 in the nucleus increasing the expression of its target genes such as NAD(P)H: quinine oxidoreductase 1 (NQO1), heme oxygenase-1 (HO-1) and glutamate cysteine ligase (GCL) (Jin et al. 2015). Importantly it was observed that phloretin could not effectively influence RAGE expression, even though activation of Nrf2 and HO-1 was not hindered, hence further study is needed to investigate if other mechanisms are underlying the activation of Nrf2. The tested bioactive compounds from apple, tea and ginger exerted an inhibitory effect against the formation of AGEs with different intensity, most likely due to their structure function relation. The structures are characterized by a two hydroxyl substituted aromatic (A & B) rings joined by a three carbon segment (C ring). The major classes of flavonoids are differentiated by the degree of unsaturation and oxidation of the C ring. It was reported that the A ring may be one of the key factors involved in trapping reactive dicarbonyl species by forming mono- and di-MGO adducts (Li, Zheng, Sang, & Lv 2014). It was also reported that positions 3 and 5 of the A rings of phloretin, as well as position 6 and 8 of EGCG A ring, were the active sites for trapping dicarbonyl species (Shao et al. 2008). This could be further explained by the findings of Peng et al. (2012) who reported that the structures of diarylphenolic (Gingerenones A) and monophenolic (shogaol) exhibited the same level of activity based on the core ring, with the extra pheno-

lic group on the gingerol not playing any additive activity, clearly indicating that the core ring is key factor for exhibiting the structure function relationship. In conclusion results obtained in this study showed that EGCG, phloretin, [6]-shogaol and [6]-gingerol could prevent MGO-induced cytotoxicity more effectively than EC, ECG, querectin and chlorogenic acid in human retinal epithelial cells. The possible mechanism involved in prevention of AGEs formation and related cytoprotective effect could be through modulation of key regulating detoxifying enzymes via regulation of Nrf2 function. This suggests that the bioactive compounds evaluated in this study may have potential therapeutic value for prevention and/or management of diabetes and its secondary complications. These compounds could be candidates for in vivo studies on their potential role as useful pharmaconutritive agents for diabetes. Furthermore, the hypothesized mechanism needs to be confirmed and the molecular basis responsible for prevention/trapping of AGEs specifically elucidated. Overall, this study provided a strong indication of the potential of the top three most active bioactive compounds as natural agents for use in interventions aimed at alleviating diabetes complications. Hence, these purified compounds could be of interest to dietary supplement makers or pharmaceutical industry. Conflict of interest The authors declare that there are no conflicts of interest.

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Acknowledgments This research was made possible by NPRP Grant #NPRP 5-2203-063 from the Qatar National Research Fund (a member of Qatar Foundation). The statements made herein are solely the responsibility of the authors. The authors are thankful to Dr. Abdelsalam Gomaa, head of Statistical Consulting Unit at the College of Arts and Sciences, for his assistance with statistical analysis and Dr. Ali Eid for his support and for providing access to his lab space during the research phase leading to this manuscript.

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