Macular degeneration or AMD – Effect of saffron eye health supplement in age-related macular degeneration:

Age-related macular degeneration or AMD is a leading cause of adult vision loss in developed countries. According to one estimate, 17,100 new cases of neovascular (wet) macular degeneration or AMD and 180,000 new cases of geographic-atrophy (dry) AMD occur in Canada each year. In 2006, the estimated cost to the Canadian gross domestic product (GDP) from vision impairment caused by macular degeneration was $2.6 billion (1).

Age-related macular degeneration or AMD is considered a major public health problem, and according to the National Coalition for Vision Health, over one million Canadians are affected by early age-related macular degeneration or AMD, with 250,000 individuals being affected with the advanced form of the disease. Unfortunately, this number is expected to double by 2031. In 2006, there were 64,000 Canadians blind due to the vision impairment caused by the eye disease age-related macular degeneration or AMD (http://www.visionhealth.ca/).

Pathology of Age-related macular degeneration or AMD:

Vision loss in age-related macular degeneration or AMD is attributable to photoreceptor death in the central retina. Growing evidence suggests a role for retinal pigment epithelial (RPE) cell damage and death, caused by different mechanisms including inflammation and oxidative stress, causing photoreceptor death and loss of vision (2).

Dry / Wet Age-related macular degeneration or AMD. There are two clinical types of AMD, the “dry” and “wet” form.

In dry age-related macular degeneration or AMD, which is the most prevalent form of this debilitating eye disease (90% of the cases), insoluble extracellular aggregates called drusen accumulate in the retina. Dry AMD causes a gradual loss of central vision, but it can eventually develop to the more aggressive wet form. In wet AMD, new immature blood vessels grow underneath the retina, leaking blood and fluid into the retina. Wet macular degeneration or wet AMD is the most severe form of the disease and can lead to rapid vision loss (2). Although vision loss due to macular degeneration or AMD is becoming a major public health problem, currently there is no therapy option for the dry form of the disease, and medication for wet AMD is expensive and invasive. Therefore, more convenient preventive or therapeutic interventions are needed for reducing this health burden. Since multiple factors are involved in the pathogenesis of AMD, a multi-faceted approach will most likely be required to prevent and treat this eye disease.

Macular degenerations studies with saffron and resveratrol:

Recent studies show that both crocin, an active compound isolated from saffron (Crocus sativus), and resveratrol possess therapeutic effects in experimental models of age-related macular degeneration or AMD via multiple mechanisms pertaining to the pathogenesis of AMD, including modulation of gene expression profile, maintaining cell membrane integrity, protecting against oxidative stress, inhibiting vascular endothelial growth factor- (VEGF) induced angiogenesis, and providing anti-inflammatory effect. Preliminary studies using combination of crocin and resveratrol demonstrated synergistic cytoprotective activities for retinal and neuronal cell lines (3,4).

Studies indicate that there is significant opportunity to achieve therapeutic effects in age-related macular degeneration or AMD with the combination of resveratrol and crocin, and therefore, there is a need to better understand the mechanism of action of this synergistic combination.

Synopsis: Age-related macular degeneration (AMD)treatment studies:

Effects of crocin from saffron in age-related macular degeneration or AMD:

Saffron (Crocus sativus) is a spice containing the antioxidant carotenoids crocin and crocetin (the aglycone derivative of crocin), and has been known for its antioxidant, anti-inflammatory, and cell-/neuro-protective functions.

Crocin from saffron has shown significant neuroprotective effects in retinal cells and in vivo models of AMD. The most important result came from a recent double-blind, placebo-controlled, cross-over clinical study, in which taking oral saffron at 20 mg/day for three months induced a significant improvement of retinal function in patients with early AMD (5). In a further 12-month follow-up study, patients continued to benefit from saffron, which led to “improvement in contrast and color perception, reading ability, and vision at low luminance, and a substantial improvement in patients’ quality of life”(6). More importantly, a recent report from the same clinical study shows that the macular benefits of saffron occur equally for both of the major genetic variations which lead to increased risk of age-related macular degeneration or AMD; the gene variations in complement factor H (CFH) and age-related maculopathy susceptibility (ARMS2) genotypes (7).

A recent time course study in a light damage model of photoreceptor degeneration in Sprague Dawley (SD) rats showed that the build-up of neuroprotection by saffron occurs over 7-10 days of supplementation (1 mg/kg/day). In macular degeneration model, saffron extract leads to reduced photoreceptor death, preservation of the population of surviving photoreceptors and reduction in upregulation of GFAP, a stress-related protein (8).

Therapeutic potential of crocin in age-related macular degeneration or AMD:

The earliest report suggesting a therapeutic potential for crocin in AMD was from Xuan et al. (9), which has shown that intraperitoneal injection of crocin analogs (10 mg/kg) significantly increases blood flow in the retina and choroids. Crocin has cell-protective effects (100% at 80mM) against the blue light- and white light-induced rod and cone death in bovine and primate retinal cell cultures (10). Exposure to light may have some impact on the incidence and progression of AMD (11), and is a recognized method used in certain animal and in vitro models of age-related macular degeneration or AMD.

Crocin inhibits caspase-3 activation in PC-12 cells under hypoxic conditions. Crocin also increases glutathione (GSH) levels, which protects cells from apoptosis-inducing agents. Crocin also suppresses activation of the c-Jun N-terminal kinases (JNK) pathway, which has a role in neural cell death (12). Retina under hypoxic conditions produces VEGF and therefore crocin has been considered for antiangiogenic effects in wet macular degeneration. Crocin has also shown anti-inflammatory effects by inhibiting TNF-α induced apoptosis and modulating Bcl-2 expression in PC-12 cells (13).

Eye health with saffron in age-related macular degeneration:

Saffron aqueous extract, containing crocin, maintains both morphology and function of retinal cells and has shown to be a regulator of apoptosis. Oral pre-treatment with saffron extract in SD rats (1mg/kg daily, 6 weeks) preserves photoreceptor layer and reduces cell death against damage caused by bright light. The effect was assessed by measuring apoptosis level with TdT-mediated dUTP nick end labeling (TUNEL) technique, histological methods, and recording electroretinographic response to a flash of light (fERG) (14).

Saffron regulates gene expression levels in retinal cells model of age-related macular degeneration or AMD:

Oral pretreatment with a saffron aqueous extract for 3 weeks (1 mg/kg/day) protects albino SD rats against light damage to photoreceptors as measured by TUNEL assay. Additionally, microarray analysis shows that saffron extract regulates expression level of a large number of genes, many of them deduced to be involved in protection and repair of photoreceptors via mechanisms influencing inflammation, cell membrane integrity, and protection against oxidative damage (15).

Crocin is a water-soluble carotenoid of saffron that after oral administration in rats (16) or in mice (17) is converted through hydrolysis to crocetin before being absorbed. Crocetin has been shown to inhibit an increase in caspase-3 and caspase-9 activities and protects retinal ganglion cells (RGC-5) against death caused by tunicamycin or hydrogen peroxide. Furthermore, crocetin inhibits photoreceptor degeneration and retinal dysfunction when administered orally to male adult ddY mice and SD rats 1 h before exposure to intense light, and then repeated once daily for 5 days afterward. Crocetin level in aqueous humor was 2mM, which is sufficient to reduce cell death in vitro (18).

Benefits of crocetin in disease models of age-related macular degeneration:

Crocetin prevents ischemia/reperfusion-induced retinal damage through the inhibition of oxidative stress mechanisms including decreasing the phosphorylation level of p38, JNK, and the redox-sensitive transcription factor nuclear factor-kappa B (NF-κB), which is present in the retina after ischemia/reperfusion (19).

Anti-angiogenic effect of crocetin in models of wet age-related macular degeneration or wet AMD:

Vascular endothelial growth factor (VEGF) plays an important role in the development of pathological angiogenesis in wet age-related macular degeneration or wet AMD. Crocetin inhibits VEGF-induced angiogenesis in both human umbilical vein endothelial cells (HUVECs) and human retinal microvascular endothelial cells (HRMECs). Crocetin has demonstrated the following effects: 1) inhibition of VEGF-induced tube formation in HUVECs; 2) suppression of the VEGF-induced HRMEC migration; 3) inhibition of VEGF-induced increase in p38 level leading to inhibition of migration); 4) prevention of VEGF-induced decrease in VE-cadherin in HUVECs and/or HRMECs (20).

Effect of saffron on amyloid beta peptide:

Amyloid beta (Aβ), which is a constituent of drusen in dry AMD, is an activator of the complement system and together are implicated in the pathogenesis of AMD. Crocin inhibits Aβ amyloid formation and disrupts amyloid aggregates (21). Similarly, a methanolic extract of saffron, which contains trans-crocin-4, inhibits Aβ fibrillogenesis in a dose-dependent manner (22).

Resveratrol for age-related macular degeneration or AMD:

Resveratrol is a natural nonflavonoid polyphenolic compound found in grapes, red wine, mulberries, knotweed, peanuts and other plants. Similar to crocin, resveratrol could modulate multiple targets involved in development and progression of AMD. It ameliorates function of retinal cells by suppressing factors involved in retinal damage such as caspases, and VEGF (23-25). It also protects retinal cells by enhancing the protective effect of targets such as catalase, heme oxygenase-1, superoxide dismutase (26), and sirtuin-1 (27).

Antioxidant effects of resveratrol in age-related macular degeneration models:

Pre-treatment with resveratrol protects human RPE cells against H2O2-induced cytotoxicity by increasing GSH levels and enhancing activities of antioxidant enzymes superoxide dismutase, glutathione peroxidase, and catalase. In retinal cells resveratrol inhibits generation of intracellular reactive oxygen species (ROS) under the H2O2-induced stress condition (28). Furthermore, resveratrol protects human RPE cells against acrolein-induced oxidative stress by increasing the mitochondrial bioenergetics (2,29).
It is widely recognized that inflammation is involved in the pathogenesis of AMD. In an EIU mouse model of ocular inflammation (endotoxin-induced uveitis), resveratrol inhibits oxidative damage and suppresses NF-kB activation, leading to the observed ocular anti-inflammatory effect (30).

Resveratrol protects against oxygen-induced retinopathy in retinal cell cultures of neonatal rat by modulating nitric oxide synthase (31).

Antiapoptotic and antioxidant properties of resveratrol in models of age-related macular degeneration or AMD:

Resveratrol due to its antiapoptotic and antioxidant properties reverses the loss of cell viability in ARPE-19 cell cultures when challenged with benzo(a)pyrene (B(e)P), a toxic compound from cigarette smoke. Resveratrol inhibits the increased activity of caspase-3/7 and caspase-9, and reduces level of reactive oxygen/nitrogen species (ROS/RNS) in B(e)P-treated cells. Activation of caspase-3/7 is capable of initiating DNA fragmentation, which leads to apoptosis. Activation of caspase-9 is associated with mitochondrial stress, leading to cellular injury (23).

Resveratrol for the treatment of macular (retinal) degeneration?

Resveratrol also inhibits tunicamycin-induced ER stress and vascular degeneration in mouse eyes via inhibiting the expression of ER stress markers, CHOP and IRE1 a(32). Another mechanism that resveratrol protects against light-induced retinal degeneration in mice is by augmenting the activity of retinal sirtuin 1 (SIRT1), and by suppressing retinal activator protein-1, which becomes up-regulated upon light exposure. For its multiple functions, resveratrol has been suggested as a therapeutic agent to prevent light-induced retinal degeneration (27).

SIRT1 is a deacylase protein that acts as energy and redox sensor and helps cells to survive under apoptosis-inducing stress conditions. It influences pathways involved in aging, inflammation, apoptosis and stress resistance. SIRT1 is present in cells forming all normal ocular structures, including the cornea, lens, and retina. Up-regulation of SIRT1 has been shown to have a potent protective effect against retinal or macular degeneration (33).

In human RPE cells amyloid β (Aβ) inhibits SIRT1, induces NF-kB signaling and leads to chronic inflammation, a key factor in development and progression of age-related macular degeneration or AMD. Resveratrol inhibits Aβ-induced expression of IL-6, IL-8, and MMP-9, which are involved in chronic inflammation. Resveratrol regulates inflammation by upstream activation of SIRT-1 leading to inhibition of Aβ-mediated activation of NF-kB (Cao et al, 2013). Activation of SIRT1 by resveratrol prevents retinal ganglion cell (RGC) loss in optic neuritis through reducing oxidative stress and promoting mitochondrial function in a neuronal cell line (34). A recent report has shown protective effects on sodium iodate-induced RPE cell toxicity via inhibition of ROS and IL-8 production (35).

In an in vitro test, the number of viable ARPE-19 cells was increased by the addition of resveratrol to the storage medium without perturbing apoptosis or differentiation. cell viability was analyzed with a microplate fluorometer. Resveratrol significantly reduced caspase-3 expression, while expression of RPE65 was maintained across groups (36).

Antiangiogenic effect of resveratrol; opportunity for treatment of wet macular degeneration or wet AMD?

In several in vitro and in vivo experimental models of macular degeneration, resveratrol inhibited proliferation and migration of vascular endothelial cells, suppressing pathological angiogenesis and providing a significant protective effect against the development and/or sustenance of choroidal neovascularization (CNV). For this, resveratrol has been suggested for treating ocular diseases with exuberant and abnormal angiogenesis including AMD(24).

Oxysterols induce VEGF secretion in human retinal cells and can trigger cytotoxic, pro-inflammatory, pro-oxidative and pro-angiogenic activities responsible for wet age-related macular degeneration or wet AMD lesions. Resveratrol downregulates VEGF synthesis, and when used at 1 mM prevents neovascularization and cell death caused by oxysterols (36). Resveratrol also inhibits VEGF secretion in hypoxia-induced choroidal vascular endothelial cell (CVEC) proliferation (37), and inhibits laser-induced choroidal neovascularization (CNV) in Brown Norway rats (29). Is resveratrol a natural treatment for macular degeneration?

Resveratrol helps patients with wet macular degeneration (wet AMD):

Recently, a resveratrol-containing oral pill has been tested in patients that refused or failed to respond to intra-vitreal anti-VEGF treatments (Lucentis®, Avastin® or Eylea). Resveratrol restored retinal structure and visual function with significant anti-VEGF type effect including anatomic restoration of retinal structure and improvement in choroidal blood flow. The retinal tissue regeneration by resveratrol is likely to be mediated by survival of endogenously-produced stem cell (38). Resveratrol at a dose of 10 μM suppressed HUVEC proliferation and migration under VEGF stimulation comparable to the effect of bevacizumab (Avastin, 39).

Resveratrol significantly reduces pathological subretinal neovascularization

in a mouse model known as, very low-density lipoprotein receptor (VLDLR) mutant (Vldlr –/-). VEGF is elevated in Vldlr_/_ retina, which shows photoreceptor degeneration. Resveratrol downregulates VEGF and attenuates response of retinal endothelial cells to angiogenic stimulation. A similar effect has been also shown in an ex-vivo aortic ring assay and in cell culture experiments (40).

Platelet-derived growth factor (PDGF) is another growth factor that exhibits strong chemotactic and proliferative effects on RPE cells in proliferative vitreoretinopathy. Another mechanism for antiangiogenic effect of resveratrol is via inhibition of PDGF-induced RPE migration (Chan et al, 2013). In disease models of wet macular degeneration, resveratrol added to drinking water showed significant inhibition of neovascularization in mouse cornea induced by VEGF and fibroblast growth factor 2 (FGF-2). (41).

Resveratrol for wet macular degeneration:

Choroidal neovascularization (CNV) is a critical step in the pathogenesis of wet age-related macular degeneration or wet AMD. In a mouse model of wet macular degeneration, the protective effects of resveratrol (RSV) supplementation against CNV as well as the underlying molecular mechanisms were studied. Mice were orally pretreated with RSV daily for 5 days, after which the mice underwent laser photocoagulation to induce CNV. One week later, CNV volume was significantly lower in the RSV-treated mice compared with vehicle-treated animals. Treatment with RSV significantly inhibited macrophage infiltration into the RPE-choroid and suppressed the expression of inflammatory and angiogenic molecules, including VEGF, monocyte chemotactic protein-1 and intercellular adhesion molecule-1. Importantly, RSV prevented the CNV-induced decrease in activated AMP-activated protein kinase, and increase in activated nuclear factor-κB in the RPE-choroid complex. Based on this most recent results, the authors suggested that resveratrol could potentially be applied in the clinic to prevent CNV development in wet age-related macular degeneration or wet AMD (42).

The synergistic combination of crocin and resveratrol for macular degeneration:

Resveratrol and crocin have shown some of the most promising neuroprotective activities against various inflammatory, oxidative, and hypoxic damaging mechanisms.

Recently, synergistic effects between resveratrol and crocin have been demonstrated in two different cell lines representing models of age-related macular degeneration and Alzheimer’s disease. Primary human RPE cells were protected (78%) in a synergistic fashion against light-induced oxidative damage when pre-treated with a combination of crocin and resveratrol (4).

A patented combination of resveratrol and crocin also synergistically protected SHSY-5Y neuroblastoma cells against oxidative damage caused by 6-hydroxydopamine (6-OHDA, 30 mM) or hydrogen peroxide (H2O2, 50 mM) in an Alzheimer’s disease model. Cell protection by the combination against 6-OHDA was over 90% (3).

Saffron carotenoids and resveratrol appear to provide more retinal protection benefits than would be expected solely based on their antioxidant properties, and therefore, further this combination presents a unique natural solution for protecting retinal RPE cells against degeneration, and for clinical management of dry and wet age-related macular degeneration or AMD. A combination therapy with saffron and resveratrol is particularly attractive considering a lack of toxicity at therapeutic levels.

A multi-targeted approach for the treatment of AMD using a combination of saffron and resveratrol is particularly important considering that there is no treatment for dry age-related macular degeneration or dry AMD. Additionally, in wet age-related macular degeneraiton or wet AMD, VEGF inhibitors are not enough to cure the disease because direct inhibition of VEGF-A (by commonly used anti-VEGF drugs) may actually decrease neuroprotection. VEGF-A is suggested to be directly involved in the maintenance and survival of normal retinal neurons, via a direct neuroprotective effect (43-45).

Macular degeneration references:

1-Brown et al,. 2005. Age-related macular degeneration: economic burden and value-based medicine analysis. Can J Ophthalmol. 2005 Jun;40(3):277-87.

2-Sheu et al,. 2010. Resveratrol protects human retinal pigment epithelial cells from acrolein-induced damage. J Ocul Pharmacol Ther. 2010 Jun;26(3):231-6.

3-Albani et al,. 2013. Synergism between resveratrol and crocin for protection of human neuroblastoma SHSY-5Y cells against oxidative stress. Natural Products at a Crossroad: Current and Future Directions. American Society of Pharmacognosy, 2013 Annual Meeting, Missouri.

4-Piraee et al,. 2012. Synergistic cytoprotective effects of crocin (CRN) and resveratrol (RSV) on primary human retinal pigmentepithelium (RPE) cells. International Congress on Natural Products Research 2012 Aug (ICNPR 2012), New York.

5-Falsini et al,. 2010. Influence of saffron supplementation on retinal flicker sensitivity in early age-related macular degeneration. Invest Ophthalmol Vis Sci. 2010 Dec;51(12):6118-6124.

6-Piccardi et al,.2012. A longitudinal follow-up study of saffron supplementation in early age-related macular degeneration: sustained benefits to central retinal function. Evid Based Complement Alternat Med. 2012 Jul:429124. doi: 10.1155/2012/429124.

7-Marangoni et al,. 2013. Functional effect of Saffron supplementation and risk genotypes in early age-related macular degeneration: a preliminary report. J Transl Med. 2013 Sep;11:228. doi: 10.1186/1479-5876-11-228.

8-Marco et al,. 2013. The time course of action of two neuroprotectants, dietary saffron and photobiomodulation, assessed in the rat retina. Am J Neurodegener Dis. 2013 Sep;2(3):208-20.

9-Xuan et al,1990. Effects of crocin analogs on ocular blood flow and retinal function. J Ocul Pharmacol Ther. 1999 Apr;15(2):143-52.

10-Laabich et al,. 2006. Protective effect of crocin against blue light- and white light-mediated photoreceptor cell death in bovine and primate retinal primary cell culture. Invest Ophthalmol Vis Sci. 2006 Jul;47(7):3156-63.

11-Thomas et al,. 2007.Visual functional effects of constant blue light in a retinal degenerate rat model. Photochem Photobiol. 2007 May-Jun;83(3):759-65.

12-Ochiai T, Soeda S, Ohno S, Tanaka H, Shoyama Y, Shimeno H. Crocin prevents the death of PC-12 cells through sphingomyelinase-ceramide signaling by increasing glutathione synthesis. Neurochem Int. 2004 Apr;44(5):321–330.

13-Soeda et al,. 2001. Crocin suppresses tumor necrosis factor-alpha-induced cell death of neuronally differentiated PC-12 cells. Life Sci. 2001 Nov;69(24):2887-98.

14-Maccarone et al,. 2008. Saffron supplement maintains morphology and function after exposure to damaging light in mammalian retina. Invest Ophthalmol Vis Sci. 2008 Mar;49(3):1254-61.

15-Natoli et al,. 2010. Gene and noncoding RNA regulation underlying photoreceptor protection: microarray study of dietary antioxidant saffron and photobiomodulation in rat retina. Mol Vis. 2010 Sep;16:1801-22.

16-Xi et al,. 2007. Pharmacokinetic properties of crocin (crocetin digentiobiose ester) following oral administration in rats. Phytomedicine. 2007 Sep;14(9):633-6.

17-Asai et al,. 2005. Orally administered crocetin and crocins are absorbed into blood plasma as crocetin and its glucuronide conjugates in mice. J Agric Food Chem. 2005 Sep;53(18):7302-6.

18-Yamauchi et al,. 2011. Crocetin prevents retinal degeneration induced by oxidative and endoplasmic reticulum stresses via inhibition of caspase activity. Eur J Pharmacol. 3011 Jan;650(1):110-119.

19-Ishizuka et al,. 2013. Crocetin, a carotenoid derivative, inhibits retinal ischemic damage in mice. Eur J Pharmacol. 2013 Mar;703(1-3):1-10. doi: 10.1016/j.ejphar.2013.02.007.

20-Umigai et al,. 2012. Crocetin, a carotenoid derivative, inhibits VEGF-induced angiogenesis via suppression of p38 phosphorylation. Curr Neurovasc Res. 2012 May;9(2):102-9.

21- Ghahghaei et al,. 2013. The protective effect of crocin on the amyloid fibril formation of Aβ42 peptide in vitro. Cell Mol Biol Lett. 2013 Sep;18(3):328-39. doi: 10.2478/s11658-013-0092-1.

22-Papandreou et al,. 2006. Inhibitory activity on amyloid-beta aggregation and antioxidant properties of Crocus sativus stigmas extract and its crocin constituents. J Agric Food Chem. 2006 Nov;54(23):8762-8.

23-Mansoor et al,. 2009. Inhibition of apoptosis in human retinal pigment epithelial cells treated with benzo(e)pyrene, a toxiccomponent of cigarette smoke. Invest Ophthalmol Vis Sci. 2010 May;51(5):2601-7. doi: 10.1167/iovs.09-4121.

24-Khan et al,. 2010. Resveratrol regulates pathologic angiogenesis by a eukaryotic elongation factor-2 kinase-regulated pathway. Am J Pathol. 2010 May;177(1):48-492.

25-Chan et al,. 2013. Inhibitory effects of resveratrol on PDGF-BB-induced retinal pigment epithelial cell migration via PDGFRβ, PI3K/Akt and MAPK pathways. PLoS One. 2013 Feb;8(2):e56819. doi: 10.1371/journal.pone.0056819.

26-Zheng et al, 2010.Resveratrol protects human lens epithelial cells against H2O2-induced oxidative stress by increasing catalase, SOD-1, and HO-1 expression. Mol Vis. 2010 Aug ;16:1467-74.

27-Kubota et al,. 2010. Resveratrol prevents light-induced retinal degeneration via suppressing activator protein-1 activation. Am J Pathol. 2010Aug;177(4):1725-31.

28-Pintea et al,. 2011. Antioxidant effect of trans-resveratrol in cultured human retinal pigment epithelial cells. J Ocul Pharmacol Ther. 2011 Aug;27(4):315-21.

29-Sheu et al,. 2013. Resveratrol stimulates mitochondrial bioenergetics to protect retinal pigment epithelial cells from oxidative damage. Invest Ophthalmol Vis Sci. 2013 Sep;54(9):6426-38. doi: 10.1167/iovs.13-12024.

30-Kubota et al,. 2009. Prevention of ocular inflammation in endotoxin-induced uveitis with resveratrol by inhibiting oxidative damage and nuclear factor-kappaB activation. Invest Ophthalmol Vis Sci. 2009 Mar;50(7):3512-9.

31-Kim and Suh,. 2010. Retinal protective effects of resveratrol via modulation of nitric oxide synthase on oxygen-induced retinopathy. Korean J Ophthalmol. 2010 Apr;24(2):108-18.

32-Li et al,.2012. Endoplasmic reticulum stress in retinal vascular degeneration: protective role of resveratrol. Invest Ophthalmol Vis Sci. 2012 May;53(6):3241-9. doi: 10.1167/iovs.11-8406.

33-Mimura et al,. 2013. The role of SIRT1 in ocular aging. Exp Eye Res. 2013 Nov;116:17-26. doi: 10.1016/j.exer.2013.07.017.

34- Khan et al,. 2012. SIRT1 activating compounds reduce oxidative stress and prevent cell death in neuronal cells. Front Cell Neurosci. 2012 Dec;6:63. doi: 10.3389/fncel.2012.00063.

35-Qin et al,. 2013. Resveratrol protects RPE cells from sodium iodate by modulating PPARα and PPARδ. Exp Eye Res. 2013 Dec;118C:100-108. doi: 10.1016/j.exer.2013.11.010.

36- Pasovic et al. 2014. Antioxidants Improve the Viability of Stored Adult Retinal Pigment Epithelial-19 Cultures. Ophthalmol Ther. 2014 Mar 29.

37-Dugas et al,. 2010. Effects of oxysterols on cell viability, inflammatory cytokines, VEGF, and reactive oxygen species production on human retinal cells: cytoprotective effects and prevention of VEGF secretion by resveratrol. Eur J Nutr. 2010 Mar; 49(7):435-46.

38-Balaiya et al,. 2013. Resveratrol inhibits proliferation of hypoxic choroidal vascular endothelial cells. Mol Vis. 2013 Nov;19:2385-92.

39-Richer et al,. 2013. Observation of human retinal remodeling in octogenarians with a resveratrol based nutritional supplement. Nutrients. 2013 Jun;5(6):1989-2005. doi: 10.3390/nu5061989.

40-Cao et al,. 2013. SIRT1 negatively regulates amyloid-beta-induced inflammation via the NF-κB pathway. Braz J Med Biol Res. 2013 Aug;46(8):659-69. doi: 10.1590/1414-431X20132903.

41-Hua et al,. 2011. Resveratrol inhibits pathologic retinal neovascularization in Vldlr-/ – Mice. Invest Ophthalmol Vis Sci. 2011 Apr;52(5):2809-26.

42-Bråkenhielm et al,. 2001.Suppression of angiogenesis, tumor growth, and wound healing by resveratrol, a natural compound in red wine and grapes. FASEB J. 2001 Aug;15(10):1798-800.

43-Nagai N1 et al., 2014. Resveratrol prevents the development of choroidal neovascularization by modulating AMP-activated protein kinase in macrophages and other cell types. J Nutr Biochem. 2014 Ju; pii: S0955-2863(14)00132-6. doi: 10.1016/j.jnutbio.2014.05.015.

44-Magnussen et al,. 2010. VEGF-A165b is cytoprotective and antiangiogenic in the retina. Invest Ophthalmol Vis Sci. 2010 Aug;51(8):4273-81. doi: 10.1167/iovs.09-4296.

45-Nishijima et al,. 2007.Vascular endothelial growth factor-A is a survival factor for retinal neurons and a critical neuroprotectant during the adaptive response to ischemic injury. Am J Pathol. 2007 Jul;171(1):53-67.