Quercetin glycosides and chlorogenic acid in highbush blueberry leaf decoction prevent cataractogenesis in vivo and in vitro: Investigation of the effect on calpains, antioxidant and metal chelating properties

Accepted Manuscript Quercetin glycosides and chlorogenic acid in highbush blueberry leaf decoction prevent cataractogenesis in vivo and in vitro: investigation of the effect on calpains, antioxidant and metal chelating properties Anastasia-Varvara Ferlemi, Olga E. Makri, Penelope G. Mermigki, Fotini N. Lamari, Constantinos D. Georgakopoulos PII:

S0014-4835(16)30011-2

DOI:

10.1016/j.exer.2016.01.012

Reference:

YEXER 6855

To appear in:

Experimental Eye Research

Received Date: 14 September 2015 Revised Date:

14 January 2016

Accepted Date: 20 January 2016

Please cite this article as: Ferlemi, A.-V., Makri, O.E., Mermigki, P.G., Lamari, F.N., Georgakopoulos, C.D., Quercetin glycosides and chlorogenic acid in highbush blueberry leaf decoction prevent cataractogenesis in vivo and in vitro: investigation of the effect on calpains, antioxidant and metal chelating properties, Experimental Eye Research (2016), doi: 10.1016/j.exer.2016.01.012. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Blueberry leaf polyphenols prevent cataractogenesis Polyphenols protect lenses Polyphenols protect lenses fromfrom oxidative stress & protein oxidative stress & protein lysishydrolysis

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20 μmol/kg BW Na2SeO3

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Rich in chlorogenic acid & quercetin glycosides Neonatal

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day 10

Quercetin inhibits SeO32- and Ca2+induced porcine lens turbidity in vitro Quercetin is oxidized by selenite anions and interacts with calcium.

days11 & 12

Na2SeO3+ blueberry leaf polyphenols 100 mg/kg BW

Quercetin glycosides inhibit μ-calpain with IC50 values in the μΜ range

ACCEPTED MANUSCRIPT Quercetin glycosides and chlorogenic acid in highbush blueberry leaf decoction prevent cataractogenesis in vivo and in vitro: investigation of the effect on calpains, antioxidant and metal chelating properties

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Anastasia-Varvara Ferlemi1, Olga E. Makri2, Penelope G. Mermigki1,

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Laboratory of Pharmacognosy & Chemistry of Natural Products, Department of

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Pharmacy, University of Patras, 26504 Patras, Greece 2

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Fotini N. Lamari1*, Constantinos D. Georgakopoulos2*

Department of Ophthalmology, Medical School, University of Patras, 26504 Patras,

Greece

*Corresponding authors:

[email protected]) or

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Dr Fotini N. Lamari (Tel.: +30 2610 962335, Fax: +30 2610 969181, E-mail:

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Dr Constantine D. Georgakopoulos (Tel.: +30 2610 999262, Fax: +30 2610 993994; E-

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mail: [email protected])

Grants: The study was supported by ‘K. Karatheodoris’ grant No C913 from the Research Committee, University of Patras, Greece.

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ACCEPTED MANUSCRIPT Abstract

The present study investigates whether highbush blueberry leaf polyphenols prevent cataractogenesis and the underlying mechanisms. Chlorogenic acid, quercetin, rutin, isoquercetin and hyperoside were quantified in Vaccinium corymbosum leaf decoction

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(BBL) using HPLC-DAD. Wistar rats were injected subcutaneously with 20 μmol selenite (Na2SeO3)/kg body weight on postnatal (PN) day 10 (Se, n=8-10/group) only or also intraperitoneally with 100 mg dry BBL/kg body weight on PN days 11 and 12 (SeBBL group, n=10). Control group received only normal saline (C). Cataract evaluation revealed

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that ΒBL significantly prevented lens opacification. It, also, protected lens from selenite oxidative attack and prevented calpain activation, as well as protein loss and aggregation.

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In vitro studies showed that quercetin attenuated porcine lens turbidity caused by SeO32- or Ca2+ and interacted efficiently with those ions according to UV-Vis titration experiments. Finally, rutin, isoquercetin and hyperoside moderately inhibited pure human μ-calpain. Conclusively, blueberry leaf extract, a rich source of bioactive polyphenols, prevents cataractogenesis by their strong antioxidant, chelating properties and through

Keywords

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direct/indirect inhibition of lens calpains.

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calpain.

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Vaccinium corymbosum, ocular cataract, lens redox system, calcium, selenite, quercetin,

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Abbreviations

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BBL: blueberry leaf decoction GAE: gallic acid equivalents QE: quercetin equivalents WSF: water soluble fraction

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WIF: water insoluble fraction GSH: reduced glutathione

CAT: catalase SOD: superoxide dismutase GPx: glutathione peroxidase

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AMC: 7-amino-4-methyl-coumarin

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GSSG: oxidized glutathione

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ACCEPTED MANUSCRIPT Introduction Cataract is the first cause of world blindness and one of the major causes of disability due to vision impairment (Pascolini and Mariotti, 2012). The only available treatment is the surgical removal of the opaque lens and its replacement with an artificial one. Nevertheless, surgical treatment is not widely available and, thus, interventions which

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will maintain the transparency of the crystalline lens are intensively sought after.

Cataractogenesis is a multifactorial process that lasts for years. Subtle posttranslational modifications in the lens structural proteins (cytoskeletal, crystallins, ion transporters) and

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mainly oxidation of sulfhydryl groups, result in their aggregation, fragmentation and precipitation and eventually in lens opacification (Stefek and Karasu, 2011). The shift of the oxidative status of lens towards the pro-oxidant state (mostly age-related) accounts for

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the abnormal lenticular calcium accumulation, which induces crystallin proteolysis through activation of calpains (Biswas et al., 2004). Ostadalova et al. (Ostadalova et al., 1978) demonstrated that an overdose of sodium selenite (Na2SeO3) to suckling rat pups induces cataractogenesis, partially mimicking senile nuclear cataract in humans. Selenite exerts its effect on lens by inducing oxidative stress and damage. More precisely,

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Na2SeO3 induces oxidation of sulfhydryl groups of proteins or ion channels, alteration of epithelial metabolism, disturbance of calcium homeostasis, calpain activation, crystallin proteolysis/precipitation and cytoskeletal loss (Shearer et al., 1997). Berries (fruits of several Vaccinium sp.) are a good source of vitamin C, dietary fiber,

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and minerals, but mainly they contain high levels of polyphenols and rank first among other fruits for their antioxidant potential. Both the fruits and the leaves of Vaccinium

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species (e.g. V. angustifolium, V. myrtillus, V. corymbosum) have been traditionally used to improve eye sight (Zafra-Stone et al., 2007) and to treat glaucoma and ocular inflammation (Miyake et al., 2012; Shim et al., 2012). Blueberry fruits are a rich source of anthocyanins and phenolic acids /esters (e.g. chlorogenic acid), and also in lesser amounts of flavanols and flavonols, proanthocyanidins and stilbenes. Βlueberry leaves contain more or less the same polyphenol compounds, albeit in different proportions and amounts. We have, previously, demonstrated that the decoction of Vaccinium corymbosum leaves is rich in hydroxycinnamic acids and quercetin glycosides and that it exerts significant beneficial effects on the selenite disturbed cerebral regions and liver of neonatal rats in a regionspecific manner (Ferlemi et al., 2015). 4

ACCEPTED MANUSCRIPT In this study we further investigated the composition of the decoction of blueberry leaves (V. corymbosum) (BBL) in quantitative terms and its potential to prevent cataractogenesis. The presence of chlorogenic acid, quercetin and its glycosides (rutin, hyperoside and isoquercetin) in high amounts in this extract adds further significance to this study due to their widespread presence in many fruits and plant extracts. Using the well-established

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model of sodium selenite-induced cataract, we investigated the effects of the administration of BBL to rat pups on cataract formation. The lens redox balance, soluble and insoluble protein content and proteolytic enzyme activity were, also, evaluated. In order to investigate the mechanisms by which those polyphenols confer protection, we

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additionally used three different in vitro experiments. Firstly, using porcine lens turbidity model, we examined whether they protect lens proteins from selenite oxidation or calciuminduced proteolysis. Furthermore, the direct effect of the above-mentioned natural

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products on μ-calpain enzymic activity was studied fluorimetrically. Finally, the UV-Vis titration was used to study the possible interactions of the phenolic compounds and selenite or calcium ions (the latter activate calpains).

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2. Methods and Materials 2.1 Phytochemical analysis of BBL

2.1.1 Plant material and extraction

Dried blueberry leaves from Vaccinium corymbosum cultivated in the area of Drama

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(East Macedonia, Greece) were kindly offered from the Greek Cooperative ”Biodrama”. The powdered leaves were refluxed at 100oC for 2h (10%w/v) in distilled

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water in the absence of light. The preparation of the decoction was repeated three times with different samples of dried blueberry leaves. The obtained decoctions were centrifuged, filtered, lyophilized and stored at -20 ºC. The animals received the dry extract, dissolved in sterilized normal saline.

2.1.2 Total phenolic content, total flavonoid content and chromatographic analysis of blueberry leaf decoction Total phenolics of blueberry leaf decoction (BBL) were measured with the FolinCiocalteau reagent method (Magalhaes et al., 2010; Singleton and Esau, 1969). The total 5

ACCEPTED MANUSCRIPT polyphenolic content was expressed as mg gallic acid equivalents (GAE)/g dry extract, using a curve of standard gallic acid at the 5 – 50 μg/mL range. The total flavonoid content was determined with the aluminum chloride colorimetric method, using a curve of standard quercetin (5-200 μg/mL) (Aiyegoro and Okoh, 2010). Total flavonoids were

experiments performed in triplicate (n=9).

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expressed as quercetin equivalents (QE)/g dry extract. The values are the result of three

The determination of the major phenolics was performed with HPLC-UV, as previously described (Tsao and Yang, 2003). The chromatographic system consisted of an Ultimate 3000 Pump (Pump LPG-3400 A, Dionex Corporation Sunnyvale, CA,

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USA) with a 20 μL Rheodyne 8125 injector (Rheodyne, Ronhert Park, CA, USA). The Column Compartment (TCC- 3100) was stabilized at 30ºC and detection was

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performed with a Diode Array Detector (DAD), Ultimate DAD-3000. Data were collected, stored and integrated on a Chromeleon v 6.80 Systems software. Separation of analytes was performed on a Phenomenex® Luna C18 column (250 mm x 4.6 mm i.d.; particle size, 5 μm) with a C18 guard column. The binary mobile phase consisted of 6% acetic acid in 2 mM sodium acetate (final pH 2.55, v/v, solvent A) and acetonitrile (solvent B). The flow rate was kept constant at 1.0 mL/min for a total run

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time of 70 min. The system was run with a gradient program: 0–15% B in 45 min, 15– 30% B in 15 min, 30–50% B in 5 min, and 50–100% B in 5 min. There was a 10 min post run at initial conditions for equilibration of the column. The detector was set at 280 (hydroxybenzoic acid derivatives, flavan-3-ols), 320 (hydroxycinnamic acid), 350

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(flavonols), and 520 nm (for anthocyanins) for simultaneous monitoring of different groups of polyphenols.

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The calibration curves were made from pure standards: chlorogenic acid (25-200 μg/mL, y= 1.0262x + 4.0157, R2=0.9979), quercetin-3-O-rutinoside (y= 0.6916x – 2.1668, R2=0.9981), quercetin-3-O-galactoside (y= 0.6477x – 2.8305, R2=0.9970), quercetin-3-O-glucoside (y= 0.6463x – 2.255, R2=0.9979) (all glycosides in the range of 5-100 μg/mL) and quercetin (2-50 μg/mL, y= 0.6159x – 1.8635, R2=0.9804). All standards [chlorogenic acid (≥99%), quercetin-3-O-rutinoside (rutin) (≥99%), quercetin3-O-galactoside (hyperoside) (≥98%), quercetin-3-O-glucoside (isoquercetin) (≥98.5%), quercetin (≥99%)] were purchased from Extrasynthese (Z.I Lyon Nord, France). Results are expressed as %w/w.

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ACCEPTED MANUSCRIPT 2.2 Selenite-induced cataract model 2.2.1 Animals Wistar rat pups weighing 12-14g on postnatal (PN) day 7 were used for the present study. They were housed along with their mothers under controlled temperature (23 ºC)

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and humidity conditions with 12h light/dark cycles. The mothers were maintained on a standard laboratory animal diet and provided tap water ad libitum throughout the experimental period. The animals were randomly divided into three groups:

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Group C: Control animals (n=8) which received only normal saline.

Group Se: Selenite-treated animals (n=8) which received a subcutaneous injection of

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selenite (20 μmol Na2SeO3/kg body weight) on PN day 10.

Group SeBBL: Selenite/blueberry leaf decoction (BBL) – treated animals (n=10), which received a subcutaneous injection of selenite (20 μmol/kg body weight) on PN day 10 and two intraperitoneal injections of BBL (100 mg dry extract/kg body weight) on PN days 11 and 12. The dose of 100 mg/kg body weight was selected as a rather

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medium "pharmaceutical" dose, which has been earlier used for studies of short duration of polyphenol rich extracts (Ren et al., 2014).

Cataract formation was evident and could be evaluated with an ophthalmoscope from the 16th P N day; both eyes were evaluated. On PN day 21, rats were weighed and

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sacrificed by cervical dislocation. The eyes were enucleated and crystalline lenses were at once excised intracapsularly through an incision 2 mm posterior to the limbus. The

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experiments were conducted in strict compliance with ARVO Statement for Use of Animals in Ophthalmic and Vision Research and Guideline Principles in the Care and Use of Animals and the EU Directive 2010/63/EU for animal experiments. The study was approved by the Bioethics Committee of the University Hospital of Patras.

2.2.2 Evaluation of cataract formation The final evaluation of cataract formation was performed on PN day 21 after dilation of pupils with instillation of a drop of tropicamide 0.5% (Tropixal, Demo SA, Greece) and removal of the lenses from the sacrificed rats. Lens opacification was evaluated by 7

ACCEPTED MANUSCRIPT examination of the lenses under a dissecting microscope and cataract staging was performed based on a scale 0 to 6 according to Hiraoka at al. (Hiraoka and Clark, 1995), as follows: Stage 0: normal transparent lens

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Stage 1: initial sign of posterior subcapsular or nuclear opacity involving tiny scatters Stage 2: slight nuclear opacity with swollen fibers or posterior subscapular scattering foci Stage 3: diffuse nuclear opacity with cortical scattering

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Stage 4: partial nuclear opacity Stage 5: nuclear opacity that does not involve lens cortex

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Stage 6: mature dense opacity involving the entire lens.

2.2.3 Lens Treatment and Protein Content Determination

Lenses were homogenized (10% w/v) in ice-cold phosphate buffer 30 mM, pH 7.6 using a glass homogenizer (Thomas Philadelphia, USA, No B 13957). The homogenates were

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centrifuged at 15,000 g for 20 min at 4oC. The supernatant contained the water-soluble fraction of lens proteins (WSF). The pellet was re-dissolved with equal volume of 1% (w/v) Triton-X 100 in sodium phosphate buffer 30 mM, pH=7.6 and centrifuged at 15,000 g for 20 min at 4oC. The supernatant obtained contained the water insoluble

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fraction of lens proteins (WIF).

Total protein content was determined with the

method of Bradford (Bradford, 1976). The results were expressed as mg protein/mg

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tissue.

2.2.4 Determination of Lipid Peroxidation Levels and Glutathione Content in WSF Lipid peroxidation, expressed as malondialdehyde levels, was determined only in the WSF due to the high solubility of MDA in water (500g/L, pH 7) (Lord et al., 2009), after reaction with thiobarbituric acid (Grotto et al., 2007; Jentzsch et al., 1996). The results, performed in triplicate, were expressed as nmol MDA equivalents/g tissue using standard concentrations of MDA (0.05-10 μΜ). The reduced (GSH) and oxidized (GSSG) glutathione were estimated fluorimetrically after reaction with o-phthalaldehyde (OPT) in WSF (Hissin and Hilf, 1976; Makri et al., 8

ACCEPTED MANUSCRIPT 2013). Standards of GSH and GSSG (0.75–15 μΜ) were used and the respective concentrations were expressed as nmol/mg wet tissue.

2.2.5 Enzyme activity assays

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Enzyme activities were determined in WSF. Catalase (CAT, EC 1.11.1.6) activity (Makri et al., 2013; Sinha, 1972), performed in triplicate, was expressed as μmol formaldehyde/min/g tissue against a standard curve (0-75 μΜ formaldehyde). Superoxide dismutase (SOD, EC 1.15.1.1), activity was determined with the

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Superoxide Dismutase Assay Kit available from the Cayman Chemical Company. The results are expressed as units/mg tissue, where one unit of SOD is defined as the

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amount of enzyme needed to exhibit 50% dismutation of the superoxide anion. Glutathione peroxidase (GPx, EC 1.11.1.9) activity (Rotruck et al., 1973), conducted in triplicate, was expressed as U/min/g tissue, using a standard curve of known concentrations of GPx (0.025-1 U/mL).

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2.2.6 Lens proteolytic activity

Tissue proteolytic activity was estimated in WSF using the fluorogenic peptide substrate Suc-Leu-Tyr-AMC (from Calbiochem®-Merck Millipore) which is used for the determination of calpain 1 and 2 and the peptidase activity of the 20S proteasome

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(Sasaki et al., 1984). Tissue homogenates were mixed with reaction buffer (100 mM Tris-HCl, 100 mM NaCl, 2 mM EDTA, 10 mM β-mercaptoethanol) and incubated at

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37oC for 10 min. Substrate (75 μΜ) and CaCl2 ( 17 mM) were added to the mixture and the samples were incubated at room temperature for 30 min and centrifuged for 10 min. Fluorescence intensity of the supernatants (excitation/emission wavelengths: 380/460 nm) was measured in a TECAN® 96-well plate reader in duplicate. A calibration curve was constructed using standard concentrations (0.01 - 100 nM) of pure 7-amino-4-methyl-coumarin (AMC).

2.2.7 Polyacrylamide gel electrophoresis Electrophoresis

was

performed

on an instrument of

Cleaver

Scientific

Ltd 9

ACCEPTED MANUSCRIPT (Warwickshire, Rugby, UK) according to the modified method of Laemmli et al. (Laemmli, 1970). Lens homogenate (30 μg WSF and 250 μg WIF per lane) were mixed with sample buffer (35% v/v glycerol, 18% v/v 2-mercaptoethanol, 230 mM Tris/HCl pH=6.8, 10% w/v sodium dodecyl sulfate, 0.3% w/v bromophenol blue), boiled for 5 min at 100oC, centrifuged for 3 min and left to cool down at room temperature.

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Then, the mix was loaded on a 12% (WSF) or on a 15% (WIF) polyacrylamide gel. Protein lanes were visualized with 0.5% v/v Coomassie Brilliant Blue and scanned.

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2.3. In vitro Lens Crystallin Turbidity Assay

Porcine lenses were obtained from a local slaughterhouse and homogenized in 50 mM

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Tris-HCl, pH 8.0, containing 0.1 M NaCl, 5 mM EDTA, 0.01% β-mercaptoethanol and 0.02% sodium azide. After centrifugation at 15,000 g for 30 min, the supernatant was collected and the protein concentration was determined (Bradford, 1976). Lens proteins (25 mg/mL, final concentration) were mixed with either Na2SeO3 or CaCl2 (10 mM, final concentration) to induce turbidity. The polyphenols were added in such concentration so that the polyphenol/Na2SeO3 or /CaCl2 molar ratio in the well was 1/2 and 1/20. Lens

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homogenate with buffer only was used as negative control. Buffers, with or without polyphenols, were used as blanks. Samples were incubated at 37oC for 5 days. The plates were carefully closed with their plastic tops and covered with parafilm and aluminum foil prior to the incubation. New covers were used after each measurement. Sodium azide was

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used in buffer in order to prevent microbial or fungal contamination of the samples. The turbidity was measured daily at 630 nm (Liao et al., 2011). Data were the result of three

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different experiments (three wells/experiment). The percentage of inhibition was calculated based on the difference between the last and the first measurement of turbidity (progress of turbidity) in comparison to the respective progress caused by selenite or calcium.

2.4. Fluorometric μ-calpain enzymic activity assay The method is based on the specific cleavage of Suc-Leu-Tyr-AMC substrate by μcalpain (Calpain 1, Human Erythrocytes-Catalogue Number 208713, Calbiochem). Chlorogenic acid, quercetin, rutin, hyperoside and isoquercetin (Extrasynthese, Z.I Lyon 10

ACCEPTED MANUSCRIPT Nord, France), which were also used for the HPLC analysis of BBL, were tested as potential inhibitors of μ-calpain. Stock solutions of flavonoids in DMSO were diluted with reaction buffer (50 mM Tris-HCl, pH 7.5, 50 mM NaCl, 1 mM EDTA, 5 mM βmercaptoethanol) so that the final concentration of DMSO was less than 1%. The concentration range for chlorogenic acid, rutin, hyperoside and isoquercetin was 1-800

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μΜ. Quercetin, which is insoluble in water, was diluted in 40% MeOH in reaction buffer, at a lower concentration range (0.001-50 μΜ) due to its intrinsic fluorescent properties. Substrate (75 μΜ), μ-calpain (100 μΜ) and polyphenols were added in a 96-well microplate for fluorescence assays. The reaction was initiated by the addition of CaCl2

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solution (2.5 mM) to a final volume of 100 μL. Reaction buffer with calcium only was the negative control. Calpain inhibitor MDL 28170 (Calbiochem) was used as positive

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control. Buffers without polyphenols were used as blanks. After 30 min incubation at RT, fluorescence intensity was measured. Excitation/emission wavelengths: 380/460nm (Kang et al., 2009). The IC50 values were calculated using the software Graph Pad Prism 6.0.

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2.5. UV-Vis titration study of polyphenol interaction with selenite or calcium Working solutions of chlorogenic acid, quercetin, rutin, hyperoside and isoquercetin (1 mM) were prepared in DMSO/H2O (3/17, v/v). Sodium selenite (40 mM) and CaCl2 (40 mM) stock solutions were prepared in the same DMSO/H2O mixture. Polyphenols (400

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μΜ, final concentration) were mixed with selenite or calcium at various ratios (1:2, 1:10, 1:20, 1:100) and 0.4 mL of each mixture was used for the monitoring of UV/Vis

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spectrum. Phenolics dissolved in DMSO/H2O without selenite or calcium ions were used as reference standards. The method was slightly modified from Liao et al. (Liao et al., 2009).

2.6 Statistical Analysis The results were analyzed by Kruskal – Wallis non-parametric one way analysis of variance (ANOVA). If the overall p-value was statistically significant, comparisons among groups were made according to Mann-Whitney U-tests. The inhibition of lens protein turbidity in vitro was analyzed by Student’s t-test. Probability values of less than 11

ACCEPTED MANUSCRIPT 0.05 were considered statistically significant. All statistical analyses were performed using the SPSS statistical software version 14.0.

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RESULTS

3.1 Phytochemical analysis of BBL

The total polyphenol content of highbush blueberry leaf decoction was 355±42 mg

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GAE/g dry extract, while total flavonoid was 69.4±1.7 mg quercetin/g dry extract. HPLC analysis of the BBL leaf decoction revealed that phenolic acids (7-17 min) and flavonoids (45-67 min) were the most abundant components, whereas anthocyanins were

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absent. The characterization of the compounds was performed based on the retention time, their UV-Vis spectra, spiked samples with standard compounds and the literature. The five major peaks were chlorogenic acid, rutin, hyperoside, isoquercetin and quercetin, and were quantified using the calibration curves of the standards (Table 1,

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figure S1).

3.2 BBL administration to selenite-treated rat pups 3.2.1 Animal development and cataract formation

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The body weight of the neonatal rats was monitored throughout the treatment period. On P N day 21, the average body weight of the control animals was 39.8 ± 0.6 g

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whereas that of the Se group was significantly lower by 14% (34.3 ±1.0 g, p<0.001). Interestingly, average body weight in the group SeBBL was higher than that in Se (38.5 ±1.2 g; 11%, p<0.01) and was not significantly different from that of controls.

Cataract classification results are presented in Table 2. Selenite injection induced cataract in all the lenses of group Se (median 4, range 2, p<0.001, compared to C group). BBL administration significantly decelerated the development of lens opacification (median 1, range 2, p<0.001) compared to group Se. Lens examples of each stage are presented in Figure 1.

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ACCEPTED MANUSCRIPT 3.2.2 Effects on Lens Proteins and Proteolytic activity Bradford analysis revealed that the total WSF of group Se lenses was statistically significant lower (35%, p<0.001) than that of Control lenses. In contrast, WIF was slightly increased (23%) in group Se compared with controls. Administration of BBL

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prevented WSF loss and WIF increase, respectively. The above results were confirmed by SDS-PAGE. In the WSF (Figure 2A) significant loss of the low molecular weight crystallin subunits (below 30 kD) in the Se-treated lenses was observed; One of the most affected band is that corresponding to ~20 kD.

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BBL administration partially prevented the loss of the polypeptides, which was triggered by selenite, as all the respective bands were more intense in the SeBBL lane than in the Se lanes. In the gel of the water-insoluble fraction (Figure 2B), we observed increased

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intensity of the bands corresponding to high molecular weights (>80 kD) in the Se lane, which indicated significant insolubilization of the proteins, as well as significant increase in the intensity of the bands less than 30 kD, corresponding to the small subunits of the insoluble crystallins. The above negative effects of selenite were prevented by BBL as both high and low molecular weight bands were less intense in the

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SeBBL lane.

Determination of calcium–induced proteolytic activity in the lens homogenate using the Suc-Leu-Tyr-AMC substrate is a good estimate of calpain activity. Selenite injection significantly induced increase of proteolytic activity in Se lenses compared to

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the controls (C: 1.7 ± 0.2 pmol AMC/min/g tissue, Se: 2.2 ± 0.5 pmol AMC/min/g tissue. Statistical difference 30%, p<0.001). On the other hand, BBL co-treatment preserved

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lens proteolytic activity at physiological levels (SeBBL: 1.3 ± 0.4 pmol AMC/min/g tissue).

3.2.3 Effects on Lens Lipid Peroxidation Levels and Antioxidant Indices Lipid peroxidation was 70% higher in group Se than in control group (Table 3). By contrast, BBL administration protected lens cell lipids from oxidation, as its levels were lower (51%) than in Se group (p<0.001) and similar to those of group C (p>0.05). As shown in Table 3, the glutathione content (ratio of reduced to the sum of reduced and oxidized) in lenses of selenite-treated animals was significantly lower in comparison with the respective controls. In group SeBBL, glutathione content was 2.5-fold 13

ACCEPTED MANUSCRIPT higher (p<0.001) than that of Se group. Remarkably, reduced glutathione as well as the ratio GSH/total glutathione in SeBBL lenses were even higher than the levels found in control group. Enzymic activity was considerably lower in the lenses of Se group in comparison to control. More specifically, SOD activity was half of the respective control values,

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while CAT and GPx were significantly lower by 37% and 27%, respectively. In the SeBBL lenses, the selenite-induced decrease of the activity of the three enzymes was

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prevented and was similar to that of the control lenses. Results are shown in Table 3.

3.3 Effect of chlorogenic acid and quercetin glycosides/aglycone on selenite or

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calcium-induced lens turbidity

The incubation of porcine lens homogenate in the presence of excess selenite concentration resulted to significant induction of turbidity. As shown in Figure 3, selenite caused gradually increasing cloudiness, even though the changes fluctuated from day to day (figure S2). All polyphenols inhibited the progress of selenite-induced turbidity in lens homogenates especially in the highest concentration. Quercetin showed

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the highest degree of inhibition during the 5 days of incubation [70% (ratio 1:2) and 50% (ratio 1:20)]. Rutin and hyperoside were almost identically effective; rutin decreased by 58% and 43% and hyperoside by 57% and 50% (ratios 1:2 and 1:20,

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respectively) the turbidity caused by selenite. Chlorogenic acid inhibited moderately cloudiness of lens homogenate [54% (1:2) and 25% (1:20)]. Finally, isoquercetin

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decreased it only by 38% (1:2) and 40% (1:20). Calcium-induced turbidity was, also, significantly inhibited by polyphenols (Figure 4). Quercetin showed, once again, the strongest inhibitory activity in both ratios. The percentage of inhibition, on the last day of the experiment, for high and low concentration of quercetin was 88% and 90%, respectively. Among glycosides, isoquercetin and rutin were so powerful as quercetin, showing percentage of inhibition in both ratios up to 80%, while the lowest inhibition was exhibited by chlorogenic acid [81% (1:2) and 60% (1:20)] and hyperoside [68% (1:2) and 64% (1:20)]. The above invitro results demonstrated that all the tested polyphenols significantly inhibited the induced cloudiness of porcine lens homogenates, but quercetin possesses the strongest inhibitory activity against both selenite- and calcium-induced turbidity. 14

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3.4. Effect of chlorogenic acid and quercetin glycosides/aglycone on μ-calpain activity The fluorometric determination of the effect of BBL phenolic compounds on μ-calpain

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showed that rutin was an effective inhibitor (IC50 96.2 μM), whereas isoquercetin and hyperoside inhibited the enzyme with IC50 values approximately 118.8 and 175.8 μM (Figure 5). Results for chlorogenic acid and quercetin revealed that they act as nonspecific inhibitors. The inhibitor MDL 28170, which was used as positive control,

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showed IC50 of 588.7 nM.

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3.5. Chlorogenic acid and quercetin aglycon/glycoside interactions with selenite and calcium ions

When we mixed quercetin with the selenite ions and prior to the analysis, we observed the formation of a brown solution. UV-Vis analysis of the SeO32-/natural product mixtures revealed that quercetin was affected to a greater extent than the other compounds (Figure 6). The reference spectrum of quercetin showed the two

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characteristic absorbance bands of flavonoids. The maximum absorbance wavelengths (λmax) were 254 nm (band I), corresponding to the ring A of the flavonoid, and 370 nm (band II), corresponding to the ring B. The lowest Q:Se ratio (1:2) showed decrease and

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red-shift of maximum absorbance values of bands I and II to 272 and 410 nm, respectively, while a new peak was observed with maximum at 320 nm. As the concentration of selenite ions increased, the peak at 272 nm was lost, the red-shift of

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band II was no further observed and the intensity of the new peak was higher. These results indicate complete oxidation of the flavonoid by selenite ions. Quercetin glycosides spectra showed only a moderate bathochromic shift and small increase of the absorbance, especially of band II, when mixed with selenite regardless of anion concentration. Similar results were obtained for the mixtures of chlorogenic acid with selenite ions (see Figure S4). Calcium interaction with quercetin resulted to moderate changes of its spectrum, while the other polyphenols were almost not affected. At ratios 1:2, 1:10 and 1:20, absorbance was lower, whereas the highest concentration of calcium ions (1:100) caused significant red shift of λmax at 396 nm, indicating changes of the ring B of the flavonoid (Figure 6). 15

ACCEPTED MANUSCRIPT Rutin, hyperoside and chlorogenic acid were almost not affected, while isoquercetin spectrum was slightly changed at the high ratio (1:100); specifically the absorbance was slightly lower and λmax, especially of the band II, was shifted to higher wavelength

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(Figure S5).

Discussion

Despite the traditional uses of blueberries, its leaf decoction is not consumed so much as the fresh blueberry fruits, and the leaves may even be considered as an agricultural

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by-product. Thus, data on the phytochemical composition of V. corymbosum leaves are limited. Nevertheless, some recent studies on other Vaccinium species showed that hydroxycinnamic acids, such as chlorogenic acid and caffeic acid, quercetin glycosides

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and anthocyanins are abundant in the leaves. In our study, polyphenolic content reached almost 35% w/w of the dry extract. HPLC analysis revealed that BBL is rich in hydroxycinnamic acids and flavonols, i.e. chlorogenic acid, quercetin and its glycosides. Quantitative analysis showed that chlorogenic acid is the major ingredient of BBL. Hyperoside is the most abundant glycoside, followed by isoquercetin and rutin,

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while quercetin concentration is the lowest. Anthocyanins were not detected in the decoction of the highbush blueberry leaves. The absence of these compounds is probably attributed to the extraction procedure; the continuous boiling in water might not be effective for their extraction, as they require polar solvents or it might have

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caused their degradation.

The study of the effects of BBL in the selenite cataract rat model showed that its

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administration protected lenses from selenite-induced opacification; the majority of the lenses of the animals treated with the decoction showed only the posterior subcapsular cataract, which is formed during the first 24 hours after the injection of sodium selenite (Shearer et al., 1983), indicating that BBL administration almost stopped the development of further opacification. In our project this inhibition was simultaneous with the elimination of the oxidative insult of selenite through reinforcement of lens antioxidants, which contributed to the significant protection against lipid oxidation. Thus, the oxidative state of the lens was more or less preserved to the physiological conditions. Calpains were not activated in SeBBL group despite the Se administration implying maintenance of the ion influx. Accordingly, the levels of soluble proteins of 16

ACCEPTED MANUSCRIPT SeBBL-treated lenses were higher than those in Se lenses demonstrating that protein loss was prevented, either via inhibition of proteolysis or through decreased oxidation. Our results are in accordance with studies of the anti–cataractogenic action of plant polyphenols in vivo and in vitro (Kim et al., 2011; Rooban et al., 2009; Rooban et al.,

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2011a, b; Sasikala et al., 2013; Wang et al., 2011). However, in the study of Sanderson et al. chlorogenic acid did not protect lens transparency and ion influx in a rat lens culture exposed to hydrogen peroxide, indicating that it could not inhibit cataractogenesis (Sanderson et al., 1999). Earlier studies about quercetin and its

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principle metabolite in the lens, 3’-O-methyl quercetin (isorhamnetin) (Cornish et al., 2002; Sanderson et al., 1999) have demonstrated that they protect lens from calcium and sodium influx, even at low micromolar concentrations, in a rat lens cultured model

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exposed to the endogenous oxidant hydrogen peroxide. In a number of studies (Orhan et al., 1999; Rooban et al., 2009; Vibin et al., 2010), quercetin was used as a reference flavonoid; uniformily, its intraperitoneal administration was reported to retard progression of lens opacity caused by selenite.

The accumulation of selenite and calcium in lenses may trigger cataract formation as

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high content of these ions has been found to be present in human lenses with increased opacification (Liao et al., 2011). Selenite accumulation might cause cataract formation either directly (Liao et al., 2009) or more possibly, during the first hours of the injection, via its indirect effects on calcium cellular homeostasis and subsequent

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induction of calcium-dependent molecules; it is known that disruption of the anterior normal epithelial cells leads to the development of posterior sub-capsular or/and slight

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nuclear opacity during the first stages of cataract (Spector, 1995). Usually, turbidity occurs after protein destruction and aggregation, caused by selenite-induced oxidation and/or proteolysis by calcium-depended calpains. The in-vitro model that we used is based on the incubation of porcine lens homogenates with excess concentration of selenite or calcium, in order to test both routes of turbidity formation (oxidation and proteolysis by calpains). We demonstrated that the quercetin aglycone was the most effective inhibitor of lens cloudiness in both selenite and calcium model. Interestingly, rutin was also greatly effective in both models, in contrast to the other glycosides which exhibited lower capacity to protect. Chlorogenic acid, the principal ingredient of the BBL, inhibited the formation of turbidity but was one of the least effective components. 17

ACCEPTED MANUSCRIPT The efficacy of quercetin (mainly) against selenite-induced opacification is attributed to its antioxidant properties including interaction with selenite ions, whereas that against calcium – induced opacification suggests that it might either inhibit calpains directly or bind excess calcium ions, or both. In order to examine the above hypotheses, we

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conducted a set of in vitro experiments. In accordance with the results from the turbidity experiment, UV-Vis titration analysis showed that quercetin was most affected when incubated with selenite than the other polyphenols. Comparing the spectra of quercetin and its glycosides, it can be

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hypothesized that the aglycone is completely oxidized by selenite ions probably forming structures, such as para-quinonomethides (Jacobs et al., 2010). In contrast, its glycosides showed only a slight red-shift of the λmax, of band II, indicating weak

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changes of the B-ring; it is likely that the conjugated sugar protects someway the A ring of the flavonoid and selenite ions may oxidize only the two –OH groups of the B ring, forming new structures such as ortho-quinones (Jacobs et al., 2010). This first report of the interaction of quercetin with selenite anions is significant as it indicates one more possible explanation of the positive results of BBL in vivo.

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the beneficial effects of BBL administration (on days 11 and 12) in Se-treated rats, i.e. stopping cataract formation at the posterior sub-capsular or slight nuclear opacity level that occurs only 24 hours after selenite injection (day 10), might also be attributed to the prevention of further damage caused by the Se indirect effects, i.e. calcium cellular

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imbalance and induction of calcium-dependent proteolysis by the polyphenol

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metabolites (including quercetin).

The inhibitory activity of the polyphenols on calcium-induced lens turbidity suggests direct inhibition of the calcium-induced proteolytic enzymes or strong interaction with the excess calcium. Ιn our project, quercetin glycosides were moderate μ-calpain inhibitors in accordance with other studies (Kim et al., 2009), but quercetin aglycone and chlorogenic acid acted as non-specific μ-calpain inhibitors. The inhibitory action of the glycosides might allude imply that the glucuronide metabolite of quercetin, might also be effective. Calcium interaction with flavonoids caused mild to moderate changes of their spectra, in comparison with selenite. Quercetin was, once again, most affected. Indeed, as ion 18

ACCEPTED MANUSCRIPT concentration increased, we observed almost loss of band I and significant decrease and red-shift of λmax intensity of band II, which indicated interactions between the –OH groups of the flavonoid rings and calcium ions. Among the glycosides, isoquercetin band II was the most affected, while the others were only slightly altered at the highest calcium concentration, indicating that the conjugated sugars allow only the interaction

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between the –OH groups of B ring and calcium ions. Chlorogenic acid did not interact with calcium.

In conclusion, blueberry leaf decoction is a neglected rich source of the common

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bioactive chlorogenic acid and quercetin glycosides, which efficiently affect the multiple key molecular mechanisms involved in the etiology of age-related cataract. After BBL i.p. administration to rats, selenite-induced cataract formation was

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prevented, lens antioxidant mechanisms were reinforced and withstood the oxidative insult of selenite, the integrity of lens crystallins was preserved and the increase of proteolytic activity was, also, prevented. After a screening of all major BBL polyphenols, it was demonstrated that quercetin was the most efficient inhibitor of selenite- and calcium- induced lens homogenate opacification, albeit chlorogenic acid was also moderately effective. We have shown in vitro that quercetin beyond its

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antioxidant properties interacts with selenite and calcium ions, and its glycosides inhibit calpain activity. Our in vitro results are consistent with other studies (Cornish et al., 2002; Rooban et al., 2009; Sanderson et al., 1999) in reporting that quercetin effectively

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inhibits cataractogenesis, although it is difficult to accurately describe which of its properties (antioxidant, metal chelating, calpain inhibition by its glycosides) is mainly responsible for the in vivo actions in the lens. Its common presence in fruits and plant

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extracts may explain the anticataractogenic action of many of those. On the other hand, plant extracts which are rich sources of quercetin glycosides could be used as food supplements or even as botanical drugs to prevent or retard the formation of cataract. Our study reveals that the decoction of blueberry leaves has anticataractogenic action in vivo and should be considered for such purposes.

Acknowledgement: The authors kindly thank the Greek Cooperative ‘Biodrama’, East Macedonia, Greece, for the offer of dried highbush blueberry leaves.

19

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Conflict of interest: Authors declare that there is no conflict of interest.

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Aiyegoro, O.A., Okoh, A.I., 2010. Preliminary phytochemical screening and in vitro antioxidant activities of the aqueous extract of Helichrysum longifolium DC. BMC Complement Altern Med 10, 21. Biswas, S., Harris, F., Dennison, S., Singh, J., Phoenix, D.A., 2004. Calpains: targets of cataract prevention? Trends Mol Med 10, 78-84. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72, 248254. Cornish, K.M., Williamson, G., Sanderson, J., 2002. Quercetin metabolism in the lens: role in inhibition of hydrogen peroxide induced cataract. Free Radic Biol Med 33, 63-70. Ferlemi, A.V., Mermigki, P.G., Makri, O.E., Anagnostopoulos, D., Koulakiotis, N.S., Margarity, M., Tsarbopoulos, A., Georgakopoulos, C.D., Lamari, F.N., 2015. Cerebral Area Differential Redox Response of Neonatal Rats to Selenite-Induced Oxidative Stress and to Concurrent Administration of Highbush Blueberry Leaf Polyphenols. Neurochem Res 40, 2280-2292. Grotto, D., Santa Maria, L.D., Boeira, S., Valentini, J., Charao, M.F., Moro, A.M., Nascimento, P.C., Pomblum, V.J., Garcia, S.C., 2007. Rapid quantification of malondialdehyde in plasma by high performance liquid chromatography-visible detection. J Pharm Biomed Anal 43, 619-624. Hiraoka, T., Clark, J.I., 1995. Inhibition of lens opacification during the early stages of cataract formation. Invest Ophthalmol Vis Sci 36, 2550-2555. Hissin, P.J., Hilf, R., 1976. A fluorometric method for determination of oxidized and reduced glutathione in tissues. Anal Biochem 74, 214-226. Jacobs, H., Moalin, M., Bast, A., van der Vijgh, W.J., Haenen, G.R., 2010. An essential difference between the flavonoids monoHER and quercetin in their interplay with the endogenous antioxidant network. PLoS One 5, e13880. Jentzsch, A.M., Bachmann, H., Furst, P., Biesalski, H.K., 1996. Improved analysis of malondialdehyde in human body fluids. Free Radic Biol Med 20, 251-256. Kang, D.H., Jun, K.Y., Lee, J.P., Pak, C.S., Na, Y., Kwon, Y., 2009. Identification of 3acetyl-2-aminoquinolin-4-one as a novel, nonpeptidic scaffold for specific calpain inhibitory activity. J Med Chem 52, 3093-3097. Kim, C.S., Kim, J., Lee, Y.M., Sohn, E., Jo, K., Kim, J.S., 2011. Inhibitory effects of chlorogenic acid on aldose reductase activity in vitro and cataractogenesis in galactosefed rats. Arch Pharm Res 34, 847-852. Kim, H.J., Lee, J.Y., Kim, S.M., Park, D.A., Jin, C., Hong, S.P., Lee, Y.S., 2009. A new epicatechin gallate and calpain inhibitory activity from Orostachys japonicus. Fitoterapia 80, 73-76. Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685. Liao, J.H., Chen, C.S., Hu, C.C., Chen, W.T., Wang, S.P., Lin, I.L., Huang, Y.H., Tsai, M.H., Wu, T.H., Huang, F.Y., Wu, S.H., 2011. Ditopic complexation of selenite anions or 20

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calcium cations by pirenoxine: an implication for anti-cataractogenesis. Inorganic chemistry 50, 365-377. Liao, J.H., Chen, C.S., Maher, T.J., Liu, C.Y., Lin, M.H., Wu, T.H., Wu, S.H., 2009. Astaxanthin interacts with selenite and attenuates selenite-induced cataractogenesis. Chem Res Toxicol 22, 518-525. Lord, H.L., Rosenfeld, J., Volovich, V., Kumbhare, D., Parkinson, B., 2009. Determination of malondialdehyde in human plasma by fully automated solid phase analytical derivatization. Journal of chromatography. B, Analytical technologies in the biomedical and life sciences 877, 1292-1298. Magalhaes, L.M., Santos, F., Segundo, M.A., Reis, S., Lima, J.L., 2010. Rapid microplate high-throughput methodology for assessment of Folin-Ciocalteu reducing capacity. Talanta 83, 441-447. Makri, O.E., Ferlemi, A.V., Lamari, F.N., Georgakopoulos, C.D., 2013. Saffron administration prevents selenite-induced cataractogenesis. Mol Vis 19, 1188-1197. Miyake, S., Takahashi, N., Sasaki, M., Kobayashi, S., Tsubota, K., Ozawa, Y., 2012. Vision preservation during retinal inflammation by anthocyanin-rich bilberry extract: cellular and molecular mechanism. Lab Invest 92, 102-109. Orhan, H., Marol, S., Hepsen, I.F., Sahin, G., 1999. Effects of some probable antioxidants on selenite-induced cataract formation and oxidative stress-related parameters in rats. Toxicology 139, 219-232. Ostadalova, I., Babicky, A., Obenberger, J., 1978. Cataract induced by administration of a single dose of sodium selenite to suckling rats. Experientia 34, 222-223. Pascolini, D., Mariotti, S.P., 2012. Global estimates of visual impairment: 2010. Br J Ophthalmol 96, 614-618. Ren, Z., He, C., Fan, Y., Guo, L., Si, H., Wang, Y., Shi, Z., Zhang, H., 2014. Immunoenhancement effects of ethanol extract from Cyrtomium macrophyllum (Makino) Tagawa on cyclophosphamide-induced immunosuppression in BALB/c mice. J Ethnopharmacol 155, 769-775. Rooban, B.N., Lija, Y., Biju, P.G., Sasikala, V., Sahasranamam, V., Abraham, A., 2009. Vitex negundo attenuates calpain activation and cataractogenesis in selenite models. Experimental Eye Research 88, 575-582. Rooban, B.N., Sasikala, V., Sahasranamam, V., Abraham, A., 2011a. Amelioration of selenite toxicity and cataractogenesis in cultured rat lenses by Vitex negundo. Graefe's archive for clinical and experimental ophthalmology = Albrecht von Graefes Archiv fur klinische und experimentelle Ophthalmologie 249, 685-692. Rooban, B.N., Sasikala, V., Sahasranamam, V., Abraham, A., 2011b. Analysis on the alterations of lens proteins by Vitex negundo in selenite cataract models. Mol Vis 17, 1239-1248. Rotruck, J.T., Pope, A.L., Ganther, H.E., Swanson, A.B., Hafeman, D.G., Hoekstra, W.G., 1973. Selenium: biochemical role as a component of glutathione peroxidase. Science 179, 588-590. Sanderson, J., McLauchlan, W.R., Williamson, G., 1999. Quercetin inhibits hydrogen peroxide-induced oxidation of the rat lens. Free Radic Biol Med 26, 639-645. Sasaki, T., Kikuchi, T., Yumoto, N., Yoshimura, N., Murachi, T., 1984. Comparative specificity and kinetic studies on porcine calpain I and calpain II with naturally occurring peptides and synthetic fluorogenic substrates. The Journal of biological chemistry 259, 12489-12494. Sasikala, V., Rooban, B.N., Sahasranamam, V., Abraham, A., 2013. Rutin ameliorates free radical mediated cataract by enhancing the chaperone activity of alpha-crystallin. Graefe's archive for clinical and experimental ophthalmology = Albrecht von Graefes 21

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Archiv fur klinische und experimentelle Ophthalmologie 251, 1747-1755. Shearer, T.R., Anderson, R.S., Britton, J.L., Palmer, E.A., 1983. Early development of selenium-induced cataract: slit lamp evaluation. Experimental eye research 36, 781-788. Shearer, T.R., Ma, H., Fukiage, C., Azuma, M., 1997. Selenite nuclear cataract: review of the model. Mol Vis 3, 8. Shim, S.H., Kim, J.M., Choi, C.Y., Kim, C.Y., Park, K.H., 2012. Ginkgo biloba extract and bilberry anthocyanins improve visual function in patients with normal tension glaucoma. J Med Food 15, 818-823. Singleton, V.L., Esau, P., 1969. Phenolic substances in grapes and wine, and their significance. Adv Food Res Suppl 1, 1-261. Sinha, A.K., 1972. Colorimetric assay of catalase. Anal Biochem 47, 389-394. Spector, A., 1995. Oxidative stress-induced cataract: mechanism of action. FASEB journal : official publication of the Federation of American Societies for Experimental Biology 9, 1173-1182. Stefek, M., Karasu, C., 2011. Eye lens in aging and diabetes: effect of quercetin. Rejuvenation Res 14, 525-534. Tsao, R., Yang, R., 2003. Optimization of a new mobile phase to know the complex and real polyphenolic composition: towards a total phenolic index using high-performance liquid chromatography. J Chromatogr A 1018, 29-40. Vibin, M., Siva Priya, S.G., B, N.R., Sasikala, V., Sahasranamam, V., Abraham, A., 2010. Broccoli regulates protein alterations and cataractogenesis in selenite models. Curr Eye Res 35, 99-107. Wang, T., Zhang, P., Zhao, C., Zhang, Y., Liu, H., Hu, L., Gao, X., Zhang, D., 2011. Prevention effect in selenite-induced cataract in vivo and antioxidative effects in vitro of Crataegus pinnatifida leaves. Biol Trace Elem Res 142, 106-116. Zafra-Stone, S., Yasmin, T., Bagchi, M., Chatterjee, A., Vinson, J.A., Bagchi, D., 2007. Berry anthocyanins as novel antioxidants in human health and disease prevention. Mol Nutr Food Res 51, 675-683.

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ACCEPTED MANUSCRIPT Figure captions

Figure 1: Anterior view photos of 21-days old rat lenses representative of the stage of each group (median score). The lenses of group C were totally clear (stage 0), those of

median score of SeBBL group in stage 1.

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group Se developed stage 4 cataract, while BBL prevented lens opacification limiting the

Figure 2: Sodium dodecyl sulphate polyacrylamide gel (SDS-PAGE) electrophoresis of

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rat lens water soluble (A) and water insoluble (B) proteins. The first left lane of each gel corresponds to the molecular weight marker (M, 17-175 kD). The three lanes in the

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right represent lens proteins of SeBBL, Se or C. the same amount of homogenate (30 μg tissue (WSF) and 250 μg tissue (WIF)) was loaded into each lane. In the WSF, BBL prevented the loss of crystallin polypeptides of 20 - 30 kD, which was observed in the Setreated group. In the WIF, BBL prevented the aggregation (>80 kD) and the

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insolubilization (<30 kD) of the proteins.

Figure 3: Inhibition of lens protein turbidity in vitro by main polyphenolic compounds of BBL. Cloudiness was induced by incubation of lens homogenate with excess concentration of Na2SeO3. The diagrams show the progress of lens turbidity during the

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incubation period. Data are the result of four measurements performed in duplicate (n=8). The values represent mean ± S.E.. A: quercetin, B: rutin, C: hyperoside, D: isoquercetin and E: Chlorogenic acid. *p<0.05 significant difference from Se. Statistical analysis was

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performed using Student’s t-test.

Figure 4: Inhibition of lens protein turbidity in vitro by main polyphenolic compounds of BBL. Cloudiness was induced by incubation of lens homogenate with excess concentration of CaCl2. The diagrams show the progress of lens turbidity during the incubation period. Data are the result of four measurements performed in duplicate (n=8). The values represent mean ± S.E. A: quercetin, B: rutin, C: hyperoside, D: isoquercetin and E: Chlorogenic acid. *p<0.05 significant difference from Se. Statistical analysis was performed using Student’s t-test. 23

ACCEPTED MANUSCRIPT Figure 5: μ-Calpain inhibitory activity of quercetin glycosides. Data are the result of two measurements performed in duplicate (n=4). Data are expressed as mean ± S.E. IC50 values of rutin (A), isoquercetin (B) and hyperoside (C).

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Figure 6: UV-Vis titration spectra of quercetin/ SeO32- (top) and quercetin/Ca2+ (bottom) mixtures. Four different ratios were measured (1:2, 1:10, 1:20 and 1:100). The maximum absorbance wavelengths (λmax) were 254 nm (band I), corresponding to the ring A of the flavonoid, and 370 nm (band II), corresponding to the ring B. The lowest ratio Q:Se (1:2)

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showed decrease and bathochromic shift of absorbance max of both bands (to 272 nm and 410 nm, respectively), while one new peak was observed at 320 nm. As the concentration of selenite ions increased, the peak at 272 nm was lost, band II returned to the initial

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wavelength (370 nm) and the intensity of the new peak was getting slightly higher. These

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results indicate complete oxidation of the flavonol by selenite ions.

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6.131±0.129

Rutin

0.316±0.007

Hyperoside

1.209±0.028

Isoquercetin

0.460±0.009

Quercetin Total polyphenols (mg GAE/g dry extract)

0.146±0.003 355±42

Total flavonoids (mg quercetin/g dry extract)

69.4±1.7

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Chlorogenic acid

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%w/w dry BBL extract

Compound

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The quantification of the phenolic compounds was based on the HPLC chromatograms of the three blueberry leaf decoctions (n=3). Total polyphenols and total flavonoids are the result of three experiments performed in triplicate (n=9).

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Values represent mean ± S.E.

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Median Number of lenses with different degree of opacification (range)

Group Stage 1

Stage 2

Stage 3

Stage 4

C (n=16)

16

0

0

0

0

Se (n=16)

0

0

0

4

10

SeBBL (n=20)

0

11

8

1

0

Stage 5

Stage 6

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Stage 0

0

0

0 (0)

2

0

4 (2) *

0

0

1 (2) ¥

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Staging of cataract formation of the isolated lenses from each group. Cataract classification was performed on PN day 21. Groups, C: Control, Se: Se-treated, ¥

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SeBBL: Se/BBL-treated animals. *p<0.05 significant difference from group C, p<0.05 significant difference from group Se. The Mann-Whitney U-test was used

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(nmol/g tissue)

(nmol/mg tissue)

C

98±19

0.36±0.08

Se

166±20*

0.19±0.05*

SeBBL

82±17¥

0.49±0.10¥* Enzymes

GSH/(GSH+GSSG) 0.24±0.06

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GSH

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Lipid peroxidation Groups

0.14±0.03*

0.32±0.09¥*

Catalase Glutathione Peroxidase

Superoxide dismutase

(U/ min/g tissue)

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(μmol formaldehyde/min/

(U/mg tissue)

g tissue)

33.04± 4.09

Se

24.11±2.96*

SeBBL

31.29±3.21¥

64.41±12.02

0.32±0.04

40.26±5.14*

0.18±0.02*

54.03±1.06¥

0.26±0.02¥

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C

Data express the results of three experiments performed in triplicate (n=9). The values represent mean±S.D. of six animals. *p<0.05 significant difference from

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group C, ¥p<0.05 significant difference from group Se. Statistical analysis was

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performed using Kruskal-Wallis non parametric analysis of Variance (ANOVA).

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ACCEPTED MANUSCRIPT Highlights

Blueberry leaf polyphenols inhibit selenite-induced cataract in rats.

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Polyphenols protect lenses from selenite oxidative stress & protein lysis.

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Quercetin inhibits SeO32- and Ca2+ -induced porcine lens turbidity in vitro.

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Quercetin is oxidized by selenite anions and interacts with calcium.

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Quercetin glycosides inhibit μ-calpain with μΜ IC50 values.

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