Journal of Pharmaceutical and Biomedical Analysis 55 (2011) 563–568

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Journal of Pharmaceutical and Biomedical Analysis

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A new validated SPE-HPLC method for monitoring in human plasma—Application after tea consumption

Dimitra G. Chryssanthi a, Fotini N. Lamari a,∗, Constantine D. Georgakopoulos b, Paul Cordopatis a a Laboratory of Pharmacognosy & Chemistry of Natural Products, Department of Pharmacy, Greece b Department of Ophthalmology, School of Medicine, University of Patras, 26504 Rion, Greece article info abstract

Article history: Saffron (stigmas of Crocus sativus L.) is a well-known spice with many attributed therapeutic uses through- Received 20 September 2010 out centuries. Although studies have demonstrated that crocetin and crocins from saffron have various Received in revised form 11 February 2011 biological functions, issues concerning the route and way of saffron administration, the absorption and Accepted 14 February 2011 metabolism of saffron in humans have not been answered yet. In the present study, an Available online 21 February 2011 isocratic reversed-phase liquid chromatographic method was developed and validated for the deter- mination of crocetin in plasma. Samples were pre-treated by solid phase extraction (recoveries >72%) Keywords: and were chromatographed on a Luna C-18 column (4.6 mm × 250 mm, 5 ␮m) with a mobile phase con- Crocus sativus −1 Saffron sisting of methanol–water–trifluoroacetic acid (75.0:24.5:0.5, v/v/v) at a flow rate of 1.0 mL min . The Crocetin HPLC method developed resulted in sharp peaks at 10.7 (trans-crocetin) and 18.6 min (cis-crocetin), HPLC whereas the calibration curve of total crocetin in plasma displayed a good linearity for concentrations of Solid-phase extraction 0.020–20 ␮M(R2 = 0.999). Specificity, precision, accuracy and stability were studied with spiked plasma Human plasma samples and were acceptable. The developed method was applied to the determination of crocetin lev- els in plasma of four healthy human volunteers before and after consumption of one cup of saffron tea (200 mg of saffron in 80 ◦C water for 5 min). Results showed that the concentration of crocetin was high after 2 h (1.24–3.67 ␮M) and still determined after 24 h (0.10–0.24). Interestingly, the percentage of the cis- ranges from 25 to 50%, suggesting in vivo isomerization. © 2011 Elsevier B.V. All rights reserved.

1. Introduction thought to be safe; at doses of 5.0 g or more, Stigma Croci may cause serious adverse reactions and overdose (12.0–20.0 g/day) may be Saffron, the dried stigmas (the term styles is also used by certain fatal [3]. Intraperitoneal administration of saffron ethanolic extract botanologists [1])ofCrocus sativus flowers, is a spice important in to Wistar rats at the high doses of 0.35, 0.70 and 1.05 g kg−1 body the Mediterranean, Indian and Chinese diet and has been contin- weight for two weeks caused mild to severe hepatic and renal tissue uously used in folk remedies for more than 90 diseases for over injuries, weight loss, significant reductions in the haemoglobin and 3000 years [2]. Recent research on its biomedical properties sup- haematocrite levels and total counts; intraperitoneal ports its use for many of these indications, although the great median lethal dose (LD50) value of ethanolic extracts was found majority of the studies are conducted in vitro and in experimental to be 3.5 g kg−1 body weight [4]. Modaghegh et al. showed that animals [3]. Its composition is also quite unique: the main con- consumption of 200 and 400 mg saffron tablets by healthy humans stituents are hydrophilic carotenoids, the crocins, which are mono- for 7 days is safe [5]. Administration of 200 mg saffron capsules and diglycosyl-esters of crocetin and are responsible for the char- to men with erectile dysfunction for 10 days was safe and had a acteristic golden yellow color they confer. positive effect on sexual function [6], whereas administration of Besides culinary uses, saffron is consumed as a herbal tea per se 30 mg capsules to patients with mild-to-moderate Alzheimer’s dis- or is added in other herbal infusions for its unique flavor charac- ease [7] and to women with symptoms of premenstrual syndrome teristics and its attributed medicinal uses. According to the WHO [8] had a favorable outcome. A reduction in lipoprotein oxidation in monograph issued in 2007, daily doses of up to 1.5 g of saffron are 20 human subjects was also reported after administration of 50 mg saffron in milk twice a day for 3–6 weeks [9]. However, issues concerning the route and way of saffron admin- istration, the absorption and metabolism of saffron carotenoids ∗ Corresponding author at: Department of Pharmacy, Laboratory of Pharmacog- in humans have not been answered. Experiments conducted in nosy & Chemistry of Natural Products, University of Patras, 26504 Rion, Greece. rodents, showed that orally administered crocins are hydrolysed Tel.: +30 2610 969335; fax: +30 2610 993278. E-mail address: fl[email protected] (F.N. Lamari). to crocetin before being incorporated into blood circulation and

0731-7085/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.jpba.2011.02.018 564 D.G. Chryssanthi et al. / Journal of Pharmaceutical and Biomedical Analysis 55 (2011) 563–568 that crocetin is likely to be metabolized to glucuronide conjugates CA, USA). The Column Compartment (TCC-3100) was stabilized at both in the intestinal mucosa and in the liver of mice [10,11]. The 40 ◦C and UV-detection was performed with a diode array detector aim of this study was the development and validation of a chro- (DAD), Ultimate DAD-3000. Data were collected, stored and inte- matographic analytical method for the determination of crocetin grated on a Chromeleon v 6.80 Systems software. Separation of in human plasma and application of that method to the determi- analytes was performed on a Luna C-18 reversed-phase column nation of plasma crocetin levels after saffron tea consumption by (250 mm × 4.6 mm I.D., 5 ␮m particle size) from Phenomenex (Tor- healthy volunteers. In this context, we screened saffron tea com- rance, CA, USA). Elution was performed with methanol–water–TFA position to investigate if tea preparation induces changes in saffron (75.0:24.5:0.5, v/v/v) for 30 min. The flow rate of the mobile phase components, i.e. isomerization. was 1 mL min−1. Spectra were monitored by the diode array detec- tor. 2. Materials and methods 2.5. Method validation 2.1. Reagents and chemicals The proposed method was validated according to the official Methanol (MeOH) and acetonitrile (AcCN) of HPLC grade were guidelines of FDA for bioanalytical methods [12]. Selectivity was supplied from Merck KGaA (Darmstadt, Germany). Dimethyl- investigated by analysis of blank samples (plasma) from at least sulphoxide (DMSO), ammonia solution, hydrochloric acid and 6 healthy individuals. The calibration curve was obtained by plot- solid reagents (ammonium acetate, chloride and sodium ting the sum of peak areas (y)oftrans- and cis-crocetin versus their phosphate), of analytical grade were also purchased from concentration (x). Nine different concentrations (0.002, 0.02, 0.2, Merck. Trifluoroacetic acid (TFA) and butylated hydroxytoluene 0.5, 1, 2, 5, 10, 20 ␮M) were used for total crocetin. Good linear- were obtained from Sigma–Aldrich (St. Louis, USA) whereas ity was determined through the correlation coefficient (R2), which trans-crocetin [(2E,4E,6E,8E,10E,12E,14E)-2,6,11,15-tetramethyl- should be better than 0.99. The LLOD was determined as the ana- 2,4,6,8,10,12,14-hexadecaheptaenedioic acid] (purity >98% for the lyte concentration yielding signal with a signal-to-noise (S/N) ratio sum of both ) was purchased from Extrasynthese (Genay of 3:1, whereas the LLOQ was defined as the analyte concentra- Cedex, France). Saffron in the form of dried styles was supplied from tion yielding signal with S/N ratio 10:1. The noise was evaluated as Cooperative de Saffron (Krokos Kozanis, Greece). The dried plant the largest deviation of detector signal of baseline. Intra-day accu- material was identified by Professor Gregoris Iatrou, Department racy and precision were estimated with five analyses on the same of Biology, University of Patras, Greece. day of plasma samples spiked with crocetin at three different con- centration levels: 0.15, 3, 15 ␮M. The accuracy was calculated by 2.2. Preparation of crocetin standards comparison of each concentration measured to the nominal con- centration of the standard solution and was expressed as % of the Stock solutions of crocetin were prepared in DMSO at an ini- relative error values whereas precision was numerically expressed tial concentration of 10 mM and diluted to working concentrations by relative standard deviation (RSD) of values. Inter-day accuracy (0.005–100 ␮M) in methanol. Methanolic solutions of the analyte and precision of the method were estimated with three analyses of were kept at −20 ◦C in a dark place, until further use. The solutions the plasma samples spiked with crocetin (0.15, 3, and 15 ␮M) ten were stable for at least one month. Pooled plasma, obtained from days later. Precision and accuracy should be less than 15% except for young healthy volunteers (25–35 years old), was used for the prepa- the LLOQ where the values should not exceed 20%. The stability of ration of spiked plasma standards. For spiked solutions of crocetin crocetin is determined by analysis of the low (0.15 ␮M) and the high in plasma, 25 ␮L of crocetin was diluted in plasma to a final volume (15 ␮M) spiked plasma standards after three freeze (at −20 ◦C and of 500 ␮L. −70 ◦C) and thaw cycles, and after 24-h storage at room tempera- ture. SPE recovery of total crocetin was calculated by comparison 2.3. Plasma sample pretreatment of the determined concentrations obtained from analysis of spiked plasma samples after SPE to those calculated from the analysis of During the optimization of plasma sample pretreatment, sam- the respective concentrations of crocetin standards in methanol (5 ples were submitted to solid phase extraction (SPE) procedure for times the concentration of spiked plasma samples). the removal of matrix interferences during the analysis. Reversed phase Strata-X cartridges (200 mg/3 mL) consisting of a surface 2.6. HPLC analysis of various saffron infusions modified with styrene–divinylbenzene polymer were obtained from Phenomenex (Torrance, CA, USA). The cartridges were con- Saffron infusion was prepared by adding 150 mL of hot water ditioned with methanol and equilibrated in loading buffer. In the (80 ◦C) to 200 mg of saffron and leaving the stigmas steep for 5 min. proposed method, 500 ␮L of plasma sample was diluted in 500 ␮L The infusion was centrifuged and the supernatant was used directly of MeOH:CH3COOH (25 mM) containing 10% NaCl (w/v) (10:90, for HPLC analysis. In order to compare the HPLC profile to that v/v), centrifuged and the supernatant was loaded onto the car- of the standard aqueous methanol extract, saffron was extracted ◦ tridges. Washing was performed with 1 mL of MeOH–CH3COOH with methanol:water (1:1, v/v) (3 mL/50 mg) for 4 h at 25 C with (25 mM) (5:95, v/v). The retained crocetin was eluted with 6 mL continuous stirring. The extract was centrifuged, filtered through NH3–MeOH–water (5:90:5, v/v/v). Finally, the eluate was evapo- a 0.2-␮m filter and evaporated to dryness. The residue was stored rated to dryness in a speed Vac system (Labconco Corp., Kansas at −20 ◦C until further use and redissolved in methanol:water (1:1, city, MO, USA). The residues were stored at −20 ◦C until the next v/v). All procedures were performed in the absence of light. day. Dry residues were redissolved in 100 ␮L of the HPLC mobile Chromatographic analysis was performed on a Supelcosil C18 phase. (5 ␮m, 25 cm × 4.6 mm, Sigma–Aldrich) column on a Mod.10 AKTA instrument (Amersham Biosciences, Piscataway USA) as described 2.4. HPLC determination of crocetin in plasma previously by minor modifications [13,14]. Elution was performed with methanol:water (20:80, v/v) for 2 min, a gradient of methanol The chromatographic system consisted of an Ultimate 3000 (20–70%) for 50 min, with a gradient of methanol (70–100%) for Pump (Pump LPG-3400 A, Dionex Corporation, Sunnyvale, CA, USA) 5 min and 100% methanol for 1 min with a flow rate 0.7 mL/min. with a 20 ␮L Rheodyne 8125 injector (Rheodyne, Ronhert Park, Both solvents (water and methanol) contained 1% (v/v) acetic D.G. Chryssanthi et al. / Journal of Pharmaceutical and Biomedical Analysis 55 (2011) 563–568 565

acid. Detection wavelength was 440 nm. Semi-quantification was carried out by multiplying the peak area of each with the respective molecular coefficient absorbance value (89,000 for trans- and 63,350 for cis-crocins) and expressed as the percentage of each crocin in relation to the total crocin content [15].

2.7. Method application: study design and biologic material

For the preparation of saffron infusions, hot water (80 ◦C, about 150 mL) was added to one cup containing 200 mg saffron stig- mas which were allowed to steep for 5 min. Four young (25–35 years old, 3 female and 1 male, normal BMI) healthy volunteers participated in the study and their written informed consent was obtained. The collection of the biologic material and the procedure followed conformed to the ethical standards of the Helsinki Dec- laration of 1975 and its latest revisions. The study was approved by the local Bioethics Committee of the University of Patras and all procedures took place in the University Hospital of Patras under medical supervision. All volunteers drank the saffron tea at about 09:00–10:00 a.m. after overnight fasting. Blood was collected in EDTA coated tubes immediately before (0 h), 2 and 24 h after tea consumption. Plasma was prepared by centrifugation of blood at 1000 × g for 15 min at 4 ◦C and then kept at −20 ◦C till analysis (next day).

3. Results and discussion Fig. 1. Crocetin determination. HPLC chromatogram of plasma spiked with crocetin (upper panel, I: trans-crocetin, II: cis-crocetin) and UV–visible absorption spectra (lower panel). Absorbance maximum of peak I appear at approximately 423 nm 3.1. Method development whereas peak II displays additional maxima at 320 nm.

Initial experiments were performed with pure crocetin stan- dards in methanol. For the determination of the mobile phase, a Purification and condensation of plasma samples were accom- number of elution systems were examined. The use of a gradient plished via SPE. All experiments were conducted with plasma of AcCN:water (15–75% of the organic phase in 25 min) showed samples spiked with crocetin. Having in mind the acid–base a very broad peak at 5 min. Addition of ammonium acetate at the balance of the carboxylic groups of crocetin at different pH ␮ final percentage of 0.1% (w/v) in both solvents resulted in two broad values, reversed phase SPE columns were loaded with 500 L and tailing peaks (a main one at 13 and a minor at 20 min). Bet- plasma diluted in water (1:1, v/v), washed with MeOH–phosphate ter HPLC profiles with good resolutions but still broad and tailing buffer (50 mM, pH 3.0) (20:80, v/v) (2 mL), and eluted with 6 mL peaks (main peak at 9 and minor at 20 min) were observed with NH3–MeOH–water (5:90:5, v/v/v). About 50% of crocetin was isocratic elution with MeOH–water–acetic acid (75:23:2, v/v/v), recovered but nearly 30% of crocetin was lost in the washing proce- as earlier suggested [11]. Decrease in the percentage of acetic dure, probably because of strong interactions with plasma proteins acid led to longer elution times. Replacing acetic acid with TFA eluted at that step. Indeed, repetition of the procedure without to the optimum mobile phase composition of MeOH–water–TFA MeOH in the washing step had no difference on recovery val- (75.0:24.5:0.5, v/v/v) resulted in sharp peaks at 10.7 and 18.6 min ues. Non-specific binding of crocetin to human serum albumin via (Fig. 1). A higher amount (>0.5%) of TFA causes broadening of the H-bonding, the stability of those complexes and the changes on peaks and increase in the peak area of the late-eluting substance. protein secondary structure have been earlier shown by Kanakis Monitoring of spectra of the two peaks by the diode array detec- et al. [18]. In order to disrupt those complexes, a high concentra- tor showed that both of them share maximum double peaks at tion of salt, a small percentage of methanol and low pH values ␮ 420–460 nm, characteristic of the carotenoids; the earlier eluting were used. In the proposed method, 500 L of plasma sample peak has another maximum at 250 nm whereas the late-eluting was diluted in 500 mL of MeOH–CH3COOH (25 mM) containing peak has two maxima at 250 nm and 310–330 nm (Fig. 1). Thus, the 10% NaCl (w/v) (10:90, v/v). Samples were loaded on cartridges peak at 10.7 min corresponds to trans-crocetin and the second one after centrifugation, and washing was performed with 1 mL of at 18.6 min to cis-crocetin. MeOH–CH3COOH (25 mM) (5:95, v/v). The retained crocetin was This is the first report stressing that during HPLC analysis of eluted with 6 mL NH3–MeOH–water (5:90:5, v/v/v), as in previ- trans-crocetin (high purity standard both commercially available ous tests. Those changes led to high recovery values; 94.8% at the ␮ ␮ ␮ and that prepared by alkaline hydrolysis of saffron extract as earlier concentration of 0.2 M, 83.3% at 0.5 M and 72.3% at 20 M. described [10,16,17]), a small amount (about 10%) of the cis-isomer always appears irrespective of the matrix (i.e. in pure crocetin stan- 3.2. Method validation dards or in plasma spiked with crocetin), and is well resolved from the trans-isomer, i.e. it elutes much later; in our optimum condi- Method’s quality parameters were studied under optimum sep- tions it elutes 8 min later. The existence of the cis-isomer is probably aration parameter conditions using plasma spiked with crocetin a result of the susceptibility of carotenoids to various factors, e.g. at final concentrations ranging from 0.002 to 20 ␮M. The calibra- light and acids. Earlier HPLC studies on determination of plasma tion curve of total crocetin (sum of areas of two peaks) displayed crocetin by Xi et al. and Asai et al. do not report the existence of the a good linearity for concentrations of 0.020–20 ␮M(R2 = 0.999, cis-isomer [10,11]. y = 1.131x − 0.054). In detail, calibration curves were linear from 566 D.G. Chryssanthi et al. / Journal of Pharmaceutical and Biomedical Analysis 55 (2011) 563–568

Table 1 Accuracy and precision data (intra- and inter-day) for analysis of plasma samples spiked with crocetin expressed as relative error (% RE) and relative standard devia- tion (% RSD), respectively.

Concentration (␮M) Accuracy (% RE) Precision (% RSD)

Intra-day 0.15 5.55 6.34 3 −1.25 2.11 15 5.72 1.05 Inter-day 0.15 12.73 16.64 3 −0.07 3.56 15 6.52 1.32

Table 2 Percentages (%) of crocins extracted by methanol–water (1:1, v/v) and by hot water (infusion).

Crocins 50% aqueous Infusion in 80 ◦C MeOH for 4 h water for 5 min

Trans-crocin-4 50.1 ± 1.4 59.5 ± 0.2 Trans-crocin-3 21.1 ± 0.5 21.0 ± 0.4 Cis-crocin-4 9.1 ± 2.9 5.0 ± 0.1 Trans-crocin-2 3.8 ± 1.3 1.3 ± 0.2 Cis-crocin-3 7.8 ± 2.2 8.2 ± 0.2 Cis-crocin-1 1.8 ± 0.4 1.1 ± 0.3

Crocin-4, crocetin-di-(␤-d-gentiobiosyl) ester; crocin-3, crocetin–(␤-d-glycosyl)- (␤-d-gentiobiosyl) ester; crocin-2, crocetin (␤-d-gentiobiosyl) ester; crocin-1, crocetin (␤-d-glycosyl) ester.

0.020 to 20 ␮M for trans-crocetin (R2 = 0.999) and from 0.569 to 10 ␮M for cis-crocetin (R2 = 0.978). LLOD values are 0.002 and 0.170 ␮M for trans- and cis-crocetin, and the respective LLOQ values are 0.020 and 0.569 ␮M. The selectivity of the method was determined by analysis of blank plasma samples of six different healthy individuals. HPLC analysis showed no endogenous peaks at the retention times of the crocetin isomers (Fig. 2A). The intra and inter-day accuracy and precision for the total amount of crocetin were less than 15% and at the LLOQ less than 20% (Table 1). Results are presented as sum of all isomers. These values show that the proposed methodology is reproducible and suitable for the quantitative determination of crocetin in human plasma samples. Peak asymmetry and theoretical plates for trans-crocetin are 1.249 ± 0.134 and 3966 ± 408, respectively, whereas for cis- crocetin the respective values are 1.126 ± 0.093 and 4327 ± 437. The short-term (up to 24 h in room temperature) and freeze–thaw (after three cycles) stability was also studied by com- paring the mean back-calculated concentrations of treated samples to those of freshly prepared ones. Low-concentration samples (0.15 ␮M) were not stable at room temperature for 24 h and after three freeze (at −20 ◦C)–thaw cycles since differences were around 30 and 48%, respectively. High concentration samples (15 ␮M) were stable in all conditions (differences < ± 15%).

3.3. Analysis of saffron infusions

Saffron infusions were analyzed by HPLC and the finger- print obtained is in complete agreement with previous reports [14,15,19]. Ten peaks could be identified: two absorbing at 250 nm (picrocrocin and HTCC) and eight absorbing at 440 nm (trans-  Fig. 2. Plasma analysis before and after saffron tea consumption. HPLC chro- crocin-4, trans-crocin-3, trans-crocin-2 , cis-crocin-5, cis-crocin- matograms of human plasma before saffron administration (A) after 2 h (B) and 4, trans-crocin-2, cis-crocin-3, cis-crocin-2). Semi-quantification after 24 h (C). I: trans-crocetin, II: cis-crocetin. showed that saffron tea preparation does not induce crocin iso- merization and hydrolysis (Table 2). Indeed, in saffron infusions, the only noteworthy changes concern trans- and cis-crocin-4: content of trans-crocin-4 is higher and of cis-crocin-4 is lower. tion of methyl esters in the presence of methanol has been put The lower percentage of trans-crocin-4 in aqueous methanolic forward [20] although Kanakis et al. showed the requirement of extracts might be explained by the higher extractability of less non-aqueous conditions for the preparation of dimethylcrocetin polar crocins in the presence of methanol; the low-degree forma- [18]. D.G. Chryssanthi et al. / Journal of Pharmaceutical and Biomedical Analysis 55 (2011) 563–568 567

Table 3 (0.10–0.24). This first study in humans confirms findings of pre- ␮ Concentration ( M) of total crocetin determined by SPE-HPLC-DAD analysis in vious pharmacokinetic studies in rodents showing that orally human plasma samples and % percentage of cis- isomers. ingested crocins are hydrolysed to crocetin before entering blood Sample Total crocetin (␮M) Cis-crocetin/total circulation. This is the first report showing the determination of the crocetin (%) cis-isomer of crocetin in small percentages (10%) in pure crocetin or 2h 24h 2h 24h spiked plasma samples due to interconversion of the trans-isomer

1 3.67 0.12 49.4 NDa during analysis to a small degree. The significance of this obser- 2 2.00 0.10 24.1 NDa vation is manifested in the determination of crocetin in healthy 3 1.24 0.24 27.5 NDa human individuals after saffron tea consumption; percentages of a 4 1.27 0.13 24.5 ND the cis-isomer range from 25 to 50% suggesting in vivo isomer- a ND, cis-crocetin was not detected. ization. At last, we showed that saffron tea preparation does not induce changes in saffron composition concerning the presence of individual crocins. This HPLC method enables the determination 3.4. Application to determination of plasma crocetin after saffron of crocetin concentrations in humans and the knowledge of actual tea consumption concentrations and the percentages of the trans- and cis-isomers after saffron consumption will aid future nutritional or pharma- The developed SPE-HPLC analytical methodology was applied cokinetic studies. for the quantification of crocetin in plasma of healthy humans. Plasma was taken before (0 h) and after (2 and 24 h) saffron tea Acknowledgements consumption. The results show that, after consumption of a cup of tea from 200 mg saffron, crocetin is detected in blood stream and The authors acknowledge with thanks the Research Commit- trace amounts are found even after 24 h (Fig. 2, Table 3). tee of the University of Patras for financial support under the “K. This is the first report showing crocetin concentrations in human Karatheodoris” Grant No. C178 and Professor Gregoris Iatrou for plasma after saffron consumption. These results confirm previous his kind assistance. studies performed in rodents, which showed that orally admin- istered crocetin is quickly absorbed into whereas References crocins are hydrolysed to crocetin in the gastrointestinal tract

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Various isomers of (mainly cis- and trans-) tiobiose ester) following oral administration in rats, Phytomedicine 14 (2007) are also found in blood samples, with the ratio of the cis-isomer 633–636. reaching the 60–80% of total lycopene [21] even though lycopene [12] Guidance for Industry – Bioanalytical Method Validation, U.S. Department of Health and Human Services – Food and Drug Administration (CDER), in foods occurs mainly in the all-trans form. The mechanisms under- 2001. lying the formation, interconversion or biological roles of various [13] P. Lozano, M.R. Castellar, M.J. Simancas, J.L. Iborra, Quantitative high- crocetin isomers remain enigmatic. performance liquid chromatographic method to analyse commercial saffron (Crocus sativus L.) products, J. Chromatogr. A 830 (1999) 477–483. [14] P.A. Tarantilis, G. Tsoupras, M. Polissiou, Determination of saffron (Crocus 4. Concluding remarks sativus L.) components in crude plant extract using high-performance liquid chromatography-UV–visible photodiode-array detection–mass spectrometry, J. Chromatogr. A 699 (1995) 107–118. An analytical SPE-HPLC method was developed for total crocetin [15] M. Carmona, A. Zalacain, J.E. Pardo, E. Lopez, A. Alvarruiz, G.L. Alonso, Influence determination in human plasma, which displays good selectiv- of different drying and aging conditions on saffron constituents, J. Agric. Food Chem. 53 (2005) 3974–3979. ity, linearity, sensitivity, accuracy and precision. The method was [16] P.A. Tarantilis, M. Polissiou, M. Manfait, Separation of picrocrocin, cis–trans- applied to the determination of crocetin in blood plasma of healthy crocins and safranal of saffron using high-performance liquid chromatography human individuals before and after saffron tea consumption. Cro- with photodiode-array detection, J. Chromatogr. A 664 (1994) 55–61. [17] C.D. Kanakis, P.A. Tarantilis, H.A. Tajmir-Riahi, M.G. Polissiou, Interaction of cetin was not detectable before tea drinking, but its concentration tRNA with safranal, crocetin, and dimethylcrocetin, J. Biomol. Struct. Dyn. 24 was high after 2 h (1.24–3.67 ␮M) and still determined after 24 h (2007) 537–546. 568 D.G. Chryssanthi et al. / Journal of Pharmaceutical and Biomedical Analysis 55 (2011) 563–568

[18] C.D. Kanakis, P.A. Tarantilis, H.A. Tajmir-Riahi, M.G. Polissiou, Crocetin [20] H. Pfander, Carotenoid glycosides, Pure Appl. Chem. 47 (1976) 121–128. dimethylcrocetin, and safranal bind human serum albumin: stability and [21] K. Wu, S.J. Schwartz, E.A. Platz, S.K. Clinton, J.W. Erdman Jr., M.G. Fer- antioxidative properties, J. Agric. Food Chem. 55 (2007) 970–977. ruzzi, W.C. Willett, E.L. Giovannucci, Variations in plasma lycopene and [19] M.A. Papandreou, C.D. Kanakis, M.G. Polissiou, S. Efthimiopoulos, P. Cordopatis, specific isomers over time in a cohort of U.S. men, J. Nutr. 133 (2003) M. Margarity, F.N. Lamari, Inhibitory activity on amyloid-beta aggregation and 1930–1936. antioxidant properties of Crocus sativus stigmas extract and its crocin con- stituents, J. Agric. Food Chem. 54 (2006) 8762–8768. JOURNAL OF MEDICINAL FOOD J Med Food 14 (9) 2011, 1060–1064 # Mary Ann Liebert, Inc. and Korean Society of Food Science and Nutrition DOI: 10.1089/jmf.2010.0187

Differential Antioxidant Effects of Consuming Tea from Sideritis clandestina subsp. peloponnesiaca on Cerebral Regions of Adult Mice

Zacharoula I. Linardaki,1 Catherine G. Vasilopoulou,1 Caterina Constantinou,1,* Gregoris Iatrou,2 Fotini N. Lamari,3 and Marigoula Margarity1

1Laboratory of Human and Animal Physiology, Department of Biology; 2Division of Plant Biology, Department of Biology; 3Laboratory of Pharmacognosy & Chemistry of Natural Products, Department of Pharmacy; University of Patras, Patras, Greece.

ABSTRACT Oxidative stress is involved in the pathophysiology of neurodegenerative diseases and aging. Many species of the genus Sideritis (mountain tea) are widely consumed in the Mediterranean region as herbal tea. This study evaluated the effect of supplementation of mice with herbal tea from Sideritis clandestina subsp. peloponnesiaca on the antioxidant status of different brain regions. To select the most bioactive herbal tea, the polyphenolic content (Folin–Ciocalteu method) and the antioxidant properties (ferric reducing antioxidant power [FRAP] and 2,2-diphenyl-1-picrylhydrazyl assays) of several taxa and different populations of the S. clandestina infusions were measured in vitro. Male adult mice had ad libitum access to water (control) or the herbal tea (4% w/v) for 6 weeks. At the end of the treatment period we assessed the total antioxidant power (FRAP assay) and the levels of malondialdehyde (indicator of lipid peroxidation) and reduced in the cerebral cortex, cerebellum, and midbrain. These biochemical measures have also been determined in liver samples used as a comparative reference peripheral tissue. Consumption of 4% herbal tea increased the total antioxidant power of the midbrain by 72% (P < .05); a significant (P < .05) decrease in malondialdehyde levels and increase in reduced glutathione content of the cerebellum (78% and 27%, respectively) and midbrain (59% and 32%, respectively) were also observed. These findings indicate that mountain tea consumption enhances the antioxidant defense of the adult rodent brain in a region-specific manner.

KEY WORDS: antioxidant capacity brain reduced glutathione lipid peroxidation mountain tea Sideritis

INTRODUCTION A moderate in vitro antioxidant action of extracts of Sideritis species has been described elsewhere,6 but to our ideritis L. (Lamiaceae), growing mainly in the knowledge no in vivo studies have examined their effect. Mediterranean region, comprises approximately 150 S Mammalian brain is particularly vulnerable to oxidative species worldwide. The dried aerial parts of Sideritis species stress,8 a factor that is considered to play a pivotal role in the are widely consumed in Greece as an herbal tea, popularly pathogenesis of several age-related neurodegenerative dis- termed mountain tea.1 It has been used extensively in folk orders.9 However, the brain areas exhibit differential vul- medicine as a calmative agent and as a treatment for in- nerability to oxidative damage, probably because of flammation, cough, and gastrointestinal disorders. Recent differences in their endogenous antioxidant defense sys- research has demonstrated that Sideritis species have anti- tem.10 Therefore, we sought to investigate the antioxidant inflammatory2 and antimicrobial3 activities, which have effect of consumption of tea composed of 4% S. clandestina been attributed to their chemical composition. Indeed, many on the cerebral cortex, cerebellum, and midbrain of adult reports indicated that these plants contain essential oils,4 normal mice. We also assessed the effects on the mice’s polyphenols5 (especially flavonoids6), and diterpenoids,7 livers, a reference peripheral tissue. This plant was chosen which are of particular pharmacologic and nutritional after in vitro evaluation of the polyphenolic content and the interest. antioxidant properties of infusions of several taxa and dif- ferent populations of S. clandestina, grown in different re- gions of the Peloponnese. *Current Address: Molecular and NMR Surgical Laboratories, Massachusetts General Hospital and Shriners Hospital for Children, Harvard Medical School, Boston, Massa- chusetts, USA MATERIALS AND METHODS Manuscript received 21 July 2010. Revision accepted 20 October 2010. Plant material and preparation of herb extracts Address correspondence to: Marigoula Margarity, Laboratory of Human and Animal Physiology, Department of Biology, University of Patras, 26500 Patras, Greece, E-mail: S. clandestina is a species endemic in Greece (specifi- [email protected] cally, the Peloponnese). It is taxonomically divided into 2

1060 ANTIOXIDANT ACTION OF GREEK MOUNTAIN TEA IN MOUSE BRAIN 1061 subspecies: S. clandestina subsp. clandestina prospers in the Aldrich), in the range of 25–800 mg/L, were used as refer- mountains of the southern Peloponnese, and S. clandestina ence standards, and total phenol content was expressed as subsp. peloponnesiaca prospers in the mountains of the gallic acid equivalents. northern Peloponnese. S. clandestina subsp. clandestina was collected between July and August 2008 from the Taygetos 2,2-Diphenyl-1-picrylhydrazyl (DPPH) radical–scavenging (southern Peloponnese), Parnon (eastern Peloponnese), and capacity assay. The antioxidant properties of the infusions Mainalo (Central Peloponnese) mountains; S. clandestina were evaluated by the DPPH free-radical method. This assay subsp. peloponnesiaca was collected from the Kyllini is based on the measurement of the reducing ability of anti- Mountain (northern Peloponnese). Taxonomic identification oxidants toward DPPH by measuring the decrease of its ab- was performed at the Division of Plant Biology, Department sorbance at 515 nm.15 The radical-scavenging activity of the of Biology, University of Patras, where herbarium vouchers samples tested, expressed as percentage inhibition of DPPH, are deposited. Green tea (blended and packed in Sri Lanka was calculated according to the following formula:16 Inhibi- under the direction of St. Dalfour, Freres & Cie, Cour tion (%) = [(AB - AA)/AB] · 100, where AB is the absorbance Cheverny, France), in 2-g packs, was used as standard. of the blank sample and AA is the absorbance of the antiox- Infusions were prepared with addition of 4 g of equal idant tested. IC50 values of the plant extracts, defined as the amounts of leaves, flowers, and stems of Sideritis taxa to concentration of the sample that causes 50% inhibition of 100 mL boiling water for 5 minutes. After 5 minutes, the DPPH, were also calculated from the kinetic curve of the solution was filtered and the filtrate volume was adjusted to percentage inhibition of DPPH toward different concentra- 100 mL. Green tea was also prepared in the same way. For tions of each sample and were expressed in mg/mL. the animal studies, the infusions were prepared daily. For the in vitro studies, deionized distilled water was used. The FRAP assay. The antioxidant activity of the plant ex- weight of the dry extract was recorded after lyophilization in tracts and the tissue samples was determined by FRAP as- a Labconco FreezeZone 6 freeze-dry system. say.12 Aqueous solutions of known FeII concentration (range, 5–1000 lMFeSO4) were used as standard solutions. The data Animals were expressed as lmol of FeII equivalents per mg dry extract Adult (3–4 months old) male Balb-c mice were kept in the for the tea infusions and as nmol of FeII equivalents per mg same room under a constant temperature (23–25C) with wet tissue weight for the brain and liver tissues (FRAP value). alternating 12-hour light/dark cycles. The mice were al- lowed free access to food. Mice were managed according to Lipid peroxidation assay. Lipid peroxidation in the brain the Greek National Laws (Animal Act, PD 160/91). regions and liver was assessed fluorometrically at 553 nm The animals were divided into 2 groups consisting of 8 (emission wavelength) and 515 nm (excitation wavelength) animals each. Group 1 mice served as controls and received by measuring the formation of MDA.17 Solutions of known water ad libitum. Group 2 mice had ad libitum access to 4% MDA concentration (0.05–10 lM) were used as standards. (w/v) tea infusion of S. clandestina subsp. peloponnesiaca Results are reported as lmol MDA per g of protein. for 6 weeks (40 days), based on the protocol of Choudhary and Verma11 with slight modifications. Body weight was Reduced glutathione determination. Reduced glutathi- measured weekly in both groups and did not significantly one content of the tissue samples was estimated fluorome- differ between them at the end of the treatment period. trically according to the procedure of Mokrasch and On the completion of the 40-day treatment period, all Teschke.18 Standard solutions of reduced glutathione were animals were sacrificed by light ether anesthesia. Liver and prepared in a range of 0.5–100 lM. The tissue reduced glu- brain were excised immediately, and cerebral cortex, cere- tathione content was expressed as lmol reduced glutathione bellum, and midbrain were separated from the whole brain. per g of protein. The tissue samples were kept at -75C until use. Protein determination. Total protein concentration was Preparation of tissue homogenates determined by the Bradford method,19 using bovine serum Brain regions and liver samples were weighed and ho- albumin as standard. mogenized (10% w/v) with a glass-Teflon homogenizer in Statistical analysis ice-cold 1.15% KCl.12 Tissue homogenates were centri- fuged for 5 minutes at 15000 g, and the supernatants were The results are expressed as mean – standard error. Sta- used to determine the antioxidant activity (by ferric reduc- tistical analysis was performed with GraphPad Instat 3 ing antioxidant power [FRAP] assay) and malondialdehyde software using the nonparametric Mann–Whitney test for (MDA) and reduced glutathione levels. evaluating statistically significant differences (P < .05) be- tween the experimental groups. Methods RESULTS AND DISCUSSION Total phenolics assay. Total polyphenols were deter- mined spectrophotometrically by using Folin–Ciocalteu To select the most bioactive population of all S. clan- reagent.13,14 Aqueous solutions of gallic acid (Sigma- destina samples among those collected, their polyphenolic 1062 LINARDAKI ET AL. content and antioxidant activity were determined in vitro. peroxidation, and reduced glutathione content of 3 brain Table 1 presents the total polyphenol content of all Sideritis regions (cerebral cortex, cerebellum, and midbrain) and a taxa infusions tested and their solid residue contents. All tea peripheral tissue (liver) in control and treated mice. As infusions of S. clandestina taxa and populations had sig- shown in Table 2, there is a hierarchy in antioxidant/re- nificantly (P < .05) lower polyphenolic composition than ducing capacity and MDA levels of the examined brain Camellia sinensis leaves, which were used as the standard. regions in control animals: cerebral cortex > cerebellum > The polyphenolic content of 4 of 5 populations of S. clan- midbrain; the differences reached significance (P < .05) be- destina subsp. clandestina was significantly lower than that tween all brain areas. However, with respect to their reduced of S. clandestina subsp. peloponnesiaca. The in vitro anti- glutathione content, the cerebellum ranks higher than the oxidant properties of the plant extracts were measured by cerebral cortex. In accordance with our observation, Feoli DPPH and FRAP assays (Table 1). The infusion of S. et al. also reported a similar hierarchy in total antioxidant clandestina subsp. peloponnesiaca displayed significantly reactivity levels, as estimated by total antioxidant reactivity (P < .05) stronger in vitro antioxidant properties than the assay.22 Other studies also reported greater lipid peroxida- other taxa of S. clandestina. However, the in vitro antioxi- tion in the cerebral cortex than in the cerebellum or midbrain dant properties of S. clandestina subsp. peloponnesiaca of young control rats23,24 and higher reduced glutathione were lower than those of green tea. Similarly, Trianta- content in rat cerebellum than in the cortex and midbrain.10 phyllou et al.20 showed that the water extract of China black These regional differences could be attributed to the dif- tea displayed higher total phenol content and antioxidant ferences in structure and physiology of the brain areas, activity than that of S. reaseri collected from a mountainous leading to differences in vulnerability to oxidative stress and region of northern Greece. activity of their endogenous antioxidant defense system. Another study showed that not only the species but also It is known that the brain regions vary with respect to their many environmental factors,21 such as the soil type, sun fatty-acid composition because white matter rich in myelin exposure, and rainfall, have a strong effect on polyphenol contains fewer polyunsaturated fatty acids (the major targets composition. This finding explains the variation observed of free radicals) than the grey matter.25 In this context, the among the different populations of S. clandestina subsp. midbrain, a heavily myelinated region, is expected to be clandestina. All S. clandestina infusions had significantly more resistant to lipid peroxidation than cerebral cortex and lower antioxidant activity than C. sinensis, in accordance cerebellum10,26 and thus demanding the development of with their lower polyphenolic content. Tunalier et al.,5 in lower degree of endogenous antioxidant defense. As shown their study of the antioxidant properties and phenolic com- in Table 2, comparison of the above biochemical measures position of 27 Sideritis species grown in Turkey, indicated a among liver and the brain regions of the controls revealed linear relationship between these 2 measures. Thus, the in- significantly higher total antioxidant capacity, significantly fusion of S. clandestina subsp. peloponnesiaca displayed lower reduced glutathione content, and a moderate level of high polyphenolic content and strong antioxidant properties lipid peroxidation in liver, in accordance with findings from in vitro, rendering it suitable for the further in vivo study. a previous study in normal rats.27 To this end, the biological effects of 4% (w/v) herbal tea Consumption of the tea significantly increased (P < .05) from S. clandestina subsp. peloponnesiaca consumption for the antioxidant capacity of midbrain by 72% but did not 6 weeks by adult control mice were further investigated. We significantly influence the antioxidant power of the cerebral assessed the total antioxidant activity, the extent of lipid cortex, cerebellum, and liver (Table 2). Furthermore, herbal

Table 1. Total Polyphenol Content and Antioxidant Activity of Sideritis Taxa Water Extracts

Antioxidant Activity

Solid residue content of Concentration of polyphenols DPPH assay—IC50 FRAP assay Sideritis samples extracts (mg/100 mL)a (lg gallic acid/mg)b (mg/mL)c (lmol FeII/mg)d S. clandestina subsp. clandestina (Taygetos I) 302 36.8 – 0.9** 7.75 – 0.55** 0.43 – 0.07** S. clandestina subsp. clandestina (Taygetos II) 332 34.6 – 1.8** 7.31 – 0.39** 0.49 – 0.12** S. clandestina subsp. clandestina (Parnon) 404 30.3 – 2.2** 7.58 – 1.12** 0.56 – 0.09** S. clandestina subsp. clandestina (Taygetos III) 262 44.7 – 0.8 16.18 – 1.69** 0.27 – 0.07** S. clandestina subsp. clandestina (Mainalo) 500 23.7 – 0.5** 8.90 – 1.55** 0.33 – 0.03** Green tea (Camellia sinensis) 744 165.0 – 8.8 0.38 – 0.12 4.84 – 0.33 S. clandestina subsp. peloponnesiaca (Kyllini) 330 45.6 – 1.7 2.79 – 0.21 1.19 – 0.09

Values are the mean of 4 or 5 independent tea preparations – standard error. aExpressed as mg dry extract per 100 mL of infusion. bExpressed as lg gallic acid equivalent per mg of dry extract. c Expressed as IC50 values corresponding to the aqueous extract concentration (mg/mL) causing 50% inhibition of DPPH radical. dExpressed as lmol FeII per mg dry extract. **P < .05 compared with S. clandestina subsp. peloponnesiaca.

DPPH, 2,2-diphenyl-1-picrylhydrazyl; FRAP, ferric reducing antioxidant power; IC50, 50% inhibitory concentration. ANTIOXIDANT ACTION OF GREEK MOUNTAIN TEA IN MOUSE BRAIN 1063

tea intake led to a significant reduction in MDA levels a a (Table 2) in the cerebellum (78%) and midbrain (59%), with

27%) 32%) simultaneous significant elevation in reduced glutathione

[ [ content of these regions (Table 2) by 27% and 32%, re- spectively, compared with the control mice. Conversely, in accordance with their total antioxidant power, the MDA and 0.683 0.159 1.109* ( 0.919* ( – – – – reduced glutathione levels of the cerebral cortex and liver remained unchanged after tea consumption. These findings clearly show that consumption of the tea infusion rein- mol/g protein)

l forces the antioxidant defense system of the mouse brain in a region-specific manner. The diverse pattern of antioxidant effects of tea con- sumption obtained in different brain areas may be attributed 0.430 12.560 0.144**** 6.836 1.085**0.493 20.612 18.737 to differences in regional antioxidant defense. This region – – – – specificity of antioxidant reinforcement in the brain by polyphenol-rich plant extracts has been also observed in 24, 28 16.265 14.145 previous studies. Ultimately, the cerebral cortex seems to have a stronger antioxidant defense system, resulting in

a a resistance to changes in its oxidant/antioxidant status. The comparison between the brain regions and liver reveals that 59%) 78%) the cerebral cortex and liver share the same antioxidant re- Y Y sponse pattern in response to the herbal tea consumption. As in the cortex, the high antioxidant power of liver, shown in 27 0.144 12.472 0.077 7.271 0.047* ( 0.011* ( the present and previous studies, might render this tissue – – – – insensitive to changes in its oxidant/antioxidant status. This finding is somewhat expected because liver executes several

mol/g protein) GSH ( vital functions of the organism. l Our findings conclusively indicate that the consumption

*** 0.555 of the herbal tea from Sideritis species induces a significant , region-specific antioxidant reinforcement in the brain, with the cerebellum and midbrain the most affected brain areas. 0.273 2.802 0.136** 1.524 0.112** 0.203** 0.426 Although further studies should address the specific bioac- – – – – tive ingredients of Sideritis infusions, their absorption and bioavailability, and their mechanism of action, the present 1.381 data strongly suggest that this herbal tea could be a good

a source of natural antioxidants. The tea might help prevent the age-related deficits and neurodegenerative disorders

72%) related to oxidative damage of the specific brain regions. [

ACKNOWLEDGMENT 0.172 3.273 0.391* ( 1.976 1.659 0.389 1.957 – – – – This research was partially supported by BIOFLORA, a network of the University of Patras. /mg wet tissue) MDA ( II AUTHOR DISCLOSURE STATEMENT *** 3.743

, No competing financial interests exist. decrease) compared with control mice. Y standard error of 8 values corresponding to 8 animals. –

FRAP (nmol Fe REFERENCES 0.408 6.047 0.281** 0.512**** 18.717 0.378** 3.744 Effect of Herbal Tea Intake on Biochemical Measures on Oxidant/Antioxidant Status of Adult Mouse Brain and Liver – – – –

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Journal of Nutritional Biochemistry 23 (2012) 961–965

Wild blueberry (V. angustifolium)-enriched diets alter aortic glycosaminoglycan profile in the spontaneously hypertensive rat☆

Aleksandra S. Kristoa, Christina J. Malavakib, Fotini N. Lamaric, Nikos K. Karamanosb, Dorothy Klimis-Zacasa,⁎

aFood Science and Human Nutrition, University of Maine, Orono, ME 04469, USA bLaboratory of Biochemistry, Department of Chemistry, University of Patras, Greece cDepartment of Pharmacy, University of Patras, Patras, Greece

Received 1 December 2010; received in revised form 28 April 2011; accepted 2 May 2011

Abstract

Glycosaminoglycans (GAGs) are essential polysaccharide components of extracellular matrix and cell surface with key roles on numerous vascular wall functions. Previous studies have documented a role of wild blueberries on the GAG profile of the Sprague–Dawley rat with a functional endothelium as well as in the vascular tone of the spontaneously hypertensive rat (SHR) with endothelial dysfunction. In the present study, the effect of wild blueberries on the composition and structure of aortic GAGs was examined in 20-week-old SHRs after 8 weeks on a control (C) or a wild blueberry-enriched diet (WB). Aortic tissue GAGs were isolated following pronase digestion and anion-exchange chromatography. Treatment of the isolated populations with specific GAG-degrading lyases and subsequent electrophoretic profiling revealed the presence of three GAG species, i.e., hyaluronic acid (HA), heparan sulfate (HS) and galactosaminoglycans (GalAGs). A notable reduction of the total sulfated GAGs and a redistribution of the aortic GAG pattern were recorded in the WB as compared to the C group:a 25% and 10% increase in HA and HS, respectively, and an 11% decrease in GalAGs. Fine biochemical analysis of GalAGs at the level of constituent disaccharides with high-performance capillary electrophoresis revealed a notable increase of nonsulfated (18.0% vs. 10.7%) and a decrease of disulfated disaccharides (2.2% vs. 5.3%) in the WB aorta. This is the first study to report the redistribution of GAGs at the level of composition and their fine structural characteristics with implications for the endothelial dysfunction of the SHR. © 2012 Elsevier Inc. All rights reserved.

Keywords: Wild blueberry; SHR; Aorta; GAGs; Heparan sulfate; Chondroitin sulfate

1. Introduction hexosamine and UA type as well as sulfation pattern, are present in the vascular wall: (a) hyaluronic acid (HA); (b) galactosaminoglycans Numerous studies suggest a protective role of dietary polyphenols (GalAGs), comprised of chondroitin sulfate (CS) and dermatan sulfate against cardiovascular disease (CVD) [1–3], due to direct antioxidant (DS); (c) heparan sulfate (HS) and (d) keratan sulfate [14]. activity [4,5] and/or signaling effects [6,7]. Wild blueberries, a rich Hyaluronic acid is the only GAG molecule that is not sulfated and source of anthocyanins and other phenolics [8–10], have demon- not linked to a protein core, whereas HS and GalAGs occur as part of strated beneficial properties for various vascular functions [11–13]. proteoglycan (PG) molecules that may contain more than one GAG Glycosaminoglycans (GAGs) are linear polysaccharides composed of type, such as HS- and CS-containing syndecan, glypican or CS- and DS- repeating disaccharide units of hexosamine, D-galactosamine or D- containing versican, decorin and bigycan [15]. glucosamine, and uronic acid (UA), D-glucuronic acid or L-iduronic Glycosaminoglycans, as prevalent components of the extracellular acid. Four major classes of GAG molecules, determined by the matrix (ECM) and cell surface, interact with numerous proteins and ligands, modulating crucial biological processes such as cell growth and development [16]. In particular, the glycocalyx associated with Abbreviations: C, control; CVD, cardiovascular disease; CS, chondroitin the vascular endothelium is increasingly gaining appreciation as a sulfate; DS, dermatan sulfate; ECM, extracellular matrix; GalAG, galacto- determinant of signaling, mechanotransduction, vascular permeabil- saminoglycan; GAG, glycosaminoglycan; HA, hyaluronic acid; HPCE, ity and inflammatory response and thereby the development of high-performance capillary electrophoresis; HS, heparan sulfate; PG, endothelial dysfunction and CVD [17,18]. Additionally, the initiation proteoglycan; SD, Sprague–Dawley; SHR, spontaneously hypertensive of atherosclerotic process by lipid accumulation and retention in the rat; UA, uronic acid; WB, wild blueberry. ☆ This work was supported by a Wild Blueberry Association of North ECM is largely mediated by CS/DS proteoglycans [19]. America grant to D.K.-Z. and a fellowship by the Greek State Scholarship Abnormalities in GAG synthesis, such as CS elevation and Foundation to A.S.K. increased sulfation, observed in the vasculature of the spontaneously ⁎ Corresponding author. Tel.: +207 581 3124. hypertensive rat (SHR) are among the early events that contribute E-mail address: [email protected] (D. Klimis-Zacas). to the vascular pathology of this animal model of endothelial

0955-2863/$ - see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.jnutbio.2011.05.002 962 A.S. Kristo et al. / Journal of Nutritional Biochemistry 23 (2012) 961–965 dysfunction [20–24]. In comparison with its normotensive genetic 4°C, samples were centrifuged for 5 min at 12,000g, supernatant was decanted, and the μ control, the Wistar Kyoto (WK) rat, the SHR aorta is characterized by a precipitate was dissolved in 2× distilled H2O, 5 l/mg aortic tissue. thicker subendothelial matrix and a higher CS synthesis leading to a 2.5. Uronic acid and total sulfated GAG determination higher concentration of total GAGs and elevated peripheral resistance and blood pressure [22]. The increased sulfate incorporation in PG Carbazole reaction modified by Bitter and Muir was applied on aliquots from each molecules in the resistance arteries of the SHR [23] is another sample to determine UA content [32]. Individual samples were also analyzed hallmark of endothelial dysfunction in this animal model [24]. colorimetrically for total sulfated GAGs (sGAGs) with Blyscan.

Although the role of bioactive dietary ingredients on ECM and GAG 2.6. GAG fractionation and identification metabolism has not been thoroughly examined to date, various plant- derived micronutrients [25], vitamins E and C [26], flavonoids [27], The intra-differences in UA and total sGAGs within each diet group were not tea polyphenols [28], sorghum phenolics [29] and genistein [30] have significant. Therefore, all GAG samples obtained from each diet group (C, n=10 and WB, n=10) were pooled due to limited tissue availability from each rat and applied to been reported to positively affect signaling pathways and enzymatic ion-exchange chromatography on a DEAE-Sephacel column (7×1.6 cm i.d.), eluted processes related to ECM and GAG metabolism. Wild blueberries with 0.1 M NaCl (3 volumes) and a NaCl linear gradient (0.1 to 0.9 M, 10 volumes), as consumed with diet have been documented to elicit reduced sulfation previously described [31]. Fractions of 0.8 ml were collected and analyzed for UA. and population redistribution of aortic GAGs in the Sprague–Dawley Uronic acid recovery from the DEAE column was higher than 90% in both C and WB (SD) rat with a normal endothelium [31]. Hence, in the present study, pooled samples. The UA-positive DEAE fractions containing distinct GAG populations were pooled, precipitated overnight with 4.5 volumes of absolute ethanol at 4°C and the effect of a wild blueberry diet on the aortic GAG profile and fine centrifuged at 12,000g for 15 min. The precipitate was then dissolved in 2× distilled structural characteristics was examined in the SHR model of H2O at the final concentration of 1 μl/μg UA. Glycosaminoglycan populations obtained endothelial dysfunction after 8 weeks of diet consumption. by DEAE fractions were identified by cellulose acetate membrane electrophoresis using known standards of HA, HS and CS/DS and a running buffer of 0.1 M pyridine–0.1 M formic acid, pH 3.1, at a constant current of 0.5 mA/cm of membrane width for 50 min. 2. Methods and materials Toluidine blue in 15% vol/vol aqueous methanol was applied for a 10-min membrane staining. Membranes were rinsed with water and scanned immediately [31]. 2.1. Chemicals

2.7. GAG degradation with specific lyases Pure NaCl, KCl, CaCl2, MgSO4,KH2PO4, NaHCO3 and dextrose for the physiologic salt solution (PSS) as well as heparinase I, II and III, chondroitinase AC and ABC, standards Digestions of pooled samples with a mixture of chondroitinases AC and ABC (0.01 for HA, HS and CS/DS analysis and standard preparations of sulfated D-disaccharides μ – from CS/DS [Δ-di-di(2,6)S, Δ-di-di(2,4)S, Δ-di-mono6S, Δ-di-mono4S and Δ-di-nonS] units/10 g UA) in 0.1 M sodium acetate 0.1 Tris-HCl buffer, pH 7.3, and a mixture of μ were purchased from Sigma (St. Louis, MO, USA). Chondroitinase AC I was purchased heparin lyases I, II and III (0.05 units/25 g UA) in 20 mM acetate buffer, pH 7.0, with μ from Seikagaku America (Ijamsville, MD, USA), the Blyscan assay kit from Accurate 1 mol calcium acetate were performed overnight at 37°C and stopped by transfer of Chemical (Westbury, NY, USA) and PRONASE Protease from Calbiochem (San Diego, samples in 100°C for 1 min [31]. CA, USA). 2.8. High-performance capillary electrophoresis (HPCE) analysis

2.2. Animal model and diets Following enzyme digestions, GAG profile analysis was conducted as previously described [31,33] on an HP3DCE instrument (Agilent Technologies, Waldronn, The animal welfare and the experimental protocols conformed to the Animal Care Germany) with a built-in diode array detector set at 232 nm, using uncoated fused- and Use Committee of the University of Maine standards (IACUC Protocol A2008-2006- silica capillary tubes (50 μm i.d., 64.5 cm total and 56 cm effective length) at 25°C. 2005). Twenty male SHRs (Charles River Laboratories, Wilmington, MA, USA) at the age Phosphate buffer, 50 mM, pH 3.0, was used as a running buffer. NaOH 0.1 M (1 min), 2× of 12 weeks were randomly assigned to one of two diets: control diet (C) (modified distilled H2O (1 min) and operating buffer (5 min) were used before each run to wash AIN-76, n=10) and wild blueberry diet (WB) (control diet+8% wt/wt freeze-dried the capillary tube. Migration of Δ-disaccharides from the cathode to the anode by wild blueberry powder substituting for dextrose, n=10), for a period of 8 weeks. The electrophoretic mobility and against the electroosmotic buffer flow was achieved by animals were housed in the Small Animal Facility at the University of Maine in applying samples at the cathodic end under 50 mbar and 30 kV for 5 s. individual stainless-steel mesh-bottomed cages in a room controlled for temperature (22°C) and light conditions (12:12-h light:dark cycle). Tap water and food were 2.9. Statistical analysis provided ad libitum. Food consumption was measured daily; and body weights, weekly. The diets were prepared in our laboratory from purified diet ingredients, Student's t test was used for comparisons of food consumption, body weights, and stored at 4°C and used within 5 to 7 days. The diet ingredients, dextrose, egg white liver and aorta weight between the diet groups. Two-way analysis of variance was solids, vitamin mix (A.O.A.C. Special Vitamin Mixture), DL-methionine, biotin, corn oil, applied in ranked observations of UA and sGAG concentration of individual aortic were purchased from Harlan Teklad (Madison, WI, USA); custom-made mineral mix specimens. Statistical analysis was performed with Sigmastat Statistical Program was purchased from MP Biomedicals (Solon, OH, USA). Wild blueberries, provided as a version 2.0 (SPSS Inc., Chicago, IL, USA). Values are given as mean±standard deviation composite by Wyman's (Cherryfield, ME, USA), were freeze-dried and powdered with (S.D.) or mean±standard error of mean (S.E.M.); differences are considered standard procedures by FutureCeuticals (Momence, IL, USA). Twenty-one different statistically significant at P≤.05. anthocyanins were detected in the wild blueberry powder with the main anthocyanins, malvidin 3-galactoside and peonidin 3-glucoside, representing approximately 13% of the total anthocyanin content (1.6±0.2 mg/100 mg) [8]. 3. Results

2.3. Aortic sample collection and preparation 3.1. Food intake and animal growth

After 8 weeks of dietary treatment, rats were anesthetized with 95%CO2/ 5%O2; the The daily food intake was similar between the control and the wild thoracic aorta was excised and transferred in PSS, composed of NaCl 118, KCl 4.7, CaCl 2 blueberry diet-fed animals: 20±0.4 g in both diet groups. Growth 2.5, MgSO4 1.2, KH2PO4 1.2, NaHCO3 12.5 and dextrose 11.1 mM. Aortas were cleaned of the adherent connective and fat tissue, frozen in liquid N2 and stored at −80°C. After rate, assessed by weekly measurement of body weight, did not differ all samples were collected, frozen specimens were powdered under liquid N2, defatted significantly between the two diet groups (data not shown). As twice with methanol/ solution (1:1, vol/vol), incubated for 16 h at 4°C, rinsed with acetone and vacuum dried.

Table 1 2.4. Tissue digestion and separation of GAGs Body weight (BW), liver weight and aortic tissue weight a of the two diet groups b Dry defatted aortas were rehydrated overnight at 4°C with 2× distilled H2O, Diet group BW (g) Liver (g) Aorta (mg) 30 μl/mg aorta. The rehydrated samples were digested overnight at 37°C with C 343.0±2.7 13.2±1.0 10.4±2.4 PRONASE (1.6 units/mg aortic tissue) in 50 mM Tris-HCl, pH 8.0. The reaction was WB 353.0±5.3 12.8±0.9 10.0±2.2 stopped with 0.6 M NaCl and transfer of vials in 100°C for 1 min. Aortic digests were centrifuged for 5 min at 12,000g, and the collected supernatant was diluted in 4.5 a Mean±S.D., n=10 rats per diet group. volumes of ethanol solution with 2.5% sodium acetate. After overnight incubation at b Aorta dry weight. A.S. Kristo et al. / Journal of Nutritional Biochemistry 23 (2012) 961–965 963 presented in Table 1, no significant difference between diet groups Table 3 a was detected in the final body weights or liver wet weight. Glycosaminoglycan-derived UA concentration in each population isolated from pooled aortic samples with DEAE-Sephacel anion-exchange chromatography in the Furthermore, thoracic aorta dry weight did not differ between the two diet groups two diet groups. CWB

HA 123.6 (5.1%) 154.2 (6.2%) 3.2. Wild blueberry diet affects total content of aortic sulfated GAGs HS 1242.2 (50.8%) 1370.3 (55.1%) GalAGs 1081.4 (44.2%) 960.8 (38.7%) Aortic samples were analyzed for UA and total sGAGs content after Total GAGs 2447.3 2485.4 tissue defatting and proteolytic digestion. No significant differences a μg UA/mg of dry defatted aortic tissue. Percentages of each type of GAG are were found in the UA concentration of the aortas isolated from either provided in parentheses. diet group: 5.2±0.3 vs. 4.6±0.3 μg/mg aorta in the WB and C group, respectively (Table 2). Notably, the concentration of total sGAGs was was observed in the SHR thoracic aortas after 8 weeks of wild significantly lower in the WB group (12.6±0.2) as compared to the blueberry consumption (Table 2). Although the total aortic GAG μ b control (13.7±0.2 g/mg aorta, P .05, Table 2). content was similar with only a slight increase (2%) in the WB group, wild blueberry consumption resulted in a redistribution of GAG 3.3. Distribution of GAG content is substantially affected in WB populations. The levels of HA and HS were 25% and 10% higher and the vs. control rats GalAGs 11% lower in the aortic tissue isolated from the WB group (Table 3, Fig. 1). The GalAG disaccharide analysis revealed that the Fractionation, isolation and identification of GAGs were performed disulfated and monosulfated GalAG disaccharides were reduced due as earlier described [31]. In brief, UA-positive populations were to the wild blueberry diet, whereas a higher percentage of separated with ion-exchange chromatography of C and WB aortic nonsulfated GalAG disaccharides was observed in the SHR aortas samples. The identity of these populations was confirmed with from the WB group (Table 4). cellulose acetate electrophoresis before and after treatment with The elevated peripheral resistance and blood pressure of the SHR specific GAG-degrading lyases (not shown). Glycosaminoglycan are related to an increase of vascular PGs [22]. Furthermore, in concentration (μg/mg aortic tissue) estimated by the amount of comparison with the parental WK strain, the SHR aorta is character- recovered UA is presented in Table 3. In both diet groups, HS was ized by a thicker subendothelial matrix and a higher CS synthesis quantitatively the major GAG population, followed by GalAGs and HA. leading to a higher concentration of total GAGs [22]. With most CSPGs Although the total GAG content between the two groups tested was contained in the media layer of the vascular wall, hypertrophy of this similar, the distribution of GAG populations was notably different layer in small and large SHR vessels agrees with the elevated CS in the between the diet groups. Hyaluronic acid concentration was SHR [34]. Hypertrophy of the aortic media observed in young SHRs significantly higher (25% increase) in the WB (154.2 vs. 123.6 μg/mg before the development of hypertension is most likely responsible for in control group). Heparan sulfate was also 10% higher in the WB the lower compliance and distensibility of the SHR aortic wall [35]. (1370.3 vs. 1242.2 μg/mg), whereas GalAGs content was 11% lower in Additionally, GalAGs seem to promote atherogenesis, since CS and the WB group (960.8 vs. 1081.4 μg/mg, Table 3, Fig. 1). DS are highly involved in low-density lipoprotein (LDL) binding [36], and especially biglycan is a critical CS/DS-containing PG for LDL 3.4. Wild blueberry diet increases nonsulfated GalAGs retention [37]. Subendothelial retention of apoB100 lipoprotein, the disaccharide content major apolipoprotein of human LDL, is an early step in atherogenesis, with the atherogenicity of the LDL particle linked to its binding Analysis of the isolated aortic GalAG population with HPCE after affinity for arterial PGs [19,38]. Another indicator of the role of GalAG combined digestion with chondroitinases AC and ABC showed that in atherosclerosis is the recently documented predictability for the Δdi-mono4S was the main GalAG disaccharide in both diet groups postoperational CS4 levels in patients undergoing coronary artery (52.4% in C and 49.5% in WB). The other monosulfated disaccharide, bypass surgery based on preoperational apoB and apoE levels [39]. Δdi-mono6S, was also slightly lower in the WB group (30.3% vs. In the present study, we observed lower GalAGs concentration 31.6%). Notably, as shown in Table 4, the content of GalAGs in after 8 weeks of WB treatment, although it was not determined nonsulfated disaccharides increased twofold in WB group (18.0% vs. whether the lower GalAGs reflected a decrease in CS or DS or both 10.7%), and that in disulfated disaccharides (Δdi-di(2,6)S) decreased types. The decrease of GalAGs due to wild blueberry consumption was by 60% (2.2% vs. 5.3%). accompanied by higher concentrations of both HA and HS in our adult

4. Discussion 1600 +10% 1400 C WB This is the first study to document that a wild blueberry-enriched diet elicited multiple alterations of aortic GAGs in the SHR model of 1200 -11% endothelial dysfunction. A significant reduction of total sulfated GAGs 1000 800 Table 2 600

a b g UA/mg aorta

Uronic acid and total sGAGs concentration in aortic tissue of the two diet groups µ 400 Diet group UA (μg/mg) a sGAGs (μg/mg) b +25% C 4.6±0.3 13.7±0.2 200 ⁎ WB 5.2±0.3 12.6±0.2 0 Individual aortas were analyzed for uronic acid concentration with carbazole reaction HA HS GalAGs and sGAG content with Blyscan. Values are given in μg/mg of dry defatted aortic tissue. a Mean±S.E.M., n=6. Fig. 1. Glycosaminoglycan-derived UA concentration (μg UA/mg of dry defatted aortic b Mean±S.E.M., n=10. tissue) in each population isolated by DEAE-Sephacel anion-exchange chromatography ⁎ Statistically significant at P≤.05. on pooled aortic samples from the two diet groups. 964 A.S. Kristo et al. / Journal of Nutritional Biochemistry 23 (2012) 961–965

Table 4 Whereas further studies are required to elucidate the mechanisms Galactosaminoglycan disaccharide composition as determined by HPCE analysis of of actions of wild blueberries on the aortic GAG profile, the possibility pooled aortic samples after combined digestion with chondroitinases AC and ABCa in the two diet groups cannot be excluded that consumption of wild blueberries alters GAG synthesis and turnover through an effect on enzymatic processes and Disaccharide C WB related signaling pathways. Phenolics from sorghum bran were Δ di-di(2,6)S 5.3 2.2 reported to inhibit hyaluronidase activity, with implications in the Δdi-di(2,4)S –– Δdi-mono6S 31.6 30.3 balance between HA synthesis and degradation in the ECM and hence Δdi-mono4S 52.4 49.5 connective tissue remodeling and homeostasis [29]. Flavonoid Δdi-nonS 10.7 18.0 compounds can inhibit the activity of HA-splitting enzymes based Dashes indicate nondetectable disaccharides. on the number of their hydroxyl groups [27]. Plant-derived a Percentage of total recovered Δ-disaccharides. compounds (ascorbic acid and quercetin) and tea extract induced alterations of the ECM composition leading to reduction of monocyte adhesion in the endothelium [25]. High concentrations of genistein SHRs. In young SHRs, an increase of HA aortic content after diuretic can inhibit GAG synthesis through an effect on epidermal growth treatment was positively correlated with total systemic compliance factor-dependent pathway in patients with mucopolysaccharidoses, a [40]. Hyaluronic acid of the arterial glycocalyx is necessary for condition of excessive GAG accumulation in lysosomes [30]. Signaling activating flow-induced (NO) release from the endothe- processes initiated by vitamins E and C [26] or tea catechins [28] that lial cells by sensing and transferring the shear stress to the modulate ECM environment with effects on vascular remodeling and endothelium [41]. Furthermore, the hydrophilicity of HA is crucial maintenance of vascular wall function also indicate a positive role of for the structural stability of the glycocalyx [17]. Heparan sulfate dietary and bioactive compounds on vascular integrity. localized on the luminal surface is another mechanosensor that Current biomedical research has been targeting the endothelium mediates NO synthesis in response to shear stress [42]. In addition to in an attempt to uncover vascular-directed agents for the prevention the key role of HS on vascular tone, several antiatherogenic of atherosclerosis and CVD [49]. Glycosaminoglycans are rendered as characteristics, such as inhibition of oxidized LDL and monocyte such molecules, with special attention given to their chain elongation binding to the ECM and inhibition of smooth muscle cell proliferation, and sulfation patterns [48]. In the present study, wild blueberries have been attributed to HSPGs and especially perlecan [43].As were involved in primary aspects of GAG metabolism such as opposed to GalAGs, HS is not involved in LDL binding [36], and population redistribution and sulfation in the SHR aorta. We are the perlecan, the main HS-containing subendothelial PG, does not co- first to document the role of a wild blueberry diet on arterial GAG localize with human apoB and therefore appears reduced in human remodeling in the SHR. Wild blueberries modulate structural atherosclerotic lesions [37]. The greater amount of HA and HS in the characteristics that potentiate cardiovascular risk, but most impor- SHR aorta observed in the WB-fed rats further supports the beneficial tantly enhance the protective features of GAG molecules associated role of wild blueberries on CVD. with endothelial dysfunction and vascular pathology in the SHR. Higher HA and HS levels might be related to our recent findings Therefore, wild blueberries may be employed as a component of that documented an NO pathway-mediated attenuation of the endothelium-targeted nutrition to prevent endothelial dysfunction vascular tone in response to adrenergic stimuli in the adult SHRs and CVD development. treated with wild blueberries [44]. The effect of wild blueberries on vascular tone, acting through the endothelium and more specifically on the NO pathway, has been also documented in the SD rat [45,46]. Acknowledgments Besides the beneficial redistribution of GAG populations, the wild blueberry diet caused a significant decrease in GAG sulfation in the The authors would like to express their gratitude to Maria I. SHR aorta. Reynertson et al. have reported a higher sulfate Krevvata for her significant contribution to this project, the Wild incorporation into aortic PG in the SHR compared to the WK rat Blueberry Association of North America (WBANA) for contributing [23], as well as in other experimental models of hypertension [47]. the wild blueberries and FutureCeuticals (Momence, IL) for proces- The higher sulfation of PG molecules from vascular smooth muscle sing them. This work was supported by a WBANA grant to D.K.-Z. and cells of mesenteric resistance arteries observed in the young a fellowship by the Greek State Scholarship Foundation to A.S.K. This prehypertensive SHRs indicates that alterations of PG synthesis is Maine Agricultural and Forest Experiment Station Publication occur before the establishment of vascular dysfunction and hyper- Number 3157. tension in this model [24]. The degree of sulfation is also positively correlated with LDL binding affinity to PGs [37]. 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Dietary manganese affects the concentration, composition and sulfation muscle cell adhesion and migration on collagen and laminin via interference with pattern of heparan sulfate glycosaminoglycans in Sprague–Dawley rat aorta. cell–ECM interaction. J Biomed Sci 2007;14:637–45. Biometals 2006;19:535–46. PHYTOTHERAPY RESEARCH Phytother. Res. 26: 956–963 (2012) Published online 15 November 2011 in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/ptr.3670

Cell-Line Specific Protection by Berry Polyphenols Against Peroxide Challenge and Lack of Effect on Metabolism of Amyloid Precursor Protein

Magdalini A. Papandreou,1 Maria Tsachaki,2 Spiros Efthimiopoulos,2 Dorothy Klimis-Zacas,3 Marigoula Margarity1 and Fotini N. Lamari4* 1Laboratory of Human and Animal Physiology, Department of Biology, University of Patras, Greece 2Division of Animal and Human Physiology, Department of Biology, University of Athens, Greece 3Department of Food Science and Human Nutrition, University of Maine, Orono, ME 04469, USA 4Laboratory of Pharmacognosy and Chemistry of Natural Products, Department of Pharmacy, University of Patras, Greece

Amyloid precursor protein (APP) altered metabolism, Ab-overproduction/aggregation and oxidative stress are implicated in the development of Alzheimer’s disease pathology. Based on our previous data indicating that administration of a polyphenol-rich (PrB) blueberry extract (from wild Vaccinium angustifolium) is memory enhancing in healthy mice and in order to delineate the neuroprotective mechanisms, this study investigated the antioxidant effects of PrB in H2O2-induced oxidative damage, Ab peptide fibrillogenesis and APP metabolism. PrB suppressed H2O2-initiated oxidation (DCF assay) and cell death (MTT assay) in SH-SY5Y cells. Protective effects were observed on Chinese hamster ovary (CHO) cells overexpressing APP770 carrying the mutation Val717Phe only at high concentrations, while further damage on HEK293 cells was induced after co-treatment with 250 mM H2O2 and PrB in comparison with H2O2 alone. Using the thioflavine T assay, blueberry polyphenols inhib- ited Ab-aggregation (~70%, 15 mg/mL) in a time-dependent manner, while in the CHOAPP770 cells it had no effect on APP metabolism as assessed by western blot. The results suggest that blueberry polyphenols exhibit antioxidant and/or pro-oxidant properties according to the cellular environment and have no effect on APP metabolism. Copyright © 2011 John Wiley & Sons, Ltd.

Keywords: antioxidant; amyloid fibrillogenesis; Vaccinium angustifolium; SH-SY5Y; CHOAPP770; APP metabolism.

or in plant extracts are good candidates since pleiotropy INTRODUCTION is their inherent characteristic. Blueberries (fruits of various Vaccinium species) con- Alzheimer’s disease, a form of senile dementia, is an tain a wide range of compounds, including the red and irreversible, progressive neurodegenerative disorder, blue colored anthocyanins, the flavonols and ellagic acid resulting from a complex pathological cascade that derivatives, and have the highest antioxidant capacity includes the accumulation of beta-amyloid (Ab) peptide among fruits and vegetables. A number of studies have aggregates or aberrant amyloid precursor protein documented that rodent diet supplementation with (APP) processing. The disease is manifested years after berries is effective in reducing not only oxidative stress its initiation with serious cognitive and neuropsychiatric but also neuronal, behavioral and cognitive defects deficits but a clear understanding of its pathogenetic associated with aging, inflammation, and factors is lacking, thus limiting the development of Alzheimer’s disease (Shukitt-Hale et al., 2008; Galli et al., adequate prevention and treatment strategies. Multiple 2006; Joseph et al., 1998, 1999, 2003). In particular, cellular/biochemical events (inflammation, oxidative blueberry-fed (4 months of age) APP + PS1 transgenic damage, aberrant signaling, high metal concentrations, mice showed no deficits in Y-maze performance (at aberrant tau/APP processing) are known to contribute 12 months of age) with no alterations in Ab burden to cognitive deficits by disruption of neuronal signal (Joseph et al., 2003). Enhancement of cognitive functions transduction pathways involved in memory and finally and antioxidant protection were also observed by Joseph neuronal and synaptic loss. There is still no effective and his co-workers after dietary supplementation for therapy for Alzheimer’s disease and pleiotropic drugs 8 weeks of aged Fischer 344 rats with blueberry extracts or cocktails have been proposed to have a better chance (Joseph et al., 1998, 1999), or of rats subjected to for halting disease progression (Cole and Frautschy, ischemia/reperfusion, with the latter exhibiting a reduction 2010). In that aspect, natural products either individually in the volume of infarction in the cerebral cortex and an increase in post-stroke locomotor activity, while they also had significantly lower caspase-3 activity in the * Correspondence to: Fotini N. Lamari, Department of Pharmacy, University ischemic hemisphere (Wang et al., 2005). Additional of Patras, 26 504 Rion, Greece. experiments have shown that blueberry supplementa- E-mail: fl[email protected] tion for 10 weeks of young and old rats subjected to an

Received 20 May 2011 Revised 01 September 2011 Copyright © 2011 John Wiley & Sons, Ltd. Accepted 02 September 2011 ANTIOXIDANT AND ANTI-AMYLOID EFFECTS OF BLUEBERRY POLYPHENOLS 957 in vitro inflammatory challenge (LPS) restored the abil- METHODS AND MATERIALS ity of the aged hippocampal cells to respond to an in- flammatory challenge with a large production of inducible heat protein 70 (HSP70) (Galli et al., Plant material and extraction. Wild blueberries were ’ fi 2006). It was shown previously that a 6-day intraper- purchased as a composite from Wyman s (Cherry eld, itoneal administration of a polyphenol-rich wild blue- ME), freeze-dried with standard procedures by American berry extract (PrB) enhanced cognitive performance Lyophilizer Inc. (Bridgeport, PA, USA) and powdered. of healthy mice, brain antioxidant markers and Polyphenols were extracted from the above as described inhibited total brain acetylcholinesterase (Papandreou previously (Lohachoompol et al., 2004; Papandreou et al., et al., 2009). Thus, blueberry extracts either through 2009). In brief, 2 g of blueberry powder was extracted in diet or administered via other routes seem to prevent the dark, under magnetic stirring with 15 mL/g of metha- characteristic cognitive deficits of senescence and nol, acetic acid and distilled water at a ratio of 25:1:24, Alzheimer’s disease and confer antioxidant protection respectively, for 2 h. The extract was evaporated to dryness in rodents. and the dry residue was stored at 20 C until further use. Some mechanisms have been postulated for the The dry residue was re-dissolved with 1 mL of 3% efficacy of the blueberry extract and its constituents, formic acid in water (w/v), centrifuged and the supernatant but many issues concerning their protective effects was absorbed on a C18 Sep-Pak cartridge. The cartridge remain unresolved. Correspondingly, polyphenol and was washed with methanol, equilibrated with 5 mL of anthocyanin treatment were also shown to be 3% formic acid in water (w/v) and eluted with 5 mL 3% effective against diverse oxidative stressors, such as formic acid in 50% methanol (w/v). The polyphenols H O -induced reactive oxygen species (ROS) gener- eluted from the cartridge were evaporated under vacuum 2 2 ation in red blood cells (Youdim et al., 2000a, until dryness and kept at 20 C until use. 2000b) and Ab-induced toxicity in COS-7 cells (Joseph Total phenolics were measured with the Folin-Ciocalteu et al., 2002). It has been recently documented that reagent method (Singleton and Rossi, 1965). The total blueberry extracts significantly enhance microglial polyphenolic content was expressed as gallic acid equiva- lents (GAE), using a standard curve with 50–600 mg/L clearance of Ab, inhibit aggregation of Ab1-42,and suppresses microglial activation, all via suppression gallic acid. Total anthocyanin content was estimated of the p44/42 MAPK module (Zhu et al., 2008). according to Giusti and Wrolstad (2001) by UV-visible Additionally, the whole blueberry extract and many spectroscopy at 538 nm after dissolution in a mixture of fractions of it (i.e. anthocyanin, pre-C18, post-C18, methanol and 0.1 M HCl at a ratio of 85:15. The total proanthocyanidins, chlorogenic acid, etc) have been anthocyanin content was expressed as cyanidin 3-rutinoside studied for their protective ability against dopamine-, equivalents. Ab42- and LPS-induced decrements in calcium buffering in primary hippocampal cells and the degree Cell culture and drug treatment. Human neuroblastoma of protection differed according to the degree of frac- SH-SY5Y and human embryonic kidney HEK293 cell tionation and the stressor applied (Joseph et al., lines were obtained from the American Type Culture 2010). Oxidative stress induces the expression and Collection (ATCC), while the Chinese Hamster Ovary misprocessing of APP, leading to the generation of (CHO) control cells (wild type, wt) or CHOAPP770 cell, amyloidogenic fragments that readily form aggregates. stably transfected to express mutant human APP770 This process can result in a potentially vicious cycle carrying the Val ! Phe mutation at residue 717, were a whereby oxidative stress leads to Ab production, and this kind gift of Professor Leonidas Stefanis from the Biomed- production in turn results in increased oxidative stress, ical Research Foundation of the Academy of Athens neuronal dysfunction and ultimately neuronal death (BRFAA). All cultures were grown in DMEM containing (Miranda et al., 2000). The effect of blueberry polyphe- 10% heat-inactivated fetal bovine serum, 4.5 g/L glucose, nols on APP metabolism has not yet been studied. 2mML-, 100 mg/mL penicillin and 100 mg/mL The aim of the present study was to examine the streptomycin sulfate. Confluent cultures were subcultured effects of the extract rich in blueberry polyphenols using 0.5 g/L trypsin/0.2 g/L EDTA into 96-well plates at a 4 (PrB) against H2O2-induced cell death in different density of 1.5 10 cells/well for SH-SY5Y cells and 5 neuronal and non-neuronal cell lines, which have not 103 cells/well for HEK293 and CHOAPP770 cells. All been previously studied, and furthermore to study experiments were carried out 24 h after the cells were the effect of PrB on APP metabolism and Ab- seeded. Prior to all treatments, the cells were incubated aggregation in vitro. For this reason, the study used in serum-free media for 24 h. In a pilot investigation, the (a) the human neuroblastoma SH-SY5Y cell line, a cells were treated with H2O2 at concentrations ranging well known model cell system for studying neuronal from 50 to 1000 mM, for 18 h, and then examined for cell cell death induced by oxidative stress (Ruffels et al., viability. Finally, the cells were co-incubated with 250 mM 2004); (b) Chinese hamster ovary (CHO) cells stably H2O2 and the tested phytochemical (1–250 mg/mL PrB), transfected to express mutant human APP770 carrying or vehicle as control (medium containing max. 0.1% v/v the Val717Phe mutation (CHOAPP770) and thus release DMSO), for 18 h. considerable amounts of the toxic, amyloidogenic Cell extracts were prepared by treating cells with Ab (Sun et al., 2002); and (c) human embryonic 50 mM Tris, 150 mM NaCl, 2 mM EDTA, pH 7.6 contain- kidney HEK293 cells. Despite the fact that the ing protease inhibitors Complete (Roche Applied HEK293 cell line was derived by transformation of Science) and 1% Triton X-100 for 30 min on ice. The primary human embryonic kidney cell and thus are lysates were then centrifuged at 10000 g for 30 min at considered a model system of peripheral cells, it has 4 C. The supernatants were collected and the amount been shown that they have unexpected neuronal of total protein was measured using the bicinchoninic progenitor properties (Shaw et al., 2002). assay (Pierce, Bonn, Germany).

Copyright © 2011 John Wiley & Sons, Ltd. Phytother. Res. 26: 956–963 (2012) 958 M. A. PAPANDREOU ET AL.

Assessment of cell viability and reactive oxygen species (SDS-PAGE). Proteins were separated on gels (12% for levels. Cell viability was estimated using 3-(4,5- media and 16% for cell lysates) and transferred to nitro- dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide cellulose membranes, which were incubated overnight at (MTT reagent) (Plumb et al., 1989). Briefly, on com- 4 Cwiththeprimaryantibodies(1:1000)(6E10:residues pletion of the treatment, the cells were washed with Ab1-16 monoclonal; R1(57): anti-APP C-terminal poly- pre-warmed phosphate-buffered saline (PBS) and clonal; 71-192wt-1147: residues APP590-596 (Parisiadou incubated with serum-free media containing MTT at a and Efthimiopoulos, 2007; Parisiadou et al., 2008). Bound final concentration of 2.5 mg/mL for 150 min. Superna- antibodies were visualized using peroxidase-conjugated tants were carefully aspirated and the resultant formazan secondary antibodies (anti-rabbit used in 1/2000 for R1 product was dissolved in DMSO and the absorbance was (57) and 1/1000 dilution for 71-192wt-1147; anti-mouse measured using a UV-Vis spectrometer (Denley WellScan, used in 1/1,000 dilution for 6E10), followed by ECL+ West Sussex, UK) at 545 nm. Wells without cells were used detection (ChemBio Ltd). as blanks and were subtracted as background from each sample. The results were expressed as a Statistical analysis. Data are presented as mean SE for percentage of control untreated cells. at least ten replicates. Statistical analysis was performed Intracellular formation of ROS was assessed using the with GraphPad Instat 3 software (GraphPad Instat probe 2′,7′-dichlorofluorescein diacetate (DCFH-DA) Software, Inc. USA) using the nonparametric Mann- (Sigma-Aldrich Corporation, St Louis, MO, USA), Whitney test. In all tests, a criterion of p ≤ 0.05 (two- which was used as a substrate (Wang and Joseph, tailed) was considered necessary for statistical 1999). All cell lines (SH-SY5Y: 1.5 104 cells/well, significance. HEK293/CHOAPP770:5 103 cells/well), growing in microtiter 96-well plates, were washed two times with pre-warmed PBS, and then incubated with 50 mM DCF-DA in PBS for 30 min at 37 C. Fluorescence RESULTS was then quantified in a microplate-reader (Bio-Tek Instruments Inc., Ville St Laurent, Que, Canada) at H2O2-induced toxicity on SH-SY5Y, HEK293 and an excitation wavelength 485 nm and emission wavelength CHOAPP770 cell lines 530 nm. To determine the effective concentrations of H2O2, all Assessment of malondialdehyde levels. CHOAPP770 cells cell lines were treated with various concentrations were incubated in serum-free media, for 24 h, and then (50–750 mM)ofH2O2 for 18 h and the viability of the treated with PrB extract (10–50 mg/mL) for 48 h. The cells cells was assessed by the MTT assay. Treatment with were then lysed, centrifuged and the collected superna- 50 mM and 100 mM H2O2 had virtually no effect on the tants were subjected to malondialdehyde (MDA) viability of all cell lines under the conditions employed, analysis. The concentration of MDA, a compound that but the viability of cells exposed to concentrations is produced during lipid peroxidation was determined higher than 100 mM showed a dose-dependent decrease. fluorometrically by the thiobarbituric acid (TBA) In the SH-SY5Y cell line, the cell viability decreased by method, as previously described (Jentzsch et al., 1996; 43.0 0.5%, 56.0 0.5% and 60.0 0.9%, compared Papandreou et al., 2009). The assay showed good linearity with untreated cells after treatment with 250 mM, – 2 (0.05 5.00 mM, y = 11.488x +0.7415, R = 0.9976) and 500 mM and 750 mM H2O2, respectively. Similarly, in the sensitivity. The level of MDA was expressed as nmol/mg HEK293 cells, in the presence of 250 mM and 500 mM protein in order to correct for any variability in cell H2O2, the percentage of cell viability decreased by number dictated by cell treatment. 30.0 2.0% and 48.0 0.9%, respectively, while at APP770 750 mM H O , by 69.0 0.8%. Treatment of CHO fl fl 2 2 Thio avine T assay. Thio avine T (ThT) measurement cells with 250 mM, 500 mM and 750 mM H2O2, resulted in was performed according to the method described 35.0 1.2%, 40.0 0.8% and 49.0 1.2% cell death, previously (Tjernberg et al., 1999; Papandreou et al., respectively. There was a significant injury in all three 2006). For Ab aggregate-formation assay, Aß1-40 cell lines tested, with the H2O2-treated cells becoming (10 mg/mL) was diluted in PBS (10 mM, 150 mM NaCl, round and some of them detaching from the plate. pH 7.4) to 40 mM and incubated at room temperature Moreover, the H2O2-induced oxidative damage to cells for 5 and 30 days, with various concentrations of the was also measured as ROS formation, as assayed by PrB extract or without (control) (Papandreou et al., the conversion of the probe DCFH-DA to DCF. ROS 2006). Within 30 min after addition of thioflavine T accumulation increased significantly in all cell lines fl (10 mM), the uorescence intensity was measured with a tested after incubation with H2O2 (250 mM), as indicated RF-1501 spectrofluorometer (Shimadzu), using an exci- by the increase in relative fluorescence units (SH-SY5Y, tation filter of 435 nm and an emission filter of 482 nm, 73400 4585; HEK293, 38529 2220; CHOAPP770, respectively. The percentage inhibition was calculated 48999 1086 DCF/MTT) compared with the untreated by comparing these fluorescence values with those found (control) cells (SH-SY5Y, 58114 2901; HEK293, in control solutions with no PrB. 18406 8433; CHOAPP770, 40861 1381 DCF/MTT values), respectively. Western blot analysis of amyloid-precursor protein (APP) metabolism. CHOAPP770 cells were treated with the plant extract (10–50 mg/mL for PrB) for 48 h. The cells PrB content in polyphenols and anthocyanins were then lysed, centrifuged and the obtained super- natant, as well as the cell culture media, were used for so- The PrB extract, obtained from solid-phase extraction dium dodecyl sulfate-polyacrylamide gel electrophoresis on Sep-Pak C18, had a total polyphenolic content of

Copyright © 2011 John Wiley & Sons, Ltd. Phytother. Res. 26: 956–963 (2012) ANTIOXIDANT AND ANTI-AMYLOID EFFECTS OF BLUEBERRY POLYPHENOLS 959

13.01 0.06 mg GAE/g dry weight and a total anthocy- or untreated cells even though viability was not different anin content of 3.78 0.05 mg/g dry weight, as reported than that of the H2O2-treated cells (Fig. 3). previously (Papandreou et al., 2009). Comparison of Furthermore, the effect of PrB on membrane lipid this composition with that reported for freeze-dried peroxidation in CHOAPP770 cells was investigated. As blueberry powder by others (Mazza et al., 2002) shows shown in Fig. 4, CHOAPP770 cells producing consider- that the solid-phase extraction concentrated the extract able amounts of Ab species, showed an increase in lipid in polyphenols about four times. peroxidation (~88%), in comparison with the wild-type CHO cells, as evidenced by determination of MDA levels (0.160 0.005 nmol/mg protein vs 0.091 0.002 nmol/mg protein). However, PrB prevented peroxida- PrB rescues viability and decreases ROS levels in tion of lipids compared with the control, by 51%, 25% H O -treated SH-SY5Y cells 2 2 and 31%, at PrB concentrations of 1, 10 and 50 mg/mL, respectively. As illustrated in Fig. 1A, treatment of SH-SY5Y cells with PrB extract (1–250 mg dry extract/mL) signif- icantly reduced the cell death caused by H2O2 in a non-concentration-dependent fashion. No difference APP metabolism is not affected by PrB was seen in cell viability between cells treated with APP770 PrB alone and controls (data not shown). Moreover, When CHO cells were treated with PrB for 48 h, co-incubation of SH-SY5Y cells with PrB extract and no alterations were observed either on sAPPa/b levels secreted into their conditioned media (Fig. 5A, B), or H2O2 not only completely abolished H2O2-induced ROS accumulation but further reduced ROS levels on cellular APP expression (Fig. 5C), as evidenced by by 57–63% compared with the untreated cells. western blot, using b-actin as an internal control.

Inhibition of Αb aggregation in vitro by PrB PrB confers additional toxicity in H2O2-treated HEK293 cells The effect of PrB on the Ab1-40 propensity to form aggregates was studied by the standard ThT-based Co-treatment of HEK293 cells with the PrB extract and fluorescence assay. The PrB extract significantly inhibited H2O2 (250 mM) further reduced the viability of HEK293 Ab-aggregation in vitro in a time-dependent manner – cells by 12 35% in comparison with the H2O2-treated (Fig. 6). Notably, the inhibitory effect at the lowest cells (Fig. 2A). In addition, the ROS levels were signif- concentration (15 mg dry extract/mL) was 67% and 62%, icantly higher (28.4–146.0%) in the presence of PrB at 5 and 30 days, respectively. and H2O2 in comparison with H2O2 alone (Fig. 2B).

APP770 DISCUSSION Antioxidant effects of PrB in CHO cells

The PrB treatment significantly reduced the cell death Hydrogen peroxide has been used extensively as an APP770 of CHO cells treated with H2O2 only at the highest inducer of oxidative stress in in vitro models. Co- applied concentrations of PrB (125 and 250 mg/mL) with treatment of cells with H2O2 and PrB showed that the a concomitant low (7–9%) but statistically significant PrB effects varied among the types of cell lines tested decrease in ROS levels in comparison with H2O2-treated (Figs 1–3). In particular, in the human neuroblastoma cells (Fig. 3). However, at concentrations of 1–50 mg/mL SH-SY5Y cell line, the PrB extract reduced H2O2- of PrB in the presence of 250 mM H2O2, the ROS levels induced cell death and generated ROS, to levels lower were significantly higher than those of the H2O2-treated than those of the untreated cells; whereas in the

Figure 1. SHSY5Y viability and ROS levels. Cells were co-treated with 250 mM H2O2 and PrB (1–250 mg dry extract/mL), for 18 h, and both cell viability (A) and ROS levels produced (B) were measured by MTT and DCF assay, respectively. *p ≤ 0.05 vs H2O2-treated cells. Percentages indicate the percentage decrease observed after incubation with PrB in the presence of H2O2. There was a statistically significant difference (p ≤ 0.05) in viability and ROS levels of all treated cells from control untreated cells.

Copyright © 2011 John Wiley & Sons, Ltd. Phytother. Res. 26: 956–963 (2012) 960 M. A. PAPANDREOU ET AL.

Figure 2. HEK293 viability and ROS levels. Cells were co-treated with 250 mM H2O2 and PrB (1–250 mg dry extract/mL), for 18 h, and both cell viability (A) and ROS levels produced (B) were measured by MTT and DCF assay, respectively. *p ≤ 0.05 vs H2O2-treated cells. Percentage indicates the percentage of cell death in the presence of the H2O2. There was a statistically significant difference (p ≤ 0.05) in viability and ROS levels of all treated cells from control untreated cells.

APP770 Figure 3. CHO viability and ROS levels. Cells were co-treated with 250 mM H2O2 and PrB (1–250 mg dry extract/mL), for 18 h, and both cell viability (A) and ROS levels produced (B) were measured by MTTand DCF assay, respectively. *p ≤ 0.05 vs H2O2-treated cells. 1p ≤ 0.05 vs untreated control.

polyphenols protected primary hippocampal neuronal cells (HNCs) against Ab-induced stress (Joseph et al., 2010). In our experiment, when CHOAPP770 cells were additionally stressed with H2O2, PrB further increased ROS levels (the higher the concentration, the lower the increase), while cell viability was enhanced in comparison with H2O2-treated cells, only at the highest applied concentrations. The current findings of PrB- induced increases in ROS levels in H2O2-stressed CHOAPP770 and HEK293 cells are in line with the recently published data by Joseph and his co-workers (Joseph et al., 2010), whereby treatment of HNC cells with whole blueberry extract or certain fractions of it (e.g. proanthocyanidins, POST-C18 and to some extent PRE-C18), resulted in increases in ROS levels, even in the absence of the stressors (i.e. dopamine, LPS or Ab42). Figure 4. Effect of PrB extract on lipid peroxidation (MDA) levels. This type of polyphenol-induced cytotoxicity is not CHOAPP770 cells were incubated with PrB (1–250 mg/mL) for 48 h. *p ≤ 0.05 vs untreated control, #p ≤ 0.05 vs wild CHO cells. uncommon in cell culture experiments and has been largely ascribed to the nature of the phenolic compounds, which can act either as pro-oxidants or as HEK293 cells it exacerbated H2O2 cytotoxicity. Since cytotoxic agents, in certain conditions (Sergediene et al., CHOAPP770 cells overproduce APP and Ab in turn, they 1999; Shamim et al., 2008). Recent findings have also can be considered as more stressed in comparison with implicated certain phenolic compounds (e.g. delphinidin) wild type cells; MDA measurements confirm this in the induction of H2O2, within commonly used cell phenomenon. Treatment of those cells with PrB culture media, possibly due to their unstable nature reduced the lipid peroxidation levels significantly. This and their ability to undergo chemical transformations, is in agreement with reports whereby blueberry resulting thus in the formation of by-products capable

Copyright © 2011 John Wiley & Sons, Ltd. Phytother. Res. 26: 956–963 (2012) ANTIOXIDANT AND ANTI-AMYLOID EFFECTS OF BLUEBERRY POLYPHENOLS 961

Figure 5. Effect of PrB extract on secretion of soluble amyloid precursor protein (sAPP) a-(A)andb- (B), and on whole APP (C) in CHOAPP770 cells.

of distinct cell surface receptors, cellular membrane composition and intracellular retention transport. For example, although quercetin-3-glucoside protects both SH-SY5Y and HEK293 cells against H2O2-induced viability loss, the underlying mechanisms are not the same: it induces sterol regulatory element-binding protein-2-mediated cholesterol biosynthesis only in SH-SY5Y cells (Soundararajan et al., 2008). Oxidative stress and ROS accumulation, apart from their implication in cellular damage, are also involved, at least in part, in the toxicity of Ab-peptide and its aggregation/accumulation (Miranda et al., 2000), and thus, with the pathogenesis of neurodegeneration in AD. The active form of b-amyloid consists of soluble – Figure 6. In vitro inhibition of Ab1-40 fibrillogenesis in the presence oligomeric fragments, composed of 39 43 amino acids; of various concentrations of PrB extract at 5 and 30 days, as assayed Ab1-40 and Ab1-42, are the predominant forms. The with the thioflavine T fluorescence assay. The fluorescence absor- latter peptidergic fragments are derived from the bance of aged Ab1-40 alone was considered as 100%. PrB exhibited proteolytic processing of the b-amyloid precursor statistically significant (p ≤ 0.05) inhibition of Ab1-40 fibrillogenesis at all concentrations. protein (APP) via two pathways, each resulting in distinct cleavage products: (1) a non-amyloidogenic which involves cleavage of APP to soluble APP (sAPP) of causing cell-specifictoxicity(Longet al., 2010). In by the a-secretase; and (b) a formation of amyloidogenic our experiments, however, treatment of cells only with b peptides by the b- and g-secretases. As the proportion PrB did not affect viability, as assessed by MTT, of APP processed by b-secretase vs a-secretase may although it is more difficult to delineate on the changes affect the amount of Ab produced, regulation of these of the phenolic compounds contained within the extract, two pathways may be critically important to the patho- due to the experimental procedure followed, i.e. co- genesis of AD. APP770 administration with H2O2. The examination of the PrB extract on CHO In addition, the lack of dose-dependency observed cells, stably transfected to express human APP, revealed in either cell-viability or ROS production after co- that the tested phytochemical had no effect on APP treatment of all three cell lines in the particular con- metabolism, as evidenced by western blot analysis centration range is not uncommon in studies of natural (Fig. 5). The former observation might provide an products. For instance, treatment of normal epithelial explanation for previous experiments performed by cells and cells derived from the salivary gland with Joseph et al. (2003) showing no effect on Ab peptide epigallocatechin gallate, resulted in a dose-independent production, deposition or amyloid load on transgenic effect in ROS production (Yamamoto et al., 2003). PS1-APP mice brains, after supplementation with blue- The fact that PrB is an extract with many different berries extracts (2% of diet) from 4 to 12 months of polyphenol entities, which act by a variety of mecha- age. Similar results were also observed after treatment nisms, implicates the principles of synergy, antago- of both transfected murine N2A (Swedish mutant form nism and pleiotropy in explaining this phenomenon. of APP, SweAPP N2a) and primary neuronal cells Taking all these data into consideration, it is con- derived from transgenic APPsw mice with the catechins, cluded that the PrB extract selectively protected ()-epigallocatechin and ()-epicatechin (Rezai-Zadeh SH-SY5Y cell viability from H2O2 stress and signifi- et al., 2005). cantly decreased ROS levels, in contrast to HEK293 However, the PrB extract significantly inhibited cells and partly CHOAPP770 cells. The observed differ- in vitro Ab-fibrillogenesis (up to 78.79 3.63%, at a ences in response could be ascribed to the existence concentration of 0.1 mg/mL, after 5 days of incubation)

Copyright © 2011 John Wiley & Sons, Ltd. Phytother. Res. 26: 956–963 (2012) 962 M. A. PAPANDREOU ET AL.

(Fig. 6) as evidenced by the thioflavin T assay. Inhibition protected SH-SY5Y at all concentrations tested and APP770 of Ab1-42 aggregation by blueberry polyphenols was also CHO only at high concentrations. On the reported by Zhu et al. (2008) and Fuentealba et al. contrary, co-treatment of HEK293 with H2O2 and (2011), whereas Guo et al. (2010) have reported a PrB exacerbated further the oxidative damage to cells. marginal inhibitory effect using two different in vitro Beyond antioxidant effects in CHOAPP770 cells (it tests. Silver staining after gel electrophoresis showed lowered MDA levels in untreated CHOAPP770 cells that a blueberry-enriched polyphenol extract signifi- compared with wild CHO cells), PrB extract did not cantly inhibited the formation of small aggregates (e.g. induce changes in APP processing. These findings 10, 40 kDa), which are considered to be toxic (Fuentealba contribute significantly to the ongoing search for com- et al., 2011). Considering the in vitro results and the results pounds with pleiotropic actions against Alzheimer’s in transgenic PS1 + APP rodents (Joseph et al., 2003), it disease and in particular to study of the neuroprotective seems that although in vitro tests document antiaggregant properties of blueberries. properties of blueberry polyphenols, these might not be enough to prevent amyloid aggregation in vivo or might be more potent against the formation of small soluble aggregates (not observed with the dye techniques usually Acknowledgement used for visualization of amyloid load). Questions on the metabolism, bioavailability in humans and the ability to The authors would like to kindly acknowledge Professor Leonidas prevent amyloid deposits at the onset of the disease Stefanis from Biomedical Research Foundation of the Academy of Athens (BRFAA) for providing the human neuroblastoma SH-SY5Y remain to be answered. APP770 In conclusion, our results demonstrated that the PrB and stably transfected Chinese Hamster Ovary CHO cell lines and the Wild Blueberry Association of North America for providing extract of wild V. angustifolium protect or further the wild blueberry powder. damage cells against H2O2-induced oxidative stress according to the cellular environment and characteristics of the particular cell line investigated. Measurements of cell viability and scavenging of ROS production Conflict of Interest after co-treatment with Η2Ο2 and various concentra- tions of PrB showed that the PrB extract significantly The authors have declared that there is no conflict of interest.

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Copyright © 2011 John Wiley & Sons, Ltd. Phytother. Res. 26: 956–963 (2012) Phytochemical composition of “mountain tea” from Sideritis clandestina subsp. clandestina and evaluation of its behavioral and oxidant/antioxidant effects on adult mice Catherine G. Vasilopoulou, Vassiliki G. Kontogianni, Zacharoula I. Linardaki, Gregoris Iatrou, Fotini N. Lamari, Alexandra A. Nerantzaki, et al.

European Journal of Nutrition

ISSN 1436-6207

Eur J Nutr DOI 10.1007/s00394-011-0292-2

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1 23 Author's personal copy

Eur J Nutr DOI 10.1007/s00394-011-0292-2

ORIGINAL CONTRIBUTION

Phytochemical composition of ‘‘mountain tea’’ from Sideritis clandestina subsp. clandestina and evaluation of its behavioral and oxidant/antioxidant effects on adult mice

Catherine G. Vasilopoulou • Vassiliki G. Kontogianni • Zacharoula I. Linardaki • Gregoris Iatrou • Fotini N. Lamari • Alexandra A. Nerantzaki • Ioannis P. Gerothanassis • Andreas G. Tzakos • Marigoula Margarity

Received: 20 September 2011 / Accepted: 5 December 2011 Ó Springer-Verlag 2011

Abstract in mice were studied by the assessment of their thigmo- Purpose The goals of this study were to monitor the tactic behavior. The oxidant/antioxidant status of brain effect of drinking of herbal tea from Sideritis clandestina (-Ce), liver and heart of adult male Balb-c mice following subsp. clandestina for 6 weeks on behavioral and oxidant/ the consumption of Sideritis tea was also evaluated via the antioxidant parameters of adult male mice and also to measurement of malondialdehyde (MDA) and reduced evaluate its phytochemical composition. glutathione (GSH) levels using fluorometric assays. Our Methods The phytochemical profile of the Sideritis tea study was further extended to determine the antioxidant was determined by liquid chromatography-UV diode array effects of the herbal tea on specific brain regions (cerebral coupled to ion-trap mass spectrometry with electrospray cortex, cerebellum and midbrain). ionization interface. The effects of two doses of the herbal Results The identified compounds were classified into sev- infusion (2 and 4% w/v, daily) intake on anxiety-like state eral natural product classes: quinic acid derivatives, iridoids, phenylethanol glycosides and flavonoids. Our results showed that only the 4% Sideritis tea exhibited anxiolytic-like prop- Catherine G. Vasilopoulou and Vassiliki G. Kontogianni contributed equally to this work. erties as evidenced by statistically significant (p \ 0.05) decrease in the thigmotaxis time and increase in the number of Electronic supplementary material The online version of this entries to the central zone in comparison with the control article (doi:10.1007/s00394-011-0292-2) contains supplementary group. Consumption of both tea doses (2 and 4% w/v) elevated material, which is available to authorized users. GSH (12 and 28%, respectively, p \ 0.05) and decreased C. G. Vasilopoulou Z. I. Linardaki M. Margarity (&) MDA (16 and 29%, p \ 0.05) levels in brain (-Ce), while liver Laboratory of Human and Animal Physiology, and heart remained unaffected. In regard to the effect of herbal Department of Biology, University of Patras, 26504 Rio, Greece tea drinking (2 and 4% w/v) on specific brain regions, it caused e-mail: [email protected] a significant increase in GSH of cerebellum (13 and 36%, V. G. Kontogianni A. A. Nerantzaki I. P. Gerothanassis respectively, p \ 0.05) and midbrain (17 and 36%, p \ 0.05). Department of Chemistry, Section of Organic Chemistry and Similarly, MDA levels were decreased in cerebellum (45 and Biochemistry, University of Ioannina, 45110 Ioannina, Greece 79%, respectively, p \ 0.05) and midbrain (50 and 63%, G. Iatrou respectively, p \ 0.05), whereas cerebral cortex remained Department of Biology, University of Patras, 26504 Rio, Greece unaffected. Conclusions Mountain tea drinking prevents anxiety-rela- F. N. Lamari ted behaviors and confers antioxidant protection to rodent’s Department of Pharmacy, University of Patras, 26504 Rio, Greece tissues in a region-specific, dose-dependent manner, and its phytochemical constituents are shown for the first time. A. G. Tzakos (&) Human Cancer Biobank Center, Department of Chemistry, Keywords LC-MS/MS Mountain tea Sideritis Section of Organic Chemistry and Biochemistry, University of Ioannina, 45110 Ioannina, Greece clandestina subsp. clandestina Oxidant/antioxidant e-mail: [email protected] parameters Thigmotaxis test 123 Author's personal copy

Eur J Nutr

Abbreviations for 50 days were protected against chemical-induced oxi- MDA Malondialdehyde dative injury [18]. We have previously found that con- GSH Reduced glutathione sumption of a herbal tea from a different Sideritis taxon TBA Thiobarbituric acid (clandestina subsp. peloponnesiaca) enhanced the antiox- BHT Butylated hydroxytoluene idant defense of the adult rodent brain in a region-specific TCA Trichloroacetic acid manner; however, the phytochemical composition remained LC/DAD/ESI-MSn Liquid chromatography/diode array unknown [19]. detection/electrospray ion-trap The aims of the present study are (1) to characterize the tandem mass spectrometry composition of the aqueous extract of the herbal tea from NMR Nuclear magnetic resonance Sideritis clandestina subsp. clandestina using LC/DAD/ ESI-MSn analysis, (2) to evaluate the effect of a 6-week consumption of 2 and 4% w/v of a herbal tea (commonly consumed tea doses) from the same taxon on the levels of anxiety and (3) to determine the oxidant/antioxidant Introduction activity of peripheral and brain tissues of adult mice by measuring malondialdehyde (MDA, index of lipid peroxi- The neurotoxic effects of oxidative stress and the conse- dation) and reduced glutathione (GSH). This is the first quent neurodegeneration in specific brain areas have been study on the phytochemical composition of S. clandestina proposed as causal factors in Alzheimer’s disease, Par- subs. clandestina ever, the first study on the composition of kinsonism and aging process [1] The brain is the most the aqueous and not organic solvent extract of the herbal susceptible organ to oxidative damage due to its high tea from this taxon and one of the few studies to investigate oxygen demand, high lipid content, especially polyunsat- its antioxidant effects in vivo after a period of drinking. urated fatty acids, the abundance of redox-active transition With regard to the latter, we aimed to investigate whether metal ions, the low activity of antioxidant defense system the beneficiary effects, recorded after the consumption of and the reduced capacity for cellular regeneration [1]. another Sideritis taxon [19], would present a similar pattern Polyphenolic compounds supplied by nutritional sources or are taxon specific and whether these are dose dependent. [2] such as fruits, vegetables, wine and tea have displayed a wide range of beneficial biological actions [3], many of which have been attributed to their strong antioxidant Materials and methods properties [4]. Anxiety is a psychological and physiological state Plant material, reagents and standards characterized by somatic, emotional, cognitive and behavioral components. Recent studies demonstrate a link The plant Sideritis clandestina subsp. clandestina used in between oxidative stress and anxiety-related behavior [5]. the study was collected from Mainalo mountain (Central Some of these studies suggest that oxidative stress causes Peloponnese) between July and August (2008). Taxonomic anxiety-related behaviors in humans with panic disorders identification of the plant material was carried out in the and especially that oxidative metabolism can affect the Division of Plant Biology, Department of Biology, Uni- regulation of anxiety [6], and others support a cause–effect versity of Patras, by Prof. G. Iatrou, and a voucher speci- relationship in humans and animal models but do not men has been deposited in the local Herbarium. explain the underlying mechanisms [7]. MDA, GSH, TBA, BHT, TCA, o-phthalaldehyde and The genus Sideritis (Lamiaceae) comprises about 140 Chromasolv Plus acetonitrile and water for HPLC (99.9%) species distributed in several countries of the Mediterra- were obtained from Sigma–Aldrich. Analytical-grade ace- nean region [8]. Sideritis species have been reported to tic acid was provided by Merck (Darmstadt, Germany), and present an array of biological activities, including anti- quinic acid (98%) was obtained from Aldrich (Steinhem, inflammatory and antinociceptive [9], antimicrobial [10] Germany). and antiulcer [11] activities. Interestingly, aqueous extracts derived from Sideritis species have been proposed to form Sample preparation and extraction the basis to design ‘‘functional foods’’ for the prevention of osteoporosis [12]. Furthermore, in vitro studies have shown All parts of dry material (flowers, leaves and stems) in moderate antioxidant activity for S. javalambrensis, equal quantities were used for the extraction. For the S. raeseri, S. euboea, S. perfoliata subsps. perfoliata and preparation of the tea infusions, 2 or 4 g of leaves, flowers S. libanotica ssp. linearis that was mainly attributed to and stems of Sideritis taxa was extracted with 100 mL of flavonoids [13–17]. Rats drinking a tea from S. caesarea boiling water for 5 min, in order to simulate actual brewing 123 Author's personal copy

Eur J Nutr conditions for tea consumed by human adults, and filtered Assignment of compounds present in the extract was after cooling. The filtrate was made to the volume of determined on the basis of 2D 1H–1H COSY, 1H–13C 100 mL. For the animal studies, the infusions were pre- HSQC and HMBC spectra. Relevant compound quantifi- pared daily. For the characterization of the phytochemical cation was performed on the basis of the recorded integrals composition of the herbal tea, it was lyophilized and a of resonance absorptions that are characteristic for specific residue (nice green powder) of 0.294 g (for the 2 g) was compounds in the 1D 1H-NMR spectrum of the extract. obtained. Animals LC-MS analysis Male, 3- to 4-month-old Balb-c mice (25–30 g BW) were To evaluate the phytochemical composition of the aqueous kept in polyacrylic cages (38 cm 9 23 cm 9 10 cm) (8 Sideritis extract, we used liquid chromatography-UV diode per cage) under constant temperature (23–25 °C) and rel- array coupled to ion-trap mass spectrometry with electro- ative 50–60% humidity, with alternating 12-h light and spray ionization interface (LC/DAD/ESI-MSn). All LC- dark cycles and ad libitum access to food. The mice were MSn experiments were performed on a quadrupole ion-trap randomly divided into three groups consisting of sixteen mass analyzer (Agilent Technologies, model MSD trap SL) animals each. Group I mice (n = 16) served as controls retrofitted to a 1,100 binary HPLC system equipped with a and received water ad libitum. Group II and III mice degasser, autosampler, diode array detector and electro- (n = 16/group) had ad libitum access to the tea from spray ionization source (Agilent Technologies, Karlsruhe, S. clandestina subsp. clandestina 2 and 4% (w/v), respec- Germany). All hardware components were controlled by tively, for a period of 6 weeks. Each of the above groups Agilent Chemstation Software. was divided into two subgroups (n = 8/subgroup), which The Sideritis extract was dissolved in acetonitrile–water were used for the isolation of a. brain, liver, heart and b. 50–50% to the desired concentration (4 mg mL-1 of specific brain tissues (cerebral cortex, cerebellum and extract). A 20-lL aliquot was filtered (0.45 lm) and midbrain), respectively. The body weight, food and water injected into the LC-MS instrument. Separation was or infusion intake were measured weekly in all the exper- achieved on a 25 cm 9 4.6 mm i.d., 5 lm Altima C18 imental groups throughout the treatment period. All pro- analytical column (Alltech, Deerfield, USA), at a flow rate cedures were in accordance with the Greek National Laws of 0.6 mL min-1, using solvent A (water/acetic acid, 99.9: (Animal Act, PD 160/91). 0.1 v/v) and solvent B (acetonitrile). The gradient used in this experiment was as follows: 0–5 min, 95–92% A; Thigmotaxis test 5–15 min 92% A; 15–20 min 92–75% A; 20–40 min 75–50% A; 40–50 min 50–20% A; 50–60 min 20–95% A. At the end of the treatment period, animals were submitted The UV/Vis spectra were recorded in the range of to the open-field behavioral thigmotaxis test in order to 200–400 nm, and chromatograms were acquired at 254, evaluate the effects of herbal tea consumption on anxiety- 280 and 330 nm. related responses [20]. Thigmotaxis (walking close the Both precursor and product (MS2 and MS3) ions scan- walls of the apparatus), which was assessed by the time that ning of the phytochemicals were monitored between m/z 50 mice spent close to the walls (\8 cm away from the walls), and m/z 1,000 in negative polarity. The ionization source and the number of entries in the central zone of the open conditions were as follows: capillary voltage, 3.5 kV; field are considered as an index of anxiety. All training and drying gas temperature, 350 °C; nitrogen flow and pres- testing sessions were carried out during the light phase sure, 12 L min-1 and 12 psi, respectively. Maximum between 08:00 and 14:00 a.m. accumulation time of ion trap and the number of MS rep- etitions to obtain the MS average spectra were set at 30 ms Tissue preparation and 3, respectively. One day after the behavioral test, all mice were killed by NMR analysis light ether anesthesia and brain (-Cerebellum) [referred as brain (-Ce)], liver, heart and specific brain regions (cerebral Nuclear magnetic resonance (NMR) experiments were cortex, cerebellum and midbrain) were rapidly removed, performed at 298 K on a Bruker AV-500 spectrometer weighted and stored at -80 °C until use. All tissues were equipped with a TXI cryoprobe (Bruker BioSpin, Rhein- homogenized (10% w/v) using a glass-Teflon homogenizer stetten, Germany). Samples were dissolved in 0.5 mL (Thomas, Philadelphia, USA, No B 13957) in ice-cold

DMSO-d6 and transferred to 5-mm NMR tubes. The NMR 30 mM Na2HPO4, pH 7.6. The homogenates were then system was controlled by the software TopSpin 2.1. centrifuged for 20 min at 15.000g at 4 °C in a Heal Force, 123 Author's personal copy

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Neofuge 13R centrifuge. Supernatants were collected and treated group from control and between the two treated used for the determination of GSH and MDA levels. Pro- groups. tein levels were evaluated using the Bradford assay.

Determination of MDA Results

MDA, one of the better-known secondary products of lipid Phytochemical profile and identification of the main peroxidation, was determined fluorometrically after reac- constituents of aqueous Sideritis extract tion with thiobarbituric acid (TBA), as previously descri- bed [21]. The assay showed good linearity (0.05–10 lL, The total ion chromatogram of the investigated extract is y = 6.6034 9-0.5959, R2 = 0.9993) and sensitivity. The illustrated in Fig. 1. The LC/DAD/ESI-MSn analysis of the level of MDA was expressed as lmol/g of tissue protein. aqueous extract led to the separation and identification of All determinations were carried out at least three times and the majority of the extract components, seventeen (17) in in triplicates. total, that belong to several classes of phytochemicals: quinic acid, melittoside, phenylpropanoids and flavonoid Determination of GSH derivatives. Specifically, two quinic acid derivatives along with two melittoside derivatives, two phenylethanoid gly- GSH content of the brain and peripheral tissues was esti- cosides (b-hydroxyverbascoside or b-hydroxyisoverbasco- mated fluorometrically after reaction with o-phthalalde- side is described for the first time in Sideritis species) and hyde, as previously described [22]. A good linearity one flavonoid 7-O-diglycoside were identified along with was obtained for GSH in the range of 0.5–100 lM six acetylated flavonoid 7-O-diglycosides of and (y = 1.2353 9-3.46, R2 = 0.9969). The levels of GSH isoscutellarein and four isomers of apigenin 7-O-(couma- were expressed as lmol/g of tissue protein. All determi- royl)glucopyranoside (See Table 1 for a detailed list of nations were carried out at least three times and in compounds and Figures S1–S9 for representative MS triplicates. spectra of selected compounds). The identification of the compounds was based on their Statistical analysis fragmentation pattern as has been documented in the lit- erature [23–29]. On the basis of this methodology, we were The results are expressed as mean ± SE. Statistical anal- not only able to determine the presence of well-known ysis was performed with the GraphPad Instat 3 software phenolic compounds in the extract, but also able to identify using the nonparametric Mann–Whitney test for evaluating the two iridoid glycosides and quinic acid derivatives that statistically significant differences (p \ 0.05) of each could not be attributed to any known compound.

Fig. 1 Total ion chromatogram of Sideritis clandestina aqueous extract acetyl]-allosyl(1 ? 2)glucoside, 10 Martynoside, 11 Isoscutellarein 1 Quinic acid derivative, 2 Quinic acid derivative, 3 Melittoside 7-O-[2000,6000-O-diacetyl]-allosyl(1 ? 2)glucoside, 12 Apigenin 7-O- derivative, 4 Melittoside derivative, 5 b-Hydroxyverbascoside or [2000,6000-O-diacetyl]-allosyl(1 ? 2)glucoside, 13 Apigenin 7-O-acetyl- b-Hydroxyisoverbascoside, 6 Apigenin 7-O-allosyl(1 ? 2)glucoside, coumaroyl-allosyl(1 ? 2)glucoside, 14–17 Apigenin 7-O-(couma- 7 Isoscutelarein 7-O-[6000-O-acetyl]-allosyl(1 ? 2)glucoside, 8 Apige- royl)glucopyranoside isomers nin 7-O-[6000-O-acetyl]-allosyl(1 ? 2)glucoside, 9 Apigenin 7-O-[600-O-

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Table 1 Compounds identified in Sideritis clandestina aqueous extract

- 2 - 3 - Peak Rt (min) [M–H] (m/z) –MS [M–H] (m/z) (%) –MS [base peak] (m/z) (%) Compounds

1 4.2 421 191 (100) 389 (100), 127 (17), 173 (14) Quinic acid derivative 2 4.2 533 191 (100) 173 (100), 127 (64), 150 (67) Quinic acid derivative 3 5.9 654, 583, 523 523 (100), 179(6), 565 (8) 179 (100), 361 (30), 343 (24), 463 (11) Melittoside derivative 4 11.9 583, 523, 665 523 (100), 565 (17), 179 (8) 179 (100), 343 (29), 361 (24), 463 (14) Melittoside derivative 10 26.7 449 389 (100), 167 (16), 329 (11) 329 (100), 161 (30), 179 (15), 251 (11) Unknown 5 27.1 639 621 (100), 459 (8) 459 (100), 179 (13) b-Hydroxyverbascoside or b-hydroxyisoverbascoside 20 28.8 579 339 (100), 327 (44) 324 (100), 161 (30), 179 (15) Unknown 6 29.3 593 269 (100), 431 (21) 269 (100) Apigenin 7-O-allosyl(1 ? 2) glucoside, 7 29.8 651 609 (100), 285 (4) 285 (100), 447 (28) Isoscutelarein 7-O-[6000-O-acetyl]- allosyl(1 ? 2)glucoside 8 30.7 635 593 (100), 269 (7) 269 (100), 431 (24) Apigenin 7-O-[6000-O-acetyl]- allosyl(1 ? 2)glucoside 9 31.9 431, 635 269 (100), 593 (51), 269 (100), 431 (26) Apigenin 7-O-[600-O-acetyl]- 515 (8), 431 (7) allosyl(1 ? 2)glucoside 30 32.1 783, 665 607 (100), 651 (53), 737 (41) 329 (100) Unknown 10 33.6 651 475 (100), 505 (31), 457 (18) 161 (100), 329 (84) Martynoside 11 34.1 693 651 (100), 633 (59) 285 (100), 489 (58), 609 (14) Isoscutellarein 7-O-[2000,6000-O- diacetyl]-allosyl(1 ? 2) glucoside 40 34.5 595 269 (100), 474 974), 513 (19) 269 (100) Unknown 50 35.4 733 698 (100), 714 (92), 634 (39) 714 (100), 673 (59) Unknown 12 36.5 677 635 (100), 269 (63) 269 (100), 593 (31), 473 (67) Apigenin 7-O-[2000,6000-O- diacetyl]-allosyl(1 ? 2) glucoside 13 37.6 781 739 (100), 721 (69), 269 (100), 593 (64), 577 (16), 431 (14) Apigenin 7-O-acetyl-coumaroyl- 269 (53), 635 (40), 575 (12) allosyl(1 ? 2)glucoside 14 38.7 577 269 (100), 431 (1), 413 (1) 225 (100), 269 (71), 149 (59), 117 (26) Apigenin 7-O-(coumaroyl) glucopyranoside 15 39.2 577 269 (100) 225 (60), 269 (100), 149 (43) Apigenin 7-O-(coumaroyl) glucopyranoside 16 40.4 577 269 (100) 225 (100), 269 (64), 149 (43) Apigenin 7-O-(coumaroyl) glucopyranoside 17 42.5 577 269 (100), 431 (15), 413 (15) 225 (42), 269 (43), 149 (100) Apigenin 7-O-(coumaroyl) glucopyranoside

Specifically, the unknown iridoid glycosides were com- while recently a new melittoside derivative was isolated pounds 3 and 4 (relevant peak fractions in Fig. 1). Com- from an extract of the species Sideritis lanata L.[31]. pound 3 gave deprotonated molecular ions [M–H]- at m/z Similarly, compound 4 eluting at 11.9 min with isocratic 654 (100%), 583 (90%), 613 (76%) and 523 (67%) (Figure conditions gave deprotonated molecular ions [M–H]- at S3). The fragmentation at m/z 583 showed the presence of m/z 583 (100%) and 523 (64%) (Figure S4). The fragmen- one main fragment at m/z 523 (100%), 565 (8%) and 179 tation at m/z 583 showed the presence of one main frag- (6%). The MS3 spectra of the ion at m/z 523 gave ions at ment at m/z 523 (100%), 565 (17%) and 179 (8%) again, m/z 179 (100%), 361 (30%), 343 (24%), 463 (11%) and 161 while the MS3 spectra of the ion at m/z 523 gave ions at m/z (11%). The ion at m/z 523 could belong to melittoside, and 179 (100%), 343 (29%), 361 (25%), 463 (14%), 439 (10%) compound 3 could be attributed to a melittoside derivative. and 161 (9%). Compounds 3 and 4 must have related In the MS3 spectra, the ion at m/z 361 corresponds to the structures, as they show similar product ions. loss of 162 amu (glucose) and the ion at m/z 179 corre- In order to ascertain the structure of compound 3, as it sponds to the loss of 182 amu, which are characteristic was the main constituent in the extract and since there is neutral losses for iridoid glycosides [25]. Melittoside has lack of available literature data regarding the fragmentation been isolated from different species of genus Sideritis [30], pattern of melittoside and its derivatives, we isolate it

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Eur J Nutr through preparative liquid chromatography. NMR investi- with the controls. The first 5 min of the testing session in gation (2D 1H–1H COSY, 1H–13C HSQC and 1H–13C both treated animal groups displayed no difference in the HMBC) of the isolated compound confirmed that the base values of the above parameters when compared with the configuration was melittoside (Table S1), in accordance control group, as this time interval is a period of accom- with literature data [32]. The full assignment is not com- modation [20]. pleted due to extensive resonance overlapping problems. Furthermore, the compound apigenin 7-O-acetyl- Evaluation of the antioxidant effects of Sideritis coumaroyl-allosyl(1 ? 2)glucoside (compound 13) was infusion intake on adult mice brain and peripheral identified and was characterized in the extract for the first tissues time. The deprotonated molecular ion [M–H]- of com- pound 13 was detected at m/z 781 (Figure S9). After MS2 Differences in the MDA and GSH levels among brain (-Ce) of the compound, the base peak at m/z 739 (100%) was and peripheral tissues as well as among the three brain detected corresponding to the loss of an acetyl group, along regions were observed in control mice. These differences with peaks at m/z 721 (69%), 269 (53%), 635 (40%) and are in accordance with previous studies [19, 33] and show 575 (12%). The MS3 spectra of the ion at m/z 721 yielded that tissues exhibit a significant variation in the distribution the ion at m/z 269 (100%), 593 (64%), 577 (16%) and 431 of antioxidant defense [34] being closely related to the (14%). The peak at m/z 593 was probably derived from the composition and the functional role of each tissue. loss of a coumaroyl or rhamnosyl moiety (146 amu). The As illustrated in Table 2, the consumption of both doses fragment at m/z 431 (14%) related to [M–H-308]- was (2 and 4%, w/v) of Sideritis herbal tea caused a significant derived from the loss of a glucose esterified with coumaric decrease in MDA levels (16 and 29%, respectively) and an acid. The base peak [(M–H)-324]- that was detected enhancement in the GSH levels (12 and 28%, respectively) indicated an O-glycosylation on a phenolic hydroxyl with a in the brain (-Ce) in comparison with the control group, 7 - dihexoside ([Y0] )[35]. The occurrence of loss of 162 amu whereas liver and heart remained unaffected. Moreover, (m/z 577, 16%) was indicative of 1 ? 2 glycosylation Sideritis-treated group that received the higher tea dose between both sugars. This compound could be tentatively (4% w/v) exhibited significant further alterations in the characterized as apigenin 7-O-acetyl-coumaroyl-allo- syl(1 ? 2)glucoside, and it is detected for the first time. Among the aforementioned compounds, the main constit- uents in the extract were found to be melittoside and quinic acid derivatives (Fig. 1). This was evident from the rele- vant integrals of characteristic resonance absorptions for the specific compounds in the 1H-NMR spectrum of the crude herbal tea (Figure S10).

Evaluation of the anxiolytic-like effects of Sideritis infusion intake on adult mice

Due to the well-studied beneficial effects in human health of the basic structures of the main phytochemical constit- uents revealed in the studied extract, we could hypothesize that the Sideritis clandestina subsp. clandestina extract could also confer anxiolytic behavioral effects and also antioxidant defence in brain. Figure 2a and b illustrates the measurement of the thigmotaxis time and the number of central entries to the open field at 5-min time intervals during the 30-min testing session (except of the first 5 min). From this, it became evident that mice receiving 4% (w/v) herbal tea displayed statistically significant Fig. 2 Evaluation of a the thigmotaxis time and b number of entries decrease (10–13%) in the thigmotaxis time and increase in the central area during the first 30 min in the open field. (70–180%) in the number of entries in the central zone Immediately after the introduction of naive mice into the open field compared with the control group. Similar but not statisti- of thigmotaxis test, the thigmotaxis time and number of central entries were measured during six consecutive periods of 5 min each. Data are cally significant alterations in the above parameters were means ± SE. of 8 mice per group. *Statistical difference between observed in Sideritis 2% (w/v)-treated group in comparison control and Sideritis 4%-treated mice 123 Author's personal copy

Eur J Nutr above oxidant/antioxidant indices of the brain (-Ce) in comparison with the control group. Neither of two tea comparison with the treated mice that received the lower doses affected the GSH content in the cerebral cortex. dose (2% w/v). Regular measurement of animal liquid and food intake Taking into consideration that brain was the only tissue, exhibited no difference between the treated animal groups which was influenced by the Sideritis tea intake, we further and the controls. Similarly, there was no difference in extended our study to determine the antioxidant effects of animal body weight between control and experimental the herbal tea on specific brain regions, such as the cerebral group throughout the 6-week treatment period. cortex, cerebellum and midbrain. Our results showed that drinking of the herbal tea from S. clandestina subsp. clandestina for 6 weeks affected significantly the oxidant/ Discussion antioxidant status of mice brain regions in a region- and dose-dependent manner. Particularly, as presented in The current study confirmed that drinking of the herbal tea Table 3, intake of both doses of the herbal tea decreased from S. clandestina subsp. clandestina for 6 weeks affected significantly the MDA levels of cerebellum (45 and 79%, significantly the oxidant/antioxidant status of mice brain respectively) and midbrain (50 and 63%, respectively), in regions in a region- and dose-dependent manner. Specifi- comparison with the control mice, whereas cerebral cortex cally, the significant alterations in MDA and GSH levels of MDA levels remained unaffected. The consumption of the the mice brain that accompanied the herbal tea intake high dose (4%) of Sideritis infusion caused significant exhibited a region specificity as cerebellum and midbrain further reduction in midbrain and cerebellum lipid perox- were significantly affected in contrast to the cerebral cortex idation levels, as compared to the low dose (2%). Fur- that showed remarkable stability. Significant stability in thermore, consumption of the low and high herbal tea dose their oxidant/antioxidant status also displayed the liver and increased significantly the GSH levels of cerebellum by 13 the heart. Accordingly, in our previous study, mice drink- and 36% and of midbrain by 17 and 36%, respectively, in ing a herbal tea (4% w/v) from another Sideritis taxon,

Table 2 Effect of herbal tea consumption (2 and 4% [w/v]) on MDA and GSH levels of mice tissues Tissues lmol MDA/g protein lmol GSH/g protein Control mice Sideritis 2% Sideritis 4% Control mice Sideritis 2% Sideritis 4% w/v mice w/v mice w/v mice w/v mice

Brain (-Ce) 5.24 ± 0.17 4.42 ± 0.13* 3.74 ± 0.16*, 11.80 ± 0.47 13.18 ± 0.41* 14.90 ± 0.38*, (;16%) (;29%) [;15%] (:12%) (:28%) [:13%] Liver 1.59 ± 0.16 1.52 ± 0.09 1.48 ± 0.09 7.06 ± 0.28 6.91 ± 0.17 6.93 ± 0.19 Heart 1.19 ± 0.16 0.95 ± 0.09 0.96 ± 0.11 5.05 ± 0.28 5.11 ± 0.15 4.77 ± 0.28 Data are mean ± SE (n = 8). Statistical analysis was performed by Mann–Whitney test. Percentage decrease (;) of MDA values or increase (:) of GSH values as to the control group. Percentage decrease [;] of MDA values or increase [:] of GSH values as to the Sideritis 2%-treated group * Statistically significant difference between control and Sideritis-treated mice (p \ 0.05) Statistically significant difference between Sideritis 2% and Sideritis 4%-treated mice (p \ 0.05)

Table 3 Effect of herbal tea consumption (2 and 4% [w/v]) on MDA and GSH levels of mice brain regions Tissues lmol MDA/g protein lmol GSH/g protein Control mice Sideritis 2% Sideritis 4% Control mice Sideritis 2% Sideritis 4% w/v mice w/v mice w/v mice w/v mice

Cerebral cortex 3.13 ± 0.22 2.95 ± 0.09 2.70 ± 0.10 12.47 ± 0.42 12.21 ± 0.41 12.28 ± 0.61 Cerebellum 2.10 ± 0.13 1.15 ± 0.04* 0.43 ± 0.008*, 15.54 ± 0.70 17.52 ± 0.75* 21.07 ± 0.98*, (;45%) (;79%) [;62%] (:13%) (:36%) [:20%] Midbrain 1.45 ± 0.08 0.72 ± 0.035* 0.53 ± 0.04*, 13.95 ± 0.44 16.27 ± 0.43* 19.01 ± 0.86*, (;50%) (;63%) [;35%] (:17%) (:36%) [:17%] Data are mean ± SE (n = 8). Statistical analysis was performed by Mann–Whitney test. Percentage decrease (;) of MDA values or increase (:) of GSH values as to the control group. Percentage decrease [;] of MDA values or increase [:] of GSH values as to the Sideritis 2%-treated group * Statistically significant difference between control and Sideritis-treated mice (p \ 0.05) Statistically significant difference between Sideritis 2% and Sideritis 4%-treated mice (p \ 0.05) 123 Author's personal copy

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S. clandestina subsp. peloponnesiaca, exhibited lower constituents identified in the current extract (quinic acid, MDA and higher GSH levels in midbrain and cerebellum melittoside (iridoid) and apigenin (flavonoid)), have been compared with their control littermates; cortex and liver extensively studied in the literature. For instance, iridoids remained unaffected [19]. The remarked stability of the have shown to enhance the antioxidant capacity in primary cerebral cortex toward changes in its oxidant/antioxidant hippocampal neurons by upregulating the antioxidative status has been also documented in the study of Haque enzyme heme oxygenase-1 via the PI3 K/Nrf2-signaling et al. [35], where green tea catechins orally administered to [39]. Furthermore, another iridoid glycoside, loganin, was rats exerted strong antioxidant effects in the hippocampus very recently found to improve learning and memory but not in the cerebral cortex. Conclusively, drinking of the impairments induced by scopolamine in mice [40]. The herbal tea exerted a dose-dependent depletion of brain iridoids E-harpagoside and 8-O-E-p-methoxycinnam- (cerebellum and midbrain) MDA levels with a simulta- oylharpagide significantly improved the impairment of neous significant elevation of its GSH content. Since MDA, reference memory in scopolamine-treated mice through the end product of tissue lipid peroxidation, is highly both anti-acetylcholinesterase and antioxidant mechanisms reactive and responsible for cytotoxic effects, whereas [41]. The iridoid 8-O-E-p-methoxycinnamoylharpagide glutathione is a tripeptide, which acts as a free radical and its aglycone, harpagide, were very potent to protect scavenger preventing tissue damage, our findings support a primary cultured neurons against glutamate-induced oxi- neuroprotective action of the herbal tea through the inhi- dative stress primarily by acting on the antioxidative bition of brain oxidative damage and the enhancement of defense system and on receptors, respec- its endogenous antioxidant defense. tively [42]. Concerning the activities of phytochemicals In addition, thigmotaxis-testing results clearly show for bearing the quinic acid basic structure, it has been shown to the first time that Sideritis tea (4% w/v) consumption by move through the blood–brain barrier and to present cog- adult mice exerts anxiolytic-like effects, although addi- nition-enhancing activities [43]. They have also demon- tional behavioral assessment is necessary. Therefore, the strated the neuroprotective effects through the upregulation observed significant reduction of the anxious behavior that of PGK1 expression and ATP production activation [44]as followed Sideritis tea consumption was accompanied by a well as due to their metal chelation properties for metal- significant antioxidant reinforcement of the mouse brain. lotoxins (aluminum) [45]. Furthermore, quinic acid deriv- There are also other reports that establish a strong link atives displayed neuroprotective action on amyloid Ab- between oxidative stress and anxiety-like behaviors in induced PC12 cell toxicity and neurotrophic activity by rodents, but the underlying mechanisms are still unclear promoting neurite outgrowth in PC12 cells [46]. Moreover, [5–7]. Despite the physiological role of cerebellum and they could effectively inhibit the formation of advanced midbrain in the control of motor functions, the anxiolytic- glycation end products (AGEs) and rat lens aldose reductase like effects of the herbal tea in the present study were [47]. In addition to these phytochemicals, the identified basic combined with a significant elevation in the antioxidant structure of apigenin in the extract has been shown to possess capacity of the specific brain areas, while cerebral cortex a variety of pharmacological actions on the central nervous remained unlikely stable. Nevertheless, the implication of system as well as antidepressant-like behavioral and neuro- cerebellum and midbrain in anxiety disorders has been chemical effects [48]. It also protects brain neurovascular recently demonstrated [36, 37]. Since behavior constitutes coupling against amyloid-b25–35-induced toxicity in mice a complex brain process, future studies examining multiple [49] and inhibits the production of nitric oxide (NO) and biochemical indices of brain oxidant status and neuro- prostaglandinE2 (PGE2) in microglia and inhibits neuronal transmitter systems may extend the current knowledge cell death in a middle cerebral artery occlusion–induced focal about anxiolytic-like and antioxidant properties of Sideritis ischemia mice model [50]. Furthermore, apigenin was found clandestina tea. to inhibit the oxidative stress–induced macromolecular In order to get a deeper understanding of the mechanism damage in N-nitrosodiethylamine (NDEA)-induced hepato- of action of the Sideritis clandestina tea, the network of cellular carcinogenesis in rats [51], and also it can confer a interactions between individual constituents and cellular protective role in the status of lipid peroxidation and anti- components needs to be characterized [38]. However, the oxidant defense against hepatocarcinogenesis in rats [52]. identification of specific basic structures of phytochemi- In conclusion, the remarked anxiolytic-like effects and cals, for which their activities have been investigated in the in vivo antioxidant capacity of Sideritis clandestina subsp. past, clearly supports the determined capacity of the stud- clandestina infusion accompanied by the phytochemical ied extract to confer enhanced antioxidant activity, cogni- analysis of its aqueous extract support the beneficial role of tion-enhancing activities and neuroprotective actions. The regular drinking of herbal mountain tea from Sideritis taxa biological and pharmacological properties of numerous in the prevention of neurobehavioral diseases and the del- derivatives bearing the same basic structure, as the main eterious effects of aging. 123 Author's personal copy

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Acknowledgments We are grateful to Dr. Garbis S. for helpful 17. Demirtas I, Ayhan B, Sahin A, Aksit H, Elmastas M, Telci I discussions and comments. This work was partially supported by (2010) Antioxidant activity and chemical composition of Sideritis BIOFLORA, network of University of Patras and Esthir-Gkani libanotica Labill ssp. linearis (Bentham) Borm. (Lamiaceae). Nat Foundation, Ioannina. Special thanks are given to the Mass Spec- Prod Res 16:1512–1523 trometry Unit of University of Ioannina for providing access to LC- 18. Celik I, Kaya MS (2011) The antioxidant role of Sideritis caes- MS/MS facilities and Mrs. Theodora Lamari for collecting the plant area infusion against TCA toxicity in rats. Br J Nutr 105:663–668 material. NMR data were recorded in the NMR center of the Uni- 19. Linardaki ZI, Vasilopoulou CG, Constantinou C, Iatrou G et al versity of Ioannina. 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34. MacEvilly CJ, Muller DPR (1996) Lipid peroxidation in neural 44. Han J, Miyamae Y, Shigemori H, Isoda H (2010) Neuroprotective tissues and fractions from vitamin E-deficient rats. Free Radical effect of 3, 5-di-O-caffeoylquinic acid on SH-SY5Y cells and Biol Med 20:639–648 senescence-accelerated-prone mice 8 through the up-regulation of 35. Haque AM, Hashimoto M, Katakura M, Tanabe Y, Hara Y, Shido phosphoglycerate kinase-1. Neuroscience 169:1039–1045 O (2006) Long-term administration of green tea catechins 45. Nday CM, Drever BD, Salifoglou T, Platt B (2010) Aluminum improves spatial cognition learning ability in rats. J Nutr does not enhance b-amyloid toxicity in rat hippocampal cultures. 136:1043–1047 Brain Res 1352:265–276 36. Baldac¸ara L, Borgio JGF, de Lacerda ALT, Jackowski AP (2008) 46. Hur JY, Soh Y, Kim BH, Suk K, Sohn N, Kim HC et al (2001) Cerebellum and psychiatric disorders. Rev Bras Psiquiatr Neuroprotective and neurotrophic effects of quinic acids from 30:281–289 Aster scaber in PC12 cells. Biol Pharm Bull 24:921–924 37. Avila MAV, Ruggiero RN, Cabral A, Branda˜o ML, Nobre MJ, 47. Jang DS, Yoo NH, Kim NH, Lee YM, Kim CS, Kim J et al Castilho VM (2008) Involvement of the midbrain tectum in the (2010) 3, 5-Di-O-caffeoyl-epi-quinic acid from the leaves and unconditioned fear promoted by morphine withdrawal. Eur J stems of Erigeron annuus inhibits protein glycation, aldose Pharmacol 590:217–223 reductase, and cataractogenesis. Biol Pharm Bull 33:329–333 38. Janga SC, Tzakos A (2009) Structure and organization of drug- 48. Yi LT, Li JM, Li YC, Pan Y, Xu Q, Kong LD (2008) Antide- target networks: insights from genomic approaches for drug pressant-like behavioral and neurochemical effects of the citrus- discovery. Mol Biosyst 5:1536–1548 associated chemical apigenin. Life Sci 82:741–751 39. Yin F, Liu J, Zheng X, Guo L, Xiao H (2010) Geniposide induces 49. Liu R, Zhang T, Yang H, Lan X, Ying J, Du G (2011) The the expression of heme oxygenase-1 via PI3K/Nrf2-signaling to flavonoid apigenin protects brain neurovascular coupling against enhance the antioxidant capacity in primary hippocampal neu- amyloid-b25–35-induced toxicity in mice. J Alzheimers Dis rons. Biol Pharm Bull 33:1841–1846 24:85–100 40. Kwon SH, Kim HC, Lee SY, Jang CG (2009) Loganin improves 50. Ha SK, Lee P, Park JA, Oh HR, Lee SY, Park J-H et al (2008) learning and memory impairments induced by scopolamine in Apigenin inhibits the production of NO and PGE2 in microglia mice. Eur J Pharmacol 619:44–49 and inhibits neuronal cell death in a middle cerebral artery 41. Jeong EJ, Lee KY, Kim SH, Sung SH, Kim YC (2008) Cognitive- occlusion-induced focal ischemia mice model. Neurochem Int enhancing and antioxidant activities of iridoid glycosides from 52:878–886 Scrophularia buergeriana in scopolamine-treated mice. Eur J 51. Jeyabal PVS, Syed MB, Venkataraman M, Sambandham JK, Pharmacol 588:78–84 Sakthisekaran D (2005) Apigenin inhibits oxidative stress- 42. Kim SR, Koo KA, Sung SH, Ma CJ, Yoon JS, Kim YC (2003) induced macromolecular damage in N-nitrosodiethylamine Iridoids from Scrophularia buergeriana attenuate glutamate- (NDEA)-induced hepatocellular carcinogenesis in Wistar albino induced neurotoxicity in rat cortical cultures. J Neurosci Res rats. Mol Carcinog 44:11–20 74:948–955 52. Singh JP, Selvendiran K, Banu SM, Padmavathi R, Sakthisekaran 43. Dimpfel W, Kler A, Kriesl E, Lehnfeld R (2007) Theogallin and D (2004) Protective role of Apigenin on the status of lipid per- L- as active ingredients in decaffeinated green tea extract: oxidation and antioxidant defense against hepatocarcinogenesis II. Characterization in the freely moving rat by means of quanti- in Wistar albino rats. Phytomedicine 11:309–314 tative field potential analysis. J Pharm Pharmacol 59:1397–1403

123 Article

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Saffron as a Source of Novel Acetylcholinesterase Inhibitors: Molecular Docking and in Vitro Enzymatic Studies † § ⊗ ⊥ George D. Geromichalos,*, Fotini N. Lamari,*, Magdalini A. Papandreou, Dimitrios T. Trafalis, ⊗ ‡ △ Marigoula Margarity, Athanasios Papageorgiou, and Zacharias Sinakos † Department of Cell Culture−Molecular Modeling and Drug Design, Symeonidion Research Center, Theagenion Cancer Hospital, Thessaloniki, Greece § Laboratory of Pharmacognosy and Chemistry of Natural Products, Department of Pharmacy, University of Patras, Rion 26504, Greece ⊗ Laboratory of Human and Animal Physiology, Department of Biology, University of Patras, Patras 26504, Greece ⊥ Department of Pharmacology, Medical School, University of Athens, Athens, Greece ‡ Department of Experimental Chemotherapy, Symeonidion Research Center, Theagenion Cancer Hospital, Thessaloniki, Greece △ Honored Professor of Haematology, Medical School, Aristotle University of Thessaloniki, Thessaloniki, Greece

*S Supporting Information

ABSTRACT: Inhibitors of acetylcholine breakdown by acetylcholinesterase (AChE) constitute the main therapeutic modality for Alzheimer’s disease. In the search for natural products with inhibitory action on AChE, this study investigated the activity of saffron extract and its constituents by in vitro enzymatic and molecular docking studies. Saffron has been used in traditional ’ ff medicine against Alzheimer s disease. Sa ron extract showed moderate AChE inhibitory activity (up to 30%), but IC50 values of crocetin, dimethylcrocetin, and safranal were 96.33, 107.1, and 21.09 μM, respectively. Kinetic analysis showed mixed-type inhibition, which was verified by in silico docking studies. Safranal interacts only with the binding site of the AChE, but crocetin and dimethylcrocetin bind simultaneously to the catalytic and peripheral anionic sites. These results reinforce previous findings about the beneficial action of saffron against Alzheimer’s disease and may be of value for the development of novel therapeutic agents based on carotenoid-based dual binding inhibitors. KEYWORDS: acetylcholinesterase, dual inhibitors, in silico, saffron, mixed inhibition, crocetin, safranal

■ INTRODUCTION serious side effects caused by activation of peripheral 4−6 Alzheimer’s disease (AD) is a multifactorial dementia cholinergic systems. Potent AChE inhibitors belong to the characterized by cerebral accumulation of extracellular class of alkaloids, due to the requirement for positively charged amyloid-β (Aβ) protein aggregates (senile plaques) and quaternary ammonium in the structure, such as GNT and intraneuronal hyperphosphorylated twisted filaments of tau physostigmine. protein (neurofibrillary tangles) in brain areas associated with AChE, apart from its involvement in cholinergic synaptic β learning and memory (e.g., cortex, hippocampus, nucleus transmission, is also known to accelerate the aggregation of A 1 during the early stages of AD, primarily via interactions through basalis of Meynhert), resulting, thus, in profound memory 7−9 disturbances and irreversible impairment of cognitive function. its peripheral anionic binding site (PAS). In light of these The latter was ascribed primarily to the loss of cholinergic data, the search for new AChE inhibitors for AD now includes neurons in those areas, due to Aβ and tau buildups, and led to the search of dual binding molecules, being able to interact the formation of “the cholinergic hypothesis” of AD.2,3 simultaneously with both the PAS and the catalytic subsite of 10−12 The abnormalities in cholinergic metabolism seen in AD are AChE. not viewed as the cause of the disorder, but cholinergic Advances in X-ray crystallography and site-directed muta- involvement is significant because it is universal, correlates with genesis have enabled the detailed mapping of AChE’s active cognitive defects, and is one of the few pathophysiologic center. Human AChE (EC 3.1.1.7) is a globular protein phenomena that can be addressed with currently approved containing a 20 Å deep groove (gorge), which includes the treatment options, such as cholinesterase inhibitors (ChEIs) following loci: (1) the acyl-binding pocket Phe295(288) and [e.g., tacrine (THA), rivastigmine, and galanthamine (GNT)]. Phe297(290) (values in parentheses represent the amino acid ChEIs enhance cognitive function by inhibiting the catabolism numbering positions in Torpedo californica AChE) at the base of the neurotransmitter acetylcholine (ACh) by acetylcholines- terase (AChE) increasing the action of ACh in the synapse. Received: February 9, 2012 Although various clinical studies document the beneficial effects Revised: May 29, 2012 of current AChE inhibitors on cognition and behavior, their Accepted: June 1, 2012 benefits are modest, suffer from short half-lives, and show Published: June 1, 2012

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Figure 1. Chemical structures of AChE’s substrate (acetylthiocholine, ATCI) and inhibitors (tacrine, THA, and galanthamine, GNT) and saffron’s bioactive constituents/metabolites (crocin, CRC; crocetin, CRT; dimethylcrocetin, DMCRT; and safranal, SFR). of the gorge; (2) the esteratic locus, which consists of two molecular modeling studies to be conducted using the subsites, the oxyanion hole [Gly120(118), Gly121(119), and coordinates of the published eel AChE X-ray structure, as Ala204(201)] and the active site catalytic hydrolase triad stated by Khabid et al.27 The chemical structures of all saffron [His447(440), Glu334(327), and Ser203(200)]; (3) the constituents tested, that is, crocin (CRC), crocetin (CRT), quaternary ammonium binding locus [Trp86(84)]; and (4) dimethylcrocetin (DMCRT)m and safranal (SFR), as well as of the PAS site [Tyr72(70), Tyr124(121), and Trp286(279)], AChE’s substrate acetylthiocholin iodide (ATCI) and of AChE situated >10 Å above the active site triad and near the gorge known inhibitors THA and GNT, are shown in Figure 1. CRC, entrance.13,14 a natural carotenoid, is the diester formed by the disaccharide Saffron, derived from the stigmas of Crocus sativus, is a well- gentiobiose and CRT. CRT is the main metabolite in humans, known spice with many reputed therapeutic uses, including its whereas the synthetic analogue of DMCRT was used to − use as a tonic, nerve sedative, and antidepressant and its use augment the study of carotenoid structure activity relation- against dementias.15,16 Its composition is rare because it is a ships, that is, to investigate the role of CRT carboxyl groups. source of crocins, that is, mono- and diglycosides of crocetin SFR is a monoterpene aldehyde and the main component of ff (CRT). We have shown that the extract has powerful the essential oil of sa ron. antioxidant properties and that the crocin constituents inhibit 17 MATERIALS AND METHODS amyloid aggregation in vitro. After oral consumption of a cup ■ of saffron infusion, CRT is determined in blood plasma as the Plant Material and Extraction. Commercially available pure red ff main metabolite in humans.18 Administration of saffron extract Greek sa ron (stigmas of C. sativus) was kindly provided by the Cooperative Association of Krokos in Kozani, in West Macedonia, to aged mice has been demonstrated to counteract age-related Greece. Saffron was extracted with methanol/water 1:1 v/v, as memory impairments and improve cerebral antioxidant previously described.17 CRT (purity > 98%) was prepared by markers, in addition to the above effects of saffron saponification of saffron extract, whereas DMCRT was prepared by administration to adult mice of significantly decreased AChE saponification of saffron extract in the presence of methanol, as activity.19 Beneficial effects against scopolamine-induced previously described,28,29 and its final purity was >98%. SFR (purity > recognition memory deficits have also been documented in 88%) was purchased from Sigma-Aldrich Corp., St. Louis, MO, USA. 20−22 In Vitro AChE Inhibition Assay. The assay for AChE activity was rats. In accordance, two recent phase II clinical studies 30 provide preliminary evidence of a possible therapeutic effect of performed with the colorimetric method of Ellman et al., utilizing ff ATCI as a substrate and AChE from the electric eel (Sigma-Aldrich sa ron extract in the treatment of patients with mild to Corp.). Briefly, in the 96-well plates were added 50 μL of 50 mM Tris- 16,23,24 ff ff moderate AD. However, the e ect of sa ron constitu- HCl, pH 8.0, containing 0.1% bovine serum albumin (BSA), 25 μLof ents/metabolites on AChE activity has not been studied, the tested samples (0−8 mM CRT, DMCRT, and SFR dissolved in 50 despite its promising in vivo effects. mM Tris-HCl, pH 8.0) or GNT (1−256 μM), 25 μL of 15 mM ATCI In the present work, in silico molecular docking studies and (dissolved in water), and 75 μL of 5 mM 5,5′-dithiobis(2-nitro- benzoate) (DTNB) (dissolved in 50 mM Tris-HCl, pH 8.0, 0.1 M in vitro enzymatic kinetic studies have been undertaken as an · attempt to explore the ability of saffron constituents to act as NaCl, 0.02 M MgCl2 6H2O). Absorbance was measured at 405 nm after 5 min of incubation at room temperature, in the dark, to subtract potent inhibitors of AChE and to elucidate the possible these values from the values after AChE addition and correct for mechanism of action. AChE from electric eel was used because nonenzymatic change of reagent absorbance. Then, 25 μL of 0.22 U/ (a) its oligomeric forms are structurally similar to those in mL of AChE from electric eel (dissolved in 50 mM Tris-HCl, pH 8.0, AChE of vertebrates, nerve, and muscles25,26 and (b) it allows containing 0.1% w/v BSA) was added, and the absorbance was

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a Table 1. Enzyme Kinetics of the Tested Compounds on AChE Activity μ μ μ μ substrate inhibitor IC50 ( M) Km ( M) Vmax ( mol/L/min) Ki ( M) type of inhibition ATCI control 23.01 ± 2.70 65 ± 3.3 (conf interval: 17.0−29.0)

ATCI galanthamine (GNT) 1.93 ± 0.05 26.3 ± 3.02 65 ± 3.3 3.4 ± 0.3 competitive (conf interval: 19.4−33.1)

ATCI safranal (SFR) 21.09 ± 0.17 18.5 ± 23.9 27 ± 2.4 90.6 ± 2.0 mixed (conf interval: 9.6−27.4)

ATCI crocetin (CRT) 96.33 ± 0.11 19.30 ± 2.6 39 ± 2.2 28.0 ± 2.3 mixed (conf interval: 13.3−25.2)

ATCI dimethylcrocetin (DMCRT) 107.1 ± 0.11 16.5 ± 1.9 38 ± 1.8 16.5 ± 2.4 mixed (conf interval: 12.1−21.0) a ff − The values of Vmax, Km, and Ki were calculated by monitoring hydrolysis of substrate (ATCI) by AChE at di erent substrate concentrations (0.125 64 μM) in the presence or absence of samples (mean results of 4 experiments, run in duplicate) (0.15−34 μM). measured again after 5 min of incubation at room temperature in the quantum-chemical calculations by utilizing the ab initio Hartree− dark. GNT (1−32 μM, final concentrations) (Sigma-Aldrich) was used Fock method with the 6-31G* basis set. as a reference inhibitor. In all experiments the final organic solvent Docking calculations were carried out via the BioMedCAChe percentage was never above 6% v/v; control experiments with the program, which is part of the CAChe package (CAChe WorkSystem same percentage of organic solvent without sample were also Pro version 7.5.0.85, Fujitsu). Docking experiments employed full conducted, and the values were subtracted. All determinations were flexibility and partial protein flexibility focused at the ligand carried out at least four times, and in duplicate, at each concentration binding site. AChE complex with various inhibitors has provided in 96-well microplates, using a UV−vis microplate reader (Molecular valuable knowledge of the interactions that mediate inhibitor Devices). Results were reported as percentage of inhibition of AChE binding.31 To identify the molecular determinants responsible for activity in the absence of inhibitor/tested compounds (positive the binding mode of the studied compounds with AChE protein, we control). The concentrations of the tested extracts that inhibited the used the determined X-ray crystallographic structure of T. californica hydrolysis of substrate (ATCI) by 50% (IC50) were determined by AChE in complex with the AChE inhibitor, THA (PDB accession no. GraphPad Prism4.0 (GraphPad Software, Inc., USA) from sigmoidal 1ACJ).32 X-ray structure was obtained from the Brookhaven Protein dose−response curves obtained by plotting the percentage of Data Bank (RCSB Protein Data Bank, operated by the Research inhibition (Y) versus the log concentration (x, μM) of the tested Collaboratory for Structural Bioinformatics).33 The produced samples, using the equation compound−protein complexes were ranked by the energy score, including their binding conformations. 3D models of the above protein LogIC −x Y =+100/(1 10(50 )) crystal structures have been developed after the deletion of the cocrystallized bound inhibitor. The docking procedure provides the fl Kinetic Analysis of AChE Inhibition. Microplate wells were filled treatment of ligand exibility within the protein binding site by means with 50 μL of 50 mM Tris-HCl, pH 8.0, containing 0.1% BSA, 25 μL of a four-point chiral pharmacophoric comparison between the ligand fi of the tested phytochemicals (0−256 μM CRT and DMCRT and 0− and the site. The nal output of the docking procedure is a set of 120 μM SFR) or GNT (0−120 μM), 25 μL of AChE (0.22 U/mL), solutions ranked according to the corresponding scoring function fi and 75 μL of 3 mM DTNB (dissolved in 50 mM Tris-HCl, pH 8.0, values, each de ned by the 3D coordinates of its atoms and expressed · as a PDB file. The accuracy of BioMedCAChe has been shown to containing 0.1 M NaCl, 0.02 M MgCl2 6H2O). The mixture of enzyme and inhibitor/or samples was mixed, and absorbance was read at 405 successfully reproduce experimentally observed binding modes, in nm after 5 min of incubation at room temperature, in the dark. terms of root-mean squared deviation (rmsd). BioMedCAChe Immediately after addition of the substrate (25 μL ATCI, 1−512 μM provided excellent result as waś observed for the low value of rmsd concentrations, dissolved in water), the change of absorbance at 405 (best docked solution of 0.43 Å) between experimental and docked nm was monitored for 5 min, and the reaction rate was calculated structures (this is also shown by superimposition of the above 30 structures). The ability to accurately predict the binding conformation according to the method of Ellman et al. Inhibition studies were fi analyzed using GraphPad Prism4.0 software. The analysis of the type of AChE inhibitors, THA and GNT, to AChE, gave con dence that of inhibition of AChE activity was determined by the Lineweaver− the BioMedCAChe could also predict, with a similar accuracy, the binding conformations of saffron’s bioactive constituents utilized in the Burk (LB) plot, whereas the kinetic parameters Km and Vmax were 34 obtained by curve fitting according to the classical Michaelis−Menten current study. The PyMol molecular graphics system was used to visualize the molecules and the results of the docking experiments. equation. The inhibition constants (Ki) of the tested samples were ± calculated according to GraphPad Prism v4.0, from linear regression Statistical Analysis. Data are presented as the mean SE. analysis of LB plots (S, hyperbolic; I, hyperbolic mixed model). Statistical analysis was performed with GraphPad Instat 3 software (GraphPad Instat Software, Inc., USA) using the nonparametric In Silico Computational Methods (Molecular Modeling and − ≤ Docking Calculations). Mann Whitney test (p < 0.05). In all tests, a criterion of p 0.05 Molecular models of all compounds fi described in this work were built in 3D coordinates and their best, (two-tailed) was considered to be necessary for statistical signi cance. most stable (lower energy) conformations were detected by geometrical optimization of their structure in the gas phase, as ■ RESULTS ‘ implemented in the Spartan 08 Molecular Modeling program suite ff (Spartan ‘08 v. 1.2.0, Wave Function Inc., Irvine, CA, USA). The In Vitro E ects on AChE Activity. To study if the tested molecules’ structures were initially optimized by conformational search phytochemicals directly inhibit AChE, the effects of saffron using the Monte Carlo method with the MMFF94 molecular extract and of CRT, DMCRT, SFR, and GNT (positive mechanics model. Geometry optimization was accomplished via control) on electric eel AChE activity were tested in vitro, and

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− ff ’ − Figure 2. Lineweaver Burk plots in the absence (a) and presence of standard GNT (b) and sa ron s constituents (c e): 1/Vmax versus 1/[ATCI]. the results are presented in Table 1 and in Figure 3 in the The topography of the calculated binding sites of the studied Supporting Information. Saffron showed moderate AChE compounds provides a clearer understanding of their potency inhibitory activity (up to 30%). Dose-dependent inhibition of differentiation in the inhibition of AChE. AChE was observed for GNT (Figure 3a of the Supporting The global energy of interaction (in kcal/mol) for each − Information), a standard AChE inhibitor, with an IC50 of 1.93 docking experiments is given as follows: CRT, 32.24; μM, as well as for the pure compounds, in the order SFR > DMCRT, −36.07; CRC, −30.63; SFR, −18.43; THA, CRT ≥ DMCRT (Figure 3c−e in the Supporting Information). −18.26; ATCI, −19.65; and GNT, −32.89. The ligand binding The determined IC50 value of GNT is in agreement with contacts of best docked poses of the known AChE inhibitors previous results.31,33 GNT and THA and the AChE substrate, ATCI, with key amino To elucidate the mechanism of AChE inhibition, kinetic acid residues and water molecules in the active site of AChE, studies of enzyme activity were performed. The relationship are shown in Tables 2−4 in the Supporting Information. between substrate concentration and reaction velocity was in CRT docked very close to ATCI (at a nearby binding good agreement with Michaelis−Menten kinetics. In the position). Its stabilization to the protein’s binding pocket was absence of inhibitors, the average Km for the substrate mainly attributed to its formed hydrophobic contacts (23 total (ATCI) was 23.01 ± 2.70 μM. When the slopes or the contacts): (a) by the hydrophobic residues Phe284, Phe290 intercepts in the Lineweaver−Burk plots were drawn in (acyl-binding pocket), Phe330, Phe331, Ile287, Leu358; (b) by GraphPad Prism 4.0 as a function of inhibitor concentration the acidic and basic charged polar residues Asp285 and Arg289; and the kinetic standards determined (Figure 2), CRT, and (c) by the polar noncharged residues Tyr121 and Ser286 DMCRT, and SFR showed mixed-type inhibition; the α values (Table 5 in the Supporting Information). Additionally, CRT of CRT and DMCRT are indicative of a mixed-type, was localized inside the binding cavity, making two hydrogen uncompetitive inhibition, whereas the α values of SFR show bond contacts with Tyr121 and Asp285. Water molecule a mixed-type competitive inhibition (Table 1). wat634 also contributes to the ligand binding by forming a In Silico Protein−ligand Docking Study. In silico with O13 of CRT. The binding contacts of molecular docking studies were also undertaken to gain insight CRT with Phe330 and wat634 were found to be common with into the interaction of CRT, DMCRT, CRC, and SFR with the those of ATCI, THA, and GNT, whereas Tyr121, Phe331, and residues of the ligand binding site of the AChE protein and to the acyl-binding residue Phe290 contributed also to the binding investigate the underlying mechanism(s) of action(s). The of THA and GNT inhibitors. As illustrated from the ligand docking simulation studies on the crystal structure of target binding site architecture (Figure 3), CRT was inserted into the protein AChE aimed to explore the ability of the studied narrow aromatic gorge of the crystal structure of AChE, compounds to act as potent inhibitors of the AChE enzyme. oriented in a way that the molecule’s polyene backbone could

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Figure 3. Ligand binding site architecture of AChE target protein (PDB accession no. 1ACJ) with the superimposed docked molecules CRT, ATCI, THA, and GNT. AChE protein is illustrated as a cartoon colored according to chainbow, with additional depiction of selected contacting amino acid residues rendered in line mode (a). The binding envelope of CRT in the target protein (b) is structured with interacting amino acid residues in stick model colored according to Figure 4. Docking of DMCRT (shown in stick representation and chainbow. Interacting water634 molecule is depicted as a blue-dotted colored according to atom type with light pink C atoms) in the crystal sphere. Docked molecules are represented in stick model and colored structure of AChE protein cocrystallized with its known inhibitor THA according to atom type (CRT carbon atoms in light blue (a) or light (hot pink C atoms) (PDB accession no. 1ACJ). (a) Target protein is magenta (b); ATCI, THA, and GNT carbon atoms in yellow, hot depicted as semitransparent ray-traced cartoon colored by secondary pink, and white, respectively). Hydrogen atoms are omitted from all fi structure (cylindrical helices, red; plank-sheet, yellow; loop, green). A molecules for clarity ( nal structure was ray traced). closeup view of the ligand binding pocket is illustrated in semitransparent surface colored by chain (in blue) (b) depicting the be stabilized via hydrophobic contacts with the amino acid binding interactions of DMCRT (light magenta C atoms) with residues of the wall of this prolonged cavity. CRT penetrated selected amino acid residues in contact highlighted yellow. Hydro- into the central cavity of the protein, where the catalytic active phobic and H-bond interactions are shown as dotted lines. Hydrogen atoms are omitted from all molecules for clarity (final structure was ray site is located, with one of its carboxylic ends to be positioned traced). near the catalytic site triad and near the position occupied by ATCI, THA, and GNT. The other end of the molecule (situated near the opening of the aromatic groove at a distance polyene backbone moiety of the compound is localized inside ∼20 Å from the catalytic active site) protrudes from the the anionic binding groove, making contacts with the following protein’s molecule and is stabilized in its position via two H- amino acid residues: Phe284, Tyr121 (PAS), Trp279 (PAS), bond contacts with the amino acid residue Asp285 and the and Tyr334. In contrast, Phe330 and Asp72 residues mainly water molecule wat634. Hydrophobic contacts between CRT’s contribute to the attachment of the molecule into the catalytic atoms of the hydrocarbon skeleton and amino acid residues of binding site (Figure 4b). The DMCRT molecule is stabilized in the peripheral anionic site contributed further to the attach- the ligand binding cavity mostly through hydrophobic ment of the molecule. interactions with residues Asp72, Phe330, Tyr334, Trp279, DMCRT superimposed with THA inhibitor is shown in Phe284, and Asp285 (Table 6 in the Supporting Information). Figure 4 to be anchored inside the ligand binding pocket of the Phe330 was a common binding residue with the ATCI protein. H-bond and hydrophobic interactions between the substrate, whereas Phe330 and Tyr121 were common with DMCRT compound and the amino acid residues of the ligand THA and GNT. The total number of binding contacts found binding cavity of the target protein are shown in Figure 4b. The between DMCRT and AChE protein was 21, of which 5 were

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H-bonds, 1 was a bridged H-bond, and the rest were hydrophobic. Docking studies of CRC on AChE demonstrated that the ligand was complexed with the protein with the incorporation of both H-bond and hydrophobic contacts. The docking studies revealed that the CRC molecule anchors inside the aromatic groove, but in a different binding locus from that of THA (binding is stabilized at the peripheral site located at the gorge rim, which encompasses binding sites for allosteric ligands). CRC has a unique orientation along the active-site gorge, extending from the anionic subsite of the active site at the bottom, to the peripheral anionic site at the top, via aromatic stacking interactions with various aromatic acid residues. The superimposed structures of CRC and THA on the target protein are illustrated in Figure 5a. The best possible docking binding mode of CRC was mediated through residues Asn230, His398, Pro232, Asp285, Ser235, and Trp524 and water molecules wat640 and wat650 (11 H-bond contacts) and Arg289, Pro361, Cys357, Asp285, and Phe284 (17 hydro- phobic contacts) (Figure 5b; Table 7 in the Supporting Information). No common binding residues with ATCI, THA, or GNT were observed. The docking procedure positioned SFR into the AChE binding site almost at the same place occupied by THA (Figure 6a,b). Through interaction analysis, it was shown that SFR makes one hydrogen bond contact with the hydroxyl group of wat634, whereas the stabilization of the compound into the pocket was additionally attributed to seven hydrophobic contacts formed by residues Phe330, Gly118, His440, and Trp84 (Figure 6c; Table 8 in the Supporting Information). All binding contacts were found to be common with the corresponding AChE−ATCI/THA complexes, whereas Gly118, His440, and Trp84 were common with the AChE− GNT complex. The former amino acid contacts of SFR are important anchoring residues for the inhibitor and are the main contributors to the inhibitor’s interaction. (Data concerning ATCI, GNT, and THA are not shown in the text or tables; they are provided as additional Supporting Information available online.) ■ DISCUSSION Figure 5. Bound CRC ligand (illustrated in stick mode and colored Our results showed that the crude aqueous methanolic saffron according to atom type with light pink C atoms) docked in crystal extract exhibited low dose-independent inhibitory values. structure of AChE protein (PDB accession no. 1ACJ) depicted as Analysis of the in vitro effect of CRT, DMCRT, and SFR on spectrum-colored cartoon (a) or solid surface colored by chain (in AChE inhibition revealed a dose-dependent inhibitory profile, purple-blue) with highlighted interacting amino acid residues of the ligand binding cavity (b) in complex with THA (hot pink C). but the IC50 values were higher than that of GNT. Kinetic analysis confirmed that GNT exerts a competitive type of Highlighted residues interacting through H-bond: Asn230, light μ orange; His398, pale green; Ser235, teal; and Trp524, yellow. inhibition, with a Ki value of 3.36 M in electric eel enzyme, but ff Hydrogen-bonded water molecule wat650 is rendered as a light blue the majority of the tested sa ron phytochemicals exhibited sphere. Hydrogen bond and hydrophobic interactions are shown as higher Ki values and a mixed type of inhibition. yellow-dotted lines. Hydrogen atoms are omitted from all molecules Molecular docking studies revealed that CRT and DMCRT for clarity (final structure was ray traced). were not docked at the same place as ATCI, TCH, and GNT inhibitors, but bound simultaneously to the catalytic and peripheral anionic sites of AChE. CRC, due to its bulky glycosidically linked sugar moieties, was unable to be inserted in possibilities for the design and synthesis of novel dual binding depth to the aromatic gorge (did not reach the active site that is AChE inhibitors, which would beneficially affect cholinergic found deep in the pocket) and attached to a different locus transmission and Aβ aggregation. from that of ATCI, THA, and GNT. The fact that the presence On the other hand, SFR seems to be a more potent inhibitor ± ± of glycosidically linked sugars alters/impairs binding character- (IC50 = 21.09 0.17, Ki = 90.6 2.0) than the carotenoids, but istics might provide an explanation for the rather low inhibitory both kinetic and in silico data show that it completely enters activity of the saffron extract in vitro. The ability of CRT to and interacts only with the catalytic center. Similar weak span the two “anionic” sites, being ∼20 Å apart, and bind to inhibitory action has been reported for many other both PAS and the catalytic site of the AChE enzyme opens new monoterpenoids,31 and it is attributed to interactions of the

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hydrocarbon skeletal terpenoids with the AChE hydrophobic active center. The hypothesis derived from the in vitro experiments revealed that the majority of the tested phytochemicals exhibited a mixed type of inhibition, as supported by the adopted in silico studies, where all saffron constituents (apart from SFR) were found to be complexed with the enzyme at a different locus (allosteric binding site) from that of ATCI and the tested inhibitors (THA and GNT). These findings showed, for the first time, that CRT, the main metabolite of CRCs in the living organism, inhibits AChE by binding at two different loci, the catalytic center and the PAS site. This finding may partly explain the beneficial effects of saffron in clinical trials against AD and provides a basis for the design and development of novel pleiotropic AChE inhibitors. The presence of glycosidi- cally linked sugars impairs this activity. Overall, our results showed that the in vitro kinetic analysis, in conjunction with molecular modeling and docking predictions, could be important initial steps toward the development of novel AChE inhibitors. ■ ASSOCIATED CONTENT *S Supporting Information The ligand binding contacts of best docked poses of the known AChE inhibitors GNT and THA, the AChE substrate ATCI, and saffron constituents CRT, DMCRT, CRC, and SFR, with key amino acid residues and water molecules in the active site of AChE, are shown in Tables 2−8. The effectiveness of GNT, saffron, and its constituents in inhibiting AChE activity is − shown in Figure 3. IC50 or pIC50 (when converted to the log IC50 scale) represents the concentration of the tested phytochemical(s) that is required for 50% inhibition in vitro and is calculated by converting the log values back to μM. This material is available free of charge via the Internet at http:// pubs.acs.org. ■ AUTHOR INFORMATION Corresponding Author *(G.D.G.) Department of Cell Culture-Molecular Modeling and Drug Design, Symeonidion Research Center, Theagenion Cancer Hospital, 2 Al. Symeonidi str., 54007 Thessaloniki, Greece. Phone: +30 231 0898237. Fax: +30 321 0845514. E- Figure 6. Cartoon diagram (a) of AChE protein with bound SFR mail: [email protected]. (F.N.L.) Department of Phar- molecule in complex with ATCI (rendered as sticks with yellow macy, University of Patras, Patras 26504, Greece. Phone:+30 carbon atoms) and the known inhibitors THA and GNT (rendered as 261 0969335. Fax: +30 261 0997714. E-mail: fl[email protected]. sticks with hot pink and blue carbon atoms, respectively). SFR (depicted as a stick with light pink carbon atoms) is docked into the Funding binding pocket of the protein (illustrated as cartoon colored according The present study is cofinanced by the EU European Social to secondary structure) at almost the same place occupied by the Fund (80%) and the Greek Ministry of Development-GSRT cocrystallized THA and GNT inhibitors (PDB ID no. 1ACJ) (20%) and by GlaxoSmithKline. (dockings of all ligands were performed individually). A closeup Notes view (b) of the ligand binding cavity of the protein is represented as fi semitransparent surface colored by chain with interacting amino acid The authors declare no competing nancial interest. residues highlighted in magenta. Wat634 molecule interacting via H- bond with SFR is depicted as a blue-dotted sphere. Interactions of ■ ABBREVIATIONS USED bound SFR and THA ligands (depicted as sticks with light pink and Aβ, amyloid-β peptide; ACh, acetylcholine; AChE, acetylcho- hot pink carbon atoms, respectively) docked in crystal structure of the ’ active site of AChE target protein are colored as chainbow (c). Amino linesterase; AD, Alzheimer s disease; ATCI, acetylthiocholine acid residues of the binding pocket are depicted in stick model and iodide; BSA, bovine serum albumin; ChAT, choline acetyl- colored according to atom type (only the contacts of SFR are shown). transferase; ChEIs, cholinesterase inhibitors; CRC, crocin; The Wat634 molecule is depicted as a magenta-dotted sphere. CRT, crocetin; DMCRT, dimethylcrocetin; DTNB, 5,5′- Hydrogen atoms are omitted from all molecules for clarity (final dithiobis(2-nitrobenzoate); GNT, galanthamine; PAS, periph- structure was ray traced). eral anionic binding site; PDB, Protein Data Bank; SAR, structure−activity relationship; SFR, safranal; THA, tacrine.

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6138 dx.doi.org/10.1021/jf300589c | J. Agric. Food Chem. 2012, 60, 6131−6138 Food and Chemical Toxicology 52 (2013) 163–170

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Food and Chemical Toxicology

journal homepage: www.elsevier.com/locate/foodchemtox

Investigation of the neuroprotective action of saffron (Crocus sativus L.) in aluminum-exposed adult mice through behavioral and neurobiochemical assessment

Zacharoula I. Linardaki a, Malvina G. Orkoula b, Alexandros G. Kokkosis a, Fotini N. Lamari c, ⇑ Marigoula Margarity a, a Laboratory of Human and Animal Physiology, Department of Biology, University of Patras, 26504 Patras, Greece b Laboratory of Instrumental Pharmaceutical Analysis, Department of Pharmacy, University of Patras, 26504 Patras, Greece c Laboratory of Pharmacognosy & Chemistry of Natural Products, Department of Pharmacy, University of Patras, 26504 Patras, Greece article info abstract

Article history: In the present study, the possible reversal effects of saffron against established aluminum (Al)-toxicity in Received 21 September 2012 adult mice, were investigated. Control, Al-treated (50 mg AlCl3/kg/day diluted in the drinking water for Accepted 8 November 2012 5 weeks) and Al + saffron (Al-treatment as previously plus 60 mg saffron extract/kg/day intraperitoneally Available online 17 November 2012 for the last 6 days), groups of male Balb-c mice were used. We assessed learning/memory, the activity of acetylcholinesterase [AChE, salt-(SS)/detergent-soluble(DS) isoforms], butyrylcholinesterase (BuChE, SS/ Keywords: DS isoforms), monoamine oxidase (MAO-A, MAO-B), the levels of lipid peroxidation (MDA) and reduced Aluminum glutathione (GSH), in whole brain and cerebellum. Brain Al was determined by atomic absorption spec- Saffron trometry, while, for the first time, crocetin, the main active metabolite of saffron, was determined in brain Memory Cholinesterases after intraperitoneal saffron administration by HPLC. Al intake caused memory impairment, significant Monoamine oxidase decrease of AChE and BuChE activity, activation of brain MAO isoforms but inhibition of cerebellar Oxidative stress MAO-B, significant elevation of brain MDA and significant reduction of GSH content. Although saffron extract co-administration had no effect on cognitive performance of mice, it reversed significantly the Al-induced changes in MAO activity and the levels of MDA and GSH. AChE activity was further signifi- cantly decreased in cerebral tissues of Al + saffron group. The biochemical changes support the neuropro- tective potential of saffron under toxicity. Ó 2012 Elsevier Ltd. All rights reserved.

1. Introduction transferrin in the blood and a subsequent transferrin receptor- mediated mechanism of brain Al influx (Yokel et al., 1999). Aluminum (Al) is the third most abundant element in nature, High brain levels of Al induce cognitive deficiency and dementia making human exposure unavoidable (Verstraeten et al., 2008). and, thus, Al is a widely accepted neurotoxin (Kawahara and The main entry sites of Al into the body are the gastrointestinal Kato-Negishi, 2011). Its implication in the pathogenesis of and respiratory tract and the skin (Verstraeten et al., 2008), en- neurodegenerative diseases has been suggested decades ago, but abling distribution and accumulation in several tissues, like spleen, is seriously debated till now (Kawahara and Kato-Negishi, 2011; lungs, liver, kidneys, heart, bone and brain (Kumar and Gill, 2009), Tomljenovic, 2011). However, some researchers consider that the through systemic circulation. It has been demonstrated that Al model of chronic Al-induced neurotoxicity best describes crosses the blood–brain barrier, implicating metal binding to Alzheimer’s disease, since it manifests many of the pathobiological hallmarks (Walton and Wang, 2009; Yokel, 2006; Zhang et al., 2003). Abbreviations: AChE, acetylcholinesterase; ATCh, acetylthiocholine iodide; Al neurotoxicity is manifested through several behavioral and BuChE, butyrylcholinesterase; BuTCh, butyrylthiocholine iodide; ce, cerebellum; neurochemical alterations, which display diversity depending on ChE, cholinesterase; DS, detergent-soluble; GSH, reduced glutathione; i.p., intra- peritoneal injections; IL, initial latency; MAO, monoamine oxidase; MDA, malondi- the animal species in question, the administration route and the aldehyde; SS, salt-soluble; STL, step-through latency. chemical form of Al administered (Erasmus et al., 1993; Kumar ⇑ Corresponding author. Tel.: +30 2610 997430; fax: +30 2610 969273. and Gill, 2009). It has been demonstrated that Al promotes E-mail addresses: [email protected] (Z.I. Linardaki), [email protected] oxidative stress in the cerebral cortex and hippocampus of young (M.G. Orkoula), [email protected] (A.G. Kokkosis), fl[email protected] and aged rats, and damage of lipids, membrane-associated proteins (F.N. Lamari), [email protected] (M. Margarity).

0278-6915/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.fct.2012.11.016 164 Z.I. Linardaki et al. / Food and Chemical Toxicology 52 (2013) 163–170

(Na+-K+ ATPase and protein kinase C) and endogenous antioxidant sativus were extracted with methanol:water (50:50, v/v, 1 g in 60 mL) for 4 h in enzymes (Sethi et al., 2008; Sharma et al., 2009; Tripathi et al., the dark, under magnetic stirring, as previously described (Papandreou et al., 2006). The extract was centrifuged and evaporated to dryness in a Speed Vac sys- 2009). Negative impacts of Al administration have been also ob- tem. The composition was screened with HPLC on a Supelcosil C-18 column as pre- served in neurotransmitter systems of rodent brain regions, includ- viously described (Papandreou et al., 2006; Tarantilis et al., 1995). The dry residue ing serotoninergic (Kumar, 2002) and cholinergic systems (Julka was stored at À30 °C until use. et al., 1995). The effects of Al on AChE activity remain controversial as both inhibition and activation have been reported (Julka et al., 2.2. Animals 1995; Sharma et al., 2009), whereas the respective data concerning Male adult (4 months-old) Balb-c mice were used in this study. The animals BuChE activity are limited, though BuChE enzyme is considered to were housed in groups in standard laboratory cages (5 mice per cage) in a temper- play a supportive functional role in acetylcholine hydrolysis (Lane ature controlled room (20 ± 2 °C) with a 12 h light/dark cycle. Food in form of dry et al., 2006). Finally, behavioral deficits assessed with different pellets (feed composition: grain and grain by-products, oil seed products, minerals, behavioral tasks, have been observed in rats following Al exposure vitamins and trace elements from Altromin Spezialfutter GmbH & Co. KG, Lage, Ger- (Julka et al., 1995; Sethi et al., 2008; Tripathi et al., 2009). many) was available ad libitum. Animals were randomly divided into three groups (n = 10/group). Control group: mice had access to normal drinking water over the Crocus sativus L. (Iridaceae) is a cultivated plant species in many experimental period. Al treated group: mice received orally AlCl3 (aluminum chlo- countries including Greece, since its styles (saffron) constitute an ride anhydrous, purity >99%, Sigma–Aldrich, St. Louis, USA) (50 mg/kg body weight/ exquisite spice. Interestingly, saffron has been used as a medicinal day) dissolved in normal drinking water for a period of 5 weeks. Body weight and agent for millenia (Schmidt et al., 2007). In recent years beneficial liquid intake were measured regularly to adjust the dose and achieve a constant in- take of Al. Al + saffron treated group: mice received orally AlCl (50 mg/kg body effects of saffron have been demonstrated in models of neuronal, 3 weight/day) dissolved in normal drinking water for 5 weeks and saffron extract cardiovascular, respiratory and other disorders (Bathaie and (60 mg/kg body weight/day, 30 lL injection volume) intraperitoneally for the last Mousavi, 2010), and are related to its unique composition; it is rich 6 days of the 5-week Al treatment. in crocins, which are mono- and di-glycosides of crocetin. Previ- AlCl3 solution was freshly prepared every 4 days over the experimental period, ously, we have reported the inhibitory activity on amyloid beta- while saffron solution was prepared fresh daily, just before administration, by dis- solving the dry extract in saline. During the 6-day saffron treatment, control and Al- peptide fibrillogenesis and protective action against H2O2-induced treated mice received intraperitoneally 30 lL of saline. Al and saffron dosage and toxicity in human neuroblastoma cells of saffron and its crocin schedule of exposure were based on the literature (Jyoti and Sharma, 2006) and constituents in vitro (Papandreou et al., 2006, 2011). Furthermore, on our previous study (Papandreou et al., 2011), respectively. All procedures were i.p. administration of saffron (60 mg/kg body weight) to normal in accordance with Greek National Laws (Animal Act, PD 160/91). and aged mice for 7 days significantly improved learning and memory as assessed by step-through passive avoidance test and 2.3. Behavioral testing: step-through passive avoidance task this was correlated with the significant cerebral antioxidant pro- This test is based on negative reinforcement to examine long-term memory tection conferred (Papandreou et al., 2011). Other studies have also (Kaneto, 1997). A two-compartment passive avoidance apparatus (white/dark, sep- demonstrated neuroprotective effects of saffron and its constitu- arated by a guillotine door) was used. Mice were subjected to single trial passive ents in vitro and in rodent models of brain disorders (amnesic avoidance task according to previously described procedures (Kaneto, 1997; Otano and ischemic) (Hosseinzadeh and Sadeghnia, 2005; Ochiai et al., et al., 1999; Papandreou et al., 2009). Briefly, on day 5 of saffron treatment an acqui- sition trial was performed 1 h after a 100 s-long habituation trial, while a retention 2007; Pitsikas and Sakellaridis, 2006). The first small-scale clinical trial was performed 24 h after the training session. Results were expressed as the trials of saffron against depression and mild Alzheimer’s disease mean initial (IL, max. time 120 s) and step-through (STL, max. time 300 s) latency have brought forward promising results, although many issues re- time values recorded once mice crossed into the dark compartment, before and main unresolved (Akhondzadeh et al., 2005, 2010; Noorbala et al., 24 h after the delivery of a mild foot shock to their paws. All training and testing sessions were carried out during the light phase (08:00–14:00 h), one hour after 2005). Yoshino et al. (2011) demonstrated the distribution of intraperitoneal administration of saffron. crocetin in brain of stroke-prone spontaneously hypertensive rats

90 min after a single oral administration of pure crocetin, but it 2.4. Preparation of tissue homogenates has not been proven yet whether crocetin, the main crocin metab- olite, crosses the blood–brain barrier after saffron administration. On the completion of the 5-week treatment period, all animals were sacrificed Taking into consideration the increasing evidence about saffron by cervical dislocation. Brain was excised immediately, and cerebellum was sepa- rated from the remaining whole brain. Liver was also removed and used as a refer- neuroprotective role we further investigated the possible ence peripheral tissue. All tissues were rinsed with saline immediately. From the neuroprotective action of saffron extract in a model of chronic tissue samples isolated, only six tissues per group were used for the biochemical Al-induced neurotoxicity. Shati et al. (2011) demonstrated the assessments. The others were assayed for Al content by atomic absorption spec- ameliorative effects of aqueous saffron extract administration trometry. The tissues for biochemical measurements and crocetin determination against Al-induced neurotoxicity, by presenting changes of brain were weighed and homogenized (10%, w/v) with a glass-Teflon homogenizer in ice-cold 30 mM Na2HPO4, pH 7.6. The homogenates were then centrifuged at antioxidant enzymes, serum tumor markers and brain expression 15,000g for 20 min at 4 °C. Supernatants constituted the salt-soluble (SS) fractions of genes (especially BCL-W, R-spondin and inositol polyphosphate and were stored at À75 °C. The pellets were re-suspended in an equal volume of 1%

4-phosphatase). However, the potential of saffron extract to re- (w/v) Triton X-100 (in 30 mM Na2HPO4, pH 7.6) and then centrifuged at 15,000g for verse the changes accompanying Al neurotoxicity has not been 20 min at 4 °C. Supernatants, the detergent-soluble (DS) fractions, were collected investigated. Therefore, the present study based on a different and stored at À75 °C. This double-extraction method was performed in order to re- cover the cytosolic (SS fraction) and membrane-bound (DS fraction) isoforms of experimental design (i.p. administration of saffron extract only cholinesterases (Das et al., 2005). Protein concentrations were determined by the for the last 6 days of the Al treatment period) starts from Bradford assay (Bradford, 1976). behavioral level, including learning and memory process, and is extended to subcellular level, focusing on determination of brain 2.5. Aluminum determination by atomic absorption spectrometry cholinergic, monoaminergic and oxidant/antioxidant indices and cerebral Al and crocetin levels. 2.5.1. Digestion of tissues The whole brain (-ce), cerebellum and liver samples from control, Al-treated and Al + saffron treated mice were analyzed for Al concentration. Glassware was 2. Material and methods not used during sample preparation in order to avoid the possible contamination.

Eppendorf tubes were used after immersing in a solution of nitric acid (HNO3): eth- 2.1. Plant material and extraction anol (1:9, v/v) for 48 h and washing with ultra-pure water (Missel et al., 2005). Sample preparation was performed as previously described (Gulya et al., 1995) with Greek saffron (pure red styles of C. sativus) was kindly provided by the Cooper- modifications. The dry weight of the tissues was measured after heating at 100 °C ative Association of Krokos in Kozani, in West Macedonia, Greece and a sample has for 20 h. Then, the samples were digested with 1 mL/100 mg wet tissue (for whole been deposited at the herbarium of the Department of Biology. Dried styles of C. brain) and 1 mL/25 mg wet tissue (for cerebellum and liver) of nitric acid: sulphuric Z.I. Linardaki et al. / Food and Chemical Toxicology 52 (2013) 163–170 165

acid (HNO3:H2SO4, 1:1 v/v) at room temperature overnight. Mixtures were centri- 2.7.3. Estimation of lipid peroxidation and reduced glutathione levels fuged at 10,000g for 10 min to remove insoluble material. Al levels were deter- Malondialdehyde (MDA) levels, an index of tissue lipid peroxidation, were mea- mined in the supernatants. sured in cerebral and liver samples (SS fractions of tissue homogenates) following a previously described fluorimetric assay (Papandreou et al., 2009). Lipid peroxida- tion levels are expressed as lmol MDA/g of tissue protein. 2.5.2. Atomic absorption spectrometry Reduced glutathione (GSH) content of brain and liver samples (SS fractions of Al content in the supernatants was measured employing Atomic Absorption tissue homogenates) was determined fluorometrically according to the procedure Spectrometry (Perkin Elmer, AAnalyst 300 equipped with a HGA 700 Graphite Fur- of Mokrasch and Teschke (1984). GSH levels are expressed as lmol/g of tissue nace and an AS-70 autosampler). The temperature of the furnace was programmed protein. as described elsewhere (Johnson and Treble, 1992). Absorbance was measured at 309.3 nm. Preparation of samples for Al analysis: All samples were prepared in eppendorf 2.8. Statistical analysis tubes and analyzed immediately after preparation. A 5% v/v HNO3 and 0.1% v/v Data are presented as mean ± S.E.M. Statistical analysis was performed with Mg solution prepared by proper dilutions from HNO3 solution (65%, Carlo Erba) 2+ GraphPad Instat 3 software using the nonparametric Mann–Whitney test for eval- and Mg stock standard solution (1000 lg/mL Mg ±2 lg to 5% HNO3, Chem-Lab), was used as a matrix modifier in order to stabilize Al in the furnace. uating statistically significant differences (p < 0.05) between the experimental The standard addition method was employed for the analysis. Four samples groups. were prepared for each aliquot, adding known amounts (spikes) of the analyte

(Al) from a stock standard solution (1000 ppm in 1 M HNO3 Fisher Scientific, UK). The spiked concentration ranged from 0 (unspiked sample) to 60 ppb. The volume 3. Results was kept equal for all samples by means of addition of the matrix modifier. Preli- minary tests assured that the overall concentration of Al was within the linear 3.1. General observations range of the technique. Analysis: Absorbance for each sample was measured three times. A calibration curve for each supernatant aliquot was constructed (OriginPro 8). The results are HPLC analysis showed that saffron extract contains a variety of expressed as lg Al/g wet tissue. crocins: trans-crocin-4 constitutes 46% of total crocin content of the crude extract, trans-crocin-3, 26%; cis-crocin-4, 12%; and trans-crocin-2, 7%, as previously described (Papandreou et al., 2.6. HPLC determination of crocetin in brain 2006; Tarantilis et al., 1995). Crocetin determination in whole brain (-ce) of Al + saffron treated mice was All animals developed normally during the experimental period performed by an isocratic reversed-phase liquid chromatographic method devel- and no mortality was recorded during Al treatment. Also, there oped by Chryssanthi et al. (2011) for plasma analysis, with slight modifications. were no differences on body weight gain and liquid intake, be- In detail, a high volume (3.5 mL) of the whole brain homogenate (100 mg/mL, SS tween the experimental groups. fraction) was submitted to solid phase extraction on reversed phase Strata-X car- tridges (200 mg/3 mL) consisting of a surface modified with styrene–divinylben- zene polymer that were obtained from Phenomenex (Torrance, CA, USA). The 3.2. Effect of saffron on learning and memory ability of Al-exposed crocetin eluate was evaporated to dryness in a speed Vac system and the dry resi- mice due was redissolved in 30 lL of the HPLC mobile phase. The samples were chro- matographed on a Luna C-18 column (4.6 Â 250 mm, 5 lm) with a mobile phase consisting of methanol–water–trifluoroacetic acid (75.0:24.5:0.5, v/v/v) at a flow Passive avoidance task was performed to examine long-term rate of 1.0 mL minÀ1. Crocetin was quantified using spiked brain standards at the memory. The initial latency (IL) time, measured at training trial, concentrations of 0.5, 1.0, 1.5, 2.0 and 3.0 lM of total crocetin (Chembiotin, purity is an indicator of motor activity, and step-through latency (STL) >98%). Results are expressed as nmol of total crocetin/g wet tissue. time, measured at testing trial, indicates memory of the aversive experience. Al intake resulted in impaired long-term memory of 2.7. Biochemical assays mice, as evidenced by the significantly lower (59%) mean STL time of Al-treated animals compared to mean STL time of the controls 2.7.1. Determination of cholinesterase activity The activity of SS and DS isoforms of AChE and BuChE in brain and liver samples, (Fig. 1). The lack of difference of mean STL time between Al + was determined spectrophotometrically based on Ellman’s assay and previously de- saffron- and Al-treated groups (Fig. 1), shows that saffron extract scribed protocols (Ellman et al., 1961; Lassiter et al., 1998; Papandreou et al., 2009). co-administered with Al during the last 6 days of the treatment Total ChE activity was measured by acetylthiocholine iodide (ATCh, Sigma–Aldrich, period was ineffective in reversing Al-induced memory UK) as substrate, whereas BuChE activity was determined by S-butyrylthiocholine iodide (BuTCh, Sigma–Aldrich, Switzerland) as substrate, since BuTCh is hydrolyzed only by BuChE (Lassiter et al., 1998). AChE activity was then calculated by subtract- ing BuChE activity from total ChE activity. Enzyme activity is expressed as lmol of substrate hydrolyzed/min/g of tissue.

2.7.2. Determination of monoamine oxidase activity The activity of both isoforms of MAO, MAO-A and MAO-B, was assessed in mouse whole brain (-ce) and cerebellum by a fluorimetric assay based on previ- ously described protocols (Mahmood et al., 1994; Xu et al., 2010). Since MAO is a mitochondrial membrane-bound enzyme, the DS fractions of brain tissues homog- enates were used. Briefly, 50 lL of brain sample was preincubated at 37 °C for 20 min with 100 lL of either 1 lM R-(À)-Deprenyl hydrochloride (Deprenyl, MAO-B inhibitor, Sigma–Aldrich, USA) or 1 lM N-Methyl-N-propargyl-3-(2,4- dichlorophenoxy)propylamine hydrochloride (Clorgyline, MAO-A inhibitor, Sig- ma–Aldrich, USA) in 100 lL30mMNa2HPO4, pH 7.6. Then 10 lL of 3.07 mM kynur- amine dihydrobromide (Sigma–Aldrich, USA) was added to the reaction mixture as substrate. The mixture was then incubated at 37 °C for 15 min again. After incuba- tion, the reaction was terminated by adding 100 lL of 0.6 M perchloric acid and centrifuging the mixture at 1500g for 10 min to remove precipitated proteins. An Fig. 1. Effect of saffron administration on initial (IL) and step-through (STL) latency aliquot of 0.3 mL of the supernatant was added to 2 mL of 1 N NaOH. The fluores- time of Al-exposed adult mice in step-through passive avoidance task. Al treated cence intensity of the produced 4-hydroxyquinoline was detected at an excitation mice displayed lower learning/memory capacity during the testing session of the wavelength of 315 nm and an emission wavelength of 380 nm, using a fluorescence task, compared to the controls, whereas saffron co-treatment was ineffective. Each spectrometer. The concentration of product was estimated from a corresponding horizontal line represents the mean latency time of ten animals in each group standard fluorescence curve of 4-hydroxyquinoline (1–100 lM) (Sigma–Aldrich, (n = 10). Individual values are presented as squares for control mice, triangles for Al USA). MAO activity is expressed as nmol of 4-hydroxyquinoline formed/g tissue/ treated and circles for Al + saffron treated ones. ap < 0.05 vs. STL time of the controls min. (non-parametric Mann–Whitney test). 166 Z.I. Linardaki et al. / Food and Chemical Toxicology 52 (2013) 163–170 impairment. The mean IL time values were not significantly differ- and 21%) and cerebellum (23%, 15% and 22%, 19%) compared to ent among all experimental groups (Fig. 1), implying no difference the controls, except of the activity of DS-BuChE of whole brain in motor activity of mice in training session. (-ce) that remained unchanged. Further significant (p < 0.05) reduction of the SS- and DS-AChE activity in whole brain (-ce) 3.3. Al and crocetin levels in cerebral tissues (15%, 28%) and cerebellum (24%, 27%) was observed in Al + saffron treated mice in comparison with Al treated group, whereas saffron The levels of Al in whole brain (-ce), cerebellum and liver of treatment did not further affect the activity of cerebral BuChE. control, Al-treated and Al + saffron treated mice are presented in Also, Al-treated mice displayed significantly (p < 0.05) decreased Table 1. Al was not detected in whole brain (-ce) of control mice, liver BuChE (11% SS, 21% DS) and SS-AChE (25%) activities whereas it was determined in high amounts in cerebellum. In compared to the controls, while Al + saffron treated mice displayed all animal groups Al levels in cerebellum were significantly further inhibition of liver BuChE (27% SS, 19% DS) and SS-AChE (p < 0.05) higher than those of the remaining brain. Also, Al con- (31%) compared to Al-treated mice. centrations were significantly (p < 0.05) increased in both cerebral tissues and liver of Al-treated mice compared to the controls and 3.5. Effect on brain MAO activity did not differ significantly (p < 0.05) in the cerebral and liver tissue of Al + saffron treated mice, compared to Al-treated group. The effect of administration of either Al alone or in the presence Pooled whole brain (-ce) homogenates of Al + saffron treated of saffron extract at the end of the treatment period, on MAO iso- and control mice were analyzed by HPLC. HPLC chromatogram forms activity of mouse whole brain (-ce) and cerebellum, is pre- (Supplementary data) of Al + saffron treated brain (chromatogram sented in Fig. 2. Al-treated mice displayed significantly (p < 0.05) B) shows a peak at 12.4 min, which is not detected in control brain higher activities of MAO-A (19%) and MAO-B (10%) in their whole (chromatogram A), and corresponds to trans-crocetin. UV–visible brain (-ce) and significantly lower MAO-B activity (14%) in cerebel- absorption spectrum of the peak presents a maximum double peak lum, compared to the controls. Short-term co-administration of at 420–450 nm, characteristic of the carotenoids. Crocetin concen- saffron restored MAO-A and MAO-B activity of whole brain (-ce) tration, was 2.0 ± 0.1 nmol total crocetin/g tissue (n = 2) in whole (decrease by 14% and 10%, respectively) and MAO-B activity (17% brain of mice treated with saffron extract and not detected in brain increase) of cerebellum, to normal levels. of control mice. 3.6. Effect on brain and liver oxidant/antioxidant indices 3.4. Effect on brain and liver ChE activity MDA and GSH levels, as indicators of lipid peroxidation and cel- The impact of Al or saffron treatment on the activities of both lular antioxidant defense, respectively, were measured in mouse isoforms (SS, DS) of AChE and BuChE in whole brain, cerebellum brain tissues and liver of all experimental groups. As shown in and liver of adult mouse is presented in Table 2. Al intake de- Fig. 3a, Al intake increased (18%) significantly (p < 0.05) MDA levels creased significantly (p < 0.05) the activity of both SS and DS iso- only in whole brain (-ce) in comparison with controls, while the forms of AChE and BuChE in mouse whole brain (-ce) (26%, 17% subsequent short-term co-administration of saffron restored (decrease by 22%) MDA levels to normal. Short-term co- administration of saffron also improved the antioxidant status of Table 1 Al concentrationsa in cerebral tissues and liver of control, Al-treated and Al + saffron cerebellum and liver by significantly lowering the levels of MDA, treated adult mice. even though these tissues remained unaffected by Al treatment (Fig. 3a). Additionally, Al-treated mice displayed remarkable Mice groups Whole brain (-ce) Cerebellum Liver reduction in GSH content of whole brain (-ce) (44%), cerebellum ** Control ND 7.77 ± 0.62 3.97 ± 0.15 (16%) and liver (29%), compared to the control animals, whereas Al treated 1.30 ± 0.45** 11.67 ± 0.63* 7.62 ± 0.53*,** Al + saffron treated 0.97 ± 0.12** 10.17 ± 1.47 10.22 ± 0.98* Al + saffron treated mice exhibited elevated levels of GSH (29%, 14% and 22%, respectively) compared to Al-exposed group (Fig. 3b). ND, not detected. a Al concentrations are expressed as lg/g wet tissue. Data are the mean ± SEM (n = 4 animals/group). 4. Discussion * p < 0.05 vs. the control group. ** p < 0.05 vs. cerebellum of the respective group (non-parametric Mann–Whitney The present study investigates the neuroprotective potential of test). a 6-day i.p. administration of saffron extract to adult mice that

Table 2 Activity of SS- (salt soluble) and DS- (detergent soluble) isoforms of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) in brain and liver of control, Al treated and Al + saffron treated adult mice.

Mice groups Whole brain (-ce) Cerebellum Liver SS-ChE DS-ChE SS-ChE DS-ChE SS-ChE DS-ChE AChE activity (lmol/min/g tissue) Control 11.17 ± 0.42 76.72 ± 0.99 6.32 ± 0.37 16.29 ± 0.61 5.93 ± 0.14 1.07 ± 0.31 Al treated 8.26 ± 0.20* 63.47 ± 3.57* 4.88 ± 0.14* 13.87 ± 0.22* 4.47 ± 0.36* 0.90 ± 0.15 Al + saffron treated 6.99 ± 0.28*,# 45.43 ± 1.32*,# 3.69 ± 0.18*,# 10.06 ± 0.17*,# 3.10 ± 0.34*,# 0.68 ± 0.17 BuChE activity (lmol/min/g tissue) Control 1.78 ± 0.08 2.49 ± 0.07 2.47 ± 0.09 1.53 ± 0.06 26.95 ± 0.68 9.64 ± 0.29 Al treated 1.40 ± 0.07* 2.50 ± 0.05 1.93 ± 0.12* 1.24 ± 0.04* 23.86 ± 0.80* 7.60 ± 0.26* Al + saffron treated 1.50 ± 0.07* 2.40 ± 0.06 1.84 ± 0.07* 1.13 ± 0.07* 17.42 ± 0.42*,# 6.17 ± 0.25*,#

Data are the mean ± SEM (n = 6 animals/group). * p < 0.05 vs. the control group. # p < 0.05 vs. Al treated group (non-parametric Mann–Whitney test). Z.I. Linardaki et al. / Food and Chemical Toxicology 52 (2013) 163–170 167

rats on passive avoidance task has been shown by Bhalla et al.

(2010) after long-term administration of a double dose of AlCl3 in drinking water, while Sethi et al. (2008) showed declined spatial learning abilities of rats in Morris-water maze test after adminis-

tration of the same dose of AlCl3 (50 mg/kg/day) in drinking water for 6 months. Saffron co-administration at the end of the treatment period failed to reverse Al-induced cognitive deficits. In our previ- ous study, short-term saffron administration enhanced learning/ memory in healthy adult and aged mice in passive avoidance task (Papandreou et al., 2011). However, in the current report the administered dosage of saffron extract and/or the duration of expo- sure (which are the same as in the study of Papandreou et al., 2011) were insufficient to counteract Al-induced memory deficit. Fig. 2. Effect of saffron administration on monoamine oxidase activity (MAO-A and Al levels were determined in whole brain (-ce), cerebellum and MAO-B) of Al-exposed adult mouse brain. Al-induced activation of both MAO isoforms in whole brain (-ce), inhibition of MAO-B in cerebellum and reversal liver of all animal groups prior to biochemical evaluation. The val- effects of saffron are evident. Data are the mean ± SEM (n = 6 animals/group). ues of Al found in cerebral tissues, are comparable with those of ⁄p < 0.05 vs. the control group, #p < 0.05 vs. Al treated group (non-parametric other studies that use Al as neurotoxic agent, although different Mann–Whitney test). Al forms, doses and routes of administration are investigated (Esparza et al., 2005; Kaizer et al., 2008; Sánchez-Iglesias et al., 2007). Our findings showed high concentrations of Al in cerebel- were previously exposed to high Al intake through their drinking lum of control mice (higher than liver), but none or below detec- water for a 5-week period. To this end, cognitive behavior and tion limits in whole brain (-ce), in accordance with Bellés et al. brain cholinergic, monoaminergic and oxidative indices were as- (1998). In the current study, long-term AlCl3 intake through drink- sessed. Our findings showed that long-term Al intake induced ing water resulted in significantly increased Al concentrations in learning/memory decline in adult mice. Impaired performance of whole brain, cerebellum and liver of adult mice. Our results are

Fig. 3. Effect of saffron administration on (a) lipid peroxidation (MDA) and (b) reduced glutathione (GSH) levels of Al-exposed adult mouse brain and liver. Al-induced increase in MDA levels of whole brain (-ce), decrease in GSH levels of whole brain (-ce), cerebellum and liver, and reversal effects of saffron are evident. Data are the mean ± SEM (n = 6 animals/group). ⁄p < 0.05 vs. the control group, #p < 0.05 vs. Al treated group (non-parametric Mann–Whitney test). 168 Z.I. Linardaki et al. / Food and Chemical Toxicology 52 (2013) 163–170

in accordance with previous studies where AlCl3 is administered to ChE activities, has not been studied before. The considerable higher rodents (Bhalla and Dhawan, 2009; El-Maraghy et al., 2001). The percentage of inhibition of DS-AChE in whole brain of Al + saffron entry of Al into the brain through the blood–brain barrier has long treated mice compared to SS-AChE, suggests greater susceptibility been established (Yokel et al., 1999) and is also confirmed by our of the enzyme’s DS isoform to saffron treatment. Geromichalos results. The accumulation of Al in brain is consistent with the ob- et al. (2012) have recently demonstrated, for the first time, that served impaired learning/memory ability of mice in passive avoid- crocetin inhibits AChE by binding at two different loci, the catalytic ance test. However, cerebral Al levels did not differ significantly center and the peripheral anionic sites, as assessed by in vitro enzy- between Al- and Al + saffron treated mice, showing that short-term matic and molecular docking studies. Thus, the observed signifi- saffron administration in the end of the treatment period did not cant cerebral AChE inhibitory activity conferred by saffron short- affect Al bioavailability. term co-administration, may be attributed to a direct interaction Additionally, for the first time, we determined crocetin, the ma- of crocetin with the enzyme, since crocetin was detected in brain jor metabolite (aglycon) of the bioactive crocin glycosides of saf- of Al + saffron treated mice and was absent in controls’ brain; how- fron, in whole brain of Al + saffron treated mice, which was ever, it remains to be investigated whether the crocetin concentra- absent from the brain of control mice. The detection of crocetin tion is sufficient for such an effect. Furthermore, other indirect in mouse brain demonstrates for the first time that this compound mechanisms cannot be excluded. crosses the blood–brain barrier when saffron extract is adminis- Monoamine oxidase (MAO, types A and B) is a mitochondrial tered for a short term through intraperitoneal route. In recent re- membrane-bound enzyme which catalyzes the oxidative deamina- port (Chryssanthi et al., 2011), the presence of crocetin in human tion of monoamines, thus regulating monoaminergic neurotrans- plasma was shown after 2 and 24 h of saffron tea consumption, mission. Increased brain MAO activity has been recorded in while only Yoshino et al. (2011) have demonstrated the distribu- neurodegenerative process (Hanish Singh et al., 2011; Mallajosyula tion of crocetin in rat brain after a single oral administration of et al., 2008). Our results showed significant increase of whole brain pure crocetin, up to now. MAO isoforms activity after Al intoxication, and an opposite effect The fact that saffron short-term co-administration failed to re- on cerebellum MAO-B activity. Bhalla et al. (2010) have also pre- verse the cognitive decline raises questions on the dosage, the per- sented increased and decreased MAO activity in rat cerebrum iod and the route of administration, before definite conclusions can and cerebellum, respectively, after long-term oral administration be drawn about its effectiveness. However, our study presents of AlCl3. Activation of MAO isotypes in rat brain by prolonged in- some remarkable brain biochemical alterations that followed saf- take of Al has been also previously reported (Huh et al., 2005), fron co-treatment. but the underlying mechanisms have not yet been clarified. Determination of ChE activity showed that the DS-AChE was Saffron co-treatment completely reversed Al-induced activation predominant in cerebral tissues, while approximately equal values of brain MAO isoforms and inhibition of cerebellar MAO-B. The ef- of the activity of the two BuChE isoforms were recorded. These fect of saffron administration on cerebral MAO activity has not observations are consistent with previous findings (Das et al., been studied before. MAO inhibitors have long been well charac- 2005; Fernández-Gómez et al., 2008). In accordance with other terized for their antidepressant properties (Bortolato et al., 2008). studies (Lassiter et al., 1998; Li et al., 2000), AChE activity consti- The first small-scale clinical trials of saffron in the treatment of tutes the major fraction of total ChE activity in whole brain (-ce) mild to moderate depression, have shown significant benefits in and cerebellum, whereas BuChE activity predominates in liver. In the mood of patients after a 6-week treatment (Akhondzadeh the current work, Al intake resulted in significant decrease of the et al., 2005; Noorbala et al., 2005). The antidepressant efficacy of activity of AChE and BuChE isoforms in mouse whole brain and cer- saffron could, at least in part, correlated with brain MAO inhibitory ebellum. Region-specific inhibition of the activity of rat cerebral activity of saffron extract and the presence of crocetin in brain that membrane-bound AChE and BuChE after Al intoxication, has been are presented in our current study. shown by others (Julka et al., 1995). Previous studies have shown Oxidative stress has long been implicated in the initial and later necrotic rat hippocampal ultrastructural changes following the stages of neuronal degeneration (Melo et al., 2011). In the present same Al administration protocol (Jyoti and Sharma, 2006), provid- study, Al intake elevated significantly lipid peroxidation in whole ing an explanation for our cholinotoxic and memory impairment brain and decreased GSH content in both cerebral tissues and liver. results. However, Al has been suggested to interfere with the ac- Other reports have also demonstrated increased lipid peroxidation tion of metabotropic glutamate receptors, enhancing excitotoxicity levels and decreased GSH content in rat brain regions after long- and neuronal injury (Blaylock, 2012). Also, Al impairs hippocampal term oral administration of AlCl3 (Jyoti and Sharma, 2006; Nehru long-term potentiation (LTP) in rats (Platt et al., 1995). Thus, be- and Bhalla, 2006). The resistance of liver to Al-induced lipid perox- sides the cholinotoxic effects of Al in the present study, the idation, indicated by us and other studies (Kaneko et al., 2004), is involvement of possible Al-induced glutamatergic neurotransmis- expected, as it constitutes the major site of detoxification in living sion disturbances in the observed memory decline, should also organism. Although Al is not a redox-active metal, there is exten- be considered. In liver, both BuChE isoforms’ activity (the main li- sive experimental evidence on oxidative stress-mediated Al neuro- ver ChE fraction) was significantly decreased following Al adminis- toxicity (Kumar and Gill, 2009). It has been demonstrated that Al tration, while only SS-AChE activity was decreased significantly. induces oxidative stress in cultured rat hippocampal neurons by However, increased activity of soluble and membrane-bound potentiating iron-mediated oxidative injury (Xie et al., 1996). Also, forms of rat liver BuChE after Al intake through diet for 100– in vitro studies (Oteiza, 1994) have shown that Al stimulates iron- 115 days (Dave et al., 2002), has been previously demonstrated, induced lipid peroxidation in isolated membrane fractions through but the Al dose and period of exposure are different. binding to the membrane and promotion of changes in the Saffron short-term co-administration caused further significant arrangement of membrane lipids. Considering the byproduct reduction of cerebral AChE and liver BuChE isoforms activity. Cere- (hydrogen peroxide) of MAO-mediated reactions, Al-induced acti- bral BuChE activity was not further affected and only SS-AChE vation of brain MAO isoforms provided by our results could, at activity of liver was further significantly decreased after saffron least in part, contribute to the observed increased brain oxidative co-treatment. The inhibitory effect of saffron on the activity of stress. brain SS- and DS-AChE in adult healthy mice has been demon- Reversal effects of saffron short-term co-administration were strated in our previous study (Papandreou et al., 2011). However, recorded against Al-induced brain lipid peroxidation and GSH con- the impact of saffron administration on brain BuChE and liver tent reduction in cerebral tissues and liver. In our previous study Z.I. Linardaki et al. / Food and Chemical Toxicology 52 (2013) 163–170 169

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Additionally, in our previous in vitro work (Papand- Blaylock, R.L., 2012. Aluminum induced immunoexcitotoxicity in neuro- reou et al., 2011) both saffron extract and its crocetin component developmental and neurodegenerative disorders. Curr. Inorg. Chem. 2, 46–53. Bortolato, M., Chen, K., Shih, J.C., 2008. Monoamine oxidase inactivation: from provided strong protection against H2O2-induced toxicity in pathophysiology to therapeutics. Adv. Drug Deliv. Rev. 60, 1527–1533. human neuroblastoma SH-SY5Y cells, by repressing reactive Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of oxygen species production. It has been reported that crocin, the microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254. di-gentiobiosyl ester of crocetin, promotes mRNA expression of Chryssanthi, D.G., Lamari, F.N., Georgakopoulos, C.D., Cordopatis, P., 2011. 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Our findings show that long-term intake of a relative high dose El-Maraghy, S.A., Gad, M.Z., Fahim, A.T., Hamdy, M.A., 2001. Effect of cadmium and of AlCl3 through drinking water resulted in metal accumulation in aluminum intake on the antioxidant status and lipid peroxidation in rat tissues. adult mouse brain tissues and exerted neurotoxic effects, as evi- J. Biochem. Mol. Toxicol. 15, 207–214. Erasmus, R.T., Savory, J., Wills, M.R., Herman, M.M., 1993. Aluminum neurotoxicity denced by the declined learning and memory capacity, metal- in experimental animals. Ther. Drug Monit. 15, 588–592. induced inhibition of ChEs, changes of MAO and the production Esparza, J.L., Gómez, M., Rosa Nogués, M., Paternain, J.L., Mallol, J., Domingo, J.L., of oxidative damage. On the other hand, short-term co-administra- 2005. Melatonin reduces oxidative stress and increases gene expression in the cerebral cortex and cerebellum of aluminum-exposed rats. J. Pineal Res. 39, tion of saffron extract at the end of the treatment period benefi- 129–136. cially affected mouse brain oxidative stress and antioxidant Fernández-Gómez, F.J., Muñoz-Delgado, E., Montenegro, M.F., Campoy, F.J., Vidal, status markers and MAO activity that were disturbed by Al. The C.J., Jordán, J., 2008. The level of butyrylcholinesterase activity increases and the content of the mRNA remains unaffected in brain of senescence-accelerated bioavailability of crocetin in mouse brain after saffron extract mouse SAMP8. Chem. Biol. Interact. 175, 332–335. administration through intraperitoneal route demonstrated in the Geromichalos, G.D., Lamari, F.N., Papandreou, M.A., Trafalis, D.T., Margarity, M., present study, supports the implication of this bioactive compo- Papageorgiou, A., Sinakos, Z., 2012. Saffron as a source of novel acetylcholinesterase inhibitors: molecular docking and in vitro enzymatic nent in the observed neurochemical alterations. However, the par- studies. 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Eleni V. Pappa,1 Aikaterini A. Zompra,1 Zoi Diamantopoulou,2 Zinovia Spyranti,1 George Pairas,1 Fotini N. Lamari,1 Panagiotis Katsoris,2 George A. Spyroulias,1 Paul Cordopatis1 1Department of Pharmacy, University of Patras, Patras 26504, Greece 2Department of Biology, University of Patras, Patras 26504, Greece

Received 3 April 2012; revised 31 May 2012; accepted 2 July 2012 Published online 17 July 2012 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/bip.22123

ABSTRACT: the well-defined b-turn conformation of GnRH-I. # Lamprey gonadotropin-releasing hormone type III 2012 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 98: (lGnRH-III) is an isoform of GnRH isolated from the sea 525–534, 2012. lamprey (Petromyzon marinus) with negligible endocrine Keywords: Lamprey GnRH-III; lGnRH-III analogs; activity in mammalian systems. Data concerning the solution structure GnRH-I; solution structure lGnRH-III; superior direct anticancer activity of lGnRH-III have antiproliferative activity been published, raising questions on the structure– activity relationship. We synthesized 21 lGnRH-III This article was originally published online as an accepted analogs with rational amino acid substitutions and preprint. The ‘‘Published Online’’ date corresponds to the preprint version. You can request a copy of the preprint by studied their effect on PC3 and LNCaP prostate cancer emailing the Biopolymers editorial office at biopolymers@wiley. cell proliferation. Our results question the importance of com the acidic charge of Asp6 for the antiproliferative activity and indicate the significance of the stereochemistry of Trp INTRODUCTION in positions 3 and 7. Furthermore, conjugation of an he decapeptide gonadotropin-releasing hormone-I (GnRH-I; pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro- acetyl-group to the side chain of Lys8 or side chain Gly-NH2) has a pivotal role in orchestrating and cyclization of amino acids 1-8 increased the maintaining normal reproductive events by regulat- antiproliferative activity of lGnRH-III demonstrating ing the secretion of pituitary gonadotropin hor- 1 6 8 Tmones. In addition, GnRH-I and many of its analogs exhibit that the proposed salt bridge between Asp and Lys is not an antiproliferative effect on various cancer cells, which can crucial. Conformational studies of lGnRH-III were be exerted by indirect or direct ways.2–4 Chronic administra- performed through NMR spectroscopy, and the solution tion of GnRH analogs desensitizes the pituitary gonadotroph structure of GnRH-I was solved. In solution, lGnRH-III cells, results in arrest of gonadotropin secretion, and thereby adopts an extended backbone conformation in contrast to suppresses ovarian and testicular function (chemical castra- tion). Thus, chemical castration is a therapeutic approach of hormone-dependent tumors such as prostate and breast can- Additional Supporting Information may be found in the online version of this cer. Thousands of synthetic peptide analogs of GnRH have article. Correspondence to: Paul Cordopatis, Laboratory of Pharmacognosy and Chemistry been synthesized since its discovery, and several of them of Natural Products, Department of Pharmacy, University of Patras, GR-265 04 (agonists or antagonists) have been used in breast and/or Patras, Greece; e-mail: [email protected] or Eleni V. Pappa, Laboratory of Phar- 4 macognosy and Chemistry of Natural Products, Department of Pharmacy, prostate cancer therapy. In addition, GnRH analogs also University of Patras, GR-265 04 Patras, Greece; e-mail: [email protected] exert anticancer activity by directly affecting the hormone-

VC 2012 Wiley Periodicals, Inc. dependent and -independent cancer cells, an effect which

PeptideScience Volume 98 / Number 6 525 526 Pappa et al.

Table I Yield of Synthesis and Physicochemical Properties of lGnRH-III Analogs

HPLCb a + c Peptide Code Sequence Yield (%) tR (min) MH obsd/(calcd)

1 2 3 4 5 6 7 8 9 10 lGnRH-III pGlu -His -Trp -Ser -His -Asp -Trp -Lys -Pro -Gly -NH2 76.1 7.09 1260.33/1259.33 Group A I [Asn6]-lGnRH-III 69.8 7.12 1258.92/1258.35 II [Asp6(OMe)]-lGnRH-III 54.8 7.58 1274.02/1273.36 III [Glu6]-lGnRH-III 71.1 7.23 1273.66/1273.36 IV [Gln6]-lGnRH-III 73.1 7.18 1272.10/1272.37 Group B V [DTrp3]-lGnRH-III 70.7 7.27 1259.70/1259.33 VI [DTrp7]-lGnRH-III 58.6 7.32 1260.23/1259.33 VII [DTrp3,DTrp7]-lGnRH-III 67.9 7.20 1259.83/1259.33 VIII [DTrp3,DTrp7]-lGnRH-III-NHEt 78.3 7.19 1287.92/1287.38 IX [Tic3, Tic7]-lGnRH-III 73.2 7.14 1205.55/1205.28 X [DTic3, DTic7]-lGnRH-III 77.8 7.23 1205.58/1205.28 XI [DTrp3, DTic7]-lGnRH-III 74.4 6.93 1231.97/1232.31 XII [DTic3,DTrp7]-lGnRH-III 78.5 6.83 1231.94/1232.31 Group C XIII [NMeGlu1]-lGnRH-III 81.3 7.73 1292.11/1291.37 XIV [cyclo(Glu1,Lys8)]-lGnRH-III 45.2 7.86 1276.17/1275.33 XV [cyclo(Ac-Glu1,Lys8)]-lGnRH-III 53.1 9.61 1318.14/1317.37 XVI [e-N-Ac-Lys8]-lGnRH-III 80.8 7.96 1302.23/1301.37 XVII [Glu1, e-N-Ac-Lys8]-lGnRH-III 67.1 7.21 1320.19/1319.38 XVIII [Ac-Glu1, e-N-Ac-Lys8]-lGnRH-III 80.4 8.11 1362.33/1361.42 XIX [Lys5, His8]-lGnRH-III 81.0 7.31 1259.71/1259.33 XX [cyclo(Glu1,Lys5), His8]-lGnRH-III 39.1 7.43 1276.08/1275.33 XXI [cyclo(Ac-Glu1,Lys5), His8]-lGnRH-III 43.7 8.60 1318.21/1317.37

a Yields were calculated on the basis of the amino acid content of the resin. All peptides were at least 98% pure. b For elution conditions, see Materials and Methods section. c Data obtained by ESI-MS, observed (obsd) and calculated (calcd) m/z values of MH+. could be mediated through the GnRH receptors (GnRHRs) GnRH analogs have been synthesized to enhance the anti- highly expressed in those cells. cancer potency of native lGnRH-III.5,13,16,17 Those studies have The discovery of lGnRH-III (pGlu-His-Trp-Ser-His-Asp- shown that modifications in positions 5-8 decrease the anti-

Trp-Lys-Pro-Gly-NH2), a native GnRH analog isolated from cancer activity of lGnRH-III. Systematic replacement of resi- sea lamprey (Petromyzon marinus)5 that stimulates the dues 5-8 of lGnRH-III by Ala19 showed that mutation of Asp6 release of estradiol and progesterone in the adult female sea or Trp7 resulted in the loss of antiproliferative effect probably lamprey,6,7 has stimulated further research on their potential due to an ionic interaction between these residues, which medicinal uses. Functional activity of lGnRH-III has been might stabilize the biologically active conformation of lGnRH- found in the hypothalamus of several (mammalian) species, III. In addition, the importance of indole rings of Trp3,7 was including rats, cows, and sheep, and it was originally suggested also proposed.16 However, further information on the struc- that lGnRH-III might act as a mammalian Follicle-Stimulating ture–activity relationships of lGnRH-III is still lacking. Hormone (FSH) releasing factor.8–11 However, the data con- To further investigate the structure–activity relationship cerning the effect of lGnRH-III on the selective secretion of of lGnRH-III, we synthesized 21 new lGnRH-III analogs and FSH are inconsistent.12–14 While questions concerning the en- studied their direct antiproliferative effect on prostate cancer docrine activity of lGnRH-III are yet not answered, studies cells. The analogs were arranged into three groups: (A) ana- concerning the direct anticancer activity of lGnRH-III have logs with single amino acid changes in position 6, (B) ana- been published in the last decade.15–18 lGnRH-III differs in the logs with modifications in positions 3 and/or 7, (C) analogs sequence 5-8 from the mammalian GnRH (GnRH-I)5 and is a with amino acid changes in positions 1, 5, 8 and cyclic ana- weak agonist of the mammalian GnRHR. The ability of logs (Table I). In particular, we studied the importance of the lGnRH-III to inhibit proliferation of cancer cells, combined Asp in position 6 by incorporation of several amino acids with the weak GnRH-I agonistic activity, makes it an excellent [Asn, Asp(OMe), Glu, Gln]. DTrp and L/D-1,2,3,4-tetrahy- starting compound for the development of peptide analogs droisoquinoline-3-carboxylic acid (Tic) were inserted in with high and selective anticancer activity. Currently, new positions 3 and/or 7 to further investigate the role of the

Biopolymers (Peptide Science) Structure–Activity Studies of lGnRH-III 527 indole ring of Trp and its stereochemistry. In the last group, protecting group on the resin was removed by treatment with 20% the importance of the amino acids in positions 1 and 8 are piperidine/N,N-dimethylformamide (DMF) and then, the coupling mainly studied. The utility of backbone cyclization has been reaction was performed by using threefold molar excesses of acti- vated Fmoc-amino acids throughout the synthesis. The amino acids well established in peptides, and it has been demonstrated to were activated essentially either in situ using diisopropylcarbodii- 20,21 increase biological activity and selectivity since they are mide/1-hydroxy-benzotriazol (HOBt) in DMF for 2.5 h or with usually more stable in metabolism than the parent linear O-benzotriazol-1-yl-1,1,3,3-tetramethyluronium tetrafluoroborate, molecules.22 Therefore, cyclopeptides are of great importance HOBt, and N,N-diisopropylethylamine (DIEA) (2.45:3:6). The and interest both in pharmaceutical and chemical respect. Fmoc deprotection step was accomplished by treatment with 20% piperidine in DMF or by 2% 1,8-diazabicyclo[5.4.0]undec-7-ene According to these considerations, we also synthesized 1-8 (DBU) in DMF containing 2% piperidine for 2–3 min24 to prevent and 1-5 side chain cyclic peptides and studied their antiproli- the potential migration of Dde and DMab groups to other func- ferative activity on prostate cancer cells. tional groups after the incorporation of Fmoc-DLys(Dde)-OH or/ Moreover, until today, some sporadic attempts have been and Fmoc-Glu(DMab)-OH. Regarding analogs XVI, XVII, and done to study the solution conformation of lGnRH-III, but XVIII after the removal of the DMab and/or Dde protecting groups 3 there is still no NMR 3D model available. According to the by 2% hydrazine in DMF (3 2 min), on-resin acetylation of the Na-amino group of Glu1 (peptide XVIII) and of Ne-amino group of results of a previous molecular dynamics simulation study by Lys8 (peptides XVI, XVII and XVIII) was performed by 0.5M Ac O 23 2 Lovas and coworkers, lGnRH-III has an extended helical con- in DMF for 1 h. The resin was washed manually with DMF, formation from residues 2 through 7, which is stabilized by dichloromethane (DCM), and successively to remove intramolecular hydrogen bonds and slightly polar interactions. the solvents excess. The peptidyl resin was treated with the splitting However, that conformational model has not been confirmed by mixture trifluoroacetic acid (TFA)/DCM/anisole/1,2-ethanedithiol 17 (EDT)/H2O (92:3:2:1:2, v/v/v/v/v), for 3 h to remove the protecting other studies. The determination of the conformational char- groups and the peptide from the resin. The resin was filtered off, acteristics of lGnRH-III is a crucial step for in depth study of and the solution was concentrated. Peptides were isolated by precip- structure–activity relationship. In that direction, we studied the itation with cold diethyl ether, centrifuged, dissolved in water with a solution structure of lGnRH-III through NMR spectroscopy, few drops of acetic acid, and lyophilized. Purification of crude pep- ¨ and a conformational model of this decapeptide is presented. tides was achieved by semipreparative HPLC (Mod.10 AKTA, Amer- sham Biosciences) on Supelcosil C18 (5 lm particle size, 8 mm 3 Moreover, the solution structure of GnRH-I (PDB 1YY1) was 250 mm, Sigma-Aldrich) Analytical HPLC (Waters, Malva) studied under the same experimental conditions to elucidate the equipped with a Waters C18 column (symmetry, 3.5 lm, 4.6 mm 3 conformational differences between GnRH-I and lGnRH-III. 75 mm) produced single peaks with at least 98% of the total peak integrals (integrated with Empower software). ESI-MS (Micromass- Platform LC instrument, Waters Micromass Technologies) was in MATERIALS AND METHODS agreement with the expected mass. The physicochemical properties of the new analogs are summarized in Table I. Peptide Synthesis The linear and the cyclic peptides (Table I) were rationally designed Cyclic Peptides. The precyclic peptides (analogs XIV, XV, XX, and synthesized manually. 9-Fluorenylmethoxycarbonyl (Fmoc)-pro- XXI) were synthesized on a Sieber amide resin (0.45 mmol/g tected amino acids and peptide reagents were obtained from CBL capacity) utilizing a standard Fmoc solid-phase synthesis protocol (Patras, Greece), Bachem (Bubendorf, Switzerland), and Novabio- described above. Fmoc-protected peptidyl resins were treated with chem (La¨ufelfingen, Switzerland). All solvents and reagents used for 2% DBU in DMF containing 2% piperidine for 2–3 min. Boc- solid-phase synthesis were of analytical quality. Purification of crude protecting group was inserted on the free terminal amino group of peptides was achieved by semipreparative high-performance liquid the XIV and XX after treatment with (Boc)2O (3 equiv.)/DIEA (3 chromatography (HPLC) (Mod.10 A¨KTA, Amersham Biosciences, equiv.) in DMF (1 h, RT) while, on-resin N-terminal acetylation of Piscataway, NJ) coupled to a UV/Vis detector from Amersham Phar- the peptides XV and XXI was performed by using 0.5M Ac2Oin macia Biotech on a Supelcosil C18 (5lm particle size, 8 mm 3 250 DMF for 1 h. After that step, Dde and DMab protecting groups mm,Sigma-Aldrich,StLouis,MO).AnalyticalHPLC(Waters,Malva, were removed by using 2% hydrazine in DMF (3 3 2 min). Then, 1 Milford, CT) equipped with a Waters C18 column (symmetry, 3.5 the amide bond between c-carboxyl group of Glu and the e-amino lm, 4.6 mm 3 75 mm) produced single peaks with at least 98% of group of Lys in position 8 (analogs XIV, XV) or in position 5 (ana- the total peak integrals (integrated with Empower software). Electro- logs XX, XXI) was carried out on the resin, using a mixture of spray ionization-mass spectrometry (ESI-MS, Micromass-Platform LC PyBoP/HOBt/DIEA (1.5 equiv./1.5 equiv./2.0 equiv, 3 3 4h,RT- instrument, Waters Micromass Technologies, Milford, MA) was in 608C), monitoring the formation of the lactam bridge by Kaiser agreement with the expected mass. The physicochemical properties of test. Cleavage from the resin and deprotection of the amino acid the new analogs are summarized in Table I. side chains of the cyclopeptides were performed as reported above. The resin was filtered off, and the solution was concentrated. The Linear Peptides. Sieber amide resin (0.45 mmol/g capacity) was crude product was precipitated with cold diethyl ether, centrifuged, used as a solid support for the linear peptides. All peptides were and lyophilized. Cyclopeptides were analyzed by analytical RP- synthesized manually following a Fmoc strategy. First, the Fmoc HPLC and characterized using the procedure described above. The

Biopolymers (Peptide Science) 528 Pappa et al. physicochemical properties of the new analogs are summarized in NOE Constraints Table I. Intensities of dipolar connectivities were mainly extracted by All peptides were purified by semipreparative HPLC on a RP C- NOESY maps acquired at 298 K and 400 ms of mixing time. 409 18 support using a linear gradient from 20% to 35% of solvent A and 299 NOESY cross-peaks were assigned in both dimensions for (0.1% TFA/H2O) and solvent B (0.1% TFA/acetonitrile) for 15 min GnRH-I and 1GnRH-III peptides, respectively, in DMSO-d6. The at a 2 mL/min flow rate and UV detection at 215 and 280 nm. number of unique cross-peaks was 198 and 150 (20 and 15 NOEs Eluted peptides were lyophilized immediately. The purity of each per residue for GnRH-I and 1GnRH-III, respectively). Their inten- peptide was verified by an analytical using a reversed-phase C18 col- sities were converted into upper limit distances through CALIBA.33 umn at a 1 mL/min flow rate with the same solvent system as in the Sequential constraints, number and range of NOEs are illustrated in preparative HPLC. The molecular weight of the peptides was con- Supporting Information Figures S1 and S2. firmed by using ESI-MS. The HPLC chromatogram showed that the purity of the peptides was >98%, while ESI-MS showed the correct molecular ion for the peptide. Structure Calculations and Refinement The NOE-derived structural information extracted from the analysis Cell Culture and Proliferation Assay of NOESY spectra acquired in DMSO-d6 solutions under identical The human prostate cancer epithelial cell lines PC3 and LNCaP experimental conditions for both peptides was introduced to (ATCC, American Type Culture Collection) were grown in RPMI- DYANA34,35 software for structure calculation. 113 and 87 NOE 1640 medium supplemented with 10% fetal bovine serum (FBS), constraints for GnRH-I and 1GnRH-III, respectively, were found 100 U/mL penicillin, and 100 lg/mL streptomycin. Cultures were meaningful and used in DYANA calculations. A force constant of maintained in 5% CO2 and 100% humidity at 378C. Cell culture 133.76 kJ mol1 A˚ 2 is applied for the distance constraints. The 20 reagents were from BiochromKG (Seromed, Germany). models’ ensembles of both GnRH peptides are illustrated in Figures Slow-growing LNCaP and fast-growing PC3 cells were seeded 4 and 5 (Figures are generated with MOLMOL36). Structural calcu- 4 into 48-well culture plates at 1 3 10 cells/well. After a 24-h incuba- lations have been performed on IBM RISC6000 and xw4100/ tion period, adherent cells were treated with the peptides in 2.5% xw4200 HP Linux workstations. (v/v) FBS-RPMI-1640. At the end of the incubation period, cells were fixed with methanol and stained with 0.5% crystal violet in 20% methanol for 20 min.25 After gentle rinsing with water, the retained dye was extracted with 30% acetic acid, and the absorbance RESULTS was measured at 590 nm. Comparison of mean values among groups was done using ANOVA and the unpaired Student t-test. Homogeneity of variance Synthesis of Peptides was tested by Levene’s test. Each experiment included at least tripli- The application of Fmoc/tBu solid phase strategy combined cate measurements for each condition tested. All results are with the appropriate side chain protecting groups resulted in expressed as the mean 6 SD of at least three independent experi- the appropriate peptides with good yields of 40–81% (Table ments. Values of P less than 0.05 were taken to be significant (*P < I). The preparation of side chain to side chain-cyclized pep- < < 0.05, **P 0.01, ***P 0.001). tides through lactam bridge formation requires orthogonal protecting groups for side chain amino and carboxylate func- NMR Spectroscopy tionalities. Our approach to acetylate or protect by the Boc Samples of synthetic GnRH-I and 1GnRH-III in deuterated di- protecting group the N-terminal amino group of the pro- methyl sulfoxide (DMSO-d6) were used for NMR studies. Data were tected precyclic peptide was effective to prevent the potential acquired in a wide range of temperatures from 298 K up to 343 K on a 1 formation of N -pyroglutamyl chain-terminated peptides, a Bruker Avance 400 MHz spectrometer. H 1D NMR spectra were 37 recorded using spectral width of 12–17 ppm with or without presatu- often favored during the deprotection step of Dmab group. 1 1 ration of the H2Osignal. H- H 2D Total Correlation Spectroscopy (TOCSY)26,27 were recorded using the MLEV-17 spin lock sequence 1 13 28,29 using sm ¼ 80–100 ms, and H- CHSQC spectra with 200.791 Effect on Prostate Cancer Cell Proliferation 1 1 ppm spectral width in F1. H- H TPPI Nuclear Overhauser Effect To study the anticancer activity of lGnRH-III and lGnRH-III Spectroscopy (NOESY)30,31 spectra were acquired using mixing time analogs, we tested their effect on the anchorage-dependent sm ¼ 400 ms applying water suppression during the relaxation delay and mixing time. All 2D spectra were acquired with 10.014 ppm spec- proliferation of the well-established prostate carcinoma cell tral width, consisting of 2K data points in the F2 dimension, 16–32 lines, LNCaP and PC3, that mimic different stages on the transients, and 512–1024 complex increments in the F1 dimension. progression of prostate cancer. LNCaP cells are androgen- Raw data were multiplied in both dimensions by a pure cosine- sensitive and express a large number of GnRHRs,16,17 and squared bell window function and Fourier-transformed to obtain PC3 cells are highly malignant, androgen-insensitive, and 2048 3 2048 real data points. A polynomial baseline correction was 6,38,39 applied in both directions. For data processing and spectral analysis, express lower number of GnRHrs. lGnRH-III inhibited the standard Bruker software (XWinNMR 3.5) and XEASY pro- the proliferation of LNCaP cells in a concentration-depend- gram32 (ETH, Zurich) were used. ent manner having a maximal effect (21.5% reduction) at the

Biopolymers (Peptide Science) Structure–Activity Studies of lGnRH-III 529

of lGnRH-III and significant (P < 0.01) effect on PC3 prolif- eration, incorporation of DTic in positions 3 and 7 (analog X) led to increased antiproliferative effect on both cancer cell lines (Figure 2). The antiproliferative effect of [DTrp3, DTic7]-lGnRH-III (analog XI) and [DTic3,DTrp7]-lGnRH- III (analog XII) was higher than that of lGnRH-III. In detail, analog XI inhibited the proliferation of LNCaP and PC3 cells by 36.5 6 0.9% (P < 0.001) and 32.4 6 4.0% (P < 0.001), respectively, and peptide XII, by 36.7 6 0.7% (P < 0.001) and 31.6 6 2.7% (P < 0.001), respectively.

Analogs with Amino Acid Changes in Positions 1, 5, 8 FIGURE 1 Effect of lGnRH-III and analogs I–IV on the prolifera- l and Cyclic Compounds (Group C) tion of LNCaP and PC3 cells at a final concentration of 20 M, after 1 144 h incubation for LNCaP cells and 96 h for PC3 cells. The cell The antiproliferative effect of [NMeGlu ]-lGnRH-III (analog number was estimated as described under Materials and Methods XIII) on LNCaP cells was 30% lower than that of lGnRH-III section. Results are presented as % inhibition relative to the control whereas it had no effect on PC3 cell proliferation. Cyclic ana- and are the mean 6 SEM of three independent experiments. ***P < logs [cyclo(Glu1,Lys8)]-lGnRH-III (XIV) and [cyclo(Ac- 0.001. Glu1,Lys8)]-lGnRH-III (XV) had increased inhibitory effect on cell proliferation compared to lGnRH-III; their effect on concentration of 20 lM. All analogs of lGnRH-III were tested LNCaP cells was 36.0 6 2.0 (P < 0.001) and 35.861.0 (P < at 20 lM concentration. 0.001), respectively, while on PC3 cells the observed effect was 33.8 6 2.0 (P < 0.001) and 30.6 6 3.0 (P < 0.001), Analogs Modified in Position 6 (Group A) respectively (Figure 3). Acetylation of Ne amino group of [Asn6]-lGnRH-III (analog I) and [Glu6]-lGnRH-III (analog Lys8 (analog XVI) led to significant increase in antiprolifera- III) did not have significant inhibitory effect on LNCaP and tive effect on both cell lines compared to lGnRH-III. Interest- PC3 cell proliferation whereas, the antiproliferative effect of ingly, further modifications on that analog in position 1 analog [Gln6]-lGnRH-III (IV) was similar to that of lGnRH- (analogs XVII and XVIII) decreased the effect on LNCaP cell III. Substitution of Asp in position 6 by Asp(OMe) (peptide proliferation compared to [e-N-Ac-Lys8]-lGnRH-III (Figure II) resulted in the most effective analog of that group (Figure 3). On PC3 cell line, analog XVII had similar and XVIII had e 1); the inhibition effect on LNCaP cell proliferation, was higher effect than analog with simple acetylation of N amino higher compared to lGnRH-III (32.1 6 0.9%, P < 0.001). group of Lys8. Analog XIX {[Lys5,His8]-lGnRH-III} resulting Furthermore, while lGnRH-III was ineffective on PC3 cell proliferation, analog [Asp6(OMe)]-lGnRH-III had significant (27.7 6 2.1%, P < 0.001) antiproliferative effect.

Analogs with Modifications in Position 3 and/or 7 (Group B) Analog [DTrp3]-lGnRH-III (V) had increased antiprolifera- tive activity on LNCaP cells and significant (P < 0.001) effect on PC3 cell proliferation compared to lGnRH-III, while sub- stitution of Trp7 by DTrp (analog VI) dramatically decreased the effect (no significant effect on both cell lines). Simultane- ous incorporation of DTrp in positions 3 and 7 (analog VII) FIGURE 2 Effect of lGnRH-III and analogs V–XII on the prolif- decreased the antiproliferative effect induced by analog V eration of LNCaP and PC3 cells at a final concentration of 20 lM, whereas, further modification of the C-terminal amide in after 144 h incubation for LNCaP cells and 96 h for PC3 cells. The 3 7 cell number was estimated as described under Materials and Meth- analog [DTrp ,DTrp]-lGnRH-III-NHEt (peptide VIII) ods section. Results are presented as % inhibition relative to the 3 7 increased it. Although analog [Tic , Tic ]-lGnRH-III (IX) control and are the mean 6 SEM of three independent experiments. caused lower inhibition of LNCaP cell proliferation than that **P < 0.01; ***P < 0.001.

Biopolymers (Peptide Science) 530 Pappa et al.

ual spin patterns of amino acids through scalar connectivities (Figure 4). Sequential, medium-, and long-range connectiv- ities were identified from NOESY maps acquired with

sm ¼ 400 ms. Proton chemical shift peptides are reported in Supporting Information Tables S1 and S2. As far as the GnRH-I peptide is concerned, NHNH con- nectivities of the type (i,i + 1) are detected for the region span- ning the residues His2-Leu7 while NHNH (i,i +2)NOE cross-peaks are detected for the residue pairs His2-Ser4,Trp3- 5 5 7 8 10 Tyr ,Tyr-Leu ,andArg-Gly . On the other hand, HaNH connectivities of the type (i,i + 1) are observed for the frag- FIGURE 3 Effect of lGnRH-III and analogs XIII–XXI on the prolif- ments pGlu1-Tyr5 and between the residues Leu7-Arg8,while eration of LNCaP and PC3 cells at a final concentration of 20 lM,af- HbNH type (i,i + 1) connectivities have been observed for ter 144 h incubation for LNCaP cells and 96 h for PC3 cells. The cell 1 4 number was estimated as described under Materials and Methods sec- the N-terminal tetra-peptide segment (pGlu -Ser )andforthe 5 6 tion. Results are presented as % inhibition relative to the control and Tyr -Gly pair. Moreover, HaNH (i,i +2)NOEsareidenti- are the mean 6 SEM of three independent experiments. ***P < 0.001. fied among amino acids almost all over the peptide sequence. More than 30 medium-range NOEs were observed in the after an exchange of positions between the two basic amino NOESY maps acquired with sm ¼ 400 ms. The most character- acids, His and Lys, had similar effect to lGnRH-III on LNCaP istics among them are HbNH type connectivities between and PC3 proliferation. The antiproliferative effect on LNCaP 4 6 2 amino acids Ser -Gly and protons of the aromatic ring of His cells of 1-5 cyclic analogs XX and XXI was lower than that of 4 with all backbone protons of Ser as well as NOEs between lGnRH-III and evidently decreased compared to counter- 7 10 5 8 Leu -Gly and Tyr -Arg . Long-range NOE signals involve the parts 1-8 cyclic analogs (XIV and XV, Figure 3). amide proton of N-terminal Serine at position 4 of the peptide sequence and the C-terminal at the position 9 (four

NMR Studies NOEs) as well as the amine protons of the CO-NH2 terminal Resonance Assignment. TOCSY maps of GnRH-I and group (one NOE). These data provide a solid experimental evi- 1GnRH-III peptides were first analyzed to assign the individ- dence for the spatial proximity of the peptide’s termini.

FIGURE 4 Characteristics 1H-1H TOCSY fingerprint regions of GnRH-I and 1GnRH-III pep- tides recorded at 400 MHz NMR and in DMSO-d6 at 298 K.

Biopolymers (Peptide Science) Structure–Activity Studies of lGnRH-III 531

ence of the HaNH and NHNH (i,i + 2) connectivities— together with the absence of HaNH (i,i + 1) type NOEs for Gly6—suggest the existence of a b-turn structure in this region. In this conformation, the N-terminal region is close to the C-terminal and the spatial proximity of the two ter- mini is manifested by long-range NOEs between: (i) the NH proton of Ser4 and various Pro9 protons, and (ii) the NH proton of the C-terminal amide. Since these long-range con- straints might be fundamental for the b-turn formation and the convergence of the two peptides’ termini, structure calcu- FIGURE 5 Backbone representation of the family of 20 energy lations were also performed without the above-mentioned minimized DYANA models calculated for the GnRH-I peptide illus- long-range NOEs. The resulted ensemble of 20 DYANA mod- trated in blue. Side chains are illustrated in yellow. Figures are gen- els (PDB 1YY1) is also characterized by the formation of the erated with MOLMOL. b-turn and the highly flexible peptide termini. This confor- Although the 1GnRH-III exhibits a lower number of NOE mation led us to conclude that the peptide preserves the hair- cross-peaks compared to the GnRH-I, the number of identi- pin-like conformation, and the NOE contacts that determine fied NOEs is satisfactory, and 1GnRH-III can be defined by a this conformation are all among the residues of the Ser4-Arg8 single well-determined structure. The NOE pattern involving loop. NHNH connectivities, spanning the residues His2-Lys8,for In the GnRH-I solution structure presented here, Gly6 is this peptide is rather similar to these of the GnRH-I analog, found in the tip of the loop formed by residues Ser4-Arg8 4 6 while HaNH and HbNH (i,i + 1) type connectivities have (Figure 5). NOEs of (i,i + 2) between Ha Ser and HN Gly 9 6 8 been identified all over the peptide sequence except for Pro . as well as Gly HaHN Arg indicate that achiral Both long- and medium-range NOEs involving backbone pro- residue in position 6 allows the turn formation and the U- tons present in GnRH-I were totally absent in the 1GnRH-III shape fold of the peptide. Additionally, the side chains of 5 7 analog. Only two HbHb (i,i + 2) type connectivities among Tyr and Leu seem to be in a short distance, while the Trp3 and His5 as well as one NOE cross-peak between the observed NOEs between the Tyr5 aryl protons and the Leu7 8 6 eNH of Lys and the Hb of Asp have been assigned. Moreover, methyl protons support the parallel orientation of their side the interaction among the aromatic rings of His5 and Trp7 is chains. Moreover, the side chains of His2 and Trp3 present manifested by the existence of two medium-range NOEs different orientation, while Trp3 orients its indole ring to the 5 7 5 between the He proton of His and the Hd proton of Trp . same direction with the Tyr aryl ring. The substitution of the GnRH-I tetra-peptide segments Tyr5-Gly6-Leu7-Arg8, which is located in the middle of the Structure Calculation and Conformational Analysis GnRH-I sequence with the His5-Asp6-Trp7-Lys8 segment in The NOE-derived structural information extracted from the 1GnRH-III, seems to impose great structural changes in the analysis of NOESY spectra acquired in DMSO under identical stereochemistry of the latter peptide. The spatial arrange- experimental conditions (s ¼ 400 ms) was introduced to m ment of both N- and C-terminal fragments is totally different DYANA for structure calculations. The average target function with respect to the peptide representing the native hormone, for the DYANA family of 20 calculated models was found 9.2 since no long-range NOEs involving the terminal residues 3 102 6 1.7 3 102 A˚ 2 for GnRH-I and 0.36 6 1.64 3 103 have been identified (Figure 6). Furthermore, there is no ex- A˚ 2 for 1GnRH-III, respectively. No consistent violations perimental evidence for a b-turn formation in the peptide existed at the final DYANA run, and no constraint violation segment 4-8, and consequently 1GnRH-III does not maintain was found larger than 0.20 A˚ . The RMSD values for the the hairpin structure obtained in many GnRH analogs.40,41 GnRH-I 20 models’ ensembles were calculated as 0.52 6 0.24 Despite the lower number of NOE cross-peaks identified in A˚ (BB) and 0.96 6 0.19 A˚ (HA). Similarly, the 1GnRH-III the NOESY spectra of 1GnRH-III, the peptide seems to DYANA 20 models’ ensembles exhibit pairwise RMSD values adopt a rather defined backbone conformation. Our NMR for all residues 0.70 6 0.18 A˚ (BB) and 1.52 6 0.60 A˚ (HA). data suggest that the molecule adopts extended backbone ge- ometry with three irregular turns/bends around amino acids 3D Solution Models Trp3, Ser4, and Pro9. The side chains of His2 and Trp3 still 3 Despite the fact that a typical HaNH (i,i + 3) connectivity occupy different region in space, with the indole ring of Trp has not been observed for the fragment Ser4-Arg8, the pres- found on the opposite site of the peptide backbone level in

Biopolymers (Peptide Science) 532 Pappa et al.

the biological activity of GnRH-I analogs, previous studies report that removal of the Asp6 in lGnRH-III resulted in complete loss of biological activity.19,43 To further investigate the significance of this position, we substituted Asp6 by Asn, Asp(OMe), Glu, and Gln. Incorporation of Asp(OMe) instead of Asp6 significantly increased the antiproliferative activity of lGnRH-III on LNCaP cell line. Furthermore, Asp(OMe)-analog was the only analog of that group, with significant effect on PC3 proliferation. Insertion of Gln was FIGURE 6 Backbone representation of the family of 20 energy well tolerated, while no antiproliferative activity on LNCaP minimized DYANA models calculated for the 1GnRH-III peptide 6 illustrated in blue. Side chains are illustrated in cyan. Figures are cells was observed when Glu or Asn substitutes Asp . These generated with MOLMOL. findings suggest that neither the acidic function of the side chain of Asp6 nor the putative salt bridge (Asp6-Lys8) are respect to the His2 side chain and pointing towards the crucial for the antiproliferative activity of lGnRH-III, and we indole ring of Trp7. Moreover, the aromatic ring of His5 is presume that different intramolecular interactions, that found within NOE distance with Trp7 indole ring supporting deserve further investigation, may occur and preserve the the parallel orientation of their side chains. bioactive conformation. According to Here´di-Szabo´ et al.,19 indole rings of Trp3,7 are crucial for the anticancer activity, probably due to inter- DISCUSSION actions of these residues with the N-terminal which stabilizes The ability of lGnRH-III to inhibit proliferation of cancer the biological active conformation of lGnRH-III. In our cells, combined with the weak GnRH-I agonistic activity, study, the antiproliferative effect of the analog with DTrp in makes it an excellent starting compound for the development position 3 was significantly higher than lGnRH-III whereas of peptide analogs with enhanced selectivity and anticancer DTrp7 analog was ineffective. Introduction of DTrp instead activity. In this study, we report the synthesis of 21 new of Trp3,7 decreased the anticancer activity while, combined lGnRH-III analogs and their impact on the proliferation of modification on the C-terminal resulted in a significant two different prostate cancer cell lines. Moreover, to investi- increase of the anticancer potency in both cancer cell lines. gate the conformational resemblance or diversity in GnRH Tic is a conformationally restrained imino acid that has been family, we studied the conformational properties of lGnRH- incorporated to GnRH antagonists as local structure deter- III and GnRH-I through NMR spectroscopy and their solu- minant.44 In addition, we have previously shown that substi- 3 tion models are presented herein. tution of Trp in GnRH-I by L-orD-Tic increased the anti- In our previous studies,41 we have shown that GnRH-I proliferative effect of the analogs.45 In this study, results does not exhibit significant antiproliferative activity on show that substitution of Trp3,7 by Tic decreases the antipro- LNCaP and PC3 cells even at the concentration of 100 lM. liferative effect of lGnRH-III on LNCaP cells and slightly Results of this study provide experimental evidence that the improves the effect on PC3 cancer cells. In contrast, the in- inhibitory effect of lGnRH-III, at the concentration of 20 hibitory activity of the analogs [DTic3,7]-lGnRH-III (X), lM, on LNCaP cell proliferation was 21.5 6 0.9% while, on [DTrp3,DTic7]-lGnRH-III (XI), and [DTic3,DTrp7]- PC3 cell it was negligible. The absence of high affinity bind- lGnRH-III (XII) was significantly higher. Based on these ing sites on PC3 cells42 and the lower number of GnRHRs findings, we presume that introduction of DTic, although expressed on them39 compared to LNCaP cells, may be lacks the indole ring, preserved the biologically active confor- related to the lack of responsiveness of PC3 cells following mation of lGnRH-III. Furthermore, the importance of the lGnRH-III treatment. However, many of the new analogs of stereochemistry of the residues in positions 3 and 7 is also lGnRH-III presented on this study had significant antiproli- indicated. ferative effect on both cancer cell lines. Our observations Conjugation of an acetyl-group via the side chain of Lys8 confirm the results of other studies that additional mecha- significantly increases the antiproliferative activity of lGnRH- nisms may mediate the biological effects of GnRH analogs III suggesting that the putative salt bridge between Asp6 and on cancer cells which have not yet been clarified.42 Lys8 in lGnRH-III, proposed in an earlier structure activity In lGnRH-III, the position 6 is occupied by Asp, whereas study16 and demonstrated by our findings, is not important mammalian GnRH (GnRH-I) bears Gly at this position. for the anticancer activity. Similar elevation of the antiproli- 6 Although Gly is often replaced by D-amino acids to enhance ferative effect was observed by analogs with side chain

Biopolymers (Peptide Science) Structure–Activity Studies of lGnRH-III 533

stitution of Leu7 with the bulky Trp7 differentiates remark- ably the conformation of the peptide. This also becomes evi- dent by the observed NOE interactions between His5 and Trp7, which do not favor the spatial proximity of the two ter- mini. As a consequence, the hydrophobic core present in GnRH-I, is lost in 1GnRH-III. Furthermore, the proposed ionic interaction between Asp6-Lys8 residues16 is in agreement with our findings. Trp7 changes its orientation pointing away from Lys8 and conse- quently no NOE interaction is found between their side FIGURE 7 Superimposition of the backbone of the two hor- chains (as in the Leu7-Arg8 case in GnRH-I), and Asp6 is mones, lGnRH-III is illustrated in yellow while GnRH-I in blue. The 8 calculated RMSD of both peptides for the segment 1-6 found to be found to be within NOE distance with Lys giving rise to one 8 6 0.815A˚ . interaction between eNH of Lys and Hb of Asp . In conclusion, we synthesized 21 new lGnRH-III analogs cyclization of amino acids 1-8 (peptides XIV, XV) probably and the structure–activity studies show that several of due to a more constrained conformation. On the other hand, them had similar or higher activity than that of the parent although exchanging places between the weakly basic His5 hormone.AnalogsII,V,VIII,X,XI,XII,XIV,XV,andXVI and the more basic Lys8 did not alter the anticancer activity had higher antiproliferative activity than lGnRH-III on of the parent hormone, simultaneous 1-5 side chain cycliza- LNCaP cells and significant activity on PC3 cell. The modi- tion in analogs XX and XXI resulted in lower antiprolifera- fications of those vary significantly but their inhibitory tive effect. effect is almost the same, hampering thus the identification In our attempt to elucidate conformational characteristics of one particular ‘‘lead compound.’’ However, we have of lGnRH-III, the solution model was determined through studied ‘‘key’’ positions of lGnRH-III, providing new infor- NMR spectroscopy in DMSO, a solvent widely used for solu- mation regarding the structure–activity relationship of bility reasons and is an acceptable medium of mimicking a lGnRH-III. Our observations are illustrating that lGnRH- prototypical hydrophobic environment. Furthermore, the 3D III is a promising molecule for the development of peptide solution model of GnRH-I was also determined to shed light analogs with increased and potentially selective anticancer on the conformational differences between the two peptides. activity. The obtained results show substantially different conforma- tional features between the 1GnRH-III and the GnRH-I (Figure 7). According to our NMR study, the network of se- REFERENCES quential NOE connectivities present in GnRH-I spectra sug- 1. Millar, R.P.; Pawson, A.J.; Morgan, K.; Rissman, E.F.; Lu, Z.L. 4 8 gest the existence of b-turn structure for the Ser -Arg seg- Front Neuroendocrinol 2008, 29, 17–35. ment. Long-range NOE signals between the N- and C-ter- 2. Morgan, K.; Stewart, A.J.; Miller, N.; Mullen, P.; Muir, M.; mini provide solid experimental evidence for the spatial Dodds, M.; Medda, F.; Harrison, D.; Langdon, S.; Millar, R.P. proximity of the two termini. On the other hand, 1GnRH-III Cancer Res 2008, 68, 6331–6340. 3. Santen, R.J.; Manni, A.; Harvey, H. Breast Cancer Res Treat does not exhibit the GnRH-I conformation (U-shape). In 1986, 7, 129–145. spite of earlier publications suggesting the existence of some 4. Limonta, P.; Moretti, R.M.; Marelli, M.M.; Motta, M. Front helical properties,23 such backbone conformation cannot be Neuroendocrinol 2003, 24, 279–295. supported by our results. In contrast, the 3D models of 5. Sower, S.A.; Chiang, Y.C.; Lovas, S.; Conlon, J.M. Endocrinol- 1GnRH-III can be characterized by the extended backbone ogy 1993, 132, 1125–1131. 6. Nozaki, M.; Ominato, K.; Gorbman, A.; Sower, S.A. Gen Comp conformation forming a kink around Asp6 and two more 3 9 Endocrinol 2000, 118, 57–67. turns/bends around Trp and Pro . The present lGnRH-III 7. Tobet, S.A.; Nozaki, M.; Youson, J.H.; Sower, S.A. Cell Tissue 17 models are in agreement with Mezo et al., where a relatively Res 1995, 279, 261–270. ordered extended like backbone conformation is proposed 8. Yu, W.H.; Karanth, S.; Sower, S.A.; Parlow, A.F.; McCann, S.M. for the central region of the 1GnRH-III peptide. Proc Soc Exp Biol Med 2000, 224, 87–92. The substitution of the small and flexible residue Gly in 9. Dees, W.L.; Hiney, K.K.; Sower, S.A.; Yu, W.H.; McCann, S.M. 6 Peptides 1999, 20, 1503–1511. position 6 of the GnRH-I sequence with Asp in 1GnRH-III 10. McCann, S.M.; Karanth, S.; Mastronardi, C.A.; Dees, W.L.; does not favor the formation of the b-type conformation in Childs, G.; Miller, B.; Sower, S.; Yu, W.H. Arch Med Res 2001, the 4-8 segment of the peptide sequence. Moreover, the sub- 32, 476–485.

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Biopolymers (Peptide Science) Publication: Molecular Vision; Type: Research Article Article ID: 999; Volume: 19; EJP Article Number: 2013MOLVIS0007

Formatted: Numbering: Continuous 1 Saffron administration prevents selenite-induced 2 cataractogenesis 3 Olga E. Makri,1 Anastasia-Varvara Ferlemi,2 Fotini N. Lamari,2 Constantine D. 4 Georgakopoulos1 5 1Department of Ophthalmology, Medical School, University of Patras, Patras, Greece 6 2Laboratory of Pharmacognosy & Chemistry of Natural Products, Department of Pharmacy, 7 University of Patras, Greece 8 Correspondence to: Constantine D. Georgakopoulos, Ophthalmology, Morfou 1 Street, 9 26441 Patras, Greece; Phone: +30 2610 999904, FAX: +30 2610 434914; email: 10 [email protected]

11 Abstract 12 Purpose: The present study sought to investigate whether Crocus. sativus stigmas (saffron) 13 extract prevents selenium-induced cataractogenesis in vivo, and to study the its possible 14 protective mechanism. 15 Methods: Wistar rat pups were randomized into 3 three groups. Group I (control) received 16 subcutaneous injection of normal saline on postnatal day 10. Groups II (selenite-treated) and 17 III (selenite+saffron-treated) received subcutaneous injection of sodium selenite (20 µmol/kg 18 bodyweight) on postnatal day 10. Group III also received intraperitoneal injections of saffron 19 extract (60 mg/kg bodyweight) on postnatal days 9 and 12. On postpartum day 21, rats were Comment [Au1]: Do you mean: postnatal? 20 sacrificed and the lenses were isolated and examined for cataract formation. Activities of 21 superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase, (CAT) and 22 glutathione levels, as markers of antioxidant defense, were measured in the isolated lenses. 23 Levels of the indicator of lipid peroxidation, malondialdehyde (MDA), and protein oxidation 24 (sulfhydryl content) in the lens were also determined. The Effect effect of the different 25 treatments on lens protein profile was evaluated with the through an estimation of the soluble 26 to insoluble protein ratio and SDS–PAGE sodium dodecyl sulfate polyacrylamide gel 27 electrophoresis analysis of the water-soluble fraction (WSF) of lens proteins. 28 Results: Saffron demonstrated significant protection against selenite-induced 29 cataractogenesis in vivo. The mean activities of superoxide dismutaseSOD, glutathione 30 peroxidaseGPx, catalase, CAT and glutathione levels were significantly increased in group 31 III compared to the selenite-treated group. Saffron significantly prevented selenite-induced 32 lipid peroxidation, protein oxidation, as well as and proteolysis and insolubilization of the 33 lens WSF. 34 Conclusions: Saffron extract prevented selenite-induced cataract formation in Wistar rats, 35 possibly by through the reinforcement of antioxidant status, reduction of the intensity of lipid 36 peroxidation, protection of the sulfhydryl groups, and inhibition of proteolysis of the lens Comment [Au2]: Do you mean: or? 37 WSF. These findings highlight the anti-cataractogenic potential of saffron by virtue of its 38 antioxidant property.

39 Introduction 40 Cataract is a degenerative condition and is the consequence of results from the loss of the 41 crystalline lens transparency. According to data provided by the World Health Organization, 42 cataract is one of the major causes of visual impairment globally and the first cause of

Page 1 of 20 Publication: Molecular Vision; Type: Research Article Article ID: 999; Volume: 19; EJP Article Number: 2013MOLVIS0007 43 blindness, accounting for 51% of cases worldwide [1]. It is estimated that about 40 million 44 people will eventually have severely reduced vision due to cataract by the year 2020 [2], 45 especially in the developing countries of Asia and Africa [3]. The only available treatment 46 for cataract is the surgical removal of the opaque lens and its replacement with an artificial 47 one to restore visual acuity. The aforementioned procedure has been optimized over the last 48 decades so as to become an everyday surgery in the developed countries. Nevertheless, 49 cataract cataract-induced blindness remains a severe health problem in the developing 50 countries, due to the low socioeconomic status of the patients and limitations in the 51 acceptability, accessibility, and affordability of cataract surgical services [4].

52 Since surgical treatment is not widely available, many researchers seek for have investigated 53 pharmaceutical agents to prevent this disease. It has been estimated that a delay of 10 years 54 in the onset of cataract, by any means, would halve the number of patients needing surgery 55 [2]. Actually, over the last few years, great emphasis has been laid placed on exploring the 56 possible protective effects of several natural products against cataract formation. Natural 57 products are privileged candidates for drug discovery, since they are often endowed with 58 multiple functions. Cataractogenesis is considered to be a multi-factorial disease, correlated 59 with various pathogenetic mechanisms, that have not been completely clarified. There is a 60 large body of evidence demonstrating that oxidative stress (i.e., measurably increased levels 61 of reactive oxygen species ROS and oxidized substrate molecules - —lipids, 62 sulphydrylssulfhydryls, nucleic acids) is compulsory to cataract development [5-9]. 63 Consequently, enhancement of the antioxidant defenses of the lens could prevent or delay the 64 onset of cataract. Based on that premise, numerous natural products, with known antioxidant 65 properties, have been evaluated during in the last decades.

66 Saffron consists of the dried stigmas of Crocus sativus L., which is a bulbous perennial plant 67 of the family Iridaceae. Saffron is has been used and consumed since antiquity, and is one of 68 the most expensive spices; it is used for flavoring and coloring food preparations, as a 69 perfume, and also as a dye. It is cultivated in many Mediterranean countries, like including 70 Spain, Greece, and Turkey, as well as in India and Iran (its major producer) [10]. Saffron’s 71 The value of saffron is attributed to its characteristic phytochemical components; , 72 specifically the unique hydrophilic carotenoids, i.e., crocins (glycosidic esters of crocetin), 73 picrocrocin (terpenoid glycoside responsible for the exquisite taste), and safranal (the main 74 essential oil component) [11-13]. Hippocrates and Dioscurides mention the use of saffron for 75 the treatment of ophthalmic disorders, among other uses. Since then, saffron has been

Page 2 of 20 Publication: Molecular Vision; Type: Research Article Article ID: 999; Volume: 19; EJP Article Number: 2013MOLVIS0007 76 constantly used in traditional medicine, as therapy of for several conditions such as insomnia, 77 depression, bronchospasm, cardiovascular diseases, gastrointestinal disorders, menstrual 78 pain, menopausal problems, as analgesic, and even against cancer [10,14]. Some of the 79 medicinal-biologic properties of saffron, or its components, are attributed to its antioxidant 80 features, which have been highlighted in several studies [15-21]. The objective of the present 81 study is to investigate, for the first time in the literature, the effect of saffron extract against 82 selenite-induced experimental cataract in vivo.

83 Methods

84 Chemicals 85 Commercially available saffron was kindly provided from by the Cooperative de Safran

86 (Krokos Kozanis), West Macedonia, Greece. Sodium selenite (44%–46%), catalase (CAT) Comment [Au3]: Please provide the manufacturer’s city. 87 from bovine liver (4966 U/mg), Purpald, N-ethylmaleimide, L-glutathione reduced, Comment [Au4]: Molecular Vision style requires a materials source and source’s location for ALL 88 glutathione oxidized, glutathione peroxidase (GPx), and N-acetyl-cysteine were purchased materials used. Please carefully check your manuscript and complete all missing materials 89 from Sigma-Aldrich (St. Louis, MO 63,103, USA). A Superoxide Dismutase Assay Kit from source information, and complete areas where materials source information is missing. 90 Cayman Chemical Company (Ann Arbor, USA) was used for the determination of Comment [Au5]: Can you say: reduced L- glutathione, oxidized glutathione? 91 superoxide dismutase (SOD) activity.

92 Preparation of saffron extract 93 Saffron stigmas powder was extracted with methanol:water (50%v/v; 18 mlL/ 250 mg) for 4 94 h at room temperature, in the absence of light and with continuous stirring. The extract was 95 centrifuged, filtered, and evaporated to dryness in using a Speed Vac System (Labconco

96 Corp., Kansas City, MO). The composition was screened with HPLC on a Supelcosil C-18 Comment [Au6]: Molecular Vision style requires that any abbreviated term be used at least three 97 column as previously described [13,20].The residues were stored at −20 °C until further use. times. For two or fewer times, the term should be spelled out in full. 98 Samples were redissolved in normal saline and sterilized though membrane filtration (0.2 μm Comment [Au7]: Please provide sufficient details of the protocols used so that readers 99 i.d.) for the injections. understand what was done.

100 Animals 101 New born suckling Wistar rat pups were used in our experiment. The pups were housed 102 along with their mothers in stainless-steel cages in well well-ventilated rooms with controlled 103 temperature (23 °C) and humidity conditions with and 12 h:12 h light-dark cycles12 h 104 dark/light cycles. The mothers were maintained on a standard laboratory animal diet in the

105 form of dry pellets and provided tap water ad libitum throughout the experimental period. Formatted: Font: Not Italic

106 Animals were randomly assigned to 3 three groups:

Page 3 of 20 Publication: Molecular Vision; Type: Research Article Article ID: 999; Volume: 19; EJP Article Number: 2013MOLVIS0007 Formatted: Bulleted + Level: 1 + Aligned at: 107  Group I (control, n=9 animals), which received only subcutaneous injection of 0.25" + Indent at: 0.5" 108 normal saline.;

109  Group II (Seleniteselenite-treated, n=8 animals), which received a subcutaneous 110 injection of sodium selenite (20 µmol/kg bodyweight) on postnatal day 10.; and

111  Group III (Seleniteselenite+Saffronsaffron-treated, n=9 animals), which received a 112 subcutaneous injection of sodium selenite (20 µmol/kg bodyweight) on postnatal day 113 10 and intraperitoneal injections of saffron extract (60 mg/kg bodyweight) on 114 postnatal days 9 and 12.

115 Cataract formation could be evaluated from the 16th day, when the pups opened their eyes, 116 with the help of an ophthalmoscope and later on with the naked eye. Bilateral cataracts were

117 observed in the animals used for this study. On postpartum day 21, rats were anaesthetized Comment [Au8]: Do you mean: postnatal?

118 with diethyl ether and then sacrificed by cervical dislocation. The eyes were enucleated and Comment [Au9]: According to Molecular Vision style, methods of animal euthanasia must be clearly 119 lenses were at once excised intracapsularly through an incision 2 mm posterior to the limbus spelled out: dosage, type, route, etc.

120 under surgical microscope magnification. Both lenses were obtained from each rat and 121 morphological examination for cataract formation was performed by gross examination of 122 lenses under the magnification of the dissecting microscope against a background of black 123 grid lines.

124 Staging of the cataract formation was conducted by an examiner, blinded regarding the 125 studied study groups, based on a scale 0 to 3 according to from Geraldine et al. [22]. The 126 degree of opacification was graded as follows:

Formatted: Bulleted + Level: 1 + Aligned at: 127  Grade 0: absence of opacification (gridlines clearly visible; Figure 1A); 0.25" + Indent at: 0.5"

128  Grade 1: a slight degree of opacification (minimal clouding of gridlines, with 129 gridlines still visible; Figure 1B);

130  Grade 2: presence of diffuse opacification involving almost the entire lens (moderate 131 clouding of gridlines, with gridlines faintly visible; Figure 1C);

132  Grade 3: presence of extensive thick opacification involving the entire lens (total 133 clouding of gridlines, with gridlines not seen at all; Figure 1D)

134 The experiments were conducted in strict compliance with the ARVO Association for 135 Research in Vision and Ophthalmology Statement for Use of Animals in Ophthalmic and 136 Vision Research and Guiding Principles in the Care and Use of Animals [23]. The study has Comment [Au10]: I would recommend stating 137 been approved by the University Hospital Bioethics Committee. this at the beginning of the section.

Page 4 of 20 Publication: Molecular Vision; Type: Research Article Article ID: 999; Volume: 19; EJP Article Number: 2013MOLVIS0007 138 Tissue treatment 139 Prior to biochemical analysis, the lenses were washed in ice-cold saline to remove blood and 140 then weighed carefully. The lenses were homogenized in 30 mM phosphate buffer, pH 7.6,

141 (10% w/v), and centrifuged at 15,000 rpm for 20 min at 4 °C. The supernatant obtained was Comment [eXtyles11]: If rpm is used in the context of centrifugation, then it needs to be replaced 142 stored at −30 °C, pending further analysis. with “g” gravity. The formula to convert rpm to g is: RCF = (1.119 × 10−5) × (rpm)2 × r, where r is the mean radius of rotation in cm, and rpm is the centrifuge speed in revolutions per minute. 143 Analytical methods

144 Assay of superoxide dismutaseSOD activity 145 Superoxide dismutase SOD activity was determined according to the Superoxide Dismutase 146 Assay Kit, Cayman Chemical Company, which is based on the formation of a tetrazolium 147 salt for the detection of superoxide radicals generated by xanthine oxidase and hypoxanthine. 148 The absorbance was read at 450 nm. The results are expressed as units/mLml, where one unit 149 of SOD is defined as the amount of enzyme needed to exhibit 50% dismutation of the 150 superoxide anion.

151 Assay of catalase activity 152 The peroxidative activity of catalase CAT was determined according to the modified method 153 of Sinha [24]. The method is based on the reaction of the enzyme with methanol in the

154 presence of an optimal concentration of H2O2. The formaldehyde produced is measured 155 colorimetrically at 540 nm with reaction with Purpald (4-amino-3-hydrozino-5-mercapto-

156 1,2,4-triazole) and oxidation with KIO4. Standard concentrations of formaldehyde (0–75 μΜ) 157 were used for the construction of a calibration curve. All determinations were performed in 158 triplicate. The results are expressed as nmol/min/mLml.

159 Glutathione Peroxidase peroxidase (GPx) activity 160 GPx activity was determined colorimetrically, according to following Rotruck et al. [25], to

161 estimate the rate of the glutathione oxidation by H2O2. Reduced glutathione was used as 162 substrate and DTNB [5,5′-dithio-bis(2-nitrobenzoic acid)] as a chromogen. Suitable aliquots 163 of standard concentrations of GPx (0.025–1 U/mLml) were, also, used for the construction of 164 the calibration curve. The color that was developed was read against a reagent blank at 412 165 nm. Results were expressed as units/mg tissue (one unit was the amount of enzyme that

166 converted 1 μmol of reduced to the oxidized form of glutathione in the presence of H2O2/ 167 min).

Page 5 of 20 Publication: Molecular Vision; Type: Research Article Article ID: 999; Volume: 19; EJP Article Number: 2013MOLVIS0007 168 Estimation of reduced and total glutathione content 169 A modified modification method of Hissin and Hilf’s method was used for the determination 170 of reduced (GSH) and oxidized glutathione (GSSG) content in the lens [26]. The two forms 171 of glutathione were estimated fluorometrically after reaction with o-phthalaldehyde (OPT).

172 The samples were treated with 50% TCA and centrifuged to eliminate the protein content. Comment [Au12]: Molecular Vision style requires that any abbreviated term be used at least 173 The supernatant was extracted three times with diethyl ether. The aqueous fraction was used three times. For two or fewer times, the term should be spelled out in full. 174 to determine the GSH and GSSG content; GSH selectively reacts reacted with OPT

175 (10 mg/mL ml in pure methanol) at pH 8.0 (500 mM Na2HPO4 buffer), whereas after the

176 addition of 5 mM NEM in the samples, only GSSG reacted with OPT at pH 12.0 (0.2 N Comment [Au13]: Molecular Vision style requires that any abbreviated term be used at least 177 NaOH). Standard concentrations of GSH and GSSG (0.75–15 μΜ) were used. The excitation three times. For two or fewer times, the term should be spelled out in full. 178 wavelength was at 340 nm and the fluorecencefluorescence intensity was determined at 420 179 nm. The respective concentrations in nmol/mg wet tissue are were calculated from the 180 respective calibration curves. The concentration of total glutathione was calculated by adding 181 GSH and GSSG.

182 Estimation of protein sulfhydryl content 183 The protein sulfhydryl content was estimated using the Ellman’s procedure as slightly 184 modified by Sedlak and Lindsay [27]. Briefly, the pellets from the samples treated with 50% 185 TCA for the determination of glutathione were redissolved with a Tris-EDTA-guanidine HCl 186 (3 mM: 3 mM: 3M) buffer, pH 8.9. In a 96-well microplate, 250 μL μl of the sample were 187 was mixed with 20 μL μl DTNB [5,5′-dithio-bis(2-nitrobenzoic acid)]. After incubation for 188 15 min at room temperature, the absorbance was read at 412 nm. The content of protein 189 sulfhydryls was calculated using a calibration curve prepared with N-acetyl-cysteine 190 (0.06 mM-–1 mM). The protein content of each sample was evaluated using the Bradford

191 assay [28]. Since NAC contains only one sulfhydryl group, the results were expressed as Comment [Au14]: Molecular Vision style requires that any abbreviated term be used at least 192 nmol -–SH/mg protein. three times. For two or fewer times, the term should be spelled out in full. Comment [Au15]: Should this be defined? 193 Determination of Lipid lipid Peroxidationperoxidation 194 Lipid peroxidation was determined by the evaluation of malondialdehyde (MDA) levels in 195 the rat lens. Due to the small volume of tissue sample, the fluorimetric method of Grotto et 196 al. [29] and Jentzsch et al. [30] was used with slight modifications to measure MDA levels 197 using a 96-well microplate. MDA was determined after the reaction with thiobarbituric acid 198 and extraction with n-butanol. The excitation wavelength was at 515 nm and the fluorescence 199 intensity was determined at 553 nm. The results were expressed as nmol MDA/g tissue 200 against a standard curve (0.05–10 μΜ MDA).

Page 6 of 20 Publication: Molecular Vision; Type: Research Article Article ID: 999; Volume: 19; EJP Article Number: 2013MOLVIS0007 201 Sodium- dodecyl- sulfate polyacrylamide gel (SDS–PAGE) electrophoresis of 202 water soluble proteins 203 Samples (30 μg protein) were loaded on a 12% polyacrylamide gel (1 mm thickness) 204 according to a modified modification of method of Laemmli’s method [31]. Before loading, 205 lens homogenate was mixed with sample buffer (35% v/v glycerol, 18% v/v 2- 206 mercaptoethanol, 230 mM Tris/HCl pH=6.8, 10% w/v sodium dodecyl sulfate [SDS], 0.3% 207 w/v bromophenol blue). Each mixture was heated at 100 °C for 5 min and then it was 208 centrifuged for 3 min. The protein mix was left to cool down at room temperature.

209 Electrophoresis was performed in using a Cleaver Scientific Ltb (Warwickshire, UK) Comment [Au16]: Do you mean: Ltd.? Comment [Au17]: Please provide the 210 apparatus, at 100V for approximately 2 h. For the visualization of protein lanes, gels were manufacturer’s city. 211 stained with 0.5% w/v Coomassie brilliant blue in methanol: acetic acid: water (3:1:6 v/v/v) 212 and destained. Image J Launcher was used for the densitometric analysis of gels (at least four 213 different experiments).

214 Determination of lens proteins 215 The determination of insoluble and soluble protein content in the rat lenses of each group 216 was estimated by using the Bradford assay, using with BSA bovine serum albumin as a 217 standard [28]; and the results were expressed as mg protein/ mg wet tissue. The ratio of 218 soluble to insoluble proteins per sample was calculated.

219 Statistical analysis 220 All variables were tested for normality with the Kolmogorov–Smirnov test. Differences in 221 clinical score between the groups were estimated with the Mann–Whitney U test. One-way 222 ANOVA analysis of variance was used to detect differences in continuous variables among 223 the three groups. The Bonferroni post hoc test was used for pairwise comparisons. A p value 224 less than 0.05 was considered statistically significant. The Analysis analysis was performed 225 using SPSS 15.0 software (SPSS, Inc., Chicago, IL).

226 Results

227 Morphological assessment of cataract formation 228 In group I, all the eyes were clear. In group II, 100% of the eyes developed moderate to 229 severe cataract, indicating success in establishing the selenite-induced cataract model in our 230 experiment. Stage 2 was the common endpoint of the cataractogenic process, accounting for 231 62.5% of the eyes; (median 2, (range 2–3). On the contraryIn contrast, saffron 232 coadministration (group III) significantly retarded selenite-induced cataractogenesis in vivo.

Page 7 of 20 Publication: Molecular Vision; Type: Research Article Article ID: 999; Volume: 19; EJP Article Number: 2013MOLVIS0007 233 In this group, 38.9% of the lenses exhibited stage 1 cataract, 44.4% of the eyes developed 234 stage 2 cataract, and only 16.7% developed dense cataract; (median 2, (range 1–3; Table 1, 235 Figure 1).The difference in exhibited cataract exhibited between in the control and selenite- 236 treated group was significant (p<0.0001, Mann–Whitney U test). The difference of in 237 cataract frequency and severity between groups II and III was statistically significant 238 (p<0.05, Mann–Whitney U test).

239 Antioxidant status in the lens

240 Superoxide dismutaseSOD activity 241 The activity of SOD (mean±standard deviation [SD]) was significantly lower in the lenses of 242 the group II compared to that of the normal lenses (p<0.001; Table 2). However, saffron co- 243 administration (group III) resulted in significantly higher SOD activity levels compared to 244 the group II, that which received only sodium selenite (p<0.05), although they are were 245 lower than those of the group I (control; p<0.05).

246 Catalase activity 247 The mean activity of CAT (mean±SD) in the lenses of group II rats was significantly lower 248 than that of the lenses in control group (p<0.01; Table 2). In the group III, CAT activity was 249 significantly higher than in group II (p<0.05). In fact, saffron coadministration resulted in the 250 prevention of the loss of CAT activity, which was measured at levels similar to those of the 251 control group (p>0.05).

252 Glutathione Peroxidase peroxidase (GPx) activity 253 The mean activity of GPx (mean±SD) in lenses of group II was significantly lower than that 254 of the lenses in the control group (p<0.001; Table 2). In the selenite+saffron treated group 255 (III), GPx activity was significantly higher than in the selenite-treated group (p<0.001).

256 Reduced and total glutathione 257 For the further evaluation of the antioxidant status of the lenses, the reduced and total 258 glutathione levels (mean±SD) were evaluated (Table 3). Selenite administration resulted in a 259 significant reduction of reduced glutathione in comparison with normal lenses (p<0.05). 260 Cotreatment with saffron (group III) restored the reduced glutathione concentration, which 261 reached to a level that was not statistically significantly different from the control group 262 (p>0.05). The Lens lens total glutathione pool was depleted after selenite administration, 263 while saffron showed a sparing effect on total glutathione content.

Page 8 of 20 Publication: Molecular Vision; Type: Research Article Article ID: 999; Volume: 19; EJP Article Number: 2013MOLVIS0007 264 Levels of lens protein sulfhydryls 265 The level of protein sulfhydryl groups is an important indicator of tissue protein oxidation. In 266 group II, protein sulfhydryl content (mean±SD) was significantly lower compared to control 267 (p<0.001; Table 4). However, in group III, lens protein sulfhydryl content was significantly 268 higher than the concentration observed in the selenite-treated group (p<0.05), demonstrating 269 its protective effect against protein oxidative damage. Sulfhydryl content in the group III 270 reached a level that was not statistically significant lower from than group I (p>0.05).

271 Levels of the indicator of lipid peroxidation in lens (malondialdehyde) 272 Malondialdehyde The MDA level (MDA; mean±SD) reflects the overall tissue lipid 273 peroxidation [32]. MDA in In group II, this was 91.6% higher than in group I (p<0.0001; 274 Table 4). In group III, MDA it was 54.8% lower compared to the selenite-treated group 275 (p<0.0001) and similar to those that of the control group (p>0.05).

276 Effect of Saffron saffron on Lens lens Proteinsproteins 277 Calculation of the soluble to insoluble ratio (mean±SD) revealed a significantly lower ratio 278 in the selenite-treated group when compared to the control group (p<0.0001; Table 4). In 279 group III, saffron resulted in a significantly higher ratio in comparison to the group II 280 (p<0.05). The ratio in group III was still significantly lower from than the that of normal 281 lenses (p<0.001).

282 The relative changes in the water-soluble fraction (WSF) of lens proteins were detected by 283 SDS–polyacrylamide gel electrophoresis (PAGE) analysis. Figure 2 shows the a significant 284 decrease of in low molecular weight proteins in the selenite-treated lens; proteins between 10 285 and 30 kDa in group II were lower (~35%) than those in group I. Saffron coadministration 286 seems to diminish the observed proteolysis of those these proteins, since the densitometric 287 analysis revealed a significant increase of protein (25–30 kDa) bands intensity (13%–27%) 288 compared to group II. Particularly, the intensity of the 19–21 kDa protein band in group III 289 was similar to that of the control (group I).

290 Discussion 291 Over the last In recent years, studies have been conducted to evaluate the possible beneficial 292 effects of saffron or its components in ophthalmology. All the published data are with make 293 reference to the pathology of the retina and its possible beneficial effects in conditions such 294 as ischemic retinopathy, age age-related macular degeneration, or other neurodegenerative 295 conditions [33-38]. However, despite the continuously increasing literature on the

Page 9 of 20 Publication: Molecular Vision; Type: Research Article Article ID: 999; Volume: 19; EJP Article Number: 2013MOLVIS0007 296 neuroprotective effects of saffron or its components in the retina, to date, no study has been 297 conducted to evaluate saffron’s its possible effects against cataract formation.

298 The purpose of our study was to examine in vivo the potential anti-cataract effects of saffron 299 extract in selenite-induced cataract. Our clinical observations of the morphological 300 examination of the lenses showed that cataract formation caused by selenite administration 301 was attenuated by the administration of saffron, suggesting its anti-cataract potential. 302 Furthermore, biochemical analyses were conducted to support the clinical observation.

303 The selenite cataract model was selected because of the for its rapid, effective, and 304 reproducible cataract formation [39,40]. Although the rate of lens opacification is much more 305 rapid than in human cataract, the selenite model shows an amount of general similarities to 306 human senile nuclear cataract, such as the formation of vesicles, increased levels of calcium, 307 proteolysis, a decrease of the in water-soluble proteins, the presence of insoluble proteins, 308 and diminished amounts of GSH. The mode of action of sodium selenite in cataractogenesis 309 has not been completely defined, but it is indicated that the main biochemical events, — 310 induced by selenite overdose, —are triggered by the selenite selenite-induced oxidative stress 311 and the generation of reactive oxygen species (ROS) in the aqueous humor in combination 312 with a decrease in the activity of antioxidant enzymes such as SOD, CAT, GPx, glutathione 313 transferase, and glutathione reductase, and of as well as GSH content in the lens [40,41]. 314 More precisely, it is assumed that oxidative damage occurs to critical sulfhydryl groups of 315 proteins of the lens epithelium membranes. The aforementionedThis, in combination with 316 oxidative stress-–induced lipid peroxidative damage of lenticular membranes, leads to the 2+ 2+ 317 inactivation of membrane proteins, such as Ca -ATPase. Inhibition of the function of Ca - Comment [Au18]: Can this be defined? 318 ATPase results in loss of calcium homeostasis and calcium accumulation in the lens. As a 319 consequence of calcium influx in rodent lenses, calpains, a family of well well-characterized 320 calcium dependent proteases, are activated. Calpain activation, mainly m-Calpain and Lp82 321 in rodent lenses, induces rapid proteolysis of the water soluble proteins, that is, lens 322 crystallins, and cytoskeletal proteins. Proteolysis exposes the hydrophobic regions of lens 323 proteins, which then interact to form insoluble aggregates [39]. Insolubilization results in 324 precipitation and aggregation of lens fragmented proteins, and finally, in loss of lens 325 transparency [40].

− 326 SOD and CAT make up a primary line of defense against superoxide anion (O2 ) and

327 hydrogen peroxide (H2O2), respectively. More precisely, SOD is a specific scavenger of

328 superoxide anion. It converts harmful superoxide radicals to H2O2, which is detoxified by Formatted: Not Superscript/ Subscript

Page 10 of 20 Publication: Molecular Vision; Type: Research Article Article ID: 999; Volume: 19; EJP Article Number: 2013MOLVIS0007 329 CAT to harmless by-products. GPx, which belongs to the family of selenoproteins, is present 330 in relatively large amounts in the epithelium of the lens [42]. It catalyzes the reduction of a 331 variety of hydroperoxides with the help of its reducing substrate, GSH. The enzyme GPx 332 maintains the integrity of the phospholipid bilayer of membranes by putting a brake on the 333 lipid peroxidation initiated by superoxide. In our experiment, administration of sodium 334 selenite resulted in significant reduction of the activities of the antioxidant enzymes SOD, 335 CAT, and GPx, whereas such a decline was prevented in the lenses of animals co-treated 336 with saffron. It could be postulated that saffron, improves the antioxidant defense 337 mechanisms of the lens, leading to restoration of the levels of SOD, CAT, and GPx activities, 338 in spite of exposure to sodium-selenite.

339 GSH, a tripeptide of glycine, , and cysteine, is found in unusually high levels in 340 the crystalline lens and is believed to play a significant role in the maintenance of the 341 reduced state in the lens. GSH (via its side sulfhydryl group) is involved in the detoxification

342 of potentially damaging oxidants such as H2O2 and dehydroascorbic acid, and in the 343 protection against oxidation of membrane –SH groups, that which are important for cation 344 transport regulation. Finally, it protects cytoskeletal proteins and prevents the cross linking of 345 soluble crystallins, and, therefore, maintenance of its levels is vital for the preservation of 346 lenticular transparency [43-45].GSH levels decrease with age and are found reduced in most 347 types of cataract [45,46]. Furthermore, selenite overdose has been proved to cause a 348 significant depletion of lens GSH levels [47,48]. Restoration of GSH and total glutathione 349 levels could be attributed to the beneficial effects of saffron treatment on the enzymes 350 involved in glutathione synthesis [49,50].

351 The crystallins are subject to oxidative changes, which include including the formation of 352 disulfide and other inter- and intramolecular cross-links, resulting to in their aggregation 353 [51]. Furthermore, one of the primary events in selenite selenite-induced cataractogenesis 354 theory is the oxidative damage that occurs to proteins of the lens epithelium membranes [39]. 355 Free sulfhydryl (–SH) groups of proteins are mainly responsible for their antioxidant 356 response, and they can serve as a sensitive indicator of oxidative stress [52]. Consequently, 357 the reduced level of protein sulfhydryl content is an indication of tissue protein oxidation. In 358 the present study, in contrast to the lower level of lens protein sulfhydryl content of the 359 selenite-treated group, saffron coadministration kept the protein sulfhydryl content near to 360 control levels, indicating its protective effect against oxidative damage.

Page 11 of 20 Publication: Molecular Vision; Type: Research Article Article ID: 999; Volume: 19; EJP Article Number: 2013MOLVIS0007 361 Free radical radical–induced lipid peroxidation is a highly destructive process and has been 362 implicated as one of the main components of the pathogenetic mechanism of selenite 363 selenite-induced cataractogenesis [47]. The accumulated peroxidation products damage vital 364 membrane structures [53]. MDA is a product of the breakdown of mainly unsaturated fatty 365 acids through the oxidation mechanism [54]. In the present study, such a disturbance of 366 membrane lipids from selenite probably resulted in the observed increase in MDA lens 367 levels, when compared to normal lenses. The observed almost twofold reduction in the MDA 368 level in the selenite+saffron-treated group suggests that saffron possibly preserved the 369 structural integrity of the lens, thereby preventing the its opacification of the lens.

370 Selenite cataract is characterized by a marked decline in water water-soluble proteins through 371 protein insolubilization either by excessive proteolysis by calpains or through structural 372 alterations resulting from sulfhydryl oxidation [40]. The selenite-induced alteration in the 373 lens protein profile has been depicted through the calculation of the soluble/insoluble 374 proteins ratio in the selenite group in our experiment. Saffron supplementation prevented 375 protein degradation and aggregation, resulting in less cataract formation. Previous studies 376 showed that many of the soluble proteins in the approximate molecular weight ranges of 23– 377 31 kDa are from β-crystallin, 19–20 kDa are from α-crystallin, and the smear of polypeptides 378 from 21 to 23 kDa are γ-crystallins [55]. Saffron coadministration resulted in considerable 379 preservation of the pattern of lens proteins with molecular size 19 ~–21 kDa, and 25–30 kDa, 380 which probably denotes inhibition of crystalline proteolysis.

381 To conclude, in the present in vivo study, we showed, for the first time in the literature, that 382 intraperitoneal administration of saffron extract could significantly protect against nuclear 383 opacity formation in selenite selenite-treated pups. This fact indicates that saffron

384 components/metabolites can penetrate the blood-aqueous barrier and reach the aqueous Comment [Au19]: Please check the change. 385 humor. It has been recently shown that crocetin, the main crocin metabolite, is determined in 386 cerebral tissue after intraperitoneal administration of saffron extract [56]. The extent of tissue 387 damage caused by selenite and the protective effect of saffron were evaluated clinically and 388 biochemically. Saffron’s The modulatory effect of saffron seems to be associated with a 389 reduction of the intensity of lipid peroxidation and protein oxidation, maintenance of 390 antioxidant status, and inhibition of proteolysis of the lens WSF. This study has focused on 391 the scientific validation of saffron as an anticataract agent, suggesting that saffron, (or its 392 components, ) exhibits a promising anti-cataractogenic effect. However, it is important to 393 emphasize that certain differences exist between human and selenite experimental cataract.

Page 12 of 20 Publication: Molecular Vision; Type: Research Article Article ID: 999; Volume: 19; EJP Article Number: 2013MOLVIS0007 394 This preliminary study is encouraging, but further studies are research is required to find out 395 determine whether a similar anti-cataractogenic potential can be demonstrated in humans.

396 Acknowledgments Comment [eXtyles20]: Please be sure to 397 The study was supported by ‘K. Karatheodoris’ Grant No C913 from the Research include funding sources and grant numbers. The 398 Committee, University of Patras, Greece. correct format for a regular award from NEI is R01EY123456. Note no spacing or dashes. 399 References 400 1. Pascolini D, Mariotti SP. Global estimates of visual impairment: 2010. Br J 401 Ophthalmol 2012; 96:614–8. 22133988 402 2. Brian G, Taylor H. Cataract blindness–challenges for the 21st century. Bull World 403 Health Organ 2001; 79:249–56. 11285671 404 3. Age-Related Eye Disease Study Research Group. Risk factors associated with age- 405 related nuclear and cortical cataract: a case-control study in the Age-Related Eye 406 Disease Study, AREDS Report No. 5. Ophthalmology 2001; 108:1400–8. 407 11470690 408 4. Foster A. Cataract and “Vision 2020-the right to sight” initiative. Br J Ophthalmol 409 2001; 85:635–7. 11371475 410 5. Taylor A, Jacques PF, Epstein EM. Relations among aging antioxidant status and 411 cataract. Am J Clin Nutr 1995; 62:1439S–47S. 7495245 412 6. Spector A. Oxidative stress-induced cataract: mechanism of action. FASEB J 1995; 413 9:1173–82. 7672510 414 7. Lou MF. Redox regulation in the lens. Prog Retin Eye Res 2003; 22:657–82. 415 12892645 416 8. Berthoud VM, Beyer EC. Oxidative stress, lens gap junctions, and cataracts. 417 Antioxid Redox Signal 2009; 11:339–53. 18831679 418 9. Michael R, Bron AJ. The ageing lens and cataract: a model of normal and 419 pathological ageing. Philos Trans R Soc Lond B Biol Sci 2011; 366:1278–92. 420 21402586 421 10. Abdullaev FI, Espinosa-Aguirre JJ. Biomedical properties of saffron and its 422 potential use in cancer therapy and chemoprevention trials. Cancer Detect Prev 2004; 423 28:426–32. 15582266 424 11. Abe K, Saito H. Effects of saffon and its constituent crocin on learning behavior 425 and long-term potentiation. Phytother Res 2000; 14:149–52. 10815004 426 12. Abdullaev FI. Cancer chemopreventive and tumoricidal properties of saffron 427 (Crocus sativus L.). Exp Biol Med 2002; 227:20–5. 11788779 428 13. Tarantilis PA, Tsoupras G, Polissiou M. Determination of saffron (Crocus sativus 429 L.) components in crude plant extract using high-performance liquid chromatography- 430 UV–visible photodiode-array detection-mass spectrometry. J Chromatogr A 1995; 431 699:107–18. 7757208 432 14. Giaccio M. Crocetin from saffron: an active component of an ancient spice. Crit 433 Rev Food Sci Nutr 2004; 44:155–72. 15239370

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560

Comment [Au21]: Molecular Vision style 561 Figure 1. Transillumination pictures of lenses representative of each grade. Lens requires that all figure legends be written in complete sentences. Please rewrite all sentence 562 opacification of experimental animals corresponding to each grade. A: Grade 0: absence of fragments in this legend and review this point for all figures. 563 opacification (gridlines clearly visible; A); B: Grade 1: a slight degree of opacification, with Comment [Au22]: Can these sentences be combined into one title? 564 minimal clouding of gridlines (B); C: Grade 2: diffuse opacification involving almost the Formatted: Font: Bold 565 entire lens, with gridlines faintly visible, (C); D: Grade 3: extensive dense opacification Formatted: Font: Bold 566 involving the entire lens (gridlines not visible; D). Formatted: Font: Bold Formatted: Font: Bold

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567 568 Figure 2. Sodium- dodecyl- sulfate PAGE polyacrylamide gel electrophoresis of the lens 569 water soluble protein fraction. An equal amount of protein (30 μg) was loaded on 12% 570 sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE). The first right lane 571 indicates the molecular weight marker; Group I lane represents the protein profile of the 572 normal untreated lens; Group II, the selenite-treated group; Group III, the selenite+saffron- 573 treated group. In the normal untreated lens water-soluble fraction (WSF), the prevalence of

Page 18 of 20 Publication: Molecular Vision; Type: Research Article Article ID: 999; Volume: 19; EJP Article Number: 2013MOLVIS0007 574 proteins with a molecular mass of 19–30 kDa (characteristic of crystallins) is shown. Selenite 575 treatment led to a significant reduction (~35%) of in those these proteins, which was 576 prevented by saffron coadministration.

577 Table 1. Morphological assessment of cataract formation of each group’s isolated lenses No of lenses with different degree of opacification

Grade Grade Grade Grade Median Group 0 1 2 3 (range) I (n=18) 18 0 0 0 0 (0) II (n=16) 0 0 10 6 2 (2–3) * III (n=18) 0 7 8 3 2 (1–3) *, ‡ 578 Incidence and staging of cataract formation of the isolated lenses in each experimental group. 579 Group I: Control, Group II: Selenite-treated, Group III: Selenite+Saffron-treated. The Mann– 580 Whitney U test was used for pairwise comparisons. Different symbols indicate statistically 581 significant difference between groups. * p<0.0001 compared with group I. ‡ p<0.05 582 compared with group II.

583 Table 2. Activities of antioxidant enzymes of the lenses of each group. Enzyme Group I Group II Group III Superoxide dismutase (unit/ml) 0.37±0.07 0.18±0.05** 0.26±0.03#, ‡ Catalase (nmol/min/ml) 0.07±0.01 0.04±0.005* 0.067±0.02‡ Glutathione peroxidase (μmol glutathione oxidized/min/mg tissue) 14.71±1.39 7.39±1.30** 12.34±1.93† 584 Activities of the enzymes superoxide dismutase, catalase and glutathione peroxidase of the 585 lenses of each experimental group. Group I: Control, Group II: Selenite-treated, Group III: 586 Selenite+Saffron-treated. Each value represents the mean ± SD of six determinations. 587 Statistical analysis was performed by one-way ANOVA with Bonferroni’s adjustment . 588 Different symbols indicate statistically significant difference between groups of each enzyme 589 activity. *p<0.01; **p<0.001; #p<0.05 compared with group I. ‡p<0.05; †p<0.001 compared 590 with group II.

591 Table 3. Total and reduced glutathione levels. Parameters Group I Group II Group III Reduced glutathione (nmol/mg wet tissue) 0.33±0.1 0.17±0.02* 0.31±0.13

Page 19 of 20 Publication: Molecular Vision; Type: Research Article Article ID: 999; Volume: 19; EJP Article Number: 2013MOLVIS0007

Total glutathione (nmol/mg wet tissue) 0.50±0.17 0.36±0.07 0.52±0.18 592 Reduced and total glutathione levels in the lens of Wistar rat pups of each group. Group I: 593 Control, Group II: Selenite-treated, Group III: Selenite+Saffron-treated. Values represent the 594 mean ± SD of six determinations. Statistical analysis was performed by one-way ANOVA 595 with Bonferroni’s adjustment. Symbol indicates statistically significant difference between 596 groups II and I (p<0.05)

597 Table 4. Levels of the indicators of protein (sulfhydryl content) and lipid (malondialdehyde) 598 oxidation and ratio of soluble/insoluble proteins in lens of each group. Component analyzed Group I Group II Group III Protein sulfhydryl (nmol -SH/mg protein) 0.93±0.1 0.67±0.08** 0.82±0.1‡ Malondialdehyde (nmol/g tissue) 56.64±12.38 108.51±13.63* 49.04±18.22† Soluble/insoluble 2.28±0.27**, proteins 2.91±0.09 1.88±0.27* ‡ 599 Quantitative analysis of protein sulfhydryl groups and malondialdehyde levels in the lenses 600 of each experimental group. Calculated soluble to insoluble proteins ratio in each group 601 lenses. Group I: Control, Group II: Selenite-treated, Group III: Selenite+Saffron-treated. 602 Data represent the mean ± SD of six determinations. Statistical analysis was performed by 603 one-way ANOVA with Bonferroni’s adjustment. Different symbols indicate statistically 604 significant difference between groups. *p<0.0001; **p<0.001 compared with group I. 605 †p<0.0001; ‡p<0.05 compared with group II.

606

Page 20 of 20 DOI 10.1515/jpem-2012-0183 J Pediatr Endocr Met 2012; aop

Nikolitsa Koutroumani , Ioanna Partsalaki , Fotini Lamari , Athina Dettoraki , Andrea Paola Rojas Gil , Alexia Karvela , Eirini Kostopoulou and Bessie E. Spiliotis * Protective mechanisms against oxidative stress and angiopathy in young patients with diabetes type 1 (DM1)

Athina Dettoraki: University of Patras School of Medicine-Research Abstract Laboratory of the Division of Pediatric Endocrinology and Diabetes, Department of Pediatrics, Patras, Achaia , Greece Objective: Advanced glycation end-products (AGEs) via Andrea Paola Rojas Gil: University of Patras School of Medicine- their receptor, RAGE, are involved in diabetic angiopathy. Research Laboratory of the Division of Pediatric Endocrinology and Diabetes, Department of Pediatrics, Patras, Achaia, Greece ; Univer- Soluble RAGE, an inhibitor of this axis, is formed by enzy- sity of Peloponnese , Faculty of Human Movement and Quality of Life matic catalysis (sRAGE) or alternative splicing (esRAGE). Sciences, Department of Nursing, Orthias Artemidos and Plateon Malondialdehyde (MDA) is an oxidative stress marker, and Sparta, Sparta, Lakonias, Greece ferric reducing ability of plasma (FRAP) is an anti-oxidant Alexia Karvela: University of Patras School of Medicine-Research capacity marker. Laboratory of the Division of Pediatric Endocrinology and Diabetes, Methods: In isolated mononuclear blood cells from 110 Department of Pediatrics, Patras, Achaia, Greece Eirini Kostopoulou: University of Patras School of Medicine- DM1-patients (P) and 124 controls (C) (4– 29 years) RAGE Research Laboratory of the Division of Pediatric Endocrinology mRNA (g) and protein expression (pe) were measured and Diabetes, Department of Pediatrics, Patras, Achaia, by RT-PCR and Western immunoblotting, respectively. Greece Plasma levels of CML (AGEs) and sRAGE were measured by ELISA, MDA by flurometry and FRAP according to ‘ Benzie and Strain ’ . Results: P showed: (i) higher g of RAGE, especially in p > 13 Introduction years of age and > 5 years DM1, (ii) increased pe of esRAGE in DM1 > 5 years and (iii) increased FRAP and MDA. Diabetes mellitus type 1 (DM1) affects up to 35 per 100,00 Conclusions: The increased esRAGE and FRAP with children, adolescents and young adults every year, increased levels of CML and MDA possibly reflects a pro- with a rising prevalence within the last decade (1) . In tective response against the formation of diabetic compli- adults it is commonly associated with microvascular cations in these young diabetic patients. complications (diabetic nephropathy, neuropathy and retinopathy) although these may rarely also develop Keywords: advanced glycation end-products (AGEs); in adolescents (2) . The development of adult diabetic diabetes mellitus type 1; oxidative stress; RAGE; soluble complications is strongly correlated to hyperglycemia RAGE. and the duration of diabetes and several pathologi- cal mechanisms have been proposed that contribute to *Corresponding author: Bessie E. Spiliotis, University of Patras these complications. School of Medicine, Research Laboratory of the Division of Pediatric One of these mechanisms is the production of Endocrinology and Diabetes, Department of Pediatrics, Patras, advanced glycation endproducts (AGE), which are prod- Achaia, Greece, E-mail : [email protected] Nikolitsa Koutroumani: University of Patras School of Medicine- ucts derived from the non-enzymatic glycation and oxi- Research Laboratory of the Division of Pediatric Endocrinology and dation of plural substrates, such as proteins, lipids and Diabetes, Department of Pediatrics, Patras, Achaia, Greece nucleotides. Intermediate products of these processes are Ioanna Partsalaki: University of Patras School of Medicine- the amadori products, the best known of which is glyco- Research Laboratory of the Division of Pediatric Endocrinology and sylated hemoglobin A1C (HbA1c) (3) . The accumulation of Diabetes, Department of Pediatrics, Patras, Achaia, Greece Fotini Lamari: University of Patras , Laboratory of Pharmacognosy AGEs can directly modify the extracellular matrix (ECM) and Chemistry of Natural Products, Department of Pharmacy, Patras, resulting in increased stiffness of the vasculature. They Achaia, Greece can also modify hormones, cytokines and free radicals

Authenticated | [email protected] author's copy Download Date | 1/25/13 9:03 PM 2 Koutroumani et al.: Protective mechanisms against oxidative stress and angiopathy that elicit oxidative stress in several tissues and subse- Patients and methods quently evoke thrombotic and inflammatory processes intracellularly, via their receptor, RAGE. A common AGE Patient population that accumulates in diabetes is N ε-(carboxymethyl)- Lysine (CML), which is believed to be lipid and protein The study population consisted of 110 patients with DM1 (P) [90 adducts that have been found to impair nitric oxide (NO) children and adolescents (4– 18 years) and 20 young adults (18– 29 production and activate the ‘ nuclear factor kappa-light- years)] and 124 age- and sex-matched (92 children and adolescents chain-enhancer of activated B cells ’ (NF-κ B), thereby and 32 young adults) normal non-diabetic control subjects (C) (Table 1 ). In all the subjects, BMI (Body Mass Index), BMI percentile increasing atherogenesis, oxidative stress and inflam- (BMI% ), Tanner stage and waist circumference (WC) were obtained, mation (4) . together with measurements of glycosylated hemoglobin (HbA1C ), to- Lipid peroxidation in diabetes has been shown tal cholesterol, HDL and LDL cholesterol, triglyceride, liver enzymes to impair LDL uptake by the endothelial cells and it is (SGOT, SGPT), and urea and creatinine plasma levels (Table 1). In all one of the proposed mechanisms involved in diabetic the P, the duration of diabetes was also noted. microangiopathy and retinopathy (5) . Malondialdehyde DM1 patients were followed-up on a regular basis in the Outpa- tient Clinic of the Division of Pediatric Endocrinology and Diabetes (MDA) has been found to be an important marker of of the University Hospital of Patras, Greece. The inclusion criteria lipid peroxidation responsible for the milieu of oxida- for the controls were: (a) absence of diabetes and any other related tive stress and its levels have been found increased in autoimmune disease and (b) absence of obesity. Informed parental adult DM1 patients (6, 7). Furthermore, in DM1 adult consent and children ’ s assent were obtained in all cases. The study patients with retinopathy the increased levels of MDA was approved by the Ethical Committee of the University Hospital of Patras (Patras, Greece). coexist with a reduction of their anti-oxidant power, as indicated by a reduction of the ferric reducing ability of plasma (FRAP) (8) . Isolation of human peripheral blood The most widely studied receptor for AGEs is RAGE. RAGE is a cell surface receptor that belongs to the immuno- mononuclear cells globin superfamily of receptors. Full-length RAGE recep- The isolation of the human peripheral blood mononuclear cells (PB- tor is comprised of one cytoplasmic domain, responsible MCs) was performed as previously reported by our research group for intracellular signaling, one transmembrane domain (10) . Specifi cally, 20 mL of venous blood were obtained from all the and two extracellular C-type domains preceded by one subjects and this was layered over a Ficoll-Paque (Histopaque 1077; Sigma Aldrich, Dorset, UK) layer at a 2:1 ratio and centrifuged at V-type immunoglobulin type domain that plays a critical 805 × g for 35 min at 20o C. PBMCs were collected from the interface role in ligand binding. Other forms of RAGE that appear and washed with RPMI (Roswell Park Memorial Institute) media 1640 to lack the cytosolic and transmembrane domains are (GIBCO Invitrogen Corporation, Carlsbad, CA, USA) in order to re- known as soluble receptors for AGEs and can be detected move the plasma and fi coll, according to the manufacturer ’ s instruc- ° in the circulating blood. These can be produced by alter- tions. The isolated pellets of PBMCs were stored at – 80 C in order to isolate total RNA and proteins subsequently. native splicing of RAGE pre-mRNA transcripts forming the endogenous secretory RAGE (esRAGE) and by enzy- matic cleavage of the full length RAGE, forming circulat- Total RNA extraction and reverse ing soluble RAGE (sRAGE). Both esRAGE and sRAGE can bind AGEs in the circulation, thus preventing the adverse transcription PCR effects of the activated AGEs-RAGE axis intracellularly Total RNA extraction from the PBMCs was performed according to (9) . the manufacturer ’ s instructions with the mammalian total RNA, Limited reports have investigated the physiological miniprep kit (Sigma, Aldrich). DNaseI (Thermoscrip RT, Invitrogen mechanisms of AGEs accumulation in relation to oxida- Corporation, Carlsbad, CA, USA) treatment of all the samples was included. Preparation of cDNA from total RNA was performed us- tive stress in a milieu of type 1 diabetic children, adoles- ing a reverse transcription system (Thermoscrip RT, Invitrogen Cor- cents and young adults. Therefore, the aim of our study poration). From the cDNA samples RAGE and β -actin mRNA were was to investigate oxidative stress (MDA) and the anti-oxi- amplifi ed with: RAGE primers: forward = 5 ′ AAC-TGC-AGG-CTC-TGT- dant capacity of the body (FRAP) in children, adolescents GGG-AGG-AT 3 ′ , reverse = 5 ′ CCT-TGC-CAA-GCT-TTC-TTC-TAC-AAC-C 3 ′ and young adults with DM1 in relation to the activation and β -actin primers: forward = 5 ′ AAG-GCC-AAC-CGT-GAA-AAG-ATG- = of the AGEs (CML)-RAGE axis and its soluble isoforms, ACC 3 ′ , reverse 5 ′ -ACC-GCT-CGT-TGC-CAA-TAG-TGA-TGA 3 ′ under the following conditions: denaturation at 95o C for 1 min, annealing at sRAGE and esRAGE, in order to investigate their possi- 55o C for 30 s and extension at 72o C for 1 min, for 35 cycles. PCR bands ble role in the predisposition for, or the protection from, were quantifi ed by densitometry with scion image (version 4.0.3.2, diabetic complications. Scion Corporation, Frederick, MD, USA).

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Diabetic patients Controls p-Value

Subjects 110 124 NS Sex, male/female 59/51 61/63 NS Age, years 14.23 ± 5.19 14.05 ± 4.97 NS Height, cm 154.39 ± 19.02 150.57 ± 16.50 NS Height SDS 0.11 ± 1 – 0.38 ± 1.18 0.002 Tanner stage I: 19 I: 11 NS II: 17 II: 11 III: 4 III: 7 IV: 18 IV: 22 V: 46 V: 32 Weight, kg 51.15 ± 17.8 44.44 ± 14.33 0.003 Body mass index, cm/kg2 20.71 ± 3.90 18.99 ± 3 0.001 Body mass index, cm/kg2 % 65.60 ± 22.88 % 47.51 ± 28.10 % 0.001 Body mass index SDS 0.29 ± 1.08 – 0.13 ± 0.93 0.003 Waist circumference, cm 70.78 ± 9.6 66.93 ± 8.9 0.019 DM1 duration, years 5.65 ± 4.29 N/A HbAlc, % 8.06 ± 1.62 5.11 ± 0.42 0.001 Cholesterol, mg/dL 164.81 ± 27.13 164.16 ± 29.16 NS LDL cholesterol, mg/dL 94.05 ± 29.1 95.18 ± 25.76 NS HDL cholesterol, mg/dL 56.69 ± 12.1 55.83 ± 12.35 NS Triglycerides, mg/dL 66.98 ± 30.71 66.83 ± 22.10 NS SCOT, U/L 18.92 ± 5.69 17.57 ± 3.82 NS SGPT, U/L 17.8 ± 9.3 16.5 ± 5.5 NS Urea, mg/dL 31.3 ± 6.83 23.57 ± 6.02 NS Creatinine, mg/dL 0.72 ± 0.18 0.8 ± 0.12 NS Micro-angiopathy– –

Table 1 Clinical and biochemical characteristics of the DM1 patients and the controls. Data are means ± SD. Statistical significance, p ≤ 0.05. NS, not significant.

Western immunoblotting Plasma assays

Protein samples of isolated PBMCs were lysed with laemnli sample The plasma levels of the total soluble RAGE (esRAGE and sRAGE) buff er (2 × SDS sample buff er) prepared in the laboratory. Protein (11) were measured by a sandwich ELISA kit (Human Immunoassay content in the samples was approximately estimated by SDS-PAGE RAGE, Quantikine, R & D Systems, Minneapolis, MN, USA). Measure- electrophoresis and staining with Coomassie Blue. Whole cell lysates ments were performed according to the manufacturer ’ s instructions were separated by SDS-PAGE and proteins were transferred to ni- and the intra-assay coeffi cient of variation for repeated measure- trocellulose membranes (Amersham Biosciences plc, Buckingham- ments was 7.5 % . shire, UK). The membranes were blocked for 1 h at room temperature The plasma levels of CML were measured by a sandwich ELISA (RT) and then incubated with IgG: anti-RAGE (Biotechnology, Santa kit (CirculexTM Anti-CML Human autoantibody, Qzzuantikine, Cyclex Cruz, CA, USA) diluted 1:150, overnight at RT. Furthermore, the pro- Co, Ohara, Japan). Measurements were performed according to the tein measurements were corrected with the expression of β -tubulin, manufacturer’ s instructions and the intra-assay precision was 5.6% , which was detected with an anti-β -tubulin antibody (Cell signaling whereas the inter-assay precision was 8.7 % . Technology, Inc., Danvers, MA, USA) diluted 1:500, overnight at RT. Malondialdehyde (MDA) was determined fl uorometrically ac- Band detection was performed with chemiluminescence reagent ECL cording to the protocol by Papandreou et al. (12) . In brief, 25 μ L of 90 (GE Healthcare Bio-Sciences AB, Sweden). The Western blot signals mM butylated hydroxytoluene (BHT) was added to 250 μ L of plasma (complexed protein bands) were quantifi ed by densitometry with samples or MDA standards (0.05– 5 μ M), to prevent further lipid perox- scion image (version 4.0.3.2, Scion Corporation). idation. Samples were hydrolysed in mild alkaline conditions at 60 ° C for 30 min in order to release bound MDA and the proteins were pre- cipitated with 10% trichloroacetic acid (TCA). Aft er being kept on ice Blood samples for 10 min, mixtures were centrifuged and the supernatants obtain- ed were incubated with 0.8% thiobarbituric acid (TBA) at 95° C for 90 Venous blood samples were obtained with heparinised syringes. The min. The supernatants were then extracted with n -butanol and fl uo- plasma was collected during the procedure of the PBMCs isolation rescence was measured at 535 nm (emission wavelength) aft er excita- and then stored at – 80o C. The plasma samples were used to measure tion at 515 nm on a Perkin-Elmer LS55 fl uorescence spectrometer. All the levels of sRAGE, CML, FRAP and MDA. determinations were carried out at least two times and in duplicates.

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The ferric reducing ability of plasma (FRAP) was determined by exhibited significantly increased height SDS, weight, body a modifi ed method of the Benzie and Strain protocol (13, 14) . In par- mass index (BMI), BMI % , BMI SDS and waist circumfer- ticular, the working FRAP reagent was produced by mixing 300 mM ence when compared to their controls, but these values were acetate buff er (pH = 3.6), 10 mM 2,4,6-tripyridil-s-triazine (TPTZ) solu- within the normal range, therefore they were not considered tion and 20 mM FeCl 6H 2O in a 10:1:1 ratio just before use. This reagent was then loaded (150 μ L) to each well of a 96-well micro titer plate as risk factors for diabetic complications. Furthermore, the μ μ together with a total of 5 L of sample and 15 L of ddH2 O. A reading diabetic patients had not developed any micro- or macro- at 595 nm was taken and also a standard linear curve of Ferrous sul- angiopathy and their glycemic profile showed that their fate (FeSO4 7H O) was produced by known concentrations (10 3 – 10 5 2 diabetes was moderately controlled (HbA1c = 8.0% ± 1.8 % ). M). FRAP values were determined according to the standard curve.

Statistical analysis RAGE mRNA and protein expression in PBMCs Our subjects were grouped according to age, DM1 duration and HbA1c levels (for DM1 patients), LDL cholesterol levels, pubertal stage, waist circumference, body mass index (BMI) and BMI % tiles. The gene expression (mRNA) of RAGE was increased in Expression levels of mean values with ± standard deviation (SD) and the overall sample of DM1 patients in comparison to the correlations were performed using the SPSS computer program (17.0 controls, (p = 0.007) (Figure 1 A). More specifically, RAGE version for Windows, Mann-Whitney or Kolmogorov 2-tailed t-test, mRNA expression was increased in the DM1 patients > 13 Pearson ’ s or Spearman ’ s correlation coeffi cients). The threshold of years of age in comparison to their respective controls statistical signifi cance was defi ned as p ≤ 0.05. (p = 0.039) (Figure 1B), and in the DM1 patients with > 5 years duration of diabetes in comparison to the controls (p = 0.002) (Figure 1C). Results The protein expression of the non-glycosylated esRAGE isoform is detected at 46 kDa (15) . The esRAGE protein Clinical and biochemical characteristics expression tended to increase in the DM1 patients in com- of the study subjects parison to their controls in the overall sample (Figure 2 A) and this tendency remained even after the samples were All the clinical and biochemical characteristics of the grouped according to their age ( ≤ or > 13 years of age) subjects are presented in Table 1. The diabetic patients (Figure 2B). esRAGE, however, was significantly increased

Controls Diabetic patients Controls ≤13 years Diabetic patients ≤13 years Controls DM1 ≤5 years DM1 >5 years Controls >13 years Diabetic patients >13 years AB C 100 100 100 90 90 90 80 80 80 70 70 70 60 60 60 50 50 50 40 40 40 30 30 30 20 20 20

Relative gene expression, % 10 10 10 0 0 0 RAGE mRNA RAGE mRNA RAGE mRNA

C1 C2P1 P2 C1 C2P1 P2 C1 C2P1 P2 RAGE RAGE RAGE β-Actin β-Actin β-Actin

Figure 1 RAGE mRNA expression in DM1 patients and their controls in (A) the overall sample, (B) grouped according to age ( ≤ or > 13 years) and (C) grouped according to DM1 duration ( ≤ or > 5 years). (A) The DM1 patients had an increased expression of RAGE in comparison to the controls (* p = 0.007) in the overall group. (B) The DM1 patients > 13 years of age had an increased RAGE mRNA expression in comparison to their respective controls (# p = 0.039), (C) the DM1 patients with > 5 years duration of diabetes had an increased RAGE mRNA expression in comparison to the controls (** p = 0.002). (Data are plotted as mean ± SE.)

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Controls ≤13 years Diabetic patients ≤13 years Controls DM1 ≤5 years DM1 >5 years Controls Diabetic patients Controls > years Diabetic patients >13 years

100 AB100 100 C 90 90 90 80 80 80 70 70 70 60 60 60 50 50 50 40 40 40 30 30 30 20 20 20 10 10 10 Relative protein expression, % 0 0 0 esRAGE protein (46 kDa) esRAGE protein (46 kDa) esRAGE protein (46 kDa)

C1 C2P1 P2 C1C2P1P2C3 C4 P3P4 C1 C2 P1 P2 P3 P4 RAGE RAGE RAGE β-Tubulin β-Tubulin β-Actin

Figure 2 Protein expression of 46 kDa isoform of RAGE (esRAGE) in DM1 patients and their controls in (A) the overall sample, (B) grouped according to age ( ≤ or > 13 years) and (C) grouped according to DM1 duration ( ≤ or > 5 years). (C) The DM1 patients with > 5 years of diabetes had an increased esRAGE protein expression in comparison to the DM1 patients with ≤ 5 years of diabetes (* p = 0.008) and the controls (** p = 0.041). (Data are plotted as mean ± SE.) in the DM1 patients with > 5 years duration of diabetes CML plasma levels when these were compared with the DM1 patients with ≤ 5 = years of diabetes (p 0.008) and also with the controls CML plasma levels tended to increase in the DM1 patients = (p 0.041) (Figure 2C). when compared to the controls in the overall sample (Figure 4 A) and this increase was significant in the DM1 patients that were ≤ 13 years of age (Figure 4B) and the DM1 sRAGE plasma levels patients with ≤ 5 years duration of diabetes (Figure 4C).

sRAGE plasma levels showed no significant differences between the DM1 patients and the controls (data not MDA plasma levels shown), but they had a tendency to increase in DM1 patients with the increasing levels of their HbA1c and MDA plasma levels were increased in DM1 patients in com- especially with HbA1c > 9 % (Figure 3 ). parison to the controls in the overall sample (p = 0.001)

Controls HbA1c <7% HbA1c 7.1-8%

HbA1c 8.1-9% HbA1c 9.1-10% HbA1c>10.1% 2.0 1.8 1.6 1.4 1.2 1.0 0.8 0.6 Plasma levels, pg/mL 0.4 0.2 0 sRAGE plasma levels

Figure 3 sRAGE plasma levels in DM1 patients and their controls, in relation to their Hemoglobin A1c (HbA1c) levels. In DM1 patients, sRAGE plasma levels tended to increase with HbA1c > 9 % . (Data are plotted as mean ± SE.)

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Controls Diabetic patients Controls ≤13 years Diabetic patients ≤13 years Controls ≤5 years DM1 >5 years DM1 Controls >13 years Diabetic patients >13 years 9 A 9 B 9 C 8 8 8 7 7 7 6 6 6 5 5 5 4 4 4 3 3

Plasma levels, ng/mL 3 2 2 2 1 1 1 0 0 0 CML plasma levels CML plasma levels CML plasma levels

Figure 4 CML plasma levels in DM1 patients and their controls in (A) the overall sample, (B) grouped according to age ( ≤ or > 13 years) and (C) grouped according to DM1 duration ( ≤ or > 5 years). (A) The DM1 patients had a tendency to increase CML in comparison to their controls. (B) The DM1 patients ≤ 13 years of age had increased CML plasma levels in comparison to their age-matched controls (* p = 0.034). (C) The DM1 patients with ≤ 5 years of diabetes had increased CML plasma levels in comparison to the controls (# p = 0.027). (Data are plotted as mean ± SE.)

(Figure 5 A). Also, MDA plasma levels were increased the FRAP plasma levels tended to increase in the DM1 in DM1 patients regardless of age [ ≤ 13 years (p = 0.001) patients regardless of age in comparison to their age and > 13 years (p = 0.012), of age] in comparison to their matched controls (Figure 6B) but they were significantly controls (Figure 5B) and regardless of the duration of dia- increased in the DM1 patients regardless of the duration betes [ ≤ 5 years (p = 0.01) and > 5 years (p = 0.01)] in com- of diabetes [ ≤ 5 years (p = 0.052) and > 5 years (p = 0.012)] parison to the controls (Figure 5C). in comparison to the controls (Figure 6C).

FRAP plasma levels Correlations

The FRAP plasma levels were significantly increased in In the overall sample of the diabetic patients: (a) the the DM1 patients in comparison to the controls in the sRAGE and MDA plasma levels were negatively correlated overall sample (p = 0.005) (Figure 6 A). More specifically, with the duration of the diabetes (r = – 0 . 2 4 7, p = 0.027 and

Controls Diabetic patients Controls ≤13 years Diabetic patients ≤13 years Controls ≤5 years DM1 >5 years DM1 Controls >13 years Diabetic patients >13 years

2.5 2.5 AB2.5 C

2.0 2.0 2.0 M μ 1.5 1.5 1.5

1.0 1.0 1.0

Plasma levels, 0.5 0.5 0.5

0 0 0 MDA plasma levels MDA plasma levels MDA plasma levels

Figure 5 MDA plasma levels in DM1 patients and their controls in (A) the overall sample, (B) grouped according to age ( ≤ or > 13 years) and (C) grouped according to DM1 duration ( ≤ or > 5 years). (A) The DM1 patients had increased MDA plasma levels in comparison to controls (* p = 0.001) in the overall group. (B) The DMI patients with ≤ and > 13 years of age had increased MDA plasma levels in comparison to their controls (** p = 0.001, *** p = 0.012). (C) The DMI patients with ≤ and > 5 years duration of diabetes had increased MDA plasma levels in comparison to the controls (# p = 0.01, ∞ p = 0.01). (Data are plotted as mean ± SE.)

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Controls ≤5 years DM1 >5 years DM1 Controls Diabetic patients Controls ≤13 years Diabetic patients ≤13 years Controls >13 years Diabetic patients >13 years 450 A 450 BC450 400 400 400 350 350 350 300 300 300 250 250 250 200 200 200 150 150 150

Plasma levels, mM 100 100 100 50 50 50 0 0 0 FRAP plasma levels FRAP plasma levels FRAP plasma levels

Figure 6 FRAP plasma levels in DM1 patients and their controls in (A) the overall sample, (B) grouped according to age ( ≤ or > 13 years) and (C) grouped according to DM1 duration ( ≤ or > 5 years). (A) The DM1 patients had increased FRAP plasma levels in comparison to the controls (* p = 0.005) in the overall group. (B) The DM1 patients with ≤ and > 13 years of age had a significant tendency to increase the FRAP plasma levels in comparison to their controls. (C) The DM1 patients with ≤ and > 5 years of diabetes had increased FRAP plasma levels in comparison to the controls (** p = 0.052, # p = 0.012). (Data are plotted as mean ± SE.) r = – 0.349, p = 0.020, respectively), (b) the protein expres- of diabetes, but were mostly associated with DM1 com- sion of the 46 kDa isoform of RAGE (esRAGE) was posi- plications (17, 18) . However, this significant increase tively correlated with the duration of the diabetes (r = 0.530, was not associated with an increase in the RAGE mRNA p = 0.005) and negatively correlated with the MDA plasma expression in these young patients, possibly indicating levels (r = – 0.661, p = 0.038). (c) The gene expression of that these increased circulating CML levels had not fully RAGE was negatively correlated with the FRAP plasma activated the AGEs-RAGE axis. Furthermore, RAGE mRNA levels (r = – 0 . 4 7 4 , p = 0.015) and (d) the CML plasma levels expression was significantly increased in the older DM1 were positively correlated with the FRAP plasma levels patients ( > 13 years of age) and in those with a longer (r = 0.306, p = 0.021) (data not shown). duration of diabetes ( > 5 years). Previous studies have also reported increased mRNA expression of RAGE with increased duration of diabetes (19) but our study has shown, for the first time, that the age cut-off of 13 years is Discussion of great significance in childhood DM1. Conversely, the protein expression of the endogenous Activation of the AGEs-RAGE axis plays a central role in inhibitor of AGEs, esRAGE (RAGE 46 kDa), increased with the development of diabetic complications. This acti- the duration of diabetes and the levels of total soluble vated axis leads to diabetic angiopathy in adults (16) . It RAGE (sRAGE and esRGE) tended to increase with the has always been of clinical interest that the younger DM1 increased levels of HbA1C ( > 9 % ) and negatively correlated patients do not develop microangiopathy frequently, even with the duration of the patients ’ diabetes. The increase in with a duration of DM1 > 5 years which is a crucial time intracellular esRAGE in the patients with increased dura- period after which adults with DM1 present with these tion of diabetes ( > 5 years) could be part of a protective complications. mechanism that is activated in these young DM1 patients In our DM1 patients, we found a tendency of the in an attempt to keep the circulating AGEs within ‘ normal ’ plasma levels of CML (AGE) to increase, a tendency that levels, as the CML levels did not significantly differ in this was still evident when the subjects were grouped accord- group from their respective controls. It is, however, of ing to age and duration of diabetes. In particular though, great interest that the sRAGE levels did not significantly the younger DM1 patients ( ≤ 13 years of age) and those change, but do show a negative correlation with the dura- with less DM1 duration ( ≤ 5 years) increased CML sig- tion of diabetes. This negative correlation may possibly nificantly. Previous reports have also shown significantly indicate that some of the AGEs’ homeostatic mechanisms increased CML levels in DM1 children and adolescents are negatively affected by the duration of the DM1, even that were not associated, though, with age and duration in these young patients. A clinical report (EURODIAB) in

Authenticated | [email protected] author's copy Download Date | 1/25/13 9:03 PM 8 Koutroumani et al.: Protective mechanisms against oxidative stress and angiopathy adults with DM1 has shown that sRAGE increases in DM1 complications. This hypothesis is also supported by patients that already show macro- and micro-angiopathy Astaneie et al. in 2005 (24) , who showed the existence when these were compared with diabetic patients with of increased anti-oxidant capacity in adult DM1 patients, no angiopathy (20) . Therefore, the tendency of sRAGE to through markers like FRAP and EGF, which also did not increase especially with increased HbA1c may also reflect exhibit changes in oxidative stress markers like the lipid a counteractive mechanism in these children attempting perodixation marker of the thiobarburitic reactive sub- to decrease their risk for diabetic complications. stances (TBARS). In addition, we found increased MDA and FRAP In conclusion, the increased protein expression of plasma levels in the DM1 subjects. Previous reports have the endogenous inhibitor of AGEs, esRAGE, in our young shown increased MDA plasma levels in children and patients with a duration of diabetes > 5 years together adults with DM1 (6, 7, 21, 22). It has also been shown that with the tendency of sRAGE to increase with HbA1c > 9 % , MDA plasma levels progressively increase in the older seem to indicate that these protective factors are possibly patients with DM1 (23) in comparison to the younger ones. recruited in the face of increased AGEs (CML), in these This increase in MDA plasma levels has not been shown young DM1 individuals, in order to decrease the risk of by other studies to be accompanied by an increase in AGE-induced complications. The counteracting effect FRAP. Nowak et al. (8) showed in 2010 that DM1 patients of the increased FRAP levels in the face of the increased who developed nephropathy have decreased antioxidant MDA levels, may also indicate that these young diabetic capacity as evaluated by their decreased FRAP plasma patients have possibly preserved their body’ s ability to levels. Also, Firoozrai et al. in 2007 (7) correlated the activate the physiological mechanisms recruited nor- decreased antioxidant defense (decreased FRAP) of their mally to reduce oxidative stress. It therefore seems that adult DM1 patients with their poor glycemic control. the younger patients with DM1 may recruit protective Therefore, it is possible that the increased FRAP in mechanisms against glycation and oxidative stress that our young DM1 patients is in response to the increased may deem them capable of reducing their risk for diabetic MDA, showing that our young DM1 patients seem to complications. still possess the ability to combat the oxidative stress mediators, which could put them in danger for vascular Received June 11, 2012; accepted August 4, 2012

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15. Yonekura H, Yamamoto Y, Sakurai S, Petrova RG, Abedin MJ, 20. Nin JW, Ferreira I, Schalkwijk CG, Prins MH, Chaturvedi N, et al. et al. Novel splice variants of the receptor for advanced glycation Levels of soluble receptor for AGE are cross-sectionally end-products expressed in human vascular endothelial cells and associated with cardiovascular disease in type 1 diabetes, and pericytes, and their putative roles in diabetes-induced vascular this association is partially mediated by endothelial and renal injury. Biochem J 2003;370:1097 – 109. dysfunction and by low-grade inflammation: the EURODIAB 16. Devaraj S, Glaser N, Griffen S, Wang-Polagruto J, Miguelino E, Prospective Complications Study. Diabetologia 2009;52: et al. Increased monocytic activity and biomarkers of 705– 14. inflammation in patients with type 1 diabetes. Diabetes 21. Ramakrishna V, Jailkhani R. Evaluation of oxidative stress in 2006;55:774 – 9. Insulin Dependent Diabetes Mellitus (IDDM) patients. Diagn 17. Berg TJ, Clausen JT, Torjesen PA, Dahl-Jorgensen K, Bangstad Pathol 2007;2:22. HJ, et al. The advanced glycation end product Nepsilon- 22. Gupta A, Tripathi AK, Tripathi RL, Madhu SV, Banerjee BD. (carboxymethyl)lysine is increased in serum from children and Advanced glycosylated end products-mediated activation adolescents with type 1 diabetes. Diabetes Care 1998;21: of polymorphonuclear neutrophils in diabetes mellitus 1997 – 2002. and associated oxidative stress. Indian J Biochem Biophys 18. Hwang JS, Shin CH, Yang SW. Clinical implications of N epsilon- 2007;44:373 – 8. (carboxymethyl)lysine, advanced glycation end product, in 23. Dominguez C, Ruiz E, Gussinye M, Carrascosa A. Oxidative children and adolescents with type 1 diabetes. Diabetes Obes stress at onset and in early stages of type 1 diabetes in children Metab 2005;7:263 – 7. and adolescents. Diabetes Care 1998;21:1736 – 42. 19. Miura J, Uchigata Y, Yamamoto Y, Takeuchi M, Sakurai S, et al. 24. Astaneie F, Afshari M, Mojtahedi A, Mostafalou S, Zamani MJ, AGE down-regulation of monocyte RAGE expression and its et al. Total antioxidant capacity and levels of epidermal growth association with diabetic complications in type 1 diabetes. factor and nitric oxide in blood and saliva of insulin-dependent J Diabetes Complications 2004;18:53 – 59. diabetic patients. Arch Med Res 2005;36:376– 81.

Authenticated | [email protected] author's copy Download Date | 1/25/13 9:03 PM Medicinal Plants are plants which have a recognized medical use. They range from plants which are used in the production of mainstream pharmaceutical products to plants used in herbal medicine preparations. The Medicinal & Aromatic Plants for health are used as herbal treatments and therapies that can be new habits for culture. Plant, drug, or medicine yielding a fragrant aroma, as sage or certain spices and oils are known as aromatic plants.

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ResearchEditorial Article OpenOpen Access Access In Search of a “Natural” Treatment of Cataract Anastasia-Varvara Ferlemi1, Olga E Makri2, Constantine D Georgakopoulos2 and Fotini N Lamari1* 1Department of Pharmacy, School of Medicine, University of Patras, 26504 Patras, Greece 2Department of Ophthalmology, School of Medicine, University of Patras, 26504 Patras, Greece

In cataract, opacification of the lens reduces visual acuity, finally their insolubilization and aggregation. Over activated calpains also leading to blindness if untreated. Cataract is the leading cause of degrade the cytoskeleton proteins, which can further elevate Ca2+ [14]. blindness worldwide – there are about 50 million blind people in the The above mechanisms compromise lens transparency and induce world and cataract formation is the cause in half of these cases [1]. cataractogenesis. There are numerous models for in vitro and in vivo study of cataractogenesis. At in vitro experiments, cataractogenic Today, cataract is treated by surgery. The cost of the surgery places agents such as galactose, glucose, naphthalene, selenite, transforming it beyond the reach of individuals in many parts of the world and there growth factor-β, methylglyoxal are added to the lens culture. The in is an urgent need for less expensive, non surgical approaches to cataract vivo models include induction of cataractogenesis especially in rodents treatment [2]. It was estimated that extra-capsular surgery used with through streptozotocin-induced diabetes, galactose feeding, ionizing 95% coverage of the population would avert more than 3.5 millions radiation, inhibition of cholesterol synthesis, steroid treatment, and disability-adjusted life years (DAILY) per year globally [3]. In 2008, a administration of selenite overdose inducing oxidative stress [5]. study showed that the mean total cost of the operation per patient was €714 (SD = €57) and ranged from €437 in Denmark to €1087 in Italy Over the last decade, an increasing number of studies indicates the [4]. The financial encumbrance is enormous considering that 28,000 possible role of some plant extracts or isolated agents against cataract new cases are reported daily [5]. Thus, pharmaceutical interventions formation. Plant extracts, like extract of Ocinum sanctum [15], aqueous that will maintain the transparency of the lens are intensively sought garlic extract [16], onion juice [17], as well as fraction of flavonoids after. extracted from Emilia sonchifolia [18], polyphenolic compounds of Clearly, to develop such treatments, a precise understanding of Camelia sinensis [19] and natural products such as curcumin [20], the underlying pathophysiological mechanism of cataract formation ellagic acid [21], luteolin [22], acetyl-L-carnitine [23,24], lycopene is required. There are three discrete categories of cataract: the “age- [25], resveratrol [26] have been proved to ameliorate selenite-induced related”, that is associated with aging, “congenital cataract” that is cataract formation by enhancing antioxidant enzyme activity and present at birth indicating pathological changes during embryonic inhibiting radical formation and lipid peroxidation. Additionally to development of the lens, and “sugar cataract” which is associated either their antioxidant potential, some plants or their compounds exhibit with diabetes mellitus [6-8] or galactosemia [9]. anticataract action via maintaining Ca2+-ATPase, prevention of Ca2+ accumulation, thus inhibiting calpain activation. Such compounds are Sun light and oxygen, that the lens is exposed to, are associated drevogenin D, an aglycone triterpene [27] and the flavonoid fraction with extensive damage to proteins and other constituents of the extracted from Vitex negundo [28]. Furthermore, it has been found that tissue. Other risk factors include environmental stress factors such as the carotenoid, , delays lens crystalin precipitation [29], smoking, excessive UV-light exposure and electromagnetic radiation, whereas ellagic acid prevents the alteration of lenticular, non-crystalin life threatening diseases like coronary heart diseases [8], renal failure and many drugs [7]. These factors contribute to the depletion of proteins [30]. antioxidants [e.g. vitamins C and E, carotenoids and glutathione Diabetic cataract and the possible preventing properties of some (GSH)] and reduction of antioxidant enzymes [e.g. superoxide plants extracts or compounds have, also, been studied, but not as dismutase (SOD), catalase (CAT) and glutathione reductase/peroxidase thoroughly as the selenite model. Zingiber officinalis (ginger) extract (GSR/GPx)]. Furthermore, they may diminish protease (i.e. caspase, [31], Allium sativum methanolic extract [32], turmeric and curcumin nuclease) activity, whose role is the second level of defense (e.g. removal [33] inhibited the polyol pathway and the advanced glycation of of obsolete proteins). proteins, and protected lens antioxidant enzymes in streptozotocin– Diabetes mellitus is another important risk factor. Hyperglycemia induced cataract model. Hydroxytyrosol and oleuropein from olive induces oxidative stress through a complicated pathway. Evidently, leaves, as well as caffeine protected lenses from oxidative stress in an during hyperglycemia the accelerated flux of sorbitol through alloxan- and a galactose-induced cataractogenesis model, respectively the polyol pathway and enhanced oxidative stress are implicated [34]. in the pathogenesis of secondary diabetic complications, such as cataractogenesis [10]. Aldose reductase (AKR1B1) catalyzes the first and rate-limiting step of the polyol pathway of glucose metabolism. *Corresponding author: Fotini N Lamari, Department of Pharmacy, School of Several biochemical mechanisms are involved in the opacification Medicine, University of Patras, 26504 Patras, Greece, Tel. 302610969335; E-mail: [email protected] of the lens and they have been thoroughly described: i) non enzymatic- glycation, ii) oxidative stress, iii) polyol pathway and iv) activation Received December 20, 2012; Accepted December 22, 2012; Published December 24, 2012 of calpain proteases. The established feature of cataract is the major structural modifications of the water soluble crystallins [11]. Ageing Citation: Ferlemi AV, Makri OE, Georgakopoulos CD, Lamari FN (2013) In process, diabetes and oxidants lead to impaired membrane function, Search of a “Natural” Treatment of Cataract . Med Aromat Plants 1: e144. doi:10.4172/2167-0412.1000e144 which results to pathologically elevated levels of intracellular Ca2+ [12,13]. Under these conditions, calpains (calpain 1 or μ-calpain, Lp85, Copyright: © 2013 Ferlemi AV, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits calpain 2 or m-calpain, calpain 10 and Lp82) are over activated and unrestricted use, distribution, and reproduction in any medium, provided the the resulting deregulated proteolysis of soluble crystallins leads to original author and source are credited.

Med Aromat Plants ISSN: 2167-0412 MAP, an open access journal Volume 2 • Issue 2 • 1000e144 Citation: Ferlemi AV, Makri OE, Georgakopoulos CD, Lamari FN (2013) In Search of a “Natural” Treatment of Cataract . Med Aromat Plants 1: e144. doi:10.4172/2167-0412.1000e144

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An increasing number of herbal extracts is documented to have 11. Harding JJ (2002) Viewing molecular mechanisms of ageing through a lens. anticataractogenic potential; whether the tested doses and dosage Ageing Res Rev 1: 465-479. schemes can be translated in effective agents that will prevent cataract 12. Manikandan R, Thiagarajan R, Beulaja S, Chindhu S, Mariammal K, et al. formation in humans is widely discussed. Most studies focus on the (2009) Anti-cataractogenic effect of curcumin and aminoguanidine agains selenium-induced oxidative stress in the eye lens of Wistar rat pups: An in vitro antioxidant properties of the natural products. Thus, members of the study using isolated lens. Chem Biol Intetact 181: 202-209. polyphenol family, i.e. flavonoids, galloyl glycosides, caffeoylquinic acids, known for their antioxidant properties are privileged candidates 13. Suryanarayana P, Saraswat M, Mrudula T, Krishna TP, Krishnaswamy K, et al. (2005) Curcumin and turmeric delay Streptozotocin-induced diabetic cataract for development of efficacious preventive agents. Beyond this approach, in rats. Invest Ophthalmol Vis Sci 46: 2092-2099. Babizhayef et al. [35-37] have shown that a natural histidine dipeptide 14. Stitt A (2001) Advanced glycation: an important pathological event in diabetic can prevent and reverse cataract. N-acetylcarnosine has been used and age related ocular disease. Br J Ophthalmol 85: 746-753. in several clinical studies as eye drops, in order to assess its ability 15. Gupta SK, Srivastava S, Trivedi D, Joshi S, Halder N (2005) Ocimum sanctum to treat senile cataract. First results were encouraging, so Innovative modulates selenite-induced cataractogenic changes and prevents rat lens Vision Products Inc. (INV) scientists developed the lubricant eye opacification. Curr Eye Res 30: 583-591. drops Can-C, designed as 1% N-acetylcarsosine (NAC) prodrug of 16. Javadzadeh A, Ghorbanihaghjo A, Arami S, Rashtchizadeh N, Mesgari M, et al. L-carnosine and tested in 75 symptomatic patients 54-78 years old for 9 (2009) Prevention of selenite-induced cataractogenesis in Wistar albino rats by months. That small-scale study showed that NAC is safe and efficacious aqueous extract of garlic. J Ocul Pharmacol Ther 25: 395-400. for prevention and treatment of senile cataract for long-term use. 17. Javadzadeh A, Ghorbanihaghjo A, Bonyadi S, Rashidi MR, Mesgari M, et The use of “omics” technologies might reveal new molecular targets al. (2009) Preventive effect of onion juice on selenite-induced experimental cataract. Indian J Ophthalmol 57: 185-189. and, thus, specificity in pharmacological targeting might reveal more effective therapeutics. Inhibition of aldose reductase is the commonest 18. Lou MF (2003) Redox regulation in the lens. Prog Retin Eye Res 22: 657-682. target in order to combat diabetic cataract. Previous studies using 19. Gupta SK, Halder N, Srivastava S, Trivedi D, Joshi S, et al. (2002) Green normal rats, dogs and mice have identified that AKR1B1 inhibitors tea (Camelia sinensis) protects agianst selenite-induced oxidative stress in are potential drugs to prevent high glucose- and galactose-induced experimental cataractogenesis. Ophthalmic Res 34: 258-263. cataracts. Nonetheless, the clinical utility of AKR1B1 inhibitors 20. Raju TN, Kanth VR, Lavanya K (2008) Effect of methanolic extract of Allium remains uncertain [38]. Another emerging molecular target is the lens sativum (AS) in delaying cataract in STZ-induced diabetic rats. J Ocul Biol Dis Infor 1: 46-54. calpains because calpain over activation is the convergence point of all implicated factors, i.e. ageing, diabetes, and oxidation. Among natural 21. Saraswat M, Suryanarayana P, Reddy PY, Patil MA, Balakrishna N, et al. (2010) Antiglycating potential of Zingiber officinalis and delay of diabetic cataract in products, chalcones were indicated as calpain inhibitors in a recent in rats. Mol Vis 16: 1525-1537. vitro study [39]. Besides that, the topical application as eye drops of those agents remains an attractive drug delivery means [35,40]. 22. Reddy AB, Tammali R, Mishra R, Srivastava S, Srivastava SK, et al. (2011) Aldose reductase deficiency protects sugar-induced lens opacification in rats. The screening of natural compounds for the identification of potent Chem Biol Interact 191: 346-350. anticataract agents is a difficult and long-term goal. However, as far 23. Geraldine P, Sneha BB, Elanchezhian R, Ramesh E, Kalavathy CM, et al. as cataract remains a significant public health problem and the only (2006) Prevention of selenite-iduced cataractogenesis by acetyl-L-carnitine: An way of treatment is, the unprofitable for the society and for individuals, experimental study. Exp Eye Res 83: 1340-1349. surgery operation, researchers have to keep seeking an agent that could 24. Elanchezhian R, Sakthivel M, Geraldine P, Thomas PA (2010) Regulatory effect prevent cataract formation. Nature has certainly arranged it for us. of acetyl-L-carnitine on expression of lenticular antioxidant and apoptotic genes in selenite- induced cataract. Chem Biol Interact 184: 346-351. References 25. Gupta SK, Trivedi D, Srivastava S, Joshi S, Halder N, et al. (2003) Lycopene 1. Johnson GJ, Foster A (2003) Prevalence, incidence and distribution of visual attenuates oxidative stress-induced experimental cataract development: an in impairment. In Johnson G, Minassian D, Weale R (eds), the epidimiology of eye vitro and in vivo study. Nutrition 19: 794-799. disease, Edward Arnold, London, UK. 26. Doganay S, Borazan M, Iraz M, Cigremis Y (2006) The effect of resveratrol 2. Biswas S, Harris F, Dennison S, Singh J, Phoenix DA (2004) Calpains: targets in experimental cataract model formed by sodium selenite. Curr Eye Res 31: of cataract prevention? Trends Mol Med 10: 78-84. 147-153.

3. Baltussen R, Sylla M, Mariotti SP (2004) Cost-effectiveness analysis of cataract 27. Biju PG, Devi VG, Lija Y, Abraham A (2007) Protection against selenite cataract surgery: a global and regional analysis. Bull World Health Organ 82: 338-345. in rat lens by drevogenin D, a triterpenoid aglycone from Dregea volubilis. J Med Food 10: 308-315. 4. Fattore G, Torbica A (2008) Cost and reimbursement of cataract surgery in Europe: a cross-country comparison. Helath Econ 17: S71-S82. 28. Sakthivel M, Geraldine P, Thomas PA (2011) Alterations in the lenticular protein profile in experimental selenite-induced cataractogenesis and prevention by 5. Lee E, Jang I, Shin MJ, Cho HJ, Kim J, et al. (2011) Chalcones as novel non- ellagic acid. Graefes Arch Clin Exp Ophthalmol 249: 1201-1210. peptidic μ-calpain inhibitors. Bull Korean Chem Soc 32: 3459-3464. 29. Lija Y, Biju PG, Reeni A, Cibin TR, Sahasranamam V, et al. (2006) Modulation 6. Kyselova Z (2010) Different experimental approaches in modelling of selenite cataract by the flavonoid fraction ofEmilia sonchifolia in exprimental cataractogenesis: An overview of selenite- induced nuclear cataract in rats. animal models. Phytother Res 20: 1091-1095. Interdiscip Toxicol 3: 3-14. 30. Sakthivel M, Elanchezhian R, Ramesh E, Isai M, Jesudasan CN, et al. 7. Cerami A, Crabbe MJC (1986) Recent advances in ocular cataract research. (2008) Prevention of selenite-induced cataractogenesis in Wistras rats by the Trends Pharmacol Sci 7: 271-274. polyphenol, ellagic acid. Exp Eye Res 86: 251-259. 8. Varma SD, Hegde KR, Kovtun S (2010) Oxidative stress in lens in vivo: 31. Sanderson J, Marcantonio JM, Duncan G (2000) A human lens model of cortical inhibitory effect of caffeine. A preliminary report. Mol Vis 16: 501-505. cataract: Ca2+-induced protein loss, vimentin cleavage and opacification. Invest 9. Bron AJ, Vrensen GF, Koretz J, Maraini G, Harding JJ (2000) The ageing lens. Ophthalmol Vis Sci 41: 2255-2261. Ophthalmologica 214: 86-104. 32. Rooban BN, Sasikala V, Gayathri Devi V, Sahasranamam V, Abraham A (2012) 10. Bosch AM (2006) Classical galactosemia revisited. J Inherit Metab Dis 34: 257- Prevention of selenite induced oxidative stress and cataractogenesis by lureolin 260. isolated from Vitex negundo. Chem Biol Interact 196: 30-38.

Med Aromat Plants ISSN: 2167-0412 MAP, an open access journal Volume 2 • Issue 2 • 1000e144 Citation: Ferlemi AV, Makri OE, Georgakopoulos CD, Lamari FN (2013) In Search of a “Natural” Treatment of Cataract . Med Aromat Plants 1: e144. doi:10.4172/2167-0412.1000e144

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33. Theodoropoulou S, Theodossiadis P, Samoli E, Vergados I, Lagiou P, et al. 37. Babizhayev MA, Deyev AI, Yermakova VN, Semiletov YA, Davydova NG, (2011) The epidimiology of cataract: A study in Greece. Acta Ophthalmol 89: et al. (2001) N-acetylcarnosine, a natural histidine-containing dipeptide, as 167-173. a potent phthalmic drug in treatment of human cataract. Peptides 22: 979- 994. 34. Jemai H, El Feki A, Sayadi S (2009) Antibiabetic and antioxidant effects of hydroxytyrosol and Oleuropein from olive leaves in alloxan-diabetic rats. J Agric 38. Rooban BN, Lija Y, Biju PG, Sasikala V, Sahasranamam V, et al. (2009) Vitex Food Chem 57: 8798-8804. negundo attenuates calpain activation and cataractogenesis in selenite models. Exp Eye Res 88: 575-582. 35. Babizhayev MA, Burke L, Micans P, Richer SP (2009) N-acetylcarnosine sustained drug delivery eye drops to control the signs of ageless vision: 39. Liao JH, Chen CS, Maher TJ, Liu CY, Lin MH, et al. (2009) Astaxanthin interacts glare sensitivity, cataract amelioration and quality of vision currently available with selenite and attenuates selenite-induced cataractogenesis. Chem Res treatment for the challenging 50,000 patient population. Clin Interv Aging 4: Toxicol 22: 518-525. 31-50. 40. Chikamoto N, Chikama T, Yamada N, Nishida T, Ishimitsu T, Kamiya A (2009) 36. Babizhayev MA, Deyev AI, Yermakova VN, Semiletov YA, Davydova NG, et Efficacy of substance P and insulin-like growth factor -1 peptides for preventing al. (2002) Efficacy of N-acetylcarnosine in the treatment of cataract. Drugs R postsurgical superficial puncate keratopathy in diabetic patients. Jpn J D 3: 87-103. Ophthalmol 53: 464-469.

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Med Aromat Plants ISSN: 2167-0412 MAP, an open access journal Volume 2 • Issue 2 • 1000e144