Role of dietary flavonoids in amelioration of sugar induced cataractogenesis

Archives of Biochemistry and Biophysics 593 (2016) 1e11

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Role of dietary flavonoids in amelioration of sugar induced cataractogenesis Kapil K. Patil a, Rohan J. Meshram a, 1, Nagesh A. Dhole a, b, Rajesh N. Gacche a, * a b

School of Life Sciences, Swami Ramanand Teerth Marathwada University, Nanded, 431 606, MS, India Department of Biochemistry, Savitribai Phule Pune University, Pune-7, MS, India

a r t i c l e i n f o

a b s t r a c t

Article history: Received 3 December 2015 Received in revised form 21 January 2016 Accepted 27 January 2016 Available online 30 January 2016

Sugar induced cataractogenesis and visual impairment is more prominent ophthalmic problem in humans suffering from diabetes. Flavonoids have been identified as one of the therapeutically important class of phytochemicals possessing myriad of biological activities. Analyzing the anti-cataract effects of flavonoids from natural sources is an important aspect owing to their bioavailability in variety of dietary sources. In the present study a panel of ten dietary flavonoids like 3, 6-dihydroxy flavone, 3, 7-dihydroxy flavone, chrysin, 3-hydroxy-7-methoxy flavone, apigenin, genistein, baicalein, galangin, Biochanin-A, and diosmin were evaluated for their anti-cataract effects in sugar induced lens model studies. Series of parameters like role of flavonoids in glycation induced lens opacity, protein aggregation measurements, carbonyl group formation: a biochemical marker of glycation reaction, non-tryptophan fluorescence: a marker of formation of advanced glycation end products (AGEs) and assessment of (experimental and in silico) aldose reductase inhibition: a key enzyme of polyol pathway involved in cataractogenesis. The results of the study clearly demonstrated the impressive anti-cataract activity of chrysin followed by significant activity by apigenin, baicalein and genistein. The results of the present study may find applications in formulation of functional foods and neutraceuticals for the management of diabetic cataract. © 2016 Elsevier Inc. All rights reserved.

Keywords: Eye lens Glycation Flavonoids Protein aggregation Cataractogenesis

1. Introduction Cataract is the opacification or cloudiness of lens. Besides ageing, humans suffering from diabetes are more prone for developing cataract mediated visual impairment leading to blindness [1]. The current estimate shows that over 366 million people are suffering from diabetes and it is projected that over 552 million may suffer from diabetes up to 2030 [2]. Cataract arising out of altered sugar homeostasis is a leading cause of blindness across the world and the incidence is more in developing countries like India: a capital of diabetes. The pathophysiology of causation of cataract indicates that the formation of insoluble lens protein aggregates resulting from abnormal proteineprotein or protein-water interactions is the main factor in imparting cloudiness or opacity in lens tissue [3]. Owing to the role of sugar induced structural changes in the lens proteins, the diabetic state has been considered as a significant cause for accelerating the process of

* Corresponding author. E-mail address: [email protected] (R.N. Gacche). 1 Author contributed equally with the first author. http://dx.doi.org/10.1016/j.abb.2016.01.015 0003-9861/© 2016 Elsevier Inc. All rights reserved.

cataractogenesis [1]. The high refractive index and the transparent nature of lens are important factors for proper focusing of the image onto the retina. About 90% of the structural lens proteins comprises of crystallins. Exhaustive information in relation to structure and functions of crystallins is described elsewhere [4,5]. The three crystallins (a-, band g) of the human lens are made up of monomeric proteins of roughly 20 kDa. Of the three classes of crystallins, a-crystallin occurs most predominantly and is consist of two types of subunits, A and B, which assemble non-covalently to form a hetero-oligomer of 800 kDa aggregate. a-crystallin is an ATP-independent chaperone protein that selectively binds to damaged or unfolded proteins, sequesters them and prevent them from undergoing aberrant conformational folding, thus plays a key role in conserving proteins in their native conformation and preserves the lens transparency [6]. The b-crystallins comprises of four acidic and three basic monomers having molecular masses in the range of 22e28 kDa, while g-crystallins are made up of 7 different types of monomers of around ~20 kDa molecular weight [4]. Because of the altered sugar homeostasis in diabetes mellitus, the increased plasma sugar concentration undergoes non

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enzymatic reaction with variety of structural and functional proteins leading to formation of covalent adducts. This reaction which basically gets triggered because of long lasting hyperglycaemic state is known as glycation or Millard reaction [7]. Although the complexity and chemistry of formation of AGEs is yet to be defined completely, the glycation reaction advances through three important stages leading to formation of AGEs, in the first stage, the sugars react with free amino groups of proteins and forms unstable Schiffs base. The Schiffs base undergoes rearrangements and forms more stable amadori products [8]. In the second stage, the degradation of the amadori products leads to formation of different reactive dicarbonyl compounds. In the late stage, these reactive compounds undergoes modifications through dehydration, oxidation, cyclization and other rearrangements and results into formation of insoluble, yellow-brown fluorescent compounds called AGEs: advanced glycation end products [8]. AGEs accumulates in proteins for longer durations, interferes and impair the normal physiological functions attributed with related proteins/enzymes [9]. A vast body of literature has accumulated in the recent past, linking the role of AGEs in diabetic complications including cataractogenesis [10]. Glycation mediated formation of AGEs are associated with conformational changes in the lens crystalline proteins, especially by altering the dielectric properties, which might trigger the aberrant proteineprotein and/or protein-water interactions resulting into decrease in the transparency of the eye lens and thereby visual impairment [11,12]. Nevertheless, the glycation induced changes in the lens crystallins also hampers the chaperone activity of a-crystallin that might accelerate the disorganization of lens proteins leading to reduction in the refractive index of the lens [5]. Considering the negative attributes of AGEs in the pathophysiology of cataractogenesis, it is advisable either to estimate the amount of AGEs or measure the intensity of their fluorescence [13]. It is well established fact that glycation of lens proteins leads to extensive cross linking resulting in formation of high molecular weight protein aggregates [10,12,14]. Due to formation of high molecular weight (HMW) protein aggregates, the scattering of light takes place which per say is the initiation phase of cataractogenesis [5]. The glycation mediated aggregation or polymerization of lens proteins into HMW complexes accounts for lens opacity and the loss of high refractive index and visual acuity [14]. The reports also states that formation of HMW a-crystallin aggregates alters the dynamic state of the lens proteins and adversely affect chaperone activity of a-crystallin leading to disorganization of lens protein assembly [14,15]. Therefore while setting up of experiments for analyzing the inhibition of glycation induced cataractogenesis it is advisable to measure the size of the protein aggregates which perhaps can be used as a useful prognostic and/or diagnostic marker both in aging and glycation induced cataractogenesis [4,16]. Aldose reductase (AR) is a key enzyme of polyol pathway that catalyzes the reduction of glucose and galactose to their polyols like sorbitol: a sugar alcohol involved in maintaining osmolarity of the cell. During normoglycemic condition, less than 3% of glucose is metabolized through polyol pathway; however, in hyperglycemic condition more than 30% of glucose enters in polyol pathway that leads to excess accumulation of sorbitol in lens and ultimately induces the opacity in the mature lenses. Excess sorbitol accumulation is also implicated in disturbing several physiological functions [17]. Accumulation of sorbitol in lens tissue induces several adverse physiological changes like osmotic swelling, protein conformation and production of oxidative stress which ultimately accelerate the process of cataractogenesis and other diabetic complications [18]. Therefore AR inhibition studies (in vitro and in silico) are considered significant for developing novel and effective agents for the management of sugar induced cataractogenesis and other

complications [19]. Flavonoids are the secondary metabolites of plants which virtually occur in all plant parts; especially the photosynthesizing plant cells are rich in flavonoids. This most studied and therapeutically important group of phytochemicals has been described to possess wide range of health promoting and disease ameliorative biological activities [20]. In the mainstream of functional foods and neutracuticals, a search for bioactive dietary flavonoids has been prioritized as it can be made bioavailable through dietary ingredients [21]. In preclinical model studies flavonoids have been reported to play a significant role in the management of diabetic cataract [22]. We have previously reported that the flavonoid fractions of series of medicinal plants like Catharanthus roseus, Ocimum sanctum, Tinospora cordifolia, Aegle marmelos, Ficus golmerata, Psoralea corlifolia, Tribulus terrestris and Morinda cetrifolia possess significant anti-cataract and AR inhibitory activities [19]. Similarly the flavonoid fractions from green/black tea, Ginkgo biloba, grape seeds Emilia sonchifolia, Vitex negundo and broccoli have been shown to possess significant anti-cataract activity in different animal model studies [22]. In the present investigation a panel of ten dietary flavonoids such as 3, 6-dihydroxy, 3, 7-dihydroxy, 5, 7-dihydroxy (chrysin), 3hydroxy-7-methoxy, 40 , 5, 7-trihydroxy flavone (apigenin), 40 , 5, 7trihydroxy isoflavone (genistein), 5, 6, 7-trihydroxy flavones (baicalein), 3, 5, 7-trihydroxy flavones (galangin), 5, 7-dihydroxy-40 methyoxy isoflavone (Biochanin-A), and 30 , 5, 7-trihydroxy-40 methoxy flavone (diosmin) have been evaluated for their activity against glycation induced lens opacity, AGE fluorescence, protein aggregation and aldose reductase inhibition (both experimental and in silico). An attempt has been made to discuss the possible structure activity relationship so as to identify the phramacophore of selected test flavonoids which perhaps can be helpful for designing novel and effective anti-cataract agents. 2. Materials and method The selected flavonoids like 3,6-dihydroxy, 3,7-dihydroxy, 3hydroxy-7-methoxy flavones, chrysin, apigenin, genistein, baicalein, galangin, biochanin-A, and diosmin were purchased from SigmaeAldrich Co. St. Louis MO USA, KRB buffer (Kreb Ringer Bicarbonate), NADPH, 2, 4-dinitrophenyl hydrazine (2, 4-DNPH), Aminoguanidine hydrochloride, penicillin, streptomycin, trichloro acetic acid (TCA), urea etc. were purchased from the Himedia Laboratories Pvt. Ltd. Mumbai (MS), India. All other chemicals, solvents and reagents used were of AR grade and were purchased from commercial sources. 2.1. Lens organ culture study Fresh goat eyeballs (from 6 to 8 month old animals) were obtained from the local slaughterhouse at Nanded city and were carried to work place immediately by transferring into a container containing KRB buffer (pH 7.5). The intact lenses were removed by performing dissection using posterior approach [23,24]. The isolated lenses were incubated individually in KRB buffer (having a baseline glucose concentration of 10 mM and osmolality of 255e295 mOsm) with addition of 30 mM glucose (a supraphysiological concentration for inducing glycation) and 50 mM individual flavonoids. A set of positive (lens in KRB buffer without further addition of glucose) and negative control (lens in KRB with added 30 mM glucose) along with a reference compound (aminoguanidine 50 mM) were also arranged in parallel for comparison purpose. Minimum four lenses (from two animals) were used in each set. The lenses in different sets were incubated in CO2 incubator (with 95% air, 5% CO2 and at 37  C temperature). The media of

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positive control was replaced with plane KRB, while the media of treated groups was replaced with fresh KRB buffer added with 30 mM glucose after every 48 h. To prevent the microbial contamination, the antibiotics penicillin and streptomycin were added to the KRB buffer. After 15 days of incubation, the lenses were observed for development of generalized opacity, haziness, disruption and other morphological changes. The digitized images of lenses in various sets were captured using Olympus make SZ 61 TR zoom trinocular microscope. 2.2. Profiling of soluble and insoluble fractions of lens proteins After 15 days of incubation, the 10% lens homogenates were prepared for measuring the amounts of soluble and insoluble fractions of lens proteins after treatment with test flavonoids [24]. In brief, the individual lens tissues from different sets were homogenized in a buffer containing, 0.025 M Tris, 0.5 mM EDTA, 0.1 M NaCl and 0.01% sodium azide (pH 8). Lens homogenates were centrifuged at 10,000 g at 4  C for 30 min. Crude lens soluble protein present in supernatant was collected and used for further analysis. Total proteins (TP) and total lens soluble proteins (TSP) were estimated by Lowry method [25]. The amount of proteins present was expressed as mg/gm of lens tissues. 2.3. Estimation of protein carbonyl groups: a marker of glycation The protein carbonyl groups were estimated as per previous method [26]. The procedure involves 1 h incubation of lens proteins with equal volume of 0.1% of 2, 4-DNPH in 2 N HCl at room temperature. After incubation, proteins were precipitated by addition of 20% trichloro acetic acid (TCA) and washed three times with ethanol/ethyl acetate (1:1) mixture. After final wash, the protein precipitate was solubilised in 133 mM Tris, 13 mM EDTA buffer, pH 7.4 containing 8 M Urea and absorbance was recorded at 365 nm. Concentration of protein carbonyl groups formed in the samples were calculated by using molar extinction coefficient (ε365 nm ¼ 21 mM1 cm1). 2.4. Measurement of intensity of AGE fluorescence AGEs formed in the reaction mixtures were detected by measuring the fluorescence intensity of TSP [23]. Protein samples (TSPs obtained from various sets) were calibrated at a concentration of 0.15 mg/ml in phosphate buffer (pH 7.4), the samples were excited at 370 nm and subsequently the fluorescence emission was recorded between 400 and 500 nm using a Jasco make spectrofluorometer (FP-8300). 2.5. Sizing of glycation induced lens protein aggregates using LM20: nanoparticle tracking and analysis (NTA) system After 15 days of incubation, the size of the glycated lens protein aggregates were measured using NTA LM 20 system. The procedure and operational settings were set as per the manufacturer's manual. The glycated protein samples exposed to various flavonoids were diluted in ultra pure water so as to achieve the particle concentration of 107e109/ml. The calibrated samples were injected into LM 20 sample laser module (Sample holder) by sterile syringes [27]. In NTA, StokeseEinstein equation is the basis for calculation of particle size [28]. The NTA software calculates the size of particles by tracking and analyzing Brownian motion of individual particles under laser light illumination. The NTA 2.3 software measure diffraction of laser light by a particles and processed and generated the data. The data generates terms of mean (average particle size measured) and mode (most frequently observed particle size)

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values [27,28]. 2.6. Preparation of aldose reductase (AR) The isolation and preparation of semipurifed AR using goat lens, was carried out as per the previously reported method [19]. The fresh eye balls from young goats were quickly removed by dissection and homogenized in 100 mM potassium phosphate buffer (pH 6.2). The homogenate was centrifuged at 15,000 rpm for 30 min at 4  C. After discarding the pellet, clear supernatant was stored at 4  C and used as source of AR enzyme. 2.7. Aldose reductase inhibition assay The AR inhibition assay was performed as per previously reported method [29]. In brief, the reaction mixture contained 1 ml of 50 mM potassium phosphate buffer (pH 6.2), 10 mM DLglyceraldehyde, 0.1 mM NADPH, 0.4 mM lithium sulfate, 5 mM 2mercaptoethanol and AR enzyme. The reaction mixture was incubated at room temperature for 10 min. NADPH was added to the reaction mixture to initiate the reaction and the fall in the extinction was recorded spectrophotometrically at 340 nm. For calculating the IC50 values, series of concentrations of selected flavonoids were added to reaction mixtures and appropriate blanks were used for corrections. AR activity without inhibitor was considered as 100%, the amount of individual flavonoids required to achieve 50% AR inhibition were considered as IC50 values. Quercetin was used as reference AR inhibitor. 2.8. In silico studies We have utilized a well established protocol in present In silico molecular docking investigation reported elsewhere [30,31]. In brief target enzyme's (AR) 3D coordinates in complex with inhibitor zopolrestat were downloaded from PDB database [32] with PDB: 2DUX [33]. The aforementioned PDB file was crystallized in monomeric state of the enzyme. Prior to actual virtual screening of flavonoids, the target protein file was appropriately edited for removal of non proteinous parts which is a mandatory step for preparing receptor in AutoDock. In order to test the validity of system, the zopolrestat molecule was individually saved in an independent file. The target system was generated in conventional pdbqt format. ChemDraw ultra software (CambridgeSoft, Cambridge, MA, USA) was initially employed to generate the 2D representations of selected flavonoids. Subsequently, SMILES notations of flavonoids were listed in separate file that was later on loaded on FROG server [34] in order to generate the 3D coordinates of the ligands in sdf format. AutoDock 4.2 implemented in Python Prescription 0.8 (PyRx) interface was used for docking investigation [35,36]. The ligands in sdf format were imported into PyRx interface and converted to pdbqt format. Conventionally, docking systems are validated for accuracy by re-docking the experimentally verified pose back into receptor file and test if software generate pose having best score with orientation similar to experimental binding mode. This fact is measured in terms of RMSD between predicted and experimental pose of ligand. If the RMSD value falls under the acceptable range, say, less than 2 Å, the prediction of binding is assumed to be successful [37]. Default parameters were employed for setting grid and further conformational search algorithm (lamarckian genetic algorithm) was used with default values in all docking runs. The RMSD reading of 0.29 Å was obtained for pose with promising binding free energy value. Structural inspection of docked zopolrestat-AR complex revealed that all the functional groups essential for biological

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activity were predicted to be oriented as required; and thus, predicted binding mode is observed nearly the same as compared to the crystallographic mode of binding (Fig. 5A). This observation thereby confirms the successful validation of docking system. Finally, the PyMol version 0.99 (www.pymol.org) was used for generating the illustrations and 2D interactions of ligands with residues from active site of AR were analyzed with LigPlotþ [38,39]. 3. Results and discussion In continuation to our efforts of deciphering the structural and functional peculiarities of series of flavonoids as possible inhibitors of glycation induced cataractogenesis, the present lens organ culture study was designed to assess the efficacy of a panel of ten flavonoids comprising of di- and tri-hydroxy substitutions. In our previous study, performed using a set of monohydroxylated flavonoids, it was demonstrated that 7-hydroxy substitution was structurally more compatible for inhibition of sugar induced cataractogenesis (unpublished data). The lens organ culture studies designed for assessing the effect of selected flavonoids on lens transparency (Fig. 1) revealed that the flavonoids like chrysin, apigenin, baicalein and genistein were impressive in maintaining the overall structural integrity and to a greater extent the transparency of glycation induced goat lenses after 15 days of incubation. The flavonoids like galangin, 3, 6- and 3, 7-dihydroxy flavonoids maintained the overall structural integrity of the lenses, however, no significant transparency of lenses was maintained using these flavonoids. It seems that methylated flavonoids demonstrate poor activity as compared to non methylated

flavonoids. Because the compounds like 3-hydroxy-7methoxyflavone and biochanin A could not even maintain the structural integrity of the lenses up to 15 days, there was clear appearance of star shaped sutures along with disruptive morphology of the treated lenses. Although not impressive, but the methylated flavonoid diosmin (30 , 5, 7-trihydroxy-40 -methoxy flavone) maintained structural integrity and to some extent the transparency of the lenses. Although the discourse on role of flavonoids in the management of diabetic cataract has long been discussed in the mainstream of cataract research [40], however, the clear molecular understandings about how flavonoids help to maintain the lens transparency and play a cytoprotective role in lens tissues is not fully understood and the research in this direction is evolving. For example, in one preclinical setting, the oral treatment of flavonoid like quercetin has been shown to ameliorate the disturbances of eye lens electrolytes and significantly restored the lens protein levels. The study concludes that the quercetin driven lens transparency might be associated with its role in maintaining the specific osmotic ion equilibrium and levels of lens proteins [41]. After 15 days of incubation, the glycation induced flavonoid treated goat lenses were also analyzed for their soluble and insoluble protein contents (Table 1). Quantification of total soluble and insoluble fractions of lens proteins can be a useful marker, as it can provide useful cues about the opacity of the lens [5]. The results obtained (Table 1) clearly shows that the lenses treated with flavonoids like chrysin (86%), apigenin (86%), genistein (85%) and baicalein (82%) retained significant amount of soluble fraction of lens proteins as compared to other test flavonoids which contained

Fig. 1. Digitized representative images of the goat lenses showing effect of selected flavonoids (50 mM) on glycation induced opacity, haziness, disruption and other morphological changes. The images of lenses were digitized after 15 days of incubation. Minimum four lenses (from two animals) were used in each set. A: Positive control (KRB buffer þ 30 mM glucose); B: Negative control (Kreb Ringer Bicarbonate buffer); C: Apigenin; D: Genistein; E: 5,7-dihydroxyflavone (chrysin); F: 3,7-dihydroxyflavone; G: 3,6-dihydroxyflavone; H: Galangin; I: Biochanin A; J: Baicalein; K: 3-hydroxy-7-methoxyflavone; L: Diosmin.

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Table 1 Summary of protein concentrations (soluble and insoluble) and size of protein aggregates in glycation induced lenses treated with different flavonoids. Sr. no.

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.

Flavonoids

3,6-DHF 3,7-DHF Chrysin Apigenin Genistein Baicalein Galangin 3-H-7-MF Biochanin A Diosmin Aminoguanidine Positive controlb (Glu 30 mM) Negative control (KRBa buffer)

Protein concentration (mg/gm of lens tissue)

Size of lens protein aggregates (nm)

Total lens protein (TP)

Soluble proteins (%)

Average size of lens protein aggregate (mean value)

63(37)c ± 3.8 55(45) ± 4.6 86(14) ± 4.2 86(14) ± 4.8 85(15) ± 6.5 82(18) ± 3.8 61(39) ± 5.9 54 (46) ± 4.1 57(43) ± 6.0 56(44) ± 3.8 88(12) ± 5.5 47(53) ± 3.5 87(13) ± 6.2

233 243 187 210 216 222 204 273 218 193 182 306 161

444 525 363 495 525 507 550 467 394 439 492 268 398

± ± ± ± ± ± ± ± ± ± ± ± ±

5.6 3.9 6.2 4.8 5.5 4.7 3.9 5.8 5.2 5.9 6.0 4.5 5.1

Lens soluble protein (TSP) 280 289 312 427 447 416 339 253 225 246 433 126 348

± ± ± ± ± ± ± ± ± ± ± ± ±

4.2 3.8 5.4 4.0 4.7 5.9 3.2 6.2 4.5 5.8 4.7 5.2 4.9

± ± ± ± ± ± ± ± ± ± ± ± ±

5.2 4.8 4.5 6.2 4.6 5.8 3.9 4.5 5.0 4.8 5.3 4.7 6.2

Actual size of lens protein aggregate (mode value) 189 201 146 162 165 171 176 244 187 178 139 300 130

± ± ± ± ± ± ± ± ± ± ± ± ±

4.0 5.6 5.8 5.0 4.2 5.4 6.4 5.2 5.0 4.9 5.5 6.2 4.5

The results summarized are the mean values n ¼ 4, ±SD. a KRB: Kreb Ringer Bicarbonate buffer. b KRB buffer þ30 mM Glucose, Aminoguanidine is a reference anti-glycation drug, DHF: dihydroxy flavone, 3-H-7-MF: 3-hydroxy-7-methoxy flavone. c Values in the parenthesis indicate the % of insoluble protein.

soluble fractions in a range of 54e63%. It is interesting to note that the methylated flavonoids like 3-hydroxy-7-methoxy flavone (54%), diosmin (56%) and biochanin-A (57%) were not found effective in arresting the glycation induced cross linking of lens proteins leading to insoluble state. Lens tissue has inbuilt water soluble (WS) and insoluble (WIS) fractions of proteins; however, the ageing and cataracts are described as the leading causes for accumulation of WIS fraction of lens proteins [5]. Comparatively the WIS fraction contains relatively more amount of cross-linked proteins, lens fibers and other cytoskeleton proteins than WS fraction [42]. As the WS fraction has clear involvement in maintaining the high refractive index while, WIS is implicated in scattering of light, therefore quantitative measurement of WS and WIS fractions can be conveniently explored as diagnostic or prognostic marker of age related or diabetic cataractogenesis. The data of the effect of selected flavonoids on the size of protein aggregates formed in the glycated lenses has been summarized in Table 1. The size measurements were carried out using NTA LM20 system (Fig. 2), as it offers direct visualization, mean and mode values (most frequently occurring particles) of sizing and counting of the protein aggregates over a wide range of intensity [27]. The smallest size of protein aggregates were observed in the glycated lenses treated with chrysin (146 nm), while the largest size of the protein aggregates was recorded in the lens samples treated with 3-hydroxy-7-methoxy flavone (244 nm) followed by 3, 7-dihydroxy flavones (201 nm). The flavonoids like apigenin (162 nm) genistein (165 nm) and baicalein (171 nm) were also found effective in inhibiting the formation glycation induced protein aggregates. Other flavonoids also had a considerable effect on inhibition of protein aggregate formation by maintaining the size in a range of 176e187 nm as compared to the glycated lens sample (300 nm) devoid of any treatment. It is interesting to note that effect of chrysin (146 nm) was almost equal to the effect demonstrated by an anti-glycating drug aminoguanidine (139 nm). Glycation reaction is known for cross linking of proteins that ultimately results into formation of aggregates [16]. It is believed that the destabilized and partially unfolded proteins contribute for the generation of aggregates. The crystallin aggregates formed in the lens tissue scatters the light and adversely affects the proper focusing of the image onto retina and thereby results into loss of visual acuity. As protein aggregation impairs the vision, its inhibition has been

considered as a target for developing novel anti-cataract agents [3]. In an in vitro experimental setting, series of flavonoids such as quercetin, morin, kaempferol, catechin and epicatechin have been described to inhibit the formation of b-amyloid protein aggregates [43]. Of note, bovine lens a-, b- and g-crystallin are also reported to form amyloid aggregated fibrils under specific denaturing conditions [3]. In general, antioxidative properties of the flavonoids have been attributed with the inhibition of amyloid protein aggregates, it is believed that the antioxidants act as chain breakers of aggregation process, confer stability to the re-folded protein conformations or disaggregate the pre-formed aggregates [3]. An additional mechanism involving aromatic interactions between the flavonoid molecule and aromatic residues in the amyloidogenic sequence has been proposed to direct the inhibitor to the amyloidogenic core and thereby facilitate interaction, but adversely affect the formation fibril (aggregate) assembly [44]. The efforts to unravel the exact molecular mechanisms of protein disaggregation using flavonoids are evolving. The profile of amount of carbonyl groups generated as a result of glycation reaction in various flavonoid treated lenses and in control sets has been summarized in Fig. 3. The results obtained clearly shows that the minimum amounts of carbonyl groups were generated in the lenses treated with chrysin (37 nmoles), genistein (53 nmoles) and apigenin (60 nmol/mg protein). While the lenses treated with galangin (97 nmoles), baicalein (76 nmoles) were found to have significant effect on inhibition of glycation induced carbonyl groups as compared to control set (495 nmoles). Maximum amount of carbonyls were generated in the glycated lenses treated with 3-hydroxy-7-methoxyflavone (245 nmoles), 3,7-dihydroxyflavone (184 nmoles) and 3,6-dihydroxyflavone (114 nmol/mg protein). Of note, the maximum amount of carbonyls (245 nmoles) was estimated from methylated flavonoid. Nevertheless, over 100 nmoles of carbonyls were found in other methylated flavonoids like biochanin A (111 nmoles) and diosmin (103 nmol/mg protein). It seems that methylated substitution reduces the anti-cataract concerns of the flavonoids. Owing to the glyco-oxidative nature of the glycation reaction, the carbonyl groups are generated on the protein side chains. Specifically, the side chains like Pro, Lys, Arg and Thr are described to be more active for undergoing sugar mediated oxidation, leaving the carbonyl groups at the oxidized port. Because of the chemical stability of

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Fig. 2. Representative image panel of effect of chrysin (50 mM) on glycation induced lens crystalline aggregate formation. The effect of selected flavonoids (50 mM) on glycation induced lens crystalline aggregate formation was analyzed by measuring the sizes of lens protein aggregates using NTA LM 20 analysis system. A: Plot representing particle size (nm) and concentration (particle/ml). Value 146 is mode size (in nm) of observed nano particles. B: Still frame from video of nano sizing experiment. C: Plot representing particle size (nm) and normalized intensity. D: A 3D plot of particle size (10 nm per division) and relative intensity. The results summarized are the mean value of four lenses (derived from two animals) each used in individual four experiments.

Fig. 3. Summary of amount of carbonyl groups generated in glycation induced bovine lenses treated with different flavonoids (50 mm). The results summarized are the mean values ± SD of four lenses (derived from two animals) each used in individual four experiments.

carbonyls, their storage and quantification is practically possible [45]. The glycation mediated extensive damage to the lens proteins is positively correlated with the amount of carbonyls generated;

therefore quantification of carbonyls have been considered as a biomarker for assessing the extent of glycation reaction [23]. The fluorescence intensities of the AGEs formed in the glycated

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lens samples has been depicted in Fig. 4. The data presented is a typical fluorescence emission spectra of AGEs recorded between 400 and 500 nm. The glycated lens protein AGEs shows the fluorescence emission peak at 430 nm [23]. It is clear from the presented data; the AGE fluorescence of lens samples treated with chrysin < baicalein < genistein < apigenin demonstrated significant reduction in the generation of AGEs as compared with the intensity of the AGEs formed in the lens sample devoid of any treatment (positive control). Although not very significant, but the remaining flvonoids also showed AGE fluorescence intensity below the values of positive control. Ultimately the progressive glycation reaction culminates into formation of more complex heterogeneous group of compounds including AGEs which acts as key players in accelerating the diabetic complications including cataract [46]. Several flavonoids such as luteolin, quercetin, rutin and its metabolites are implicated in inhibition of fluorescent and nonfluorescent AGEs [47,48]. Muthenna et al. have proposed two possible mechanisms for flavonoid-mediated inhibition of AGEs formation in lens proteins. In the first mechanism it has been proposed that the antioxidant activity of flavonoids terminates the free radical mediated chain reaction of AGE formation, while the second mechanism is related with metal chelating abilities of the flavonoids forming complexes with intermediates and thereby partly inhibits postAmadori product formation [49]. However the clear understandings regarding the molecular mechanisms of flavonoid mediated inhibition of AGEs is evolving. The potential of selected panel of flavonoids towards the inhibition of AR is shown in Table 2. Amongst the selected flavonoids, apigenin (IC50 3.5 mM) and chrysin (IC50 7.0 mM) demonstrated significant inhibition of AR activity. All other test flavonoids showed the AR inhibition in an IC50 range of 9.5e36 mM). AR is the key enzyme of polyol pathway. It has been described that around tenfold amount of sorbitol is generated during diabetic condition owing to up regulation of AR activity. The excess amount of sorbitol generated in the lens tissue causes extensive oxidative damage to

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Table 2 Profile of AR inhibition potential of selected flavonoids. Sr. no.

Flavonoids

AR inhibition IC50 (mM)

1. 2. 3. 4. 5.

3,6-DHF 3,7-DHF 3-H-7-MF Chrysin (5, 7-DHF) Apigenin (40 , 5, 7-THF) Genistein (40 , 5, 7-THIF) Baicalein (5, 6, 7-THF) Galangin (3, 5, 7-THF) Biochanin A (5, 7-DH-40 -MIF Diosmin (30 , 5, 7-TH-40 -MF) Quercetin (Reference compound)

36 34.5 41 7.0. 3.5

6. 7. 8. 9. 10. 11.

± ± ± ± ±

1.4 1.5 0.7 0.4 0.2

18 ± 1.5 13.2 ± 0.6 14.2 ± 0.2 20.9 ± 2.1 24.0 ± 1.2 3.2 ± 0.3

The AR inhibition results summarized are the mean values of n ¼ 3, ±SD. D: di; F: flavone; H: hydroxy; I: iso; M: methoxy; T: tri.

the lens proteins and alters the osmolarity of lens tissue which ultimately leads to formation of cataract [17]. Several reports describe the inhibitory action of active flavonoid compounds from different natural sources against rat lens or human recombinant aldose reductase [22]. While defining the structure activity relationship of AR inhibition using flavonoids, previous report states that the presence of 7-hydroxy substitution and catechol group in B ring is an effective pharmacophore for inhibition of AR activity; however the presence of 3-hydroxy group reduces the activity [50]. To the greater extent, the results of the present study are in agreement with the previous investigation, wherein the 7-hydroxy substituted flavonoids such as chrysin, apigenin, genistein, baicalein and galangin demonstrated the AR inhibition in an IC50 range

Fig. 4. Effect of selected flavonoids (50 mM) on glycation induced non-tryptophan AGEs fluorescence of lens proteins. The result summarized in Fig. 4 is the representative image of individual four experiments performed with four lenses (derived from two animals).

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of 3.5e18 mM. It is interesting to note that, although biochanin A and diosmin have 7-hydroxyl substitution but the IC50 values are 20 and 24 mM. It has been reported that the methylation of 40 -hydroxyl group reduces the AR inhibition [50], it seems that the 40 -methoxy substitution present in these flavonoids might be a cause in reducing the AR inhibition as compared to other test flavonoids. Of note, the flavonoids possessing 3-hydroxy substitution such as 3, 6dihydroxy (IC50 36 mM), 3, 7-dihydroxy (IC50 34.5 mM) and 3hydroxy-7-methoxy (IC50 41 mM) flavones showed reduced inhibition of AR as compared to all other flavonoids. The result summarized in Table 3 comprehensively illustrates the results of the docking experiments carried out to understand the interactions of the selected flavonoids with active site of AR. The presented docking data evidently demonstrates that test flavonoids interact with the residues at the active site of AR with polar as well as non polar interactions. The binding free energy values calculated during the docking experiment may prove be a very good start towards better understanding the ligand protein interactions [51]. The effective binding of a ligand to active site of an enzyme is generally expressed in terms of loose or tight binding. This fact can be inferred from length and number of hydrogen bonds. Lower hydrogen bonding is reported to correspond tighter binding of a ligand [52]. Chrysin and Apigenin can be observed to have potential binding free energy values (9.53 and 9.15 kcal/ mol respectively) in comparison to other test flavonoids. Correspondingly, the same two compounds can be observed to engage multiple, short distance hydrogen bonds with residues at the active site of AR. This in silico observation exactly correlates with the experimental data in fact that the same two compounds demonstrates promising AR inhibitory potential (IC50 values 7 and 3.5 mM

respectively) as compared to other flavonoids. Table 3 also summarizes the list of residues in hydrophobic contact or van der Waal's interactions with the flavonoids. Decent amount of literature has recently piled up that clearly demonstrate the fact that these non polar interactions first come in to play for appropriate orienting the ligand in the active site before they form firm polar interactions in form of hydrogen bonds that stabilize the ligand protein interaction [53]. Therefore, such interactions usually play an important role in defining selectivity of ligand towards proteins. All of the flavonoid compounds used to evaluate AR inhibition in this study appears to have conserved polar interaction (a hydrogen bond) with back bone atoms of Leu-300 (Fig. 5BeD). Recent crystallographic study of drug JF0064 dependent AR inhibition verified such interaction as an important feature in abolishing AR activity [54]. Both of the potential flavonoids like Chrysin and Apigenin are observed to form a hydrogen bond with side chain hydroxy group of Thr-113. Experimental structural studies conducted on inhibition of AR activity by compound IDD388 clearly demonstrate the imperative role in AR inhibition mechanism [55]. Furthermore, biochemical mutagenesis analysis at this location revealed that substitution of threonine with alanine resulted in significant down fall in binding free energy resulting in reduced inhibitory activity of the inhibitors [56]. In addition, Chrysin as well as Apigenin seems to be hydrogen bonded with sulfhydryl group of side chain of Cys-303. Biochemical inhibitory and crystallographic report suggests the critical role of this hydrogen bond with Cys-303 in inhibition of AR by pyridazinone group of compounds [57]. Among all the tested flavonoids for AR inhibition in this study, diosmin is a sole compound that possesses an extra sugar moiety attached to parent flavone

Table 3 Summary of the virtual screening and structural analysis data of various flavonoids in complex with enzyme aldose reductase. Ligand 3,6-DHF

Binding free energy (Kcal/mol)

bb

8.49

Leu300(N ):: Lig(C):C4 OH

3,7-DHF

8.86

3-H-7MF

8.45

Chrysin

9.53a

Apigenin

9.15a

Genistein

8.0

Baicalein

9.11

Galangin

8.89

Biochanin A

8.39

Diosmin

5.17

Hydrogen bonding pair (HBD in Å) (HD::HA)

a

Ox

sc

(2.92)

Lig(A)C5 :: Thr113(O ):OG1 (2.81) Leu300(Nbb):: Lig(C):C4Ox (2.67) Lig(A)C5OH:: Cys303 (Ssc):SG (3.13) Cys303(Ssc):: Lig(C): C4Ox (3.31) Leu300(Nbb):: Lig(C):C3OH (2.89) Lig(A):C7OH:: Thr113(Osc):OG1 (2.82) Leu300(Nbb):: Lig(C): C4Ox (2.73) Lig(A):C5OH::Leu300(Nbb):N (3.03) Lig(A):C7OH:: Cys303 (Ssc):SG (3.11) Lig(A):C7OH:: Thr113(Osc):OG1 (2.83) Leu300(Nbb):: Lig(C): C4Ox (2.81) Lig(A):C5OH::Leu300(Nbb):N (2.96) Lig(A):C7OH:: Cys303 (Ssc):SG (3.10) Leu300(Nbb):: Lig(C): C4Ox (3.20) Lig(A):C5OH::Leu300(Nbb):N (3.06) Lig(A):C7OH:: Thr113(Osc):OG1 (2.83) Leu300(Nbb):: Lig(C): C4Ox (2.83) Lig(A):C5OH:: Leu300(Nbb):N (3.18) Lig(A):C7OH:: Cys303 (Ssc):SG (3.09) Lig(A):C7OH:: Thr113(Osc):OG1 (2.82) Leu300(Nbb):: Lig(C): C4Ox (2.76) Lig(A):C5OH:: Leu300(Nbb):N (2.95) Lig(A):C7OH:: Cys303 (Ssc):SG (3.11) Leu300(Nbb):: Lig(C): C4Ox (2.95) Lig(A):C5OH:: Leu300(Nbb):N (3.18) Lig(A):C7sug:: Val47(Obb):O (2.51) Lig(A):C7sug:: Val47(Obb):O (2.96) Trp20(Nsc):: Lig(A):C7sug: O (3.0)

Residues in hydrophobic interaction or van der Waals contact Trp20, Trp79, Cys80, Trp111, Thr113, Phe115, Phe122, Cys298, Ala299, Cys303, Tyr309 Trp20, Trp79, Trp111, Phe115, Phe122, Cys298, Ala299

Trp20, Val47, Tyr48, Trp79, Cys80, Trp111, Thr113, Phe115, Phe122, Trp219, Ala299, Cys303 Trp20, Trp79, Trp111, Phe115, Phe122, Cys298, Ala299

Trp20, Val47, Tyr48, Trp79, Trp111, Phe115, Phe122, Cys298, Ala299

Trp20, Trp79, Trp111, Thr113, Phe115, Phe122, Trp219, Ala299, Cys303, Tyr309, Pro310 Trp79, Trp111, Phe115, Phe122, Cys298, Ala299, Tyr309, Pro310

Trp79, Trp111, Phe115, Phe122, Cys298, Ala299, Tyr309

Trp20, Trp79, Trp111, Thr113, Phe115, Phe122, Trp219, Ala299, Cys303, Tyr309, Pro310 Tyr48, Gln49, Trp79, His110, Trp111, Phe121, Phe122, Pro218, Trp219, Leu300

D: di; F: flavone; H: hydroxy; M: methoxy; a: flavonoid compounds showing best binding free energy value towards Aldose reductase, Lig: ligand, sc: side chain, bb: back bone, : Sugar, Ox: oxo oxygen, HB: hydrogen bond, HBD: hydrogen bond distance, Å: Angstrom, HD: hydrogen donor group, HA: hydrogen acceptor group. The interaction Lig(A)C5OH:: Thr113(Osc):OG1 is to be inferred as: Hydroxyl group at C5 position in A ring of flavonoid ligand hydrogen bonds with OG1 atom of side chain oxygen of Threonine113. sug

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Fig. 5. Docking result of promising flavonoids A: comparison of predicted pose (Green sticks) to experimentally verified pose (Cyan Sticks) of inhibitor zopolrestat obtained during validation experiment. BeD: Docking results of promising flavonoids (yellow sticks) in aldose reductase (cartoon representation). Residues in hydrophobic contacts are depicted in green color lines. Atoms of residues engaging hydrogen bonds (green dotted lines) with flavonoid molecules are shown as cyan sticks. B: Chrysin, C: Baicalein, D: Apigenin. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

scaffold. Structural data obtained from current docking evaluation support the fact that owing to the sugar group, this flavonoid might have differentially oriented in active site of AR acquiring a unique pose different than other test flavonoids. This orientation might have resulted in interaction of diosmin with AR residues that are not found in the interaction list of other test compounds. For example, among all the test flavonoids, only diosmin shows hydrogen bonding interaction with Trp-20. Hydrophobic support provided by Trp-20 is experimentally verified to strongly influence co-factor binding to AR. Numerous mutagenesis experiments at this position suggest that interaction with Trp-20 plays a crucial role in inhibitor binding [58,59]. Reports describing dual Alrestatin-AR complexes suggest that stacking interaction of Trp-20 over Alrestatin might be one of the factors that determine AR specificity [60,61]. Complex of AR with hexonic acid and phenylacetic acid class of inhibitors demonstrated the significance of PiePi orbital interaction between Trp-20 and various ring systems of these inhibitors. Moreover, van der Waal's contribution of this residue with lipoic acid and hexanoic acid mediate inhibitory effect via interaction with Trp-20 [62]. Moreover, recently proposed drug JF0064 is also known to mediate its AR inhibitory effect via interaction with Trp-20 [54]. Structural analysis of docked poses of all the test flavonoids reveals the fact that flavonoid compounds under investigation mediate piepi stacking interaction with the aromatic side chain of Trp-111. Such stacking interaction is also known to play a key role in inhibition of AR by compounds like zopolrestat [59] and IDD594 [33]. Besides interaction with these conserved residues we propose involvement of Val-47 in flavonoid based inhibition of AR. Function of this residue is rarely reported in literature and its exact structural role still remains elusive. In view of the observed in vitro AR inhibition data and in light of structural observation made from docking study carried out here together with already published supportive crystallographic and biochemical data, an assumption

can be made that residues like Leu-300, Thr-113, Trp-111, Cys-303, Trp-20, Val-47 might have involved in flavonoid based inhibition of AR. The flavonoids (chrysin, apigenein, genistein and baicalein) showing promising activity in various parameters in the present study are available in variety of fruits, vegetables and many medicinal plants [20]. For example sufficient amount of apigenin glycosides (5.36 mg/gm sample) have been recorded in Apium graveolens, commonly called Chinese celery [63]. Honey has been described as a rich source of chrysin with a total flavonoid content of 122e5482 mg/100 g of honey [64], while over 4.6e18.2 mg of genistein have been detected in per gram of soynut [65]. The medicinal herbs like Scutellaria baicalensis and Paeonia lactiflora are described as rich sources of flavonoid baicalein [66]. Using dietary natural resources it is possible to design functional foods and neutraceuticals for the amelioration of sugar induced cataractogenesis. Although fruits, vegetables, tea and wine are the main dietary sources of flavonoids for humans; however the bioavailability, biological activity and metabolism of flavonoids also depends on the structural configuration, total number and substitution of hydroxyl and other functional groups onto flavonoid nucleus [20]. Comprehensive studies have been carried out for addressing the bioavailability of flavonoids administered orally in series of clinical settings [67]. For example, in clinical studies involving human subjects it has been reported that, the intake of a single oral bolus of 2 g/kg of body weight of blanched parsley (equivalent to 65.8 mmol apigenin) resulted in accumulation of a maximum plasma concentration of 127 nmol/l of apigenin after 7.2 h and the decline in plasma concentration started after 28 h [68]. One possible approach of increasing the bioavailability of flavonoids is the glycosylation of aglycone flavonoids as the previous reports state that the sugar moiety of the flavonoids is the main determinant of bioavailability, nevertheless the bioavailability is sometimes enhanced by a glucose moiety [67] however the

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biological activity and metabolism of such modified flavonoids opens a new window for further investigation. 4. Conclusion The basic aim of the present study was to assess the efficacy of a panel of ten selected dietary flavonoids as possible inhibitors of glycation, glycation induced lens opacity, AGEs, AR and lens protein aggregation. It can be concluded from the results of the present study that the selected flavonoids have considerable potential to act as anti-cataract agents. Amongst the test flavonoids chrysin, apigenin, baicalein and genistein can be considered as lead molecules for developing novel and effective anti-cataract agents. In general, in the context of studied parameters it was observed that the methylated flavonoids were less effective as compared to non methylated. Owing to the growing incidence of diabetes and related complications, it is important to assess the efficacy of the natural dietary ingredients like flavonoids as possible agents for the management of diabetes related complications like cataractogenesis, as such bioactive molecules can be made bioavailable through dietary sources. Identifying and designing functional foods and neutraceuticals for the management of diabetic complications has gained momentum because of several health benefits from such fortified foods. The results of the present investigation may also contribute significantly towards the efforts of developing and formulating functional foods/neutraceuticals for the effective amelioration of sugar induced cataractogenesis. Acknowledgments Authors are thankful DST-SERB, New Delhi, for financial assistance (F. No. SR/SO/BB-0088/2012) and to Professor Pandit Vidyasagar, Vice Chancellor, SRTM University, for providing facilities and encouragement. KKP thanks DST-SERB for JRF. References [1] A. Pollreisz, U. Schmidt-Erfurth, Diabetic cataract-pathogenesis, epidemiology and treatment, J. Ophthalmol. 2010 (2010) 608751, http://dx.doi.org/10.1155/ 2010/608751. [2] International Diabetes Federation, Diabetes Atlas, fifth ed., 2011. [3] K.L. Moreau, J.A. King, Protein misfolding and aggregation in cataract disease and prospects for prevention, Trends. Mol. Med. 18 (2012) 273e282, http:// dx.doi.org/10.1016/j.molmed.2012.03.005. [4] H. Bloemendal, W. de Jong, R. Jaenicke, N.H. Lubsen, C. Slingsby, A. Tardieu, Ageing and vision: structure, stability and function of lens crystallins, Prog. Biophys. Mol. Biol. 86 (2004) 407e485. [5] K.K. Sharma, P. Santhoshkumar, Lens aging: effects of crystallins, Biochim. Biophys. Acta 1790 (2009) 1095e1108, http://dx.doi.org/10.1016/ j.bbagen.2009.05.008. [6] A. Laganowsky, J.L. Benesch, M. Landau, L. Ding, M.R. Sawaya, D. Cascio, Q. Huang, C.V. Robinson, J. Horwitz, D. Eisenberg, Crystal structures of truncated alphaA and alphaB crystallins reveal structural mechanisms of polydispersity important for eye lens function, Protein. Sci. 19 (2010) 1031e1043, http://dx.doi.org/10.1002/pro.380. [7] M. Brownlee, Biochemistry and molecular cell biology of diabetic complications, Nature 414 (2001) 813e820. [8] V.M. Monnier, R.H. Nagaraj, M. Portero-Otin, M. Glomb, A.H. Elgawish, D.R. Sell, M.A. Friedlander, Structure of advanced Maillard reaction products and their pathological role, Nephrol. Dial. Transpl. 11 (1996) 20e26. [9] A. Lapolla, P. Traldi, D. Fedele, Importance of measuring products of nonenzymatic glycation of proteins, Clin. Biochem. 38 (2005) 103e115. [10] V.P. Singh, A. Bali, N. Singh, A.S. Jaggi, Advanced glycation end products and diabetic complications, Korean. J. Physiol. Pharmacol. 18 (2014) 1e14, http:// dx.doi.org/10.4196/kjpp.2014.18.1.1. [11] M. Chen, T.M. Curtis, A.W. Stitt, Advanced glycation end products and diabetic retinopathy, Curr. Med. Chem. 20 (2013) 3234e3240. [12] N. Ahmed, Advanced glycation end productserole in pathology of diabetic complications, Diabetes. Res. Clin. Pract. 67 (2005) 3e21. [13] M. Saraswat, P. Suryanarayana, P.Y. Reddy, M.A. Patil, N. Balakrishna, G.B. Reddy, Antiglycating potential of Zingiber officinalis and delay of diabetic cataract in rats, Mol. Vis. 16 (2010) 1525e1537. [14] J.J. Harding, Viewing molecular mechanisms of ageing through a lens, Ageing.

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