J Nutr Health Aging
THE JOURNAL OF NUTRITION, HEALTH & AGING©
GRAPE SEED EXTRACT AND ZINC CONTAINING NUTRITIONAL FOOD SUPPLEMENT PREVENTS ONSET AND PROGRESSION OF AGE-RELATED CATARACT IN WISTAR RATS S. MANI SATYAM1, L. KURADY BAIRY2, R. PIRASANTHAN2 R. LALIT VAISHNAV2 1. Department of Pharmacology, Melak Manipal Medical College, Manipal University Manipal; 2. Department of Pharmacology, Kasturba Medical College, Manipal University, Manipal-576104, Karnataka (India). Corresponding author: Dr. K. L. Bairy, Professor & Head of Pharmacology, Kasturba Medical College, Manipal University, Manipal-576104, Karnataka (India). Phone number- 0820-2922365, Fax number- 0820-2922083, E-mail- [email protected]
Abstract: Objective: To study possible inhibition of oxidative stress and cataract formation by single combined formulation of grape seed extract and Zincovit tablets against sodium selenite-induced age-related cataract in Wistar rat pups. Methods: Oxidative stress and consequent cataract formation was induced by subcutaneous administration of a single dose of sodium selenite (10 µmoles/kg) to Wistar rat pups on day 7 post-natally. In experiments designed to inhibit such cataract formation, the pups were pretreated subcutaneously with combined formulation of grape seed extract and Zincovit tablets (40, 80 and 160 mg/kg), one day prior to the administration of selenite and continuing such treatment till day 20, when the experiments were terminated. The extent of tissue damage caused by the selenite was assessed biochemically by measurements of the levels of reduced glutathione, glutathione peroxidase, glucose-6-phosphate dehydrogenase, protein thiol, catalase, superoxide dismutase, malondialdehyde, aldose reductase, sorbitol dehydrogenase and adenosine triphosphate in the isolated lenses. Cataract formation and its prevention were monitored by examining the eye with pen light illumination and subsequent photography of the isolated lenses. Results: Injection of selenite led to a significant loss of lens clarity due to cataract formation. In the group treated with combined formulation of grape seed extract and Zincovit tablets, the formation of cataract was significantly prevented. In the normal and selenite induced senile cataract control group, the levels of lens oxidative stress markers, G6PD and ATP were substantially lower than in the grape seed extract with Zincovit tablets treated group (p < 0.05). Conclusion: Over all, the results suggest that single combined formulation of grape seed extract and Zincovit tablets may offer a prophylactic measure against onset and progression of age- related cataract of human subjects as nutritional food supplement. Key words: Selenite cataract, oxidative stress, ATP, grape seed extract, zincovit tablets.
surgery in 45%, saving millions (6). Therefore, there is a search for pharmacological intervention that will maintain the transparency of the lens. Zincovit tablet is an advanced combined formulation of vitamins, minerals and grape seed extract (Table 1). Long-term daily administration of grape seed extract offers enhanced antioxidant potential and protection against tissue lipid peroxidation and protein oxidation (7). The biologically active constituents of grape seed extracts are proanthocyanidins, which represent a variety of polymers of flavan-3-ol, such as catechin and epicatechin and have a strong antioxidative effect in aqueous systems (8). “One of the studies suggests the anticataract activity of grape seed extract (GSE, which contains 38.5% procyanidins) in hereditary cataractous rats (ICR/f rats) (9).” Studies suggest that Zinc prevents diabetes-induced GSH loss in the retina (10), vitamin C and E prevents inhibition of glutathione peroxidase, glutathione reductase and superoxide dismutase activities in retina (11-13). Increased oxygen free-radical production lowers the intracellular magnesium concentration and in light of such evidence, vitamin E administration might also regulate the intracellular magnesium concentration (14). A synergistic effect of vitamins C and E along with Zinc could be expected based on the different environments in which they act (14). Vitamin C acts in the hydrophilic milieu, scavenging reactive oxygen species, zinc located in the interphase of the bilayer prevents
Introduction Cataract, a visual impairment causing disturbance in lens transparency occurs mainly due to opacification or optical dysfunction of crystallin lens. Senile cataract, also called age related cataract, is the commonest type of cataract affecting equally persons of either sex usually above the age of 50 years. The total number of persons with cataracts is estimated to rise to 30.1 million by 2020 and it can vary from country to country (1). World Health Organization launched Vision 2020, to eliminate cataract as priority diseases. Various experimental models have been used to study the etiology of cataracts and to investigate different therapeutic modalities. Free radicalinduced oxidative stress is postulated to be perhaps the major factor leading to senile cataract formation (2). Sodium selenite is a strong sulfhydryl oxidant and is considered as a convenient model for the study of senile nuclear cataractogenesis (3, 4). The reliability and extensive characterization of selenite cataract makes it a useful rodent model for rapid screening of potential anti-cataract agents (5). Currently, surgical removal of the opacified lens is the mainstay of the management of cataracts, but there continues to be a backlog in the services provided in many parts of the world. It is estimated that a delay of 10 years in the development of the cataract would reduce the necessity for Received September 17, 2013 Accepted for publication October 24, 2013
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SELENITE CATARACT iron or copper binding to the membrane and alpha-tocopherol in the hydrophobic domains of the bilayer inhibits the lipid oxidation free-radical chain reaction (14). Magnesium inhibits Malondialdehyde (MDA) formation in endothelial cells and low Magnesium oxide induced lipid peroxidation (14). Table 1 Composition of Zincovit tablet Ingredients
per tablet contains
Vitamin C Vitamin B3 Vitamin E Vitamin B1 Vitamin B2 Vitamin B5 Vitamin B6 Folic acid Vitamin A Vitamin D3 Biotin Vitamin B12 Zinc Magnesium Silica Manganese Copper Iodine Boron Selenium Chromium Molybdenum Grape Seed Extract
75 mg 50 mg 15 mg 10 mg 10 mg 10 mg 2 mg 1 mg 5000 IU 400 IU 150 mcg 7.5 mcg 22 mg 18 mg 1 mg 0.9 mg 0.5 mg 150 mcg 150 mcg 50 mcg 25 mcg 25 mcg 50 mg
In previous study, we found the strong in vitro antioxidant potential of the combined formulation of grape seed extract and Zincovit tablets [15]. Therefore, the present study was undertaken to investigate effects of combined formulation of grape seed extract and Zincovit tablets (Nutritional food supplement) against sodium selenite induced cataract in Wistar rat pups. Materials and Methods Drugs and Reagents Single combined formulation of grape seed extract and Zincovit tablets (Nutritional food supplement) was obtained as kind gift from Apex Laboratories Private Ltd., Chennai (India). One Zincovit tablet (850 mg) which contains grape seed extract in addition to other constituents (Table 1) was crushed and dissolved in 100 ml of normal saline (0.9% Sodium chloride solution). The solution was injected subcutaneously to different treatment groups (40, 80 and 160 mg/kg). Sodium selenite, 2
Glutathione reductase, Reduced glutathione (GSH), NADPH, Thiobarbituric acid (TBA), Trichloroacetic acid (TCA) and 5, 5’-Dithiobis (2-nitrobenzoic acid) (DTNB) were procured from Sigma Aldrich, Mumbai (India). One touch glucometer (AccuChek Active) with glucose oxidase-peroxidase reactive strips was purchased from Roche Diagnostics, (USA). Aldose reductase, Sorbitol dehydrogenase and Catalase assay kits were purchased from Cusabio (USA), Uscn Life Science Inc. (USA) and Bioassay Systems (USA). Both ATP and Glucose-6phosphate dehydrogenase assay kits were procured from Abcam Inc. (USA). Sodium azide, Potassium chloride, Sodium chloride, Sodium hydroxide, Ethylene-di-amine-tetra-acetic acid (EDTA) and all other chemicals were obtained from Merck Chemicals, Mumbai (India). All reagents were analytical grade. All reagents except for the phosphate buffers were prepared every day and stored in a refrigerator at +4˚C. The reagents were equilibrated at room temperature for 30 minutes before use, either at the start of analysis or when reagent containers were refilled. Phosphate buffers were stable at +4˚C for one month. Animals 7 days old Wistar rat pups with their mother were housed in separate polypropylene cages, maintained under standard conditions with temperature (22–240C), 12-h light/12-h dark cycle and relative air humidity 40–60%. Rats had continuous access to normal calorie standard rat pellet diet (Hindustan Lever Ltd., Mumbai, India) and to tap water. After grouping, the animals were acclimatized to the laboratory conditions for one week before the start of the experiment. Animals described as fasted were separated from their mother overnight and deprived of food for 16-h but had allowed free access to water. The experimental protocol was approved by the Institutional Animal Ethics Committee (IAEC/KMC/06/2012) and experiments were conducted according to the ethical norms approved by Ministry of Social Justices and Empowerment, Government of India and Committee for the Purpose of Control and Supervision on Experiments on Animals (CPCSEA) guidelines and Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Research. Experimental design In the experiment, 30 Wistar rat pups were divided into five groups (n= 6). The corresponding doses of Zincovit tablets with grape seed extract were administered one day prior to the sodium selenite (10 µmoles/kg; subcutaneously) in addition to the regular treatment. Treatment was continued from day 7 to day 20 of their post-natal life as followGroup I: Normal control rat pups were given 0.1 ml of normal saline; subcutaneously Group II: Sodium selenite induced cataract control rat pups were given 0.1 ml of normal saline; subcutaneously Group III: Sodium selenite induced cataract rat pups were given Zincovit tablets with grape seed extract 40 mg/kg/day;
J Nutr Health Aging
THE JOURNAL OF NUTRITION, HEALTH & AGING© subcutaneously Group IV: Sodium selenite induced cataract rat pups were given Zincovit tablets with grape seed extract 80 mg/kg/day; subcutaneously Group V: Sodium selenite induced cataract rat pups were given Zincovit tablets with grape seed extract 160 mg/kg/day; subcutaneously
incubated for 10 minutes at room temperature and absorbance was read at 412 nm by using an ELISA reader Bio Tek Instruments ELx800- MS, (USA). Determination of Glutathione peroxidase (GPx) activity 550 µl phosphate buffer (100 mM) containing 0.1 mM EDTA (pH 6.5), 50 µl Sodium azide (2 mM), 50 µl lens tissue homogenate, 100 µl glutathione reductase (2.5 U/ml) and reduced glutathione (GSH, 100 mM) were added together and incubated at 37ºC for 10 minutes. Then, 100 µl NADPH (2.5 mM) was added in the above mixture. Reaction was started by adding 100 µl hydrogen peroxide (1.5 mM) and optical density was read at 340 nm at one minute interval for five minutes by using UV-2450 spectrophotometer, Shimadzu Corporation, Tokyo (Japan).
Blood, Lens Collection and Processing The rat pups first opened their eyes, approximately 16 days after birth, cataract formation and its prevention were monitored by examining the eye (cloudy area in the lens) with pen light illumination thereafter till the end of experiment and subsequent photography of the isolated lenses was done on 21st day. Body weight of each rat pup was checked before start and at the end of the experiment. On 21st day, all the rat pups were sacrificed by administering overdose of ketamine, i.p. according to the annexure-6 of euthanasia of laboratory animals in the Committee for the Purpose of Control and Supervision on Experiments on Animals (CPCSEA) guidelines for Laboratory Animal Facility. The eye lenses were dissected by the posterior approach and stored at -70°C until further analysis. Lens weight of each rat pup was taken. Fasting blood samples were drawn on from retro-orbital plexus of all the experimental animals using capillary tube for the estimation of blood glucose with the help of glucose oxidase-peroxidase reactive strips (Accu-Chek, Roche Diagnostics, USA). Lens homogenates (10% w/v) were prepared from three to five pooled lenses in 50 mM potassium phosphate buffer (pH 7.4) using a Remi homogenizer. The unbroken cells and cell debris were removed by centrifugation at 10000 rpm for 20 minutes using a Remi C-24 refrigerated centrifuge. The resulting supernatant was stored at -80˚C. All biochemical parameters were analyzed in the soluble fraction of the lens homogenate except for malondialdehyde (MDA) which was determined in the total homogenate. The following biochemical analysis was done in triplicate manner and optical density was also read for reagent and sample blank-
Determination of Protein thiol (PT) level 20 µl of lens tissue homogenate sample was added in the mixture of 180 µl disodium edetate (2mM disodium edetate in 0.2 M disodium hydrogen phosphate) buffer solution and 4 µl DTNB solution (10mM DTNB in 0.2 M disodium hydrogen phosphate) in 96-wells of micro test plate. Then, optical density was read at 412 nm by using an ELISA reader Bio Tek Instruments ELx800- MS, (USA). Determination of Superoxide dismutase (SOD) activity To 25 µl of lens tissue homogenate sample, 925 µl sodium carbonate buffer (0.1M, pH 10-11) and 50 µl of adrenaline bitartarate (1mM) was added and absorbance (A0s-A60s,) was read at 480 nm by using UV-2450 spectrophotometer, Shimadzu Corporation, Tokyo (Japan). Determination of Catalase (CAT) activity Catalase activity in lens homogenate was measured according to the standard protocol given along with the Catalase assay kit of Bioassay Systems, Hayward (USA) by using an ELISA reader Bio Tek Instruments ELx800- MS, (USA).
Determination of Malondialdehyde (MDA) level To 20 µl lens homogenate sample, 200 µl 0.67% thiobarbituric acid and 100 µl 20% trichloroacetic acid were added and incubated at 100˚C for 20 minutes. Then, it was centrifuged at 12000 rpm for 5 minutes and 100 µl of supernatant was transferred to 96- wells of micro test plate. Optical density of supernatant was read at 540 nm by using an ELISA reader Bio Tek Instruments ELx800- MS, (USA).
Determination of Glucose-6-phosphate dehydrogenase (G6PD) activity Glucose-6-phosphate dehydrogenase activity in lens homogenate was measured according to the standard protocol given along with the Glucose-6-phosphate dehydrogenase assay kit of Abcam Inc., (USA) by using an ELISA reader Bio Tek Instruments ELx800- MS, (USA). Determination of Adenosine triphosphate (ATP) level Adenosine triphosphate level in lens homogenate was measured according to the standard protocol given along with the Adenosine triphosphate assay kit of Abcam Inc., (USA) by using an ELISA reader Bio Tek Instruments ELx800- MS, (USA).
Determination of Reduced glutathione (GSH) level Mixture of 100 µl of lens tissue homogenate and 100 µl of 5% trichloro acetic acid (TCA) solution was centrifuged at 5000 rpm for 5 minutes. Then, 25 µl of tissue supernatant, 150 µl sodium phosphate buffer (PBS 0.2 M, pH 8.0) and 25 µl DTNB (0.6mM) was added together in 96-wells of micro test plate and 3
J Nutr Health Aging
SELENITE CATARACT Determination of Aldose reductase (AR) level Aldose reductase concentration in lens homogenate was measured according to the standard protocol given along with the Aldose reductase assay kit of Cusabio Inc., (USA) by using an ELISA reader Bio Tek Instruments ELx800- MS, (USA).
mg/kg, 80 mg/kg and 160 mg/kg; s.c, respectively) exhibited mild lenticular opacification (Figure 1, 2C-2E). Effect on body weight and isolated lens weight There was no significant change in body as well as isolated lens weight among the experimental groups.
Determination of Sorbitol dehydrogenase (SDH) level Sorbitol dehydrogenase level in lens homogenate was measured according to the standard protocol given along with the Sorbitol dehydrogenase assay kit of Uscn Life Science, (USA) by using an ELISA reader Bio Tek Instruments ELx800MS, (USA).
Figure 1 Effect of different doses of combined formulation of grape seed extract and Zincovit tablets on prevalence of sodium selenite induced senile cataract among experimental groups
Statistical analysis Using Statistical Package for the Social Sciences (SPSS version 16.0; SPSS Inc., Chicago, USA), normally distributed data were expressed as mean ± standard error of mean and analyzed by one way analysis of variance (ANOVA) followed by post hoc Tukey test. Data with non-uniform distribution were expressed as median, Quartile (Q1, Q3) and analyzed by non-parametric K Independent samples test followed by Kruskal-Wallis H test. A level for p ≤ 0·05 was considered to be statistically significant (two-sided). Results Effect on lens morphology Cataract formation and its prevention were monitored on 16th day onwards of their post natal life after first opening of eyes. All six rat pups in normal control group (which received normal saline; 0.1 ml, s.c) exhibited complete transparency of the lens (Figure 1, 2A). “Out of 12 eye lenses of six rat pups in Effect on biochemical parameters sodium selenite induced senile cataract control group (which There was significant increase in reduced glutathione (p = received normal saline; 0.1 ml, s.c), 10 eye lenses exhibited 0.005), glutathione peroxidase (p = 0.019), glucose-6dense opacification of the lenses (Figure 1, 2B).” In contrast, phosphate dehydrogenase (p = 0.030), protein thiol (p = 0.022) only 6, 2 and 1 among 12 lenses (which received combined and adenosine triphosphate (p = 0.046) in the lens of rat pups formulation of grape seed extract and Zincovit tablets 40 treated with combined formulation of grape seed extract and Table 2 Effect of combined formulation of grape seed extract and Zincovit tablets on reduced glutathione (µmoles/mg) and glutathione peroxidase (µmoles/ml) and Glucose-6-phosphate dehydrogenase (µmoles/min/ml) in lens tissue homogenate Groups (n=6) I- Normal control (2% gum acacia) II- Selenite control (2% gum acacia) III- Selenite + ZVT (40 mg/kg/day) IV- Selenite + ZVT (80 mg/kg/day) V- Selenite + ZVT (160 mg/kg/day)
GSH (Q1, Q3)
GPx (Q1, Q3)
G6PD (Q1, Q3)
73.38 (47.71, 111.43)
361.83 (220.31, 494.98)
43.47 (28.04, 82.79)
27.48 (24.41, 66.62)
97.08 (82.08, 292.51)
17.79 (15.74, 47.26)
22.60 (16.60, 28.22)
98.02 (63.04, 126.84)
16.58 (11.60, 20.26)
18.12 (15.45, 61.44)
95.50 (61.97, 257.21)
20.74 (11.50, 42.27)
350.28 (171.44, 389.45)
56.09 (45.46, 84.14)
p = 0.019
p = 0.030
82.98 (63.18, 114.08) p = 0.005
n- Number of rats in each group. GSH- Reduced glutathione, GPx- Glutathione peroxidase and G6PD- Glucose-6-phosphate dehydrogenase. Data are expressed as the median (quartilesQ1,Q3) and different treatments were analyzed by non-parametric K Independent sample test followed by Kruskal-Wallis H test.
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THE JOURNAL OF NUTRITION, HEALTH & AGING© Table 3 Effect of combined formulation of grape seed extract and Zincovit tablets on protein thiol (µmoles/mg), adenosine triphosphate (mmoles/ml) and malondialdehyde (nmoles/g) in lens tissue homogenate Groups (n=6)
I- Normal control (2% gum acacia) II- Selenite control (2% gum acacia) III- Selenite + ZVT (40 mg/kg/day) IV- Selenite + ZVT (80 mg/kg/day) V- Selenite + ZVT (160 mg/kg/day)
PT (Q3, Q3)
ATP (Q3, Q3)
MDA (Q3, Q3)
890.69 (524.43, 1178.84)
1.00 (0.16, 2.12)
27.47 (21.97, 32.05)
397.97 (336.20, 908.85)
0.29 (0.16, 1.60)
27.47 (24.72, 58.60)
304.96 (226.24, 342.82)
0.20 (0.07, 0.56)
36.62 (21.06, 53.11)
237.55 (223.29, 434.78)
0.21 (0.13, 0.30)
23.80 (21.97, 26.55)
1171.78 (511.08, 1673.37)
0.91 (0.68, 1.87)
20.14 (17.39, 21.97)
p = 0.022
p = 0.046
p = 0.024
n- Number of rats in each group. PT- Protein thiol, ATP- Adenosine triphosphate and MDA- Malondialdehyde. Data are expressed as the median (quartiles- Q1, Q3) and different treatments were analyzed by non-parametric K Independent sample test followed by Kruskal-Wallis H test. .
Figure 2 Photographs of experimental rats on 21st day: (A). Normal control rat (B). Sodium selenite induced cataract control rat (C). Sodium selenite induced cataract rat treated with combined formulation of grape seed extract and Zincovit tablets 40 mg/kg (D). Sodium selenite induced cataract rat treated with combined formulation of grape seed extract and Zincovit tablets 80 mg/kg (E). Sodium selenite induced cataract rat treated with combined formulation of grape seed extract and Zincovit tablets 160 mg/kg
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SELENITE CATARACT Zincovit tablets in dose dependent manner when compared with sodium selenite induced senile cataract control rat pups (Table 2, 3). Lens malondialdehyde level was significantly lower (p = 0.024) in rat pups treated with combined formulation of grape seed extract and Zincovit tablets in comparison with sodium selenite induced senile cataract control rat pups (Table 3). There were no significant changes in fasting blood glucose level and lens superoxide dismutase, catalase, aldose reductase and sorbitol dehydrogenase among the experimental groups.
in Wistar rat pups. “A consistent finding in the present study was significant increase of lens ATP levels in the rats treated with combined formulation of grape seed extract and Zincovit tablets in a dose dependent fashion in comparison with selenite-induced cataract control rats (Table 3).” The decrease in ATP level in lens of selenite-induced cataract control rats could be due to irreversible damage of the lens. ATP is needed as a cofactor for the conversion of glucose to phosphorylated glucose by hexokinase. Decrease in ATP might have affected glucose 6phosphate generation and thereby diverting glucose through the polyol pathway. Similarly, ATP also affects enzymes involved in the glutathione synthesis. The energy needed for the biosynthesis of proteins in lens is supplied in the form of ATP derived from carbohydrate metabolism. Decrease in ATP affects the biosynthesis of protein in the lens. The decreased supply of energy, as a result of altered glucose metabolism results in pronounced change in the cytoplasmic composition of the lens, cellular deterioration and eventually loss of transparency. “One of the studies also suggests significant decreased level of lens ATP in selenite induced cataract control rats compared to normal control rats (20)”. The significant increase in the activity of lens glucose-6phosphate dehydrogenase in senile cataractous rats treated with combined formulation of grape seed extract and Zincovit tablets in comparison with selenite-induced cataract control rats may enhance formation of ribose for nucleic acids synthesis and also increase the renewal of lens protein (Table 2). The pentose phosphate pathway supplies NADPH which is the main component of the glutathione system. It has been reported that when the intensity of a stress increases, GSH concentrations usually decline and redox state becomes more oxidized, leading to deterioration of the system (24). “A decrease in GSH level may disorganize and disorient the mitochondria of the cell resulting in reduced supply of ATP, thereby limiting optimal cellular and physiological function in the senile cataract state.” In the present study, there was decrease in reduced form glutathione (GSH) and protein thiol level in the selenite-induced cataract control group whereas Zincovit tablets with grape seed extract treated group especially at the dose of 160 mg/kg restored the level of reduced glutathione and protein thiol (Table 2, 3). Decrease in the activity of glutathione peroxidase in lenses of selenite-induced cataract control rats may be due to either direct inactivation by reactive oxygen species or by decreased aerobic metabolism in lens. In this study, the significant increase in the activity of glutathione peroxidase following the administration of the combined formulation of grape seed extract and Zincovit tablets may be adduce to the presence of vitamin C, E, riboflavin and minerals like zinc that might have enhanced the synthesis of this enzyme and preserved the glutathione level (Table 2). Increased lenticular MDA may be the result of increased lipid peroxidation of the lenticular cell membranes, or may represent the consequence of migration of MDA from the
Discussion Age-related cataract is the major cause of blindness globally. Yet, a pharmacological treatment for cataract has not been achieved. Although it is a multifactorial disease associated with several risk factors, oxidative stress has been suggested as a common underlying mechanism of cataractogenesis, and augmentation of the antioxidant defenses of the lens has been shown to prevent or delay experimental cataract (16). The selenite cataract model was selected because of its rapid, effective and reproducible cataract formation. “As a model for senile nuclear cataract, it has been extensively characterized histologically and biochemically (17).” Most pronounced biochemical mechanisms occur during development of seleniteinduced cataract are altered epithelial metabolism, calcium accumulation, calpain-induced proteolysis, crystalline precipitation, phase transition, and cytoskeletal loss (5). “Although selenite cataract shows no high molecular weight covalent aggregates or increased disulfide formation, it has many similarities to human age related cataract such as vesicle formation, increased calcium, insoluble protein, proteolysis, decreased water soluble proteins, and glutathione (GSH) (5).” “Selenite induced conversion of GSH to GSSG caused directly by its reaction with sodium selenite (18) as or through an initial formation of seleno-diglutathione (19).” The latter is rapidly converted to GSSG with the generation of selenium dioxide. This in turn restarts the thiol depleting reaction. The GSSG so formed is extruded out the lens (20). Selenite induces bilateral nuclear cataract within 4 to 6 days when administered to suckling rat pups before completion of the critical maturation period of the lens (21). Any strategy that prevents or slows the progression of cataract has a significant health impact. The potential role of vitamins in preventing cataract is well documented, especially vitamin C or ascorbic acid which plays an important part in lens biology, both as an antioxidant and as a UV filter. “Some of the studies suggest that routine consumption of grape seed proanthocyanidins extract in the form of food or a dietary supplement may offer a prophylactic measure against onset and progression of cataract (21-23), which supports our findings in this study.” The combination of multivitamin-multimineral and grape seed extract Zincovit tablets may produce synergistic anticataractogenic potential (Table 1). Hence, we set out to investigate the role of combined formulation of grape seed extract and Zincovit tablets in the prevention or delay of progression and maturation of selenite-induced senile cataract 6
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THE JOURNAL OF NUTRITION, HEALTH & AGING© readily peroxidizable retina or from the central body compartment (24-30). In the present investigation, such a disruption of membrane lipids possibly accounted for the observed increase in MDA levels in the lenses of cataractuntreated (Group II) rat pups when compared to normal (Group I) rat pups (Table 3). The observed lower mean MDA level in the cataract-treated with combined formulation of grape seed extract and Zincovit tablets relative to selenite-induced cataract control rats suggests that grape seed extract with Zincovit tablets possibly preserved the structural integrity of the lens, thereby preventing lenticular opacification. The ability of the combined formulation of grape seed extract and Zincovit tablets (Nutritional food supplement) to delay the progression and maturation of selenite-induced agerelated cataract might be attributed to synergistic interplay of constituents of Zincovit tablets, such as- grape seed extract proanthocyanidins which comprise only procyanidins [subunits constituted of (+)-catechin (C) and (−)-epicatechin (EC)], Vitamins A, B, C, D, E, folic acid, biotin and minerals like zinc, copper, selenium, magnesium, manganese, chromium and molybdenum mainly, which are promoters of antioxidant activity (Table 1). Vitamin C, E (mainly tocopherols) and a variety of carotenoids are present in lens tissue and in the fluid that surrounds it and have an important part to play in lenticular antioxidant status. Riboflavin is a precursor to flavin-adenine dinucleotide (FAD), which is a coenzyme for the biosynthesis of glutathione reductase. Selenium is an integral part of the enzyme, glutathione peroxidase. Both the lens of the eye and the aqueous humor contain protective enzymes that breakdown the damaged proteins that clump together and cause cataracts. This combined formulation due to its strong antioxidant potential may keep these enzymes from being destroyed. From the present study it can be concluded that the combined formulation of grape seed extract and Zincovit tablet is the potential functional nutritional food supplement that can prevent the onset, progression and maturation of seleniteinduced age-related cataract in Wistar rat pups. The therapeutic effect seen in animal studies cannot always be entirely extrapolated to humans. Hence, clinical evaluation should be performed to precisely define the anti-cataractogenesis role of Zincovit tablets with grape seed extract in humans. Our study opens the perspective to clinical studies specifically designed to evaluate the impact of the single combined formulation of grape seed extract and Zincovit tablets on the onset and progression of age-related cataract of human subjects as nutritional food supplement. “This information would eventually complement our findings, opening the way to sustain age-related cataract development in human population”.
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Acknowledgments: All the authors declare that they do not have any conflict of interest. This work was financially supported by Apex Laboratories Private Ltd., Chennai (India) for which authors are grateful.
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Nakamura Y, Tsuji S, Tonogai Y (2003) Analysis of Proanthocyanidins in Grape seed extracts, Health foods and Grape seed oils. Journal of Health Science 49:45-54. Yamakoshi J, Saito M, Kataoka S, Tokutake S (2002) Procyanidin-Rich Extract from Grape Seeds Prevents Cataract Formation in Hereditary Cataractous (ICR/f) Rats. J Agric Food Chem 50(17):4983-4988. Moustafa SA (2004) Zinc might protect oxidative changes in the retina and pancreas at the early stage of diabetic rats. Toxicol Appl Pharmacol 20:149-155. Mustata GT, Rosca M, Biemel KM, Reihl O, Smith MA, et al (2005) Paradoxical effects of green tea (Camellia sinensis) and antioxidant vitamins in diabetic rats: improved retinopathy and renal mitochondrial defects but deterioration of collagen matrix glycoxidation and cross-linking. Diabetes54:517-526. Kowluru RA, Tang J, Kern TS (2001) Abnormalities of retinal metabolism in diabetes and experimental galactosemia. VII. Effect of long-term administration of antioxidants on the development of retinopathy. Diabetes 50:1938-1942. Penn JS, Madan A, Caldwell RB, Bartoli M, Caldwell RW, et al. Vascular endothelial growth factor in eye disease (2008) Prog Retin Eye Res 27:331-371. Farvid MS, Jalali M, Siassi F et al (2005) Comparison of the effects of vitamins and/or mineral supplementation on glomerular and tubular dysfunction in type 2 diabetes. Diabetes Care 28:2458-464. Satyam SM, Bairy KL (2013) Antioxidant activity of combination of Grape seed extract and Zincovit tablets (nutritional food supplement) on free radical scavenging in vitro models. Jokull Journal 63:360-369. Spector A (1995) Oxidative stress induced cataract: mechanism of action. FASEB J 9:1173–1182. Shearer TR, Anderson RS, Britton JL (1983) Influence of selenite and fourteen trace elements on cataractogenesis in the rat. Invest Ophthalmol Vis Sci 24:417–423. Sankui S, Arai K, Kojima T, Nagaoka S, Majima H (2007) Photocatalytic reduction of selenate and selenite solutions using TiO2 powders. Metallurgical Material Trans 30:15-20. Tsen CC, Tappel AL (1958) Catalytic oxidation of GSH and other sulfhydryl compounds by selenite. J Biol Chem 233:1230-1232. Varma SD, Hegde KR, Kovtun S (2010) Inhibition of Selenite Induced Cataract by Caffeine Acta Ophthalmol 88(7):e245-e249. Durukan AH, Evereklioglu C, , Hurmeric V, Hurkan Kerimoglu H, Erdurman C, Bayraktar MZ, Mumcuoglu T (2006) Ingestion of IH636 grape seed proanthocyanidin extract to prevent selenite-induced oxidative stress in experimental cataract. J Cataract Refract Surg 32:1041-1045. Xuan Z, Yizhen HU (2012) Inhibitory effects of grape seed proanthocyanidin extract on selenite induced cataract formation and possible mechanism. J Huazhong Univ Sci Technol 32(4):613-619. Jia Z, Song Z, Zhao Y, Wang X, Liu P (2011) Grape seed proanthocyanidin extract protects human lens epithelial cells from oxidative stress via reducing NF-кB and MAPK protein expression. Mol Vis 17:210-217. Tausz T, Sircelj H, Grill D (2004) The glutathione system as a stress marker in plant ecophysiology: Is a stress-response concept valid? J Exp Bot 55:1955-1962. Isai M, Sakthivel M, Ramesh E, Thomas PA, Geraldine P (2009) Prevention of selenite-induced cataractogenesis by rutin in Wistar rats Molecular Vision 15:25702577. Babizhayev MA (1989) Accumulation of lipid peroxidation products in human cataracts. Acta Ophthalmol 67:281-7. Babizhayev MA, Deyev AI (1989) Lens opacity induced by lipid peroxidation products as a model of cataract associated with retinal disease. Biochim Biophys Acta 1004:124-133. Simonelli F, Nesti A, Pensa M, Romano L, Savastano S, Rinaldi E (1989) Lipid peroxidation and human cataractogenesis in diabetes and severe myopia. Exp Eye Res 49:181-187. Altomare E, Vendemiale G, Grattagliano I, Angelini P, Ferrari MT, Cardia L (1995) Human diabetic cataract: role of lipid peroxidation. Diabete Metab 21:173-179. Altomare E, Grattagliano I, Vendemaile G, Ferrari MT, Signorile A, Cardia L (1997) Oxidative protein damage in human diabetic eye: evidence of a retinal participation. Eur J Clin Invest 27:141-147.
THE JOURNAL OF NUTRITION, HEALTH & AGING©
GRAPE SEED EXTRACT AND ZINC CONTAINING NUTRITIONAL FOOD SUPPLEMENT PREVENTS ONSET AND PROGRESSION OF AGE-RELATED CATARACT IN WISTAR RATS S. MANI SATYAM1, L. KURADY BAIRY2, R. PIRASANTHAN2 R. LALIT VAISHNAV2 1. Department of Pharmacology, Melak Manipal Medical College, Manipal University Manipal; 2. Department of Pharmacology, Kasturba Medical College, Manipal University, Manipal-576104, Karnataka (India). Corresponding author: Dr. K. L. Bairy, Professor & Head of Pharmacology, Kasturba Medical College, Manipal University, Manipal-576104, Karnataka (India). Phone number- 0820-2922365, Fax number- 0820-2922083, E-mail- [email protected]
Abstract: Objective: To study possible inhibition of oxidative stress and cataract formation by single combined formulation of grape seed extract and Zincovit tablets against sodium selenite-induced age-related cataract in Wistar rat pups. Methods: Oxidative stress and consequent cataract formation was induced by subcutaneous administration of a single dose of sodium selenite (10 µmoles/kg) to Wistar rat pups on day 7 post-natally. In experiments designed to inhibit such cataract formation, the pups were pretreated subcutaneously with combined formulation of grape seed extract and Zincovit tablets (40, 80 and 160 mg/kg), one day prior to the administration of selenite and continuing such treatment till day 20, when the experiments were terminated. The extent of tissue damage caused by the selenite was assessed biochemically by measurements of the levels of reduced glutathione, glutathione peroxidase, glucose-6-phosphate dehydrogenase, protein thiol, catalase, superoxide dismutase, malondialdehyde, aldose reductase, sorbitol dehydrogenase and adenosine triphosphate in the isolated lenses. Cataract formation and its prevention were monitored by examining the eye with pen light illumination and subsequent photography of the isolated lenses. Results: Injection of selenite led to a significant loss of lens clarity due to cataract formation. In the group treated with combined formulation of grape seed extract and Zincovit tablets, the formation of cataract was significantly prevented. In the normal and selenite induced senile cataract control group, the levels of lens oxidative stress markers, G6PD and ATP were substantially lower than in the grape seed extract with Zincovit tablets treated group (p < 0.05). Conclusion: Over all, the results suggest that single combined formulation of grape seed extract and Zincovit tablets may offer a prophylactic measure against onset and progression of age- related cataract of human subjects as nutritional food supplement. Key words: Selenite cataract, oxidative stress, ATP, grape seed extract, zincovit tablets.
surgery in 45%, saving millions (6). Therefore, there is a search for pharmacological intervention that will maintain the transparency of the lens. Zincovit tablet is an advanced combined formulation of vitamins, minerals and grape seed extract (Table 1). Long-term daily administration of grape seed extract offers enhanced antioxidant potential and protection against tissue lipid peroxidation and protein oxidation (7). The biologically active constituents of grape seed extracts are proanthocyanidins, which represent a variety of polymers of flavan-3-ol, such as catechin and epicatechin and have a strong antioxidative effect in aqueous systems (8). “One of the studies suggests the anticataract activity of grape seed extract (GSE, which contains 38.5% procyanidins) in hereditary cataractous rats (ICR/f rats) (9).” Studies suggest that Zinc prevents diabetes-induced GSH loss in the retina (10), vitamin C and E prevents inhibition of glutathione peroxidase, glutathione reductase and superoxide dismutase activities in retina (11-13). Increased oxygen free-radical production lowers the intracellular magnesium concentration and in light of such evidence, vitamin E administration might also regulate the intracellular magnesium concentration (14). A synergistic effect of vitamins C and E along with Zinc could be expected based on the different environments in which they act (14). Vitamin C acts in the hydrophilic milieu, scavenging reactive oxygen species, zinc located in the interphase of the bilayer prevents
Introduction Cataract, a visual impairment causing disturbance in lens transparency occurs mainly due to opacification or optical dysfunction of crystallin lens. Senile cataract, also called age related cataract, is the commonest type of cataract affecting equally persons of either sex usually above the age of 50 years. The total number of persons with cataracts is estimated to rise to 30.1 million by 2020 and it can vary from country to country (1). World Health Organization launched Vision 2020, to eliminate cataract as priority diseases. Various experimental models have been used to study the etiology of cataracts and to investigate different therapeutic modalities. Free radicalinduced oxidative stress is postulated to be perhaps the major factor leading to senile cataract formation (2). Sodium selenite is a strong sulfhydryl oxidant and is considered as a convenient model for the study of senile nuclear cataractogenesis (3, 4). The reliability and extensive characterization of selenite cataract makes it a useful rodent model for rapid screening of potential anti-cataract agents (5). Currently, surgical removal of the opacified lens is the mainstay of the management of cataracts, but there continues to be a backlog in the services provided in many parts of the world. It is estimated that a delay of 10 years in the development of the cataract would reduce the necessity for Received September 17, 2013 Accepted for publication October 24, 2013
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SELENITE CATARACT iron or copper binding to the membrane and alpha-tocopherol in the hydrophobic domains of the bilayer inhibits the lipid oxidation free-radical chain reaction (14). Magnesium inhibits Malondialdehyde (MDA) formation in endothelial cells and low Magnesium oxide induced lipid peroxidation (14). Table 1 Composition of Zincovit tablet Ingredients
per tablet contains
Vitamin C Vitamin B3 Vitamin E Vitamin B1 Vitamin B2 Vitamin B5 Vitamin B6 Folic acid Vitamin A Vitamin D3 Biotin Vitamin B12 Zinc Magnesium Silica Manganese Copper Iodine Boron Selenium Chromium Molybdenum Grape Seed Extract
75 mg 50 mg 15 mg 10 mg 10 mg 10 mg 2 mg 1 mg 5000 IU 400 IU 150 mcg 7.5 mcg 22 mg 18 mg 1 mg 0.9 mg 0.5 mg 150 mcg 150 mcg 50 mcg 25 mcg 25 mcg 50 mg
In previous study, we found the strong in vitro antioxidant potential of the combined formulation of grape seed extract and Zincovit tablets [15]. Therefore, the present study was undertaken to investigate effects of combined formulation of grape seed extract and Zincovit tablets (Nutritional food supplement) against sodium selenite induced cataract in Wistar rat pups. Materials and Methods Drugs and Reagents Single combined formulation of grape seed extract and Zincovit tablets (Nutritional food supplement) was obtained as kind gift from Apex Laboratories Private Ltd., Chennai (India). One Zincovit tablet (850 mg) which contains grape seed extract in addition to other constituents (Table 1) was crushed and dissolved in 100 ml of normal saline (0.9% Sodium chloride solution). The solution was injected subcutaneously to different treatment groups (40, 80 and 160 mg/kg). Sodium selenite, 2
Glutathione reductase, Reduced glutathione (GSH), NADPH, Thiobarbituric acid (TBA), Trichloroacetic acid (TCA) and 5, 5’-Dithiobis (2-nitrobenzoic acid) (DTNB) were procured from Sigma Aldrich, Mumbai (India). One touch glucometer (AccuChek Active) with glucose oxidase-peroxidase reactive strips was purchased from Roche Diagnostics, (USA). Aldose reductase, Sorbitol dehydrogenase and Catalase assay kits were purchased from Cusabio (USA), Uscn Life Science Inc. (USA) and Bioassay Systems (USA). Both ATP and Glucose-6phosphate dehydrogenase assay kits were procured from Abcam Inc. (USA). Sodium azide, Potassium chloride, Sodium chloride, Sodium hydroxide, Ethylene-di-amine-tetra-acetic acid (EDTA) and all other chemicals were obtained from Merck Chemicals, Mumbai (India). All reagents were analytical grade. All reagents except for the phosphate buffers were prepared every day and stored in a refrigerator at +4˚C. The reagents were equilibrated at room temperature for 30 minutes before use, either at the start of analysis or when reagent containers were refilled. Phosphate buffers were stable at +4˚C for one month. Animals 7 days old Wistar rat pups with their mother were housed in separate polypropylene cages, maintained under standard conditions with temperature (22–240C), 12-h light/12-h dark cycle and relative air humidity 40–60%. Rats had continuous access to normal calorie standard rat pellet diet (Hindustan Lever Ltd., Mumbai, India) and to tap water. After grouping, the animals were acclimatized to the laboratory conditions for one week before the start of the experiment. Animals described as fasted were separated from their mother overnight and deprived of food for 16-h but had allowed free access to water. The experimental protocol was approved by the Institutional Animal Ethics Committee (IAEC/KMC/06/2012) and experiments were conducted according to the ethical norms approved by Ministry of Social Justices and Empowerment, Government of India and Committee for the Purpose of Control and Supervision on Experiments on Animals (CPCSEA) guidelines and Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Research. Experimental design In the experiment, 30 Wistar rat pups were divided into five groups (n= 6). The corresponding doses of Zincovit tablets with grape seed extract were administered one day prior to the sodium selenite (10 µmoles/kg; subcutaneously) in addition to the regular treatment. Treatment was continued from day 7 to day 20 of their post-natal life as followGroup I: Normal control rat pups were given 0.1 ml of normal saline; subcutaneously Group II: Sodium selenite induced cataract control rat pups were given 0.1 ml of normal saline; subcutaneously Group III: Sodium selenite induced cataract rat pups were given Zincovit tablets with grape seed extract 40 mg/kg/day;
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THE JOURNAL OF NUTRITION, HEALTH & AGING© subcutaneously Group IV: Sodium selenite induced cataract rat pups were given Zincovit tablets with grape seed extract 80 mg/kg/day; subcutaneously Group V: Sodium selenite induced cataract rat pups were given Zincovit tablets with grape seed extract 160 mg/kg/day; subcutaneously
incubated for 10 minutes at room temperature and absorbance was read at 412 nm by using an ELISA reader Bio Tek Instruments ELx800- MS, (USA). Determination of Glutathione peroxidase (GPx) activity 550 µl phosphate buffer (100 mM) containing 0.1 mM EDTA (pH 6.5), 50 µl Sodium azide (2 mM), 50 µl lens tissue homogenate, 100 µl glutathione reductase (2.5 U/ml) and reduced glutathione (GSH, 100 mM) were added together and incubated at 37ºC for 10 minutes. Then, 100 µl NADPH (2.5 mM) was added in the above mixture. Reaction was started by adding 100 µl hydrogen peroxide (1.5 mM) and optical density was read at 340 nm at one minute interval for five minutes by using UV-2450 spectrophotometer, Shimadzu Corporation, Tokyo (Japan).
Blood, Lens Collection and Processing The rat pups first opened their eyes, approximately 16 days after birth, cataract formation and its prevention were monitored by examining the eye (cloudy area in the lens) with pen light illumination thereafter till the end of experiment and subsequent photography of the isolated lenses was done on 21st day. Body weight of each rat pup was checked before start and at the end of the experiment. On 21st day, all the rat pups were sacrificed by administering overdose of ketamine, i.p. according to the annexure-6 of euthanasia of laboratory animals in the Committee for the Purpose of Control and Supervision on Experiments on Animals (CPCSEA) guidelines for Laboratory Animal Facility. The eye lenses were dissected by the posterior approach and stored at -70°C until further analysis. Lens weight of each rat pup was taken. Fasting blood samples were drawn on from retro-orbital plexus of all the experimental animals using capillary tube for the estimation of blood glucose with the help of glucose oxidase-peroxidase reactive strips (Accu-Chek, Roche Diagnostics, USA). Lens homogenates (10% w/v) were prepared from three to five pooled lenses in 50 mM potassium phosphate buffer (pH 7.4) using a Remi homogenizer. The unbroken cells and cell debris were removed by centrifugation at 10000 rpm for 20 minutes using a Remi C-24 refrigerated centrifuge. The resulting supernatant was stored at -80˚C. All biochemical parameters were analyzed in the soluble fraction of the lens homogenate except for malondialdehyde (MDA) which was determined in the total homogenate. The following biochemical analysis was done in triplicate manner and optical density was also read for reagent and sample blank-
Determination of Protein thiol (PT) level 20 µl of lens tissue homogenate sample was added in the mixture of 180 µl disodium edetate (2mM disodium edetate in 0.2 M disodium hydrogen phosphate) buffer solution and 4 µl DTNB solution (10mM DTNB in 0.2 M disodium hydrogen phosphate) in 96-wells of micro test plate. Then, optical density was read at 412 nm by using an ELISA reader Bio Tek Instruments ELx800- MS, (USA). Determination of Superoxide dismutase (SOD) activity To 25 µl of lens tissue homogenate sample, 925 µl sodium carbonate buffer (0.1M, pH 10-11) and 50 µl of adrenaline bitartarate (1mM) was added and absorbance (A0s-A60s,) was read at 480 nm by using UV-2450 spectrophotometer, Shimadzu Corporation, Tokyo (Japan). Determination of Catalase (CAT) activity Catalase activity in lens homogenate was measured according to the standard protocol given along with the Catalase assay kit of Bioassay Systems, Hayward (USA) by using an ELISA reader Bio Tek Instruments ELx800- MS, (USA).
Determination of Malondialdehyde (MDA) level To 20 µl lens homogenate sample, 200 µl 0.67% thiobarbituric acid and 100 µl 20% trichloroacetic acid were added and incubated at 100˚C for 20 minutes. Then, it was centrifuged at 12000 rpm for 5 minutes and 100 µl of supernatant was transferred to 96- wells of micro test plate. Optical density of supernatant was read at 540 nm by using an ELISA reader Bio Tek Instruments ELx800- MS, (USA).
Determination of Glucose-6-phosphate dehydrogenase (G6PD) activity Glucose-6-phosphate dehydrogenase activity in lens homogenate was measured according to the standard protocol given along with the Glucose-6-phosphate dehydrogenase assay kit of Abcam Inc., (USA) by using an ELISA reader Bio Tek Instruments ELx800- MS, (USA). Determination of Adenosine triphosphate (ATP) level Adenosine triphosphate level in lens homogenate was measured according to the standard protocol given along with the Adenosine triphosphate assay kit of Abcam Inc., (USA) by using an ELISA reader Bio Tek Instruments ELx800- MS, (USA).
Determination of Reduced glutathione (GSH) level Mixture of 100 µl of lens tissue homogenate and 100 µl of 5% trichloro acetic acid (TCA) solution was centrifuged at 5000 rpm for 5 minutes. Then, 25 µl of tissue supernatant, 150 µl sodium phosphate buffer (PBS 0.2 M, pH 8.0) and 25 µl DTNB (0.6mM) was added together in 96-wells of micro test plate and 3
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SELENITE CATARACT Determination of Aldose reductase (AR) level Aldose reductase concentration in lens homogenate was measured according to the standard protocol given along with the Aldose reductase assay kit of Cusabio Inc., (USA) by using an ELISA reader Bio Tek Instruments ELx800- MS, (USA).
mg/kg, 80 mg/kg and 160 mg/kg; s.c, respectively) exhibited mild lenticular opacification (Figure 1, 2C-2E). Effect on body weight and isolated lens weight There was no significant change in body as well as isolated lens weight among the experimental groups.
Determination of Sorbitol dehydrogenase (SDH) level Sorbitol dehydrogenase level in lens homogenate was measured according to the standard protocol given along with the Sorbitol dehydrogenase assay kit of Uscn Life Science, (USA) by using an ELISA reader Bio Tek Instruments ELx800MS, (USA).
Figure 1 Effect of different doses of combined formulation of grape seed extract and Zincovit tablets on prevalence of sodium selenite induced senile cataract among experimental groups
Statistical analysis Using Statistical Package for the Social Sciences (SPSS version 16.0; SPSS Inc., Chicago, USA), normally distributed data were expressed as mean ± standard error of mean and analyzed by one way analysis of variance (ANOVA) followed by post hoc Tukey test. Data with non-uniform distribution were expressed as median, Quartile (Q1, Q3) and analyzed by non-parametric K Independent samples test followed by Kruskal-Wallis H test. A level for p ≤ 0·05 was considered to be statistically significant (two-sided). Results Effect on lens morphology Cataract formation and its prevention were monitored on 16th day onwards of their post natal life after first opening of eyes. All six rat pups in normal control group (which received normal saline; 0.1 ml, s.c) exhibited complete transparency of the lens (Figure 1, 2A). “Out of 12 eye lenses of six rat pups in Effect on biochemical parameters sodium selenite induced senile cataract control group (which There was significant increase in reduced glutathione (p = received normal saline; 0.1 ml, s.c), 10 eye lenses exhibited 0.005), glutathione peroxidase (p = 0.019), glucose-6dense opacification of the lenses (Figure 1, 2B).” In contrast, phosphate dehydrogenase (p = 0.030), protein thiol (p = 0.022) only 6, 2 and 1 among 12 lenses (which received combined and adenosine triphosphate (p = 0.046) in the lens of rat pups formulation of grape seed extract and Zincovit tablets 40 treated with combined formulation of grape seed extract and Table 2 Effect of combined formulation of grape seed extract and Zincovit tablets on reduced glutathione (µmoles/mg) and glutathione peroxidase (µmoles/ml) and Glucose-6-phosphate dehydrogenase (µmoles/min/ml) in lens tissue homogenate Groups (n=6) I- Normal control (2% gum acacia) II- Selenite control (2% gum acacia) III- Selenite + ZVT (40 mg/kg/day) IV- Selenite + ZVT (80 mg/kg/day) V- Selenite + ZVT (160 mg/kg/day)
GSH (Q1, Q3)
GPx (Q1, Q3)
G6PD (Q1, Q3)
73.38 (47.71, 111.43)
361.83 (220.31, 494.98)
43.47 (28.04, 82.79)
27.48 (24.41, 66.62)
97.08 (82.08, 292.51)
17.79 (15.74, 47.26)
22.60 (16.60, 28.22)
98.02 (63.04, 126.84)
16.58 (11.60, 20.26)
18.12 (15.45, 61.44)
95.50 (61.97, 257.21)
20.74 (11.50, 42.27)
350.28 (171.44, 389.45)
56.09 (45.46, 84.14)
p = 0.019
p = 0.030
82.98 (63.18, 114.08) p = 0.005
n- Number of rats in each group. GSH- Reduced glutathione, GPx- Glutathione peroxidase and G6PD- Glucose-6-phosphate dehydrogenase. Data are expressed as the median (quartilesQ1,Q3) and different treatments were analyzed by non-parametric K Independent sample test followed by Kruskal-Wallis H test.
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THE JOURNAL OF NUTRITION, HEALTH & AGING© Table 3 Effect of combined formulation of grape seed extract and Zincovit tablets on protein thiol (µmoles/mg), adenosine triphosphate (mmoles/ml) and malondialdehyde (nmoles/g) in lens tissue homogenate Groups (n=6)
I- Normal control (2% gum acacia) II- Selenite control (2% gum acacia) III- Selenite + ZVT (40 mg/kg/day) IV- Selenite + ZVT (80 mg/kg/day) V- Selenite + ZVT (160 mg/kg/day)
PT (Q3, Q3)
ATP (Q3, Q3)
MDA (Q3, Q3)
890.69 (524.43, 1178.84)
1.00 (0.16, 2.12)
27.47 (21.97, 32.05)
397.97 (336.20, 908.85)
0.29 (0.16, 1.60)
27.47 (24.72, 58.60)
304.96 (226.24, 342.82)
0.20 (0.07, 0.56)
36.62 (21.06, 53.11)
237.55 (223.29, 434.78)
0.21 (0.13, 0.30)
23.80 (21.97, 26.55)
1171.78 (511.08, 1673.37)
0.91 (0.68, 1.87)
20.14 (17.39, 21.97)
p = 0.022
p = 0.046
p = 0.024
n- Number of rats in each group. PT- Protein thiol, ATP- Adenosine triphosphate and MDA- Malondialdehyde. Data are expressed as the median (quartiles- Q1, Q3) and different treatments were analyzed by non-parametric K Independent sample test followed by Kruskal-Wallis H test. .
Figure 2 Photographs of experimental rats on 21st day: (A). Normal control rat (B). Sodium selenite induced cataract control rat (C). Sodium selenite induced cataract rat treated with combined formulation of grape seed extract and Zincovit tablets 40 mg/kg (D). Sodium selenite induced cataract rat treated with combined formulation of grape seed extract and Zincovit tablets 80 mg/kg (E). Sodium selenite induced cataract rat treated with combined formulation of grape seed extract and Zincovit tablets 160 mg/kg
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SELENITE CATARACT Zincovit tablets in dose dependent manner when compared with sodium selenite induced senile cataract control rat pups (Table 2, 3). Lens malondialdehyde level was significantly lower (p = 0.024) in rat pups treated with combined formulation of grape seed extract and Zincovit tablets in comparison with sodium selenite induced senile cataract control rat pups (Table 3). There were no significant changes in fasting blood glucose level and lens superoxide dismutase, catalase, aldose reductase and sorbitol dehydrogenase among the experimental groups.
in Wistar rat pups. “A consistent finding in the present study was significant increase of lens ATP levels in the rats treated with combined formulation of grape seed extract and Zincovit tablets in a dose dependent fashion in comparison with selenite-induced cataract control rats (Table 3).” The decrease in ATP level in lens of selenite-induced cataract control rats could be due to irreversible damage of the lens. ATP is needed as a cofactor for the conversion of glucose to phosphorylated glucose by hexokinase. Decrease in ATP might have affected glucose 6phosphate generation and thereby diverting glucose through the polyol pathway. Similarly, ATP also affects enzymes involved in the glutathione synthesis. The energy needed for the biosynthesis of proteins in lens is supplied in the form of ATP derived from carbohydrate metabolism. Decrease in ATP affects the biosynthesis of protein in the lens. The decreased supply of energy, as a result of altered glucose metabolism results in pronounced change in the cytoplasmic composition of the lens, cellular deterioration and eventually loss of transparency. “One of the studies also suggests significant decreased level of lens ATP in selenite induced cataract control rats compared to normal control rats (20)”. The significant increase in the activity of lens glucose-6phosphate dehydrogenase in senile cataractous rats treated with combined formulation of grape seed extract and Zincovit tablets in comparison with selenite-induced cataract control rats may enhance formation of ribose for nucleic acids synthesis and also increase the renewal of lens protein (Table 2). The pentose phosphate pathway supplies NADPH which is the main component of the glutathione system. It has been reported that when the intensity of a stress increases, GSH concentrations usually decline and redox state becomes more oxidized, leading to deterioration of the system (24). “A decrease in GSH level may disorganize and disorient the mitochondria of the cell resulting in reduced supply of ATP, thereby limiting optimal cellular and physiological function in the senile cataract state.” In the present study, there was decrease in reduced form glutathione (GSH) and protein thiol level in the selenite-induced cataract control group whereas Zincovit tablets with grape seed extract treated group especially at the dose of 160 mg/kg restored the level of reduced glutathione and protein thiol (Table 2, 3). Decrease in the activity of glutathione peroxidase in lenses of selenite-induced cataract control rats may be due to either direct inactivation by reactive oxygen species or by decreased aerobic metabolism in lens. In this study, the significant increase in the activity of glutathione peroxidase following the administration of the combined formulation of grape seed extract and Zincovit tablets may be adduce to the presence of vitamin C, E, riboflavin and minerals like zinc that might have enhanced the synthesis of this enzyme and preserved the glutathione level (Table 2). Increased lenticular MDA may be the result of increased lipid peroxidation of the lenticular cell membranes, or may represent the consequence of migration of MDA from the
Discussion Age-related cataract is the major cause of blindness globally. Yet, a pharmacological treatment for cataract has not been achieved. Although it is a multifactorial disease associated with several risk factors, oxidative stress has been suggested as a common underlying mechanism of cataractogenesis, and augmentation of the antioxidant defenses of the lens has been shown to prevent or delay experimental cataract (16). The selenite cataract model was selected because of its rapid, effective and reproducible cataract formation. “As a model for senile nuclear cataract, it has been extensively characterized histologically and biochemically (17).” Most pronounced biochemical mechanisms occur during development of seleniteinduced cataract are altered epithelial metabolism, calcium accumulation, calpain-induced proteolysis, crystalline precipitation, phase transition, and cytoskeletal loss (5). “Although selenite cataract shows no high molecular weight covalent aggregates or increased disulfide formation, it has many similarities to human age related cataract such as vesicle formation, increased calcium, insoluble protein, proteolysis, decreased water soluble proteins, and glutathione (GSH) (5).” “Selenite induced conversion of GSH to GSSG caused directly by its reaction with sodium selenite (18) as or through an initial formation of seleno-diglutathione (19).” The latter is rapidly converted to GSSG with the generation of selenium dioxide. This in turn restarts the thiol depleting reaction. The GSSG so formed is extruded out the lens (20). Selenite induces bilateral nuclear cataract within 4 to 6 days when administered to suckling rat pups before completion of the critical maturation period of the lens (21). Any strategy that prevents or slows the progression of cataract has a significant health impact. The potential role of vitamins in preventing cataract is well documented, especially vitamin C or ascorbic acid which plays an important part in lens biology, both as an antioxidant and as a UV filter. “Some of the studies suggest that routine consumption of grape seed proanthocyanidins extract in the form of food or a dietary supplement may offer a prophylactic measure against onset and progression of cataract (21-23), which supports our findings in this study.” The combination of multivitamin-multimineral and grape seed extract Zincovit tablets may produce synergistic anticataractogenic potential (Table 1). Hence, we set out to investigate the role of combined formulation of grape seed extract and Zincovit tablets in the prevention or delay of progression and maturation of selenite-induced senile cataract 6
J Nutr Health Aging
THE JOURNAL OF NUTRITION, HEALTH & AGING© readily peroxidizable retina or from the central body compartment (24-30). In the present investigation, such a disruption of membrane lipids possibly accounted for the observed increase in MDA levels in the lenses of cataractuntreated (Group II) rat pups when compared to normal (Group I) rat pups (Table 3). The observed lower mean MDA level in the cataract-treated with combined formulation of grape seed extract and Zincovit tablets relative to selenite-induced cataract control rats suggests that grape seed extract with Zincovit tablets possibly preserved the structural integrity of the lens, thereby preventing lenticular opacification. The ability of the combined formulation of grape seed extract and Zincovit tablets (Nutritional food supplement) to delay the progression and maturation of selenite-induced agerelated cataract might be attributed to synergistic interplay of constituents of Zincovit tablets, such as- grape seed extract proanthocyanidins which comprise only procyanidins [subunits constituted of (+)-catechin (C) and (−)-epicatechin (EC)], Vitamins A, B, C, D, E, folic acid, biotin and minerals like zinc, copper, selenium, magnesium, manganese, chromium and molybdenum mainly, which are promoters of antioxidant activity (Table 1). Vitamin C, E (mainly tocopherols) and a variety of carotenoids are present in lens tissue and in the fluid that surrounds it and have an important part to play in lenticular antioxidant status. Riboflavin is a precursor to flavin-adenine dinucleotide (FAD), which is a coenzyme for the biosynthesis of glutathione reductase. Selenium is an integral part of the enzyme, glutathione peroxidase. Both the lens of the eye and the aqueous humor contain protective enzymes that breakdown the damaged proteins that clump together and cause cataracts. This combined formulation due to its strong antioxidant potential may keep these enzymes from being destroyed. From the present study it can be concluded that the combined formulation of grape seed extract and Zincovit tablet is the potential functional nutritional food supplement that can prevent the onset, progression and maturation of seleniteinduced age-related cataract in Wistar rat pups. The therapeutic effect seen in animal studies cannot always be entirely extrapolated to humans. Hence, clinical evaluation should be performed to precisely define the anti-cataractogenesis role of Zincovit tablets with grape seed extract in humans. Our study opens the perspective to clinical studies specifically designed to evaluate the impact of the single combined formulation of grape seed extract and Zincovit tablets on the onset and progression of age-related cataract of human subjects as nutritional food supplement. “This information would eventually complement our findings, opening the way to sustain age-related cataract development in human population”.
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Acknowledgments: All the authors declare that they do not have any conflict of interest. This work was financially supported by Apex Laboratories Private Ltd., Chennai (India) for which authors are grateful.
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