Evaluation of skeletal muscle changes during aging

The present study investigated the effect of supplementation of quercetin on changes in body weight, lipid peroxidation and antioxidant enzymes activities gastrocnemius muscular tissues in different rat ages

The present study investigated the effect of supplementation of quercetin on changes in body weight, lipid peroxidation and antioxidant enzymes activities gastrocnemius muscular tissues in different rat ages (1M & 10M). Average body weight of experimental rats was as follows: 1M (28.37±2.52g), & 10M (211.45±12.14g). Male rats were divided into two groups: control rats (C1M, C10M), fed on laboratory regular diet, and quercetin treatments (Q1M꞊0.108 mg, Q10M꞊0.86mg). It was observed that the daily weight gain in quercetin group was higher than that of control group. ln addition to normal growth, food consumption, and muscle mass, rat treated with the quercetin enriched diet did not exhibit obvious signs of toxicity including failure to groom or lethargy. Importantly, supplementation with quercetin did not result in unexpected deaths. Quercetin treatment increases the content of GPx in (1M) by 22.45%, but decreasing GPx by 25.32% for 10M, when compared with values of the control group. Quercetin treatment increases the content of SOD at different ages by 16.2% & 68.15%, for 1M & 10M, respectively, when compared with values of the control group. It was found that tissue MDA was increased by 168.8% & 110.0% at 1M & 10M, respectively, when compared with value of control 1M. When comparing quercetin treatments at different ages, it is noted the MDA content of muscular tissues decrease by 35.9% at 10M, when compared with values of quercetin treatment 1M. Skeletal muscle of non-treated animal (10M) showing marked hyalinization and fragmentation of muscle fibers whereas Skeletal muscle of Quercetintreated animal (10M) showing focal hyalinization of muscle fibers while the remaining bundles were within normal. Conclusion: Quercetin could be used as anti-aging modulator against changes in gastrocnemius muscle resulted in aging.

Introduction
Quercetin (3,3',4',5,7-pentahydroxyflavone) is a natural polyphenolic flavonoid commonly found in a sugar bonding form such as quercetin-3-glucoside (Q3G) or quercetin-4glucoside (Q4G). Flavonoids are found in leaves, flowers, roots, seeds, nuts, and barks and fulfill many biological functions including UV protection, pigmentation and antimicrobial defense. Quercetin levels in plants positively correlated with exposure to UVB radiation and its accumulation has been considered a natural protection against UV induced damage.

2001).
Quercetin expresses several physiological functions including antioxidant, anti-hyperglycemic, anti-inflammatory, cytoprotective, hepatoprotective and inhibition of lipopolysaccharide-induced nitric oxide production. In the gut quercetin is formed through hydrolysis of rutin by intestinal microorganisms. Studies in syndrome and diabetes have suggested its potential in the treatment of metabolic syndrome and diabetes. Flavonoid antioxidants may act by a variety of ways including trapping of oxygencentered radicals, inhibition of enzymes involved in their production, chelation of transition metal ions involved in radicalforming processes such as the Fenton reaction, and regeneration of membrane-bound antioxidants such as α-tocopherol (Bors et al. 1990, Harborne, 1994. The purpose of this study was to investigate the effect of short-term supplementation of quercetin (14 days) on body weight gain, and oxidative markers the gastrocnemius muscles of rats of different ages.

Sampling
Random tissue specimens were collected from gastrocnemius muscles of treated and control groups under strict hygienic conditions to minimize the contamination or autolysis of the samples, then quickly either frozen in liquid nitrogen before storing at -80 °C to be used for RNA extraction or preserved in neutral buffered formalin solution (10%) for immunohistochemistry.

2.3.Biochemical analysis
The biomarkers, MDA levels, SOD, and GPx activities, were evaluated. Superoxide dismutase (SOD) activity was determined by measuring the decrease in hydrogen peroxide (H2O2) concentration, with a method described by (Nishikimi et al. 1972). Results were expressed as mU/ ml. Glutathione peroxidase (GPx) enzyme activity whose main biological role is to protect the organism from oxidative damage was measured according to the method described by (Beutler et al. 1963

Body weight gain
Average body weight gain in control groups were 47.85 ± 3.28 g& 25.42 ± 3.59 g for 1M&10M, respectively. The daily weight gain follows the same trend (1.06 ± 0.07 g& 0.59 ± 0.19 g/day) ( Table 1, Fig 1).  Fig 2). It was observed that the daily weight gain in quercetin group was higher than that of control group. In addition to normal growth, food consumption, and muscle mass, rats treated with the quercetin enriched diet did not exhibit obvious signs of toxicity including failure to groom or lethargy. Importantly, supplementation with quercetin did not result in unexpected deaths.

Glutathione peroxidase (GPx)
Results show a slight increase in glutathione peroxidase GPx enzyme activity in muscular tissues in control groups at different ages. Quercetin treatment increase the content of GPx in (1M) by 22.45%, but decreasing GPx by 25.32% for 10M, when compared with values of the control group (Table 2, Fig. 3).

Superoxide dismutase (SOD)
Results show a slight increase in superoxide dismutase SOD enzyme activity in muscular  Fig. 4).

Total Protein
The total protein concentration in the muscular tissues of rats was found to be slightly increase in the control group at different ages of rats. Quercetin treatments increased the total protein concentration in muscular tissues of rats at different ages.  Fig 6).

Tissue cytological changes
Representative (H&E stained) histology of gastrocnemius muscle of rats treated for 15 days with different doses of quercetin at the end of experiment (Fig. 7-10).

Discussion
The results of this study show that he body weight in control and quercetin treated animals at different ages increased consistently and no decrease in body weight was observed throughout the experimental period. It was observed that the daily weight gain in quercetin treatment group was higher than that of control group by 148% &275% for 1M& 10M, respectively. In addition to normal growth, food consumption, and muscle mass, rats treated with the quercetin enriched diet did not exhibit obvious signs of toxicity including failure to groom or lethargy. Importantly, supplementation with quercetin did not result in unexpected deaths.
In this study, the effect of dietary quercetin at a rate of 4 mg / kg body weight per day on growth performance and oxidative markers body weight per day on growth performance and oxidative markers in the gastrocnemius muscular tissues of rats of different ages were in the gastrocnemius muscular tissues of rats of different ages were examined. Several studies have promoted quercetin as excellent antioxidants in vivo ( Sanders et al. (2001) also shows that a dose of 10 mg/kg/day quercetin lead to an increase of renal and cardiac GPx activity.
Another study conducted on rats also shows the improved oxidative status in liver by dietary quercetin treatment ( Skeletal muscles of non-treated rats at age 1M& 10M show marked Caspace3 immunostaining (degree ++& (degree +++++), respectively; whereas quercetin treatment rats at age 1M& 10M showing decrease of Caspace3 immunostaining (degree +) & (degree +++) respectively when compared to normal (untreated) rats at the same age.

Conclusion
From a critical evaluation of the available literature on the biological effects of quercetin, including data related to safety, it may be concluded that quercetin, at estimated dietary intake levels, would not produce adverse health effects.