EXTRACT ON FREE RADICAL METABOLISM OF LIVER IN MICE DURING ENDURANCE EXERCISE

This study investigated the effect of Ginkgo biloba extract on Free Radical Metabolism of Liver in mice during endurance exercise. Forty-eight mice were divided into the quiet group and the exercised group. And the two groups were both grouped again, including the control group and the drug-treated group. After exhaustive exercise, the exercised groups were subdivided into the immediate group and the recovery group. The swimming time to exhaustion significantly prolonged in the exercised drug-treated group as compared with the exercised control group (P <0.05); The SOD activity of drug-treated groups significantly increased (P <0.05) as compared with the control groups and MDA content was significantly lower (P <0.05). The SOD activity and MDA content of exercised control groups significantly increased (P <0.05) as compared with the quiet control group. The SOD activity and MDA content of exercised drug-treated groups significantly increased (P <0.05) as compared with the quiet drug-treated group. The results indicated that Ginkgo biloba extract can obviously increase the body's endurance exercise capacity in mice and delay fatigue; Ginkgo biloba extract can help to increase the activity of the antioxidant enzymes in liver tissue, reduce the lipid peroxidation injury in liver tissue caused by free radicals, improve athletic ability, and promote the recovery process after exercise in mice.


Introduction
Ginkgo biloba (Ginkgoaceae) is a native tree from China that has been exhaustively cultivated in Europe, Australia, Japan, Korea, and the USA, because of its health-promoting properties (Shinozuka et al., 2002;Vilar et al., 2009).It has been used for 5000 years in traditional Chinese medicine.The leaves of the Ginkgo biloba tree, also known as maidenhair, are the source of this herb.Ginkgo biloba extract (EGB) is from green leaves of the Ginkgo biloba tree and the main ingredients of EGB contain 24% flavonoids (ginkgo-flavone glycosides) and 6 % terpenoids (including ginkgolides A, B, C, J and bilobalide) ( Peng et al., 2003;Zhou et al., 2006 ).EGB is well known for its antioxidant property due to its ability to scavenge free radicals and to neutralize ferry lion-induced peroxidation ( Bridi et al., 2001;He et al., 2006).
In the organism, free radicals always come from the redox intermediates.Under normal physiological conditions, the generation and removal of free radicals remained dynamic balance at low levels (Xu et al., 2002;Packer et al., 2008).
Strenuous exercise can make the body ischemia and hypoxia, enhance the oxidation effect, make the free radicals rapidly increase, triggering a chain reaction and destruction of the cell structure, then cause lipid peroxidation and lead to body damage finally (Benderitter et al., 1996;Zhao et al., 2007).Both exercise and exercise-induced fatigue can cause an increase in free radicals in liver tissues of the body and cause liver cell's damage (Voces et al., 1999;Gul et al., 2006).Therefore, screening natural substances to obtain effective and non-toxic free radical scavengers is becoming a hot study spot in the field of sports biology.The purpose of this study was to investigate the scavenge free radicals effect of EGB in order to explore the mechanism that this substance can delay the occurrence of exercise-induced fatigue and speed up the recovery process by swimming exercise.

Experimental animals
Purebred male Kun-Ming mice (20 ± 2 g) were purchased from the Experimental Animal Center of Shandong Province (Jinan,China).The mice were allowed to adapt to our laboratory environment for one week before the beginning of the experiment.They were housed in standard cages with free access to tap water and maintained in a room under standard conditions of feeding and temperature with a 12 h : 12 h light-dark cycle.This animal study was approved by the Ethical and Research Committee of the Dezhou University.

Reagents
EGB were provided by Guizhou Xinbang Pharmaceutical Company, China.The contents of flavonoids and terpenoids were used as quality control standard (24 %±1 % and 6.0%±0.5 % , respectively).SOD and MDA detection kits were provided by Rongsheng Biotechnology Company, China.Test methods and formulas were used in strict accordance with the instructions of the kits.

Experimental design
Forty-eight mice were randomly divided into two groups, the quiet group (Q group, 16 mice) and the exercised group (E group, 32 mice).The mice of quiet group were not exercised, while the mice of exercised group were exercised to swim for 4 weeks.The swimming exercise was carried out in a tank (30×50×30cm), filled with water to 25 cm depth and maintained at a temperature of 30± 1 ℃.During the first week, the swimming exercise time was 30 min every day; during the following three weeks, the swimming exercise time was respectively 35 min, 40 min and 45min.
The two groups (Q group and E group) were both grouped again, including the control group (C group) and the drug-treated group (D group).The 4 groups were the quite control group (QC group, 8 mice), the exercised control group (EC group, 16 mice), the quite drug-treated group (QD group, 8 mice), and the exercised drug-treated group (ED group, 16mice).
The drug-treated groups received EGB in doses of 100 mg/kg by stomach every morning.In the control groups of mice, saline was given instead of EGB at the same dose for 4 weeks.
At the end of a four-week period, the mice were killed by using ether anesthesia.Before being killed the mice were weighed and then were forced to swim without a load until being exhausted and the mice were considered to be exhausted when they failed to rise to the surface of the water to breathe within a 7-s period ( Fushiki et al., 1995).After exhaustive exercise, the exercised group (E group) were subdivided into two groups, including the immediate group (IE group, 16 mice) and the recovery group (IR group, 16 mice).The 4 groups were the immediate exercised control group (IEC group, 8 mice), the recovery exercised control group (REC group, 8 mice), the immediate exercised drug-treated group (IED group, 8 mice), and the recovery exercised drug-treated group (RED group, 8 mice).The mice of immediate group were killed immediately after exhaustive exercise and the mice of recovery group were killed after recovering for 24h after exhaustive exercise.Liver was excised from the mice and placed in ice-saline to clear off blood, then the liver were dried with filter paper and weighed.
Then the liver was homogenized in ice-cold 0.15M Tris-KCl buffer (pH 7.4) to yield a 10% (w/v) homogenate.The latter was next subjected to high-speed centrifugation at 15000 r/min for 30 minutes at 4 ℃.The resulting supernatant was used as such for assaying Superoxide dismutase (SOD) and Malondialdehyde (MDA).

Statistical analyses
All the data is expressed as a mean ± standard deviation (S.D.).A one-way ANOVA using SPSS ver.10.0 software was used for multiple comparisons.A value of p < 0.05 was considered to be significant.

Effects of EGB on forced swimming capacity
The forced swimming capacities are shown in Fig. 1.There are significant differences in the swimming time to exhaustion between the ED group and the EC group(P <0.05).

Effects of EGB on the SOD activity and MDA content of Liver in mice
The effects of EGB on the SOD activity and MDA content of Liver in mice are shown in Table 1.The SOD activity of D groups (QD, IED, RED) significantly increased (P <0.05) as compared with their respective control groups (QC, IEC, REC) and MDA content were significantly lower (P <0.05).The SOD activity and MDA content of EC groups (IEC, REC) significantly increased (P <0.05) as compared with the QC group.The SOD activity and MDA content of ED groups (IED, RED) significantly increased (P <0.05) as compared with the QD group.

Figure 1 :
Figure 1: Effects of EGB on swimming time to exhaustion in mice n =16; Mean ± S.D. # p<0.05 compared with the EC group.

Table 1 :
Effects of EGB on the SOD activity and MDA content of Liver in mice n =8; Mean ± S.D. a p<0.05 compared with the control groups (QC, IEC, REC); b p<0.05 compared with the QC group; c p<0.05 compared with the QD group.