Long-Term Dietary Supplementation with a Yang-Invigorating Chinese Herbal Formula Increases Lifespan and Mitigates Age-Associated Declines in Mitochondrial Antioxidant Status and Functional Ability of Various Tissues in Male and Female C57BL/6J Mice

Chemicals

Reduced glutathione (GSH), oxidized GSH, GSH reductase, xanthine oxidase, xanthine, cytochrome c, α-tocopherol (α-TOC), ATP, and adenosine diphosphate (ADP) were purchased from Sigma Chemical Co. (St. Louis, MO). Luciferase and 2′,7′-dichlorofluorescein diacetate (DCFDA) were obtained from Fluka (Basel, Switzerland) and Perkin Elmer (Boston, MA), respectively. All other chemicals were of analytical grade. Solvents used for high-performance liquid chromatography (HPLC) were of HPLC grade.

Animal care and treatment

Male and female C57BL/6J mice were bred in the Animal Care Facility at the Hong Kong University of Science and Technology (HKUST), Hong Kong. All animals, born within a 2- week interval, were housed in an air-conditioned room with humidity control, under a 12-h dark/light cycle at about 22°C, and allowed food and water ad libitum. Experimental protocols were approved by the Research Practice Committee of HKUST. Animals were randomly divided into longevity and sacrifice groups. In each of the two groups, animals were assigned to control or VI-28–supplemented subgroups. In the supplemented subgroups, starting from 36 weeks of age, animals were fed a diet containing VI-28 at a concentration of 0.05% (D1) or 0.5% (wt/wt) (D2) until death or sacrifice. During the first 9 months of VI-28 supplementation, the average daily intake of 3–5 g of VI-28–supplemented diet (0.05%, wt/wt, VI-28) would yield a daily VI-28 dose of 50–80 mg/kg. Male and female mice in the longevity group (n = 41–60 in each subgroup) were monitored separately for survival changes during aging, whereas mice in the sacrifice group (n = 30 in each subgroup) were killed at 72, 96, and 120 weeks of age, and tissue mitochondrial parameters were measured.

Herbal preparation

VI-28, a multicomponent herbal formula composed of Radix Ginseng, Cornu Cervi, Cordyceps, Radix Salviae, Semen Allii, Fructus Cnidii, Fructus Evodiae, and Rhizoma Kaempferiae, was prepared as described. ⁶ A commercial sample was manufactured and supplied by Vigconic (International) Ltd., Hong Kong, with standardized contents of total saponins (1.2%, wt/wt), total flavonoids (0.5%, wt/wt), and total lignans (1.0%, wt/wt). ⁴

Preparation of mitochondrial fractions

Tissue homogenates were prepared from various tissues (brain, heart, liver, and kidney) as previously described. ^4,5 Mitochondrial pellets were isolated from tissue homogenates by differential centrifugation. Tissue homogenates were centrifuged at 600 × g at 4°C for 20 min. The resulting supernatants were then centrifuged at 9200 × g at 4°C for 30 min to obtain the mitochondrial pellets. Mitochondrial pellets were resuspended in 1 mL of the homogenizing buffer appropriate for the particular tissue and constituted mitochondrial fractions.

Biochemical analyses

Protein level

The protein concentration of mitochondrial fractions was measured using a Bio-Rad (Hercules, CA) protein assay kit.

Statistical analysis

Data on survival rates were analyzed by the log rank test, and median lifespan values were estimated. Mitochondrial and behavioral parameters were analyzed by one-way analysis of variance (ANOVA). Post-hoc multiple comparisons were performed using the least significant difference (LSD) test. p values < 0.05 were regarded as statistically significant.

Survival of control and VI-28-supplemented male and female mice

VI-28 dietary supplementation (0.05 or 0.5%, both wt/wt, starting at 36 weeks of age) significantly extended the lifespan of male mice, with the median lifespans being 127 or 126 weeks (p = 0.0356 and p = 0.0083), respectively, compared to the control value (111 weeks) (Fig. 1 A). The largest difference in survival rate decline between control and VI-28–supplemented male mice was observed from 100 to 120 weeks of age. VI-28 dietary supplementation at 0.05% (wt/wt) significantly extended the lifespan of female mice, with the median lifespan being 123 weeks (p = 0.0035), compared to the control value (113 weeks) (Fig. 1A). However, female mice fed a diet supplemented with VI-28 at 0.5% (wt/wt) showed only a marginal increase in median lifespan (119 weeks, p = 0.3161). The largest difference in survival rate decline between control and VI-28-supplemented female mice was seen from 80 to 100 weeks of age.

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FIG. 1.

Survival curves (A) and changes in mitochondrial antioxidant status and functional ability in brain tissue (B) of control and VI-28–supplemented male and female mice. Commencing at 36 weeks of age, male and female mice in the longevity and sacrifice groups were fed a diet supplemented with VI-28 [0.05% (D1) or 0.5% (D2), (both wt/wt)] until death or sacrifice at 72 weeks of age. Control animals were given a normal diet. The number of animals in the longevity groups was: male, control = 54, VI-28 D1 = 61, VI-28 D2 = 50; female, control = 56, VI-28 D1 = 58, VI-28 D2 = 41. Animal mortality was checked on a weekly basis. In the sacrifice groups, animals were sacrificed at 72 weeks of age, and reduced glutathione (GSH) and α-tocopherol (α-TOC) levels, manganese-superoxide dismutase (Mn-SOD) activity, as well as adenosine triphosphate generation capacity (ATP-GC), were measured in brain mitochondria. Data are expressed as a percentage of 36-week control value (mean ± standard error of the mean [SEM], with n = 5). (*) Significantly different from the 36 weeks control; (#) significantly different from the 72 weeks control.

Age-associated changes in mitochondrial antioxidant status and functional abilities of various tissues in control and VI-28-supplemented mice

Aging caused progressive decreases in the levels and activities of mitochondrial antioxidant components (GSH, α-TOC, and Mn-SOD) in all tested tissues of male and female mice, up to the age of 120 weeks, with the overall mean extents of depletion (encompassing three antioxidant components in four tissues) being larger in male mice (15–60% versus 11–43%) and the rate of depletion being higher between 72 and 96 weeks or 96 and 120 weeks of age (data not shown). Aging also caused decreases in mitochondrial ATP-GC of all tested tissues in male and female mice, with the mean degrees of impairment (in four tissues) being larger in female than male mice (7–35% versus 7–46%) (data not shown). A higher rate of decrease in mitochondrial ATP-GC was observed between 72 and 96 weeks or 96 and 120 weeks of age.

VI-28 dietary supplementation dose-dependently counteracted the age-associated depletion in mitochondrial antioxidant components of various tissues in male and female mice, with the mean degrees of protection diminishing with increasing age from 72 to120 weeks (male, 39% to 7% and 65% to 10%; female, 34% to 8% and 59% to 18%, in mice fed VI-28 at 0.05% and 0.5% [both wt/wt], respectively) (data not shown). The age-associated decrease in mitochondrial ATP-GC in various tissues of male and female mice was also inhibited by VI-28 supplementation, with protection diminishing with age and the effect being more prominent in male mice (male, 31% to13% and 60% to 22%; female, 22% to 7% and 40% to 13%) (data not shown). Figure 1B shows the changes in mitochondrial antioxidant and functional parameters at 72 weeks of age in control and VI-28–supplemented male and female mice.