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No significant (P > 0.05) differences in the levels of glucose, HDLC, and LDLC were observed among the five groups (Table 1). However, the levels of cholesterol and triglycerides were significantly (P < 0.05) higher in positive control mice than in vehicle control mice. SMPH significantly decreased the level of triglycerides at 400 mg/kg. However, the level of cholesterol in positive control mice was significantly decreased only at a dose of 800 mg/kg of SMPH. Since pharmacological effects of a complex mixture are dependent on the concentration of its individual components, we observed that a dose of 800 mg/kg was effective in reducing cholesterol levels.
Table 1. Levels of Glucose, Cholesterol, Triglycerides, High-density Lipoprotein Cholesterol (HDLC), and Low-density Lipoprotein Cholesterol (LDLC) in the Serum
Groups Glucose
(mmol/L)Cholesterol
(mmol/L)Triglycerides
(mmol/L)HDLC
(mmol/L)LDLC
(mmol/L)Vehicle control 4.32 ± 0.79a 2.06 ± 0.22a 2.28 ± 0.71a 1.95 ± 0.22a 0.24 ± 0.04a Positive control 4.87 ± 0.71a 2.56 ± 0.58b 2.99 ± 1.08b 2.11 ± 0.43a 0.21 ± 0.06a SMPH (400 mg/kg) 4.25 ± 0.87a 2.23 ± 0.22ab 2.29 ± 0.44a 1.96 ± 0.24a 0.27 ± 0.21a SMPH (800 mg/kg) 4.23 ± 0.36a 2.11 ± 0.26a 1.62 ± 0.31a 1.94 ± 0.19a 0.19 ± 0.05a SMPH (1, 200 mg/kg) 4.70 ± 0.78a 2.31 ± 0.30ab 2.12 ± 0.50a 2.15 ± 0.35a 0.22 ± 0.05a Note. Data are expressed as mean ± SD (n = 10); different letters indicate a significant difference (P < 0.05); Positive Control: D-galactose-treated mice; SMPH: D-galactose-treated mice receiving SMPH. -
To determine whether SMPH attenuates oxidative damage in the livers of D-galactose-treated mice, we measured the activities of GSH-Px and SOD along with T-AOC and MDA level in the liver (Table 2). SOD and GSH-Px activities as well as T-AOC were significantly lower in positive-control mice than in vehicle control mice. Moreover, D-galactose treatment reduced the level of MDA. However, supplementation with SMPH effectively countered the effect of D-galactose.
Table 2. Activities of Glutathione Peroxidase (GSH-Px) and Superoxide Dismutase (SOD), Total Antioxidant Capacity (T-AOC), and Malondialdehyde (MDA) Level in the Liver
Groups SOD
(U/mg protein)GSH-Px
(U/mg protein)T-AOC
(U/mg protein)MDA
(nmol/mg protein)Vehicle control 33.27 ± 4.09a 415.91 ± 27.60a 1.02 ± 0.15a 7.14 ± 0.53ac Positive control 24.37 ± 6.05b 273.66 ± 43.57b 0.62 ± 0.13b 11.52 ± 0.29b SMPH (400 mg/kg) 32.37 ± 5.18a 388.96 ± 22.89c 0.64 ± 0.06b 6.11 ± 0.47a SMPH (800 mg/kg) 36.11 ± 5.45a 412.37 ± 33.01a 0.89 ± 0.21c 8.11 ± 0.89c SMPH (1, 200 mg/kg) 32.17 ± 3.88a 359.88 ± 28.52c 0.81 ± 0.07c 7.30 ± 1.06c Note. Data are expressed as mean ± SD (n = 10); different letters indicate a significant difference (P < 0.05); Positive Control: D-galactose-treated mice; SMPH: D-galactose-treated mice receiving SMPH. -
The activities of GSH-Px and SOD as well as T-AOC were significantly lower, and MAO and MDA levels were significantly higher in the brains of positive control mice than in the brains of vehicle control mice (Table 3). However, SMPH treatment significantly increased the activities of GSH-Px and SOD, and decreased both MAO and MDA levels in the brain. No significant differences in the levels of T-AOC were observed between mice treated with SMPH and positive control mice. Further, the levels of GSH-Px, SOD, MAO, and MDA in the brains of vehicle control mice did not significantly differ from those in the brains of SMPH-treated mice.
Table 3. Activities of Glutathione Peroxidase (GSH-Px) and Superoxide Dismutase (SOD), Total Antioxidant Capacity (T-AOC), and Levels of Monoamine Oxidase (MAO) and Malondialdehyde (MDA) in the Brain
Groups SOD
(U/mg protein)GSH-Px
(U/mg protein)T-AOC
(U/mg protein)MAO
(U/mg protein)MDA
(nmol/mg protein)Vehicle control 65.53 ± 9.16a 16.17 ± 4.11a 0.40 ± 0.08a 1.20 ± 0.27a 3.52 ± 0.25a Positive control 41.06 ± 2.64b 7.33 ± 3.14b 0.23 ± 0.07b 1.94 ± 0.31b 5.06 ± 0.64b SMPH (400 mg/kg) 62.25 ± 9.83a 17.29 ± 3.14a 0.25 ± 0.06b 1.49 ± 0.34a 3.92 ± 0.20a SMPH (800 mg/kg) 62.69 ± 3.45a 19.61 ± 3.52a 0.24 ± 0.05b 1.16 ± 0.24a 3.43 ± 0.47a SMPH (1, 200 mg/kg) 58.54 ± 7.37a 18.56 ± 2.22a 0.24 ± 0.04b 1.28 ± 0.12a 3.98 ± 0.21a Note.Data are expressed as mean ± SD (n = 10); different letters indicate a significant difference (P < 0.05); Positive Control: D-galactose-treated mice; SMPH: D-galactose-treated mice receiving SMPH. -
The level of Bcl-2 was significantly lower, while the levels of 8-OHdG and caspase-3 were significantly higher in positive control mice than in vehicle control mice (Figure 1). By contrast, SMPH-treated mice had significantly higher levels of Bcl-2 and lower levels of 8-OHdG and caspase-3 in the brains and livers than positive control mice. No significant differences in the levels of Bcl-2 and caspase-3 were observed between vehicle control and SMPH-treated mice.
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The effect of SMPH on membrane fluidity was demonstrated by reduced η and P values in SMPH-treated mice relative to those in positive control mice (Table 4). η and P values in the liver mitochondrial membrane were significantly higher in positive control mice than in vehicle control mice. However, SMPH significantly decreased these values. The liver ΔΨm was significantly lower in positive control mice than in vehicle control mice. Treatment with SMPH significantly increased the ΔΨm.
Table 4. Liver Mitochondrial Membrane Potential (ΔΨm) and Fluidity
Groups ΔΨm Fluorescence polarization (P) Microviscosity (η) Vehicle control 44.46 ± 0.48a 0.16 ± 0.02a 1.05 ± 0.15a Positive control 24.94 ± 1.54b 0.32 ± 0.02b 4.49 ± 0.69b SMPH (400 mg/kg) 38.33 ± 1.61c 0.27 ± 0.03c 2.82 ± 0.62c SMPH (800 mg/kg) 39.61 ± 0.73c 0.22 ± 0.02d 1.89 ± 0.26d SMPH (1, 200 mg/kg) 39.50 ± 0.89c 0.21 ± 0.02d 1.76 ± 0.22d Note. Data are expressed as mean ± SD (n = 10); different letters indicate a significant difference (P < 0.05); Positive Control: D-galactose-treated mice; SMPH: D-galactose-treated mice receiving SMPH. -
The effect of SMPH on the activities of Na+-K+‑ATPase and Ca2+-Mg2+‑ATPase are presented in Table 5. The activities of Na+-K+‑ATPase and Ca2+-Mg2+-ATPase were significantly lower in positive control mice than in vehicle control mice. In contrast, their activities were significantly increased in SMPH-treated mice compared with those in positive control mice.
Table 5. Activities of Na+-K+-ATPase and Ca2+-Mg2+-ATPase in Liver Mitochondria
Groups Na+-K+-ATPase (U/mg protein) Ca2+-Mg2+-ATPase (U/mg protein) Vehicle control 6.92 ± 0.55a 5.35 ± 0.32a Positive control 3.06 ± 0.14b 2.62 ± 0.39b SMPH (400 mg/kg) 4.03 ± 0.26c 3.66 ± 0.87c SMPH (800 mg/kg) 4.59 ± 0.31cd 4.23 ± 0.38c SMPH (1, 200 mg/kg) 4.62 ± 0.38d 3.79 ± 0.29c Note.Data are expressed as mean ± SD (n = 10); different letters indicate a significant difference (P < 0.05); Positive Control: D-galactose-treated mice; SMPH: D-galactose-treated mice receiving SMPH. -
Ultrastructural changes in liver mitochondria were observed by TEM (Figure 2). The results showed that D-galactose treatment led to liver mitochondrial oxidative damage. Mitochondria in positive control mice exhibited swelling and vacuolization or disruption of mitochondrial cristae (Figure 2B). Treatment with SMPH ameliorated these morphological changes, and mitochondria in SMPH-treated mice were less swollen with intact membranes.
doi: 10.3967/bes2017.083
Antioxidant and Anti-aging Activities of Silybum Marianum Protein Hydrolysate in Mice Treated with D-galactose
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Abstract:
Objective In the present study, we investigated the antioxidant and anti-aging effects of Silybum marianum protein hydrolysate (SMPH) in D-galactose-treated mice. Methods D-galactose (500 mg/kg body weight) was intraperitoneally injected daily for 7 weeks to accelerate aging, and SMPH (400, 800, 1, 200 mg/kg body weight, respectively) was simultaneously administered orally. The antioxidant and anti-aging effects of SMPH in the liver and brain were measured by biochemical assays. Transmission electron microscopy (TEM) was performed to study the ultrastructure of liver mitochondria. Results SMPH decreased triglyceride and cholesterol levels in the D-galactose-treated mice. It significantly elevated the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), and total antioxidant capacity (T-AOC), which were suppressed by D-galactose. Monoamine oxidase (MAO) and malondialdehyde (MDA) levels as well as the concentrations of caspase-3 and 8-OHdG in the liver and brain were significantly reduced by SMPH. Moreover, it increased Bcl-2 levels in the liver and brain. Furthermore, SMPH significantly attenuated D-galactose-induced liver mitochondrial dysfunction by improving the activities of Na+-K+-ATPase and Ca2+-Mg2+-ATPase as well as mitochondrial membrane potential (ΔΨm) and fluidity. TEM showed that the degree of liver mitochondrial damage was significantly decreased by SMPH. Conclusion The results indicated that SMPH protects against D-galactose-induced accelerated aging in mice through its antioxidant and anti-aging activities. -
Key words:
- Silybum marianum protein hydrolysate /
- Antioxidant /
- Anti-aging /
- D-galactose
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Table 1. Levels of Glucose, Cholesterol, Triglycerides, High-density Lipoprotein Cholesterol (HDLC), and Low-density Lipoprotein Cholesterol (LDLC) in the Serum
Groups Glucose
(mmol/L)Cholesterol
(mmol/L)Triglycerides
(mmol/L)HDLC
(mmol/L)LDLC
(mmol/L)Vehicle control 4.32 ± 0.79a 2.06 ± 0.22a 2.28 ± 0.71a 1.95 ± 0.22a 0.24 ± 0.04a Positive control 4.87 ± 0.71a 2.56 ± 0.58b 2.99 ± 1.08b 2.11 ± 0.43a 0.21 ± 0.06a SMPH (400 mg/kg) 4.25 ± 0.87a 2.23 ± 0.22ab 2.29 ± 0.44a 1.96 ± 0.24a 0.27 ± 0.21a SMPH (800 mg/kg) 4.23 ± 0.36a 2.11 ± 0.26a 1.62 ± 0.31a 1.94 ± 0.19a 0.19 ± 0.05a SMPH (1, 200 mg/kg) 4.70 ± 0.78a 2.31 ± 0.30ab 2.12 ± 0.50a 2.15 ± 0.35a 0.22 ± 0.05a Note. Data are expressed as mean ± SD (n = 10); different letters indicate a significant difference (P < 0.05); Positive Control: D-galactose-treated mice; SMPH: D-galactose-treated mice receiving SMPH. Table 2. Activities of Glutathione Peroxidase (GSH-Px) and Superoxide Dismutase (SOD), Total Antioxidant Capacity (T-AOC), and Malondialdehyde (MDA) Level in the Liver
Groups SOD
(U/mg protein)GSH-Px
(U/mg protein)T-AOC
(U/mg protein)MDA
(nmol/mg protein)Vehicle control 33.27 ± 4.09a 415.91 ± 27.60a 1.02 ± 0.15a 7.14 ± 0.53ac Positive control 24.37 ± 6.05b 273.66 ± 43.57b 0.62 ± 0.13b 11.52 ± 0.29b SMPH (400 mg/kg) 32.37 ± 5.18a 388.96 ± 22.89c 0.64 ± 0.06b 6.11 ± 0.47a SMPH (800 mg/kg) 36.11 ± 5.45a 412.37 ± 33.01a 0.89 ± 0.21c 8.11 ± 0.89c SMPH (1, 200 mg/kg) 32.17 ± 3.88a 359.88 ± 28.52c 0.81 ± 0.07c 7.30 ± 1.06c Note. Data are expressed as mean ± SD (n = 10); different letters indicate a significant difference (P < 0.05); Positive Control: D-galactose-treated mice; SMPH: D-galactose-treated mice receiving SMPH. Table 3. Activities of Glutathione Peroxidase (GSH-Px) and Superoxide Dismutase (SOD), Total Antioxidant Capacity (T-AOC), and Levels of Monoamine Oxidase (MAO) and Malondialdehyde (MDA) in the Brain
Groups SOD
(U/mg protein)GSH-Px
(U/mg protein)T-AOC
(U/mg protein)MAO
(U/mg protein)MDA
(nmol/mg protein)Vehicle control 65.53 ± 9.16a 16.17 ± 4.11a 0.40 ± 0.08a 1.20 ± 0.27a 3.52 ± 0.25a Positive control 41.06 ± 2.64b 7.33 ± 3.14b 0.23 ± 0.07b 1.94 ± 0.31b 5.06 ± 0.64b SMPH (400 mg/kg) 62.25 ± 9.83a 17.29 ± 3.14a 0.25 ± 0.06b 1.49 ± 0.34a 3.92 ± 0.20a SMPH (800 mg/kg) 62.69 ± 3.45a 19.61 ± 3.52a 0.24 ± 0.05b 1.16 ± 0.24a 3.43 ± 0.47a SMPH (1, 200 mg/kg) 58.54 ± 7.37a 18.56 ± 2.22a 0.24 ± 0.04b 1.28 ± 0.12a 3.98 ± 0.21a Note.Data are expressed as mean ± SD (n = 10); different letters indicate a significant difference (P < 0.05); Positive Control: D-galactose-treated mice; SMPH: D-galactose-treated mice receiving SMPH. Table 4. Liver Mitochondrial Membrane Potential (ΔΨm) and Fluidity
Groups ΔΨm Fluorescence polarization (P) Microviscosity (η) Vehicle control 44.46 ± 0.48a 0.16 ± 0.02a 1.05 ± 0.15a Positive control 24.94 ± 1.54b 0.32 ± 0.02b 4.49 ± 0.69b SMPH (400 mg/kg) 38.33 ± 1.61c 0.27 ± 0.03c 2.82 ± 0.62c SMPH (800 mg/kg) 39.61 ± 0.73c 0.22 ± 0.02d 1.89 ± 0.26d SMPH (1, 200 mg/kg) 39.50 ± 0.89c 0.21 ± 0.02d 1.76 ± 0.22d Note. Data are expressed as mean ± SD (n = 10); different letters indicate a significant difference (P < 0.05); Positive Control: D-galactose-treated mice; SMPH: D-galactose-treated mice receiving SMPH. Table 5. Activities of Na+-K+-ATPase and Ca2+-Mg2+-ATPase in Liver Mitochondria
Groups Na+-K+-ATPase (U/mg protein) Ca2+-Mg2+-ATPase (U/mg protein) Vehicle control 6.92 ± 0.55a 5.35 ± 0.32a Positive control 3.06 ± 0.14b 2.62 ± 0.39b SMPH (400 mg/kg) 4.03 ± 0.26c 3.66 ± 0.87c SMPH (800 mg/kg) 4.59 ± 0.31cd 4.23 ± 0.38c SMPH (1, 200 mg/kg) 4.62 ± 0.38d 3.79 ± 0.29c Note.Data are expressed as mean ± SD (n = 10); different letters indicate a significant difference (P < 0.05); Positive Control: D-galactose-treated mice; SMPH: D-galactose-treated mice receiving SMPH. -
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