Research investigating antioxidation of astaxanthin extracted from Haematoccus pluvialis in mice

*Corresponding Author:

Shaolei Yu
Yunnan Green A Biological Project Co., LTD,1088 Haiyuan Zhong Road Kunming High-Develop Zone, Yunnan Kunming 650106, China.
Telephone 13529390463
E-mail 15980819@163.com

This open-access article is distributed under the terms of the Creative Commons Attribution Non-Commercial License (CC BY-NC) (http://creativecommons.org/licenses/by-nc/4.0/), which permits reuse, distribution and reproduction of the article, provided that the original work is properly cited and the reuse is restricted to noncommercial purposes. For commercial reuse, contact support@pulsus.com

Keywords

Antioxidation; Astaxanthin; Haematococcus pluvialis; Mice

Haematococcus pluvialis is a major source of natural astaxanthin [1]. Production of astaxanthin can be stimulated by dry, high temperatures, ultraviolet light, nutritional deficiency and other conditions. It produces strong antioxidants, which can remove oxides and restore balance [2]. Modern research demonstrates that astaxanthin has a strong antioxidant effect, along with other multiple effects in the body [3]. Therefore, using Haematococcus pluvialis to produce astaxanthin to develop astaxanthin-containing drugs, cosmetics and health food has been a hot scientific research topic. We extracted natural astaxanthin from Haematococcus pluvialis using supercritical CO₂ extraction technology, and evaluated its antioxidant effect.

Methods

Test substance

Astaxanthin oil (Yunnan Green A Bioligical Project Co Ltd, China) was diluted with vegetable oil.

Experimental animals

Fifty healthy female Kunming mice, weighing between 18 g and 22 g, were provided by the Experimental Animal Center of Zhongshan University (China). The mice were quarantined for one week before the experiment. The mice were fed in a barrier level animal room, which was mainted at a temperature between 20°C and 23°C, and a relative humidity between 55% and 65% for the duration of the experiment. Mice were provided with food and water ad libitum.

Equiptment and reagents

For the present study, the following equiptment was used: 722 spectrophotometer; water bath maintained at a constant temperature; microadjustable pipette; high- and low-speed centrifuges; vortex mixers; a DY89-1 electric glass homogenizer; and malondialdehyde (MDA), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) assay kits (Nanjing Jiancheng Bioengineering Institute, China). Chloroform, ethanol, acetic acid and other chemical reagents were analytical grade.

Dose group

Fifty mice were randomly divided into five groups (high-dose [astaxanthin 4.0 mg/kg], medium dose [astaxanthin 2.0 mg/kg], low-dose [astaxanthin 1.0 mg/kg], model control and blank control). The amount of astaxanthin administered to the groups receiving astaxanthin, repectively, were equivalent to 20×, 10× and 5× the human recommended amount.

Experimental methods

The three groups receiving aztaxanthin were administered treatment according to the corresponding dose for 30 days. The model control group and blank control group were given equal volumes of vegetable oil for 30 days. After 30 days, blood samples taken from the tail were tested for antioxidant enzyme activity. In addition to the blank control group, each group was exposed to 8 Gy 60Coγ ray exposure once. Four days after irradiation, all animals were euthanized to test for MDA content, SOD activity and GSH-Px in liver tissue according to the corresponding kit instructions.

Statistical analysis

ANOVA was performed using PEMS 3.0 (Chinese Medical Encyclopedia Medical Statistics, China).

Results

Effect of astaxanthin on body weight of mice

There were no adverse effects on body weight of the mice (P>0.05) (Table 1).

Table 1 Effect of astaxanthin on the body weight of mice

SOD activity in mice before irradiation

There was no significant difference among any of the groups in SOD activity before irradiation (P>0.05) (Table 2).

Table 2: Superoxide dismutase (SOD) activity in mice before irradiation

Effect of astaxanthin MDA, SOD and GSH-Px activity in liver tissue

The activity of MDA in the model group was significantly higher than that of the control group (P<0.05), and MDA content of the three groups that received antaxanthin were significantly lower than in the control group (P<0.05). SOD activity in the model group was significantly lower than in the control group (P<0.05), and SOD activity of the three groups that received astaxanthin were significantly higher than in the control group (P<0.05). The GSH-Px activity in the model group was significantly lower than in the control group (P<0.05), and the GSH-Px activity in the high-dose group was significantly higher than in the model control group (P<0.05) (Table 3).

Table 3: Effect of astaxanthin on malondialdehyde (MDA), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) activities in liver tissue

Discussion

Astaxanthin (3,3′-hydroxy-β, β′-carotene-4, 4′-dione) is a type of shortchain antioxidant that has strong antioxidant properties. It can provide an electron or a radical to react with another radical, which can then absorb excess energy, making the radical transform to a nonactive or more stable compound, thereby interrupting the radical chain reaction process. The main activity of its antioxidant mechanism has several aspects [4-6]:

1. Quenching singlet oxygen and scavenging oxygen-free radicals: astaxanthin has a strong ability to quench singlet oxygen capacity. Its molecular structure, which contains both hydroxyl and keto groups, may also promote keto-hydroxyl hydrogen transfer to peroxide radicals;

2. Stabilizing membrane structure and reducing membrane fluidity: astaxanthin contains polar ends, which act as bridge-like molecules across the cell membrane, which can increase its stability and mechanical strength, and can reduce membrane permeability, limiting penetration of intracellular oxidants and protecting cells from oxidative damage;

3. Increasing antioxidant enzyme activity: astaxanthin can act as an antioxidation system by activating the cells to decrease MDA production, reducing its concentration in the body, and increase SOD and GSH-Px activity, protecting cells from free radicalinduced oxidative damage;

4. Reducing the oxidative damage to mitochondria: after hydrogen peroxide damage, the contents of MDA and NO increased, while the activities of GSH, SOD, ATP enzyme decreased in mitochondria. Astaxanthin can reverse these changes.

5. Inhibition of lipid peroxidation: astaxanthin inhibits unsaturated fatty-acid methyl ester peroxide, phosphatidylcholine protection from oxidation, delay time phosphatidylcholine single large bubble (liposome) peroxidation.

Astaxanthin extracted from Haematoccus pluvialis using supercritical CO₂ extraction technology, compared with synthetic astaxanthin, has more stable and secure advantages. The results of the present study demonstrate that SOD activity in the three groups that received astaxanthin were significantly higher than in the model control group. MDA activity in the three groups that received astaxanthin was significantly lower than in the model group. Finally, GSH-Px activity in the high-dose group was significantly higher than the model control group. According to the Inspection and Assessment Standard for Health Food (2003 edition), the results produced by astaxanthin extracted from Haematoccus pluvialis suggest that it produced antioxidant effects.

References

1. Huang W, Hong B, Yi R. Astaxanthin production methods and biological activity research progress. Chinese Food Additives 2012;6:214-8.
2. Wang C, Han S, Chen Z. Haematococcus pluvialis antioxidant system of active oxygen scavenge mechanism. Aquatic Biology 2012;36:804-8.
3. Xu H, Cao B, Chen J. Biological function of astaxanthin and its application. Feed Wide-Angle 2012;11:23-5.
4. Leite MF, Massuyama MM, Otton R. Astaxanthin restores the enzymatic antioxidant profile in salivary gland of alloxan-induced diabetic rats. Archives of Oral Biology 2010;55:479-85.
5. Lee DH, Kim CS, Lee YJ. Astaxanthin protects against MPTP/ MPP+-induced mitochondrial dysfunction and ROS production in vivo and in vitro. Food Chem Toxicol 2011;49:271-80.
6. Yang Y, Zhou Y, Xu H. Animal studies astaxanthin antioxidant. Modern Preventive Medicine 2009;36:2432-3.

*Corresponding Author:

Shaolei Yu
Yunnan Green A Biological Project Co., LTD,1088 Haiyuan Zhong Road Kunming High-Develop Zone, Yunnan Kunming 650106, China.
Telephone 13529390463
E-mail 15980819@163.com

This open-access article is distributed under the terms of the Creative Commons Attribution Non-Commercial License (CC BY-NC) (http://creativecommons.org/licenses/by-nc/4.0/), which permits reuse, distribution and reproduction of the article, provided that the original work is properly cited and the reuse is restricted to noncommercial purposes. For commercial reuse, contact support@pulsus.com

Abstract

Objective: To evaluate the antioxidative effect of astaxanthin extracted from Haematoccus pluvialis in mice. Methods: Fifty mice were randomly divided into five groups (high-dose [astaxanthin 4.0 mg/kg]; medium dose [astaxanthin 2.0 mg/kg]; low dose [astaxanthin 1.0 mg/kg]; model control; and blank control). The experimental substance was administered every day. Mice in the model group and the control group received an equal volume of vegetable oil for 30 days. After 30 days, blood antioxidant enzyme activity was measured in tail blood. Except for the blank control group, the other groups were exposed to 8 Gy 60Coγ irradiation. On the fourth day after irradiation, all animals were euthanized and liver tissue was tested for malondialdehyde, superoxide dismutase and glutathione peroxidase activity levels. Results : Superoxide dismutase levels in all three groups that received astaxanthin were significantly higher than in the model control group, and malondialdehyde levels in all three groups that received astaxanthin were significantly lower than in the control group. Glutathione peroxidase enzyme activity in the high-dose group was significantly higher in the model control group. Conclusion: According to the evaluation standards in Inspection and Assessment Standard for Health Food, 2003 edition, the present study demonstrated that astaxanthin extracted from Haematoccus pluvialis has antioxidative effects in a mouse model.

Keywords

Antioxidation; Astaxanthin; Haematococcus pluvialis; Mice

Haematococcus pluvialis is a major source of natural astaxanthin [1]. Production of astaxanthin can be stimulated by dry, high temperatures, ultraviolet light, nutritional deficiency and other conditions. It produces strong antioxidants, which can remove oxides and restore balance [2]. Modern research demonstrates that astaxanthin has a strong antioxidant effect, along with other multiple effects in the body [3]. Therefore, using Haematococcus pluvialis to produce astaxanthin to develop astaxanthin-containing drugs, cosmetics and health food has been a hot scientific research topic. We extracted natural astaxanthin from Haematococcus pluvialis using supercritical CO₂ extraction technology, and evaluated its antioxidant effect.

Methods

Test substance

Astaxanthin oil (Yunnan Green A Bioligical Project Co Ltd, China) was diluted with vegetable oil.

Experimental animals

Fifty healthy female Kunming mice, weighing between 18 g and 22 g, were provided by the Experimental Animal Center of Zhongshan University (China). The mice were quarantined for one week before the experiment. The mice were fed in a barrier level animal room, which was mainted at a temperature between 20°C and 23°C, and a relative humidity between 55% and 65% for the duration of the experiment. Mice were provided with food and water ad libitum.

Equiptment and reagents

For the present study, the following equiptment was used: 722 spectrophotometer; water bath maintained at a constant temperature; microadjustable pipette; high- and low-speed centrifuges; vortex mixers; a DY89-1 electric glass homogenizer; and malondialdehyde (MDA), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) assay kits (Nanjing Jiancheng Bioengineering Institute, China). Chloroform, ethanol, acetic acid and other chemical reagents were analytical grade.

Dose group

Fifty mice were randomly divided into five groups (high-dose [astaxanthin 4.0 mg/kg], medium dose [astaxanthin 2.0 mg/kg], low-dose [astaxanthin 1.0 mg/kg], model control and blank control). The amount of astaxanthin administered to the groups receiving astaxanthin, repectively, were equivalent to 20×, 10× and 5× the human recommended amount.

Experimental methods

The three groups receiving aztaxanthin were administered treatment according to the corresponding dose for 30 days. The model control group and blank control group were given equal volumes of vegetable oil for 30 days. After 30 days, blood samples taken from the tail were tested for antioxidant enzyme activity. In addition to the blank control group, each group was exposed to 8 Gy 60Coγ ray exposure once. Four days after irradiation, all animals were euthanized to test for MDA content, SOD activity and GSH-Px in liver tissue according to the corresponding kit instructions.

Statistical analysis

ANOVA was performed using PEMS 3.0 (Chinese Medical Encyclopedia Medical Statistics, China).

Results

Effect of astaxanthin on body weight of mice

There were no adverse effects on body weight of the mice (P>0.05) (Table 1).

Table 1 Effect of astaxanthin on the body weight of mice

SOD activity in mice before irradiation

There was no significant difference among any of the groups in SOD activity before irradiation (P>0.05) (Table 2).

Table 2: Superoxide dismutase (SOD) activity in mice before irradiation

Effect of astaxanthin MDA, SOD and GSH-Px activity in liver tissue

The activity of MDA in the model group was significantly higher than that of the control group (P<0.05), and MDA content of the three groups that received antaxanthin were significantly lower than in the control group (P<0.05). SOD activity in the model group was significantly lower than in the control group (P<0.05), and SOD activity of the three groups that received astaxanthin were significantly higher than in the control group (P<0.05). The GSH-Px activity in the model group was significantly lower than in the control group (P<0.05), and the GSH-Px activity in the high-dose group was significantly higher than in the model control group (P<0.05) (Table 3).

Table 3: Effect of astaxanthin on malondialdehyde (MDA), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) activities in liver tissue

Discussion

Astaxanthin (3,3′-hydroxy-β, β′-carotene-4, 4′-dione) is a type of shortchain antioxidant that has strong antioxidant properties. It can provide an electron or a radical to react with another radical, which can then absorb excess energy, making the radical transform to a nonactive or more stable compound, thereby interrupting the radical chain reaction process. The main activity of its antioxidant mechanism has several aspects [4-6]:

1. Quenching singlet oxygen and scavenging oxygen-free radicals: astaxanthin has a strong ability to quench singlet oxygen capacity. Its molecular structure, which contains both hydroxyl and keto groups, may also promote keto-hydroxyl hydrogen transfer to peroxide radicals;

2. Stabilizing membrane structure and reducing membrane fluidity: astaxanthin contains polar ends, which act as bridge-like molecules across the cell membrane, which can increase its stability and mechanical strength, and can reduce membrane permeability, limiting penetration of intracellular oxidants and protecting cells from oxidative damage;

3. Increasing antioxidant enzyme activity: astaxanthin can act as an antioxidation system by activating the cells to decrease MDA production, reducing its concentration in the body, and increase SOD and GSH-Px activity, protecting cells from free radicalinduced oxidative damage;

4. Reducing the oxidative damage to mitochondria: after hydrogen peroxide damage, the contents of MDA and NO increased, while the activities of GSH, SOD, ATP enzyme decreased in mitochondria. Astaxanthin can reverse these changes.

5. Inhibition of lipid peroxidation: astaxanthin inhibits unsaturated fatty-acid methyl ester peroxide, phosphatidylcholine protection from oxidation, delay time phosphatidylcholine single large bubble (liposome) peroxidation.

Astaxanthin extracted from Haematoccus pluvialis using supercritical CO₂ extraction technology, compared with synthetic astaxanthin, has more stable and secure advantages. The results of the present study demonstrate that SOD activity in the three groups that received astaxanthin were significantly higher than in the model control group. MDA activity in the three groups that received astaxanthin was significantly lower than in the model group. Finally, GSH-Px activity in the high-dose group was significantly higher than the model control group. According to the Inspection and Assessment Standard for Health Food (2003 edition), the results produced by astaxanthin extracted from Haematoccus pluvialis suggest that it produced antioxidant effects.

References

1. Huang W, Hong B, Yi R. Astaxanthin production methods and biological activity research progress. Chinese Food Additives 2012;6:214-8.
2. Wang C, Han S, Chen Z. Haematococcus pluvialis antioxidant system of active oxygen scavenge mechanism. Aquatic Biology 2012;36:804-8.
3. Xu H, Cao B, Chen J. Biological function of astaxanthin and its application. Feed Wide-Angle 2012;11:23-5.
4. Leite MF, Massuyama MM, Otton R. Astaxanthin restores the enzymatic antioxidant profile in salivary gland of alloxan-induced diabetic rats. Archives of Oral Biology 2010;55:479-85.
5. Lee DH, Kim CS, Lee YJ. Astaxanthin protects against MPTP/ MPP+-induced mitochondrial dysfunction and ROS production in vivo and in vitro. Food Chem Toxicol 2011;49:271-80.
6. Yang Y, Zhou Y, Xu H. Animal studies astaxanthin antioxidant. Modern Preventive Medicine 2009;36:2432-3.