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Saponin-Based Nanoemulsification Improves the Antioxidant Properties of Vitamin A and E in AML-12 Cells

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ABSTRACT

Our work aimed to investigate the protective effects of saponin-based nanoemulsions of vitamin A and E against oxidative stress-induced cellular damage in AML-12 cells. Saponin nanoemulsions of vitamin A (SAN) and vitamin E (SEN) were prepared by high-pressure homogenization and characterized in terms of size, zeta potential, and polydispersity index. SEN and SAN protect AML-12 cells against oxidative stress-induced cellular damage more efficiently via scavenging reactive oxygen species (ROS), and reducing DNA damage, protein carbonylation, and lipid peroxidation. These results provide valuable information for the development of nanoemulsion-based delivery systems that would improve the antioxidant properties of vitamin A and E.

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Protective effect of nanoemulsion against oxidative damage in AML-12 cells after H2O2 exposure. (a) Lipid peroxidation in AML-12 cells was detected with polyclonal anti-HNE antibody and DAB counterstain; (b) Protein carbonyl content of AML-12 cells was detected with DNP-specific antibody; (c) 8-OH-dG levels in AML-12 cells, which reflected the binding of avidin-TRITC, were analyzed using fluorescence microscopy; (d) The mean fluorescence intensity of avidin was quantified using ImageJ software; (e) Effect of nanoemulsion on intracellular ATP level was measured using FITC-labeled antibody; (f) ATP fluorescence intensity was quantified by ImageJ software. C, control; SN, empty saponin nanoemulsion; VA, vitamin A; SAN, saponin nanoemulsion of vitamin A; VE, vitamin E; SEN, saponin nanoemulsion of vitamin E. All values are represented as mean ± SEM from three or more independent studies. *** p < 0.001; * p < 0.05.
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ijms-17-01406-f004: Protective effect of nanoemulsion against oxidative damage in AML-12 cells after H2O2 exposure. (a) Lipid peroxidation in AML-12 cells was detected with polyclonal anti-HNE antibody and DAB counterstain; (b) Protein carbonyl content of AML-12 cells was detected with DNP-specific antibody; (c) 8-OH-dG levels in AML-12 cells, which reflected the binding of avidin-TRITC, were analyzed using fluorescence microscopy; (d) The mean fluorescence intensity of avidin was quantified using ImageJ software; (e) Effect of nanoemulsion on intracellular ATP level was measured using FITC-labeled antibody; (f) ATP fluorescence intensity was quantified by ImageJ software. C, control; SN, empty saponin nanoemulsion; VA, vitamin A; SAN, saponin nanoemulsion of vitamin A; VE, vitamin E; SEN, saponin nanoemulsion of vitamin E. All values are represented as mean ± SEM from three or more independent studies. *** p < 0.001; * p < 0.05.

Mentions: Oxidative DNA damage, protein oxidation, and lipid peroxidation are well-known markers that indicate cellular oxidative damage. An increase in cellular oxidative stress is directly proportional to lipid peroxidation, which leads to accumulation of potent cytotoxic lipid peroxides, such as HNE and MDA [35]. As shown in Figure 4a, the endogenous level of HNE adducts in proteins was significantly increased when AML-12 cells were exposed to H2O2, and the increase of lipid peroxidation was markedly reduced in emulsified vitamin samples compared with that in non-emulsified vitamin samples. For further investigation of whether emulsified vitamin treatment also decreased H2O2-induced protein damage, we evaluated protein oxidation by measuring the DNP-adduct of protein carbonyl content that corresponds to protein oxidation and hence protein damage after exposure to oxidative stress. As shown in Figure 4b, the endogenous level of carbonyl groups in proteins was significantly increased when cells were exposed to H2O2. Moreover, protein oxidation was reduced in SEN and SAN samples compared with that in SN samples.


Saponin-Based Nanoemulsification Improves the Antioxidant Properties of Vitamin A and E in AML-12 Cells
Protective effect of nanoemulsion against oxidative damage in AML-12 cells after H2O2 exposure. (a) Lipid peroxidation in AML-12 cells was detected with polyclonal anti-HNE antibody and DAB counterstain; (b) Protein carbonyl content of AML-12 cells was detected with DNP-specific antibody; (c) 8-OH-dG levels in AML-12 cells, which reflected the binding of avidin-TRITC, were analyzed using fluorescence microscopy; (d) The mean fluorescence intensity of avidin was quantified using ImageJ software; (e) Effect of nanoemulsion on intracellular ATP level was measured using FITC-labeled antibody; (f) ATP fluorescence intensity was quantified by ImageJ software. C, control; SN, empty saponin nanoemulsion; VA, vitamin A; SAN, saponin nanoemulsion of vitamin A; VE, vitamin E; SEN, saponin nanoemulsion of vitamin E. All values are represented as mean ± SEM from three or more independent studies. *** p < 0.001; * p < 0.05.
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ijms-17-01406-f004: Protective effect of nanoemulsion against oxidative damage in AML-12 cells after H2O2 exposure. (a) Lipid peroxidation in AML-12 cells was detected with polyclonal anti-HNE antibody and DAB counterstain; (b) Protein carbonyl content of AML-12 cells was detected with DNP-specific antibody; (c) 8-OH-dG levels in AML-12 cells, which reflected the binding of avidin-TRITC, were analyzed using fluorescence microscopy; (d) The mean fluorescence intensity of avidin was quantified using ImageJ software; (e) Effect of nanoemulsion on intracellular ATP level was measured using FITC-labeled antibody; (f) ATP fluorescence intensity was quantified by ImageJ software. C, control; SN, empty saponin nanoemulsion; VA, vitamin A; SAN, saponin nanoemulsion of vitamin A; VE, vitamin E; SEN, saponin nanoemulsion of vitamin E. All values are represented as mean ± SEM from three or more independent studies. *** p < 0.001; * p < 0.05.
Mentions: Oxidative DNA damage, protein oxidation, and lipid peroxidation are well-known markers that indicate cellular oxidative damage. An increase in cellular oxidative stress is directly proportional to lipid peroxidation, which leads to accumulation of potent cytotoxic lipid peroxides, such as HNE and MDA [35]. As shown in Figure 4a, the endogenous level of HNE adducts in proteins was significantly increased when AML-12 cells were exposed to H2O2, and the increase of lipid peroxidation was markedly reduced in emulsified vitamin samples compared with that in non-emulsified vitamin samples. For further investigation of whether emulsified vitamin treatment also decreased H2O2-induced protein damage, we evaluated protein oxidation by measuring the DNP-adduct of protein carbonyl content that corresponds to protein oxidation and hence protein damage after exposure to oxidative stress. As shown in Figure 4b, the endogenous level of carbonyl groups in proteins was significantly increased when cells were exposed to H2O2. Moreover, protein oxidation was reduced in SEN and SAN samples compared with that in SN samples.

View Article: PubMed Central - PubMed

ABSTRACT

Our work aimed to investigate the protective effects of saponin-based nanoemulsions of vitamin A and E against oxidative stress-induced cellular damage in AML-12 cells. Saponin nanoemulsions of vitamin A (SAN) and vitamin E (SEN) were prepared by high-pressure homogenization and characterized in terms of size, zeta potential, and polydispersity index. SEN and SAN protect AML-12 cells against oxidative stress-induced cellular damage more efficiently via scavenging reactive oxygen species (ROS), and reducing DNA damage, protein carbonylation, and lipid peroxidation. These results provide valuable information for the development of nanoemulsion-based delivery systems that would improve the antioxidant properties of vitamin A and E.

No MeSH data available.


Related in: MedlinePlus