Abstract: Oxidation and carbonylation of bovine serum albumin (BSA) were induced by Cu2+/H2O2 and 2,2'-azobis(2-amidinopropane)hydrochloride (AAPH); oxidation and carbonylation of linoleic acid (linoleic acid) were induced by Fe2+, azobis(2,4-di-methyl- valeronitrile) (AMVN), respectively. The oxidation and carbonylation of bovine serum albumin (BSA) induced by Fe2+ and azobis (2,2'-azobis (2,4-di-methyl-valeronitrile), AMVN) and oxidative damage of herring sperm DNA (hsDNA) induced by AAPH were investigated to determine the effects of rosemary extract on the oxidation of proteins and lipids as well as DNA in the free radicals, The inhibitory effects of rosemary extract on the oxidative damage of protein, lipid and DNA under the attack of free radicals were investigated. The results showed that 25 μg/mL~500 μg/mL of rosemary extract significantly inhibited the radical-induced carbonylation of BSA and the production of malondialdehyde (MDA), a product of LA oxidation (p<0.05); 100 μg/mL~500 μg/mL of rosemary extract effectively reduced the production of conjugated diene products of LA induced by AMVN; 100 μg/mL~500 μg/mL of rosemary extract effectively reduced the production of conjugated diene products of LA induced by AMVN. Conjugated diene production induced by AMVN was effectively reduced by 100 μg/mL-500 μg/mL of rosemary extract. 25 μg/mL-500 μg/mL of rosemary extract significantly inhibited the production of hsDNA oxidized products induced by AAPH. Conclusion: Rosemary extract can effectively inhibit the oxidation of proteins, lipids and DNA induced by free radicals, and its inhibitory effect increases with the increase of the added concentration.
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Reactive oxygen species (ROS) are a group of oxygen atoms and groups containing unpaired electrons, including superoxide anion (O2-), hydroxyl radical (-OH), and hydrogen peroxide (H2O2), etc.[1] . Studies have shown that the excessive production of ROS can trigger protein oxidation, lipid peroxidation, and DNA oxidative damage, which can lead to a variety of human diseases [2-3]. For example, Hu et al. [4] found that oxidative stress may induce pemphigus, and Ram et al. [5] found that ROS-induced DNA oxidative damage is closely related to the occurrence of neurodegenerative diseases such as Alzheimer's disease. In order to reduce the damage caused by oxidative stress, some natural antioxidants have received extensive attention from researchers. Studies have shown that catechins, gallic acid and plant polyphenols have strong free radical scavenging ability, anti-aging, anti-cancer, anti-inflammatory and antioxidant functions [6-8], and can effectively prevent and mitigate the occurrence of oxidative stress.
Rosemary (Rosmarinus officinalis L.) is a natural spice plant native to the Mediterranean coast and is widely used as a food additive[9] . Rosemary contains rosemarinic acid, rosemarinol, rhamnetin and rhamnol, which have been proved to have physiological activities such as antioxidant, antibacterial, anti-inflammatory and antitumor functions, but relatively few studies have been conducted on the inhibition of oxidative damage of biomolecules and lipids [9-12]. In this study, oxidative damage to bovine serum albumin (BSA) was induced by Cu2+/H2O2, 2,2'-azobis(2 -amidinopropane) hydrochloride (AAPH), FeSO4, azobis(2 -isoheptanedinitrile) [2,2'-AZO, AAPH], and FeSO4, azobis(2 -isoheptanedinitrile) (AAPH). The oxidative damage of linoleic acid (LA) induced by FeSO4, azobis (2,2'-azobis (2,4-di-methyl-valeronitrile), AMVN) and herring sperm DNA (hsDNA) induced by AAPH was investigated to determine the inhibitory effect of rosemary extract on the oxidative damage. The inhibitory effect of rosemary extract on oxidative damage was investigated to provide a scientific theoretical basis for the application of rosemary in antioxidant functional foods.
1 Materials and Methods
1.1 Materials and reagents
Rosemary extract: homemade by the Food Nutrition and Safety Laboratory; linoleic acid (LA), 2,2'-azodiisobutylamidine dihydrochloride (AAPH) and azodiisoheptanenitrile (AMVN): Aladdin Reagent (Shanghai) Co: Ltd.; β-mercaptoethanol, sodium dodecyl sulfate (SDS), acrylamide, N,N'-methylenebisacrylamide, glycine, N,N,N'-tetramethyldiethylamine (TEMED), isopropanol, trimethylolamine methane, bromophenol blue, ammonium persulfate, and dimethyl sulfoxide (DMSO). DMSO: Shanghai McLean Biochemical Technology Co. trichloroacetic acid (TCA), 2,4-dinitrophenylhydrazine (DNPH): Beijing Bailing Wei Technology Co. CuSO4-5H2O, 30% hydrogen peroxide, glacial acetic acid and FeSO4-7H2O: Tianjin Damao Chemical Reagent Factory; Malondialdehyde (MDA) kit: Nanjing Jianjian Bioengineering Institute.
1.2 Instruments and equipment
PowerPacTM universal electrophoresis power supply, Mini-PROTEAN R Tetra electrophoresis tank, ChemiDocTM XRS+ chemiluminescence imaging system: Bio-Rad, USA; 5427R high-speed refrigerated centrifuge: Ep-pendorf, Germany; Multiskan GO full-wavelength enzyme labeling instrument: Thermo Fisher Scientific, USA; 2450 UV-Vis spectrophotometer: Shimadzu, Japan; HH-8J constant temperature water bath: Changzhou Runhua Electric Co. Multiskan GO full-wavelength enzyme labeler: Thermo Fisher Scientific, USA; 2450 UV-Vis spectrophotometer: Shimadzu, Japan; HH-8J thermostatic water bath: Changzhou Runhua Electric Co.
1.3 Test methods
1.3.1 Effect of rosemary extract on Cu2+/H2O2 induced protein oxidation and carbonylation
In the final concentration of 2 mg/mL BSA solution (pH 6.0, 0.2 mol/L PBS dissolved), 100 μL of different concentrations (0, 25, 50, 100, 200, 500 μg/mL) of rosemary extract was added, and after mixing, the final concentration of 400 μmol/L and 10 mmol/L CuSO4 solution and H2O2 solution were added sequentially. The total volume of the reaction was 1 mL, and the reaction was sealed and placed in a water bath at 37 ℃ for 90 min. At the end of the reaction, the proteins were separated by SDS-PAGE using 10% separator gel, and then stained with Cauloblue R-250 staining to detect the degree of oxidative damage of the BSA, and the carbonyl content of the BSA was detected by the DNPH colorimetric method.
1.3.2 Effect of rosemary extract on AAPH-induced protein oxidation and carbonylation
In the final concentration of 2 mg/mL BSA solution was added 100 μL of different concentrations of rosemary extract solution, mixed well, then added the final concentration of 100 mmol/L AAPH solution (pH 7.4, PBS dissolved), the total volume of the reaction was 1 mL, sealed and placed in a water bath at 37 ℃ for 4 h. After the reaction, the degree of oxidative damage and the carbonyl content of BSA were determined. The total volume of the reaction was 1 mL, and the reaction was sealed and placed in a water bath at 37 ℃ for 4 h. At the end of the reaction, the degree of oxidative damage and carbonyl content of BSA were determined.
1.3.3 Determination of the degree of oxidative damage of BSA
Take 50 μL of reaction samples, add 50 μL of 5×sampling buffer and mix well, and then boil for 5 min. 10 % separator gel was used to separate the proteins, which were stained with Caulobacter Brilliant Blue R-250 solution (1 g/L) for 30 min, and then decolorized with decolorizing solution overnight. The protein bands were semi-quantitatively analyzed by Image Lab software.
1.3.4 Determination of BSA carbonyl content
1 mL of the reaction solution was mixed with 3 mL of 0.01 mol/L DNPH solution, and the reaction was kept at room temperature (25 ℃) for 30 min. 4 mL of 20% TCA solution was added to the reaction solution, and the reaction was centrifuged at 4,000 r/min for 10 min. 3 mL of ethanol-ethyl acetate (1:1, v/v/v) was added to the reaction solution to wash the reaction solution and the process was repeated three times. After the last centrifugation, the supernatant was discarded, and 2 mL of 6 mol/L guanidine hydrochloride solution was added, and the absorbance at 370 nm was measured by UV-Vis spectrophotometer after mixing. The protein carbonyl content (nmol/mg) was calculated using a molar absorption coefficient of 22 000 mol/(L-cm).
1.3.5 Effect of rosemary extract on FeSO4 induced oxidation of linoleic acid
In the final concentration of 1 mmol/L LA (dissolved in ethanol), 100 μL of different concentrations of rosemary extract was added, and after mixing, 100 μmol/L FeSO4 solution was added to the final concentration, the total volume of the reaction was 1 mL, and then the reaction was sealed at 37 ℃ in a water bath and kept away from the light for 24 h. The levels of lipid oxidation of linoleic acid were expressed as the levels of conjugated diene and MDA at the end of the reaction. The conjugated diene content was determined by the method reported by Esterbauer et al [13]. The absorbance of the samples at 233 nm was measured by UV-Vis spectrophotometer, and the conjugated diene content was calculated based on the molar absorbance coefficient of conjugated diene, 2.8×104 L/(mol-cm), and the results were expressed as μmol/L. The MDA kit was used to determine the lipid oxidation level of linoleic acid in the samples. The MDA content of the samples was determined by using MDA kit and the results were expressed as nmol/mL.
1.3.6 Effect of rosemary extract on AMVN-induced linoleic acid oxidation
In the final concentration of 1 mmol/L LA, 100 μL of different concentrations of rosemary extract was added, and then 1 mmol/L AMVN solution (dissolved in methanol) was added after mixing, the total volume of reaction was 1 mL, and then the reaction was sealed in a water bath at 37 ℃ and protected from light for 12 h. At the end of the reaction, the conjugated diene and MDA contents were used as indicators to evaluate the levels of linoleic acid lipid oxidation.
1.3.7 Effect of rosemary extract on AAPH-induced oxidative DNA damage
In the final concentration of 2 mg/mL hsDNA (pH 6.0, 0.2 mol/L PBS solubilization), 100 μL of different concentrations of rosemary extract solution was added, and then 40 mmol/L AAPH solution was added, the total volume of the reaction was 1 mL, and the reaction was mixed well and placed in a 37 ℃ water bath for 12 h. The oxidative damage level of hsDNA was determined by using the MDA kit of Nanjing Jianjian Research Institute, and the inhibition rate of AAPH-induced MDA production was calculated according to the formula (1). The oxidative damage level of hsDNA was determined by using the MDA kit of Nanjing Institute of Building Research, and the inhibition rate of rosemary extract on AAPH-induced MDA production was calculated according to the formula (1). Where: A0 is the absorbance of the blank group (without inducer and rosemary extract); A1 is the absorbance of the inducer group; A2 is the absorbance after adding rosemary extract and inducer at the same time.
1.3.8 Statistical analysis
All the experiments were repeated three times and the results were expressed as mean ± standard deviation. SPSS21.0 statistical software was used for data processing, and the least significant difference (LSD) method was used for significance analysis, with p<0.05 indicating a significant difference, and GraphPad Prism 7.0 was used for plotting.
2 Results and Discussion
2.1 Effect of rosemary extract on Cu2+/H2O2 induced protein oxidation
The effect of rosemary extract on Cu2+/H2O2 induced BSA oxidation is shown in Figure 1.
Where: A0 is the absorbance of the blank group (without inducer and rosemary extract); A1 is the absorbance of the inducer group; A2 is the absorbance after adding both rosemary extract and inducer.
As shown in Figure 1A, the color of the BSA bands in the experimental group was significantly lighter after Cu2+/H2O2 induction compared with the control group, and the color of the BSA bands in the experimental group was significantly darker with the increase in the concentration of rosemary extract compared with the model group. As shown in Figure 1B, the relative grayness of the BSA bands in the test group was significantly reduced after Cu2+/H2O2 induction compared with the control group (p<0.05), and the relative grayness of the BSA bands in the model group was significantly increased with the increase in the concentration of rosemary extract compared with the model group (p<0.05), which is in agreement with the results of Zhang Yue et al [14]. This result was consistent with the results of Zhang Yue et al [14]. The reason for this result may be that Cu2+/H2O2 can induce the oxidative degradation of BSA by generating hydroxyl radicals through the Fenton reaction, resulting in the decrease in the gray level of the protein bands, and the polyphenols contained in rosemary extract can scavenge the hydroxyl radicals, thus inhibiting the protein oxidative degradation [15].
2.2 Effect of rosemary extract on AAPH-induced protein oxidation
The effect of rosemary extract on AAPH-induced BSA oxidation is shown in
Figure 2.
As shown in Figure 2, compared with the control group, the color of BSA bands in the AAPH-treated group became lighter, and the relative grayscale of BSA bands decreased significantly (p<0.05); compared with the model group, the relative grayscale of BSA bands increased significantly with the increase of the extract concentration (p<0.05), which was consistent with the results of the oxidative damage of protein induced by Cu2+/H2O2. The above results indicated that rosemary extract could effectively inhibit the oxidative degradation of BSA induced by AAPH.
2.3 Effect of rosemary extract on free radical-induced protein carbonylation
The effect of rosemary extract on free radical-induced carbonylation of BSA is shown in Fig. 3.
Protein carbonylation is an important indicator of protein oxidative damage[16] . As shown in Figure 3A, the carbonyl content of BSA in the control group was very low (3.80 nmol/mg), while the carbonyl content of BSA increased significantly (p<0.05) to 30.09 nmol/mg after 90 min of Cu2+/H2O2 treatment. The rosemary extract was able to inhibit the increase of carbonyl content in BSA induced by Cu2+/H2O2 (p<0.05), and its inhibitory effect was enhanced with the increase of the added concentration of the extract. As shown in Figure 3B, the carbonyl content of BSA in the AAPH-treated group was significantly higher than that in the control group (p<0.05); rosemary extract effectively inhibited the AAPH-induced increase in the carbonyl content of BSA (p<0.05), and its inhibitory effect was enhanced with the increase in the concentration of the added extract, which was similar to that of the results of the Cu2+/H2O2-induced increase in the carbonyl content of BSA.
Rosemary extract contains a variety of polyphenols such as rosmarinic acid, rosmarinol, rhamnolic acid and rhamnol [10, 17], which are able to inhibit the carbonylation modification of BSA through the removal of hydroxyl radicals and alkoxyl radicals generated by the Cu2+/H2O2 and AAPH reaction systems.
2.4 Inhibition of FeSO4-induced oxidation of linoleic acid by rosemary extracts
The effect of rosemary extract on FeSO4 induced oxidation of linoleic acid is shown in Figure 4.
LA is a common polyunsaturated fatty acid and is susceptible to lipid oxidation [18]. As shown in Figure 4A, the concentration of LA-conjugated diene in the FeSO4-treated group was 152.20 μmol/L, which was significantly higher than that in the control group (p<0.05). The inhibitory effect of the extracts of Dieffenbachia officinalis was enhanced with the increase in the concentration of the added extracts, and the increase in the concentration of the added extracts of Dieffenbachia officinalis was also effective in the inhibition of the increase in the concentration of LA-conjugated diene induced by FeSO4 (p<0.05).
MDA is a secondary oxidation product resulting from the oxidative decomposition of unsaturated fatty acids in fats and oils[19] . As shown in Figure 4B, in the control group, the amount of MDA produced by LA oxidation was very low, while in the model group, the amount of MDA produced by LA oxidation was significantly higher (p<0.05) after treatment with FeSO4 alone, and the amount of MDA produced by LA oxidation was gradually decreased with the increase of the concentration of rosemary extract in the treated group. This suggests that rosemary extract can effectively inhibit LA oxidation, and the inhibitory effect increases with the increase of its concentration. This is consistent with the results of Yao et al. [20], who investigated the effect of haematoxylin on FeSO4-induced LA oxidation.
2.5 Inhibition of AMVN-induced linoleic acid oxidation by rosemary extracts
The effect of rosemary extract on AMVN-induced linoleic acid oxidation is shown in Figure 5.
Under thermal decomposition conditions, AMVN generates alkoxyl radicals, which trigger the chain reaction of LA fat oxidation, accelerating the oxidation of oils and fats. As shown in Figure 5A, the lower concentration of rosemary extract in the treatment group failed to significantly inhibit the production of conjugated diene products, while the concentration of rosemary extract in the range of 100 μg/mL~500 μg/mL significantly inhibited LA lipid oxidation (p<0.05); and as shown in Figure 5B, the lipid oxidation of LA in the model group was significantly (p<0.05) inhibited under the action of AMVN, whereas LA oxidation of LA in the control group was lower; it can be concluded that, the lipid oxidation of LA in the model group could be significantly (p<0.05) inhibited under the action of AMVN. It can be concluded that rosemary extract significantly (p<0.05) inhibited the production of MDA, and its inhibitory effect gradually increased with the increase in the concentration of the extract (25 μg/mL-500 μg/mL).
2.6 Effect of rosemary extract on AAPH-induced DNA oxidative damage
The inhibitory effect of rosemary extract on AAPH-induced DNA oxidative damage is shown in Figure 6.
DNA is the carrier of genetic information in the organism, and the free radicals generated by AAPH will attack the DNA and cause oxidative damage to the DNA, and the degree of oxidative damage can be determined by measuring the absorbance of the oxidized DNA products at 532 nm. As shown in Figure 6, rosemary extract could effectively inhibit the oxidation of hsDNA induced by AAPH, and its inhibition rate increased significantly with the increase of the added concentration (p<0.05).
3 Conclusion
The present study showed that rosemary extract significantly inhibited the oxidative degradation of proteins, carbonylation modification, lipid oxidation and DNA oxidative damage induced by different free radicals, and the inhibitory effect increased with the concentration of rosemary extract; the reason for this may be that rosemary extract blocked the free radical chain reaction induced by the inducers, such as Cu2+/H2O2, AAPH, FeSO4 and AMVN, thus inhibiting the oxygenation damage of biomolecules and LA. This may be due to the fact that rosemary extract blocked the free radicals induced by the inducers such as Cu2+/H2O2, AAPH, FeSO4 and AMVN, thus inhibiting the oxygenation damage of biomolecules and LA. This study provides a theoretical basis for the prevention of certain diseases related to oxidative damage of proteins and the development of functional foods with rosemary extracts. The inhibitory effect of rosemary extract on free radical-induced oxidation of proteins, lipids and DNA and its mechanism should be further evaluated in future experiments using animal studies.
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