Antioxidant And Anti-aging Potential Of A Peptide Formulation (Gal2–Pep) Conjugated With Gallic Acid Ⅱ
May 04, 2023
3. Results and discussion
3.1. Synthesis and conjugation of gallic acid with peptide
The synthesized peptides TPPTTP, galloyl–TPPTTP (Gal–Pep), and Gal2–Pep were obtained and purified using semi-preparative RP-HPLC. Fig. 1a and Scheme 1 show the conjugation strategy of gallic acid with KTPPTTP as Gal2–Pep. In the SPPS method, the impurities generated during peptide synthesis usually include side-reacted peptides, amino acid-protecting groups, and solvents used for synthesis and purification. The HOBt used in peptide synthesis is known to inhibit racemization and improve the efficiency of peptide synthesis.17–19 In order to remove amino acid-protecting groups and solvents used for synthesis, we performed washing several times using solvents such as DMF and DCM during the peptide synthesis process. Even after the last washing step, impurities including amino acid-protecting groups are still present in trace amounts. In order to meet the peptide purity of more than 90% reported by several researchers,20–22 semi-prep HPLC was performed as a polishing step to obtain the nal peptide of 99.2% purity as a result of HPLC analysis (see Fig. S1†). The purified peptide product was analyzed using Q-TOF to reconfirm their successful synthesis (Fig. 1b–d).
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3.2. Cell viability assays
3.3. Antioxidant activity of synthesized peptides
TPPTTP had no radical scavenging activity at any concentration, while GA, Gal–Pep, and Gal2–Pep exhibited higher radical scavenging activity in a concentration-dependent manner.
In particular, Gal2–Pep demonstrated the most effective antioxidant activity; at a concentration of 10 mM, GA, Gal–Pep, and Gal2–Pep had a radical scavenging activity of 13.6%, 24.23%, and 26.0%, respectively. This indicates that the binding of TPPTTP to GA does not inhibit the radical scavenging activity. Fig. 3b confirms the stability of the samples as confirmed by assessing their radical scavenging activity when stored at room temperature. GA retained its radical scavenging activity for up to one week but, after two weeks, this activity decreased by more than 60%. On the other hand, Gal–Pep and Gal2–Pep maintained their scavenging activity for over two weeks. In particular, Gal2–Pep had a radical scavenging activity of 50% even in its fourth week. These results suggest that binding GA to TPPTTP stabilizes its radical scavenging activity. Some studies have shown that ascorbic acid is stabilized by binding it with peptides.12,25 We believe that GA may be stabilized by binding it with a peptide in the same way.
The antioxidant effect of the peptides was also tested using DCFDA assays in the presence of intracellular oxidative stress induced by H2O2 (Fig. 4). Aer 24 h of treating the samples on the cells at a concentration of 100 mM, DCF-DA was loaded onto the cells, and the cells were then treated with H2O2 for 30 min and the fluorescence intensity of DCF was observed at an excitation wavelength of 485 nm and an emission wavelength of 535 nm. Cells subjected to oxidative stress with 1 mM H2O2 exhibited fluorescence intensity that was more than three times higher than the negative control; however, Gal2–Pep treatment produced a fluorescence that was only 67% of that produced by cells treated with H2O2 only. In the presence of TPPTTP, which did not exhibit radical scavenging activity (Fig. 3a), the ROS stress caused by H2O2 was reduced by about 20%, supporting previous research that has reported that TPPTTP decreases ROS levels in cells.24 When the cells were treated with GA only, there was no significant change in ROS levels. Taken together, these results suggest that Gal2–Pep, which combines two GA molecules with a peptide, is a stable and effective antioxidant.
3.4. Effect of synthesized peptides on the mitochondria membrane potential
In this study, JC-1 dye was used to investigate the effect of the synthesized peptides on the MmP (DJm). The MmP acts as an important parameter for mitochondrial function and is known to be associated with several diseases such as Alzheimer's and Huntington's disease.26 JC-1 dye produces a redshirt from green emissions by accumulating in mitochondria and forming J-aggregates. After treating the HaCaT and fibroblast cells with the synthesized peptides for 24 h, the cells were exposed to JC-1 dye for 10 min, and then the fluorescence intensity was measured using a microplate reader (green lEx ¼ 475 nm and lEm ¼ 530 nm, red lEx ¼ 475 nm and lEm ¼ 590 nm). The ratio of the red to green fluorescence was expressed as the fold-change compared to the negative control (Fig. 5). HaCaT cells treated with GA and the peptide independently did not exhibit any significant change compared to the negative control group, while Gal2–Pep exhibited a significant difference, increasing 1.45-fold compared to the control. The fibroblast cells treated with Gal2–Pep also significantly
Fig. 4 Intracellular antioxidant activity assays using DCF-DA at gallic acid and peptide concentrations of 100 mM. After 24 hours of treatment, H2O2 was added and incubated for 30 minutes, followed by measurement of DCF fluorescence intensity. Asterisks indicate statistically significant differences (*p < 0.05, **p < 0.005, ***p < 0.001).
3.5. Elastase inhibitory activity
3.6. Expression of type I collagen, MMP-1, and PGC-1a genes
Fig. 7 Gene expression analysis in dermal fibroblasts using RT-qPCR: (a) type I collagen, (b) MMP-1, and (c) PGC-1a.
The expression of PGC-1a was highest after 8 h for all treatments, with expression rates for GA, TPPTTP, Gal–Pep, and Gal2–Pep increasing 2.06-, 1.41-, 1.07-, and 2.05-fold, respectively. However, unlike the other treatments, Gal2–Pep increased PGC-1a expression 1.27-fold after 24 h. PGC-1a regulates the synthesis and antioxidant effects of mitochondria and is reported to decrease with aging.29–32 Therefore, Gal2–Pep has the potential to be used as an effective antioxidant that can effectively protect against ROS.
Taken all together, Gal2–Pep increased the expression of PGC-1a and collagen compared to the effects of GA and TPPTTP alone, while the expression of MMP-1 tended to decrease, thus illustrating its potential for use in anti-aging cosmetics.
4. Conclusion
Conflicts of interest
There are no conflicts to declare.
Acknowledgments
This study was supported by a grant of the Korea Industrial Complex Corp. (KICOX, NTIS No. 1415165722) and the Inha University Research Grant.
References
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22 Z. Zhai, K. Xu, L. Mei, C. Wu, J. Liu, Z. Liu, L. Wan and W. Zhong, So Matter, 2019, 15, 8603–8610.
23 D. J. Tobin, J. Tissue Viability, 2017, 26, 37–46.
