Antioxidant And Anti-aging Potential Of A Peptide Formulation (Gal2–Pep) Conjugated With Gallic Acid
May 04, 2023
Skin is highly vulnerable to premature aging due to external stress, therefore, in this study, a peptide formulation, (galloyl)2–KTPPTTP (Gal2–Pep) was synthesized by combining TPPTTP peptide, and gallic acid (GA). All peptides were synthesized on 2-chlorotrityl chloride resin using solid-phase peptide synthesis (SPPS), and analyzed on an electrospray ionization (ESI)/quadrupole-time-of-flflight (Q-TOF) tandem mass spectroscopy (MS) system. Initially, Gal2–Pep showed no toxicity below the concentration 100 mM with a cell survival rate of 88% for keratinocytes and fibroblasts. The reactive oxygen species (ROS) scavenging activity of Gal2–Pep was more stable compared to GA alone; and after four weeks at room temperature, its ROS scavenging activity remained higher than 50%. Moreover, the peptide formulation, Gal2–Pep also exhibited an elastase inhibitory effect in CCD-1064Sk fibroblast cells. Based on the results of RT-qPCR, it was proved in this study that Gal2–Pep increased the expression of PGC-1a to prevent oxidative stress, and validated its potential as an anti-aging agent by increasing the expression of type I collagen and by decreasing the expression of matrix metalloproteinase-1 (MMP1). The findings obtained reinforce the suggestion that the peptide formulation synthesized in this study could be used as a natural antioxidant and anti-aging agent for its cosmetic applications.
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1. Introduction
Skin aging occurs as a result of the gradual decrease in and eventual halting of the cell division of keratinocytes and bro blasts within the skin. Wrinkles on the skin develop as the number of cells decreases, with the resulting denaturation of the extracellular matrix leading to dryness and a loss of elasticity in the skin.1 Damage to intracellular mitochondrial DNA and a reduction in the rate of protein synthesis also occurs during the skin aging process.2 Skin aging has two main pathways, intrinsic and extrinsic, with ultraviolet (UV) exposure
a major driver of extrinsic aging. The UV light reacts with major skin components, including lipids, proteins, and nucleic acids, to produce reactive oxygen species (ROS) that lead to cell death.3–5 Excessive ROS levels also lead to the cleavage and abnormal binding of collagen or elastin chains and promote the expression of matrix metalloproteinases (MMPs), such as MMP-1, that break down collagen, leading to the wrinkling of the skin and accelerating skin aging.6,7 Therefore, antioxidant therapy to remove ROS and to activate mitochondrial potential has the merits to protect the skin from aging.
In addition to ROS, elastase has a significant role in the loss of skin elasticity, which breaks down elastin, an important protein of the extracellular matrix.8 Elastin, due to its significant elastic recoil characteristics, provide elasticity to the skin, whereas elastase has the ability to cleave elastin and other proteins.8 Therefore, inhibition of elastase enzyme could be a key strategy for skin sagging via preventing the loss of skin elasticity.
In this study, we designed a new material that combines the peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1a)-derived peptide TPPTTP and gallic acid (GA) for use in anti-aging cosmetics. GA is a phytochemical found in various fruits and vegetables, including grapes, tomatoes, and green tea, that is known to have high antioxidant and antibacterial effects.9 Though GA is used in the cosmetics and food industries because of these beneficial effects, its formulation tends to be unstable.10 We thus developed (galloyl)2–KTPPTTP (Gal2–Pep) in order to increase the stability of GA by binding it to the PGC-1a-derived peptide TTPTTP.
In this study, we synthesized a (galloyl)2–KTPPTTP (Gal2– Pep) peptide formulation in conjugation with gallic acid and confirmed its potential as an antioxidant and antiaging agent.
2. Materials and methods
2.1. Materials
Dulbecco's Modified Eagle's Medium (DMEM), penicillin/streptomycin, fetal bovine serum (FBS), phosphate-buffered saline (PBS), and trypsin were purchased from Gibco (Carls
bad, CA). Hydroxybenzotriazole (HOBt), diisopropyl carbodiimide (DIC), 1,8-diazabicyclo[5.4.0]undec-7-en (DBU), N, N-diisopropylethylamine (DIEA), gallic acid and N-succinyl-tri-alanyl-p nitroanilide were obtained from Sigma-Aldrich (St. Louis, MO). L-Form amino acids (Fmoc-Lys (Boc)–OH, Fmoc-Thr (tBu)–OH, and Fmoc-Pro–OH) were purchased from Bead-Tech (Seoul,Korea).
2.2. Synthesis of peptides and gallic acid-coupled peptides
All peptides were synthesized on 2-chlorotrityl chloride resin (200 mmol scale, substitution value ¼ 1.46 mmol g 1) using HOBt–DIC mediated solid-phase peptide synthesis (SPPS).
Peptide synthesis using the SPPS method was carried out with slight modications referring to previous studies.11–13 The 2-chlorotrityl chloride (CTC) resin was swelled in dichloro
methane (DCM, 8 mL) for 30 min. The resin was washed with dimethylformamide (DMF). All reactions were carried out at room temperature. Fmoc protected amino acid derivatives (2 equivalent (Equiv.), for the first attached amino acid or 5 equiv., for the other amino acids) were coupled with either DIEA (5 equiv., for an attached amino acid) or HOBt/DIC (6 equiv./5 equiv.) in DMF after performing deprotection of Fmoc using 2% (v/v) DBU in DMF (2 times, 2 min a time, 8 mL). And all steps were washed 5 times with DMF. Final gallic acid was coupled with lysine-attached peptides. After the coupling of the gallic acid, the resin was altered, washed, and dried under a high vacuum. The cleavage solution (TFA: deionized [DI] water: TIS ¼ 95 : 2.5 : 2.5, v/v/v%) was treated for 2 h to obtain the crude peptides. The crude peptides were precipitated and washed three times with cold diethyl ether.
Analytical reverse-phase high-performance liquid chromatography (RP-HPLC) was conducted on a Waters 2695 Separations Module with a Capcell Pak C18 column (4.6 mm 250 mm, 5 mm, Shiseido). The mobile phase consisted of 0.05% TFA in H2O (mobile phase A) and 0.05% TFA in acetonitrile (mobile phase B).
Elution was achieved by employing a linear gradient for mobile phase B of 5% to 65% over 30 min at a ow rate of 1.0 mL min 1. The peptide peaks were detected at a wavelength of 230 nm. Semi-preparative RP-HPLC was conducted on a Waters HPLC system (Pump 600E, Detector UV-484) with a Gemini RP-C18 column (21.2 mm 250 mm, 5 mm, Phenomenex). The mobile phase consisted of 0.05% TFA in H2O (mobile phase A) and 0.05% TFA in acetonitrile (mobile phase B). Elution was achieved by employing a linear gradient for mobile phase B of 7% to 22% over 15 min at a low rate of 10 mL min 1. The peptide peaks were detected spectrophotometrically at a wavelength of 230 nm.
The synthesized peptides were dissolved in 5% formic acid in water and analyzed on an electrospray ionization (ESI)/quadrupole-time-of-light (Q-TOF) tandem mass spectroscopy (MS) system (TripleTOF6600, ABSciex, Foster City, CA). The samples were injected into the Q-TOF system equipped with a nano-spray source at a low rate of 1 mL min 1. The ESI ion source parameters were as follows: an ion spray voltage of 1.5 kV, curtain gas at 10 psi, and sheath gas at 5 psi. The spectra in full-scan mode and MS/MS were collected in the range of m/z
100–1200 Da at an accumulation time of 25 ms per spectrum. The collision energy was ramped from 10 to 80. The resultant data were acquired using Analyst TF soware and automatically assigned by centroid 80% with a minimum peak, noise reduction of 10%, medium deisotoping with a 3% threshold, no noise reduction, and no smoothing. The synthesized peptides were identified with a mass tolerance of 0.05 Da and manually sequenced using an MS/MS tolerance of 0.01 Da.
2.3. Cell cultures and viability assays
Human, adult, low calcium, high temperature (HaCaT) cells and CCD-1064Sk (normal human skin fibroblast) cells were cultured in DMEM with 10% FBS and 1% penicillin/streptomycin. The cells were incubated at 37 C in a humidified air chamber with 5% CO2.For the viability assays, cell counting kit-8 (CCK-8, Dojindo Co. Ltd. Beijing, China) was used. HaCaT and CCD-1064Sk cells were seeded in 96-well microplates (5 103 cells per well) and incubated at 37 C in 5% CO2 for 24 h. The cultures were then exposed to different concentrations of the synthesized peptides or GA and incubated at 37 C in 5% CO2 for 24 h. Cell viability was subsequently measured using a CCK-8 following the manufacturer's guidelines.
2.4. Determination of antioxidant activity
2.4.1. DPPH assay. The antioxidant activity of the synthesized peptides and GA was evaluated individually using DPPH assay. DPPH assays were performed following a previously reported
methodology with minor modication.12,14–16 First, a 0.12 mg mL 1 DPPH solution was prepared in methanol, then 100 mL of DPPH solution and 100 mL of the synthesized peptides or GA were mixed at different concentrations (0.1 mM, 0.05 mM, 0.01 mM, and 1 mM) in 96-well microplates. In an orbital shaking incubator, the mixtures were reacted for 30 min at 37 C and 100 rpm without light. The absorbance was then measured at 517 nm and the antioxidant capacity was calculated.
2.4.2. ROS scavenging activity.
Intracellular ROS scavenging activity was evaluated using a 20,70-dichlorofluorescein diacetate kit (DCF-DA, Abcam, USA). HaCaT cells were seeded in 96-well microplates (5 103 cells per well) and incubated at 37 C for 24 h. After incubation, the cultures were exposed to the synthesized peptides or GA (100 mM) and incubated at 37 C for 24 h. The cells were then exposed to a 10 mM DCF-DA solution and incubated for 30 min. After incubation, the solution was washed with PBS. The cells were analyzed using a microplate reader at excitation and
emission wavelengths of 485 nm and 530 nm, respectively.
Fig. 1 Synthetic step and sequence confirmation of peptides using quadrupole-time of flight (Q-TOF) mass spectrometry: (a) detailed synthetic procedure (b) TPPTTP, (c) galloyl–TPPTTP, and (d) (galloyl)2–KTPPTTP.
2.5. Mitochondrial membrane potential assay
JC-1 dye (Thermo Fisher, USA) was used for the mitochondrial membrane potential (MmP) assay. CCD-1064Sk and HaCaT cells were seeded in 96-well microplates (5 10 3cells per well)
and incubated at 37 C and in 5% CO2 for 24 h. After incubation, the cultures were exposed to different concentrations of the synthesized peptides and incubated at 37 C in 5% CO2 for 24 h. JC-1 dye was added to the CCD-1064Sk and HaCaT cells to produce a final concentration of 10 mM. After 10 min of incubation, the cells were washed twice with 37 C PBS. Green and red fluorescence were measured using a Varioskan LUX Multimode Microplate Reader (ThermoFisher, USA) at an excitation (lEx)/emission (lEm) of 475/530 nm and a lEx/lEm of 475/590 nm, respectively.
2.6. Elastase inhibitory activity assay
CCD-1064Sk cells were seeded in 6-well plates and incubated at 37 C in 5% CO2 for 24 h. The cultures were then exposed to a concentration of 100 mM of the synthesized peptides or GA and incubated at 37 C in 5% CO2 for 24 h. Cells were washed with dPBS twice and we collected each cell using cell scraper. Aer adding 0.2 M Tris–HCl (pH 8.0) with 0.1% Triton-X to the collected cells, cells were homogenized by ultrasonicator. The homogenized solution was centrifuged for 20 min at 3000 rpm and 4 C. Then, after taking the supernatant, protein quanti-cation was performed using BCA protein assay. Protein for each sample was dispensed at the final concentration of 100 mgmL 1, and N-succinyl-tri-alanyl-p-nitroanilide was added at the final concentration of 1.6 mM and reacted at 36 C for 1 h. Then, the absorbance was measured at 405 nm using a microplate reader.
2.7. RT-qPCR
The mRNA levels of anti-aging markers in CCK-1064Sk cells were assessed using RT-qPCR. Total RNA was collected using TRIzol reagent (Life Technologies, Carlsbad, CA) and reverse
transcribed using the PrimeScript RT reagent kit (Takara Bio Inc., Kusatsu, Japan). A CFX96 system (Bio-Rad Laboratories, Hercules, CA) and iQ SYBR Green Supermix (Bio-Rad Labora
tories) were used for RT-qPCR. b-Actin was used to normalize the mRNA levels of type I collagen, MMP-1, and PGC-1a.
2.8. Statistical analysis
The data are presented as the mean standard deviation from three independent measurements and analyzed using Student's t-tests, with a p < 0.05 considered to be a signicant difference.
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