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Evaluation of Natural Bioactive-Derived Punicalagin Niosomes in Skin-Aging Processes Accelerated by Oxidant and Ultraviolet Radiation

Authors Mohamad EA, Aly AA, Khalaf AA, Ahmed MI, Kamel RM, Abdelnaby SM, Abdelzaher YH, Sedrak MG, Mousa SA 

Received 17 April 2021

Accepted for publication 4 June 2021

Published 17 July 2021 Volume 2021:15 Pages 3151—3162

DOI https://doi.org/10.2147/DDDT.S316247

Checked for plagiarism Yes

Review by Single anonymous peer review

Peer reviewer comments 2

Editor who approved publication: Dr Tuo Deng



Ebtesam A Mohamad,1 Aya A Aly,2 Aya A Khalaf,2 Mona I Ahmed,2 Reham M Kamel,2 Sherouk M Abdelnaby,2 Yasmine H Abdelzaher,2 Marize G Sedrak,2 Shaker A Mousa3

1Biophysics Department, Faculty of Science, Cairo University, Giza, Egypt; 2Biotechnology/Biomolecular Chemistry Program, Faculty of Science, Cairo University, Giza, Egypt; 3The Pharmaceutical Research Institute, Albany College of Pharmacy and Health Sciences, Rensselaer, NY, USA

Correspondence: Shaker A Mousa
The Pharmaceutical Research Institute, Albany College of Pharmacy and Health Sciences, Rensselaer, NY, 12144, USA
Tel +1-518-694-7397
Fax +1-518-694-7567
Email [email protected]

Introduction: Skin aging is a normal process that might be accelerated or delayed by altering the balance between antioxidants and free radicals due to increase in the exposure to reactive oxygen species (ROS) into skin cells via UV radiation. Antioxidants can neutralize the harmful effects of ROS, and secondary plant metabolites might help protect against UV radiation.
Methods: In this study, punicalagin was extracted from pomegranate, and concentrations of total polyphenolics and flavonoids were determined, and antioxidant activities were measured. Punicalagin was loaded onto niosomes, and its morphology and release were studied. An in vitro study was performed on human fibroblast cell line HFB4 cells with aging induced by H2O2 and UV radiation. Cell cycle arrest was studied, and different genes (MMP3, Col1A1, Timp3, and TERT) involved in the skin aging process were selected to measure punicalagin’s effect.
Results: Punicalagin succeeded in reducing the growth arrest of HFB4 cells, activated production of the Col1A1 and Timp3 genes, maintained collagen level, and lowered MMP3. Punicalagin increased human TERT concentration in skin cells.
Discussion: Punicalagin is promising as a natural antioxidant to protect human skin from aging.

Keywords: skin aging, punicalagin, niosomes, UV radiation, collagen

Introduction

During the human life cycle, the skin is exposed to various harmful factors.1 Our skin is the largest organ, accounting for one-sixth of total body weight, and it acts as a barrier to protect the body from water loss and environmental factors such as chemicals, pathogens, and ultraviolet (UV) rays.2

Human skin is divided into three layers: epidermis, dermis, and hypodermis. In the dermis, collagen, fibers and elastin fibers are found. Aging skin shows thinning of the skin, loss of elasticity, dryness, wrinkles, and delayed wound healing. In general, with aging skin becomes pale in appearance.2

Intrinsic aging is defined as the aging process that occurs naturally and begins when a person is in their mid-twenties. In the skin, the collagen production slows down, and elastin has a little less spring.3 In contrast, extrinsic aging is a phenomenon that can be avoided to some extent. Many extrinsic or external factors often work in conjunction with the natural aging process to prematurely age our skin. Extrinsic factors are frequent facial expressions, gravity, sun, and smoking. The most harmful environmental factor that leads to extrinsic aging is repeated UV exposure, which is referred to as photoaging.3 UV A radiation (UVA) (400–320 nm) accounts for about 95–98% of the total UV rays that reach the surface of the earth. Besides damaging the epidermis, UVA rays penetrate deep into the dermis to break down collagen and elastin.2 Exposure to a high amount of UV radiation makes young people’s skin prematurely aged due to the occurrence of mutations unless damaged skin cells are repaired.2–4 UV radiation exposure leads to the generation of ROS and its accumulation in the skin cells, which results in skin aging.

Many genes were proved to have influence on the aging process. The group of genes known as the Collagens (COL) Gene group5 are responsible for collagen synthesis, in addition to the matrix metalloproteinases (MMPs) that play an important role in inflammation, tumor invasion, and skin aging. MMPs degrade dermal collagen and elastin during the aging process.6,7 Previous research has also demonstrated the importance of tissue inhibitors of metalloproteinase (TIMPs), which play a considerable role in the inhibition of protein degradation of the extracellular matrix.7

Skin as a self-renewing tissue goes through massive proliferation during life and telomere shortening acts to control mitotic division and prevent abnormal proliferation such as cancer. And as a result, cellular ageing occurs. The telomerase enzyme complex (Telomerase Reverse Transcriptase TERT) ensures telomere length maintenance in germ and cancer cells. Telomerase is also active in some somatic cells as in the epidermis but is hardly detectable in the dermis. Shortening of telomeres in skin cells may be accelerated due to proliferation and DNA-damaging agents as reactive oxygen species (ROS).8

Oxidation processes in cell metabolism produce ROS normally to produce the energy required to fuel other biological processes, and this explains the inevitable production of free radicals through aerobic chemical reactions of cells in chronological aging that lead to mitochondrial damage, altering cell function, and if the damage is excessive, infected cells may die.1,2 Although a strong antioxidant defense system exists in skin cells, excessive production of ROS will alter the balance between free radicals and antioxidants, a process known as oxidative stress that may damage cells because ROS affects cellular constituents such as membranes, enzymes, proteins, lipids, RNA, and DNA in addition to reducing levels of antioxidants in the skin, which leads to many diseases and disorders as well as aging.1,2,9

It has been shown that hydrogen peroxide (H2O2) enhances the development of skin injury and aging like other free radicals because it increases the expression of inflammation-related proteins and downregulates collagen I/III formation and expression of antioxidative proteins.10 Previous studies showed that treating human skin fibroblasts with 0–500 µM H2O2 decreased the viability of cells in a concentration-dependent manner.

Antioxidants are any substances that can directly scavenge ROS or indirectly act to enhance antioxidant defenses or inhibit ROS output.11–13 They are produced to counteract oxidative stress generated by the presence of excess ROS.14 Also, antioxidants are distinguished by their ability through intramolecular hydrogen bonding to further oxidize to form a more stable radical.11

Plants’ secondary metabolites can be used as natural antioxidants to protect human skin from photoaging.11 The natural antioxidants from plant secondary metabolites can protect human skin from photoaging.11 They mainly contain polyphenols (phenolic acids, anthocyanins, flavonoids), carotenoids, and vitamins (vitamin E and C).15 Pomegranate (Punica granatum L.) is a fruit that belongs to the family Punicaceae and consists of several substances such as fiber, vitamins, minerals, and bioactive plant compounds and sugar that are useful in reducing the risk of disease.16,17

Pomegranate fruits are divided into anatomical origins: peel (husk), arils, and seeds, and each has valuable compounds. The juice, which can be obtained from arils or from whole fruit, is also an important product. The edible part as well as seeds contain biologically active compounds like flavonoids and phenolics, principally anthocyanins capable of suppressing the effect of ROS on the body.18 The peel weighs 50% of the total fruit weight and it is considered an important source of bioactive compounds such as flavonoids, phenolics, highly polymerized compounds as lignins, melanins, tannins, and proanthocyanidin compounds, complex polysaccharides, and minerals.16 It was shown in previous studies that the peel extracts are more effective than juice and seed extracts as a potential source of natural antioxidants.19,20 The peel extract has antioxidant ability significantly higher than other known natural antioxidant plants such as green tea, and this is the main reason peel extract was used in this study.

To improve the therapeutic performance of the peel extract, niosomes can be used to protect them from the biological environment. Niosomes became popular and practical in the field of topical drug delivery because of its unique characteristics.21–23 Its bilayer is formed by mixing non-ionic surfactants at variable combinations and molar ratios and may have cholesterol added.24–26 Niosome is spherical in shape and consists of microscopic lamellar structures. Niosomes can deliver insoluble agents into vesicular bilayer membranes and hydrophilic agents in aqueous compartments, to the target region with lower doses of the drug to reduce side effects, increase the stability of the drug, enhance the therapeutic effects, prolong the blood circulation time in the body, and enhance the absorption of the drugs into the target area. The toxicity of the drug is reduced due to the decreased absorption of drug by nonspecific tissues.22,27–32

In this study, we extracted punicalagin from pomegranate peel and then encapsulated it in niosomes to treat the HFB4 cell line as an anti-aging agent. We evaluated the antioxidant and anti-aging effects of punicalagin coated in niosomes by studying cell cycle arrest and different skin aging genes (MMP3, Col1A1, Timp3, and TERT).

Materials and Methods

Materials

The fruits of pomegranate were purchased from a local market, Giza. Methanol, ethanol, and Tween 80 were supplied from Sigma-Aldrich Co. (Schnelldorf, Germany). Cell line HFB4 (Human fibroblast cells) was provided by the Tissue Culture Department, Vaccines & Sera (VACSERA), Egypt, and fetal bovine serum (FBS) was purchased from Invitrogen Corp. (Carlsbad, CA, USA). Penicillin-streptomycin, trypsin, EDTA, and Hank’s buffer were purchased from Gibco, New York, NY, USA. Dimethyl sulfoxide (DMSO) was obtained from Merck (Darmstadt, Germany). Propidium Iodide Flow Cytometry Kit for cell cycle analysis was from Abcam, Cairo, Egypt. Qiagen® RNA extraction kit, BioRad SYBR® green PCR MMX kit, and TERT (Human) ELISA kit were from BioVision, Inc., Milpitas CA, USA. A punicalagin reference standard was obtained from Aktin Chemicals Inc., Chengdu, China.

Methods

Preparation of Methanol Extract from Peels of Pomegranate

The fruits were washed and peeled, and then the peels were dried in a solar oven with temperature ranging from 27°C to 30°C (according to climate temperature) for nearly 10 days. The dried peels were then collected and ground in a grinding mixer until the powder form was obtained.

Powdered peels were macerated with 20% methanol for 7 days in a dark place to avoid degradation by the effect of light. After the soaking process was completed, the thick mixture was squeezed by hand and filtered through a muslin cloth to obtain a dark brown aqueous extract. The extract was then centrifuged, and the supernatant contained the extract.33

Isolation of Punicalagin from the Extract with HPLC

An HPLC system was used to isolate punicalagin and consisted of an HPLC pump, an automatic sampler cooled to 4°C (Thermo Finnigan, San Jose, CA, USA), and a PDA detector (scanning 200 to 700 nm). The HPLC column was C18, 5 µm, 4.6×250 mm (Thermo Finnigan). For the chromatographic conditions, ChemStation software (Agilent Technologies, Inc., Santa Clara, CA, USA) was used. The mobile phase was solvent A (2% acetic acid in distilled water) and solvent B (0.5% acetic acid in 50% acetonitrile in water). The flow rate was 1 mL/min. Samples (50 µL) were injected onto the HPLC column (25°C).34 The chromatogram peak regions were quantified at 260 nm using a calibration curve obtained with a punicalagin reference standard.

Determination of Total Polyphenolics

The phenolic compounds were complexed with Folin-Ciocalteau reagent, and the intensity of the blue color formed was measured using gallic acid as a standard.35 Total phenolics concentration was calculated. The absorbance of the resultant blue color was read at 765 nm using a UV-visible spectrophotometer (UVD-2960 LABOMED Inc., Los Angeles, CA, USA). For each concentration, three replicates were carried out and the average was calculated.

Determination of Total Flavonoids

The flavonoids were combined with aluminum chloride and the produced color intensity was measured. The concentration of flavonoids was calculated as quercetin equivalent (QE) with reference to a pre-established standard calibration curve. The content was incubated for 30 min at room temperature and the absorbance was measured at 415 nm. Total content of flavonoids in the extracts was calculated as QE.

Determination of Antioxidant Activity (DPPH Method)

The extract stock solution was diluted by methanol to concentrations of 250, 125, 50, 25, 10, and 5 µg/mL. One mL of 0.3 mM DPPH was added to 2.5 mL of the extracts or standard, and the mixture was incubated at room temperature in the dark for 30 min. Absorbance was measured at 518 nm and converted to antioxidant activity percentage (AA %).36,37

Preparation of Niosomes

Using a thin film hydration method, the mixture of 10 μL Tween 80, 3 mg cholesterol and punicalagin was dissolved in ethanol in a round flask. Ethanol was evaporated using a rotary evaporator at 50°C, and a thin solid mixture appeared on the flask wall.38 To form multilamellar niosomes, that layer was rehydrated with 10 mL phosphate buffered saline (PBS, pH 7.4) with bath sonication.39

Niosomes Entrapment Efficiency

The capacity of niosomes to entrap punicalagin extract was determined by the centrifugation method.40 Briefly, samples were centrifuged at 12,000 rpm (VS-18000 M, Korea, power 220 V/50 Hz) for 30 min, to separate the free drug (supernatant) from the encapsulated one (pellet). The clear supernatant was then collected and vortexed to obtain a homogeneous solution. The absorbance of punicalagin was measured at different concentrations using a UV spectrophotometer (JENWAY 6405, UK) at 255 nm (the resonance absorption of punicalagin). The calibration curve of punicalagin was made by plotting the absorbance against the concentration. The absorbance of free punicalagin in the supernatant was determined at 255 nm; then, its concentration was calculated from the calibration curve made for punicalagin and the encapsulation efficiency for noisome was calculated from the following equation:

Study of Niosome Morphology

The morphology of free and punicalagin loaded niosomes was examined using a transmission electron microscope (TEM) (JEOL JEM.1230, Shizuoka, Japan.)

Release Test for Free Punicalagin and Punicalagin Entrapped into Niosomes

Two dialysis sacs were washed and soaked in distilled water. Each sample (free punicalagin and punicalagin entrapped in niosomes) was pipetted into a sac and the sac was sealed. Then, the sacs were placed each in a 250 mL beaker containing 200 mL PBS with constant shaking at 37°C. At different time intervals, the buffer was analysed for punicalagin content at 255 nm using a UV spectrophotometer.

In vitro Cell Culture Study

Preparation of Cell Culture

HFB4 (human dermal fibroblast) cell lines were preserved as a culture of monolayer in Dulbecco′s Modified Eagle′s Medium (DMEM) containing 10FBS, L-glutamate, sodium pyruvate, and 1% penicillin-streptomycin and then incubated at 37°C. DMEM medium was added to cells in a 15 mL tube and then centrifuged to get rid of any freezing medium. The pellet was resuspended in DMEM, then incubated at 37°C in a T-75 flask for 2 days. When the cells reached at least 80% confluence, they were washed twice with PBS and were detached, then the HFB4 cells were collected and counted. The cell line used is human dermal fibroblast commercially available.41 Melanocytes can be generated directly from fibroblasts.42

MTT (Cytotoxicity) Assay

The MTT test was used to assess the cell viability. The test depends on the reduction of MTT. The cells (cell count was 1×106) were suspended in 10 mL DMEM + 10% FBS and seeded in 96-well plates at 8–10 ×103/well for 24 hrs. Then, treatment with two-fold dilution was applied on punicalagin and H2O2 to determine the concentration for each. The plates were incubated for 24 hrs at 37°C and then filtered from the medium and 10 μL MTT was added to each well. After incubation for 4 hrs, the supernatants were removed, and the absorbance was analyzed with an ELISA reader (Exert 96, Asys Hitch, Ec, Neudorf, Austria) at 570 nm.

Effect of H2O2 and Punicalagin on HFB4

Subculture of HFB4 cells was placed into 3 T-75 flasks and in a 6-well plate, and all were incubated at 37°C until the cells attained at least 80% confluence. The cells then were distributed into three groups in T-75 flasks: untreated cells (CC), cells treated with concentration (0.11 mM) of H2O2 (H2O2/HFB4), and cells treated with punicalagin (0.27 Mm) in addition to hydrogen peroxide (0.11 mM) (s + H2O2). Another two groups of cells were placed in 6-well plates: untreated cells + exposing to UV (cR), and cells treated with concentration (0.27Mm) of punicalagin + exposing to UV (s.R), then the two 6-well plates were exposed to UV (365 nm for 15 min at height 12.5 cm2). After 24 hrs incubation at 37°C, cells were taken from the 6-well plates and put into 5 tubes (15 mL), then centrifuged to get rid of any remaining medium. The pellet was resuspended and washed twice with PBS, then dispersed in 0.25% trypsin/EDTA in Hank’s buffer. Then, the HFB4 cell lines were collected in five tubes to be ready for the next tests.

Cell Cycle Analysis (DNA Content)

Cell cycle analysis was performed using a Propidium Iodide Flow Cytometry Kit from Abcam®. The cells were prepared and stored at 4°C until use, then they were transferred to the bench-top to equilibrate to room temperature, and then resuspended by gentle inversion. The cell pellet was resuspended in 200 μL 1X propidium iodide + RNase staining solution, then incubated for 20 min in the dark at 37°C. The tube was placed in ice (in the dark) and the cells resuspended by gentle inversion. Then, the samples were analyzed using flow cytometry. To exclude debris and cell aggregates, FSC vs SSC gates were set, and the propidium iodide fluorescence was collected in FL2.

RT-PCR Assay

RT-PCR was performed to detect the fold change in the expression of the MMP3, CoL1A1, and Timp3 genes with the aid of beta actin as housekeeping gene. mRNA isolation was performed using RNeasy® extraction kit. Cells were torn and homogenized in Buffer RLT. Then, ethanol was added to the mixture, creating conditions that enhance selective binding of RNA to the RNeasy membrane. The sample was then added to the RNeasy Mini spin column. Total RNA was bound to the membrane, contaminants were efficiently isolated, and high-quality RNA was filtered in RNase-free water. Ligation, washing, and rinsing steps were carried out by centrifugation in a micro-centrifuge. Then, the Master Mix was prepared, and the amplification was performed by incubating the reaction mix in the RT-PCR device.

Telomerase Reverse Transcriptase (TERT) Enzyme Assay

The reagents, samples, and standards were prepared from TERT (Human) ELISA kit from BioVision. The clean plate was washed 2 times using 1X wash solution, 100 μL of each sample and standard was added, and the wells were covered for 1.5 hrs incubation at 37°C. The plate content was discarded without washing or letting the wells completely dry. Biotin-detection antibody work solution (0.1 mL) was added into the wells, then the plate was sealed and left for 60 min for incubation. The solution was discarded, and the plate washed 3 times by filling the wells with the buffer solution and soaked for 1–2 min, decanted, and clapped by absorbent filter papers. SABC working solution (0.1 mL) was added to the wells and then the plate was incubated at 37°C for 30 min, then the solution discarded and washed 5 times. TMB substrate (90 µL) was added, and the plate was incubated in a dark place for 15–30 min at 37°C. At the end, 50 μL of stop solution was added and the results were read at 450 nm within 20 min.

Statistical Analysis

Data were examined using SPSS v. 15.0. Significant variations between groups were estimated using one-way ANOVA analysis. p ≤ 0.05 was considered significant.

Results

Isolation of Punicalagin with HPLC

Punicalagin was isolated with HPLC by comparing the pomegranate methanol extract with a punicalagin reference standard (Figure 1A and B).

Figure 1 HPLC chromatograms. (A) Punicalagin standard. (B) Pomegranate total extract. It has mainly α-punicalagin (1.04 min) and β-punicalagin (3.49 min).

Two peaks appeared, representing α-punicalagin and β-punicalagin, and they were both isolated. A calibration curve was made and the concentration of the isolated punicalagin was determined as 7.34 mg/g (Figure 2).

Figure 2 Calibration curve for beta punicalagin.

Total Polyphenolics

The total polyphenolics were determined by using a standard curve between different standard concentrations of gallic acid and the absorbance at 765 nm. Total polyphenolics were 138 mg/g.

Total Flavonoids

The total flavonoids were determined by using a standard curve between different concentrations of quercetin. Total flavonoids were 58 mg/g.

Antioxidant Activity

The scavenging value of DPPH was 73.33% and was closely related to the total polyphenol contents. It can be assumed that these compounds have significant oxidation capacity. This result is compatible with the previous studies43,44 as the total phenolic content in punicalagin reflects high antioxidant activity.

Niosomes Entrapment Efficiency

Niosomes exhibit a high efficiency for loading punicalagin extract (92±3%).

Study of the Morphology Using TEM

TEM examination displayed the morphology of the niosomes and the difference in size between the free ones (135–200 nm) and those loaded with punicalagin (185–335 nm) (Figure 3). Also, it showed that the sizes are in the nano-range.

Figure 3 Transmission electron microscope (TEM) images of (A) Free niosomes, (B) Niosomes entrapping punicalagin.

Release Test for Free Punicalagin and Niosomes Loaded with Punicalagin

The entrapment of punicalagin in the niosomes caused a decrease in the release rate of the punicalagin from the sac to the medium (Figure 4).

Figure 4 The release of free punicalagin and niosomes loaded with punicalagin.

MTT (Cytotoxicity) Assay

Cytotoxicity assay showed the safe doses to be used on the HFB4 cells. For H2O2 the concentration was determined between dilution factor 4096 and 8192, while the punicalagin was at dilution factor 128 (Figure 5).

Figure 5 Effect of different concentrations of punicalagin and H2O2 on the percentage of cell viability.

Cell Growth Rate

The cell percentage arrested in the G2/M phase increased in the case of cR and H2O2/HFB4 samples; however, the percentage of cells arrested in the G0/G1 phase diminished in the case of H2O2/HFB4 sample. The samples (s + H2O2 and s.R) that were pretreated with punicalagin showed an effect that opposed the effect resulting from H2O2 and UV radiation (Figure 6).

Figure 6 The variation of % arrest for cell cycles G0/G1, S, G2/M, and Pre-G1. CC is the control HFB4 cell lines, cR is cells exposed to UV (365 nm for 15 min at height 12.5 cm2), s.R is cells treated with punicalagin in addition to UV exposure, H2O2/HFB4 is cells treated with H2O2, and s + H2O2 is cells treated with punicalagin in addition to H2O2. The error bars represent standard deviation.

RT-PCR Assay

MMP3 expression increased in cR and H2O2/HFB4 samples, and the expression of Col1A1 and Timp3 decreased in those two samples. The s + H2O2 and s.R samples showed an effect that opposed that resulted from H2O2 and UV radiation (Figure 7).

Figure 7 Fold change in the expression of different genes on the samples. CC is the control HFB4 cell lines, cR is cells exposed to UV (365 nm for 15 min at height 12.5 cm2), s.R is cells treated with punicalagin in addition to UV exposure, H2O2/HFB4 is cells treated with H2O2, and s + H2O2 is cells treated with punicalagin in addition to H2O2. The error bars represent standard deviation.

TERT Assay (Telomerase)

Telomere shortening occurred significantly in both H2O2/HFB4 and cR samples by 64.20% and 14.22%, respectively, while punicalagin treated samples showed telomere shortening in s.R and s + H2O2 samples by only 31.19% and 6.5%, respectively, and cells were protected from telomere shortening-mediated by oxidative stress via H2O2 or UV exposure (Figure 8).

Figure 8 Quantitative measurement of human TERT in the samples. CC is the control HFB4 cell lines, cR is cells exposed to UV (365 nm for 15 min at height 12.5 cm2), s.R is cells treated with punicalagin in addition to UV exposure, H2O2/HFB4 is cells treated with H2O2, and s + H2O2 is the cells treated with punicalagin in addition to H2O2. The error bars represent standard deviation.

Discussion

The use of natural bioactive compounds and essential oils in skin aging and skin diseases are well documented in the literatures.45,46 Furthermore, essential oil and their constituents were utilized as a skin penetration enhancer for transdermal delivery.47 Our current report examined the effect of punicalagin as a strong antioxidant against skin aging. Total polyphenolics and flavonoids were 138 mg/g and 58 mg/g, respectively. Furthermore, the scavenging activity percentage showed high activity (73.33%), proving the high antioxidant activity of pomegranate husk extract.18 Those active compounds work to protect the skin from absorbing UV radiation, inhibiting UV-induced free radical reactions, or modifying endogenous antioxidant and inflammatory systems.11

TEM analysis showed that the ultra-structure of the arranged niosomes and the niosomes loaded with punicalagin were non-aggregated circular-shaped particles. The morphology demonstrated the homogeneity of the molecule, and the niosomes appeared spherical. The sizes ranged from 180 nm to 220 nm for the free niosomes, which is smaller than those loaded with niosomes (218–437 nm). This shows that the sizes were within a reasonably large unilamellar vesicle size range.25 The electrostatic charge between the comparable charged particles prevents accumulation and so guarantees a scattered state of the nanosuspension in tests.

Release tests showed a decrease in the absorbance of punicalagin because the niosomes dragged the release of punicalagin slightly in the medium compared to its free state. The target of a long-time sustained drug release inside the body was achieved here.

The cytotoxicity (MTT) assay was done to ensure that a safe dose was used and that the percentage of cell viability was more than 90%.

The cR sample showed higher number of cells in phase of G2/M, while the s.R sample showed a low number of cells in phase of G2/M. As for the H2O2/HFB4 sample showed a higher value of cells in the phase of G2/M and a low number of cells in the G0/G1 phase, while the s + H2O2 sample showed a lower number of HFB4 cells in phase of G2/M and a high number of cells in G0/G1 phase. This proved the protection ability of punicalagin to cells versus the effects of UV radiation and H2O2.48

RT-PCR assay was carried out to determine the level of expression of 3 genes that are responsible for skin-aging process and considered as markers for aging. The MMPs degrade collagen in the dermal layer and proteins in the extracellular matrix, so the upregulation of MMP3 accompanied by the decrease in Col1A1 and Timp3 compared to the control was shown in the cR and H2O2/HFB4 samples. In s + H2O2 and s.R samples, there was an increase in both Col1A1 and Timp3 and a decrease in MMP3 compared to the other samples.

In the TERT assay it was observed that H2O2 and UV-irradiation affected telomerase activity and promoted telomerase shortening, which leads to cell senescence and skin aging, but the samples pretreated with punicalagin showed an increase in the concentration of human TERT.

Because of complexity of skin aging, future studies are required to use human skin and in vivo swine skin model of UV-mediated skin damage and aging to define the efficacy of punicalagin niosomes in skin aging.

Conclusion

There are many factors that increase the rate of skin aging by changing the balance between free radicals and antioxidants in the body. The solution to counteracting negative changes is to increase antioxidants. This study demonstrated the high antioxidant effect of punicalagin extracted from pomegranate and its loading into niosomes as a carrier. Punicalagin protects HFB4 cells from cell growth arrest caused by UV radiation and H2O2, activates production of the Col1A1 and Timp3 genes, maintains collagen level, and reduces MMP3. Punicalagin increases human TERT concentration. We conclude that punicalagin treatment efficiently protects cells from aging caused by oxidative stress.

Abbreviations

ROS, reactive oxygen species; HFB4, human skin fibroblast cell line; UV, ultraviolet rays; COL, collagens; MMPs, matrix metalloproteinases; TIMPs, metalloproteinase; TERT, telomerase reverse transcriptase; FBS, fetal bovine serum; DMSO, dimethyl sulfoxide; QE, quercetin equivalent; DPPH, determination of antioxidant activity; AA%, antioxidant activity percentage; DMEM, Dulbecco′s Modified Eagle′s Medium; CC, untreated cells; H2O2/HFB4, cells treated with concentration (0.11 mM) of H2O2; s + H2O2, cells treated with punicalagin (0.27 Mm) in addition to hydrogen peroxide (0.11 mM); cR, untreated cells + exposing to UV; s.R, cells treated with concentration (0.27Mm) of punicalagin+ exposed to UV.

Compliance with Ethical Standards

The study was conducted in accordance with the international guidelines.

Disclosure

The authors declare that they have no conflicts of interest for this work.

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