1. INTRODUCTION:

Sunlight's UV consist of 95% UV A and 5% UV B in which UV B has a lower wavelength range of 280–320 nm, while UVA has wavelengths in the range of 320–400nm. With the different wavelengths, UV A is able to reach deeper into the dermis layer and UV B can only reach in the epidermis layer. The production of ROS is initiated by UV A radiation, and these ROS might lead to the process of photoaging¹and skin cancer²_. Consequently, it is believed that substances possessing UV photoprotection are crucial for combating photoaging and deseases such as scin cancer. The majority of such substances are found in nature.

One such naturally occurring molecule is curcumin which is yielded by Turmeric plant (Curcuma longa L.). Numerous biological effects of curcumin have been documented, including hepatoprotective, anti-inflammatory, anticancer, and antioxidant properties^3-7. In addition, several compounds known as curcumin analogs have been created. The following are a few examples of curcumin analogs: Tetrahydropentagamavunon-0 (THPGV-0), Tetrahydropentagamavunon-5 (THPGV-5), Hexagamavunon-5 (HGV-5), Pentagamavunon-6 (PGV-6), and Tetrahydrohexagamavunon-0 (THHGV-5)^3,5-8_. According to the previous biological activity in vitro test, HGV-5, THPGV-5, THHGV-5, and THPGV-0 have more antioxidant activity than vitamin E_. It is also known that THPGV-0 and THPGV-1 have bactericidal action⁹_. Some of the curcumin analogs have also been shown to have anti-inflammatory, anti-tuberculosis, anti-cancer, and antibacterial properties^10–19. In regard to increase the biological activities, some dosage forms of curcumin and its analog have been developed for example in form of nanoparticle, microparticles, liposome^20-22. Nevertheless, the development of the curcumin analog as a UV A and UV B radiation protector agent has received little study attention. The chemical structures of curcumin and a few of its analogs are depicted in Figure 1.

Fig. 1: Chemical structures of curcumin and its several analogs

A: Curcumin, B: PGV-5, C: THPGV-5, D: HGV-5, E: THHGV-5.

PGV-5 and HGV-5, two curcumin analogs, are recognized to possess the highest level of antioxidant activity within their respective chemical series, cyclopentanone and cyclohexanone. The enhanced ROS brought on by UV A radiation-induced skin cell damage might be prevented by this antioxidant activity. Furthermore, THPGV-5 and THHGV-5, two of their derivative chemicals, are being developed as antiaging cosmetic products^3,23–24_. Several important factors may be used to explain the link between THPGV-5's (Tetrahydropentagamavunon-5) structure and antioxidant activity, notably in terms of trapping reactive oxygen species (ROS). Based on its structure, each THPGV-5 and THHGV-5 are curcumin analogs that have been tweaked to improve their antioxidant capabilities. To increase the molecule's capacity to neutralize free radicals, functional groups are usually added or substituted. Therefore, it is essential that THPGV-5/THHGV-5 has hydroxyl groups. By giving hydrogen atoms to ROS, these groups can change them into molecules that are less reactive and more stable. One of the main ways that antioxidants counteract free radicals is by this hydrogen transfer. Effective electron delocalization is made possible by the structure of THPGV-5/THHGV-5. This indicates that the radical state of THPGV-5/THHGV-5 that results from THPGV-5/THHGV-5 donating an electron to neutralize a ROS is sustained by the delocalization of electrons throughout the molecule. Sustaining antioxidant activity requires this stability. THPGV-5/THHGV-5's structural changes are intended to make it more stable in physiological settings. This guarantees that the substance continues to be active and efficient in absorbing ROS for an extended amount of time. Together, these structural characteristics increase THPGV-5's and THHGV-5’s antioxidant activity, which makes them more potent than its unaltered equivalents in neutralizing ROS that is produced by UVA radiation. Accordingly, THPGV-5 and THHGV-5 can shield cells from harm and lower oxidative stress. THPGV-5 and THHGV-5 have the potential to lessen UVA-induced skin damage since they are meant to be an active element in antiaging cosmetics. These compounds do this by scavenging ROS produced by UVA. However, the assessment of these four compounds as antiaging was limited to their Sun Protection Factor (SPF) value, which alone measures their protection against UVB rays. This indicates that there has not yet been any testing done on curcumin analog as a UV A radiation photoprotectant. This study is therefore to investigate the potential of the curcumin analogs (PGV-5, HGV-5, THPGV-5, and THHGV-5) as antiphotoaging and UB protective possibilities, with a focus on their impact on the intracellular ROS buildup and proliferation of human dermal fibroblasts caused by UV A.

2. MATERIALS AND METHODS:

2.1 Materials:

Curcumin analogs (PGV-5, HGV-5, THPGV-5, and THHGV-5) were collected from the Curcumin Research Center (CRC), Faculty of Pharmacy, Gadjah Mada University. Curcumin with 81% purity (Sigma-Aldrich, USA) and dimethyl sulfoxide (DMSO; Merck KGaA, Germany) were obtained from Integrated Research and Testing Laboratory, Gadjah Mada University (LPPT UGM). Other chemicals used in this study: Dulbecco’s modified Eagle medium (DMEM; Sigma-Aldrich, USA), fetal bovine serum (FBS; Gibco, USA), 2 mmol/L glutamine (Gibco, USA), sodium bicarbonate (NaHCO₃₎, hydrogen chloride (HCl), sodium hydroxide (NaOH), fungizone, phosphate-buffered saline (PBS), penicillin-streptomycin antibiotic, L-glutamine, trypsin-EDTA, MTT, sodium dodecyl sulfate (SDS) 10% in 0,01 N HCl, trypan blue, and dichlorodihydrofluorescein diacetate (H₂DCFDA).

2.2 Curcumin analogs preparation:

Curcumin analogs were dissolved in DMSO as a 10mM stock solution. These solutions were then diluted in complete DMEM to reach desired five concentrations (1, 2, 4, 8, and 16µM). Curcumin was also used as a control in this study which dissolved in DMSO.

2.3 Preparation of Cell culture:

Human dermal fibroblast (HDF) is a primary culture cell isolated from the human foreskin, obtained from the Department of Dermatology and Venereology, Fakultas Kedokteran, Kesehatan Masyarakat, dan Keperawatan (FK-KMK), Universitas Gadjah Mada (UGM). The use of primary HDFs in this study has received ethical approval from the Medical and Health Research Ethics Committee (MHREC), FK-KMK UGM with the number KE/FK/0494/EC/2023. Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS, 100 IU/mL penicillin, 100 μg/mL streptomycin, and 2 mmol/L glutamine were used as medium for subculturing HDFs in the flasks. The cells were incubated in a 5% CO₂ incubator at 37℃ and subcultured every 80% confluence. Then, cells were detached with trypsin-EDTA following cell counting with trypan blue. Cells were seeded at several 1,4×10⁵ cells/well in 6-well plates and 1×10⁴ cells/well in 96-well plates. Cells were incubated for a minimal 24 hours²⁵.

2.4 Optimization of UVA irradiation doses:

The culture medium in 96-well plates containing cells was replaced with a thin layer of PBS before UV A irradiation. For optimization of UV A irradiation, the irradiation was performed using a UV A lamp at three doses that were 1 J/cm² (11 min), 2 J/cm² (22 min), and 3 J/cm² (33 min). The UV A exposure doses can be converted into exposure duration with a radiation distance of 45 cm. Cell viability the each doses was measured using MTT assay to determine the lethal dose of ±50% HDFs death.

2.5 Pretreatment of culture cells with curcumin analogs:

The 1×104 cells/well in the 96-well plates were pretreated with concentration series of curcumin analogs (0, 1, 2, 4, 8, and 16µM) and followed by incubation for 2 h. This pretreatment duration refers to a similar study by Liu et al. (2018) using curcumin as sample²⁶_. After the pretreatment, the cells were washed with PBS twice and followed by UV A irradiation at the observed lethal dose. Whilst, in the other 96-well plate was not irradiated with UVA and used for toxicity evaluation. The final solution in the plates was replaced with complete DMEM and followed by MTT procedures²⁷.

2.6 MTT assay procedure:

The cells were washed with PBS and replaced with 100 µL complete DMEM. Cells were incubated for 1h following the MTT procedure. Ten mL MTT reagents 5 mg/mL in PBS were added to the cells. Incubation was performed for 4h. Then, cells were added with SDS 10% in 0.01 N HCl for 100µL to dissolve the formazan crystals. Absorbance was measured at λ595nm²⁸.

2.7 Intracellular ROS analysis using Flow cytometry:

The cells in 6-well plates were pretreated with 1μM curcumin analogs and followed by 2h incubation for intracellular ROS analysis. Pretreatment with curcumin, vitamin E and ascorbic acid at concentration of 1μM was also done as control for comparison of the protective activity. Next, the cells were exposed to UVA radiation at a dose of 3 J/cm² for 33 minutes. Then, cells were harvested to round bottom tubes by trypsinization and then centrifuged at 200xg. The pallet was added with 50μL H₂DCFDA 20μM following incubation for 30 min. About 300μL supplemented buffers containing 10% FBS in PBS were added to the cell suspension. Flow cytometry was performed at λ535nm and λ485nm, respectively, as emission and excitation wavelengths^29-30.

2.8 Molecular docking analysis:

A molecular docking analysis was conducted to evaluate the potential activity of curcumin and its analogs as Keap1 inhibitors. The docking process was performed using the CB-Dock2 platform (cadd.labshare.cn/cb-dock2), a web-based tool designed to predict the active binding sites of target molecules and calculate ligand-protein interaction affinities. The Keap1 with the PDB ID: 1U6D as the target protein in this study was retrieved from the Protein Data Bank (www.rcsb.org). Curcumin and its analogs were modeled to interact with Keap1, and the Gibbs free energy (ΔG) was calculated to assess the stability of the complexes formed. Lower ΔG values indicate stronger interactions between the compounds and the target protein³¹.

2.9 Data analysis:

The percentage of cell viability in the MTT assay test was calculated using Equation 1. The data is presented as a graph of the percentage of cell viability versus curcumin analogs concentration in the treatment with and without exposure to UVA radiation. Then, the intracellular ROS levels were analyzed based on the mean value of the fluorescence intensity fold to control (FITC) which was calculated using Equation 2.

Absorbace of treatment – Absorbance of media control

Cell viability = ------------------------------------------------------ x100 (1)

Absorbance of cell control – Absobance of media control

Mean fluorescence treatment

Mean fluorescence fold to control (FITC) = -------------------------- (2)

Mean fluorescence control cells

Data were processed using the Shapiro-Wilk Test and Levene Test to ensure data distribution and homogeneity, respectively. This analysis is useful for ensuring the validity of the test where the data must be homogeneous and normally distributed (significance value> 0.05). Furthermore, statistical analysis was continued using the one-way ANOVA test with a Post Hoc LSD test. This statistical test was operated using a 95% confidence level with an acceptance parameter in the form of a significance value of less than 0.05, indicating a significant difference among treatment groups. All statistical operations were obtained using the SPSS version 22 program.

3. RESULT AND DISCUSSION:

3.1 UV A irradiation doses optimization:

The HDF cells were exposed to UV A radiation at 1 J/cm2, 2 J/cm2, and 3 J/cm2, respectively and cell viability was measured using MTT assay. The results demonstrated that the cell viability significantly decreased in each successive UV A radiation exposure when compared to the control group (100%, without irradiation) (Figure 2).

Figure 2: Cell viability of HDFs irradiated with UVA at 0, 1, 2 and 3 J/cm²

Cell viability data were displayed in the form of the average percent of cell viability ±standard error. The same asterisk (*) indicates no significantly decrease in cell viability with a 95% confidence level (p<0.05).

As shown in Figure 2, the three UV A irradiation dosages (1, 2, and 3 J/cm2) were able to dropp the cell viability. The level of the cell viability was changed into approximately 87.37±0.98%, 88.56±0.94%, and 88.00±2.10%, respectively. This result is in consistent with research by Chang et al. (2021) and Lin et al. (2022)^32-33, which found that the HDF viability was only maintained at 80% by a single dose of 3 J/cm² UV A irradiation. However, in this study, the 1–3 J/cm² range of doses result in the cell viability in the range 87 – 88%. The UV A dosages have an effect on maintaining the HDF viability at about 88%. The morphology of HDFs alters following UV A irradiation, as seen in Figure 3. After three J/cm² of UV A exposure, adherence HDFs in the shape of a longated spindle can be separated to form round-shaped cells.

Figure 3: HDF morphology after UVA Irradiation at various doses. Following UV A exposure, the number of HDFs dropped into about 80% viability.

The findings demonstrated that HDFs could not be killed by up to 50% by a single UV A radiation dosage in the 1–3 J/cm² range. However, a 12% difference from the control group was observed in the reduction of cell viability caused by the maximum single dosage of UV A irradiation, 3 J/cm². This dose was then used to examine the cytoprotective effect of the curcumin analogs in the subsequent study. Therefore, to examine the photoprotective properties of curcumin analogs, a UV A irradiation dose of 3 J/cm² will be employed.

3.2 Determination of the concentration range of toxicity of curcumin analogs using the MTT assay:

The purpose of this test was to determine how curcumin analogs affect to HDF viability. Five concentration levels were employed: 1, 2, 4, 8, and 16 µM. Figure 4 illustrates the impacts of the curcumin analogs PGV-5, HGV-5, THPGV-5, and THHGV-5 on the viability of HDFs (without UV A irradiation dose of 3 J/cm2). When curcumin analogs, spesifically PGV-5, HGV-5, THPGV-5, and THHGV-5 were administered to HDFs at concentrations ranging from 1 to 4 µM, no harmful effect was detected.

Figure 4: Cell viability of HDFs following treatment with curcumin analog at the concentration range of 1, 2, 4, 8, and 16 µM (without UV A irradiation). The percentage of viable HDFs varies following treatment with curcumin analogs at these concentrations. The same asterisk (*) indicates no significant different in the cell viability among groups with a 95% confidence level (p<0.05).

The percentage of viable HDFs was demonstrated to not drop noticeably following treatment with curcumin analogs at these concentrations. HGV-5 and THHGV-5 appeared to have the ability to boost HDF proliferation by raising the percentage of cell viability at higher concentrations—8 and 16µM. On HDFs, however, 8µM and 16µM of PGV-5 and THPGV-5 started to exhibit a cytotoxic impact. The PGV-5 treatment at these doses did not result in a statistically significant difference in viability values when compared to the control group, despite there was a drop in HDF viability. When compared to DEQA (3,5-dicafeoyl-epi-qinic acid), another polyphenolic molecule in another study, the molecule shows cytotoxic action to cell lines. At a concentration of 25µM, the DEQA preserved the viability of HDFs at about 80%³⁴. The result shows that DEQA has strong antioxidant activity, particularly in the area of radical scavenging³⁵.

The results of testing many curcumin analogs at high doses and their cytotoxic effects reveal a different phenomenon than antioxidant activity. When curcumin analogs, particularly PGV-5 and THPGV-5, are supplied at high concentrations (>16 µM), it explains why prooxidant activity is present. Prooxidants are substances that might cause oxidative stress by suppressing the antioxidant system or generating reactive oxygen species (ROS). It is recognized that a number of antioxidant substances, including polyphenols, vitamin E, and ascorbic acid, exhibit prooxidant action. High concentrations or the presence of transition metals are the primary conditions for the prooxidant activity to occur³⁶. At a dosage of 5 µM, curcumin was shown to have a prooxidant action that decreased the glutathione (GSH) level in rat hepatocytes by up to 40%. The structure-activity connection suggests that this is caused by the unsaturated α and β-ketone groups, which can react through Michael addition with GSH and protein thiols³⁷.

The cytotoxicity of PGV-5 on HDFs appears to be influenced by Michael's addition and may occur at concentrations higher than 16 µM. PGV-5 possesses an unsaturated α,β-ketone group that can react with GSH and function as a Michael acceptor (Figure 5).

Figure 5: Thiol-Michael’s addition reaction of PGV-5 with GSH might occur at concentrations higher than 16 µM. The final product can cause cytotoxicity effect on cells.

The level of the naturally occurring antioxidant GSH will drop as a result of this interaction. The cell eventually experiences oxidative death as a result of ROS's diminished ability to neutralize them. In HDFs, HGV-5, on the other hand, does not function as a prooxidant due to its unsaturated α,β-ketone group. Its lack of a prooxidant impact is most likely caused by cyclohexanone's steric hindrance, which makes addition processes difficult to happen. Subsequently, like its parent chemical PGV-5, THPGV-5 has a prooxidant action despite lacking an unsaturated α,β-ketone group. This discovery is explained by autoxidation in the THPGV-5 compound's side chain, which prolongs the conjugated double bond by generating a radical molecule. This process is analogous to the unsaturated carbon chain of vitamin E³⁸. In addition to producing ROS-induced oxidative death, this process will decrease the body's own antioxidants.

3.3 Determination of cytoprotective of curcumin analogs using the MTT assay:

The range concentration of curcumin analog in protecting HDF from UV A-induced oxidative stress was determined by cytoprotective experiments using the MTT assay. The UV A irradiation dose was 3 J/cm2 UV A irradiation and five concentration levels of curcumin analogs were also employed: 1, 2, 4, 8, and 16µM.

The results (Figure 6) show that pretreatment of PGV-5 and THPGV-5 in the 1µM–16µM concentration range was able to keep HDF viability over 90% following of UV A irradiation 3 J/cm². With 1 µM PGV-5 dose, HDF viability was 97.74±3.47%. Additionally, PGV-5 produced cell viability values of 101.76±2.62% and 99.58±2.33% at concentrations of 2µM and 4µM, respectively. The normal level of cell viability of 101.68 ±3.05% and 103.10±2.27% was maintained by PGV-5 compounds at concentrations of 8µM and 16 µM, respectively. With THPGV-5 pretreatment, the HDFs viability was 90.62±2.27%, 93.24±1.22%, 108.27± 8.74%, 123.72±6.22%, and 104.57±6.47%, respectively, in the concentration range of 1µM, 2µM, 4µM, 8µM, and 16µM.

In the meantime, pretreatment with UV A 3 J/cm² and curcumin analogs of HGV-5 and THHGV-5 in the concentration range of 1µM, 2µM, and 4µM was able to preserve the viability of HDF cells in over 90%, more than control without a sample. The viability of HDFs was 90.94±2.58%, 90.65±3.08%, and 106.46±0.44%, respectively, at that concentration of HGV-5. However, HDF cell viability was lower than the control at 81.68± 3.91% and 76.18±3.06% at concentrations of 8 µM and 16µM.

Figure 6: Cell viability of HDFs (treated with UV A irradiation) following treatment with curcumin analog and UVA at several concentrations. The percentage of viable HDFs at high concentration dropped noticeably following treatment with curcumin analogs at concentration of higher than 8 µM. The same asterisk (*) indicates no significant different in the cell viability among groups with a 95% confidence level (p<0.05).

This phenomenon might be caused by the persistent senescence of HDF cells. A cell aged and permanently ceased dividing throughout this phase, although it did not die. Subsequently, THHGV-5 pretreatment yields a comparable outcome to that of HGV-5. The viability of HDFs was found to be 97.88±1.20%, 102.78 ± 2.91, 94.20±1.5, 84.23±6.10%, and 18.46±2.29% for the concentrations of THHGV-5. A 95% confidence interval statistical analysis revealed a significant difference (p<0.05) between all of these values and the UV A control group.

3.4 Inhibition of intracellular ROS accumulation:

Intracellular ROS was measured based on their fluorescence intensity value which was then converted into value of the fluorescence intensity fold to control (FITC).

Figure 1: Intracellular ROS of HDFs described with FITC values following treatment with curcumin analogs, vitamin E and C.

The FITC (mean ± standard error) was obtained from 3 replications. The same asterisk (*) symbolizes no significant difference of FITC (p<0.05).

As seen in Fig. 7, FITC of control group (cells without UV A irradiation) has a value of 1.00. While, in the UV A treatment group was 1.6 times higher than that of the control cell group. This finding demonstrates that intracellular ROS buildup in HDFs that can be brought on by UV A radiation. Moreover, the pretreated HDFs with curcumin and its analogs resulted in FITC value in the range of 0.7-0.8 (significant; p<0.05). In contrast, pretreatment with α-tocopherol (vitamin E) in the cells did not significantly alter intracellular ROS levels. These findings clarified that 1 µM curcumin analogs might lower the intracellular ROS levels but no that effect for vitamin E.

The inhibition of intracellular ROS generation in HDFs is thought to be an indication of curcumin analogs' antioxidant properties, particularly with regard to neutralizing radical chemicals. It is thought that curcumin analogs work by neutralizing ROS molecules that are created in cells as a result of UVA's impact on endogenous photosensitizers. The production of ROS, including superoxide, hydroxyl radicals, singlet oxygen, and hydrogen peroxide, is inhibited by this neutralization. A phenolic hydroxy group present in PGV-5, HGV-5, THPGV-5, and THHGV-5 is crucial to their ability to scavenge radicals. This is due to curcumin's additional hydroxy phenolic group, which donates H atoms to radical molecules in order to snare them³⁹. Figure 8 illustrates how curcumin, curcumin analogs, and vitamin E work to neutralize ROS. Curcumin's phenolic hydroxy group effect is correlated with its antioxidant action. Methoxy groups in position carbon number 3' of phenol in PGV-5, HGV-5, THPGV-5, and THHGV-5 can boost their antioxidant activity². An electron-withdrawing group that can contribute the lone pairs of electrons on the ortho position is a methoxy group. This state abstracts the H atoms from the phenolic hydroxy groups, which makes it easier to neutralize molecular oxygen radicals. Furthermore, it is thought that curcumin analogs' middle ring strengthens the radical resonance. In the meantime, lipid radical molecules tend to be neutralized by vitamin E because of its sufficiently long saturated carbon chain (nonpolar). Vitamin E molecules' lipophilic property is linked to poor skin penetration^40-41. According to this study, at low doses of 1 µM, vitamin E is thought to have poor penetration into HDFs. Because of this, curcumin analogs, which have a tendency to be more polar, are more effective than vitamin E at reducing the buildup of intracellular ROS in HDFs.

The inhibition of intracellular ROS formation by curcumin analogs is also thought to be mediated by the UV filter mechanism. The colorful compounds for examples PGV-5 and HGV-5 are the result of sufficiently long conjugated double bonds. PGV-5 has a maximum absorbance (λmax) of 415nm⁴². It demonstrates the high absorption of visible light by PGV-5 and maybe HGV-5. Though they do not have maximal absorption in the UV light band, it is thought that both of these compounds can function as UV filters.

4. CONCLUSION:

PGV-5, HGV-5, THPGV-5, and THHGV-5, which are curcumin analogs, shown cytoprotective effect on UVA-induced human dermal fibroblasts. After being exposed to UVA radiation of 3 J/cm², curcumin analog pretreatment can keep HDF viability over 90%, particularly in the concentration range of 1–4 μM. Further research is necessary to determine the cytoprotective analysis of curcumin analogs at concentrations lower than 1 μM. It was also demonstrated that curcumin analogs, at 1 µM, exhibited superior protective efficacy than curcumin and even vitamin E against intracellular ROS buildup in HDFs caused by a single dose of UVA radiation 3 J/cm².

5. ACKNOWLEDGEMENTS:

The authors are grateful to Curcumin Research Center (CRC), Faculty of Pharmacy, Gadjah Mada University for the sample of PGV-5, HGV-5, THPGV-5, and THHGV-5 and Department of Dermatology and Venereology FK-KMK UGM for giving their cell collection of human dermal fibroblasts (HDFs)

6. CONFLICTS OF INTEREST:

The authors confirm that this article’s content has no conflicts of interest.

7. FUNDING SOURCE:

This research was funded by Rekognisi Tugas Akhir (RTA) Programme, No.: 5722/UN1.P.III/Dit-Lit/PT.01.05/2022, 18 July 2022, from Gadjah Mada University, Yogyakarta, Indonesia.

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Received on 01.02.2024 Revised on 16.08.2024

Accepted on 15.01.2025 Published on 02.05.2025

Available online from May 07, 2025

Research J. Pharmacy and Technology. 2025;18(5):1951-1958.

DOI: 10.52711/0974-360X.2025.00279

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