Santamarine was isolated from the combination of methanolic and chloride extracts of A. scoparia. The leaves and stems of A. scoparia were air dried and ground to powder subsequently.

Powdered material (100 g) was extracted with 1 ℓ methylene chloride (CH₂Cl₂) and 3 ℓ methanol (MeOH) separately for 24 h each at room temperature. Obtained extracts were combined and dried with a rotary evaporator. Santmarine was isolated from the future separation of this extract as described earlier (Oh et al., 2021).

Characterization of santamarine was carried out via comparison of spectral data with published literature. NMR spectral data were recorded on a Bruker Avance II NMR 900 spectrometer (Billerica, MA, USA) and obtained at the Korean Basic Science Institute (Daejeon, Korea).

HaCaT immortal keratinocyte was purchased from Cell Line Service (Eppelheim, Germany). Cells were cultured in Dulbecco’s modified Eagle medium (DMEM, Gibco-BRL, Gaithersburg, MD, USA) containing 100 ㎍/㎖ penicillin-streptomycin antibiotics, 10% fetal bovine serum (FBS) (Gibco-BRL, Gaithersburg, MD, USA) and maintained at 37℃ in a humidified atmosphere of 5% carbon dioxide.

For UVB irradiation, the culture medium was removed, and the cells were washed with phosphate-buffered saline (PBS). Then the cells were exposed to UVB using a Bio-Sun UV Irradiation System (Vilber Lourmat, Marine, France) with a spectral peak of 312 ㎚. After the UVB irradiation at the range of 10 mJ/㎠ - 100 mJ/㎠, cells were maintained with DMEM without FBS for 24 h. UVB exposure at higher than 30 mJ/㎠ dose significantly decreased the cell viability, therefore intensity of UVB was selected at 20 mJ/㎠ for the current study (Fig. 1).

Fig. 1.  The effect of UVB (A) and santamarine (B) on the viability of HaCaT cells.

The viability of the cells was assessed using MTT assay. Cell viability following santamarine treatment was measured by examining the ability to form MTT-formazan crystals. Viability was quantified as a relative percentage of the untreated group. Data are shown as the means ± standard deviation (n = 3). ^* p < 0.05, ^** p < 0.01 vs. non-treated group.

The viability of cells was analyzed by MTT assay. Briefly, cells were counted and seeded in 96-well plates and incubated for 24 h. Then, the cells were treated with five different concentrations (1, 5, 10, 20 and 25 μM) of santamarine and introduced with serum-free medium. After incubation for 24 h, the cell culture supernatant was removed and replaced with 100 ㎕/well of 1 ㎎/㎖ MTT solution (AMRESCO, Solon, OH, USA), followed by incubation for 4 h. Cells were then rinsed in PBS and dimethyl sulfoxide was added to dissolve the formazan crystals.

The absorbance was read on a GENios^® microplate reader (Tecan Austria GmbH, Grödig, Austria) at 540 ㎚. The viability of cells was quantified as a relative percentage compared with the untreated control group. Each sample group was tested by three independent repeats.

The generation of intracellular ROS was determined using an oxidation-sensitive dye, 2′,7′-dichlorofluorescein diacetate (DCFH-DA). The cells were seeded in fluorescence microtiter 96-well plates and cultured for 24 h. The cells were stained with 20 μM DCFH-DA for 30 min and treated with samples for 2 h in the dark. Immediately after washing three times with PBS, 500 μM H₂O₂ was added to the wells.

The 2′,7-dichlorofluorescin (DCF) fluorescence intensity was measured at the excitation wavelength at 495 ㎚ and the emission wavelength at 630 ㎚ using a fluorometer (Tecan Group Ltd., Mannedorf, Switzerland).

UVB-induced changes in the MMP-1 production in HaCaT cells were determined by ELISA. HaCaT cells were preincubated in six-well plates, and after 24 h of treatment with or without santamarine, the culture medium was collected.

The amount of MMP-1 in the culture medium was calculated with the help of a commercial ELISA kit (R&D systems, Minneapolis, MN, USA) in accordance with the manufacturer′s protocol.

Total RNA was isolated using Trizol reagent from HaCaT cells treated with or without santamarine for 24 h following UVB irradiation (Invitrogen Co., Carlsbad, CA, USA).

Complementary DNA (cDNA) was synthesized from 2 μg RNA using cell Script All-in-One cDNA Master Mix (CellSafe, Yongin, Korea).

PCR amplification of the cDNA template was performed with PCR premix (Bioneer, Daejeon, Korea) and the following gene-specific primer pairs: forward 5'-GAT-GTG-GAG-TGC-CTG-ATG-TG-3' and reverse 5'-TGC-TTG-ACC-CTC-AGA-GAC-CT-3' for MMP-1; forward 5'-ATT-CAG-TCC-CTC-TAT-GGA-CCT-CC-3' and reverse 5'-CTC-CAG-TAT-TTG-TCC-TCT-AC-3' for MMP-3; forward 5'-AGC-CAT-GTA-CGT-AGC-CAT-CC-3' and reverse 5'-TCC-CTC-TCA-GCT-GTG-GTG-GT-3' for β-actin.

Amplification conditions consisted of 30 cycles as follows: 2 min denaturation at 95℃, 45 sec annealing at 60℃, and 1 min extension at 72℃. The reaction was performed with the T100 Thermo Cycler (Bio-Rad Laboratories Ltd., Watford, England). PCR products were separated by 2.0% agarose gel and stained with ethidium bromide.

Photographs were detected using a Davinch-Chemi imager^TM (Davinch K, Seoul, Korea). mRNA levels were then divided by the corresponding â-actin band, respectively.

Total cellular protein was collected with the RIPA buffer (Sigma-Aldrich Co., Saint Louis, MO, USA) containing protease inhibitor cocktail (Thermo Scientific Inc., Waltham, MA, USA). Protein concentrations were determined by bicinchoninic acid (BCA) method. Aliquots of 25 ㎍ total proteins were separated by 10% SDS-PAGE.

Proteins on gels were transferred onto a nitrocellulose membrane (Amersham Pharmacia Biotech UK Ltd., Amersham, England) and hybridized at 4℃ overnight with primary antibodies against the following proteins: MMP-1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), MMP-3 (Santa Cruz biotechnology, Santa Cruz, CA, USA), p38 (Cell Signaling Technology Inc., Danvers, MA, USA), phospho(p)-p38 (Cell Signaling Technology Inc., Danvers, MA, USA), ERK (Cell Signaling Technology Inc., Danvers, MA, USA), p-ERK (Cell Signaling Technology Inc., Danvers, MA, USA), JNK (Thermo Scientific Inc., Waltham, MA, USA), pJNK (Cell Signaling Technology Inc., Danvers, MA, USA), TIMP-1 (Cell Signaling Technology Inc., Danvers, MA, USA), TIMP-2 (Cell Signaling Technology Inc., Danvers, MA, USA) and β-actin (Cell Signaling Technology Inc., Danvers, MA, USA).

After hybridization with HRP-labeled secondary antibodies for 1 h, the protein bands were visualized with an ECL illuminate (Amersham Pharmacia Biotech UK Ltd., Amersham, England) using a Davinch-Chemi imager^TM (Davinch K, Seoul, Korea).

To detect collagen I expression in UVB-irradiated HaCaT cells, immunofluorescence staining was conducted using an Immunofluorescence Application Solutions kit (Cell Signaling Technology Inc., Danvers, MA, USA) as follows.

HaCaT cells were cultured on the glass cover slips and exposed to UVB. Then, the cells were treated with the santamarine for 24 h. After the treatment, HaCaT cells were fixed with 4% paraformaldehyde, permeabilized with methanol and stained with anti-collagen I (Abcam, Cambridge, England) antibody at 4℃ overnight. The cells were then incubated with an Alexa Fluor 488-conjugated secondary antibody at room temperature in the dark and mounted with a drop of mounting medium containing the 4',6-diamidino-2-phenylindole (DAPI) (Cell Signaling Technology Inc., Danvers, MA, USA).

Data werepresented as means ± SD. All experiments were conducted at least three times. The statistical analysis of the result performed by One-way Analysis of Variance (ANOVA), followed by Duncan's Multiple Rage Tests (DMRT) using the SPSS 12.0 (SPSS Inc., Chicago, IL, USA). The significance of differences was defined significant when ^*p < 0.05 or ^**p < 0.01.

Prior to in vitro analysis of protective effect of santamarine against UVB-induced photoaging, the cytotoxic effects of treatment with various concentrations of santamarine and UVB intensity were examined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay.

To determine the UVB-induced phototoxic effects on HaCaT cell line, UVB irradiation was carried out within a range of 10 mJ/㎠ - 100 mJ/㎠. UVB radiation at higher than 20 mJ/㎠ significantly reduced the cell viability (Fig. 1B).

Therefore, an intensity at 20 mJ/㎠ was selected as appropriate level for further experiments. When the cells were treated with santamarine, the cell viability was dose-dependently reduced. However, treatment with santamarine did not have any significant cytotoxicity up to a concentration of 10 μM (Fig. 1C).

Excessive exposure to UVB causes oxidative stress, generates reactive oxygen species (ROS) including singlet oxygen, hydrogen peroxide, superoxide and hydroxyl free radicals and induces the activation of the MMP, which is directly related to the skin’s collagen degradation.

The first aim of the study was to determine whether santamarine modulates the UVB-induced ROS production. Therefore, the intracellular ROS changes were evaluated using DCFH-DA, a ROS sensitive dye. Based on the results in Fig. 2, exposure to UVB considerably enhanced ROS generation by 77.0% compared with the non-irradiated cells, whereas treatment with santamarine greatly reduced the UVB-induced ROS generation in a dose-dependent manner. This result demonstrates that santamarine has antioxidant effect through its own ROS scavenging ability.

Fig. 2.  The effect of santamarine on the intracellular ROS production in HaCaT cells treated with H₂O₂.

ROS scavenging effects of santamarine was evaluated after 2 h treatment by the DCFH-DA assay. Data are shown as the means ± standard deviation (n = 3). ^** p < 0.01 vs. H₂O₂ treated group. ^## p < 0.01 vs. non-treated group.

Since santamarine showed antioxidant effect on UVB-induced oxidative stress, the effect of santamarine on the MMPs, which are provoked by ROS generation was addressed next.

As exhibited in Fig. 3A, exposure to UVB expectedly elevated MMP-1 secretion to 21,481.67 pg/㎖ from 11,021.67 pg/㎖ of non-treated blank group. Santamarine decreased the secretion of MMP-1 in UVB-irradiated cells in a dose-dependent manner. In particular, santamarine at 10 μM concentration lowered UVB-mediated MMP-1 release to 8,865.8 pg/㎖, which was a 58.7% decrease compared with the UVB-treated control group.

Fig. 3.  The effect of santamarine on MMP-1 secretion (A), the mRNA levels (B) and the protein expression levels of MMPs and TIMPs (C and D) in UVB-irradiated cells, respectively.

(A) MMP-1 secretion in UVB-irradiated HaCaT cells was analyzed by conducting ELISA following 24 h treatment. The mRNA and protein expression levels were detected by conducting reverse transcription-polymerase chain reaction and western blot, respectively. β-actin was used as an internal standard. Data are shown as the means ± standard deviation (n = 3). ^** p < 0.01 vs. UVB treated group. ^## p < 0.01 vs. non-treated

The result obtained from ELISA analysis was consistent with the mRNA and protein expression results. UVB irradiation stimulated the mRNA (Fig. 3B) and protein (Fig. 3C) levels of MMPs. The expression levels of MMP-1 and MMP-3 mRNA were dose-dependently inhibited by treatment with santamarine in UVB-irradiated cells. In parallel with the mRNA, santamarine downregulated the induction of MMP-1, MMP-2, MMP-3 and MMP-9 proteins in a dose-dependent fashion.

These findings speculated santamarine suppressed the UVB-mediated activation of MMPs through depletion of oxidative stress. Moreover, cells treated with 10 ìMsantamarine recovered the downregulated level of TIMP-1 and TIMP-2 proteins, evidencing the photoprotector capacity of santamarine.

In order to study the action mechanism behind the suppression of UVB-induced MMP regulation, the effect of santamarine on MAPKs signaling pathway was investigated with western blotting. Notably, santamarine downregulated the phosphorylation of p38, JNK and ERK compared to only UVB-irradiated cells.

This indicates that santamarine might inhibit the UVB-induced MMPs expression by suppressing the MAPKs pathway (Fig. 4A). UV radiation promotes MAPK pathway, which then activates its downstream transcription factor, AP-1.

Fig. 4.  The effect of santamarine on the phosphorylation of MAPKs (A) and AP-1 (B) signaling pathway in UVB-irradiated HaCaT cells.

The protein expression levels were detected by western blot. β-actin was used as an internal standard.

The results seen in Fig 4B show that treatment with santamarine significantly reduced the protein levels of phosphorylated c-Fos and phosphorylated c-Jun, which form the AP-1 transcription factor complex. Thus, the inhibition of UVB-induced p38, JNK and ERK phosphorylation by santamarine is involved in the suppression of AP-1 activity in HaCaT cells, indicating that santamarine can suppress the photoaging-related signaling pathways.

Next, to confirm the capability of santamarine to increase collagen production in UVB-irradiated HaCaT cells, fluorescent staining of cellular collagen was conducted. As seen in Fig. 5, UVB irradiation resulted in a considerable decrease in the collagen amount compared with the UVB-untreated group. However, treatment with 10 μM santamarine effectively enhanced the UVB-induced decrease in collagen amount.

Fig. 5.  The effect of santamarine on the collagen Ι in UVB-irradiated HaCaT cells.

Intracellular production of collagen was observed by immunofluorescence through an overlay of green collagen Ⅰ staining, and blue DAPI staining. Scale bar, 100 μm.