Sesquiterpenes From Oplopanax elatus Stems and Their Anti-Photoaging Effects by Down-Regulating Matrix Metalloproteinase-1 Expression via Anti-Inflammation

In the process of continuing to investigate ultraviolet b (UVB) irradiation protective constituents from Oplopanax elatus stems, nine new sesquiterpenes, named as eurylosesquiterpenosides A–D (1–4), eurylosesquiterpenols E–I (5–9), and ten known ones (10–19) were gained. Their structures were established by analysis of their NMR spectroscopic data, and electronic circular dichroism calculations were applied to define their absolute configurations. In addition, UVB induced HaCaT cells were used to study their anti-photoaging activities and mechanism. The results consolidated that compounds 7, 11, and 14 could improve the survival rate of HaCaT cells in concentration dependent manner at 10, 25, and 50 μM. Furthermore, western blot assay suggested that all of them could inhibit the expression of matrix metalloproteinase-1 (MMP-1), and increase the level of type I collagen markedly. Compounds 11 and 14 could reduce the phosphorylation of extracellular signal-regulated kinase and p38, respectively. Besides, compounds 7, 11, and 14 could significantly down-regulate the expression of inflammation related protein, such as tumor necrosis factor-α and cyclooxygenase-2, which indicated that they played anti-photoaging activities by reducing MMP-1 expression via down-regulating the production of inflammatory mediators and cytokines in UVB-induced HaCaT cells.

Oplopanax elatus Nakai belongs to Oplopanax genus (Araliaceae family). It was reviewed to contain various constituents such as volatile oil, phenolic acids, lignans, quinic acid esters, steroids, and aliphatic acids, and the stem of it was reported to exhibit anti-aging effect . Moreover, our previous study demonstrated that phenolic acids obtained from it had anti-photodamage activity, too (Han et al., 2021). We hypothesize there are other components may exhibit benefits for the skin photodamage. Then, the other constituents in the stems of O. elatus, along with their activities and mechanisms against photoaging induced by UVB irradiation in HaCaT cells were continue to be investigated.
Then, the absolute configuration of eurylosesquiterpenol E (5) was elucidated here firstly.
As we introduced previously, the level of MMP-1 will be increased after UVB irradiation, and the degradation of COL1A1 will be caused at the same time in HaCaT cells. The process is related to the upregulation of inflammatory mediator, MAPKs, and inflammatory cytokines such as TNF-α and COX-2. Therefore, the expressions of above proteins were evaluated by using western blot assay to study the anti-photoaging mechanism of compounds 7, 11, and 14.
Comparing with normal group (Nor), the level of MMP-1 was increased and COL1A1 was decreased significantly in Con after UVB irradiation. While, the expression of MMP-1 was significantly decreased by 30, 21, and 16%, and the level of COL1A1 was up-regulated by 24, 36, and 29% in pretreatment of compounds 7, 11, and 14, respectively (Figure 9).  Nor; ***p < 0.001, **p < 0.01, and *p < 0.05 vs. Con). Frontiers in Chemistry | www.frontiersin.org November 2021 | Volume 9 | Article 766041 8 Meanwhile, the phosphorylations of MAPKs were upregulated in varying levels in Con comparing with Nor. However, the p-ERK was markedly reduced to 0.62-fold by compound 11; and the p-p38 could be inhibited to 0.74-fold by 14. Nevertheless, none of active compounds could prevent upregulation of p-JNK ( Figure 10).
Basing on the above results, the anti-photoaging mechanism of compounds 7, 11, and 14 might be related to inhibiting collagen degradation via anti-inflammation.

General Experimental Procedures
NMR spectra were performed on Bruker ascend 600 MHz and/or Bruker ascend 500 MHz NMR spectrometer (Bruker BioSpin AG Industriestrasse 26 CH-8117) with tetramethylsilane as an internal standard. Negative-ion mode ESI-Q-Orbitrap MS were determined on a Thermo ESI-Q-Orbitrap MS mass spectrometer connected to the UltiMate 3000 UHPLC instrument via ESI interface (Thermo Scientific). Optical rotations, UV, IR, and ECD spectra were run on a Rudolph Autopol ® IV automatic Dichloromethane (CH 2 Cl 2 ), methanol (MeOH), acetonitrile (CH 3 CN), acetic acid (HAc), and other reagents (chromatographically pure or analytical pure) were purchased from Tianjin Concord Technology Co., Ltd.

Plant Material
The stems of Oplopanax elatus Nakai were collected from Tonghua city, Jilin province, China, identified by Professor Junyi Zhu (Tonghua Normal University). The voucher specimen (2018121001) was deposited at the Academy of Traditional Chinese Medicine of Tianjin University of TCM.

Computations
Relative configurations of compounds 4-7 were deduced by analyses of their 1D and 2D NMR data assisted by Chem3D modeling. Conformation search was then firstly accomplished under the MMFF94 force field by using CONFLEX 8 software (Takanawa, 2019), and the low energy conformers, which meet the requirements of NOESY analysis, were selected out for further computations. To verify the stabilities of the selected conformers, geometry optimizations and the frequencies pre-calculations were finished by DFT method at the APFD/6-311+G(2d,p) basis set level in methanol (for 4) or acetonitrile (for 5-7), using Gaussian 16 package (Revision C.01) (Frisch et al., 2019). By TD-SCF/DFT method, energies of one hundred excitation states of the optimized conformers were then calculated at the APFD/6-311+G(2d,p) level with a IEFPCM solvent model in MeOH or acetonitrile. With a half bandwidth of ∼0.2 eV, the calculation results were Boltzmann averaged to simulate the ECD spectra after UV correction, which were finally extracted by GaussView 6.0 and Origin Pro 2016 software before comparing with those experimental data. United States); MTT and dimethyl sulfoxide (DMSO) were gained from Sigma-Aldrich (St. Louis, MO, United States); Vitamin C (Vc) were purchased from Shanghai Yuanye Bio-Technology Co., Ltd. (Shanghai, China); BCA protein quantification kit was ordered from Thermo Fisher Scientific (Waltham, United States); PVDF membrane was purchased from Merch/Millipore (Schwalbach, Germany); Immobilon western chemilumescent HRP substrate was gained from Millipore (Massachusetts, United States); TNFα (ab6671), COX-2 (ab52237), β-actin (ab8227) JNK (ab208035), and p-JNK (ab4821) were ordered from abcam (Cambs, United Kingdom); p38 (8690S), p-p38 (4511S), ERK (4695S), p-ERK (4370S), and COL1A1 (72026S) were purchased from CST (Massachusetts, United States); MMP-1 (SC-137044) was obtained from Santa Cruz Biotech.INC. (United States).

Cell Viability Assay
MTT assay was applied to test cell viability. HaCaT cells were incubated at 96-well plates and treated with or without test samples for 24 h, respectively. The culture condition was similar to "Cell Culture." The medium was removed, then 1% MTT (5 mg/ml) were added into wells to format formazan. After incubating 4 h, the supernatant was removed, then 100 μL dimethyl sulfoxide (DMSO) was added in each well to dissolve the formazan. The absorbance was measured with a microplate reader at 490 nm.

Selection of Ultraviolet B Radiation Dose
After being cultured with MEM medium containing 10% FBS, streptomycin (100 μg/ml), penicillin (100 U/ml) in 96-well plates until grown to 70% confluence, the HaCaT cells were covered with fresh medium for 24 h. Then, the fresh medium was replaced with 100 μL/well PBS, and the cells were exposed to 50, 75, 100, 125, and 150 mJ/cm 2 of UVB, respectively. After irradiation, 100 μL/well PBS was removed, and the cells were cultured with 100 μL/well fresh medium for 24 h again. The cell viability was tested in line with "Cell Viability Assay."

Cell Viabilities of Ultraviolet B Induced HaCaT Cells Pretreated With Compounds
HaCaT cells were seeded in 96-well culture plates with complete medium until grown to 70% confluence, and then treated with fresh medium containing various concentrations of samples (10, 25, and 50 μM) for 24 h. Then, the cells were irradiated with UVB at 125 mJ/cm 2 (UVB-irradiated with 0.46 mW cm −2 s −1 for approximately 272 s) in 100 μL PBS. After irradiation, the PBS was immediately replaced by 100 μL fresh medium and incubated for 24 h. Finally, the cell viability was measured by using the same method as that described in the part of "Cell Viability Assay."

Statistical Analysis
All experimental results were presented as the means ± standard error of mean (SEM). SPSS 26.0 was used to conduct the statistics of all data. Unpaired Student's t-test (when two groups were analyzed) and one-way analysis of variance (ANOVA) (for > 3 groups) were used to analyze results. p < 0.05 was considered to indicate a statistically significant difference.

CONCLUSION
In summary, in the process of investigating photoprotective constituents from natural products, nine new sesquiterpenes, named as eurylosesquiterpenosides A-D (1-4), eurylosesquiterpenols E-I (5-9), together with ten known ones were obtained and identified from the 70% EtOH extract of O. elatus stems. Though the diverse ingredients such as volatile oil, phenolic acids, lignans, quinic acid esters, anthraquinones, steroids, and aliphatic compounds had been reported from the medicine , the sesquiterpenes were rarely found in it, which enriched its material base.
Furthermore, our study suggested that the underlying mechanism of active-sesquiterpenes might be relevance with down-regulating MMP-1 expression via the decreasing production of inflammatory mediators and cytokines in UVBirradiated HaCaT cells.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding authors.