Sarmentosamide, An Anti-Aging Compound From A Marine-Derived Streptomyces Sp. APmarine042

Keywords: cistanche; Streptomyces sp. APmarine042; anti-aging; MMP-1; UVB; TNF-α

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1. Introduction 

Skin aging is induced by two main processes, which result from intrinsic and extrinsic factors. Extrinsic aging is primarily caused by exposure to environmental factors such as air pollution [1] and ultraviolet (UV) radiation [2]. UV light is composed of UVA (315–400 nm), UVB (280–315 nm), and UVC (200–280 nm), of which UVB rays are known to penetrate the epidermis and contribute to skin photoaging [3]. UVB-induced skin photoaging causes DNA damage [4] and reactive oxygen species (ROS) generation [5] and disrupts the extracellular matrix [6]. Intrinsic aging is caused by the natural consequences of physiological change, such as genetic factors, hormones, and metabolic processes [7]. Moreover, inflflammatory cytokines, such as tumor necrosis factor-alpha (TNFα), interleukin 1 alpha/beta (IL-1α/β), and interleukin 6 (IL-6), are chronically increased with age. In particular, tumor necrosis factor-α(TNFα) is known to accelerate the degradation of extracellular matrix (ECM) components via upregulating expression and activity of matrix metalloproteinases (MMPs) in aged skin [8]. MMP-1 is mainly released by fibroblasts in the dermis, and secreted MMP-1 is responsible for the degradation of dermal collagen, which supports skin structure and function. Therefore, modulation of MMP-1 expression in dermal fibroblasts could be a promising target for the development of anti-aging cosmetic ingredients.

Previous studies have reported that UVB radiation induces ROS (reactive oxygen species production and leads to the activation of intracellular signaling pathways and transcription factors(e.g., AP-1, NF-kB) [9. These transcription factors are regulated by mitogen-activated protein kinasesMAPKs), which induce MMP-1 and proinflammatory cytokines. There are three distinct MAPKamilies involved in UVB/ROS stimuli: extracellular signal-regulated kinases (ERKs), c-Jun N-terminal

kinase (NK), and p38.Our study identified a Streptomyces sp. APmarine042 extract that exhibited a potent inhibitory effect on MMP-1 expression. Afterward, an MMP-1 inhibitory compound was identified in this Streptomyces sp. APmarine042 extract and its chemical structure were identified via NMR analysis assistance (1), a compound with a hexadienamide moiety 101. We then further investigate whether this compound-inhibited MMP-1 expression in UVB-irradiated and TNFa-induced human dermal fibroblasts as well as its role in the regulation of underlying signaling pathways. Additionally, we examined the antioxidant activity of cistanche (1) via a 2,2-diphenyl-1-picrylhydrazyl(DPPHI) radical scavenging assay.

2. Results and Discussion

2.1. cistanche (1) Isolation and Identification

cistanche (1) was isolated through a combination of HPLC-UV and bioactivity-guided isolation. Based on the comparison between the NMR data (Supplementary Materials) of the compound identified in this study (hereinafter referred to as compound 1") to those of previously reported compounds, compound 1 was identified as cistanche. The optical rotation value of compound 1 was also consistent with previous reports, which further supported that compound 1 assistance (Figure 1).

2.2. Cytotoxic Effects of cistanche (1) on Human Dermal Fibroblasts

To investigate the cytotoxic effects of cistanche, normal human dermal fibroblasts (NHDFor human foreskin fibroblast cells (Hs68) were treated with cistanche at different concentrations(6, 12, 25, 50, 100 ug/mL) for 24 h, after which the cell counting kit-8 (CCK-8) assay was performed to quantify cell viability. Water-soluble tetrazolium salt (WST-8) in CCK-8 solution was reduced by dehydrogenase activities in cells to give a yellow-color formazan dye, the amount of which was proportional to the number of living cells. As shown in Figure 2, cistanche did not cause observable cytotoxic effects in either NHDF or Hs68 cells at concentrations ranging from 0-100 ug/ml

Figure 2. Cell viability test of cistanche on fibroblasts. (ab) The cell viability of cistanche-treated normal human dermal fibroblasts (NHDF) (a) and human foreskin fibroblast cells (Hs68) (b) cells at 24 h was measured by CCK (cell counting kit) assay. * p < 0.05, ** p < 0.01vs. untreated group ).

2.3. cistanche (1) Inhibits MMP-1 Expression and Secretion in UVB-Irradiated NHDF Cells

Next, NHDFs in the absence and the presence of UVB radiation (25 mJ/cm2) were treated with cistanche (2.5 ug/mL) for short periods, and relative MMP-1 mRNA expression levels were quantified via quantitative real-time PCR analysis. cistanche was found to downregulate MMP-1 mRNA levels by up to 16% and 17% in the absence (Figure 3a) and the presence (Figure 3bof UVB, respectively. UVB radiation, a major extrinsic skin aging risk factor, can penetrate the epidermis and reach the upper dermis, resulting in the generation of reactive oxygen species (ROS)MMP production, and collagen synthesis downregulation. Therefore, our study sought to investigate whether cistanche-inhibited MMP-1 secretion in UVB-irradiated NHDF cells. The secretion levels of MMP-1 in cell culture media were measured via ELISA, and the relative amounts of MMP-1secretion were normalized to otal protein in whole cell lysates, which were measured via a bicinchoninic acid (BCA) protein quantification assay. UVB irradiation (25 mJ/cm2) remarkably increased MMP-1secretion (8.3-fold) compared to the non-UVB-irradiated group (Figure 3c) and cistanche-inhibited MMP-1 secretion in UVB-irradiated NHDF cells in a dose-dependent manner.

Figure 3. Effect of cistanche on metalloproteinase-1 (MMP-1) expression in UVB-irradiatedNHDFs. (a,b) The relative mRNA levels of MMP-1 in NHDF cells irradiated UVB (25 mJ/cm2) or treated with cistanche (2.5 ug/mL) for 16 h. RPLPO gene was used as an internal control. * p < 0.05.*** p < 0.001 (C) After UVB irradiation (25 mJ/cm2), cistanche in serum-free media was treated for 48 h, and then the relative secretion levels of MMP-1 into cell culture media were analyzed by ELISA### p < 0.001 (vs. non-UVB control group); ** p < 0.01; *** p < 0.001 (UVB-irradiated control group).

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