Protective Effect Of Djulis (Chenopodium Formosanum) Extract Against UV- And AGEs-Induced Skin Aging Via Alleviating Oxidative Stress And Collagen Degradation Part 1

Abstract: Skin aging is a complex process involving photoaging and glycation stress, which share some fundamental pathways and have common mediators. They can cause skin damage and collagen degradation by inducing oxidative stress and the accumulation of reactive oxygen species (ROS). Chenopodium formosanum (CF), also known as Djulis, is a traditional cereal in Taiwan. This study investigated the protection mechanisms of CF extract against ultraviolet (UV) radiation and advanced glycation end products (AGEs)-induced stress. The results indicated that CF extract had strong antioxidant and free radical scavenging effects. It could reduce UV-induced intracellular ROS  generation and initiate the antioxidant defense system by activating the nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) signaling pathway in human skin fibroblasts. CF extract modulated mitogen-activated protein kinase (MAPK) and transformed growth factor-beta (TGF-β) signaling pathways to alleviate oxidative stress-induced skin aging. Moreover, the results revealed that CF extracts not only promoted collagen synthesis but also improved aging-induced collagen degradation. CF extract attenuated AGEs-induced ROS production and the upregulation of receptors for AGEs (RAGE). The overall results suggest that CF extract provides an effective anti-aging strategy by preventing skin damage from oxidative stress and collagen loss with potent antioxidant,  anti-photoaging, and antiglycation activities.

Glycoside of cistanche can also increase the activity of SOD in heart and liver tissues, and significantly reduce the content of lipofuscin and MDA in each tissue, effectively scavenging various reactive oxygen radicals (OH-, H₂O₂, etc.) and protecting against DNA damage caused by OH-radicals. Cistanche phenylethanoid glycosides have a strong scavenging ability of free radicals, a higher reducing ability than vitamin C, improve the activity of SOD in sperm suspension, reduce the content of MDA, and have a certain protective effect on sperm membrane function. Cistanche polysaccharides can enhance the activity of SOD and GSH-Px in erythrocytes and lung tissues of experimentally senescent mice caused by D-galactose, as well as reduce the content of MDA and collagen in lung and plasma, and increase the content of elastin, have a good scavenging effect on DPPH, prolong the time of hypoxia in senescent mice, improve the activity of SOD in serum, and delay the physiological degeneration of lung in experimentally senescent mice With cellular morphological degeneration, experiments have shown that Cistanche has the good antioxidant ability and has the potential to be a drug to prevent and treat skin aging diseases. At the same time, echinacoside in Cistanche has a significant ability to scavenge DPPH free radicals and has the ability to scavenge reactive oxygen species and prevent free radical-induced collagen degradation, and also has a good repair effect on thymine free radical anion damage.

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【For more info:george.deng@wecistanche.com / WhatApp:8613632399501】

Keywords: Chenopodium formosanum; Djulis; ultraviolet radiation; advanced glycation end products;  glycation stress; reactive oxygen species

1. Introduction

Aging is defined as the result of a progressive decline in the physiological function of cells and tissues of the body. During aging, the accumulation of damaged products gradually affects the organization and repair capacity of the tissue. Skin, being the protective barrier for the body, is often subjected to intrinsic and extrinsic factors of aging [1]. The predominant external factor that contributes to skin aging is exposure to UV radiation in sunlight, termed photoaging [2]. Besides environmental stimuli, AGEs accumulation is considered to be an internal factor of chronological aging that triggers the skin aging process through glycation stress [3].

Both environment ROS produced by UV light and endogenous ROS formed by oxidative metabolism regulate cellular oxidative stress and destroy the collagen-rich extracellular matrix (ECM), which is not only the hallmark of skin connective tissue aging but also the major cause of skin deterioration [4]. Damage to collagen cross-links weakens the dermal structural integrity and increases the stiffness and brittleness of the collagen network,  thereby promoting wrinkle formation, loss of elasticity, and dryness of the skin [5].

UVB rays are the most damaging waveband of solar radiation reaching the earth,  which results in oxidative stress by stimulating ROS formation and is involved in hyperpigmentation, skin inflammation, immunosuppression, photoaging, and carcinogenesis [6,7]. It is well-known that Nrf2 acts as a major regulator of cellular antioxidant defense against cutaneous photodamage mediated by UV radiation. Cells can eliminate oxidative stress and prevent injury by regulating the Nrf2/Kelch-like ECH-associated protein 1 (Keap1)  signaling pathway [8,9]. After UV exposure, excessive ROS production activates MAPK  and nuclear factor-kappa B (NF-κB), which, in turn, induces the upregulation of matrix metallopeptidases (MMPs), culminating in the degradation of collagen and elastin. In addition, activator protein 1 (AP-1) inhibits TGF-β signaling and leads to a reduction in procollagen synthesis [10,11].

AGEs are a heterogeneous group of molecules formed as a result of glycation, a  spontaneous non-enzymatic reaction between the reducing sugars and amino groups of proteins [3]. The interaction between AGEs and a specific receptor of AGEs (RAGE)  triggers the generation of ROS and alters gene expression in AGEs-related disorders, such as diabetes and renal failure [12,13]. AGEs accumulate in ECM proteins upon aging, and Nε -(1-Carboxymethyl)-L-lysine (CML) produced by oxidative modification is the major glycated protein in humans [14,15]. During intrinsic aging, AGEs are expressed in the epidermis and dermis, and significantly increased AGEs accumulation in the sun-exposed areas of the skin can be observed [16]. Additionally, UV enhances the deposition of AGEs via glycation of the elastic fiber network [17].

Chenopodium formosanum (CF), also known as Djulis, is an edible plant in Taiwan and is usually cooked with rice or millet as a staple food. CF is considered a high nutritional value food and a source of bioactive compounds for human health [18]. Besides abundant dietary fiber, starch, grain-limited essential amino acids, and polyphenols [19,20], CF also contains a class of compounds with a long history of medicinal use, called phytoecdysteroids [21–24]. Previous studies have reported that CF displayed several biological activities, including, anti-obesity [21], antihypertensive [22], antihyperglycemic [25,26],  hepatoprotective [27–29], anticarcinogenic [23,24,30,31], and anti-aging properties [19,32]. Nevertheless, neither the detailed skin protective effects of CF extract nor the underlying mechanisms against skin intrinsic and extrinsic aging associated with oxidative and glycation stress have been revealed to date. The purpose of this study was to clarify the antioxidant effects of CF extract and investigate the underlying anti-aging defense mechanisms of CF  extract against collagen degradation by UV and AGEs in human skin fibroblasts.

2. Results 

2.1. HPLC-ESI-MS/MS and MRM Quantitative Analysis

The content analysis of target bioactive compounds in CF extract was conducted by high-performance liquid chromatography (HPLC)-Mass/MS and the multiple reaction monitoring (MRM) methods. The MRM ion chromatogram and mass spectrum on electrospray ionization (ESI) positive mode of 20-hydroxyecdysone and rutin standard were shown in Supplementary Materials. The transition from the precursor ion at m/z 481 to the production at m/z 371 for 20-hydroxyecdysone (Figure S1a) and the transition from the precursor ion at m/z 611 to the production at m/z 303 for rutin (Figure S1b) are shown. Figure S2 showed the total ion chromatogram (TIC) and extracted ion chromatogram (EIC)  of 20-hydroxyecdysone (m/z 481) and rutin (m/z 611) in CF extract. The content of rutin and 20-hydroxyecdysone in CF extract was 14.5 ± 0.4 mg/g and 19.5 ± 0.6 mg/g, respectively.

2.2. Antioxidant Activity and Free Radical Scavenging Effect of CF Extract 

2.2.1. DPPH Free Radical Scavenging Activity

DPPH is a commonly used reagent to estimate the total free radical scavenging activity of antioxidants by detecting the ability of extracts to provide hydrogen protons. The result showed that CF extract exhibited strong scavenging activity of 40.2% ± 3.3% at 50 µg/mL and 89.1% ± 2.6% at 500 µg/mL, while the scavenging activity of ascorbic acid was 96.6% ± 2.3% at 10 µg/mL (Figure 1a). The half maximal inhibitory concentration (IC50) of CF extract on DPPH free radical scavenging was 84.7 ± 13.0 µg/mL.

2.2.2. Reducing Capability 

The reducing capability of the CF extract observed in the present study is shown in Figure 1b. The reducing capacity of CF extract was 51.3% ± 1.0% at 100 µg/mL and 66.9% ± 1.7% at 1000 µg/mL. The reducing capacity of the comparative control ascorbic acid (100 µg/mL) was 79.7% ± 1.2%.

2.2.3. Superoxide Anion Radical (O2 −) Scavenging Activity

The superoxide anion radical scavenging activity was 82.2% ± 6.1% for 250 µg/mL BHA (comparative control) and ranged from 59.6% ± 3.5% to 73.1% ± 2.6% for 100–1000 µg/mL CF extract (Figure 1c).

2.2.4. Hydrogen Peroxide (H2O2) Scavenging Activity

The hydrogen peroxide scavenging activity ranged from 10.3% ± 1.1% to 61.1% ± 2.3%  for 200–1500 µg/mL CF extract and was 96.3% ± 2.0% for BHA (250 µg/mL) (Figure 1d). The IC50 of CF extract was 956.5 ± 59.2 µg/mL for hydrogen peroxide scavenging activity.

2.2.5. Hydroxyl Radical (· OH) Scavenging Activity

The hydroxyl radical scavenging activities of CF extract (100–1000 µg/mL) and the comparative control mannitol (µg/mL) are shown in Figure 1e. The hydroxyl radical scavenging activity of 1000 µg/mL CF extract was 63.3% ± 3.5%, and that of mannitol (2500 µg/mL) was 69.7% ± 2.2%. The scavenging effect of the CF extract was similar to that of the comparative control, and the IC50 of CF for hydroxyl radical scavenging activity was 240.7 ± 24.3 µg/mL.

2.2.6. Ferrous Ion (Fe2+) Chelating Activity 

Figure 1f showed the metal chelating activity of the CF extract and the comparative control EDTA. The activity ranged from 6.5% ± 1.4% to 67.6% ± 2.5% for 100–1000 µg/mL CF  extract, and 97.5% ± 2.0% for 100 µM EDTA. The IC50 of CF extract was 625.5 ± 46.0 µg/mL for metal chelation.

2.3. CF Extract Alleviated UV-Induced Cytotoxicity in Hs68 Cells

To evaluate the cytotoxic effect of CF extract in human skin fibroblasts (Hs68 cells),  the viability of cells treated with CF extract was assessed by MTT assay. As shown in Figure 2a, the CF extract did not induce cytotoxicity in Hs68 cells in the concentration range of 50–250 µg/mL; thus, these concentrations were chosen for the following experiments.

Hs68 cells were exposed to UVB radiation, and the cell viability was determined to evaluate the protective effect of CF extract against photodamage. The results showed that UVB (20–100 mJ/cm2 ) radiation-induced cytotoxicity in Hs68 cells in a dose-dependent manner (Figure 2b). Nevertheless, Hs68 cells were exposed to UVB (80 mJ/cm2 ) and subsequently treated with CF extract (100, 150, 200, and 250 µg/mL) for 24 h; CF extract significantly alleviated UV-induced cytotoxicity (Figure 2c).

2.4. CF Extract Reduced UV-Induced Intracellular ROS Generation in Hs68 Cells

ROS can induce skin photoaging-related protein and downstream signal transduction by causing intracellular oxidative stress in human skin. A fluorogenic dye measures the amount of oxidative free radicals within the cells. After radiation with UVB (80 mJ/cm2 ) for 2h, ROS production in Hs68 cells was induced 2.16-fold compared with that in the non-irradiated group. However, treatment with 100–250 µg/mL CF extract significantly decreased ROS generation. ROS were downregulated 1.64-fold compared with the radiation group by CF extract at 250 µg/mL (Figure 3).

2.5. CF Extract Initiated the Antioxidant Defense System by Activating the Nrf2/HO-1 Signaling Pathway in Hs68 Cells

2.5.1. The Effects of CF Extract on UV-Induced Expression of Nrf2/HO-1

Under normal conditions, Keap1 forms a complex with Nrf2 and promotes the ubiquitination and degradation of Nrf2. However, the Nrf2 signaling pathway is initiated when cells are stimulated by environmental oxidative stress. In this study, Hs68 cells were treated with CF extract to observe its effects on the Nrf2 signaling pathway after UV radiation. As shown in Figure 4, UVB (40 mJ/cm2 ) radiation-induced Nrf2 expression and decreased Keap1 expression, whereas CF extract (250 µg/mL) significantly induced Nrf2 expression by 2.4-fold compared with the radiation group. After UV radiation and treatment with CF extract at 250 µg/mL the protein expression of HO-1 increased by 2.5-fold. The results indicated that CF extract could initiate the antioxidant defense mechanism through the Nrf2/HO-1 signaling pathway and protect cells from oxidative stress in a biological system.

2.5.2. The Effects of CF Extract on UV-Induced Nuclear Translocation of Nrf2

UV radiation promotes Nrf2 activation and translocation into the nucleus of cells. In this study, the Nrf2 immunofluorescence staining assay in human skin fibroblasts was performed to determine the level of Nrf2 activation. The translocation of Nrf2 from the cytoplasm to the nucleus increased after UVB radiation, whereas the translocation was also enhanced after CF extract treatment (Figure 5). The results of the immunofluorescence staining assay were consistent with Nrf2 expression in Figure 4.

2.6. CF Extract Modulated Skin Photoaging-Associated Cellular Signaling Pathway in Hs68 Cells 

2.6.1. The Effects of CF Extract on UV-Induced Expression of MMP-1, -3, -9 and TIMP-1 

UV radiation induces the phosphorylation of MAPK, which triggers downstream signal transduction to activate transcription factor AP-1 and enhance MMP expression. When the skin is exposed to oxidative stress, MMPs are upregulated, which then induces collagen degradation and wrinkle formation. As shown in Figure 6, UVB radiation significantly elevated MMP-1, -3, and -9 expression (2.2-fold, 1.3-fold, and 1.5-fold, respectively,  compared with the control group), whereas CF extract attenuated the expression of MMPs. CF extract treatment at 250 µg/mL significantly suppressed the levels of UVB-induced expression of MMP-1, MMP-3, and MMP-9 by 1.1-fold, 0.9-fold, and 1.1-fold, respectively,  compared with the control group.

Tissue inhibitors of metalloproteinases (TIMPs) are inhibitors of MMPs expressed in the skin connective tissue. TIMP-1 expression was decreased after UVB radiation and enhanced by CF extract (100–250 µg/mL) (Figure 6). These results demonstrated that CF  extract treatment prevented the UV-induced elevation of MMP-1, -3, and -9 levels and the reduction of TIMP-1; therefore, CF extract may decrease UV-induced skin extracellular matrix breakdown and maintain structural integrity in the dermis.

2.6.2. The Effects of CF Extract on UV-Induced Expression of AP-1 and MAPK

UVB radiation upregulated the expression of p-c-Jun and c-Fos (2.4- and 1.2-fold  compared with the control group), whereas they were downregulated by CF extract (100–250 µg/mL). Treatment with CF extract significantly decreased the expression of p-c-Jun and c-Fos at 250 µg/mL (Figure 7a). Figure 7b showed that UVB exposure increased the expression of phosphorylation of p38 and ERK to 2.1- and 1.5-fold relative to total protein levels in Hs68 cells. However, treatment with CF extract (100–250 µg/mL) significantly inhibited the UVB-induced MAPK overexpression. The phosphorylation of p38  and ERK was suppressed to basal levels with a 250 µg/mL concentration of CF extract. Moreover, CF extract could inhibit UV-induced overexpression of AP-1, protecting the skin from photoaging.

2.6.3. The Effects of CF Extract on UV-Inhibited Expression of TGF-β and Smad3

TGF-β plays a central role in extracellular matrix synthesis and controls collagen homeostasis in the human dermis. Increased ROS in fibroblasts impairs TGF-β signaling by downregulating TGF-β and Smad3 expression, which leads to the loss of collagen and destruction of dermal tissue in aged skin. Similarly, in our study, UVB radiation downregulated the expression of TGF-β and Smad3 to inhibit collagen synthesis. Compared with the control group, TGF-β, and Smad3 expression were lower in the UVB-irradiated group; however, CF extract (100–250 µg/mL) significantly restored protein expression (Figure 8).

2.7. CF Extract Attenuated AGEs-Induced ROS Production and RAGE Expression in Hs68 Cells

To investigate the effect of CF extract on glycation stress, Hs68 cells were treated with CML. After treating the cells with CML (100 µg/mL) for 6 h, the amount of ROS  generated was 1.92-fold higher than that in the non-treated group. However, treatment with CF extract (100–250 µg/mL) significantly decreased ROS production. ROS was downregulated 1.30-fold compared with the CML-treated group by CF extract at 250 µg/mL (Figure 9). Furthermore, CML (100 µg/mL) significantly increased RAGE expression to 2.2-fold compared with the control group, but it was attenuated by treatment with CF  extract (Figure 10), indicating that CF extract reduced the induction mainly caused by CML.

2.8. CF Extract Promoted Collagen Synthesis and Improved Aging-Induced Collagen Degradation  in Hs68 Cells

2.8.1. The Effects of CF Extract on Collagen Synthesis 

Hs68 cells were treated with CF extract (50–250 µg/mL) for 24 h, and type I procollagen expression was measured using western blotting. After treatment with CF extract, type I procollagen expression was significantly upregulated (1.7-fold compared with the control group) (Figure 11a). The total collagen content in the control group was 14.93 ± 1.27 µg/mL which increased to 39.00 ± 2.39 µg/mL after treatment with 250 µg/mL CF extract (Figure 11b). The results of type I procollagen expression and total collagen content indicated that CF extract promoted collagen synthesis and production.

2.8.2. The Effects of CF Extract on UV and AGEs-Induced Collagen Degradation

Hs68 cells exposed to UVB (40 mJ/cm2 ) radiation or treated with CML (100 µg/mL)  were treated with CF extract for 24 h. As shown in Figure 12a, the total collagen content was significantly decreased after UVB radiation, and treatment with CF extract restored collagen and significantly increased collagen formation at 250 µg/mL. Similar results were shown in collagen production treated with CML and a series of CF extracts. CF extract attenuated the CML-treated reduction in total collagen (Figure 12b).

The immunofluorescence staining assay was used to determine the collagen level in fibroblast cells (Figure 12c). Both UVB radiation and CML treatment reduced collagen content, while CF extracts improved collagen degradation. The results of the immunofluorescence staining assay of CF extract on collagen were consistent with the collagen content in Figures 11b and 12a,b.

【For more info:george.deng@wecistanche.com / WhatApp:8613632399501】