Protective Effect Of Djulis (Chenopodium Formosanum) Extract Against UV- And AGEs-Induced Skin Aging Via Alleviating Oxidative Stress And Collagen Degradation Part 2

3. Discussion

Fibroblasts play a crucial role in the turnover of the dermal extracellular matrix, including the synthesis of collagen, elastic fiber, and glycosaminoglycan to form dense connective tissue [33]. The accumulation of UV and AGEs stress causes collagen degradation and interferes with each other during intrinsic and extrinsic aging [3,34]. The development of natural products or materials with antioxidant, anti-photoaging, and antiglycation activities for the amelioration of skin aging has been ongoing in recent years.

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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Chenopodium formosanum (CF) is a native grain plant grown in Taiwan, mainly cultivated in aboriginal residential areas. Apart from cereal use, the application of CF based on antioxidant properties has attracted increasing attention [28–30,35]. Polyphenols are well-known antioxidants and skin protectors by possessing strong free radical scavenging activity [36], reducing inflammation, and absorbing UV radiation to provide skin photoprotection [37,38]. Rutin is a flavonoid widely distributed in fruits and vegetables and the representative polyphenol in CF indicated by previous studies [21,22]; several reports have demonstrated the biological effects of rutin on ROS-induced skin aging [39] and suggested that rutin effectively inhibits the formation of AGEs on collagen synthesis [40]. CF has been indicated to protect skin against UV-induced epidermal damage and inflammation in keratinocytes and mouse models, and rutin is the major component in CF that contributed to the protective effect [19]. In this study, the results showed that HaCaT cells treatment with CF had a higher survival rate and less production of interleukin-6, MMP-1, and ROS in UVB-irradiated conditions. A previous study used keratinocytes exposed to UVB radiation to mimic a skin photoaging model, and the fibroblasts were used in the present study to explore the mechanisms and effects of intrinsic factors (AGEs) and extrinsic factors (UV) on dermis skin aging. The results of this study showed that CF decreased UV-induced cytotoxicity and provided a good ROS scavenging activity, and the results were consistent with Hong’s study [19]. This study considered more factors of skin aging and investigate the anti-glycation effect of CF on intrinsic aging, and the findings further confirmed that CF has excellent skin protection efficacy via alleviating oxidative stress and collagen degradation.

20-Hydroxyecdysone is a bioactive compound belonging to phytoecdysteroids, commonly found in Chenopodium formosanum [21–24]. Microsorum grossum (Polypodiaceae) is one of the most commonly used ferns in Polynesian traditional medicine, and its frond and rhizome extracts contain 20-hydroxyecdysone as the main bioactive components. Microsorum grossum was known to have UVB-protective effects on human dermal fibroblasts by upregulating the antioxidant enzyme HO-1 and inhibiting stress-induced premature senescence [41]. Chenopodium quinoa (Amaranthaceae) is the seed crop known as the “golden grain” by the native Andean people in South America, including 20-hydroxyecdysone,  which has been shown to inhibit intracellular ROS production and MMPs activity [42]. In addition, 20-hydroxyecdysone isolated from C. quinoa seeds possess strong inhibition activity against collagenase and the DPPH free radicals, and a potent ability to chelate iron ions [43]. Many previous studies have reported that CF extract contains rutin and 20-hydroxyecdysone as its rich bioactive ingredient, other plants containing these compounds also possess similar activities and skin care potential. The primary advantage of botanicals is their complex composition and the synergistic effect of related compounds with multiple activities to obtain greater efficacy [44]. Plant extracts pose a promising future in skin care due to their appeal as natural products, perception as safe, and abundance and sustainability.

ROS are byproducts of regular oxidative cell function in the electron transport chain of aerobic metabolism reaction. Excessive UV exposure and other environmental stressors overwhelm the cutaneous antioxidant capacity further inducing ROS production, DNA  fragmentation, lipid peroxidation, and apoptosis [45,46]. High levels of ROS initiate aging signal cascades in skin cells, thereby promoting cell senescence, even death [47]. According to the results of the cell assay, CF extract with no cytotoxic effects on fibroblasts significantly rescued UV-induced cell cytotoxicity and showed good photoprotective activity. Furthermore, ROS formed in fibroblasts was significantly increased either by UV radiation or by AGEs treatment and both were effectively inhibited by CF extract. The strong radical scavenging activity of CF was also verified by different antioxidant assays. Polyphenols are capable of donating electrons to free radicals and converting them into more stable non-reactive species owing to the presence of hydroxyl groups in the ring structure [48,49]. CF rich in polyphenols may prevent ROS-induced skin injury by terminating the free radicals chain reaction. The multiple antioxidant-related compounds in CF extract provide a synergistic effect to enhance the overall activity of CF extract and make CF extract a strong antioxidant to protect skin from oxidative stress.

The skin possesses an endogenous antioxidant defense system to deal with oxidative stress induced by aging. Nrf2 is a transcription factor that acts against oxidative stress,  and Keap1 is a pressure sensor that regulates Nrf2 expression [8]. When the intracellular pressure imbalance causes the configuration change of Keap1 and loses its ability to bind to Nrf2, Nrf2 translocates into the nucleus and enhances the level of intrinsic antioxidant enzymes to prevent oxidative damage and exert photoprotective activity [50,51]. The results demonstrated that CF extract could initiate the Nrf2 signaling pathway and antioxidant defense system when cells were stimulated by UV-induced oxidative stress. CF extract upregulated the expression of Nrf2 and HO-1. The cellular defensive properties of CF  extracts can be explained by their ability to either directly neutralize ROS or indirectly upregulate the expression of cellular defensive proteins. Based on the above results, CF  extract can reduce intracellular oxidative stress and be used as a photo protectant. In addition to its direct effect on ROS generation, CF extract also protected fibroblasts against UV damage by enhancing intracellular antioxidant defense mechanisms via Nrf2 and its target factor, HO-1.

Photoaging is caused by an imbalance between the accumulation and degradation of ECM after repetitive UV absorption. UV radiation damages alternations of cutaneous collagen and hastens the generation of ROS, which in turn act on cells and matrix components to mediate further turnover [33,47]. ROS and oxidative stress result in damaged skin via activation of AP-1 signaling and elevation of multiple MMPs. During normal tissue maintenance, collagen undergoes continuous turnover. However, UV radiation is known to induce three different MMPs, collagenase (MMP-1), stromelysin (MMP-3),  and gelatinase (MMP-9), to break collagen and elastic fibers, resulting in fragmentation and disorganization [52,53]. TIMPs are inhibitors of MMPs, and the homeostasis between these two proteins plays an important role in the functional and structural integrity of the ECM [54–56]. In this study, CF extract inhibited UVB-induced AP-1 and phosphorylation of ERK and p38 proteins in human skin fibroblasts. Furthermore, treatment with CF extract blocked UVB-induced collagen degradation by inhibiting MMP-1, -3, and -9 expression in human skin fibroblasts and inducing TIMP-1 expression against MMPs activity. The TGF-β pathway is the major pathway regulating procollagen biosynthesis and ROS production [57]. Oxidative stress inhibits TGF-β signaling by downregulating Smad3, which contributes to the loss of collagen content in aged skin [4]. The results showed that CF  extract increased TGF-β and Smad3 expression to enhance the total collagen content. Based on the above results, CF extract ameliorated UVB-induced collagen degradation through ROS scavenging, MMP inhibition, and TGF-β upregulation.

One of the factors of intrinsic skin aging is the progressive accumulation of AGEs throughout the lifespan. RAGE belongs to the immunoglobulin superfamily of cell surface receptors, which interacts with several ligands, especially CML, one of the glycation products of the Maillard reaction [58]. RAGE is highly expressed in skin keratinocytes and fibroblasts by interaction with AGEs followed by UVB radiation [59] and upregulated in sun-exposed skin [60]. UV-induced oxidation accelerates the formation of human skin elastin in actinic elastosis, and AGEs accumulate on skin collagen and elastin, which interfere with normal skin function [61]. Activation of RAGE decreases type I collagen synthesis and matrix production in fibroblasts [62]. A previous study mentioned that CF extract inhibited the formation of AGEs in the cell-free system [32], but the detailed antiglycation mechanism in dermal fibroblasts remains unclear. Based on the results of this study, we presumed that preventing collagen degradation by blocking AGE–RAGE interaction and decreasing ROS production inside the cells are the possible antiglycation pathways of CF extract.

Glycation-induced biological products are known to be associated with skin aging,  diabetes, and the progression of some tumors. During the aging process, the accumulation of AGEs changes the mechanical properties of the skin by increasing stiffness [34]. Therefore,  the presence of AGEs is considered to be one of the factors for delayed wound healing and loss of elasticity of the healed skin. The cross-talk between RAGE and TGF-β1 affects wound healing in diabetes by regulating collagen turnover and cytokine production in AGEs-treated fibroblast cells [63]. In this study, CF extract not only promotes pro-collagen synthesis in fibroblasts but also improves the degradation of collagen caused by intrinsic and extrinsic skin aging. Moreover, CF extract upregulated TGF-β expression and downregulated RAGE expression. Based on the above research results, the role of CF in glycation stress-related wound healing mechanisms can be further explored in the future.

4. Materials and Methods 

4.1. Materials and Chemicals

Reagents used in the cell culture, including Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin-streptomycin, and trypsin-EDTA, were obtained from Gibco, Thermo Fisher Scientific, Inc. (Waltham, MA, USA). CML was purchased from Cayman Chemical (Ann Arbor, MI, USA). Sodium dodecyl sulfate (SDS), 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), and potassium phosphate-potassium hydroxide (KH2PO4-KOH) were purchased from USB Corporation (Cleveland, OH, USA). 2,2-Diphenyl-1-picrylhydrazyl (DPPH), potassium ferricyanide(III) (K3Fe(CN)6), ferric chloride (FeCl3), ascorbic acid, phenazine methosulfate (PMS), β-  nicotinamide adenine dinucleotide, reduced disodium salt hydrate (NADH), nitrotetrazolium blue chloride (NBT), butylated hydroxyanisole (BHA), 2-deoxy-D-ribose, thiobarbituric acid (TBA), trichloroacetic acid (TCA), mannitol, ferrous chloride (FeCl2), ethylenediaminetetraacetic acid (EDTA), 20, 70 -dichlorofluorescin-diacetate (H2DCFDA), 2-propanol  and 20-hydroxyecdysone were purchased from Sigma-Aldrich (Saint Louis, MO, USA). Rutin was obtained from ACROS Organics (Morris Plains, NJ, USA). HPLC-grade acetonitrile and formic acid were obtained from J.T. Baker, Thermo Fisher Scientific, Inc. (Waltham, MA, USA). All other chemicals used were of analytical grade or higher grade.

4.2. Preparation of CF Extract

CF used in this study was purchased from Pingtung County, Taiwan, and the CF  extract was supplied by TCI Co., Ltd. (Taipei, Taiwan). The preparation method of CF  extract has been reported previously [32]. Briefly, the unhulled Chenopodium formosanum was added to water at the ratio of 1:10 (w/v) and extracted at three different temperatures,  first at 25 ◦C, then at 50 ◦C, and finally at 70 ◦C. The mixture was centrifuged at 4600× g  for 20 min and the supernatant was collected. The powder of CF extract was obtained after removing solvent from the supernatant by freeze-drying and stored at room temperature.

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

The HPLC-ESI mass spectrometric analysis was performed using Dionex Ultimate 3000 liquid chromatography (Thermo Scientific, Waltham, MA, USA) coupled with an ion trap mass spectrometer (HCT Ultra, Bruker Daltonics, Bremen, Germany). The separation method was performed as described by Chen et al., with minor modifications [22]. CF  extract was injected into the Atlantis T3 C18 column (2.1 mm ID × 150 mm, 3 µm particle size, Waters Corp., Milford, MA, USA), which was connected with a guard column (SecurityGuard™ C18 2.0 mm ID × 4.0 mm, Phenomenex Inc., Torrance, CA, USA). The mobile phases employed were: 0.1% formic acid in water (solvent A) and 0.1% formic acid in acetonitrile (solvent B) at a flow rate of 0.3 mL/min. Multi-step gradient elution was carried out with 5–45% B in 15 min, 45–95% B in 5 min, and finally 95% B isocratic elution for 5 min.

Mass spectrometry was carried out by the program Esquire Control (V6.2) and LC/MS  was controlled by Hystar (V3.2) (Bruker Daltonics, Bremen, Germany). The mass spectrometer operating parameters were set as follows: nebulizer pressure 30 psi, dry gas 12 L/min,  dry temperature 300 ◦C, capillary voltage of 3600 V with an end plate of −500 V. MS/MS  was applied to all major ions in the selected mass range. Multiple reaction monitoring (MRM)-profiling was performed by analyzing the following ion transitions: the transition from the precursor ion at m/z 481 to the production at m/z 371 for 20-hydroxyecdysone and the transition from the precursor ion at m/z 611 to the production at m/z 303 for rutin. The ion isolation width was set to 4 m/z with a fragmentation amplitude of 0.5 V. The compound was identified and quantified by comparing the mass spectra obtained by ESI-MS/MS with commercial reference standards.

4.4. Antioxidant Capacity Measurement 

4.4.1. DPPH Free Radical Scavenging Assay

Reaction mixtures containing a methanol solution of DPPH and serial CF extract dilutions of 50–500 µg/mL were placed in a 96-well microplate and incubated for 30 min. The absorbance was determined at 517 nm using a microplate reader (Sunrise, Tecan, Salzburg, Austria). Ascorbic acid (10 µg/mL) was used as a comparative control [64].

4.4.2. Reducing Power Assay

Serial concentrations of CF extract (100–1000 µg/mL) were mixed with phosphate buffer and K3Fe(CN)6 and then incubated at 50 ◦C for 20 min. TCA was added, and the reaction mixture was centrifuged at 3000 rpm for 10 min. FeCl3 solution was mixed with supernatant and the absorbance was measured at 700 nm using a multi-mode reader (Synergy HTX, BioTek Instruments, Winooski, VT, USA). Ascorbic acid (100 µg/mL) was used as a comparative control [64].

4.4.3. Superoxide Anion Radical (O2 −) Scavenging Assay 

Reaction mixtures containing PMS, NADH, and NBT were prepared in phosphate buffer and serial CF extract dilutions of 100–1000 µg/mL. The mixture was reacted for 5 min, and the absorbance was read at 560 nm using a microplate reader (Sunrise, Tecan, Salzburg, Austria). BHA (250 µg/mL) was used as a comparative control [65].

4.4.4. Hydrogen Peroxide (H2O2) Scavenging Assay

The activity of CF extract to scavenge H2O2 was performed as previously described [65]. Various concentrations of CF extract (200–1500 µg/mL) were added to the H2O2 solution and reacted in the dark for 10 min. The absorbance of the reaction mixture was recorded at 230 nm using a multi-mode reader (Synergy HTX, BioTek Instruments, Winooski, VT, USA), and BHA (250 µg/mL) was used as a comparative control.

4.4.5. Hydroxyl Radical (· OH) Scavenging Assay

The assay was conducted by mixing CF extract dilutions (100–1000 µg/mL), KH2PO4- KOH buffer, 2-deoxy-D-ribose, FeCl3, ascorbic acid, EDTA, H2O2, and deionized water. After incubation at 37 ◦C for 1 h, TBA and TCA were added to the mixture, and the mixture was incubated at 100 ◦C for 15 min and then centrifuged at 4000 rpm and 25 ◦C for 10 min. Subsequently, the absorbance of the supernatant was measured at 532 nm using a microplate reader (Synergy HTX, BioTek Instruments, Winooski, VT, USA). Mannitol (2500 µg/mL) was used as a comparative control [64].

4.4.6. Ferrous Ion (Fe2+) Chelating Assay

The chelation of ferrous ions by CF extract was carried out according to the procedure described previously [65]. CF extract (100–1000 µg/mL) was mixed with FeCl2 solution and the reaction was initiated by adding ferrozine. After the mixture had reached equilibrium,  the Fe2+-chelating activity of the CF extract was monitored by measuring the absorbance of the Fe2+-ferrozine complex at 562 nm using a multi-mode reader (Synergy HTX, BioTek Instruments, Winooski, VT, USA). EDTA (100 µM) was used as a comparative control.

4.5. Cell Culture

Hs68 cells were purchased from the Bioresource Collection and Research Center (BCRC, Hsinchu, Taiwan). Cells were maintained in 10-cm cell culture dishes and grown in DMEM supplemented with 10% FBS, 100 U/mL penicillin, and 100 µg/mL streptomycin at 37 ◦C in an incubator containing 5% CO2. At near confluence (80–90%), cells were disaggregated in trypsin-EDTA and subcultured [55].

4.6. UV Exposure and AGEs Treatment

The UV exposure dose selection for Hs68 cells referred to previous studies [55,56]. Before treatment, cells were rinsed with PBS twice and irradiated with different doses of UVB (mJ/cm2 ). The UVB energy spectrum (280–320 nm) was supplied by UV Crosslinker CL-1000M (UVP, Upland, CA, USA) with an emission peak at 302 nm and the exposure time was 15–30 s. The UVB radiation doses in the cell viability assay were 20–100 mJ/cm2  to evaluate the appropriate UVB exposure dose to induce cytotoxicity. Hs68 cells were radiated with a high dose UVB (80 mJ/cm2 ) for 2 h to investigate the rapid radical scavenging ability of CF extract against short-term overproduced intracellular ROS. Other experiments use UVB radiation (40 mJ/cm2 ) in Hs68 cells for 24 h to mimic cells under long-term oxidative stress conditions. CML was supplied as a crystalline solid and aqueous solutions of CML  were prepared by dissolving the compound in PBS buffer. The cells were treated with 100 µg/mL CML.

4.7. Cell Viability Assay 

Hs68 cells were seeded into 24-well plates (4 × 104  cells/well), allowed to attach overnight, and treated with 1 mL of various concentrations of CF extract (50–250 µg/mL)  dissolved in DMEM for 24 h. MTT solution was added to the plates followed by incubation at 37 ◦C for 4 h. Then, 2-propanol was added to dissolve the MTT formazan crystals, and the absorbance was measured at 570 nm using a microplate reader (Sunrise, Tecan, Salzburg, Austria) [56].

4.8. Measurement of Intracellular ROS

The assay is based on the use of an established non-fluorescent (H2DCFDA)/fluorescent (DCF) system that measures ROS activity within the cell. Hs68 cells were seeded into 24-well plates (4 × 104  cells/well) and allowed to attach overnight. Cells were incubated in a serum-free medium in the presence of various concentrations of CF extract (100–250 µg/mL) after UVB radiation or CML treatment. The cells were washed with PBS,  and the ROS detection reagent DCFDA was added to each well for 30 min. Images were captured using a fluorescence microscope (Leica DM IL LED, Leica Microsystems, Wetzlar, Germany), and the fluorescence (excitation/emission: 488 nm/520 nm) was measured using a microplate reader (Synergy HTX, BioTek Instruments, Winooski, VT, USA) [55].

4.9. Protein Expression by Western Blot Analysis

After the indicated treatments, Hs68 cells (5 × 105  cells/dish) were collected and homogenized with protein extraction buffer on ice. The cell lysates were vortexed for 30 min at 4 ◦C and centrifuged to obtain the supernatant as an intracellular protein. Prepared samples containing equal amounts of total protein were separated by electrophoresis on SDS-polyacrylamide gels and transferred to polyvinylidene difluoride blotting membranes. Nonspecific binding was blocked with non-fat milk in TBST buffer. The membrane was incubated with primary antibodies against, c-Jun, c-Fos, MMP-1, MMP-3, TGF-β, Smad3,  and pro-COL1A1 (Santa Cruz Biotechnology, Inc., Dallas, TX, USA), MMP-9, TIMP-1, and HO-1 (GeneTex, Inc., Irvine, CA, USA). Nrf2, Keap1, RAGE, p-c-Jun, p-p38, p-ERK, and β-  actin (Cell Signaling Technology, Inc., Danvers, MA, USA) at 4 ◦C overnight. Subsequently,  the membranes were washed with TBST buffer several times, and the corresponding anti-immunoglobulin G-horseradish peroxidase was used as the secondary antibody. The blots were incubated with a chemiluminescence detection reagent. Immunoreactive bands were detected with the ECL western blotting detection system (LAS-4000, Fujifilm, Tokyo, Japan),  and the signal intensity was quantified using ImageJ software (National Institutes of Health, Bethesda, MD, USA) [56].

4.10. Immunofluorescence Staining 

The cells were seeded on slides in a 6-well plate (Hs68 cells: 1 × 105  cells/well) and allowed to attach overnight. Cells were irradiated with UVB (40 mJ/cm2 ) and treated with 1 mL CF extract dissolved in DMEM. The cells were washed with PBS, fixed with paraformaldehyde for 30 min, and blocked using non-fat milk with Triton X-100/PBS buffer (Thermo Fisher Scientific, Inc., Waltham, MA, USA) for 60 min. Samples were incubated with primary rabbit anti-Nrf2 antibody overnight at 4 ◦C. Slides were washed with PBS  and then incubated with an anti-rabbit secondary antibody for 2 h at room temperature. Slides were mounted using ProLong® Gold Antifade Mountant with DAPI (Invitrogen, Waltham, MA, USA). All samples were analyzed using a confocal spectral microscope imaging system (Leica Microsystems, Wetzlar, Germany) [51].

4.11. Determination of Total Collagen Content

The total collagen content was quantified using the Sirius Red Collagen Detection kit (Chondrex Inc., Redmond, WA, USA). The cell culture medium was collected and mixed with the concentration reagent. The mixture was vortexed and incubated at 4 ◦C for 24 h. After incubation, the mixture was centrifuged at 10,000 rpm, and the supernatant was discarded. Following this, acetic acid was added to the microtube to dissolve the pellet,  and the obtained solution was used as the test sample. Sirius Red solution, for collagen staining, was added to each tube, vortexed, incubated for 20 min at room temperature, and centrifuged. The supernatant was removed by carefully pipetting without disturbing the pellet. The washing solution was added to each tube, and the above steps were repeated once. Finally, an extraction buffer was added and the absorbance was measured at 540 nm using a microplate reader (Sunrise, Tecan, Salzburg, Austria).

4.12. Statistical Analysis 

Data are presented as mean ± standard deviation in at least three independent experiments. All statistical analyses were performed using GraphPad Prism 5 (GraphPad Software Inc., San Diego, CA, USA). Significant differences between groups in the experiments were analyzed using one-way ANOVA, followed by Tukey’s post-hoc test. Differences with p < 0.05 were considered statistically significant.

5. Conclusions 

The present study reveals the molecular mechanisms responsible for the protective effect of CF against UV and AGEs-induced intracellular ROS generation and collagen degradation in skin fibroblasts. As a consequence, our study provides current understanding and information on the role of CF extract in the cellular defense system against oxidative stress via activation of the Nrf2/HO-1 signaling pathway. Additionally, CF not only modulates MAPK/AP-1/MMPs and TGF-β signaling pathways to prevent oxidative stress-induced collagen loss but also attenuates the upregulation of receptors for AGEs. CF  extract can be developed into healthy foods or applied to skincare products and cosmetics as an antioxidant, anti-photoaging, and antiglycation agent to delay aging in the future. The regulatory mechanisms of CF extract in Hs68 cells are shown in Figure 13.

Supplementary Materials: The following supporting information can be downloaded on the website, Figure S1: The MRM ion chromatogram and mass spectrum on ESI positive mode of 20-hydroxyecdysone and rutin standard. Figure S2: The total ion chromatogram (TIC) and extracted ion chromatogram (EIC) of 20-hydroxyecdysone (m/z 481) and rutin (m/z 611) in CF extract under MRM monitoring.

Author Contributions: Conceptualization, J.-L.L., K.-C.W., C.-Y.C., Y.-H.L. and H.-M.C.; Methodology, J.-L.L., C.-Y.C. and H.-M.C.; Investigation, J.-L.L. and Y.-J.L.; Resources, Y.-H.L.; Data curation, J.-L.L. and Y.-J.L.; Writing—original draft, J.-L.L. and H.-M.C.; Writing—review & editing, J.-L.L., Y.- J.L.; K.-C.W., C.-Y.C. and H.-M.C.; Visualization, J.-L.L.; Supervision, K.-C.W.; Project administration, H.-M.C. All authors have read and agreed to the published version of the manuscript.

Funding: This research was funded by the China Medical University (CMU106-ASIA-20, CHM106-5- 2 and CMU108-MF-38).

Institutional Review Board Statement: Not applicable. 

Informed Consent Statement: Not applicable. 

Data Availability Statement: Not applicable. 

Acknowledgments: The Chenopodium formosanum extract was supplied by TCI Co., Ltd. (Taipei, Taiwan). The experiments and data analysis were performed partially through the use of the Medical Research Core Facilities, Office of Research and Development at China Medical University (Taichung, Taiwan). Mass spectrometric analysis was performed by the Proteomics Research Core Laboratory, Office of Research and Development at China Medical University (Taichung, Taiwan).

Conflicts of Interest: The authors declare no conflict of interest. 

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