Search for


TEXT SIZE

search for



CrossRef (0)
Protective Effect of Aqueous Extract from Erigeron annuus Against Cell Death Induced by Free Radicals
Biomed Sci Letters 2022;28:307-311
Published online December 31, 2022;  https://doi.org/10.15616/BSL.2022.28.4.307
© 2022 The Korean Society For Biomedical Laboratory Sciences.

Myeongguk Jeong1,*, Hyeokjin Kwon1,*, Youngdon Ju1,*, Go-Eun Choi1,** and Kyung-Yae Hyun2,†,**

1Department of Biomedical Laboratory Science, College of Health Sciences, Catholic University of Pusan, Busan 46252, Korea
2Department of Clinical Laboratory Science, Dong-Eui University, Busan 47340, Korea
Correspondence to: Kyung-Yae Hyun. Department of Clinical Laboratory Science, Dong-Eui University, Busan 47340, Korea.
Tel: +82-51-890-2683, Fax: +82-0505-182-6877, e-mail: kyhyun@deu.ac.kr
*Graduate student, **Professor.
Received November 21, 2022; Accepted December 22, 2022.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
 Abstract
The extract of EA lacks studies showing its efficacy other than that it contains caffeic acid, an active compound that has antioxidant and neuroprotective effects on nerve cells. Therefore, in this study, we attempted to determine the effectiveness of EA extraction. In this study, we performed a DPPH assay to determine the antioxidant potential of EA. And then, the cytotoxic concentration of EA in HaCaT keratinocytes was determined, and the antioxidant effect was determined by measuring the malondialdehyde (MDA). The results of DPPH, a chemical antioxidant assay, clearly demonstrated the antioxidant capacity of EA extracted with distilled water. In addition, cell-based assays provide useful information on the protective effect of EA on oxidative stress-induced apoptosis.
Keywords : Erigeron annuus, Protective effect, 2,2-diphenyl-1-picrylhydrazyl (DPPH), Malondialdehyde (MDA), Free radicals
Preparation of EA extracts.

Erigeron annuus (EA) is exactly an annual plant. EA comes from the Greek words meaning that the plant blooms in late spring and forms fuzzy white seed heads while still producing new flowers. Only the leaves of EA are edible. You can use it anywhere you cook with vegetables. The extract of EA lacks studies showing its efficacy other than that it contains caffeic acid, an active compound that has antioxidant and neuroprotective effects on nerve cells. Therefore, in this study, we attempted to determine the effectiveness of EA extraction. There are many chemical methods that can be used to measure the activity of natural products. Among them, antioxidant activity is known to play an important role in many cellular activities, including signaling pathways, cell proliferation and differentiation (Wellen and Thompson, 2010). Excessive increases in reactive oxygen species (ROS) can lead to loss of cell function and eventually to apoptosis (Maynard et al., 2009; Circu and Aw, 2010; Ayala et al., 2014; Volpe et al., 2018). Antioxidant activity is based on free radical scavenging and competition with free radicals. Although limited, free radical scavenging assays have been widely used to determine the antioxidant capacity of natural products due to their ease and sensitivity (Olszowy and Dawidowicz, 2018). Moreover, Oxidative stress is assessed by detecting markers of lipid or protein oxidation. Lipid peroxidation is determined by the measurement of the final lipid peroxidation product, MDA. It is a good indicator of cellular antioxidant status (Szychta et al., 2014). Therefore, in this study, we performed a DPPH assay to determine the antioxidant potential of EA. And then, the cytotoxic concentration of EA in HaCaT keratinocytes was determined, and the antioxidant effect was determined by measuring the MDA.

EA was purchased Daum International (Hanam, Korea). The EA was washed and dried at room temperature for about 1 week. The dried EA was ground into a fine powder in a blender. About 10 g of EA powder was extracted with distilled water at 60℃. for 15 hours. The extract was filtered with filter paper. The filtrate was concentrated under reduced pressure at 45℃ using a rotary evaporator. The concentrated extract was lyophilized and quantified. The stock solution of the extract was stored frozen at a concentration of 1 mg/mL and used for further experimental analysis.

2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay.

DPPH analysis was performed with modifications as previously described (Lee et al., 2017). Briefly, 100 μL of two-fold serial dilutions of extracts from various concentration ranges were placed in 96-well microplates. Then, 100 μL of 200 μM DPPH (Sigma, St. Louis, USA) in methanol was added to each sample. For blank samples, the extract and DPPH solutions were replaced with distilled water. Assay controls contained 100 μL ascorbic acid and 100 μL DPPH solution. After incubating the reaction mixture for 15 min at room temperature, absorbance was recorded at 515 nm.

Cell culture.

HaCaT keratinocytes, a human keratinocyte cell line used in this study, were purchased from American Type Culture Collection (ATCC, VA, USA), and HaCaT cell lines were cultured in Dulbeccós modified Eagles medium (Gibco, Paisley, UK). A medium containing 10% fetal bovine serum (Gibco, Paisley, UK) and 1% penicillin (100 IU/mL)/streptomycin (100 μg/mL) (Gibco, Paisley, UK) was used. Culture conditions were maintained at 37℃ and 5% CO2.

Cell viability assay.

A methylthiazolyldiphenyl-tetrazolium bromide (MTT) colorimetric assay was performed to evaluate the cytotoxicity of the extract. Various concentrations of EA were exposed to HaCaT keratinocytes for 24 h. After removal of the medium, the cells were incubated with a 0.5 mg/mL MTT (Sigma, ST. Louis, USA) solution prepared in serum and phenol red-free culture medium for 4 hours. The resulting formazan crystals were dissolved in DMSO and absorbance was measured at 570 nm using a multi-plate reader.

MDA assay.

The level of the lipid peroxidation product MDA in cell lysates was determined according to the instructions in a commercial assay kit (Cayman, MI, USA). MDA was analyzed using the thiobarbituric acid (TBA) assay, which is based on the release of color complexes resulting from the reaction of TBA with MDA. The level of the lipid peroxidation products were measured spectrophotometrically.

Statistical analysis.

Data was expressed as mean ± standard error and compared by one-way analysis of variance. Significant differences between groups used Duncan's test. It was considered statistically significant when P < 0.05.

Extraction yield.

In this study, distilled water was used as the extraction solvent. After removing the extraction solvent using a rotary evaporator, the extract was lyophilized and quantified. The yield was 20.0%.

DPPH radical scavenging activity.

DPPH is a stable radical and is mainly used to determine the antioxidant potential of natural and synthetic compounds. First, the concentration of the substance that reduced the absorbance of the DPPH radical solution by 50% and the ascorbic acid standard was calculated. Then, the antioxidant capacity of the extract was shown graphically (Fig. 1). It was confirmed that the extract increased in a concentration-dependent manner compared to the ascorbic acid standard up to 800 g/mL.

Fig. 1. DPPH radical scavenging activity.

Cytotoxic effects of EA extracts on HaCaT keratinocytes.

We determined the effect of EA on the viability of HaCaT keratinocytes according to the MTT colorimetric assay method. EA affected the viability of HaCaT cells in a dose-dependent manner. At concentrations up to 200 μg/mL, cell viability was greater than 90% (Fig. 2). Therefore, we limited the concentration of the extract to 200 μg/mL for further experimental analysis.

Fig. 2. Cytotoxic effects of EA extracts on HaCaT keratinocytes.

Protective Effect of EA extracts against H<sub>2</sub>O<sub>2</sub>-Induced HaCaT keratinocytes.

EA extract shows cytoprotective effect on HaCaT keratinocytes against oxidative damage caused by H2O2. Exposure of HaCaT keratinocytes to 0.4 mM H2O2 for 2 h significantly reduced cell viability. Pretreatment of HaCaT keratinocytes with EA extract at a concentration of 200 μg/mL significantly reduced the cytotoxicity induced by H2O2 exposure (Fig. 3).

Fig. 3. Protective Effect of EA extracts against H2O2-induced HaCaT keratinocytes.

Effect of EA extracts against H<sub>2</sub>O<sub>2</sub>-induced MDA depletion in HaCaT keratinocytes.

MDA is considered as one of the key molecules in cellular antioxidant defense mechanisms. Thus, the production of MDA mainly depends on the amount of H2O2 produced by cells under stress conditions. In this study, we confirmed that pretreatment of cells with EA significantly rescued the H2O2-induced the production of MDA depletion (Fig. 4).

Fig. 4. Effect of EA extracts against H2O2-induced MDA depletion in HaCaT keratinocytes.

Oxidative stress results from overproduction of ROS or failure of cellular endogenous antioxidant defense mechanisms (Ray et al., 2012). In this respect, dietary supplementation of exogenous antioxidants such as plant extracts is very important to maintain a balanced cellular antioxidant status. In this study, we determined the antioxidant potential of EA using widely considered chemical radical scavenging assays such as DPPH. In addition, the antioxidant capacity of natural products is generally expressed as compared to the well-established standard antioxidant ascorbic acid. Furthermore, because even the same antioxidants have different effects in chemical and cell-based assays, we further investigated the antioxidant potential of EA extracts using a cell-based model. HaCaT keratinocytes, a human keratinocyte line, have a stable antioxidant defense system and are widely used to screen for beneficial effects of various antioxidants (Alía et al., 2006). The H2O2, a non-radical derivative of oxygen, is widely used as an exogenous oxidant to induce oxidative stress in cell-based antioxidant assays (Chen et al., 2007; Jiang et al., 2014; Han et al., 2017). Based on this background, in this study, the antioxidant efficacy of EA extract was confirmed by inducing oxidative stress in HaCaT keratinocytes using the H2O2.

In conclusion, the results of DPPH, a chemical antioxidant assay, clearly demonstrated the antioxidant capacity of EA extracted with distilled water. In addition, cell-based assays provide useful information on the protective effect of EA on oxidative stress-induced apoptosis.

ACKNOWLEDGEMENT

This work was supported by the Busan Innovation Institute of Industry, Science & Technology Planning (BISTEP) grant funded by the Busan MetropolitanCity (Project Name: Open Laboratory Business Meeting Market Demands).

CONFLICT OF INTEREST

No potential conflict of interest relevant to this article was reported.

References
  1. Al챠a M, Ramos S, Mateos R, Granado-Serrano AB, Bravo L, Goya L. Quercetin protects human hepatoma hepg2 against oxidative stress induced by tert-butyl hydroperoxide. Toxicology and Applied Pharmacology. 2006. 212: 110-118.
    Pubmed CrossRef
  2. Ayala A, Mu챰oz MF, Arg체elles S. Lipid peroxidation: Production, metabolism, and signaling mechanisms of malondialdehyde and 4-hydroxy-2-nonenal. Oxidative Medicine and Cellular Longevity. 2014.
    Pubmed KoreaMed CrossRef
  3. Chen CH, Liu TZ, Chen CH, Wong CH, Chen CH, Lu FJ, Chen SC. The efficacy of protective effects of tannic acid, gallic acid, ellagic acid, and propyl gallate against hydrogen peroxide릋nduced oxidative stress and DNA damages in imr90 cells. Molecular Nutrition &. Food Research. 2007. 51: 962-968.
    Pubmed CrossRef
  4. Circu ML, Aw TY. Reactive oxygen species, cellular redox systems, and apoptosis. Free radical biology and medicine. 2010. 48: 749-762.
    Pubmed KoreaMed CrossRef
  5. Han D, Chen W, Gu X, Shan R, Zou J, Liu G, Shahid M, Gao J, Han B. Cytoprotective effect of chlorogenic acid against hydrogen peroxide-induced oxidative stress in mc3t3-e1 cells through pi3k/akt-mediated nrf2/ho-1 signaling pathway. Oncotarget. 2017. 8: 14680.
    Pubmed KoreaMed CrossRef
  6. Jiang J, Yu S, Jiang Z, Liang C, Yu W, Li J, Du X, Wang H, Gao X, Wang X. N-acetyl-serotonin protects hepg2 cells from oxidative stress injury induced by hydrogen peroxide. Oxidative Medicine and Cellular Longevity. 2014.
    Pubmed KoreaMed CrossRef
  7. Lee J-S, Kim H-G, Lee H-W, Kim W-Y, Ahn Y-C, Son C-G. Pine needle extract prevents hippocampal memory impairment in acute restraint stress mouse model. Journal of Ethnopharmacology. 2017. 207: 226-236.
    Pubmed CrossRef
  8. Maynard S, Schurman SH, Harboe C, de Souza-Pinto NC, Bohr VA. Base excision repair of oxidative DNA damage and association with cancer and aging. Carcinogenesis. 2009. 30: 2-10.
    Pubmed KoreaMed CrossRef
  9. Olszowy M, Dawidowicz AL. Is it possible to use the dpph and abts methods for reliable estimation of antioxidant power of colored compounds? Chemical Papers. 2018. 72: 393-400.
    CrossRef
  10. Ray PD, Huang B-W, Tsuji Y. Reactive oxygen species (ros) homeostasis and redox regulation in cellular signaling. Cellular Signalling. 2012. 24: 981-990.
    Pubmed KoreaMed CrossRef
  11. Szychta P, Zadrozny M, Lewinski A, Karbownik-Lewinska M. Increased oxidative damage to membrane lipids following surgery for breast cancer. Neuroendocrinology Letters. 2014. 35.
    Pubmed
  12. Volpe CMO, Villar-Delfino PH, Dos Anjos PMF, Nogueira-Machado JA. Cellular death, reactive oxygen species (ros) and diabetic complications. Cell Death &. Disease. 2018. 9: 1-9.
    Pubmed KoreaMed CrossRef
  13. Wellen KE, Thompson CB. Cellular metabolic stress: Considering how cells respond to nutrient excess. Molecular Cell. 2010. 40: 323-332.
    Pubmed KoreaMed CrossRef