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Effects of Helichrysum bracteatum flower extracts on UVB irradiation-induced inflammatory biomarker expression
Biomedical Dermatology volume 3, Article number: 9 (2019)
The present study aimed to investigate the anti-inflammatory activity of Helichrysum bracteatum (H. bracteatum) flower extracts in vitro.
H. bracteatum flowers were extracted with water, ethanol and 1,3-butylene glycol, and the anti-oxidative activities of the extracts were measured using a 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay. The inhibition of the expression of inflammation-related genes, including tumour necrosis factor-alpha (TNF-α), interleukin-6 (IL-6) and cyclooxygenase-2 (COX-2), was evaluated in vitro using reverse transcription-PCR in ultraviolet B (UVB)-irradiated human epidermal keratinocytes (HEKa cells). To investigate the inhibitory effects of H. bracteatum flower extracts on UVB-induced inflammatory responses in HEKa cells, the production of nitric oxide (NO) and TNF-α was measured using enzyme-linked immunosorbent assays. Results were expressed as the mean ± standard deviation; statistical significance was calculated using the Student’s t-test.
The DPPH assay results showed that H. bracteatum flower extracts have good anti-oxidative effects and inhibited the expression of inflammation-related genes IL-6, COX-2 and TNF-α. Moreover, the production of NO and TNF-α was inhibited by H. bracteatum flower extracts.
These findings indicate that H. bracteatum flower extracts have efficacy against UVB-induced inflammation-related gene expression.
Skin serves as a barrier between the human body and environmental stresses, such as pollution, ultraviolet (UV) radiation and other factors. These stresses trigger inflammatory and immune responses in the skin by stimulating the biosynthesis and release of pro-inflammatory cytokines, such as tumour necrosis factor (TNF) and interleukins (ILs) (Kwon et al. 2001; Parrado et al. 2019; Takashima and Bergstresser 1996; Pupe et al. 2002; Nedoszytko et al. 2014; Striz et al. 2014). It has been reported that chronic inflammation leads to the development and progression of skin diseases (Benedetto et al., 2012; Masuoka et al. 2012; Lee and Chang 2012). For example, cyclooxygenase-2 (COX-2) and inducible nitric oxide (NO) synthase, which are enzymes produced during inflammatory responses induced by UV damage, produce prostaglandin E2 and NO, respectively (Giuliano and Warner 2002; Schafer and Werner 2008; Hong et al. 2002; Jean et al. 2008). Meanwhile, prolonged UV exposure promotes photoageing, which is primarily mediated by reactive oxygen species (ROS) that deplete anti-oxidant defence systems in the skin. At a histological level, photoaged skin is characterized by a loss of collagen and elastin fibres (Rhie et al. 2001).
As a result of environmental stress-mediated ROS synthesis, a wide array of signalling pathways can be aberrantly activated, leading to the abnormal expression of inflammatory genes (Varga et al. 2013). Upon their activation, macrophages express an increased level of NO (Kim et al. 2001; Nathan and Hibbs Jr 1991).
Increasing levels of pollution introduce considerable amounts of hazardous chemicals into the environment, which subsequently cause skin irritation and inflammation from prolonged exposure. Therefore, corticosteroids have been widely used for the treatment of inflammatory skin diseases to effectively ameliorate inflammation. However, prolonged treatment with corticosteroid preparations may lead to side effects, such as skin atrophy and cutaneous reactivity (Hughes et al. 1995). Thus, the development and use of more effective anti-inflammatory agents are desired.
In the field of herbal medicine, extensive evidence has shown that plants synthesize anti-oxidants and anti-inflammatory compounds to suppress UV radiation-mediated ROS. Helichrysum bracteatum (H. bracteatum) is an annual herb that has been naturalized at high altitudes. H. bracteatum was originally found in Australia and is now widely grown in China (Liu et al. 2007). Helichrysum spp. are rich in secondary metabolites that have medicinal value (Kelmanson et al. 2000). However, the anti-inflammatory activity of H. bracteatum flower extracts has not been widely studied. Therefore, the present study was undertaken to investigate the anti-oxidative and anti-inflammatory effects of H. bracteatum flower extracts in vitro on the expression of inflammatory biomarkers (TNF-α, IL-6 and COX-2).
Preparation of H. bracteatum flower extracts
Dried H. bracteatum flowers were ground and extracted in 20 volumes of extraction solvent at room temperature for 24 h with vigorous shaking. For extraction purposes, water, 50% ethanol, 95% ethanol, 50% 1,3-butylene glycol and 100% 1,3-butylene glycol were used. The mixtures were then filtered using filter paper (No. 2 qualitative filter papers, Whatman, England), and the resulting filtrates were concentrated under vacuum using a rotary evaporator (N-1110, EYELA, USA). The concentrated extracts were stored at −20 °C until further use.
Human epidermal keratinocytes (HEKa cells) were cultured in Dulbecco’s modified Eagle’s medium (Gibco, Thermo Fisher Scientific, USA) containing 1% of penicillin/streptomycin (Sigma Aldrich, USA) and 10% of foetal bovine serum (Gibco, USA) at 37 °C in a 5% CO2 incubator.
Cytotoxicity was determined using the modified Mosmann’s method (Mosmann 1983). Approximately 1 × 105 HEKa cells were seeded into a 96-well plate and incubated for 24 h. After 24 h, the cells were then treated with H. bracteatum flower extracts at the following concentrations: 10, 20, 50, 100 and 500 μg/mL. After treatment for 24 h, the cells were washed twice with phosphate-buffered saline, and 0.5 mg/mL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was subsequently added to each well. After incubation for 4 h with MTT, 150 μL dimethyl sulfoxide was added to each well and mixed thoroughly to dissolve the dark blue crystals. After 20 min, when all the crystals were dissolved at room temperature, the absorbance at 570 nm was measured using spectrophotometry.
DPPH free radical-scavenging activity assay
The free radical-scavenging activity of H. bracteatum flower extracts and the effects of the extraction solvents on this activity were determined using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay. Briefly, 50 μL of the diluted H. bracteatum flower extracts were mixed with 100 μL of 0.1 mM DPPH solution. DPPH solution without the test sample was used as a control. Next, the absorbance was measured at 515 nm after the mixture was incubated for 30 min at room temperature. The anti-oxidative activity was calculated using the formula below and expressed as the percentage of DPPH radical elimination:
where Ablank is the absorbance of the blank DPPH solution and Asample is the absorbance of the DPPH solution after the addition of H. bracteatum flower extracts.
Approximately 1 × 105 HEKa cells were seeded into a 96-well plate and cultured for 18 h. Next, the cells were treated with 100 μg/mL H. bracteatum flower extracts. After 24 h of culture, the cells were irradiated with 200 mJ/cm2 UVB for 10 min, and the supernatant was harvested to determine the TNF-α concentration. The human TNF-α ELISA kit (Abcam, England) was used, according to the manufacturer’s instructions. Absorbance was measured at 450 nm using a microplate reader (Epoch2C, BioTek, USA).
A NO detection kit (Intron biotechnology, Korea) was used to determine the NO concentration. Approximately 1 × 105 HEKa cells were seeded into a 96-well plate and cultured for 18 h. Next, the cells were treated with different concentrations of the H. bracteatum flower extracts. After 24 h of treatment, the cells were irradiated with 200 mJ/cm2 UVB for 10 min, and the supernatants were used to determine the NO concentrations, according to the manufacturer’s instructions. Absorbance at 540 nm was measured using a microplate reader (Epoch2C, BioTek). Sodium nitrate was used to obtain a standard curve, and the obtained absorbance values were used to calculate the NO concentration in the cell cultures.
RNA isolation and reverse transcription (RT)-PCR
The total RNA of treated and untreated cells was isolated using the RNAiso reagent (Takara, Japan), according to the manufacturer’s instructions. Following the validation of purified RNA using spectrophotometry, the RNA was converted into cDNA using the First-Strand cDNA Synthesis SuperMix kit (Tragen, China). The converted cDNA was used as a template for amplification using PCR primers (Bioneer, Korea). The specific forward and reverse primers for each gene are presented in Table 1. Subsequently, the PCR products were loaded on a 1% agarose gel and evaluated using the Gel Documentation system (GelDoc EZ system, BioRad, Korea).
All experiments were conducted in triplicate; data are shown as the mean ± standard deviation. Statistical analysis was performed with Microsoft Excel (Microsoft, USA) using the Student’s t-test.
Anti-oxidative effects of H. bracteatum flower extracts
The effects of H. bracteatum flower extracts on free radical scavenging were confirmed using a DPPH assay. The results showed that the anti-oxidative effects of H. bracteatum flower extracts increased in a concentration-dependent manner. Among the five different solvents examined, a 50% ethanolic extract of H. bracteatum flowers exhibited the highest free radical-scavenging effects (84%), which was higher than that of quercetin (the positive control) (Fig. 1).
Cell viability after treatment with H. bracteatum flower extracts
The viability of HEKa cells was measured after treatment for 24 h with H. bracteatum flower extracts at concentrations of 10, 20, 50, 100 and 500 μg/mL. The results showed that cell viability decreased when higher concentrations of the extract were added: 91.14% with 10 μg/mL extract, 88.61% with 20 μg/mL extract, 83.12% with 50 μg/mL extract and 82.28% with 100 μg/mL extract. At 500 μg/mL, the H. bracteatum flower extract reduced cell viability to 77.64%; hence, it was considered cytotoxic. Therefore, 100 μg/mL was the maximum concentration that was used for subsequent experiments (Fig. 2).
Inhibition of TNF-α production
The effects of H. bracteatum flower extracts on the inhibition of TNF-α production were assessed using an ELISA kit. The results showed that the expression of the TNF-α gene in HEKa cells was inhibited in a dose-dependent manner. TNF-α production levels were measured as 302.35 pg/mL with 5 μg/mL extracts, 274.65 pg/mL with 10 μg/mL extracts, 266.52 pg/mL with 20 μg/mL extracts, 266.52 pg/mL with 50 μg/mL extracts and 188.60 pg/mL with 100 μg/mL extracts, whereas 10 μM hydrocortisone resulted in the production of 122.77 pg/mL TNF-α (Fig. 3).
Inhibition of NO production
To determine the effects of H. bracteatum flower extracts on NO production in HEKa cells, cells were treated with H. bracteatum flower extracts at concentrations of 5, 10, 20, 50 and 100 μg/mL. The results showed that the production of NO in HEKa cells was inhibited by H. bracteatum flower extracts in a dose-dependent manner. The amounts of NO were measured as 30.97 μM with 5 μg/mL extracts, 30.56 μM with 10 μg/mL extracts, 30.28 μM with 20 μg/mL extracts, 28.19 μM with 50 μg/mL extracts and 24.72 μM with 100 μg/mL extracts, whereas 10 μM hydrocortisone treatment resulted in the production of 19.72 μM NO (Fig. 4).
Gene expression levels of cytokines (IL-6, TNF-α and COX-2)
The effects of H. bracteatum flower extracts on the inhibition of IL-6 gene expression were studied using RT-PCR. The results showed that the expression of the IL-6 gene in HEKa cells was inhibited in a dose-dependent manner. IL-6 expression levels were measured as 82.73% with 5 μg/mL extracts, 73.56% with 10 μg/mL extracts, 51.42% with 20 μg/mL extracts, 50.45% with 50 μg/mL extracts and 41.27% with 100 μg/mL extracts, whereas 10 μM hydrocortisone resulted in an IL-6 expression level of 29.34% (Fig. 5a).
Similarly, H. bracteatum flower extracts inhibited the expression of the TNF-α gene in HEKa cells in a dose-dependent manner. TNF-α expression levels were measured as 72.21% with 5 μg/mL extracts, 69.83% with 10 μg/mL extracts, 49.93% with 20 μg/mL extracts, 48.32% with 50 μg/mL extracts and 40.55% with 100 μg/mL extracts, whereas 10 μM hydrocortisone resulted in a TNF-α expression level of 27.05% (Fig. 5b).
H. bracteatum flower extracts also inhibited the expression of the COX-2 gene in HEKa cells in a dose-dependent manner. COX-2 expression levels were measured as 79.78% with 5 μg/mL extracts, 72.96% with 10 μg/mL extracts, 64.72% with 20 μg/mL extracts, 53.54% with 50 μg/mL extracts and 50.81% with 100 μg/mL extracts, whereas 10 μM hydrocortisone resulted in a COX-2 expression level of 36.83% (Fig. 5c).
H. bracteatum flower extracts prepared using five different solvents had anti-oxidative effects greater than 50% as determined by the DPPH assay. Specifically, the 50% ethanolic extract showed the highest anti-oxidative effect of all the tested solvents. Liu et al. reported that H. bracteatum extracts contain several well-known anti-oxidants, including quercetin, chrysoeriol and isoorientin (Liu et al. 2007; Yuan et al. 2016; Mishra et al. 2003; Zheng et al. 2017). In this study, the anti-oxidative effects of H. bracteatum flower extracts prepared with 50% ethanol were higher than those of quercetin.
Exposure of the skin to environmental or physiological stresses can upregulate TNF-α, ILs and other cytokines that cause inflammation. These inflammatory factors retard the growth of skin cells while aggravating skin inflammation and ageing. TNF-α is a prime inducer and driver of inflammatory skin diseases (Han et al. 2001). In this study, the anti-inflammatory effects of H. bracteatum flower extracts were studied on human skin cells in vitro. As shown in Fig. 3, HEKa cells treated with 100 μg/mL H. bracteatum flower extracts produced 188.6 pg/mL TNF-α, which is approximately 45% of levels generated after UVB irradiation. Effects of H. bracteatum flower extracts on NO production were also similar. UV irradiation caused the production of 31.11 μM NO, whereas 100 μg/mL H. bracteatum flower extracts caused the production of 24.72 μM NO, which was approximately 20% lower than that of the control. For quercetin, a 37% reduction of NO production was observed compared to the control (Fig. 4).
HEKa cells irradiated with 200 mJ/cm2 UVB exhibited increases in the expression levels of TNF-α, IL-6 and COX-2. However, H. bracteatum flower extracts reduced these expression levels in a dose-dependent manner (Fig. 5).
Liu et al. (2007) reported that prunin (a hypocholesterolaemic agent), quercetin (an inhibitor of tumours and lipid peroxidation), luteolin (an antimicrobial agent), isoorientin (an anti-oxidative, anti-nociceptive and anti-inflammatory agent) and 4-hydroxymethyl-1-methoxycabonylazulene (an anti-inflammatory and antimicrobial agent) were found in ethanolic extracts of H. bracteatum flowers. Hence, it is possible that the anti-inflammatory effects of H. bracteatum are contributed by isoorientin, 4-hydroxymethyl-1-methoxycabonylazμLene and quercetin. Thus, chemical analyses and an evaluation of the active compounds in H. bracteatum flower extracts are required.
Overall, our results provide the potential role of H. bracteatum flower extracts in the inhibition of UVB-induced inflammatory responses in human keratinocytes. Using various biochemical assays including qRT-PCR and ELISA assays, we show that H. bracteatum flower extracts have beneficial functions on keratinocytes. The extracts have the ROS-scavenging effect and also can promote the UV irradiation-induced cellular inflammatory responses, including production of cytokines (TNF-a and IL-6), NO and expression of COX-2 gene in human keratinocytes. Since UV irradiation induces acute inflammation in the skin by increasing pro-inflammatory cytokine production in keratinocytes (Lee et al. 2017), our data suggest that H. bracteatum flower extracts can potentially be used as the novel dermatological and cosmetic ingredient for reduction of skin inflammation.
The current findings suggest that a 50% ethanolic extract of H. bracteatum flowers elicits the strongest DPPH free radical-scavenging activity among the other solvents used for extraction; this extract also ameliorates skin inflammation at the mRNA level, as evidenced by the downregulated expression of IL-6, TNF-α and COX-2 in HEKa cells. In addition, the inhibition of TNF-α and NO production further confirms the anti-inflammatory effects of H. bracteatum flower extracts. In conclusion, the findings of this study indicate that H. bracteatum flower extracts have efficacy against the expression of inflammation-related genes.
Availability of data and materials
Enzyme-linked immune sorbent assay
Human epidermal keratinocyte
Reactive oxygen species
Reverse transcription polymerase chain reaction
Tumour necrosis factor
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This study was supported by Mannay Asia R&D Center (Anyang, Korea) and a part of the project (Grant No. 20190070), funded by the Ministry of Oceans and Fisheries, Republic of Korea.
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Kim, Y.J., Seok, J.H., Cheung, W. et al. Effects of Helichrysum bracteatum flower extracts on UVB irradiation-induced inflammatory biomarker expression. biomed dermatol 3, 9 (2019). https://doi.org/10.1186/s41702-019-0049-8
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