Antimicrobial and Antioxidant Activities of Perilla frutescens var. acuta Extract and Its Fraction and Their Component Analyses

자소엽 추출물의 항균 및 항산화 효과와 성분분석

Abstract

In this study, antimicrobial and antioxidative activities of Perilla frutescens var. acuta were investigated with 50% ethanol and the ethyl acetate fraction and also the components were analyzed. The minimum inhibitory concentration (MIC) of the ethyl acetate fraction for both Staphylococcus aureus and Pseudomonas aeruginosa were $78{\mu}g/mL$, indicating high antimicrobial effects. The free radical scavenging activity ($FSC_{50}$) and the reactive oxygen species (ROS) scavenging activity ($OSC_{50}$) in $Fe^{3+}-EDTA/H_2O_2$ system values of the ethyl acetate fraction were $25.90{\mu}g/mL$ and $1.40{\mu}g/mL$, respectively. After the cell damage induced by $400mJ/cm^2$ UVB irradiation, the cytoprotective effect of the ethyl acetate fraction of P. frutescens var. acuta showed the concentration dependent manner ranging from 2.0 to $16.0{\mu}g/mL$. The intracellular ROS inhibitory activity in HaCaT cells decreased to 28.6% and 40.7% for the 50% ethanol extract and ethyl acetate fraction, respectively at the concentration of $32{\mu}g/mL$. Components of rosmarinic acid, luteolin, apigenin, caffeic acid and ethyl caffeate were identified in the ethyl acetate fraction. These results suggest that the extract and fraction of P. frutescens var. acuta may be applied to the field of cosmetics as a natural material that protects the skin from an external environment by having antimicrobial and antioxidative activities.

본 연구에서는 자소엽의 50% 에탄올 추출물 및 에틸아세테이트 분획을 제조하여 항균 및 항산화 활성을 평가하고 성분 분석을 진행하였다. 피부 상재균에 대한 항균 활성을 측정한 결과, S. aureus 및 P. aeruginosa에 대해서 에틸아세테이트 분획의 최소저해농도(minimum inhibitory concentration, MIC) 값은 모두 $78{\mu}g/mL$로 나타나 항균 활성이 매우 우수함을 확인하였다. 2,2-diphenyl-1-picrylhydrazyl (DPPH) 라디칼 소거 활성($FSC_{50}$)과 $Fe^{3+}-EDTA/H_2O_2$계에서의 활성산소 소거 활성($OSC_{50}$) 측정 결과 50% 에탄올 추출물 보다 에틸아세테이트 분획에서 높은 활성을 보였고 각각 25.90, $1.40{\mu}g/mL$로 나타났다. HaCaT 세포에서 세포 내 ROS 억제 효능은 최고 농도인 $32{\mu}g/mL$에서 50% 에탄올 추출물과 에틸아세테이트 분획 각각 PC군 대비 28.6, 40.7%의 감소율을 보였고 UVB에 대한 세포보호효과는 에틸아세테이트 분획의 $2.0-16.0{\mu}g/mL$ 농도에서 농도 의존적으로 나타났다. 자소엽 추출물의 에틸아세테이트 분획을 이용한 성분 분석 결과 rosmarinic acid, luteolin, apigenin, caffeic acid 및 ethyl caffeate가 있음을 확인하였다. 이상의 결과들은 자소엽 추출물 및 분획이 항균 및 항산화 효과를 가짐을 보여주며 외부 환경으로부터 피부를 보호할 수 있는 천연 소재로써 화장품 분야에서 응용 가능성이 있음을 시사한다.

Figure 1. Fractionation scheme of P. frutescens var. acuta 50% EtOH extract and ethyl acetate fraction.

Figure 2. Free radical scavenging activities of 50% EtOH extract and EtOAc fraction from P. frutescens var. acuta and (+)-α-tocopherol. Data are presented as mean ± S.D. *p < 0.05 compared with (+)-α- tocopherol.

Figure 3. ROS scavenging activities of 50% EtOH extract and EtOAc fraction from P. frutescens var. acuta and L-ascorbic acid in Fe³⁺-EDTA/H₂O₂ system by luminol-dependent chemiluminescence assay. Data are presented as mean ± S.D. *p < 0.05 and **p < 0.01 compared with L-ascorbic acid.

Figure 4. Effects of 50% EtOH extract and EtoAc fraction from P. frutescens var. acuta on HaCaT cells viability. HaCaT cells were treated with different concentration of samples for 24 h, and cytotoxicity was then determined by the MTT assay. Data are presented as mean ± S.D.

Figure 5. Effects of 50% EtOH extract and EtOAc fraction from P. frutescens var. acuta on UVB-induced ROS generation in HaCaT cells. HaCaT cells were treated with different concentration of 50% EtOH extract and EtOAC fraction for 24 h, after being stimulated with UVB. ROS generation was measured by the fluorescence intensity of H₂DCF-DA. Data are presented as mean ± S.D. ^*p < 0.05 compared with UVB treated control in 50% EtOH extract dose-treated group, ^#p < 0.05 compared with UVB treated control in EtOAc fraction dose-treated groups.

Figure 6. 50% EtOH extract and EtOAc fraction from P. frutescens var. acuta treatment protect HaCaT cells against UVB-mediated decreased cell viability. HaCaT cells were treated with different concentration of samples for 24 h after being exposed to oxidative stress. Data are presented as mean ± S.D. ^*p < 0.05 compared with untreated control in 50% EtOH extract dose-treated group, ^#p < 0.05 compared with untreated control in EtOAc fraction dose-treated groups.

Figure 7. TLC chromatogram of 50% EtOH extract and EtOAc fraction from P. frutescens var. acuta and references. Eluent system; hexane : ethyl acetate : acetic acid = 24 : 14 : 5 (v/v), ① rosmarinic acid ② luteolin ③ 50% ethanol extract ④ ethyl acetate fraction ⑤ caffeic acid ⑥ apigenin ⑦ ethyl caffeate.

Figure 8. (a) Chromatogram of standards, (b) 50% EtOH extract, (c) EtOAc fraction from P. frutescens var. acuta extract at 254-400 nm. Peak 1, caffeic acid; peak 2, rosmarinic acid; peak 3, ethyl caffeate; peak 4, luteolin; peak 5, apigenin.

Table 1. HPLC Condition for Separation of 50% EtOH Extract and EtOAc Fraction of P. frutescens var. acuta Extract

Table 2. Minimum Inhibitory Concentration (MIC, μg/mL) and Minimum Bactericidal Concentration (MBC, μg/mL) of 50% EtOH Extract and Ethyl Acetate Fraction from P. frutescens var. acuta and Reference

Table 3. DPPH Radical Scavenging Activity of 50% EtOH Extract and Ethyl Acetate Fraction from P. frutescens var. acuta and Reference

Table 4. Reactive Oxygen Species Scavenging Activity of 50% EtOH Extract and Ethyl Acetate Fraction from P. frutescens var. acuta and Reference in Fe³⁺-EDTA/H₂O₂ System by Luminol-Dependent Chemiluminescence Assay

Table 5. Bioactive Compounds Contents of P. frutescens var. acuta 50% EtOH Extract and Ethyl Acetate Fraction

Table 6. LC/ESI-MS and UV Spectrum Characteristics of Ethyl Acetate Fraction from P. frutescens var. acuta Extract