Applied Chemistry for Engineering (공업화학)

The Korean Society of Industrial and Engineering Chemistry (한국공업화학회)
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Antioxidant, Antimicrobial and Cytoprotective Effects of the Extract and Its Fraction Obtained from Rhizomes of Belamcanda chinensis (L.) DC

범부채 뿌리 추출물 및 분획물의 항산화, 항균 및 세포 보호 효과

  • Song, Ba Reum (Department of Fine Chemistry, Cosmetic R&D Center, Seoul National University of Science and Technology) ;
  • Lee, Sang Lae (Department of Fine Chemistry, Cosmetic R&D Center, Seoul National University of Science and Technology) ;
  • Lee, Yun Ju (Department of Fine Chemistry, Cosmetic R&D Center, Seoul National University of Science and Technology) ;
  • Shin, Hyuk Soo (Department of Fine Chemistry, Cosmetic R&D Center, Seoul National University of Science and Technology) ;
  • Park, Soo Nam (Department of Fine Chemistry, Cosmetic R&D Center, Seoul National University of Science and Technology)
  • 송바름 (서울과학기술대학교 정밀화학과, 화장품종합기술연구소) ;
  • 이상래 (서울과학기술대학교 정밀화학과, 화장품종합기술연구소) ;
  • 이윤주 (서울과학기술대학교 정밀화학과, 화장품종합기술연구소) ;
  • 신혁수 (서울과학기술대학교 정밀화학과, 화장품종합기술연구소) ;
  • 박수남 (서울과학기술대학교 정밀화학과, 화장품종합기술연구소)
  • Received : 2018.09.04
  • Accepted : 2018.10.15
  • Published : 2018.12.10
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Abstract

In this study, we investigated antioxidant, antimicrobial and cytoprotective effects of 50% ethanol extract and ethyl acetate fraction from rhizomes of Belamcanda chinensis (L.) DC. 1,1-Diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging activities ($FSC_{50}$) of the 50% ethanol extract and ethyl acetate fraction were 621.5 and $253.0{\mu}g/mL$, respectively. Total antioxidant capacities ($OSC_{50}$) of the extract and fraction were 13.6 and $3.0{\mu}g/mL$, respectively. Minimum inhibitory concentrations (MIC) of the ethyl acetate fraction for Staphylococcus aureus and Candida albicans were 156, $1,250{\mu}g/mL$, respectively, indicating similar or higher levels of those of using methyl paraben. Cytoprotective effects of the 50% ethanol extract against $^1O_2$-induced cellular damage (${\tau}_{50}$) showed in a dose dependent manner at 4 to $64{\mu}g/mL$. ${\tau}_{50}$ of the 50% ethanol extract, ethyl acetate fraction and (+)-${\alpha}$-tocopherol at $16{\mu}g/mL$ were 36.4, 45.0 and 45.8 min respectively, and the ethyl acetate fraction showed cytoprotective effects similar to (+)-${\alpha}$-tocopherol. In ultraviolet B radiation-induced HaCaT cell damage, the ethyl acetate fraction decreased intracellular reactive oxygen species (ROS) up to 45.9% at $8{\mu}g/mL$. Also in $H_2O_2$-induced HaCaT cell damage, the ethyl acetate fraction significantly increased the cell viability at $0.5{\sim}8.0{\mu}g/mL$. As a result of chemical analyses of the ethyl acetate fraction, the presence of flavonoids and polyphenol such as irisflorentin, irigenin, tectorigenin, resveratrol, iridin and tectoridin were identified. In conclusion, the extract/fraction from rhizomes of B. chinensis can be applied as a natural antioxidant and antimicrobial material to cosmetics.

본 연구에서는 범부채 뿌리 50% 에탄올 추출물 및 에틸아세테이트 분획물을 제조하고 이들의 항산화 및 항균 활성, 세포 보호 효능을 평가하였다. 1,1-Diphenyl-2-picrylhydrazyl (DPPH) 자유라디칼 소거 활성($FSC_{50}$) 측정 결과, 50% 에탄올 추출물은 $621.5{\mu}g/mL$, 에틸아세테이트 분획물은 $253.0{\mu}g/mL$이었다. Luminol 발광법을 이용한 총 항산화능($OSC_{50}$)은 추출물과 분획물에서 각각 13.6 및 $3.0{\mu}g/mL$이었다. 항균 활성 측정에서 Staphylococcus aureus 및 Candida albicans에 대한 에틸아세테이트 분획물의 최소저해농도(minimum inhibitory concentration, MIC)는 각각 156 및 $1,250{\mu}g/mL$으로 나타났으며, 화장품에 사용하는 기존 방부제인 methyl paraben보다 유사하거나 더 높은 활성을 보여주었다. $^1O_2$로 유도된 세포 손상에 대한 보호 효과(${\tau}_{50}$)에서 50% 에탄올 추출물은 $4{\sim}64{\mu}g/mL$ 농도 범위에서 농도 의존적으로 세포 보호 활성을 나타냈다. $16{\mu}g/mL$ 농도에서 50% 에탄올 추출물, 에틸아세테이트 분획물 및 (+)-${\alpha}$-tocopherol의 ${\tau}_{50}$은 각각 36.4, 45.0 및 45.8 min이었으며, 에틸아세테이트 분획물은 $^1O_2$로 유도된 세포 손상에서 (+)-${\alpha}$-tocopherol과 유사한 세포 보호 활성을 나타냈다. UVB로 유도된 HaCaT 세포 손상에서 에틸아세테이트 분획물은 $8{\mu}g/mL$에서 세포 내 활성산소종(reactive oxygen species, ROS)을 최대 45.9%까지 감소시켰다. 과산화수소로 유도된 HaCaT 세포 손상에서도 에틸아세테이트 분획물은 $0.5{\sim}8.0{\mu}g/mL$에서 세포 생존율을 유의적으로 증가시켰다. 범부채 뿌리 에틸아세테이트 분획물의 성분 분석 결과, irisflorentin, irigenin, tectorigenin, resveratrol, iridin 및 tectoridin 등의 플라보노이드 및 폴리페놀 성분이 확인되었다. 결론적으로 범부채 뿌리 추출물 및 분획물은 화장품의 천연 항산화 및 항균 소재로서 적용 가능성이 있음을 시사한다.

Keywords

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Figure 1. Preparation of 50% EtOH extract and EtOAc fraction from rhizomes of B. chinensis.

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Figure 2. FSC50 of 50% EtOH extract and EtOAc fraction from rhizomes of B. chinensis and (+)-α-tocopherol. Data are indicated as mean ± S.D. *p < 0.05 compared with (+)-α-tocopherol.

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Figure 3. OSC50 of 50% EtOH extract and EtOAc fraction from rhizomes of B. chinensis and L-Ascorbic acid in Fe3+-EDTA/H2O2 system by luminol-dependent chemiluminescence assay. Data are indicated as mean ± S.D. *p < 0.05 compared with L-ascorbic acid.

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Figure 5. Effects of 50% EtOH extract and EtOAc fraction from rhizomes of B. chinensis on HaCaT cell viability. HaCaT cells were treated with different concentration of samples for 24 h and cell viability was determined using the MTT assay. Data are presented as mean ± S.D.

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Figure 6. Effects of 50% EtOH extract and EtOAc fraction from rhizomes of B. chinensis on UVB-induced oxidative stress in HaCaT cell. 50% EtOH extracts and EtOAc fraction scavenged UVB-induced upregulation of intracellular ROS production. The H2DCF-DA probe was used to investigate intracellular ROS levels. Data are presented as mean ± S.D. *p < 0.05 compared with UVB treated control in EtOAc fraction groups by one-way ANOVA.

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Figure 7. Cell protective effects of 50% ethanol extract and EtOAc fraction from rhizome of B. chinensis on H2O2-induced damaged HaCaT cell. HaCaT cells were treated with different concentration of samples for 2 h before being exposed to oxidative stress. Data are presented as mean ± S.D. *p < 0.05 compared with H2O2 treated control in EtOAc fraction groups by one-way ANOVA.

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Figure 8. TLC chromatogram of EtOAc fraction from rhizomes of B. chinensis extract and reference. Eluent system; toluene : chloroform : methanol : formic acid = 1 : 7 : 1 : 1 (v/v), ① ethyl acetate fraction, ② resveratrol.

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Figure 9. HPLC chromatogram of the EtOAc fraction from rhizomes of B. chinensis extract at λ = 254~400 nm. 1: tectoridin, 2: iridin, 3: resveratrol, 4: tectorigenin, 5: irigenin, 6: irisflorentin.

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Figure 4. Antimicrobial activity of 50% EtOH extract and EtOAc fraction from rhizomes of B. chinensis against bacteria and fungi. A: S. aureus, B: E. coli, C: P. aeruginosa, D: C. albicans, E: A. niger, a: methyl parben (control), b: EtOAc fraction, c: 50% EtOH extract.

Table 1. List of Strains and Cultivation Condition Used for Antimicrobial Experiment

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Table 2. TLC Mobile Phase for Separation of EtOAc Fraction from Rhizomes of B. chinensis Extracts

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Table 3. HPLC Condition for Separation of EtOAc Fraction from Rhizomes of B. chinensis Extracts

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Table 4. Antimicrobial Activity of 50% EtOH Extract and EtOAc Fraction from rhizomes of B. chinensis against Bacteria and Fungi

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Table 5. Minimum Inhibitory Concentration (MIC, μg/mL) of 50% EtOH Extract and EtOAc Fraction from Rhizomes of B. chinensis against Bacteria and Fungi

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Table 6. Cellular Protective Effects of 50% EtOH Extract and EtOAc Fraction from Rhizomes of B. chinensis and (+)-α-Tocopherol on Rose-bengal Sensitized Photohemolysis of Human Erythrocytes

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Table 7. TLC, HPLC, and LC-MS Data of EtOAc Fraction from Rhizomes of B. chinensis Extract

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