Journal of Life Science (생명과학회지)

Korean Society of Life Science (한국생명과학회)
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Antibacterial and Antibiofilm Activities of Diospyros malabarica Stem Extract against Streptococcus mutans

Streptococcus mutans에 대한 인도감나무 줄기 추출물의 항균활성 및 생물막 형성 억제 효과

  • Kim, Hye Soo (Department of Pharmaceutical Engineering, Gyeongnam National University of Science and Technology) ;
  • Lee, Sang Woo (International Biological Material Research Center, Korea Research Institute of Bioscience and Biotechnology) ;
  • Sydara, Kongmany (Institute of Traditional Medicine, Ministry of Health, Vientiane Capital) ;
  • Cho, Soo Jeong (Department of Pharmaceutical Engineering, Gyeongnam National University of Science and Technology)
  • Received : 2018.12.06
  • Accepted : 2019.01.09
  • Published : 2019.01.30
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Abstract

The objective of this study was to evaluate the potential of Diospyros malabarica stem extract, a natural materials, in oral health material. With this aim in mind, thin layer chromatography (TLC), TLC-bioautography, high-performance liquid chromatography (HPLC), electrospray ionization-mass spectrometry (ESI-MS), scanning electron microscopy (SEM), and real-time qPCR were performed. The antibacterial activity of D. malabarica stem extract against Streptococcus mutans KCTC3065 was confirmed in an n-hexane fraction with low polarity. The molecular weight of the antibacterial compound was estimated to be 188 by ESI-MS analysis. The inhibitory effects of the extract on biofilm formation and gene expression related to biofilm formation of S. mutans were determined by SEM and real-time PCR analysis. The extract inhibited the formation of S. mutans biofilms at D. malabarica stem extract concentrations of 1 mg/ml, as shown by SEM. The real-time PCR analysis showed that the expression of the gtfC gene, which is associated with biofilm formation, was significantly decreased in a dose-dependent manner. Based on the above results, it can be concluded that D. malabarica stem extracts, a natural materials, can be used in oral health products to suppress the formation of biofilms by inhibiting tooth adhesion of S. mutans, a causative agent of dental caries.

본 연구에서는 천연물 유래 구강 건강소재로써 인도감나무 줄기 추출물의 이용 가능성을 평가하기 위해 TLC, TLC-bioautography, HPLC, ESI-MS 등을 이용하여 인도감나무 줄기 추출물로부터 S. mutans KCTC3065에 대해 항균활성이 있는 항균물질을 분리하고 주사전자현미경과 real-time PCR을 이용하여 추출물이 S. mutans의 생물막에 미치는 영향을 조사하였다. S. mutans에 대한 인도감나무 줄기 추출물의 항균활성은 극성이 낮은 n-hexane 분획물에서 확인되었고 TLC, TLC-bioautography, HPLC에 의해 분리된 항균물질의 분자량은 ESI-MS분석 결과 188로 추정되었다. 인도감나무 줄기 추출물(1 mg/ml) 처리에 따른 S. mutans의 바이오필름 바이오매스 변화는 주사전자현미경으로 관찰하였으며 추출물을 처리하지 않은 대조구는 추출물 처리구에 비해 세포가 군집을 이루고 모여 있었으며 세포 주변에서 바이오필름이 관찰되었지만 추출물을 처리한 처리구의 세포 주변에서는 바이오필름을 관찰할 수 없었다. Real-time PCR을 이용하여 바이오필름 생성 과정에서 치면 부착에 필수적인 GTFs의 발현 양상을 조사한 결과, 인도감나무 추출물이 0.2-1.0 mg/ml의 농도로 처리된 배양액에서 gtfB 유전자 발현은 추출물의 농도에 따라 큰 변화가 없었지만 gtfC 유전자 발현은 추출물의 농도가 높아질수록 감소하는 경향을 나타내었다. 이상의 결과를 종합하면 인도감나무 줄기 추출물은 바이오필름 형성 단계에서 치아우식증 원인균인 S. mutans의 초기 치면 부착을 저해함으로서 바이오필름 생성을 억제할 수 있는 천연물 유래 구강 건강소재로써 이용 가능성이 높을 것으로 판단된다.

Keywords

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Fig. 1. Isolation procedure of putative antibacterial compound from D. malabarica stems.

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Fig. 2. Antibacterial activity of partitioned fraction (1 mg/ disc) with solvents of D. malabarica stems against S. mutans KCTC3065. a: Methanol as a negative control, b: Methanol extracts from D. malabarica stems as a positive control, c: n-Hexane fraction, d: Chloroform fraction, e: Ethyl acetate fraction, f: n-Butanol fraction, g: Water soluble fraction.

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Fig. 3. TLC (A) and TLC-bioautography (B) of n-hexane fraction from Diospyros malabarica stems.

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Fig. 4. HPLC chromatogram of hexane fraction from D. malabarica stems (A) and antibacterial activity against S. mutans KCTC3065 of HPLC fraction (1 mg/ml) (B). a: Methanol as a negative control, b: Methanol extracts from D. malabarica stems as a positive control, c: HPLC fraction F1, d: HPLC fraction F2, e: HPLC mobile phase (chloroform ethyl acetate=4:1) a negative control.

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Fig. 5. Electrospray ionization–mass spectrometry (ESI-MS) spectrum of D. malabarica stems extract.

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Fig. 6. Scanning electron microscopy (SEM) micrographs of Streptococcus mutans KCTC3065 biofilms in the absence (A, B) and presence (C, D) of D. malabarica stems extract (1 mg/ml). Magnification is shown by the bar (1 um). Two different magnifications are shown for each surface; ×5,000 for the upper (A, C) and ×10,000 for the lower images (B, D).

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Fig. 7. Effects of Diospyros malabarica stems extract on the genes in relation to the biofilm formation of S. mutans KCTC 3065 by real-time PCR analysis. The genes in relation to the biofilm formation of S. mutans KCTC3065 in the absence and presence of extract of D. malabarica stems. Results are shown as the SD of five replicates. *p<0.05, as compared with control.

Table 1. Oligonucleotides used for real-time PCR in this study

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