Original Article
Investigation of the antimicrobial activity of Bilberry (Vaccinium myrtillus L.) extract against periodontopathic bacteria

https://doi.org/10.1016/j.job.2020.01.009Get rights and content

Abstract

Objective

The purpose of this study was to isolate the active antibacterial compounds from Bilberry (Vaccinium myrtillus L.) against periodontopathic bacteria.

Methods

The acetone soluble fraction of Bilberry was extracted from the oil layer by oil/water separation. The extract was then purified by a silica gel open column chromatography. The minimum inhibitory concentration (MIC) of the total extract or purified fractions against bacteria was measured at each step.

Results

The MIC of the total extract against Porphyromonas gingivalis was 500 μg/mL. The fraction exhibiting antibacterial activity against P. gingivalis was called NU4-TDC, and its MICs against P. gingivalis, Fusobacterium nucleatum, and Prevotella intermedia were 26.0 ± 7.8 μg/mL, 59.0 ± 10.4 μg/mL, and 45.1 ± 16.5 μg/mL, respectively. The MIC against Streptococcus mutans was >62.5 μg/mL.

Conclusion

Bilberry contains antibacterial components against periodontopathic bacteria, such as P. gingivalis, F. nucleatum, and P. intermedia.

Introduction

Dental caries and periodontitis are infectious diseases caused by oral microorganisms [1]. In recent years, these bacteria have been suggested to participate in many systemic diseases, such as diabetes, cardiovascular disease, and infective endocarditis [[2], [3], [4]]. These reports emphasized that maintenance of the oral microbiome is required to maintain systemic health. Antimicrobials have often been employed for the treatment of periodontitis [5,6]. However, they may cause opportunistic infections and induce drug resistance in microorganisms. Owing to this perspective, an alternative regimen for the control of the oral microbiome is required.

Previously, the antibacterial activity of berries in the genus Vaccinium, including bilberries (Vaccinium myrtillus L.), was reported [7,8]. The polyphenols contained in berry fruits have been suggested to exhibit antibacterial activity; however, other components have not been thoroughly evaluated [[9], [10], [11]]. Further, the effects of berries on oral microorganisms have not been adequately examined. Therefore, we focused on the organic compounds of V. myrtillus L., a type species of the genus Vaccinium, and aimed to extract the active antibacterial substances.

In this study, bilberry fruits were crushed under liquid nitrogen and extracted with acetone. Additionally, ethyl acetate was employed for oil/water separation. The extract was purified using open column chromatography. The antimicrobial activities of the fractions collected at each step of the purification were then determined.

Section snippets

Bilberry preparation

Dried bilberries were purchased from Blueberry Fields Kinokuniya (Shiga, Japan). Bilberries (30 g) were crushed under liquid nitrogen. Thereafter, 150 mL of acetone (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) was added. The mixture was sonicated for 5 min and incubated overnight at room temperature. After acetone was removed, another 150 mL of acetone was added to the bilberry residue for a 5-min sonication. Thereafter, acetone was removed. The above steps were repeated seven times.

Characteristics of the components extracted from bilberry

A total of 5.0 × 102 mg of extract was obtained from 30 g of dried bilberry fruit. The extract was separated by TLC, and 12 bright spots, including those that remained at the origin, were obtained (Fig. 1). The MIC of the total extract against P. gingivalis was 500 μg/mL.

Separation of the extract by primary silica gel column chromatography

The components contained in the extract were separated into fractions between Fc 1–31 and Fc 1–70 (Fig. 2a–d). After the fractions were combined into Cf A to Cf H, they were used to determine the MICs against P. gingivalis. The

Discussion

In this study, methods, such as the crushing of bilberry fruits under liquid nitrogen, compound extraction with acetone, oil/water separation using EtOAc, and purification using open column chromatography, were employed to determine the activity of different compounds without explicitly specifying their type.

The implemented open column method enabled the elution of compounds with slight differences in polarity owing to the slow increase in acetone concentration in the developing solvent. In

Funding

This work was supported by the Private University Research Branding Project from MEXT of Japan.

CRediT authorship contribution statement

Yutaroh Satoh: Investigation, Data curation, Methodology, Writing - original draft. Kazuyuki Ishihara: Supervision, Funding acquisition, Writing - review & editing.

Conflicts of interest

The authors declare no conflict of interest.

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