
The leaves of
Many microorganisms inhabit the oral cavity, but among these, the disease-causing ones are limited by a few strains9,10). Caries and periodontal diseases, are caused by infection of pathogenic bacteria isolated from dental plaques11).
The purpose of this study was to identify the useful components of EEPF and to determine their inhibitory effects on oral microbial activity and production of nitric oxide (NO) and prostaglandin E2 (PGE2_ in lipopolysaccharides (LPS)-stimulated Raw264.7 macrophages, consequently, to confirm the possibility of using EEPF as a functional substance for improving the oral environment and preventing inflammation
Fresh
The powder from the extract was transferred to a 50-ml flask, adjusted to a volume of 70% methanol, and filtered through a syringe filter (0.2 mm; Altech, Beerfield, IL, USA). The filtrate (10 ml) was injected into the high- performance liquid chromatography (HPLC) for quantitative analysis of RA. Ultraviolet detection of RA was made at 330 nm wavelength of the HPLC (Table 1). Elution was performed at a 1.0 ml/min flow rate at 30°C. Mobile phase A was 0.1% (v/v) formic acid in water and mobile phase B was acetonitrile. A ratio of 88% mobile phase A and 12% B was applied for the first 10 minutes. After 50 minutes, a ratio of 60% A and 40% B was used for 5 minutes. Finally, 88% A and 12% B was used after 5 minutes for 15 minutes.
Results of High-Performance Liquid Chromatography (HPLC) Analysis for 70% Ethanol Extracted
No. | Compound | UV (nm) | Concentration (mg/L) | Regression equation (y=ax+b) | Correlation coefficient (r2) |
---|---|---|---|---|---|
1 | EEPF | 330 | 25, 50, 100 | Y=152057x−164534 | 0.999306 |
2 | Y=157516x−365061 | 0.999762 | |||
3 | Y=151398x−171408 | 0.999895 |
According to the standardized method16), colonies from each strain were cultured for 24 hours, diluted to 5×106 CFU/ml with saline solution, and then 100 ml spread on an agar plate. After absorption of each concentration of EEPF on sterilized paper discs (f6 mm; Advantec Toyo Kaisha Ltd., Tokyo, Japan), these discs were placed on a microorganism coated agar plate. After incubation for 24 hours, the diameter of the clear zone formed around the discs was measured. Ampicillin (10 IU; Oxoid Ltd., Hampshire, United Kingdom) and penicillin G (10 mcg, Oxoid Ltd.) antibiotic discs were used as controls.
The RAW264.7 macrophages (KCLB, Seoul, Korea) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco-BRL, Seoul, Korea) containing 10% fetal bovine serum (Gibco-BRL) and 1% antibiotic- antimycotic solution (Gibco-BRL) in a 5% CO2 incubator at 37°C. The prepared cells were treated with LPS (100 ng/ml) or EEPF (50, 100, 200, 300, 400 mg) and were incubated.
Cell viability as an EEPF effect was elucidated using the methylthiazolydiphenyl-tetrazolium (MTT) assay. LPS and EEPF treated Raw264.7 macrophages were added to 0.5 mg/ml of MTT (Sigma-Aldrich Chemical Co., St. Louis, MO, USA) and were wrapped in foil to block light and then reacted in an incubator for 2 hours. The cells were treated with DMSO and 200 ml of the solution was used for measurement at 540 nm using an enzyme-linked immunosorbent assay (ELISA) reader (Molecular Devices, Sunnyvale, CA, USA).
Prepared cells were fixed with 2.5% glutaraldehyde (MERCK, Frankfurter, Germany) in phosphate-buffered saline (PBS, pH 7.4), and washed thrice with PBS, and then examined using an inverted microscope (Olympus, Japan) to explore the morphological variation of the cells.
NO was measured at an absorbance of 540 nm with an ELISA reader after treatment according to the manufacturer's method using a NO assay kit (R&D Systems, Minneapolis, MN, USA). PGE2 was measured at an absorbance of 490 nm after treatment according to the manufacturer's method using a PGE2 ELISA kit (R&D Systems).
All data from triplicate experiments are expressed as mean±standard deviation and were analyzed using SPSS 16.0 (SPSS Inc., Chicago, IL, USA). Statistically significant differences (p<0.05) were analyzed using the Student’s t-test.
One kg of Perilla leaves was extracted with 70% ethanol and the crude extract was used for HPLC analysis. The HPLC chromatogram showed the highest peak at 23.270 minutes retention time, which was RA (Fig. 1). The peak area of RA was based on the HPLC analysis shown in Table 1, and the RA extracted from EEPF was 49.967± 0.153 g/kg (Table 2).
Results from High-Performance Liquid Chromatography (HPLC) Analysis of 70% Ethanol Extract of
Compound | Trial | Sample weight (g) | Retention time (min) | Area | Sample Con (g/kg) | Mean±standard deviation (g/kg) |
---|---|---|---|---|---|---|
Rosmarinic acid | 1 | 0.1021 | 23.138 | 7617032 | 50.1 | 49.967 |
2 | 0.1000 | 23.250 | 7535870 | 49.8 | ±0.153 | |
3 | 0.1040 | 23.268 | 7709009 | 50.0 |
In order to confirm the antimicrobial activity of EEPF against
Anti-Microbial Activity of 70% Ethanol Extract of
Microorganism | EEPF (mg) | Ampicillin (10 IU) | Penicillin G (10 mcg) | |||||
---|---|---|---|---|---|---|---|---|
0 | 1 | 2 | 3 | 4 | 5 | |||
|
- | - | + | + | + | + | +++ | ++ |
|
- | - | - | - | + | + | +++ | ++ |
|
- | - | - | - | + | + | ++ | + |
|
- | - | - | - | - | + | - | - |
-: resistrant (<5 mm), +: susceptible (5∼14 mm), ++: more susceptible (15∼24 mm), +++: most susceptible (>25 mm).
In order to confirm the change in cell viability due to EEPF, the MTT assay was conducted on cells treated with LPS and EEPF (Fig. 3A).
Raw264.7 macrophages were treated with LPS (100 ng/ml) for 1 hour and then treated with EEPF according to the concentration. Cell viability of the EEPF-treated groups was similar to that of the control, and that of the 300 mg-treated group was slightly increased, confirming that EEPF had no cytotoxic effects. Based on this result, the highest concentration of EEPF for the experiment was 300 mg. The variations in cell morphology of Raw264.7 cells treated with LPS and each concentration of EEPF were confirmed using a microscope (Fig. 3B). In the LPS-treated group, Raw264.7 cells had increased sharp protrusions, but the formation of sharp protrusions gradually decreased as the concentration of EEPF increased. From the above results, EEPF was not toxic to Raw264.7 macrophages and alleviated the morphological variations of LPS-stimulated cells.
Changes in the production of NO and PGE2 were confirmed in Raw264.7 macrophages following treatment with LPS and EEPF (Fig. 4). The production of NO and PGE2 was significantly increased in the LPS-treated group, but significantly decreased as the concentration of EEPF increased in the group treated with LPS and EEPF. Therefore, EEPF inhibited the LPS-induced production of NO and PGE2, inflammatory mediators in RAW264.7 cells.
Dental caries and periodontal diseases, which are important diseases of the oral cavity, are caused by infection of pathogenic bacteria that are isolated from dental plaque11).
LPS from gram-negative oral bacteria is an important component of the outer membrane of bacteria17), and induces the up-regulation and secretion of pro-inflammatory enzymes, such as nitric oxide synthase (NOS) and cyclooxygenase (COX) from immune cells (monocytes, macrophages and neutrophils)17,18). iNOS and COX-2, as inflammatory inducers, induce the secretion of large amounts of inflammatory mediators, such as NO and PGE2, and inflammatory cytokines such as tumor necrosis factor alpha and interleukin 1 beta, respectively18,19). They play an important role in the destruction of periodontal tissue during the progression of periodontal disease and induce activation of immune and inflammatory responses15,17,20). Plant-derived phytochemicals capable of modulating inflammatory mediators are used for the relief and treatment of acute and chronic inflammatory diseases21,22). Form our results, the main component of EEPF was RA (Fig. 1, Table 2) and EEPF reduced the morphological variations and the production of NO and PGE2, inflammatory mediators induced bu LPS, in Ras264.7 macrophages (Fig. 4).
Therefore, EEPF component RA was confirmed to be an important element in the HPLC analysis that had anti-bacterial and anti-fungal activity against microorganisms living in the oral cavity. In addition, EEPF has an inhibitory effect on the morphological variation and the production of NO and PGE2 in LPS-stimulated Raw264.7 macrophages. These results indicate that EEPF can be used as a functional substance for improving the oral environment and preventing inflammation.
No potential conflict of interest relevant to this article was reported.
This article is not necessary for IRB screening.
Conceptualization: Moon-Jin Jeong, Soon-Jeong Jeong, Data acquisition: Do-Seon Lim, Myoung-Hwa Lee, Kyungwon Heo, Han-Hong Kim. Formal analysis: Do-Seon Lim, Myoung-Hwa Lee, Kyungwon Heo, Han-Hong Kim. Funding: Moon-Jin Jeong. Supervision: Soon-Jeong Jeong. Writing-original draft: Soon-Jeong Jeong. Writing-review & editing: Moon-Jin Jeong, Soon-Jeong Jeong.