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Inhibitory Effects on Oral Microbial Activity and Production of Lipopolysaccharides-Induced Pro-Inflammatory Mediators in Raw264.7 Macrophages of Ethanol Extract of Perilla flutescens (L.) Britton
J Dent Hyg Sci 2020;20:213-20
Published online December 31, 2020;  https://doi.org/10.17135/jdhs.2020.20.4.213
© 2020 Korean Society of Dental Hygiene Science.

Moon-Jin Jeong1 , Do-Seon Lim2 , Myoung-Hwa Lee1 , Kyungwon Heo1 , Han-Hong Kim3 , and Soon-Jeong Jeong4,†

1Department of Oral Histology and Developmental Biology, School of Dentistry, Chosun University, Gwangju 61452, 2Department of Dental Hygiene, Graduate School of Public Health Science, Eulji University, Seongnam 13135, 3Department of Dental Hygiene, Hyejeon College, Hongseong 32244, 4Department of Dental Hygiene & Institute of Basic Science for Well-Aging, Youngsan University, Yangsan 50510, Korea
Correspondence to: † Soon-Jeong Jeong, https://orcid.org/0000-0002-8959-4663
Department of Dental Hygiene & Institute of Basic Science for Well-Aging, Youngsan University, Junam-ro 288, Yangsan 50510, Korea
Tel: +82-55-380-9453, Fax: +82-55-380-9305, E-mail: jeongsj@ysu.ac.kr
Received November 4, 2020; Revised November 18, 2020; Accepted November 26, 2020.
Abstract
Background: The leaves of Perilla frutescens, commonly called perilla and used for food in Korea, contain components with a variety of biological effects and potential therapeutic applications. The purpose of this study was to identify the components of 70% ethanol extracted Perilla frutescens (EEPF) and determine its 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 component for improving the oral environment and preventing inflammation.
Methods: One kg of P. frutescens leaves was extracted with 70% ethanol and dried at −70oC. EEPF was analyzed using high-performance liquid chromatography analysis, and antimicrobial activity against oral microorganisms was revealed using the disk diffusion test. Cell viability was elucidated using a methylthiazolydiphenyl-tetrazolium bromide assay, and the effect of EEPF on LPS-induced morphological variation was confirmed through microscopic observation. The effect of EEPF on LPS-induced production of pro-inflammatory mediators, NO and PGE2 was confirmed by the NO assay and PGE2 enzyme-linked immunosorbent assay.
Results: The main component of EEPF was rosemarinic acid, and EEPF showed weak anti-bacterial and anti-fungal effects against microorganisms living in the oral cavity. EEPF did not show toxicity to Raw264.7 macrophages and had inhibitory effects on the morphological variations and production of pro-inflammatory mediators, NO and PGE2 in LPS-stimulated Raw264.7 macrophages.
Conclusion: EEPF can be used as a functional material for improving the oral environment through the control of oral microorganisms and for modulating inflammation by inhibiting the production of inflammatory mediators.
Keywords : Oral microbial activity, Perilla frutescens, Pro-Inflammatory Mediator, Raw264.7 cells, Rosmarinic acid
Introduction

The leaves of Perilla frutescens, commonly called perilla or Korean perilla, are plants used for food in Asia, especially in Korea, and belong to the mint family Lamiacea1). P. frutescens contains antioxidant, anti-tumor and anti-allergic components, and is used for treatment in traditional medicine2-5). The important components that can be extracted from this plant are rosmarinic acid (RA), apigenin, and luteolin6). One of the chemical components of perilla leaves, RA (a-o-caffeoyl-3,4-dihydroxyphenyl-lactic acid), has various effects (anti-oxidative, anti-inflammatory, anti-mutagen, anti-bacterial and anti-viral), and has the potential for use as a therapeutic drug for disease7,8).

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). Streptococcus mutans plays an important role in the formation of dental plaques and is known to cause tooth caries12,13). Aggregatibacter actinomycetemcomitans is isolated from the dental plaque of healthy individuals and those with periodontal disease. It is one of the few strains causing aggressive periodontal disease characterized by the rapid destruction of periodontal tissue14,15). Escherichia coli accounts for 15% of the oral flora and is known to be associated with dry mouth. Candida albicans is a representative opportunistic fungus in the oral cavity13). The control of these oral microorganisms is important to prevent infectious diseases, such as tooth decay and periodontal disease. Some broad-spectrum antibiotics, such as penicillin, ampicillin, clindamycin, chlorohexidine, and metronidazole, are effective in controlling dental caries and periodontal disease, but have various adverse effects, such as discoloration of teeth, increased calculus, diarrhea, and oral flora imbalance11). Therefore, it is important to consider safe and useful chemicals from plant extracts with low adverse effects for the control of infection-causing microorganisms and infectious diseases11). Despite various therapeutic properties and interest in perilla cultivated in large quantities in the Korean peninsula, studies on the chemical components of the ethanol extracted P. frutescens (EEPF) extract and their anti-microbial activity against oral microorganisms causing oral disease and pro-inflammatory mediators are rare.

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

Materials and Methods

1. Preparation of EEPF leaves

Fresh P. frutescens leaves were purchased from the herb markets in Busan, Korea, and one kg was used for ethanol extraction. For ethanol extraction, the perilla leaves were immersed in 1 L of 70% (v/v) ethanol, and ultrasonication was performed thrice in 30 minutes at room temperature. The extracted solvent was filtered with whatman No. 2 filter paper, concentrated at 60°C with a rotary evaporator (Eyela A-1000, Eyela, Tokyo, Japan), and then freeze- dried at −70°C. The powder from the extract was preserved at −20°C until use, and was reconstituted to an appropriate concentration using dimethyl sulfoxide (DMSO, Sigma- Aldrich Chemical Co., St. Louis, MO, USA).

2.High-performance liquid chromatography analysis

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 Perilla frutescens (EEPF)

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


3. Oral microorganisms

S. mutans (KCCM 40105), A. actinomycetemcomitans (KCTC 2581), E. coli (KCTC 1039), and C. albicans (KCCM 11282) were purchased from the Korea Microbiological Conservation Center (KCCM) and the gene bank (KCTC) for experiments. S. mutans was cultured in Brain Heart Infusion (MB cell Ltd., Seoul, Korea) agar and broth, A. actinomycetemcomitan s in MRS (MB cell Ltd.) agar and broth, E. coli in Luria Bertani (MB cell Ltd.) agar and broth, and C. albicans in Potato Dextrose (MB cell Ltd.) agar and broth.

4. Disk diffusion test

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.

5. Cell culture and EEPF treatment

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.

6. Methylthiazolydiphenyl-tetrazolium assay

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).

7. Microscopic observation

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.

8. NO assay and PGE2 ELISA assay

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).

9. Statistical analysis

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.

Results

1. Contents of bioactive materials of EEPF

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 Perilla frutescens Leaves for Rosmarinic Acid

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

Fig. 1. High performance liquid chromatography (HPLC) profile of ethanol extracts of Perilla frutescens (EEPF) Leaves. (A) The chromatogram of standard of rosmarinic acid (RA). (B) Cromatograms from EEPF. The quantitative data chromatogram form HPLC analysis of EEPF showed the highest peak in RA.

2.Anti-microbial activity of EEPF against oral microorganisms

In order to confirm the antimicrobial activity of EEPF against S. mutans, A. actinomycetemcomitans, E. coli, and C. albicans, microorganisms living in the oral cavity, a disk diffusion test was performed (Fig. 2, Table 3). EEPF (4 and 5 mg) had weak anti-bacterial activity against S. mutans, A. actinomycetemcomitans, and E. coli. In addition, 5 mg EEPF showed a weak antifungal effect against C. albicans. From the above results, EEPF has weak anti-bacterial and anti-fungal effects against microorganisms living in the oral cavity.

Anti-Microbial Activity of 70% Ethanol Extract of Perilla frutescens (EEPF) Leaves against Oral Microorganisms

Microorganism EEPF (mg) Ampicillin (10 IU) Penicillin G (10 mcg)

0 1 2 3 4 5
Streptococcus mutans - - + + + + +++ ++
Aggregatibacter actinomycetemcomitans - - - - + + +++ ++
Escherichia coli - - - - + + ++ +
Candida albicans - - - - - + - -

-: resistrant (<5 mm), +: susceptible (5∼14 mm), ++: more susceptible (15∼24 mm), +++: most susceptible (>25 mm).


Fig. 2. Inhibitory effects on microbial activity of ethanol extracts of Perilla frutescens (EEPF) against oral bacteria and fungi. A: ampicillin, P: penicillin G, DMSO: dimethyl sulfoxide.

3.Effects of EEPF on the cell viability and morphology of LPS-stimulated Raw264.7 cells

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).

Fig. 3. Effects of EEPF on cell viability and morphological variation of RAW264.7 macrophages. Raw264.7 macrophages were treated with LPS (100 ng/ml) before 1 hour and then treated with EEPF according to the concentration. MTT assay was performed to evaluate cell viability (A) and observed using a microscope to evaluate morphological variation (B). Cell viability of the EEPF-treated groups was similar to that of the control and those of 300 mg-treated group was slightly increased, confirming that EEPF had not cytotoxic effects. The LPS-stimulated Raw264.7 cells had increased sharp protru-sions, but as the treatment concentration of EEPF increased, the formation of sharp protrusions gradually decreased. EEFP: ethanol extracts of Perilla frutescens, LPS: lipopolysaccharides, MTT: methylthiazolydiphenyl-tetrazolium.

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.

4.Effects of EEPF on NO and PGE2 production in LPS-stimulated Raw264.7 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.

Fig. 4. Inhibition effects of EEPF on production of LPS-induced pro-inflammatory mediators production in Raw264.7 macrophages. (A) NO, (B) PGE2. The production of NO and PGE2 was significantly increased in the LPS-treated group compared to the control, but the production of NO and PGE2 in the group treated with LPS and EEPF significantly decreased as the concentration of EEPF increased, compared to the group treated with only LPS (***p<0.001). EEFP: ethanol extracts of Perilla frutescens, PGE2: prostaglandin E2, NO: nitric oxide, LPS: lipopolysaccharides.
Discussion

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). S. mutans plays an important role in the formation of dental plaque which is the basis for colonization of pathogenic bacteria that can cause oral disease and induce dental caries12,13). Periodontal tissues, including the cementum, periodontal ligaments, and alveolar bone surrounding the tooth root, are plagued by bacterial infection as acute and chronic periodontal diseases17). If periodontal disease is left untreated, excessive inflammatory reactions and alveolar bone resorption progress, leading to tooth loss17). Periodontal disease is initiated by the overgrowth of special gram- negative anaerobic bacteria such as A. actinomycetemcomitans, Porphyromonas gingivalis and Fusobacterium nucleatum, and the number increases significantly during the inflammatory conditions of periodontal tissues15). A. actinomycetemcomitans, isolated from the dental plaque of healthy individuals and those with periodontitis, is one of the representative bacteria causing aggressive periodontal disease characterized by rapid destruction of periodontal tissue12,14,15). E. coli, although transiently present, accounts for 15% of the oral flora, and the frequency increases with age and is known to be related to the induction of dry mouth13).

C. albicans is a representative opportunistic fungus that causes candidiasis in people with weak immunity13). The control of these microorganisms in the oral cavity is important for the treatment of infectious diseases such as tooth caries and periodontal disease. EEPF, which contains RA as a major component, weakly inhibited the growth of oral pathogenic bacteria S. mutans, A. actinomycetemcomitans, E. coli, and C. albicans belonging to the fungus, and showed anti-bacterial and anti-fungal activities (Fig. 2, Table 3). Although there is paucity of data on the effect of perilla on oral microorganisms, RA has weak antimicrobial activity against oral streptococci and relatively strong antimicrobial activity against P. gingivalis 9) and has no antimicrobial activity against fungi (data not shown). The above results showed that another component of EEPF can control oral fungi. Therefore, it is necessary to analyze other components of the EEPF in future studies.

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.

Conflict of interest

No potential conflict of interest relevant to this article was reported.

Ethical approval

This article is not necessary for IRB screening.

Author contributions

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.

References
  1. Liu J, Wan Y, Zhao Z, Chen H: Determination of the content of rosmarinic acid by HPLC and analytical comparison of volatile constituents by GC-MS in different parts of Perilla frutescens (L.) Britt. Chem Cent J 7: 61, 2013.
    https://doi.org/10.1186/1752-153X-7-61.
    Pubmed KoreaMed CrossRef
  2. Lee HA, Han JS: Anti-inflammatory effect of Perilla frutescens (L.) Britton var. frutescens extract in LPS-stimulated RAW 264.7 macrophages. Prev Nutr Food Sci 17: 109-115, 2012.
    https://doi.org/10.3746/pnf.2012.17.2.109.
    Pubmed KoreaMed CrossRef
  3. Meng L, Lozano YF, Gaydou EM, Li B: Antioxidant activities of polyphenols extracted from Perilla frutescens varieties. Molecules 14: 133-140, 2008.
    https://doi.org/10.3390/molecules14010133.
    Pubmed KoreaMed CrossRef
  4. Makino T, Furuta Y, Wakushima H, Fujii H, Saito K, Kano Y: Anti-allergic effect of Perilla frutescens and its active constituents. Phytother Res 17: 240-243, 2003.
    https://doi.org/10.1002/ptr.1115.
    Pubmed CrossRef
  5. Ueda H, Yamazaki C, Yamazaki M: Inhibitory effect of Perilla leaf extract and luteolin on mouse skin tumor promotion. Biol Pharm Bull 26: 560-563, 2003.
    https://doi.org/10.1248/bpb.26.560.
    Pubmed CrossRef
  6. Kangwan N, Pintha K, Lekawanvijit S, Suttajit M: Rosmarinic acid enriched fraction from Perilla frutescens leaves strongly protects indomethacin-induced gastric ulcer in rats. Biomed Res Int 2019: 9514703, 2019.
    https://doi.org/10.1155/2019/9514703.
    Pubmed KoreaMed CrossRef
  7. Moon DO, Kim MO, Lee JD, Choi YH, Kim GY: Rosmarinic acid sensitizes cell death through suppression of TNF- alpha-induced NF-kappaB activation and ROS generation in human leukemia U937 cells. Cancer Lett 288: 183-191, 2010.
    https://doi.org/10.1016/j.canlet.2009.06.033.
    Pubmed CrossRef
  8. Parnham MJ, Kesselring K: Rosmarinic acid. Drugs Future 10: 756-757, 1985.
    CrossRef
  9. Yamamoto H, Ogawa T: Antimicrobial activity of perilla seed polyphenols against oral pathogenic bacteria. Biosci Biotechnol Biochem 66: 921-924, 2002.
    https://doi.org/10.1271/bbb.66.921.
    Pubmed CrossRef
  10. Petit MD, van Steenbergen TJ, Timmerman MF, de Graaff J; , van der Velden U: Prevalence of periodontitis and suspected periodontal pathogens in families of adult periodontitis patients. J Clin Periodontol 21: 76-85, 1994.
    https://doi.org/10.1111/j.1600-051x.1994.tb00283.x.
    Pubmed CrossRef
  11. Bernardes WA, Lucarini R, Tozatti MG, et al.: Antimicrobial activity of Rosmarinus officinalis against oral pathogens: relevance of carnosic acid and carnosol. Chem Biodivers 7: 1835-1840, 2010.
    https://doi.org/10.1002/cbdv.200900301.
    Pubmed CrossRef
  12. Pokrowiecki R, Mielczarek A, Zaręba T, Tyski S: Oral microbiome and peri-implant diseases. Ther Clin Risk Manag 13: 1529-1542, 2017.
    https://doi.org/10.2147/TCRM.S139795.
    Pubmed KoreaMed CrossRef
  13. Yang SH, Park SJ, Lee RU, et al.: Antibacterial effects on oral microorganism and inhibitory effects of halitosis of Omija (Schizandra chinensis baillon) extract. J Korean Soc Oral Health Sci 4: 93-98, 2016.
  14. Eswar P, Devaraj CG, Agarwal P: Anti-microbial activity of Tulsi {Ocimum sanctum (Linn.)} extract on a periodontal pathogen in human dental plaque: an invitro study. J Clin Diagn Res 10: ZC53-ZC56, 2016.
    https://doi.org/10.7860/JCDR/2016/16214.7468.
    Pubmed KoreaMed CrossRef
  15. Kim MY, Bo HH, Choi EO, et al.: Induction of apoptosis by Citrus unshiu peel in human breast cancer MCF-7 cells: involvement of ROS-dependent activation of AMPK. Biol Pharm Bull 41: 713-721, 2018.
    https://doi.org/10.1248/bpb.b17-00898.
    Pubmed CrossRef
  16. Bauer AW, Kirby WM, Sherris JC, Turck M: Antibiotic susceptibility testing by a standardized single disk method. Am J Clin Pathol 45: 493-496, 1966.
    https://doi.org/10.1093/ajcp/45.4_ts.493.
    Pubmed CrossRef
  17. Lee SY, Nho TH, Choi BD, Jeong SJ, Lim DS, Jeong MJ: Secretory leukocyte protease inhibitor reduces inflammation and alveolar bone resorption in LPS-induced periodontitis in rats and in MC3T3-E1 preosteoblasts. Anim Cells Syst 20: 344-352, 2016.
    https://doi.org/10.1080/19768354.2016.1250817.
    CrossRef
  18. Jeong JW, Lee HH, Han MH, et al.: Ethanol extract of Poria cocos reduces the production of inflammatory mediators by suppressing the NF-kappaB signaling pathway in lipopo-lysaccharide-stimulated RAW 264.7 macrophages. BMC Complement Altern Med 14: 101, 2014.
    https://doi.org/10.1186/1472-6882-14-101.
    Pubmed KoreaMed CrossRef
  19. Kwon DH, Cha HJ, Choi EO, et al.: Schisandrin A suppresses lipopolysaccharide-induced inflammation and oxidative stress in RAW 264.7 macrophages by suppressing the NF-κB, MAPKs and PI3K/Akt pathways and activating Nrf2/HO-1 signaling. Int J Mol Med 41: 264-274, 2018.
    https://doi.org/10.3892/ijmm.2017.3209.
    CrossRef
  20. Huang N, Hauck C, Yum MY, et al.: Rosmarinic acid in Prunella vulgaris ethanol extract inhibits lipopolysaccharide- induced prostaglandin E2 and nitric oxide in RAW 264.7 mouse macrophages. J Agric Food Chem 57: 10579-10589, 2009.
    https://doi.org/10.1021/jf9023728.
    Pubmed KoreaMed CrossRef
  21. Na HS, Jeong SY, Park MH, Kim S, Chung J: Nuclear factor-κB dependent induction of TNF-a and IL-1b by the Aggregatibacter actinomycetemcomitans Lipopolysaccharide in RAW 264.7 cells. Int J Oral Biol 39: 15-22, 2014.
    https://doi.org/10.11620/IJOB.2014.39.1.015.
    CrossRef
  22. Kang HJ, Jeong JS, Park NJ, et al.: An ethanol extract of Aster yomena (Kitam.) Honda inhibits lipopolysaccharide- induced inflammatory responses in murine RAW 264.7 macrophages. Biosci Trends 11: 85-94, 2017.
    https://doi.org/10.5582/bst.2016.01217.
    Pubmed CrossRef


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