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Research article
Synergistic Antimicrobial Effect of Lonicera japonica and Magnolia obovata Extracts and Potential as a Plant-Derived Natural Preservative
Department of Fine Chemistry, Cosmetic R&D Center, Cosmetic Industry Coupled Collaboration Center, Seoul National University of Science and Technology, Seoul 01811, Republic of Korea
J. Microbiol. Biotechnol. 2018; 28(11): 1814-1822
Published November 28, 2018 https://doi.org/10.4014/jmb.1807.07042
Copyright © The Korean Society for Microbiology and Biotechnology.
Abstract
Keywords
Introduction
The skin is the outermost barrier of the human body. It plays a role in protecting the body from external stimuli such as ultraviolet rays, antigens, microorganisms, and chemical substances [1]. The skin preserves the body in various ways by controlling the pH on its surface, moisture content, and through secretion by the sebaceous glands and sweating. If the function of the skin barrier is compromised, pathogens can penetrate the skin and cause many skin diseases [2]. Therefore, people use cosmetics to maintain skin homeostasis and general skin health [3]. As interest in cosmetics has increased, the demand for these products has also grown rapidly. This eventually led to the development of plant-derived and eco-friendly products [4].
According to the United States Federal Food, Drug, and Cosmetic Act, cosmetics are defined as “articles intended to be rubbed, poured, sprinkled, or sprayed on, introduced into, or otherwise applied to the human body...for cleansing, beautifying, promoting attractiveness, or altering the appearance.” Cosmetics contain large amounts of water, which could allow the survival of microorganisms and subsequently cause harmful skin diseases, even though their intended use is for skin protection [5]. Therefore, preservatives are added to cosmetics to prevent microbial contamination [6]. However, recent studies have reported that preservatives, especially parabens, may have side effects. Consequently, the demand for plant-derived natural cosmetics has increased, thereby spurring developments in studies on plant-derived preservatives [7, 8].
In this study, we evaluated the antimicrobial activities of
Materials and Methods
Equipment and Reagents
The UV-visible spectrophotometer used for experiments was a Cary 50 from Varian (Australia). Various solvents such as ethanol, methanol, and ethyl acetate were purchased from Daejung Chemicals & Metals Co. (Korea). The caffeic acid and luteolin used as standards for comparison were purchased from Sigma Chemical Co. (USA). Magnolol and honokiol were purchased from ActivON.
Extraction and Fractionation
Four hundred grams of dried
Three hundred grams of dried
Microbial Strains and Culture Conditions
The strains used to evaluate the antimicrobial effect of the extracts were
Evaluation of Antimicrobial Activity
Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). To determine the minimum inhibitory concentration and the minimum bactericidal concentration for each extract, each strain was used at 3 to 5 × 106 colony-forming units (CFU)/ml. Methylparaben was used as a control. The samples were diluted with DMSO using a 2-fold dilution method [17]. Then, 20 μl of the extract, 20 μl of the microbial suspension (inocula), and 160 μl of the medium (bacteria and yeast - Tryptic Soy Broth, mold - Potato Dextrose Broth) were added to the wells on a 96-well plate. The cells were then observed with the naked eye after cultivation (Bacteria and yeast were incubated for 18 h at 37°C and mold was incubated for 48 h at 30°C.), and the concentration at which the microorganisms did not proliferate was determined to be the minimum inhibitory concentration. The concentration at which colonies were not formed upon inoculation of the culture on an agar plate with a sterilized cotton swab was determined to be the minimum bactericidal concentration.
Disc diffusion assay. The antimicrobial activity of each extract and each fraction obtained with different extraction conditions were determined through a disc diffusion assay. The cultured strains were used at 3 ~ 5 × 106 CFU/ml. Then, 100 μl of each strain was plated using a sterile spreader. Solutions containing 0.05 mg (luteolin) or 5 mg of the samples were absorbed slowly by paper discs (diameter: 8 mm, Toyo Roshi Kaisha Ltd., Japan). The discs were then dried to volatilize the solvent and then incubated with the previously inoculated plates. The clear zone (mm) formed around each disc was measured to compare the antimicrobial activities of the extracts.
Synergistic Antimicrobial Effects of the Extracts
Checkerboard test. The synergistic effect is generally determined in the same manner as the minimum inhibitory concentration, except that 10 μl each of two different samples are mixed [17]. That is, in the checkerboard test, two samples in different concentration are added to each well. Sample A was diluted 2-fold, and then sample B diluted 2-fold was added. The resulting mixture was incubated with one of the six microorganisms. After incubation, the ratio between Sample A and Sample B at which the growth of the microorganism was not observed visually was determined. The fractional inhibition concentration (FIC) and FIC index were then calculated.
If the FIC index ≤ 0.5, the effects are synergistic; if 0.5 <FIC index ≤ 1, the effects are additive; if 1 <FIC index ≤ 4 there is no difference between individual and combined effects; and if FIC index> 4, the effects are antagonistic [17].
Kill-time analysis. Kill-time analysis is a method used to measure the change in the number of bacterial colonies in the culture medium upon the addition of the sample over time [17]. The experiment was conducted using a specific sample concentration selected based on the result of the checkerboard test. For the analysis, 0.5 ml of the sample was added to 4.5 ml of the bacterial suspension (105 CFU/ml). This was then incubated at 3-h intervals for 18 hours. Between each interval, the change in the number of bacterial colonies was determined.
Componential Analysis of Each Extract
The ethyl acetate fraction from each of the two extracts was dissolved in 100% ethanol and filtered using a syringe filter (Millipore, USA, 0.45 μm). The filtered extract solution was then subjected to nonpolar HPLC (C18) analysis. HPLC analysis of the
-
Table 1 . HPLC conditions for separation of EtOAc fraction from
L. japonica .Condition of HPLC Analysis Column Shim-pack VP-ODS C18 Column (L : 250 mm, LD : 4.6 mm, 5 μm) Detector UVD 170s DIONEX Detection Wavelength 254 - 400 nm Flow Rate 1.0 mL/min Injection Volume 20 μL Mobile Phase Conditions for HPLC Gradient-elution Program order Time (min) 2% AA1) in water (%) 0.5% AA1) in 50% ACN2) (%) 1 0 100 0 2 10 100 0 3 150 50 50 4 200 30 70 5 210 30 70 6 215 100 0 7 220 100 0 1) AA : Acetic acid, 2) ACN : Acetonitrile
-
Table 2 . HPLC conditions for separation of EtOAc fraction from
M. obovata .Condition of HPLC Analysis Column Shim-pack VP-ODS C18 Column (L : 250 mm, LD : 4.6 mm, 5 μm) Detector UVD 170s DIONEX Detection Wavelength 254 - 400 nm Flow Rate 1.0 mL/min Injection Volume 20 μL Mobile Phase Conditions for HPLC Gradient-elution Program order Time (min) DW1) (%) 100% ACN2) (%) 1 0 75 25 2 40 50 50 3 60 5 95 4 70 5 95 5 75 95 5 1) DW : Distilled Water, 2) ACN : Acetonitrile
Statistical Analysis
All experiments were conducted in triplicate, averaged and presented followed by the standard deviation.
Results and Discussion
Extraction Yields of L. japonica and M. obovata
The yields of the 50% ethanol extract and the ethyl acetate fraction from
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Table 3 . Yields of
L. japonica andM. obovata extractions.Solvent Yield (%, w/w)* Lonicera japonica 50% EtOH Extract 4.85 EtOAc Fraction 0.41 Magnolia obovata 70% EtOH Extract 14.52 EtOAc Fraction 4.47 *Yield (%, w/w) = (weight of dried extract / weight of dried raw material) × 100
Antimicrobial Activity of L. japonica and M. obovata Extracts
The antimicrobial activities of
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Table 4 . Minimum Inhibitory Concentration (MIC, ug/ml) and Mininum Bactericidal Concentration (MBC) of
L. japonica andM. obovata extracts and fractions.Strains MIC (MBC) (μL/ml) Gram positive bacteria Gram negative bacteria Yeast Mold S. aureus B. subtilis E. coli P. aeruginosa C. albicans A. brasiliensis Lonicera japonica 50% EtOH extract5000 (10000) - ( - ) - ( - ) 5000 ( - ) - ( - ) - ( - ) Lonicera japonica EtOAc fraction78 (312) 10000 (10000) 5000 (5000) 312 (1250) 1250 (10000) 10000 ( - ) Magnolia obovata 70% EtOH extract78 (156) 2500 (2500) 2500 (5000) 1250 (1250) 1250 (5000) 10000 ( - ) Magnolia obovata EtOAc fraction39 (78) 2500 (5000) 2500 (2500) 625 (625) 625 (625) 2500 ( - ) Methyl paraben 2500 (5000) 2500 (10000) 1250 (5000) 1250 (1250) 1250 (5000) 625 (5000) Methylparaben used as a control. - no inhibition at 10,000 ug/ml.
The 50% ethanol extract from
The 70% ethanol extract and ethyl acetate fraction from
Synergistic Antimicrobial Effects of L. japonica and M. obovata
The synergistic antimicrobial effect of the two extracts was evaluated using the ethyl acetate fractions from
Checkerboard test. The checkerboard test was used to confirm the synergistic effect of the ethyl acetate fractions from the two extracts, which showed antimicrobial activity against all six strains. The results are shown in Fig. 1. A synergistic effect was observed against
-
Fig. 1. Checkerboard test showing the synergistic antimicrobial effects of
L. japonica andM. obovata fractions against six microorganisms. (A)S. aureus , (B)B. subtilis , (C)E. coli , (D)P. aeruginosa , (E)C. albicans , (F)A. brasiliensis . Colored wells indicate microbial growth and white wells indicate no microbial growth. The number in the well is the FIC index. Wave patterns indicate the synergistic effect (FIC index ≤ 0.5) and dotted patterns mean an addition effect (0.5 <FIC index ≤ 1).
A synergistic antimicrobial effect was observed against
Kill-time analysis. In the previous experiment, we confirmed that the combination of
-
Fig. 2. The synergistic antimicrobial effects of
L. japonica andM. obovata fractions againstB. subtilis by kill-time analysis. Control (○) was treated with DMSO only and the concentration of the ethyl acetate fraction fromL. japonica (□) and M. obovate (■) was 2,500 ppm and 625 ppm, respectively. Combination (▲) indicates a mixture ofL. japonica andM. obovata fractions. The experiments were conducted at 3-h intervals for 18 h and in triplicate. Data are presented as the mean ± SD.
We found that upon treatment with the
Componential Analysis of L. japonica and M. obovata
Since we have confirmed that the extracts have antimicrobial activities against the six strains, we used HPLC to determine the effective components of each extract. The results are shown in Figs. 3 and 4.
-
Fig. 3. The HPLC chromatogram of ethyl acetate fraction of
L. japonica and the components in detection wavelength range from 254 to 400 nm. (A) Ethyl acetate fraction, caffeic acid; 1, luteolin; 2. (B) Caffeic acid. (C) Luteolin.
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Fig. 4. The HPLC chromatogram of ethyl acetate fraction of M. obovate and the components in detection wavelength range from 254 to 400 nm. (A) Ethyl acetate fraction, honokiol; 1, magnolol; 2. (B) Magnolol and honokiol (standard).
HPLC analysis of the
Analysis of the
Antimicrobial Effects of the Components of L. japonica and M. obovata Extracts
The antimicrobial activity of each component of each extract was evaluated, and the relationship between the antimicrobial activity of the extract and that of each component was determined. The antimicrobial activity was evaluated through a disc diffusion assay and the minimum inhibition concentration.
Disc diffusion assay. To evaluate the antimicrobial activity of each component of the
As shown in Fig. 5, the results were most effective against
-
Fig. 5. Antimicrobial activities of
L. japonica ,M. obovata extracts/fractions and the components against P. aerusinosa by disc diffusion assay. (A) Methylparaben (as a control); 1,L. japonica 50% EtOH extract; 2,L. japonica EtOAc fraction; 3, caffeic acid; 4, luteolin; 5. (B) Methylparaben (as a control); 1,M. obovata 70% EtOH extract; 2,M. obovata EtOAc fraction; 3, magnolol and honokiol; 4.
Minimum inhibitory concentration (MIC). To accurately evaluate the antimicrobial activity of each component of the
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Table 5 . Minimum Inhibitory Concentration (MIC, ug/ml) of the components of
L. japonica andM. obovata extracts.Strains MIC (μL/ml) Gram positive bacteria Gram negative bacteria Yeast Mold S. aureus B. subtilis E. coli P. aeruginosa C. albicans A. brasiliensis Methyl paraben 1250 1250 1250 1250 1250 625 Caffeic acid 312 5000 5000 2500 5000 > 10000 Luteolin 25 200 200 50 200 200 Magnolol, Honokiol 31.2 10000 10000 1250 5000 39 Methylparaben used as a control.
Caffeic acid showed high antimicrobial activity against
Magnolol and honokiol showed antimicrobial activity against all strains, especially
In this study, we evaluated the antimicrobial activities of
We also evaluated the synergistic antimicrobial activity of the ethyl acetate fractions from
To determine the extract components that have antimicrobial activity, we performed HPLC analysis and detected the presence of caffeic acid and luteolin in the
Conflict of Interest
The authors have no financial conflicts of interest to declare.
References
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Related articles in JMB

Article
Research article
J. Microbiol. Biotechnol. 2018; 28(11): 1814-1822
Published online November 28, 2018 https://doi.org/10.4014/jmb.1807.07042
Copyright © The Korean Society for Microbiology and Biotechnology.
Synergistic Antimicrobial Effect of Lonicera japonica and Magnolia obovata Extracts and Potential as a Plant-Derived Natural Preservative
Ye Seul Lee , Yun Ju Lee and Soo Nam Park *
Department of Fine Chemistry, Cosmetic R&D Center, Cosmetic Industry Coupled Collaboration Center, Seoul National University of Science and Technology, Seoul 01811, Republic of Korea
Abstract
Most people use cosmetics to protect their skin. Preservatives are often used to prevent their
contamination upon use. There has been a great demand for natural preservatives due to recent
reports on the side effects of parabens. Therefore, we evaluated the antimicrobial activities of
Lonicera japonica and Magnolia obovata extracts and determined their potential as natural
preservatives. We found that the 50% ethanol extract from L. japonica had antibacterial activity
only against S. aureus and P. aeruginosa, while the ethyl acetate fraction showed antimicrobial
activity against all six microbial strains tested. On the other hand, the 70% ethanol extract and
the ethyl acetate fraction from M. obovata showed antimicrobial activity against all six strains.
A synergistic effect against S. aureus, B. subtilis, and C. albicans was confirmed when two ethyl
acetate fractions having antimicrobial activity against all six strains were used in combination.
Synergistic activity against B. subtilis was also confirmed through kill-time analysis. Highperformance
liquid chromatography was performed to identify the components of each extract.
Based on the minimum inhibitory concentration and the results of a disc diffusion assay, we
confirmed that caffeic acid and luteolin influenced the antimicrobial activity of L. japonica and
that the antimicrobial activity of M. obovata was influenced by the interaction of magnolol and
honokiol with other components. Therefore, this study suggests that the combination of
L. japonica and M. obovata extracts may be used as a plant-derived natural preservative.
Keywords: Lonicera japonica, Magnolia obovata, antimicrobial activity, synergistic effect
Introduction
The skin is the outermost barrier of the human body. It plays a role in protecting the body from external stimuli such as ultraviolet rays, antigens, microorganisms, and chemical substances [1]. The skin preserves the body in various ways by controlling the pH on its surface, moisture content, and through secretion by the sebaceous glands and sweating. If the function of the skin barrier is compromised, pathogens can penetrate the skin and cause many skin diseases [2]. Therefore, people use cosmetics to maintain skin homeostasis and general skin health [3]. As interest in cosmetics has increased, the demand for these products has also grown rapidly. This eventually led to the development of plant-derived and eco-friendly products [4].
According to the United States Federal Food, Drug, and Cosmetic Act, cosmetics are defined as “articles intended to be rubbed, poured, sprinkled, or sprayed on, introduced into, or otherwise applied to the human body...for cleansing, beautifying, promoting attractiveness, or altering the appearance.” Cosmetics contain large amounts of water, which could allow the survival of microorganisms and subsequently cause harmful skin diseases, even though their intended use is for skin protection [5]. Therefore, preservatives are added to cosmetics to prevent microbial contamination [6]. However, recent studies have reported that preservatives, especially parabens, may have side effects. Consequently, the demand for plant-derived natural cosmetics has increased, thereby spurring developments in studies on plant-derived preservatives [7, 8].
In this study, we evaluated the antimicrobial activities of
Materials and Methods
Equipment and Reagents
The UV-visible spectrophotometer used for experiments was a Cary 50 from Varian (Australia). Various solvents such as ethanol, methanol, and ethyl acetate were purchased from Daejung Chemicals & Metals Co. (Korea). The caffeic acid and luteolin used as standards for comparison were purchased from Sigma Chemical Co. (USA). Magnolol and honokiol were purchased from ActivON.
Extraction and Fractionation
Four hundred grams of dried
Three hundred grams of dried
Microbial Strains and Culture Conditions
The strains used to evaluate the antimicrobial effect of the extracts were
Evaluation of Antimicrobial Activity
Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). To determine the minimum inhibitory concentration and the minimum bactericidal concentration for each extract, each strain was used at 3 to 5 × 106 colony-forming units (CFU)/ml. Methylparaben was used as a control. The samples were diluted with DMSO using a 2-fold dilution method [17]. Then, 20 μl of the extract, 20 μl of the microbial suspension (inocula), and 160 μl of the medium (bacteria and yeast - Tryptic Soy Broth, mold - Potato Dextrose Broth) were added to the wells on a 96-well plate. The cells were then observed with the naked eye after cultivation (Bacteria and yeast were incubated for 18 h at 37°C and mold was incubated for 48 h at 30°C.), and the concentration at which the microorganisms did not proliferate was determined to be the minimum inhibitory concentration. The concentration at which colonies were not formed upon inoculation of the culture on an agar plate with a sterilized cotton swab was determined to be the minimum bactericidal concentration.
Disc diffusion assay. The antimicrobial activity of each extract and each fraction obtained with different extraction conditions were determined through a disc diffusion assay. The cultured strains were used at 3 ~ 5 × 106 CFU/ml. Then, 100 μl of each strain was plated using a sterile spreader. Solutions containing 0.05 mg (luteolin) or 5 mg of the samples were absorbed slowly by paper discs (diameter: 8 mm, Toyo Roshi Kaisha Ltd., Japan). The discs were then dried to volatilize the solvent and then incubated with the previously inoculated plates. The clear zone (mm) formed around each disc was measured to compare the antimicrobial activities of the extracts.
Synergistic Antimicrobial Effects of the Extracts
Checkerboard test. The synergistic effect is generally determined in the same manner as the minimum inhibitory concentration, except that 10 μl each of two different samples are mixed [17]. That is, in the checkerboard test, two samples in different concentration are added to each well. Sample A was diluted 2-fold, and then sample B diluted 2-fold was added. The resulting mixture was incubated with one of the six microorganisms. After incubation, the ratio between Sample A and Sample B at which the growth of the microorganism was not observed visually was determined. The fractional inhibition concentration (FIC) and FIC index were then calculated.
If the FIC index ≤ 0.5, the effects are synergistic; if 0.5 <FIC index ≤ 1, the effects are additive; if 1 <FIC index ≤ 4 there is no difference between individual and combined effects; and if FIC index> 4, the effects are antagonistic [17].
Kill-time analysis. Kill-time analysis is a method used to measure the change in the number of bacterial colonies in the culture medium upon the addition of the sample over time [17]. The experiment was conducted using a specific sample concentration selected based on the result of the checkerboard test. For the analysis, 0.5 ml of the sample was added to 4.5 ml of the bacterial suspension (105 CFU/ml). This was then incubated at 3-h intervals for 18 hours. Between each interval, the change in the number of bacterial colonies was determined.
Componential Analysis of Each Extract
The ethyl acetate fraction from each of the two extracts was dissolved in 100% ethanol and filtered using a syringe filter (Millipore, USA, 0.45 μm). The filtered extract solution was then subjected to nonpolar HPLC (C18) analysis. HPLC analysis of the
-
Table 1 . HPLC conditions for separation of EtOAc fraction from
L. japonica ..Condition of HPLC Analysis Column Shim-pack VP-ODS C18 Column (L : 250 mm, LD : 4.6 mm, 5 μm) Detector UVD 170s DIONEX Detection Wavelength 254 - 400 nm Flow Rate 1.0 mL/min Injection Volume 20 μL Mobile Phase Conditions for HPLC Gradient-elution Program order Time (min) 2% AA1) in water (%) 0.5% AA1) in 50% ACN2) (%) 1 0 100 0 2 10 100 0 3 150 50 50 4 200 30 70 5 210 30 70 6 215 100 0 7 220 100 0 1) AA : Acetic acid, 2) ACN : Acetonitrile.
-
Table 2 . HPLC conditions for separation of EtOAc fraction from
M. obovata ..Condition of HPLC Analysis Column Shim-pack VP-ODS C18 Column (L : 250 mm, LD : 4.6 mm, 5 μm) Detector UVD 170s DIONEX Detection Wavelength 254 - 400 nm Flow Rate 1.0 mL/min Injection Volume 20 μL Mobile Phase Conditions for HPLC Gradient-elution Program order Time (min) DW1) (%) 100% ACN2) (%) 1 0 75 25 2 40 50 50 3 60 5 95 4 70 5 95 5 75 95 5 1) DW : Distilled Water, 2) ACN : Acetonitrile.
Statistical Analysis
All experiments were conducted in triplicate, averaged and presented followed by the standard deviation.
Results and Discussion
Extraction Yields of L. japonica and M. obovata
The yields of the 50% ethanol extract and the ethyl acetate fraction from
-
Table 3 . Yields of
L. japonica andM. obovata extractions..Solvent Yield (%, w/w)* Lonicera japonica 50% EtOH Extract 4.85 EtOAc Fraction 0.41 Magnolia obovata 70% EtOH Extract 14.52 EtOAc Fraction 4.47 *Yield (%, w/w) = (weight of dried extract / weight of dried raw material) × 100.
Antimicrobial Activity of L. japonica and M. obovata Extracts
The antimicrobial activities of
-
Table 4 . Minimum Inhibitory Concentration (MIC, ug/ml) and Mininum Bactericidal Concentration (MBC) of
L. japonica andM. obovata extracts and fractions..Strains MIC (MBC) (μL/ml) Gram positive bacteria Gram negative bacteria Yeast Mold S. aureus B. subtilis E. coli P. aeruginosa C. albicans A. brasiliensis Lonicera japonica 50% EtOH extract5000 (10000) - ( - ) - ( - ) 5000 ( - ) - ( - ) - ( - ) Lonicera japonica EtOAc fraction78 (312) 10000 (10000) 5000 (5000) 312 (1250) 1250 (10000) 10000 ( - ) Magnolia obovata 70% EtOH extract78 (156) 2500 (2500) 2500 (5000) 1250 (1250) 1250 (5000) 10000 ( - ) Magnolia obovata EtOAc fraction39 (78) 2500 (5000) 2500 (2500) 625 (625) 625 (625) 2500 ( - ) Methyl paraben 2500 (5000) 2500 (10000) 1250 (5000) 1250 (1250) 1250 (5000) 625 (5000) Methylparaben used as a control. - no inhibition at 10,000 ug/ml..
The 50% ethanol extract from
The 70% ethanol extract and ethyl acetate fraction from
Synergistic Antimicrobial Effects of L. japonica and M. obovata
The synergistic antimicrobial effect of the two extracts was evaluated using the ethyl acetate fractions from
Checkerboard test. The checkerboard test was used to confirm the synergistic effect of the ethyl acetate fractions from the two extracts, which showed antimicrobial activity against all six strains. The results are shown in Fig. 1. A synergistic effect was observed against
-
Figure 1. Checkerboard test showing the synergistic antimicrobial effects of
L. japonica andM. obovata fractions against six microorganisms. (A)S. aureus , (B)B. subtilis , (C)E. coli , (D)P. aeruginosa , (E)C. albicans , (F)A. brasiliensis . Colored wells indicate microbial growth and white wells indicate no microbial growth. The number in the well is the FIC index. Wave patterns indicate the synergistic effect (FIC index ≤ 0.5) and dotted patterns mean an addition effect (0.5 <FIC index ≤ 1).
A synergistic antimicrobial effect was observed against
Kill-time analysis. In the previous experiment, we confirmed that the combination of
-
Figure 2. The synergistic antimicrobial effects of
L. japonica andM. obovata fractions againstB. subtilis by kill-time analysis. Control (○) was treated with DMSO only and the concentration of the ethyl acetate fraction fromL. japonica (□) and M. obovate (■) was 2,500 ppm and 625 ppm, respectively. Combination (▲) indicates a mixture ofL. japonica andM. obovata fractions. The experiments were conducted at 3-h intervals for 18 h and in triplicate. Data are presented as the mean ± SD.
We found that upon treatment with the
Componential Analysis of L. japonica and M. obovata
Since we have confirmed that the extracts have antimicrobial activities against the six strains, we used HPLC to determine the effective components of each extract. The results are shown in Figs. 3 and 4.
-
Figure 3. The HPLC chromatogram of ethyl acetate fraction of
L. japonica and the components in detection wavelength range from 254 to 400 nm. (A) Ethyl acetate fraction, caffeic acid; 1, luteolin; 2. (B) Caffeic acid. (C) Luteolin.
-
Figure 4. The HPLC chromatogram of ethyl acetate fraction of M. obovate and the components in detection wavelength range from 254 to 400 nm. (A) Ethyl acetate fraction, honokiol; 1, magnolol; 2. (B) Magnolol and honokiol (standard).
HPLC analysis of the
Analysis of the
Antimicrobial Effects of the Components of L. japonica and M. obovata Extracts
The antimicrobial activity of each component of each extract was evaluated, and the relationship between the antimicrobial activity of the extract and that of each component was determined. The antimicrobial activity was evaluated through a disc diffusion assay and the minimum inhibition concentration.
Disc diffusion assay. To evaluate the antimicrobial activity of each component of the
As shown in Fig. 5, the results were most effective against
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Figure 5. Antimicrobial activities of
L. japonica ,M. obovata extracts/fractions and the components against P. aerusinosa by disc diffusion assay. (A) Methylparaben (as a control); 1,L. japonica 50% EtOH extract; 2,L. japonica EtOAc fraction; 3, caffeic acid; 4, luteolin; 5. (B) Methylparaben (as a control); 1,M. obovata 70% EtOH extract; 2,M. obovata EtOAc fraction; 3, magnolol and honokiol; 4.
Minimum inhibitory concentration (MIC). To accurately evaluate the antimicrobial activity of each component of the
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Table 5 . Minimum Inhibitory Concentration (MIC, ug/ml) of the components of
L. japonica andM. obovata extracts..Strains MIC (μL/ml) Gram positive bacteria Gram negative bacteria Yeast Mold S. aureus B. subtilis E. coli P. aeruginosa C. albicans A. brasiliensis Methyl paraben 1250 1250 1250 1250 1250 625 Caffeic acid 312 5000 5000 2500 5000 > 10000 Luteolin 25 200 200 50 200 200 Magnolol, Honokiol 31.2 10000 10000 1250 5000 39 Methylparaben used as a control..
Caffeic acid showed high antimicrobial activity against
Magnolol and honokiol showed antimicrobial activity against all strains, especially
In this study, we evaluated the antimicrobial activities of
We also evaluated the synergistic antimicrobial activity of the ethyl acetate fractions from
To determine the extract components that have antimicrobial activity, we performed HPLC analysis and detected the presence of caffeic acid and luteolin in the
Conflict of Interest
The authors have no financial conflicts of interest to declare.
Fig 1.

Fig 2.

Fig 3.

Fig 4.

Fig 5.

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Table 1 . HPLC conditions for separation of EtOAc fraction from
L. japonica ..Condition of HPLC Analysis Column Shim-pack VP-ODS C18 Column (L : 250 mm, LD : 4.6 mm, 5 μm) Detector UVD 170s DIONEX Detection Wavelength 254 - 400 nm Flow Rate 1.0 mL/min Injection Volume 20 μL Mobile Phase Conditions for HPLC Gradient-elution Program order Time (min) 2% AA1) in water (%) 0.5% AA1) in 50% ACN2) (%) 1 0 100 0 2 10 100 0 3 150 50 50 4 200 30 70 5 210 30 70 6 215 100 0 7 220 100 0 1) AA : Acetic acid, 2) ACN : Acetonitrile.
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Table 2 . HPLC conditions for separation of EtOAc fraction from
M. obovata ..Condition of HPLC Analysis Column Shim-pack VP-ODS C18 Column (L : 250 mm, LD : 4.6 mm, 5 μm) Detector UVD 170s DIONEX Detection Wavelength 254 - 400 nm Flow Rate 1.0 mL/min Injection Volume 20 μL Mobile Phase Conditions for HPLC Gradient-elution Program order Time (min) DW1) (%) 100% ACN2) (%) 1 0 75 25 2 40 50 50 3 60 5 95 4 70 5 95 5 75 95 5 1) DW : Distilled Water, 2) ACN : Acetonitrile.
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Table 4 . Minimum Inhibitory Concentration (MIC, ug/ml) and Mininum Bactericidal Concentration (MBC) of
L. japonica andM. obovata extracts and fractions..Strains MIC (MBC) (μL/ml) Gram positive bacteria Gram negative bacteria Yeast Mold S. aureus B. subtilis E. coli P. aeruginosa C. albicans A. brasiliensis Lonicera japonica 50% EtOH extract5000 (10000) - ( - ) - ( - ) 5000 ( - ) - ( - ) - ( - ) Lonicera japonica EtOAc fraction78 (312) 10000 (10000) 5000 (5000) 312 (1250) 1250 (10000) 10000 ( - ) Magnolia obovata 70% EtOH extract78 (156) 2500 (2500) 2500 (5000) 1250 (1250) 1250 (5000) 10000 ( - ) Magnolia obovata EtOAc fraction39 (78) 2500 (5000) 2500 (2500) 625 (625) 625 (625) 2500 ( - ) Methyl paraben 2500 (5000) 2500 (10000) 1250 (5000) 1250 (1250) 1250 (5000) 625 (5000) Methylparaben used as a control. - no inhibition at 10,000 ug/ml..
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Table 5 . Minimum Inhibitory Concentration (MIC, ug/ml) of the components of
L. japonica andM. obovata extracts..Strains MIC (μL/ml) Gram positive bacteria Gram negative bacteria Yeast Mold S. aureus B. subtilis E. coli P. aeruginosa C. albicans A. brasiliensis Methyl paraben 1250 1250 1250 1250 1250 625 Caffeic acid 312 5000 5000 2500 5000 > 10000 Luteolin 25 200 200 50 200 200 Magnolol, Honokiol 31.2 10000 10000 1250 5000 39 Methylparaben used as a control..
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