Research Article

Korean Journal of Plant Resources. 31 December 2018. 719-724
https://doi.org/10.7732/kjpr.2018.31.6.719

ABSTRACT


MAIN

  • Introduction

  • Materials and Methods

  •   Plant material and sample preparation

  •   Test Microorganisms

  •   Antibiotics

  •   Minimum Inhibitory Concentration

  •   Checkerboard dilution test

  •   Colorimetric assay using MTT test

  • Results

  • Discussion

Introduction

Methicillin-resistant Staphylococcus aureus (MRSA) has been a problem since the 1960s as its infection is associated with higher mortality and increase cost in the hospitals (Klevens et al., 2007; Joung et al., 2012). It becomes more and more evident that bacteria, when faced with a new developed drug, respond with clever mechanisms of resistance (Tenover, 2006). Today, with this emergence of antibiotic resistant pathogens like MRSA, a new approach to natural products must be taken. These natural products are increasingly in demand due to their non-side effect benefit (Ghosh et al., 2008). Therefore, our ongoing efforts to find bioactive natural products have led us to study the antibacterial activity of Lysimachia clethroides Duby. The primary purpose of this study was to investigate the in vitro effect against MRSA. Lysimachia clethroides Duby, one of the species of genus Lysimachia, is a traditional folk Chinese medicine, distributed widely in many provinces of China. This plant has been used widely for treatment of throat ache, edema, and menoschesis (Bae, 1998).

It has also been shown to have antimicrobial activity on food-borne microorganisms (Han et al., 2001). Chemical study showed Astragalin, Isoquercitrin, Kaempferol-3-rutinoside, kaempferol-3-0-(2,6-di-o-rhamnopyranosylglucopyranoside), Kaempferol-3-0-(rhamnopyranosylglucopyranoside) (Yasukawa et al., 1986). Flavonoids and saponins (Zou et al., 2004; Ren et al., 2001) were present in this plant and the flavonoids were proved to be the main biological constituents, with the activities of anti-tumor, anti-bacterial and anti-platelet aggregation (Xu et al., 2003). However, little is known about its antimicrobial effects on MRSA. Thus, we present the current study demonstrating the antimicrobial activity of Lysimachia clethroides Duby against MRSA and methicillin-sensitive (MSSA) strains, as well as its synergistic effect.

Materials and Methods

Plant material and sample preparation

Lysimachia clethroides Duby roots were collected from Sunchon, southern Republic of Korea, in June, 2017. A voucher specimen was deposited in the Laboratory of Oriental Pharmacology (N.1369). Lysimachia clethroides Duby root was air-dried, and boiled in ethanol (2L for 3h). The ethanol extract of Lysimachia clethroides Duby root (5.67% w/w) was partitioned with organic solvents of different polarities to yield n-hexane, EtOAc, n-BuOH and water fractions, in sequence. The samples were stored at 4℃.

Test Microorganisms

Fourteen Clinical isolates (MRSA) were obtained from fourteen different patients at Wonkwang University Hospital (Iksan, South Korea). The Other 2 strains were S. aureus ATCC 33591 (Methicillin-resistant strain) and S. aureus ATCC 25923 (Methicillin-susceptible strain). Before use, all of the bacteria were stored in 30% glycerol and frozen at -70℃. The bacteria were cultured in Mueller-Hinton Broth (MHB) and Mueller-Hinton Agar (MHA) (Difco Laboratories, Baltimore, MD, USA). The bacteria were suspended in Mueller-Hinton Broth and then incubated at 37℃ for 24 hr.

Antibiotics

Ampicillin (AM) and Oxacillin (OX) (Sigma Chemical Co. St. Louis, M0, USA) were used.

Minimum Inhibitory Concentration

The Minimum Inhibitory Concentration (MIC) was determined using the broth microdilution method according to the clinical and Laboratory standards Institute guideline (CLSI., 2000). Briefly, a preparation of the microorganisms inoculated were done on 24 hr Broth cultures, and the suspensions were adjusted to a 0.5 McFarland standard turbidity (approximately 1.5×108 CFU/㎖). Final inoculums were adjusted to the 1.5×106 CFU/㎖. These serially diluted cultures were then incubated at 37℃ for 18 hr. MIC was defined at the lowest concentration of AM, OX, Lysimachia clethroides Duby extracts, Fractions (n-hexane, EtOAc, n-BuOH, H2O). At the end of the incubation period, the well plates were visually examined for turbidity. Cloudiness indicates that bacterial growth has not been inhibited by the concentration of antimicrobial agents contained in the medium. A colorimetric assay for rapid detection of the presence of bacteria was also performed (see below, Colorimetric assay using 3-4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide [MTT] test).

Checkerboard dilution test

The synergistic combinations were investigated in the preliminary checkerboard method performed using the MRSA, MSSA and the five isolate strains came from fourteen patients via MIC determination, according to the CLSI guidelines (Mazumdar et al., 2005). The MIC was defined as the lowest concentration of drug alone or in combination that inhibited the visible growth. The in vitro interaction was quantified by determining the fractional inhibitory concentration (FIC). The FIC index was calculated as follows: FIC = (MIC of drug A in combination/MIC of drug A alone) + (MIC of drug B in combination/MIC of drug B alone). FIC indices (FICI) were interpreted as follows: <0.5, synergy; 0.5-0.75, partial synergy; 0.76-1.0, additive effect; >1.0-4.0, indifference; and >4.0, antagonism. All experiments were independently repeated three times.

Colorimetric assay using MTT test

A colorimetric assay based on MTT for rapid detection of the presence of bacteria was performed as previously described (Luis et al., 2014; Joung et al., 2015; Shi et al., 2008). Briefly, a stock solution of 5 ㎎/㎖ MTT (Sigma) was prepared in phosphate-buffered saline and kept at -70℃. A final concentration of 1 ㎎/㎖ of MTT was used in the assay. After 24hrs of incubation a 37℃, 20 ㎕ of the yellow MTT was added to the 96-well microtiter plate and incubated for an additional 20 min. The presence of a blue color indicates the presence of bacteria.

Results

Ethanol extract had a MIC of 250 ㎍/㎖ against S. aureus ATCC 33591 under dark, and had a MIC of 250 ㎍/㎖ against S. aureus ATCC 25923 in the same condition. Antimicrobial activity of n-hexane fraction was remarkable, and had a MIC of from 15.62 ㎍/㎖ to 62.5 ㎍/㎖ against S. aureus strains (Table 1 and Table 2). n-hexane fraction of Lysimachia clethroides Duby root (HFL) lowered the MICs against the MRSA strain and MSSA but FICI values for HFL+AM and HFL+OX were 0.12-1 and 0.25-0.75, showing the increase of synergistic effect (Table 3 and 4).

Table 1. The S. aureus strains used in the experiments

S. aureus strains Class mecA gene Antibiotic resistance pattern
ATCC25923 MSSA -z -
ATCC33591 MRSA +z AMy, OXy
DPS -1x MRSA + AM, OX
DPS -2 MRSA + AM, OX
DPS -3 MRSA + AM, OX
DPS -4 MRSA + AM, OX
DPS -5 MRSA + AM, OX
DPS -6 MRSA + AM, OX
DPS -7 MRSA + AM, OX
DPS -8 MRSA + AM, OX
DPS -9 MRSA + AM, OX
DPS -10 MRSA + AM, OX
DPS -11 MRSA + AM, OX
DPS -12 MRSA + AM, OX
DPS -13 MRSA + AM, OX
DPS -14 MRSA + AM, OX
z(+), positive; (-), negative.
yAM, ampicillin; OX, oxacillin.
xDPS-1 indicates Staphylococcus aureus strains from the Department of Plastic Surgery, Wonkwang University Hospital.

Table 2. Antimicrobial activity of Lysimachia clethroides Duby root ethanol extract, n-hexane, EtOAc, n-BuOH and water fractions against S. aureus strains under dark

Minimal Inhibitory Concentration(MIC) (㎍/㎖)
S. aureus
strain
Ethanol
extract
Fractions
n-hexane EtOAc n-BuOH H20 Ampicillin Oxacillin
ATCC33591 250 31.25 250 NDy ND 1000 250
ATCC25923 250 31.25 250 ND ND 7.8 7.8
DPS -1z 250 31.25 250 ND ND 31.25 500
DPS -2 250 15.62 125 ND ND 1000 500
DPS -3 250 31.25 250 ND ND 31.25 500
DPS -4 250 31.25 250 ND ND 31.25 500
DPS -5 125 31.25 250 ND ND 31.25 500
DPS -6 250 62.5 250 ND ND 31.25 250
DPS -7 250 62.5 250 ND ND 250 500
DPS -8 250 62.5 250 ND ND 250 500
DPS -9 250 31.25 250 ND ND 125 500
DPS -10 250 31.25 250 ND ND 250 500
DPS -11 250 31.25 250 ND ND 250 500
DPS -12 250 31.25 250 ND ND 250 500
DPS -13 250 31.25 250 ND ND 31.25 1000
DPS -14 250 31.25 250 ND ND 250 500
zDPS1 indicates staphylococcus strains from the Department of Plastic Surgery, Wonkwang University Hospital. yND; no detected activity at this concentration.

Table 3. Result of the combined effect of n-hexane fraction of Lysimachia clethroides Duby root and AM against S. aureus

MICs (㎍/㎖)
S. aureus strain bHFL Alone With AM AM Alone With HFL cFICI
ATCC 25923 31.25 1.95 7.8 0.48 0.12
ATCC 33591 31.25 7.8 1000 250 0.5
DPS-1a 31.25 7.8 31.25 3.9 0.37
DPS-2 15.62 7.8 1000 62.5 0.56
DPS-3 31.25 7.8 31.25 7.8 0.5
DPS-4 31.25 7.8 31.25 7.8 0.5
DPS-5 31.25 7.8 31.25 7.8 0.5
DPS-6 62.5 7.8 31.25 15.62 0.62
DPS-7 62.5 15.62 250 31.25 0.37
DPS-8 62.5 15.62 250 31.25 0.37
DPS-9 31.25 15.62 125 7.8 0.56
DPS-10 31.25 7.8 250 62.5 0.5
DPS-11 31.25 7.8 250 62.5 0.5
DPS-12 31.25 15.62 250 62.5 0.75
DPS-13 31.25 15.62 31.25 15.25 1
DPS-14 31.25 7.8 250 62.5 0.5
cFICI; fractional inhibitory concentration index.
bHFL; n-hexane fraction of Lysimachia clethroides Duby root.
aDPS; indicates Staphylococcus aureus strains from the Department of Plastic Surgery, Wonkwang University Hospital.

Table 4. Result of the combined effect of n-hexane fraction of Lysimachia clethroides Duby root and OX against S. aureus

MICs(㎍/㎖)
S. aureus strainbHFL Alone With OX OX Alone With HFL cFICI
ATCC 25923 31.25 3.9 7.8 0.97 0.25
ATCC 33591 31.25 7.8 250 62.5 0.5
DPS-1a 31.25 1.95 500 250 0.56
DPS-2 15.62 3.9 500 125 0.5
DPS-3 31.25 15.62 500 125 0.75
DPS-4 31.25 15.62 500 125 0.75
DPS-5 31.25 7.8 500 125 0.5
DPS-6 62.5 15.62 250 62.5 0.5
DPS-7 62.5 15.62 500 62.5 0.37
DPS-8 62.5 15.62 500 62.5 0.37
DPS-9 31.25 7.8 500 125 0.5
DPS-10 31.25 7.8 500 125 0.5
DPS-11 31.25 7.8 500 125 0.5
DPS-12 31.25 15.62 500 62.5 0.62
DPS-13 31.25 7.8 1000 125 0.37
DPS-14 31.25 7.8 500 31.25 0.31
cFICI; fractional inhibitory concentration index
bHFL; n-hexan fraction of Lysimachia clethroides Duby root
aDPS; indicates Staphylococcus aureus strains from the department of plastic surgery Wonkwang University Hospital.

Discussion

The most effective method is to develop antibiotics from the natural products without having any toxic or side effects. Therefore, there is a need to develop alternative antimicrobial drugs for the treatment of infections diseases. Combination therapy is the most commonly recommended empirical treatment for bacterial infections in intensive care units, where monotherapy may not be effective against all potential pathogens, and for preventing the emergence of resistant mutants (Drago et al., 2007; Joung et al., 2016). When combined together, these antibiotic effects were dramatically increased. Different drug combinations are reported to treat infections caused by pathogens (Miranda-Novales et al., 2006; Drago et al., 2007; Liu et al., 2000). The (Methicillin-resistant) of 15 MRSA strains and S. aureus ATCC 25923 (Methicillin-susceptible strain) to the tested antibiotics. Antimicrobial activity of n-hexane fraction was remarkable, and had a MICs ranging from 31.25 ㎍/㎖ to 62.5 ㎍/㎖ and checkerboard dilution test was performed to determine the action of HFL alone as well as its synergistic action with AM, or OX against the 16 strains. When tested against ATCC 33591, our data indicated that HFL alone only had moderate inhibitory effect on the growth of MRSA. However, in the presence of a nongrowth inhibitory dose of HFL (31.25 ㎍/㎖) or AM (1000 ㎍/㎖), HFL together with AM was highly effective with a FICI of 0.5. Similar effects were also observed in MSSA strain. These results showed that HFL in combination with these antibiotics could effectively inhibit MRSA growth. It may be partly due to the fact that they had abundant flavonoids which contributed to their antimicrobial activity and should be further studied. In conclusion, we found that Lysimachia clethroides Duby root extracts and n-hexane fraction have an antibacterial effect on MRSA and MSSA, and showing the increase of synergistic effect.

Acknowledgements

This study was supported by Sunchon National University Research Fund in 2018.

References

1
Bae, K.H. 1998. The Medicinal Plant of Korea. Kyo Hak Pub. Co., Ltd. Seoul, Korea. p. 391.
2
CLSI. 2000. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically: Approved Standard: In Wayne, USA.
3
Drago, L., E. De Vecchi, L. Nicola and M.R. Gismondo. 2007. In vitro evaluation of antibiotics' combinations for empirical therapy of suspected methicillin resistant Staphylococcus aureus severe respiratory infections. BMC Infectious Diseases 7:111.
10.1186/1471-2334-7-11117888153PMC2025599
4
Ghosh, A., B.K. Das, A. Roy, B. Mandal and G. Chandra. 2008. Antibactrial activity of some medicinal plant extracts. J. Natl. Med 62:259-262.
10.1007/s11418-007-0216-x18404337
5
Han, J.S., D.H. Shin. 2001. Antimicroial activity of Lysimachia clethroides Duby extracts on food-borne Microorganisms. Korea J. Food SCI. Technol 33(6):774-783.
6
Joung, D.K., D.Y. Shin, D.Y. Kwon and D.W. Shin. 2016. Antibacterial activity and synergism of Hydnocarpi Semen extracts with ampicillin or oxacillin against methicillin-resistant Staphylococcus aureus. Korea J. Plant Res. 29(6):699-703.
10.7732/kjpr.2016.29.6.699
7
Joung, D.K., S.H. Choi, O.H. Kang, S.B. Kim, S.H. Mun, Y.S. Seo, D.H. Kang, R. Gong, D.W. Shin, Y.C. Kim and D.Y. Kwon. 2015. Synergistic effects of oxyresveratrol in conjunction with antibiotics against methicillin-resistant Staphylococcus aureus. Molecular Medicine Reports 12(1):663-667.
10.3892/mmr.2015.334525683461
8
Klevens, R.M., M.A Morrison, J. Nadle, S. Petit, K. Gershman, S. Ray, L.H. Harrison, H. Lynfield, G. Dumyati, J.M. Townes, A.S. Craig, E.R. Zell, G.E. Fosheim, L.K. McDougal, R.B. Carey and S.K. Fridkin. 2007. Invasive methicillin-resistant Staphylococcus aureus infections in the United States. J. Am. Med. Assoc 298:1763-1771.
10.1001/jama.298.15.176317940231
9
Liu, I.X., D.G. Durham and R.M. Richards. 2000. Baicalin synergy with beta-lactam antibiotics against methicillin-resistant Staphylococcus aureus and other beta-lactam-resistant strains of S. aureus. J Pharm Pharmacol. 52:361-366.
10.1211/002235700177392210757427
10
Luis, A., L. Breitenfeld, S. Ferreira, A.P. Duarte and F. Domingues. 2014. Antimicrobial, antibiofilm and cytotoxic activities of Hakea sericea Schrader extracts. Pharmacognosy Magazine 10(1):S6-S13.
24914310PMC4047586
11
Mazumdar, K., N.K. Dutta, K.A. Kumar and S.G. Dastidar. 2005. In vitro and in vivo synergism between tetracycline and the cardiovascular agent oxyfedrine HCl against common bacterial strains. Biological & Pharmaceutical Bulletin 28(4):713-717.
10.1248/bpb.28.713
12
Miranda-Novales, G., B.E. Leanos-Miranda, M. Vilchis-Perez and F. Solorzano-Santos. 2006. In vitro activity effects of combinations of cephalothin, dicloxacillin, imipenem, vancomycin and amikacin against methicillin-resistant Staphylococcus spp. strains. Annals of Clinical Microbiology and Antimicrobials 5:25.
10.1186/1476-0711-5-2517034644PMC1617116
13
Ren, F.Z. Ren, J.K. Qie, H.H. Qu, X.H. Luan and Y.M. Zhao. 2001. Pharm. J. Chin. PLA 17:178–180.
14
Shin, S.W., J.H. Lee and K.S. Bang. 2012. Antioxidant and antimicrobial activities of Xanthium sibiricum. Korea J. Plant Res. 25(4):372-378.
10.7732/kjpr.2012.25.4.372
15
Shi, Y.J. Chen and M. Xu. 2008. A new method for antimicrobial susceptibility testing of in vitro-cultured bacteria by means of resonance light scattering technique. Journal of Microbiology and Biotechnology 18(1):118-123.
18239427
16
Tenover, F.C. 2006. Mechanisms of antimicrobial resistance in Bacteria. Am. J. Med 119:3-10.
10.1016/j.amjmed.2006.03.01116735149
17
Xu, X.Y., L.H. Tang, Z.Q. Liang and Z.L. Gu. 2003. Chin. Wild Plant Resour. 22:31-34.
18
Yasukawa, K. and M. Takido. 1986. Studies on the chemical constituents of genus Lysimachia. I. on the whole parts of Lysimachia japonica Thunb. and Lysimachia chethrides Duby. Yakugaku Zasshi 106(10):939
10.1248/yakushi1947.106.10_939
19
Zou, H.Y. and P.F. Tu. 2004. Antioxidant and a-glucosidase inhibitory compounds in Lysimachia clethroides. Chin. J. Nat. Med. 2:59-61.
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