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Phytochemical profile of ethanolic extracts of Chimonanthus salicifolius S. Y. Hu. leaves and its antimicrobial and antibiotic-mediating activity
Industrial Crops & Products 125 (2018) 328–334
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Phytochemical profile of ethanolic extracts of Chimonanthus salicifolius S. Y. Hu. leaves and its antimicrobial and antibiotic-mediating activity Ning Wang, Hui Chen, Lei Xiong, Xin Liu, Xiang Li, Qi An, Ximei Ye, Wenjun Wang
T
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Key Lab for Natural Products and Functional Foods of Jiangxi Province, College of Food Science and Engineering, Jiangxi Agricultural University, Nanchang 330045, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Chimonanthus salicifolius S. Y. Hu. leaves Antibacterial activity Flavonoids Antibiotics
The aim of this study was to determine the phytochemical profiles in the ethanol elutions of Chimonanthus salicifolius S. Y. Hu. leaves (EECS) and study its antibacterial activity. The ethanol extracts of C. salicifolius S. Y. Hu. leaves were prepared by ethanol gradient elution orderly and analyzed by HPLC-DAD. Three bacterial and four fungal species were used to evaluate the antimicrobial activities of the EECS by the broth microdilution method. Results showed that the MIC values against bacteria ranged from 0.35 mg/mL to 11.25 mg/mL (Escherichia coli CVCC1490, Staphylococcus aureus, and Bacillus subtilis). The C. salicifolius S. Y. Hu. leaves exhibited MIC values ranging from 2.81 mg/mL to 45 mg/mL against both the strains of yeast (Saccharomyces cerevisiae CICC 1540, Saccharomyces cerevisiae CICC 1340) and against strains of Cephalckiscus Fiagans (Penicillium digitatum AS3.5752, Aspergillus niger) with the MIC values ranging from 2.81 mg/mL to 11.25 mg/ mL, respectively. Remarkable antibacterial potential was marked with low minimal inhibitory concentration (MIC) for the 50%, 70% and 95% ethanolic fractions against bacteria. The HPLC analysis revealed quercetin and kaempferol as major ingredients in the screening EEs. Interestingly, the content of quercetin and kaempferol varied with the concentration of elution ethanol. The synergistic and additive effects of the selected EEs were frequently observed against E. coli, S. aureus and B. subtilis when combined with standard antibiotics (aminoalcohol, aminoglycosides and β-lactam antibiotics). In conclusion, the EECS possessed the property of modifying antibiotics against bacteria. Synergistic combination can be used to reduce the use of antibiotics or look forward to dealing with drug-resistant bacteria, and also provide theoretical underpinning as natural food preservatives and medicinal plants.
1. Introduction About 50 000 deaths per annum in Europe and the United States were due to antibiotic resistant infections, which predicted that the problem would exceedingly increase over the next decade (Mayor, 2016). Resistance crisis has eventually been noticed to almost all antibiotics developed, which was due to inappropriate prescribing, extensive agricultural use and availability of few new antibiotics et al (Ventola, 2015). On the other hand, synthetic and semi-synthetic chemical preservatives pose potential health risks to humans due to the unreasonable use (Xiong et al., 2013). Medicinal plants were abundant in polysaccharides, triterpenes, phenolic and flavonoid compounds etc., which attracted widespread interest toward development natural antimicrobial agents as novel preservatives and functional supplementations. Furthermore, there are about 250,000–500,000 plant species on the earth and only about 5000 species of these have been researched so far (Borris, 1996).
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Many researches have focused on developing plant natural products as novel antimicrobial, which could also combat bacterial resistance in cooperation with standard antibiotics, due to its safety and efficiency (Ambrosio et al., 2017; Cristo et al., 2016). For instance, Eumkeb et al. showed that synergistic FIC indices (FIC index, < 0.02–0.11) were observed against Penicillin-resistant S. aureus in combination with all selected β-lactam antibiotics (methicillin, ampicillin) and test flavonoids from Alpinia officinarum Hance (Eumkeb et al., 2010). Chimonanthus salicifolius S. Y. Hu. leaves also known as Shi liang tea and Xiang feng tea, is an excellent functional green tea and medicinal plant, mainly distributed in the Southern Anhui, Hubei and Jiangxi provinces etc. (Bi et al., 2013). Investigations were performed on bioactive substances of C. salicifolius S. Y. Hu leaves including coumarin, volatile oil, essential oil and flavonoids. (Ouyang, 2009; Ouyang et al., 2010; Yang et al., 2012). Our previous studies have demonstrated that C. salicifolius S. Y. Hu leaves presented notable α-glucosidase inhibitory activity (flavonoids function) antihyperglycemic and antihyperlipidemic (Chen
Corresponding author. E-mail address: [email protected] (W. Wang).
https://doi.org/10.1016/j.indcrop.2018.09.021 Received 17 May 2018; Received in revised form 7 September 2018; Accepted 11 September 2018 0926-6690/ © 2018 Elsevier B.V. All rights reserved.
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Table 1 Antibacterial activities of different ethanol eluates from Chimonanthus salicifolius S. Y. Hu. leaves. Bacteria
MICs / MBCs obtained by different extracts / (μg / μL)
Type of strain
CEE E. coli (cvcc1490) S. aureus B. subtilis Saccharomyces cerevisiae (CICC 1340) Saccharomyces cerevisiae (CICC 1540) Penicillium digitatum (AS3.5752) Aspergillus niger
WE
G−
11.25 / 45
G+ G+ yeast yeast Cephalckiscus Fiagans Cephalckiscus Fiagans
11.25A/ > 45A 5.63B/ > 45A 45A/ > 45A 45A/ 45A 2.81C/11.2B 5.63B/ 45A
A
A
30% EE A
11.25 / > 45
A
11.25A/ > 45A 11.25A / > 45A 5.63C/ > 45A 5.63C/45A 2.81C/5.63C 2.81C/ > 45A
B
50% EE A
C
70% EE B
C
95% EE A
0.70D/ > 45A
5.63 / > 45
2.81 /5.63
2.81 /45
11.25A/ > 45A 5.63B/ > 45A 11.25B/ > 45A 11.25B/ > 45A 11.25A/11.25B 11.25A/ > 45A
2.81B/45A 0.7D/ > 45A 2.81D/45A 5.63C/22.5B 5.63B/11.25B 2.81C/ 45A
0.70C/ 45A 2.81C/ 45A 2.81D/ > 45A 5.63C/ > 45A 5.63B > 45A 5.63B/5.63C
0.35D/5.63B 0.35E/ > 45A 2.81D/11.25B 2.81D/11.25C 2.81C/2.81D 2.81C/ 22.5B
Note: G + refers to Gram - positive; G − refers to Gram-negative. A − E: Averages within the same row with different superscripts are significantly different by the Tukey test (P < 0.001). Table 2 Flavonoids composition of different ethanol eluates from Chimonanthus salicifolius S. Y. Hu. leaves. Compounds
Calibration curve
R2
ethanol eluates from C. salicifolius S. Y. Hu. leaves (μg/mL) 50% EE
Rutin Hyperin Isoquercitrin Luteoloside Astragalin Luteolin-5-Oglucoside Quercetin Kaempferol
70% EE
95% EE
LOD
LOQ
Y = 18.19x - 88.422 Y = 31.508x + 94.029 Y = 27.098x + 1185.8 Y = 18.928x + 193.19 Y = 28.368x - 46.802 Y = 14.642x + 165.91
0.9989 0.9986 0.9957 0.9989 0.9984 0.9993
160.36 ± 0.03 19.62 ± 0.01E 78.76 ± 0.18D 229.03 ± 0.05A 55.45 ± 0.02D 68.20 ± 0.02D
– – – – – –
– – – – – –
0.020 0.011 0.013 0.019 0.013 0.024
0.059 0.034 0.040 0.057 0.038 0.074
Y = 13.209x - 104.25 Y = 19.144x - 160.18
0.9980 0.9980
176.70 ± 0.06BC 197.24 ± 0.05B
719.33 ± 0.38*** 917.54 ± 0.52***
1362.31 ± 0.23*** 1270.09 ± 0.21***
0.027 0.019
0.082 0.056
C
Results are expressed as mean ± S.D. of three determinations. LOD: limit of detection and LOQ: limit of quantification. Averages followed by different letters differ by the Tukey test at P < 0.01, ***P < 0. 001 compared to the fraction of 50% ethanol eluate.
Fig. 2. High performance liquid chromatography profile of 70% EE of Chimonanthus salicifolius S. Y. Hu. leaves. Quercetin (peak 1); kaempferol (peak 2).
Fig. 1. Representative high performance liquid chromatography profile of 50% EE of Chimonanthus salicifolius S. Y. Hu. leaves. Rutin (peak 1); hyperin (peak 2); isoquercitrin (peak 3); luteoloside (peak 4); luteolin-5-O-glucoside (peak 5); astragalin (peak 6); unknown (peak 7); unknown (peak 8); quercetin (peak 9); kaempferol (peak 10).
plant flavonoids possessed excellent antimicrobial activity and corresponding mechanisms (Cushnie and Lamb, 2011; Cushnie and Lamb, 2005). The flavonoid aglycones, quercetin and kaempferol were the two important ingredients in C. salicifolius S. Y. Hu leaves. Because of affluent secondary metabolites in C. salicifolius S. Y. Hu leaves, it is proposed to have bacteriostatic activity. In the present study, we primitively investigated the antimicrobial properties of the EECS using bacteria, yeast and molds. Identification and quantification of the antimicrobial phytochemicals from C. salicifolius S. Y. Hu leaves was done by high performance liquid chromatography-diode array detector (HPLC-DAD) in sequence. This work
et al., 2017a, b). Wu et al., proved that the dichloromethane fraction of C. salicifolius S. Y. Hu leaves presented best antimicrobial activity against four common bacteria (Wu et al., 2017), but the specific chemical composition of the fraction was lack of further exploration. Flavonoids contain over 4000 biologically active compounds, which belong to polyphenols, abundant in tea, wine, honey, nuts etc (Fathima and Rao, 2016). Flavonoids were biosynthesized mainly through two pathways including the shikimic acid and acetic acid pathways (Cazarolli et al., 2008). There are several reviews demonstrated that 329
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Fig. 3. High performance liquid chromatography profile of 95% EE of Chimonanthus salicifolius S. Y. Hu. leaves. Quercetin (peak 1); kaempferol (peak 2).
Fig. 4. The selected ethanol extracts of Chimonanthus salicifolius S. Y. Hu.leaves in association with antibiotics against E. coli.
Table 3 Antibiotics potentiating effect of Chimonanthus salicifolius S. Y. Hu.leaves extracts. Antibiotics
Bacteria E. Coli (G-)
MIC (ug/mL) alone
50%EE
70%EE
95%EE
Chloramphenicol Streptomycin Imipenem Chloramphenicol Streptomycin Imipenem Chloramphenicol Streptomycin Imipenem Chloramphenicol Streptomycin Imipenem
S. Aureus(G+)
3.91 3.91 7.81 7.81 7.81 0.98 ƩFIC of plant extracts: 0.50 0.75 0.28 0.38 0.75 2.25 0.75 1.25 0.31 0.63 1.25 – 1.06 0.56 0.13 0.13 1.06 –
B. Subtilis(G+)
3.91 7.81 0.98 0.50 0.28 0.50 0.75 0.53 1.25 0.56 0.31 –
Note: No antimicrobial activity is indicated by-, refer to antagonism (ΣFIC > 2); Additive effects are indicated with figures underlined (0.5 < ΣFIC ≤1); Indifference are marked with nothing (1 < ΣFIC ≤ 2); Synergy is indicated with figures in bold figures underlined (ΣFIC ≤0.5).
Fig. 5. The selected ethanol of Chimonanthus salicifolius S. Y. Hu.leaves in association with antibiotics against S. aureus.
focused on evaluating in vitro the antimicrobial and antibiotic modulatory potential of the selected ethanol eluates (EEs) against S. aureus, P. subtilis and E. coli alone, or in association with aminoalcohol (Chloramphenicol), aminoglycosides (Streptomycin) and β-lactam antibiotics (Imipenem).
Medium with Chloramphenicol; Yeast Malt Agar (YMA) and Yeast Malt Broth (YMB) were purchased from Qingdao Hope Bio-Technology Co., Ltd (Qingdao, China). Analytical grade chemicals were employed in all assays. 2.2. Microorganisms and cultures
2. Materials and methods
Staphylococcus aureus, Bacillus subtilis, Aspergillus niger were obtained from the Lab of Light Chemical Engineering Teaching and Research Department, College of Food Science and Engineering, Jiangxi Agricultural University, China. Saccharomyces cerevisiae (CICC 1540), Saccharomyces cerevisiae (CICC 1340), Escherichia coli (CVCC1490) and Penicillium digitatum (AS3.5752) were purchased from Guangdong Microbial Culture Center (GDMCC). Saccharomyces cerevisiae cultures were grown statically in YMB. Molds were cultivated in Potato Dextrose liquid medium.
2.1. Chemicals and regents Acetonitrile (HPLC grade) was purchased from TEDIA Company Inc., (Shanghai, China). Flavonoids Standards including luteoloside, kaempferol, astragalin, luteolin-5-O-glucoside, rutin, hyperin, isoquercitrin, and quercetin were obtained from Beijing Solarbio Science & Technology Co., Ltd (Beijing, China). Dimethyl sulfoxide (DMSO) was obtained from Tianjin Wingtai Chemical Co., Ltd., (Tianjin, China). The sample solutions and mobile phase were filtered and sonicated before HPLC analysis. The analytical HPLC system is described as the previous reference (Chen et al., 2017a). Mueller-Hinton Agar (MHA); Mueller Hinton broth (MHB); Potato Dextrose Agar (PDA); Potato Liquid
2.3. Plant collection and identification The dried C. salicifolius S. Y. Hu leaves were purchased from a local 330
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incubation temperature of yeast and molds was 28 ℃. The A. niger and P. digitatum were made of spore suspension, which was counted by the plate line and diluted to corresponding quantity. 100 μL of a set of twofold serial dilutions of EECS in MHB were added to 100 μL of the microorganism suspension together in a sterile 96-well microplate, giving final concentrations from 0.35 to 45 μg/μL. The five ethanol elutions and CEE in pure DMSO (90 μg/μL) were thinned in MHB by using flatbottomed sterile 96-well microplates in order to gain a concentration ranging from 0.35 to 45 μg/μL. The plate was again incubated at corresponding temperature for 18 h. We used sterility (no inoculum added), inoculum viability (no flavonoids added) as positive and negative control, respectively. After cultivation, 20 μL of resazurin solution (0.05% w/v) was poured into all holes, followed by final incubation of 1 h at appropriate conditions. Holes were measured visually: a color change from blue to pink or mauve, was taken as representative of bacterial reproduction; while the highest dilution which remained blue was used to represent the MIC in contrast. The MIC was defined as the minimum concentration, at which microorganisms were unable to grow in MHB supplemented with ethanol elutions. Streak plate method was applied to determine MBC from the uncolored holes, in which no bacteria growth in the above MIC assay, then incubated for 24 h at 37 ℃ on MHA. Yeast and molds were streaked plate from the uncolored wells and cultured for 48 h at 28℃ on PDA. The MBC was defined as the lowest concentration at which microorganisms were all killed with ethanol elutions. This experiment was repeated in triplicate. DMSO at a non-toxic concentration was applied in the experiment.
Fig. 6. The selected ethanol of Chimonanthus salicifolius S. Y. Hu.leaves in association with antibiotics against B. Subtilis.
farmer in Lishui City (Zhejiang, China). All voucher specimens were dried in the constant temperature oven until the weight remained stable. The dried plant material was ground into fine powder, passed through a 60-meshsieve and stored in a desiccator until next use. 2.4. Extraction procedures
2.6. High performance liquid chromatography (HPLC)
The powder of C. salicifolius S. Y. Hu leaves were extracted twice at 40℃ followed our previous research: ultrasonic treatment time was 30 min, ultrasonic power was 57.59 W, ethanol concentration was 52.03%, and solid-liquid ratio was 1:23.85. After filtering, the filtrates were pooled, concentrated and further purified by polyamide resin. The adsorbed resin was eluted with gradient aqueous ethanols. The process leaded to in five fractions remained including, 95% ethanol eluate (95% EE), 70% EE and 50% EE, 30% EE, water eluate (WE) at last. Detailed experimental procedures followed previous literature (Chen et al., 2017a).
The EECS were injected into a Waters Symmetry C18 (250 × 4.6 mm, 5 μm, Waters, Ireland) column. Corresponding mobile phases and solvent conditions were used as described by previous literature (Chen et al., 2017a). The C. salicifolius S. Y. Hu leaves extracts and the mobile phase were filtered through a 0.22 μm microporous organic membrane, and degassed by ultrasonic bath before using. The EEs dissolved in methanol were tested at a concentration of 10 mg/mL. Comparing the retention time of reference standards with the HPLC chromatography peaks of the EECS was obtained by DAD spectra. The phytochemicals would have been confirmed. Calibration curves of flavonoid standards were supplemented in Table 2. All chromatography operations were in triplicates. The LOD and LOQ were calculated as 3.3 and 10 δ/S, respectively, where δ represents the standard deviation of the response, and S represents the slope of the calibration curve.
2.5. Determination of the minimum inhibitory concentration (MIC) and minimum bacterial concentration (MBC) MIC was conducted according to the standard microbroth dilution technique following the guideline of National Committee for Clinical Laboratory Standards with some modifications (Ferraro, 2004). The bacteria was cultured in MHB at 37℃ overnight to give the logarithmic phase bacteria and adjusted to 0.5 McFarland standards (Guangdong Huankai Microbial Sci. & Tech. CO., Ltd, Guangzhou, China) and further diluted with MHB to achieve approximately 1 × 106 CFU/mL, so do S. cerevisiae (CICC 1540) and S. cerevisiae (CICC 1340). The
2.7. Modulation of the antibiotic activity of aminoalcohol, aminoglycosides and β-lactam The antibiotic modulating effects of the 50% EE, 70% EE and 95% EE were determined by a checkerboard method with a few adjustments (Taukoorah et al., 2016). The extracts, the antibiotic and the inoculum were distributed into 36 wells (6 × 6) so that the final volume of the
Table 4 Antibacterial activities of Quercetin and Kaempferol. Compounds
Quercetin Kaempferol
MICs / MBCs obtained by Quercetin and Kaempferol / (μg / mL) E. coli G−
S. aureus G+
B.subtilis G+
Saccharomyces cerevisiae (CICC 1340) yeast
Saccharomyces cerevisiae (CICC 1540) yeast
Penicillium digitatum (AS3.5752) Cephalckiscus Fiagans
Aspergillus niger Cephalckiscus Fiagans
2.8A/ > 2500D 317.5B/ > 2500D
317.5A/ > 2500
1250B/ > 2500
156A/317.5B
156A/317.5B
156A/156A
156A/1250C
D
D
317.5B/1250C
317.5A/ > 2500D
156A/317.5A
156A/1250C
156A/625B
156A/1250C
Note: G + refers to Gram - positive; G − refers to Gram-negative. A–D: Averages within the same row with different superscripts are significantly different by the Tukey test (P < 0.001). 331
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et al., 2010). For instance, the MIC value of quercetin was 6.25 μg/mL and 100 μg/mL both for S. aureus and E.coli, whereas the MIC value of isoquercitrin (its corresponding glycosides) was 100 μg/mL and > 400 μg/mL, respectively. Hong et al. demonstrated that quercetin inhibited the growth and protein utilization of the E. coli through the disruption of cell wall and the loss of physiological function of bacterium (Hong et al., 2017). Kaempferol was proved to inhibit the growth of S. aureus by disturbing reproduction of genetic material (Bernard et al., 1997). The bactericidal mechanisms of flavonoids were in several different paths on literature published: (a) Destroy of cytoplasmic membrane by perforating and decrease in membrane fluidity. (b) Inhibitory energy metabolism. (c) Suppression of synthesis of genetic material. (d) Neutralization of bacterial toxins (Ahmad et al., 2015). (e) Metal chelation by flavonoids (Cowan, 1999) (f) Inhibition the formation of biofilm. (g) Inhibition the carrier protein of cell membrane (Xie et al., 2015). Second, the interactions among phytochemicals resulted in either superposition or antagonism effects. For example, quercetin (inhibitory zone diameter 13.5 ± 0.21 mm) was demonstrated to be more active than the combination (inhibitory zone diameter 0) between quercetin and rutin against S. aureus (Amin et al., 2015). Studies indicated that strong activity was defined as MIC < 5 mg/mL (Bussmann et al., 2010). Thus, the criterion of MIC (< 5 mg/mL) classified 50% EE, 70% EE and 95% EE as having outstanding competence than the others against bacteria. Hence, it was important to demonstrate the constituents and the regulative effects of different kinds of antibiotics for the selected fractions in coming future.
mixture was 200 μL according to 1: 1: 2. The antibiotic was serially diluted along the Y axis while the EECS was serially diluted along the X axis so that both of them with the variation range of the final concentration was 0–1 MIC. All combinations were tested. 100 μL of prepared inoculums was poured into individual wells as the mixture was incubated overnight in a suitable condition. After the incubation was complete, resazurin solution was used as an indicator to determine the growth of microorganisms. 2.7.1. Fractional inhibitory concentration index (ΣFIC) and interpretation The fractional inhibitory concentration index (ΣFIC) of the combination was counted according to the following equation:
∑ FIC =
MIC of EEs in combination MIC of EEs in alone MIC of antibiotic in combination + MIC of antibiotic in alone
The combined effects of ethanol elutions and antibacterial agents were determined by ΣFIC according to the following criteria: synergy ΣFIC ≤0.5, additive effects 0.5 < ΣFIC ≤1, indifference 1 < ΣFIC ≤ 2, antagonism ΣFIC > 2 (Guendouze-Bouchefa et al., 2015). The selection of holes to calculate the final ΣFIC was highly disputed. 2.8. Determination of the MIC and MBC of quercetin and kaempferol The MICs and MBCs of quercetin and kaempferol were done according to the Section 2.5. The standard products of quercetin and kaempferol (purity > 99%) were used.
3.2. HPLC analysis
2.9. Statistical analysis
The HPLC analysis of the 50%, 70%, and 95% EE obtained from C. salicifolius S. Y. Hu. leaves were presented in Figs. 1–3, respectively. All chromatographic peaks represented substances were as following: In Fig. 1, rutin (tR = 11.59 min, Peak 1); Hyperin (tR = 12.45 min, Peak 2); Isoquercitrin (tR = 13.58 min, Peak 3); Luteoloside (tR = 14.56 min, Peak 4); Astragalin (tR = 15.62 min, Peak 5); Luteolin-5-O-glucoside (tR = 16.85 min, Peak 6); Unknown (tR = 17.88 min, Peak 7); Unknown (tR = 18.96 min, Peak 8); Quercetin (tR = 25.52 min, Peak 9); Kaempferol (tR = 33.12 min, Peak10). In Fig. 2, Quercetin (tR = 25.54 min, Peak 1); Kaempferol (tR = 33.18 min, Peak2). In Fig. 3, Quercetin (tR = 25.54 min, Peak 1); Kaempferol (tR = 33.18 min, Peak 2). From Table 2, as we could see, luteoloside was the most abundant component in 50% EE, followed with kaempferol and quercetin. Interestingly, the content of flavonoid aglycones (quercetin and kaempferol) followed with the augment in the concentration of elution ethanol, which was in accordance with the tendency of antimicrobial activity from C. salicifolius S. Y. Hu. leaves.
All tests were performed at least in triplicate. Significant difference analysis was conducted using Tukey’s multi-comparison test, which was carried out by processor of SPSS Statistics 17.0. Differences between means were statistically considered significant if the p value was less than 0.05. 3. Results and discussion 3.1. Antimicrobial activity All the eluted fractions were tested for antimicrobial effectiveness using a microwell dilution assay, and the corresponding MIC values were summarized in Table 1. It was well known that the lower value of MIC or MBC was, the more sensitive of the microorganism was to the ethanol eluates. Antimicrobial effect (MIC) of the EECS ranged from 0.35 μg/μL to 45 μg/μL. Bactericidal activity (MBC) of the EECS ranged from 2.81 μg/μL to > 45 μg/μL, from which we implied that the inhibitory effect of the EECS was bacteriostatic rather than bactericidal against all the strains. Among EEs, the 95% EE was proved to possess the highest activity, with MIC values ranging from 0.35 μg/μL to 2.81 μg/μL against microorganisms. Wheares, the CEE was the most inactive against all microorganisms except for P. digitatum (MIC 2.81 μg/μL). It was notable that the traditional way of drinking, referred to WE, merely had a remarkable antibacterial effect on P. digitatum (2.81 μg/μL) and A. niger (2.81 μg/μL). Our results were consistent with the previous reports (Bi et al., 2013; Cheng et al., 2015; Li et al., 1996). The antimicrobial potency of the EECS mainly was attributed to the existence of abundant flavonoids and the interaction among phytochemicals. First, flavonoids such as quercetin, rutin, luteoloside, and other phytochemicals have been shown to have significant antimicrobial activities (Xiong et al., 2013; Butkhup et al., 2016; Rauha et al., 2000). In addition, the activity of the 50% EE mostly was due to flavonoids aglycones quercetin and kaempferol, which comprised only 19% of 50%EE (data not show), but the aglycones with relative low polarity had stronger bioactivity than their glycosides (Hao
3.3. Modulation of the antimicrobial activity of antibiotics The antibiotics modulation activity of the selected extracts was determined using calculating the ΣFICs by an adapted checkerboard method. The MICs of Chloramphenicol, Streptomycin and Imipenem, and the effects of the combination of the selected EEs with antibiotics against E. coli, S. aureus and B. subtilis were summarized in Table 3. The synergistic effectiveness was determined in the all combinations when associated with Streptomycin as demonstrated by the ΣFIC values, except when associated with the 70% EE against S. aureus and B. subtilis. The synergy and additive properties were always observed among the 50% EE – antibiotics combinations, apart from the combination of 50% EE and Imipenem (50% EE – Imipe) against S. aureus, which displayed antagonism effects (ΣFIC = 2.25). It was observed that the 50% EE – Strep combinations exhibited outstanding synergistic properties against all bacteria (ΣFIC ≤0.38). The 70% EE – Chlo combination showed additive properties or indifference against E. coli (ΣFIC = 0.75), B. subtilis (ΣFIC = 0.75) and S. aureus (ΣFIC = 1.25), severally. The 70% EE – Strep association showed addition effects against B. subtilis 332
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3.5. The MIC and MBC of quercetin and kaempferol
(ΣFIC = 0.53) and S. aureus (ΣFIC = 0.63), whereas synergistic property against E. coli (ΣFIC = 0.31). The 70% EE – Imipe combination revealed indifference activity against B. subtilis (ΣFIC = 1.25) and E. coli (ΣFIC = 1.25). When the color of combinations turned pink, it was indicated that the combinations were antagonistic against indicators. The 95% EE – Strep association exhibited dramatically synergistic effects for bacteria (ΣFIC ≤ 0.31). The 95% EE– Chlo association exhihited additive effects when used to fight against B. subtilis (ΣFIC = 0.56) and S. aureus (ΣFIC = 0.56), indifference against E. coli (ΣFIC = 1.06). The 95% EE – Imipe combination indicated indifference against E. coli (ΣFIC = 1.06), however, which were antagonistic on neither B. subtilis nor S. aureus. On the whole, the 50% EE exhibited the best synergy and superposition effects, then 95% EE, and last was 70% EE. The constituents from C. salicifolius S. Y. Hu leaves belong of flavonol and homologous glycosides. The antibiotics modulation mechanisms of flavonol have rarely been explored till today, such as destruction cytoplasmic membrane, β-lactamase inhibition (Eumkeb et al., 2010; Liu et al., 2009). Comparing their compositions and antibiotics potentiating activity, we proposed that the phytochemicals of plant extracts affected multiple targets of bacteria; the interaction among components improved the solubility, hence promoted the bioavailability of the extracts; themselves neutralized the unfavorable factors in the ingredients (Wagner and Ulrich-Merzenich, 2011). The above mechanisms involved have led to an outstanding improvement in antimicrobial effects. On the contrary, compared their constitutes and antibiotics antagonism activity, the plant extracts caused excretion of antibiotics or bound to the molecular structure of antibiotics (Cristo et al., 2016), which have reduced the pharmacology effectiveness of drugs. In the past few years, lots of works have been performed to prove efficacy and safe of plant materials, which could acted as excellent natural food additives (Caleja et al., 2016; Shan et al., 2009). The EECS had different regulatory effects on antibiotics, which provided a theoretical basis to be an antibiotic regulator for C. salicifolius S. Y. Hu. leaves, and the antibacterial mechanisms of synergistic combination need further exploration.
The MICs and MBCs of quercetin and kaempferol were presented in Table 4. Antimicrobial effect (MIC) of the quercetin ranged from 2.8 μg/mL to 1250 μg/mL and bactericidal activity (MBC) of the quercetin ranged from 156 μg/mL to > 2500 μg/mL. E. coli was the most sensitive to quercetin whereas B.subtilis was the most insensitive to quercetin. With regarding to kaempferol, the MIC was ranged from 156 μg/mL to 317.5 μg/mL against indicators. Our results were in contradiction with the previous reports on the antibacterial potency of quercetin and kaempferol, due to different test methods, the concentration and volume of inoculated bacteria etc (Amin et al., 2015; Hamed et al., 2015; Rauha et al., 2000; Wong et al., 2012). According to the structure-activity relationship, the hydroxylation of the A ring at the 5th and 7th positions and the hydroxyl groups of B ring all contributed to the antibacterial activity of flavonoids (Cushnie and Lamb, 2011). Both of quercetin and kaempferol were satisfied with the chemical characteristics and had antimicrobial activity acting on multiple targets, such as inhibition cell wall synthesis etc (Wu et al., 2008). For instance, Andreja Plaper et al. showed that quercetin inhibited DNA gyrase of Escherichia coli by two different mechanisms, which were based on interaction with DNA gyrase and inhibition ATPase activity of DNA gyrase (Plaper et al., 2003). Significant inhibition activity of the C. salicifolius S. Y. Hu. leaves were observed against microorganisms, supporting the hypothesis that C. salicifolius S. Y. Hu. Leaves possessed bacteriostatic activity. 4. Conclusion All of the ethanol elutions displayed commendable antibacterial properties against indicators and the antibacterial properties of fractions were in the sequence of 95% EE, 70% EE, 50% EE, 30% EE, WE, CEE. Among EEs, 95% EE had the most considerable antibacterial activity. The 50% EE, 70% EE and 95% EE possessed remarkable antibacterial action from the initial screening process. The selected fractions modulated the activity of antibiotics against common food-borne pathogenic bacteria (S. aurous, E. coli and B. subtitles), and the 50% EE showed very promising significant synergies with antibiotics. In addition, Streptomycin was proved to have the prominent cooperation effect with the selected EEs. The results showed that the flavonoids can be an alternative tool to extend the antibacterial potential of aminoglycosides. The interactions of several compounds lead to synergistic or antagonistic activities, which included luteolin, rutin and quercetin, et al. In conclusion, C. salicifolius S. Y. Hu. leaves were an alternative source to be applied as natural food preservatives and resistance-modifying agents. The antibacterial mechanisms of synergistic combination need intensive study.
3.4. Comparison the effects of C. salicifolius S. Y. Hu. leaves on Chloramphenicol, Streptomycin, and Imipenem The Chloramphenicol, Streptomycin, and Imipenem belong to amphenicols, aminoglycoside and β-lactam antibiotics, respectively. The selected EEs have been observed to lower the dose of three antibiotics to varying degrees, according to ΣFIC (Figs. 4–6). Thus, comparing their ΣFICs and continuously discovering that the associations with Streptomycin inclined to present lower ΣFIC values, followed with Chloramphenicol and Imipenem. Streptomycin was a kind of aminoglycoside drug, the mechanism of which was to bind the ribosomes in bacteria, disturbing the synthesis of bacterial proteins and destroying the integrity of bacterial cytoplasmic membrane (Davies and Davis, 1968). Imipenem was classified as β-lactam antibiotics, which exhibited superior affinity to a mass of bacterial penicillin binding proteins (PBPs), which were the specicific target sites of antibiotics (Hoffman, 2001). The inhibitory mechanisms of the associations were conjectured to inhibit efflux systems and mutation cell permeability (Abreu et al., 2012). In most situations, the EECS possessed stronger antibacterial effect on gram-negative bacteria than gram-positive bacteria in line with MICs (Table 1). Nevertheless the associations demonstrated significantly synergy effectiveness against gram-negative bacteria in contrast. The phenomenon was probably related to the diversities of photochemical constituents, antibiotic type and the membrane structure of bacterial cell et al. In a word, the EECS could be combined with aminoglycoside antibiotics to reduce both the dosage of antibiotics and resistant bacteria. Further investigations are needed regarding the involved mechanisms of synergy effectiveness and the service regulations of natural food preservatives.
Conflicts of interest The authors declare no conflict of interest related to this work. Acknowledgments The authors gratefully acknowledge the financial supports by National Natural Science Foundation of China (grant numbers 31560459); Modern Agricultural Research Collaborative Innovation Special Funds of Jiangxi Province (grant numbers JXXTCX201703-1); the Science Funds of Educational Commission of Jiangxi Province, China (grant numbers KJLD13027). References Abreu, A.C., Mcbain, A.J., Simões, M., 2012. Plants as sources of new antimicrobials and resistance-modifying agents. Nat. Prod. Rep. 29, 1007–1021. Ahmad, A., Kaleem, M., Ahmed, Z., et al., 2015. Therapeutic potential of flavonoids and
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Phytochemical profile of ethanolic extracts of Chimonanthus salicifolius S. Y. Hu. leaves and its antimicrobial and antibiotic-mediating activity Ning Wang, Hui Chen, Lei Xiong, Xin Liu, Xiang Li, Qi An, Ximei Ye, Wenjun Wang
T
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Key Lab for Natural Products and Functional Foods of Jiangxi Province, College of Food Science and Engineering, Jiangxi Agricultural University, Nanchang 330045, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Chimonanthus salicifolius S. Y. Hu. leaves Antibacterial activity Flavonoids Antibiotics
The aim of this study was to determine the phytochemical profiles in the ethanol elutions of Chimonanthus salicifolius S. Y. Hu. leaves (EECS) and study its antibacterial activity. The ethanol extracts of C. salicifolius S. Y. Hu. leaves were prepared by ethanol gradient elution orderly and analyzed by HPLC-DAD. Three bacterial and four fungal species were used to evaluate the antimicrobial activities of the EECS by the broth microdilution method. Results showed that the MIC values against bacteria ranged from 0.35 mg/mL to 11.25 mg/mL (Escherichia coli CVCC1490, Staphylococcus aureus, and Bacillus subtilis). The C. salicifolius S. Y. Hu. leaves exhibited MIC values ranging from 2.81 mg/mL to 45 mg/mL against both the strains of yeast (Saccharomyces cerevisiae CICC 1540, Saccharomyces cerevisiae CICC 1340) and against strains of Cephalckiscus Fiagans (Penicillium digitatum AS3.5752, Aspergillus niger) with the MIC values ranging from 2.81 mg/mL to 11.25 mg/ mL, respectively. Remarkable antibacterial potential was marked with low minimal inhibitory concentration (MIC) for the 50%, 70% and 95% ethanolic fractions against bacteria. The HPLC analysis revealed quercetin and kaempferol as major ingredients in the screening EEs. Interestingly, the content of quercetin and kaempferol varied with the concentration of elution ethanol. The synergistic and additive effects of the selected EEs were frequently observed against E. coli, S. aureus and B. subtilis when combined with standard antibiotics (aminoalcohol, aminoglycosides and β-lactam antibiotics). In conclusion, the EECS possessed the property of modifying antibiotics against bacteria. Synergistic combination can be used to reduce the use of antibiotics or look forward to dealing with drug-resistant bacteria, and also provide theoretical underpinning as natural food preservatives and medicinal plants.
1. Introduction About 50 000 deaths per annum in Europe and the United States were due to antibiotic resistant infections, which predicted that the problem would exceedingly increase over the next decade (Mayor, 2016). Resistance crisis has eventually been noticed to almost all antibiotics developed, which was due to inappropriate prescribing, extensive agricultural use and availability of few new antibiotics et al (Ventola, 2015). On the other hand, synthetic and semi-synthetic chemical preservatives pose potential health risks to humans due to the unreasonable use (Xiong et al., 2013). Medicinal plants were abundant in polysaccharides, triterpenes, phenolic and flavonoid compounds etc., which attracted widespread interest toward development natural antimicrobial agents as novel preservatives and functional supplementations. Furthermore, there are about 250,000–500,000 plant species on the earth and only about 5000 species of these have been researched so far (Borris, 1996).
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Many researches have focused on developing plant natural products as novel antimicrobial, which could also combat bacterial resistance in cooperation with standard antibiotics, due to its safety and efficiency (Ambrosio et al., 2017; Cristo et al., 2016). For instance, Eumkeb et al. showed that synergistic FIC indices (FIC index, < 0.02–0.11) were observed against Penicillin-resistant S. aureus in combination with all selected β-lactam antibiotics (methicillin, ampicillin) and test flavonoids from Alpinia officinarum Hance (Eumkeb et al., 2010). Chimonanthus salicifolius S. Y. Hu. leaves also known as Shi liang tea and Xiang feng tea, is an excellent functional green tea and medicinal plant, mainly distributed in the Southern Anhui, Hubei and Jiangxi provinces etc. (Bi et al., 2013). Investigations were performed on bioactive substances of C. salicifolius S. Y. Hu leaves including coumarin, volatile oil, essential oil and flavonoids. (Ouyang, 2009; Ouyang et al., 2010; Yang et al., 2012). Our previous studies have demonstrated that C. salicifolius S. Y. Hu leaves presented notable α-glucosidase inhibitory activity (flavonoids function) antihyperglycemic and antihyperlipidemic (Chen
Corresponding author. E-mail address: [email protected] (W. Wang).
https://doi.org/10.1016/j.indcrop.2018.09.021 Received 17 May 2018; Received in revised form 7 September 2018; Accepted 11 September 2018 0926-6690/ © 2018 Elsevier B.V. All rights reserved.
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Table 1 Antibacterial activities of different ethanol eluates from Chimonanthus salicifolius S. Y. Hu. leaves. Bacteria
MICs / MBCs obtained by different extracts / (μg / μL)
Type of strain
CEE E. coli (cvcc1490) S. aureus B. subtilis Saccharomyces cerevisiae (CICC 1340) Saccharomyces cerevisiae (CICC 1540) Penicillium digitatum (AS3.5752) Aspergillus niger
WE
G−
11.25 / 45
G+ G+ yeast yeast Cephalckiscus Fiagans Cephalckiscus Fiagans
11.25A/ > 45A 5.63B/ > 45A 45A/ > 45A 45A/ 45A 2.81C/11.2B 5.63B/ 45A
A
A
30% EE A
11.25 / > 45
A
11.25A/ > 45A 11.25A / > 45A 5.63C/ > 45A 5.63C/45A 2.81C/5.63C 2.81C/ > 45A
B
50% EE A
C
70% EE B
C
95% EE A
0.70D/ > 45A
5.63 / > 45
2.81 /5.63
2.81 /45
11.25A/ > 45A 5.63B/ > 45A 11.25B/ > 45A 11.25B/ > 45A 11.25A/11.25B 11.25A/ > 45A
2.81B/45A 0.7D/ > 45A 2.81D/45A 5.63C/22.5B 5.63B/11.25B 2.81C/ 45A
0.70C/ 45A 2.81C/ 45A 2.81D/ > 45A 5.63C/ > 45A 5.63B > 45A 5.63B/5.63C
0.35D/5.63B 0.35E/ > 45A 2.81D/11.25B 2.81D/11.25C 2.81C/2.81D 2.81C/ 22.5B
Note: G + refers to Gram - positive; G − refers to Gram-negative. A − E: Averages within the same row with different superscripts are significantly different by the Tukey test (P < 0.001). Table 2 Flavonoids composition of different ethanol eluates from Chimonanthus salicifolius S. Y. Hu. leaves. Compounds
Calibration curve
R2
ethanol eluates from C. salicifolius S. Y. Hu. leaves (μg/mL) 50% EE
Rutin Hyperin Isoquercitrin Luteoloside Astragalin Luteolin-5-Oglucoside Quercetin Kaempferol
70% EE
95% EE
LOD
LOQ
Y = 18.19x - 88.422 Y = 31.508x + 94.029 Y = 27.098x + 1185.8 Y = 18.928x + 193.19 Y = 28.368x - 46.802 Y = 14.642x + 165.91
0.9989 0.9986 0.9957 0.9989 0.9984 0.9993
160.36 ± 0.03 19.62 ± 0.01E 78.76 ± 0.18D 229.03 ± 0.05A 55.45 ± 0.02D 68.20 ± 0.02D
– – – – – –
– – – – – –
0.020 0.011 0.013 0.019 0.013 0.024
0.059 0.034 0.040 0.057 0.038 0.074
Y = 13.209x - 104.25 Y = 19.144x - 160.18
0.9980 0.9980
176.70 ± 0.06BC 197.24 ± 0.05B
719.33 ± 0.38*** 917.54 ± 0.52***
1362.31 ± 0.23*** 1270.09 ± 0.21***
0.027 0.019
0.082 0.056
C
Results are expressed as mean ± S.D. of three determinations. LOD: limit of detection and LOQ: limit of quantification. Averages followed by different letters differ by the Tukey test at P < 0.01, ***P < 0. 001 compared to the fraction of 50% ethanol eluate.
Fig. 2. High performance liquid chromatography profile of 70% EE of Chimonanthus salicifolius S. Y. Hu. leaves. Quercetin (peak 1); kaempferol (peak 2).
Fig. 1. Representative high performance liquid chromatography profile of 50% EE of Chimonanthus salicifolius S. Y. Hu. leaves. Rutin (peak 1); hyperin (peak 2); isoquercitrin (peak 3); luteoloside (peak 4); luteolin-5-O-glucoside (peak 5); astragalin (peak 6); unknown (peak 7); unknown (peak 8); quercetin (peak 9); kaempferol (peak 10).
plant flavonoids possessed excellent antimicrobial activity and corresponding mechanisms (Cushnie and Lamb, 2011; Cushnie and Lamb, 2005). The flavonoid aglycones, quercetin and kaempferol were the two important ingredients in C. salicifolius S. Y. Hu leaves. Because of affluent secondary metabolites in C. salicifolius S. Y. Hu leaves, it is proposed to have bacteriostatic activity. In the present study, we primitively investigated the antimicrobial properties of the EECS using bacteria, yeast and molds. Identification and quantification of the antimicrobial phytochemicals from C. salicifolius S. Y. Hu leaves was done by high performance liquid chromatography-diode array detector (HPLC-DAD) in sequence. This work
et al., 2017a, b). Wu et al., proved that the dichloromethane fraction of C. salicifolius S. Y. Hu leaves presented best antimicrobial activity against four common bacteria (Wu et al., 2017), but the specific chemical composition of the fraction was lack of further exploration. Flavonoids contain over 4000 biologically active compounds, which belong to polyphenols, abundant in tea, wine, honey, nuts etc (Fathima and Rao, 2016). Flavonoids were biosynthesized mainly through two pathways including the shikimic acid and acetic acid pathways (Cazarolli et al., 2008). There are several reviews demonstrated that 329
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Fig. 3. High performance liquid chromatography profile of 95% EE of Chimonanthus salicifolius S. Y. Hu. leaves. Quercetin (peak 1); kaempferol (peak 2).
Fig. 4. The selected ethanol extracts of Chimonanthus salicifolius S. Y. Hu.leaves in association with antibiotics against E. coli.
Table 3 Antibiotics potentiating effect of Chimonanthus salicifolius S. Y. Hu.leaves extracts. Antibiotics
Bacteria E. Coli (G-)
MIC (ug/mL) alone
50%EE
70%EE
95%EE
Chloramphenicol Streptomycin Imipenem Chloramphenicol Streptomycin Imipenem Chloramphenicol Streptomycin Imipenem Chloramphenicol Streptomycin Imipenem
S. Aureus(G+)
3.91 3.91 7.81 7.81 7.81 0.98 ƩFIC of plant extracts: 0.50 0.75 0.28 0.38 0.75 2.25 0.75 1.25 0.31 0.63 1.25 – 1.06 0.56 0.13 0.13 1.06 –
B. Subtilis(G+)
3.91 7.81 0.98 0.50 0.28 0.50 0.75 0.53 1.25 0.56 0.31 –
Note: No antimicrobial activity is indicated by-, refer to antagonism (ΣFIC > 2); Additive effects are indicated with figures underlined (0.5 < ΣFIC ≤1); Indifference are marked with nothing (1 < ΣFIC ≤ 2); Synergy is indicated with figures in bold figures underlined (ΣFIC ≤0.5).
Fig. 5. The selected ethanol of Chimonanthus salicifolius S. Y. Hu.leaves in association with antibiotics against S. aureus.
focused on evaluating in vitro the antimicrobial and antibiotic modulatory potential of the selected ethanol eluates (EEs) against S. aureus, P. subtilis and E. coli alone, or in association with aminoalcohol (Chloramphenicol), aminoglycosides (Streptomycin) and β-lactam antibiotics (Imipenem).
Medium with Chloramphenicol; Yeast Malt Agar (YMA) and Yeast Malt Broth (YMB) were purchased from Qingdao Hope Bio-Technology Co., Ltd (Qingdao, China). Analytical grade chemicals were employed in all assays. 2.2. Microorganisms and cultures
2. Materials and methods
Staphylococcus aureus, Bacillus subtilis, Aspergillus niger were obtained from the Lab of Light Chemical Engineering Teaching and Research Department, College of Food Science and Engineering, Jiangxi Agricultural University, China. Saccharomyces cerevisiae (CICC 1540), Saccharomyces cerevisiae (CICC 1340), Escherichia coli (CVCC1490) and Penicillium digitatum (AS3.5752) were purchased from Guangdong Microbial Culture Center (GDMCC). Saccharomyces cerevisiae cultures were grown statically in YMB. Molds were cultivated in Potato Dextrose liquid medium.
2.1. Chemicals and regents Acetonitrile (HPLC grade) was purchased from TEDIA Company Inc., (Shanghai, China). Flavonoids Standards including luteoloside, kaempferol, astragalin, luteolin-5-O-glucoside, rutin, hyperin, isoquercitrin, and quercetin were obtained from Beijing Solarbio Science & Technology Co., Ltd (Beijing, China). Dimethyl sulfoxide (DMSO) was obtained from Tianjin Wingtai Chemical Co., Ltd., (Tianjin, China). The sample solutions and mobile phase were filtered and sonicated before HPLC analysis. The analytical HPLC system is described as the previous reference (Chen et al., 2017a). Mueller-Hinton Agar (MHA); Mueller Hinton broth (MHB); Potato Dextrose Agar (PDA); Potato Liquid
2.3. Plant collection and identification The dried C. salicifolius S. Y. Hu leaves were purchased from a local 330
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incubation temperature of yeast and molds was 28 ℃. The A. niger and P. digitatum were made of spore suspension, which was counted by the plate line and diluted to corresponding quantity. 100 μL of a set of twofold serial dilutions of EECS in MHB were added to 100 μL of the microorganism suspension together in a sterile 96-well microplate, giving final concentrations from 0.35 to 45 μg/μL. The five ethanol elutions and CEE in pure DMSO (90 μg/μL) were thinned in MHB by using flatbottomed sterile 96-well microplates in order to gain a concentration ranging from 0.35 to 45 μg/μL. The plate was again incubated at corresponding temperature for 18 h. We used sterility (no inoculum added), inoculum viability (no flavonoids added) as positive and negative control, respectively. After cultivation, 20 μL of resazurin solution (0.05% w/v) was poured into all holes, followed by final incubation of 1 h at appropriate conditions. Holes were measured visually: a color change from blue to pink or mauve, was taken as representative of bacterial reproduction; while the highest dilution which remained blue was used to represent the MIC in contrast. The MIC was defined as the minimum concentration, at which microorganisms were unable to grow in MHB supplemented with ethanol elutions. Streak plate method was applied to determine MBC from the uncolored holes, in which no bacteria growth in the above MIC assay, then incubated for 24 h at 37 ℃ on MHA. Yeast and molds were streaked plate from the uncolored wells and cultured for 48 h at 28℃ on PDA. The MBC was defined as the lowest concentration at which microorganisms were all killed with ethanol elutions. This experiment was repeated in triplicate. DMSO at a non-toxic concentration was applied in the experiment.
Fig. 6. The selected ethanol of Chimonanthus salicifolius S. Y. Hu.leaves in association with antibiotics against B. Subtilis.
farmer in Lishui City (Zhejiang, China). All voucher specimens were dried in the constant temperature oven until the weight remained stable. The dried plant material was ground into fine powder, passed through a 60-meshsieve and stored in a desiccator until next use. 2.4. Extraction procedures
2.6. High performance liquid chromatography (HPLC)
The powder of C. salicifolius S. Y. Hu leaves were extracted twice at 40℃ followed our previous research: ultrasonic treatment time was 30 min, ultrasonic power was 57.59 W, ethanol concentration was 52.03%, and solid-liquid ratio was 1:23.85. After filtering, the filtrates were pooled, concentrated and further purified by polyamide resin. The adsorbed resin was eluted with gradient aqueous ethanols. The process leaded to in five fractions remained including, 95% ethanol eluate (95% EE), 70% EE and 50% EE, 30% EE, water eluate (WE) at last. Detailed experimental procedures followed previous literature (Chen et al., 2017a).
The EECS were injected into a Waters Symmetry C18 (250 × 4.6 mm, 5 μm, Waters, Ireland) column. Corresponding mobile phases and solvent conditions were used as described by previous literature (Chen et al., 2017a). The C. salicifolius S. Y. Hu leaves extracts and the mobile phase were filtered through a 0.22 μm microporous organic membrane, and degassed by ultrasonic bath before using. The EEs dissolved in methanol were tested at a concentration of 10 mg/mL. Comparing the retention time of reference standards with the HPLC chromatography peaks of the EECS was obtained by DAD spectra. The phytochemicals would have been confirmed. Calibration curves of flavonoid standards were supplemented in Table 2. All chromatography operations were in triplicates. The LOD and LOQ were calculated as 3.3 and 10 δ/S, respectively, where δ represents the standard deviation of the response, and S represents the slope of the calibration curve.
2.5. Determination of the minimum inhibitory concentration (MIC) and minimum bacterial concentration (MBC) MIC was conducted according to the standard microbroth dilution technique following the guideline of National Committee for Clinical Laboratory Standards with some modifications (Ferraro, 2004). The bacteria was cultured in MHB at 37℃ overnight to give the logarithmic phase bacteria and adjusted to 0.5 McFarland standards (Guangdong Huankai Microbial Sci. & Tech. CO., Ltd, Guangzhou, China) and further diluted with MHB to achieve approximately 1 × 106 CFU/mL, so do S. cerevisiae (CICC 1540) and S. cerevisiae (CICC 1340). The
2.7. Modulation of the antibiotic activity of aminoalcohol, aminoglycosides and β-lactam The antibiotic modulating effects of the 50% EE, 70% EE and 95% EE were determined by a checkerboard method with a few adjustments (Taukoorah et al., 2016). The extracts, the antibiotic and the inoculum were distributed into 36 wells (6 × 6) so that the final volume of the
Table 4 Antibacterial activities of Quercetin and Kaempferol. Compounds
Quercetin Kaempferol
MICs / MBCs obtained by Quercetin and Kaempferol / (μg / mL) E. coli G−
S. aureus G+
B.subtilis G+
Saccharomyces cerevisiae (CICC 1340) yeast
Saccharomyces cerevisiae (CICC 1540) yeast
Penicillium digitatum (AS3.5752) Cephalckiscus Fiagans
Aspergillus niger Cephalckiscus Fiagans
2.8A/ > 2500D 317.5B/ > 2500D
317.5A/ > 2500
1250B/ > 2500
156A/317.5B
156A/317.5B
156A/156A
156A/1250C
D
D
317.5B/1250C
317.5A/ > 2500D
156A/317.5A
156A/1250C
156A/625B
156A/1250C
Note: G + refers to Gram - positive; G − refers to Gram-negative. A–D: Averages within the same row with different superscripts are significantly different by the Tukey test (P < 0.001). 331
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et al., 2010). For instance, the MIC value of quercetin was 6.25 μg/mL and 100 μg/mL both for S. aureus and E.coli, whereas the MIC value of isoquercitrin (its corresponding glycosides) was 100 μg/mL and > 400 μg/mL, respectively. Hong et al. demonstrated that quercetin inhibited the growth and protein utilization of the E. coli through the disruption of cell wall and the loss of physiological function of bacterium (Hong et al., 2017). Kaempferol was proved to inhibit the growth of S. aureus by disturbing reproduction of genetic material (Bernard et al., 1997). The bactericidal mechanisms of flavonoids were in several different paths on literature published: (a) Destroy of cytoplasmic membrane by perforating and decrease in membrane fluidity. (b) Inhibitory energy metabolism. (c) Suppression of synthesis of genetic material. (d) Neutralization of bacterial toxins (Ahmad et al., 2015). (e) Metal chelation by flavonoids (Cowan, 1999) (f) Inhibition the formation of biofilm. (g) Inhibition the carrier protein of cell membrane (Xie et al., 2015). Second, the interactions among phytochemicals resulted in either superposition or antagonism effects. For example, quercetin (inhibitory zone diameter 13.5 ± 0.21 mm) was demonstrated to be more active than the combination (inhibitory zone diameter 0) between quercetin and rutin against S. aureus (Amin et al., 2015). Studies indicated that strong activity was defined as MIC < 5 mg/mL (Bussmann et al., 2010). Thus, the criterion of MIC (< 5 mg/mL) classified 50% EE, 70% EE and 95% EE as having outstanding competence than the others against bacteria. Hence, it was important to demonstrate the constituents and the regulative effects of different kinds of antibiotics for the selected fractions in coming future.
mixture was 200 μL according to 1: 1: 2. The antibiotic was serially diluted along the Y axis while the EECS was serially diluted along the X axis so that both of them with the variation range of the final concentration was 0–1 MIC. All combinations were tested. 100 μL of prepared inoculums was poured into individual wells as the mixture was incubated overnight in a suitable condition. After the incubation was complete, resazurin solution was used as an indicator to determine the growth of microorganisms. 2.7.1. Fractional inhibitory concentration index (ΣFIC) and interpretation The fractional inhibitory concentration index (ΣFIC) of the combination was counted according to the following equation:
∑ FIC =
MIC of EEs in combination MIC of EEs in alone MIC of antibiotic in combination + MIC of antibiotic in alone
The combined effects of ethanol elutions and antibacterial agents were determined by ΣFIC according to the following criteria: synergy ΣFIC ≤0.5, additive effects 0.5 < ΣFIC ≤1, indifference 1 < ΣFIC ≤ 2, antagonism ΣFIC > 2 (Guendouze-Bouchefa et al., 2015). The selection of holes to calculate the final ΣFIC was highly disputed. 2.8. Determination of the MIC and MBC of quercetin and kaempferol The MICs and MBCs of quercetin and kaempferol were done according to the Section 2.5. The standard products of quercetin and kaempferol (purity > 99%) were used.
3.2. HPLC analysis
2.9. Statistical analysis
The HPLC analysis of the 50%, 70%, and 95% EE obtained from C. salicifolius S. Y. Hu. leaves were presented in Figs. 1–3, respectively. All chromatographic peaks represented substances were as following: In Fig. 1, rutin (tR = 11.59 min, Peak 1); Hyperin (tR = 12.45 min, Peak 2); Isoquercitrin (tR = 13.58 min, Peak 3); Luteoloside (tR = 14.56 min, Peak 4); Astragalin (tR = 15.62 min, Peak 5); Luteolin-5-O-glucoside (tR = 16.85 min, Peak 6); Unknown (tR = 17.88 min, Peak 7); Unknown (tR = 18.96 min, Peak 8); Quercetin (tR = 25.52 min, Peak 9); Kaempferol (tR = 33.12 min, Peak10). In Fig. 2, Quercetin (tR = 25.54 min, Peak 1); Kaempferol (tR = 33.18 min, Peak2). In Fig. 3, Quercetin (tR = 25.54 min, Peak 1); Kaempferol (tR = 33.18 min, Peak 2). From Table 2, as we could see, luteoloside was the most abundant component in 50% EE, followed with kaempferol and quercetin. Interestingly, the content of flavonoid aglycones (quercetin and kaempferol) followed with the augment in the concentration of elution ethanol, which was in accordance with the tendency of antimicrobial activity from C. salicifolius S. Y. Hu. leaves.
All tests were performed at least in triplicate. Significant difference analysis was conducted using Tukey’s multi-comparison test, which was carried out by processor of SPSS Statistics 17.0. Differences between means were statistically considered significant if the p value was less than 0.05. 3. Results and discussion 3.1. Antimicrobial activity All the eluted fractions were tested for antimicrobial effectiveness using a microwell dilution assay, and the corresponding MIC values were summarized in Table 1. It was well known that the lower value of MIC or MBC was, the more sensitive of the microorganism was to the ethanol eluates. Antimicrobial effect (MIC) of the EECS ranged from 0.35 μg/μL to 45 μg/μL. Bactericidal activity (MBC) of the EECS ranged from 2.81 μg/μL to > 45 μg/μL, from which we implied that the inhibitory effect of the EECS was bacteriostatic rather than bactericidal against all the strains. Among EEs, the 95% EE was proved to possess the highest activity, with MIC values ranging from 0.35 μg/μL to 2.81 μg/μL against microorganisms. Wheares, the CEE was the most inactive against all microorganisms except for P. digitatum (MIC 2.81 μg/μL). It was notable that the traditional way of drinking, referred to WE, merely had a remarkable antibacterial effect on P. digitatum (2.81 μg/μL) and A. niger (2.81 μg/μL). Our results were consistent with the previous reports (Bi et al., 2013; Cheng et al., 2015; Li et al., 1996). The antimicrobial potency of the EECS mainly was attributed to the existence of abundant flavonoids and the interaction among phytochemicals. First, flavonoids such as quercetin, rutin, luteoloside, and other phytochemicals have been shown to have significant antimicrobial activities (Xiong et al., 2013; Butkhup et al., 2016; Rauha et al., 2000). In addition, the activity of the 50% EE mostly was due to flavonoids aglycones quercetin and kaempferol, which comprised only 19% of 50%EE (data not show), but the aglycones with relative low polarity had stronger bioactivity than their glycosides (Hao
3.3. Modulation of the antimicrobial activity of antibiotics The antibiotics modulation activity of the selected extracts was determined using calculating the ΣFICs by an adapted checkerboard method. The MICs of Chloramphenicol, Streptomycin and Imipenem, and the effects of the combination of the selected EEs with antibiotics against E. coli, S. aureus and B. subtilis were summarized in Table 3. The synergistic effectiveness was determined in the all combinations when associated with Streptomycin as demonstrated by the ΣFIC values, except when associated with the 70% EE against S. aureus and B. subtilis. The synergy and additive properties were always observed among the 50% EE – antibiotics combinations, apart from the combination of 50% EE and Imipenem (50% EE – Imipe) against S. aureus, which displayed antagonism effects (ΣFIC = 2.25). It was observed that the 50% EE – Strep combinations exhibited outstanding synergistic properties against all bacteria (ΣFIC ≤0.38). The 70% EE – Chlo combination showed additive properties or indifference against E. coli (ΣFIC = 0.75), B. subtilis (ΣFIC = 0.75) and S. aureus (ΣFIC = 1.25), severally. The 70% EE – Strep association showed addition effects against B. subtilis 332
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3.5. The MIC and MBC of quercetin and kaempferol
(ΣFIC = 0.53) and S. aureus (ΣFIC = 0.63), whereas synergistic property against E. coli (ΣFIC = 0.31). The 70% EE – Imipe combination revealed indifference activity against B. subtilis (ΣFIC = 1.25) and E. coli (ΣFIC = 1.25). When the color of combinations turned pink, it was indicated that the combinations were antagonistic against indicators. The 95% EE – Strep association exhibited dramatically synergistic effects for bacteria (ΣFIC ≤ 0.31). The 95% EE– Chlo association exhihited additive effects when used to fight against B. subtilis (ΣFIC = 0.56) and S. aureus (ΣFIC = 0.56), indifference against E. coli (ΣFIC = 1.06). The 95% EE – Imipe combination indicated indifference against E. coli (ΣFIC = 1.06), however, which were antagonistic on neither B. subtilis nor S. aureus. On the whole, the 50% EE exhibited the best synergy and superposition effects, then 95% EE, and last was 70% EE. The constituents from C. salicifolius S. Y. Hu leaves belong of flavonol and homologous glycosides. The antibiotics modulation mechanisms of flavonol have rarely been explored till today, such as destruction cytoplasmic membrane, β-lactamase inhibition (Eumkeb et al., 2010; Liu et al., 2009). Comparing their compositions and antibiotics potentiating activity, we proposed that the phytochemicals of plant extracts affected multiple targets of bacteria; the interaction among components improved the solubility, hence promoted the bioavailability of the extracts; themselves neutralized the unfavorable factors in the ingredients (Wagner and Ulrich-Merzenich, 2011). The above mechanisms involved have led to an outstanding improvement in antimicrobial effects. On the contrary, compared their constitutes and antibiotics antagonism activity, the plant extracts caused excretion of antibiotics or bound to the molecular structure of antibiotics (Cristo et al., 2016), which have reduced the pharmacology effectiveness of drugs. In the past few years, lots of works have been performed to prove efficacy and safe of plant materials, which could acted as excellent natural food additives (Caleja et al., 2016; Shan et al., 2009). The EECS had different regulatory effects on antibiotics, which provided a theoretical basis to be an antibiotic regulator for C. salicifolius S. Y. Hu. leaves, and the antibacterial mechanisms of synergistic combination need further exploration.
The MICs and MBCs of quercetin and kaempferol were presented in Table 4. Antimicrobial effect (MIC) of the quercetin ranged from 2.8 μg/mL to 1250 μg/mL and bactericidal activity (MBC) of the quercetin ranged from 156 μg/mL to > 2500 μg/mL. E. coli was the most sensitive to quercetin whereas B.subtilis was the most insensitive to quercetin. With regarding to kaempferol, the MIC was ranged from 156 μg/mL to 317.5 μg/mL against indicators. Our results were in contradiction with the previous reports on the antibacterial potency of quercetin and kaempferol, due to different test methods, the concentration and volume of inoculated bacteria etc (Amin et al., 2015; Hamed et al., 2015; Rauha et al., 2000; Wong et al., 2012). According to the structure-activity relationship, the hydroxylation of the A ring at the 5th and 7th positions and the hydroxyl groups of B ring all contributed to the antibacterial activity of flavonoids (Cushnie and Lamb, 2011). Both of quercetin and kaempferol were satisfied with the chemical characteristics and had antimicrobial activity acting on multiple targets, such as inhibition cell wall synthesis etc (Wu et al., 2008). For instance, Andreja Plaper et al. showed that quercetin inhibited DNA gyrase of Escherichia coli by two different mechanisms, which were based on interaction with DNA gyrase and inhibition ATPase activity of DNA gyrase (Plaper et al., 2003). Significant inhibition activity of the C. salicifolius S. Y. Hu. leaves were observed against microorganisms, supporting the hypothesis that C. salicifolius S. Y. Hu. Leaves possessed bacteriostatic activity. 4. Conclusion All of the ethanol elutions displayed commendable antibacterial properties against indicators and the antibacterial properties of fractions were in the sequence of 95% EE, 70% EE, 50% EE, 30% EE, WE, CEE. Among EEs, 95% EE had the most considerable antibacterial activity. The 50% EE, 70% EE and 95% EE possessed remarkable antibacterial action from the initial screening process. The selected fractions modulated the activity of antibiotics against common food-borne pathogenic bacteria (S. aurous, E. coli and B. subtitles), and the 50% EE showed very promising significant synergies with antibiotics. In addition, Streptomycin was proved to have the prominent cooperation effect with the selected EEs. The results showed that the flavonoids can be an alternative tool to extend the antibacterial potential of aminoglycosides. The interactions of several compounds lead to synergistic or antagonistic activities, which included luteolin, rutin and quercetin, et al. In conclusion, C. salicifolius S. Y. Hu. leaves were an alternative source to be applied as natural food preservatives and resistance-modifying agents. The antibacterial mechanisms of synergistic combination need intensive study.
3.4. Comparison the effects of C. salicifolius S. Y. Hu. leaves on Chloramphenicol, Streptomycin, and Imipenem The Chloramphenicol, Streptomycin, and Imipenem belong to amphenicols, aminoglycoside and β-lactam antibiotics, respectively. The selected EEs have been observed to lower the dose of three antibiotics to varying degrees, according to ΣFIC (Figs. 4–6). Thus, comparing their ΣFICs and continuously discovering that the associations with Streptomycin inclined to present lower ΣFIC values, followed with Chloramphenicol and Imipenem. Streptomycin was a kind of aminoglycoside drug, the mechanism of which was to bind the ribosomes in bacteria, disturbing the synthesis of bacterial proteins and destroying the integrity of bacterial cytoplasmic membrane (Davies and Davis, 1968). Imipenem was classified as β-lactam antibiotics, which exhibited superior affinity to a mass of bacterial penicillin binding proteins (PBPs), which were the specicific target sites of antibiotics (Hoffman, 2001). The inhibitory mechanisms of the associations were conjectured to inhibit efflux systems and mutation cell permeability (Abreu et al., 2012). In most situations, the EECS possessed stronger antibacterial effect on gram-negative bacteria than gram-positive bacteria in line with MICs (Table 1). Nevertheless the associations demonstrated significantly synergy effectiveness against gram-negative bacteria in contrast. The phenomenon was probably related to the diversities of photochemical constituents, antibiotic type and the membrane structure of bacterial cell et al. In a word, the EECS could be combined with aminoglycoside antibiotics to reduce both the dosage of antibiotics and resistant bacteria. Further investigations are needed regarding the involved mechanisms of synergy effectiveness and the service regulations of natural food preservatives.
Conflicts of interest The authors declare no conflict of interest related to this work. Acknowledgments The authors gratefully acknowledge the financial supports by National Natural Science Foundation of China (grant numbers 31560459); Modern Agricultural Research Collaborative Innovation Special Funds of Jiangxi Province (grant numbers JXXTCX201703-1); the Science Funds of Educational Commission of Jiangxi Province, China (grant numbers KJLD13027). References Abreu, A.C., Mcbain, A.J., Simões, M., 2012. Plants as sources of new antimicrobials and resistance-modifying agents. Nat. Prod. Rep. 29, 1007–1021. Ahmad, A., Kaleem, M., Ahmed, Z., et al., 2015. Therapeutic potential of flavonoids and
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