16
To investigate the antimicrobial effects of extracted polyphenols from green tea (Camellia sinensis) and banana (Musa sp.) leaves Qin Xiang Ng In recent years, there is an emergence of antibiotic-resistant bacterial strains due to the indiscriminate use of antibiotics, commonly prescribed to treat infectious diseases. The increasing occurrence of antibiotic resistance has undoubtedly made bacterial infections a greater health risk and threat than before. In this project, the use of natural polyphenolic compounds, extracted from green tea (Camellia sinensis) and banana (Musa sp.) leaves, as a novel antimicrobial chemotherapeutic agent was investigated. This study examined their bactericidal effects on Escherichia coli and Micrococcus luteus, and also evaluated the potential synergistic ability of these polyphenol-rich extracts when used alongside the beta-lactam antibiotic ampicillin. For the same dry mass of plant material used (3g), the prepared green tea extract had a higher total polyphenol concentration than that of banana leaves extract (495.1mg/L as compared to 221.6mg/L respectively). Subsequently, bacterial broth of 10 5 colony forming units/ml was mixed with varying concentrations of the different test compounds before plating 10µl of the mixture and quantifying the compounds’ bactericidal effect by doing a viable count after overnight incubation. The green tea extract showed the greatest bactericidal effect on E. coli and M. luteus as compared to the banana leaves extract and ampicillin. Against E. coli, this difference was confirmed to be significant (p<0.05) using one-way ANOVA and Tukey’s post-hoc analysis. Furthermore, by applying ampicillin in close association with polyphenol-rich extracts of green tea or banana leaves, there was an enhanced bactericidal effect on the tested bacterial strains. This interaction was categorized as synergistic by the fractional bactericidal concentration index calculated (≤0.5). Ultimately, the plant polyphenols studied in this research could provide a potential adjuvant to currently-produced antibiotics and help to reduce antibiotic application concentrations; as well as prolong the effective lifespan of antibiotics through its synergistic action. PeerJ PrePrints | https://doi.org/10.7287/peerj.preprints.1580v1 | CC-BY 4.0 Open Access | rec: 10 Dec 2015, publ: 10 Dec 2015

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To investigate the antimicrobial effects of extractedpolyphenols from green tea (Camellia sinensis) and banana(Musa sp.) leavesQin Xiang Ng

In recent years, there is an emergence of antibiotic-resistant bacterial strains due to theindiscriminate use of antibiotics, commonly prescribed to treat infectious diseases. Theincreasing occurrence of antibiotic resistance has undoubtedly made bacterial infections agreater health risk and threat than before. In this project, the use of natural polyphenoliccompounds, extracted from green tea (Camellia sinensis) and banana (Musa sp.) leaves,as a novel antimicrobial chemotherapeutic agent was investigated. This study examinedtheir bactericidal effects on Escherichia coli and Micrococcus luteus, and also evaluatedthe potential synergistic ability of these polyphenol-rich extracts when used alongside thebeta-lactam antibiotic ampicillin. For the same dry mass of plant material used (3g), theprepared green tea extract had a higher total polyphenol concentration than that ofbanana leaves extract (495.1mg/L as compared to 221.6mg/L respectively). Subsequently,bacterial broth of 105 colony forming units/ml was mixed with varying concentrations ofthe different test compounds before plating 10µl of the mixture and quantifying thecompounds’ bactericidal effect by doing a viable count after overnight incubation. Thegreen tea extract showed the greatest bactericidal effect on E. coli and M. luteus ascompared to the banana leaves extract and ampicillin. Against E. coli, this difference wasconfirmed to be significant (p<0.05) using one-way ANOVA and Tukey’s post-hoc analysis.Furthermore, by applying ampicillin in close association with polyphenol-rich extracts ofgreen tea or banana leaves, there was an enhanced bactericidal effect on the testedbacterial strains. This interaction was categorized as synergistic by the fractionalbactericidal concentration index calculated (≤0.5). Ultimately, the plant polyphenolsstudied in this research could provide a potential adjuvant to currently-producedantibiotics and help to reduce antibiotic application concentrations; as well as prolong theeffective lifespan of antibiotics through its synergistic action.

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1

2

3

4

5

To investigate the antimicrobial effects of extracted 6

polyphenols from green tea (Camellia sinensis) and 7

banana (Musa sp.) leaves 8

9

10

Qin Xiang, Ng1 * 11

12 1National University of Singapore, Yong Loo Lin School of Medicine, 13

Singapore 117597 14

15

16

* Address correspondence to [email protected] 17

18

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INTRODUCTION 19

According to a study done by CDC (Center for Disease Control and Prevention), in the United 20

States alone, food-borne diseases account for 76 million diseases, 325,000 hospitalizations and 21

5,000 deaths each year; and bacterial infections make up for approximately 5 million of these 22

diseases, which is around 13% of the 38.6 million food-related diseases caused by known 23

pathogenic bacteria [1]. 24

25

Modern medicine relies heavily on the use of antibiotics to kill pathogenic bacteria. However, 26

in the recent years, the emergence of antibiotic resistant bacteria has become a global concern 27

as seen in the recent emergence of multiple drug-resistant ‘superbugs’ found in hospitals in 28

countries like India, London and Nottingham. These ‘superbugs’ are resistant to even the most 29

powerful of modern antibiotics [2]. Scientific research has shown that tetracycline and 30

erythromycin resistance among bacteria are encoded by over 100 different genetic 31

mechanisms, several of which are readily transferable to other bacteria via conjugation [3]. 32

Hence, many bacterial strains have gained resistance to the popular beta-lactam antibiotics, 33

which include ampicillin and oxacillin [4]. 34

35

Polyphenolic antioxidants such as catechins occur most abundantly in green tea (leaves of 36

Camellia sinensis that have undergone minimal oxidation during processing). Banana (Musa 37

sp.) leaves, though commonly thrown away, also contain large amounts of polyphenols, 38

including epigallocatechin gallate (EGCG), similar to green tea [5]. EGCG is 100 times more 39

potent than vitamin C and 25 times more potent than vitamin E in terms of antioxidant power 40

[6]. The extraction of polyphenolic compounds from banana leaves and green tea leaves for 41

use as an antimicrobial agent would surely be a natural and low-cost treatment method. 42

Furthermore, due to the complicated biochemical structure of these polyphenolic compounds, 43

it is therefore difficult for bacteria to develop resistance against it [7]. This study therefore 44

investigates the antimicrobial properties of banana leaves and green tea extracts. The two main 45

objectives are: (1) to assay the antimicrobial effect of the polyphenol-rich extracts on 46

Micrococcus luteus (gram-positive bacteria) and Escherichia coli (gram-negative bacteria), 47

and (2) to evaluate the ability of these polyphenol compounds to work synergistically with β-48

lactam antibiotic ampicillin (a widely-prescribed moderate-spectrum antibiotic) to render M. 49

luteus and E. coli more susceptible to ampicillin. 50

51

52

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MATERIALS AND METHODS 53

I. Culturing Bacteria 54

E. coli (ATCC 25922) and M. luteus (ATCC 10240) were purchased from American Type 55

Culture Collection (ATCC). 56

57

II. Extraction and Quantification of Polyphenols from Banana Leaves/ Green Tea 58

3g of oven dried banana leaves or green tea were brewed using 50ml of Millipore ultrapure 59

water, and placed inside an electric water bath at 80°C for 40min. Filtration was then carried 60

out to obtain the aqueous extract. Using a separation funnel, the aqueous layer was partitioned 61

with an equal volume of diethyl ether (50ml) twice, keeping only the aqueous layer. The 62

retained aqueous layer was then partitioned with 50ml of ethyl acetate. The ethyl acetate phase 63

was collected and concentrated using a rotary evaporator (mainly to evaporate off the ethyl 64

acetate solvent) and the final extract collected was stored in a 4oC refrigerator. 65

After which, the total polyphenol content of the extracts was measured using the Folin-66

Ciocalteu (F-C) reagent [8]. Briefly, prepare solutions with phenol concentrations of 0, 50, 100, 67

150, 250, and 500 mg/L gallic acid. For each calibration solution, sample, or blank, 20µl sample 68

was added to a clean cuvette containing 1.58 mL water, 100µl of the 2N F-C reagent and 300µl 69

of 20% sodium carbonate solution. The absorbance of the solution was determined at 765nm 70

against the ‘blank’ (the “0 mg/L gallic acid” solution), and a standard curve of absorbance 71

(ABS) against concentration was plotted. 72

III. Antimicrobial assay (Minimum Bactericidal Concentration) 73

The Minimum Bactericidal Concentration (MBC) is defined as the lowest concentration of 74

antimicrobial compound required to kill an organism [9]. The bacterial isolates were tested for 75

their minimum bactericidal concentration (MBC) when exposed to the green tea or banana 76

leaves extract, antibiotic ampicillin (30mcg/mL), or ethyl acetate. The bacteria was cultured in 77

nutrient broth overnight and then adjusted to 0.50 McFarland Standard by dilution with sterile 78

0.8% NaCl saline, followed by UV-VIS spectrophotometric detection at 600 nm (and checking 79

for 0.132 ABS). The adjusted broth was then diluted one thousand times (3 consecutive ten-fold 80

dilutions) to give a final bacterial suspension concentration of around 105 cfu/ml. Briefly, 1000 81

µl of 105 cfu/ml bacteria broth culture was combined with 100 µl of green tea or banana leaves 82

extract, antibiotic ampicillin, or ethyl acetate of different concentrations (obtained by 83

performing 2-fold serial dilution with sterile 0.8% NaCl saline until 1:128) and left to incubate 84

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for 10 minutes. The resultant mixture’s bactericidal concentration was determined by 85

depositing 10 µl of the mixture onto a new sterile LB agar plate. The agar plate was left to 86

culture overnight to count the number of bacteria colonies while the minimum bactericidal 87

concentration (MBC) was determined by subculture of the mixture showing no apparent 88

growth. 89

90

IV. Synergistic Effects of Polyphenol Extracts with Ampicillin 91

After determining the MBC values of the polyphenol-rich extracts, the standard LB agar was 92

enhanced with 100 µl of sub-bactericidal (defined as half of the MBC value as determined 93

previously) concentration of the green tea or banana leaves extract and allowed to cure. The 94

same procedure as described in the antimicrobial assay of ampicillin was then repeated with 95

the enhanced agars in place of normal LB agar plates. 96

97

V. Statistics 98

Table 1: List of formulae used in data processing and discussion of results 99

Statistical Test Formulae Interpretation

Average/Mean

xx

n

x = mean of sample

x = Sum of all values

𝑛 = sample size

Calculated using Microsoft Excel 2007 ®,

employing the formula “=AVERAGE (first

number: last number)”. Gives the mean value

of the 5 repeats done, minimizing the

influence of random errors; a more accurate

reflection of data.

𝒔, Standard

Deviation 𝒔 = √

∑(𝒙 − �̅�)𝟐

𝒏 − 𝟏

Calculated using Microsoft Excel 2007 ®,

employing the formula “=STDEV (first

number: last number)”. The calculated

Standard Deviation value gives a measure of

the spread of the data, ±1 Standard Deviation

accounts for 68% of the data about the mean

in a Gaussian distribution.

One-way

ANOVA test for

variance

This test is done on Microsoft

Excel 2007® using the Data

Analysis Pack with a level of

significance set at

ANOVA test is essentially a generalization of

the independent-samples t-test designed to

determine the significance of the differences

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0.05:

Data → Data analysis →

Anova: single factor → input

the range of data by selecting

the range containing the data

as well as the heading (select

‘Labels in first row) → Leave

Alpha as 0.05

among three or more, rather than just two,

group means. ANOVA test is used as doing

repeated or multiple two-sample t-tests

would result in an increased chance of

committing a type I error, i.e. a “false

positive”. ANOVA test enabled the

comparison of the antimicrobial efficacy of

all three compounds tested to be done all at

once. The F-statistic and p-value produced

then helps to confirm whether there is a

significant difference in the level of

antimicrobial activity among the various

compounds tested.

Tukey’s

Honestly

Significant

Difference

(HSD) test

This test is done using

KaleidaGraph® v3.6 software

with a level of significance

set at

0.05.

Tukey’s HSD test is based on

what is called the studentized

range distribution. To test all

pairwise comparisons among

means using Tukey HSD,

compute ts for each pair of

means using the formula:

Where Mi - Mj is the

difference between the ith and

jth means, MSE is the Mean

Square Error (an estimate of

the population variance in the

analysis of variance), and n is

the mean of the sample sizes

of groups i and j.

The produced p-value can

then be used to determine

As ANOVA test does not specify which

means differ significantly. Thus, it was

necessary to conduct post-hoc Tukey’s HSD

which allows for multiple pair-wise

comparisons.

Tukey’s HSD test basically avoids the need

to perform multiple independent-sample t-

tests to test all possible pairs of treatments as

it can determine which of three or more

sample means are significantly different. The

problem with doing multiple t-tests is that the

type I error (“false positive” results) rate will

increase with the number of comparisons

made.

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whether the means tested are

significantly different:

- When p>0.05 (or 5%),

then the difference is not

significant (and might be

due to chance).

- When p<0.05 (or 5%),

then the difference is

significant.

- When p<0.01 (or 1%),

then the difference is

very significant.

- When p<0.001 (or 0.1%),

then the difference is

extremely significant.

100

101

Table 2: One-way ANOVA (analysis of variance) for average number of E. coli colonies 102

counted after mixing bacterial broth with 2-7 (or 1/128) full concentration of the various 103

compounds tested and after 24-h incubation 104

105

Sum of Squares df Mean Square F P-value F-critical

Between Groups 296.133 2 148.0667 21.459 0.00011 3.885

Within Groups 82.8 12 6.9

Total 378.933 14

106

Given that the F-statistic (21.459) > F-critical value (3.885), and that the calculated p-value 107

of 0.00011 is <0.05, we can safely conclude that there is a significant difference between the 108

number of bacterial colonies counted for the various test compounds applied at 2-7 109

concentration against E. coli. However, ANOVA test does not specify which means differ 110

significantly. Thus, it was necessary to conduct post-hoc multiple pair-wise comparisons. 111

Hence, Tukey’s Honestly Significant Difference (HSD) test was used to determine which of 112

the sample means were significantly different. 113

114

115

116

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Table 3: Tukey’s all pairs comparison for average number of E. coli colonies counted after 117

mixing bacterial broth with 2-7 (or 1/128) full concentration of the various compounds tested 118

and after 24-h incubation 119

Pair for Comparison

P-value Test Compound 1 Test Compound 2

Green Tea Extract Ampicillin 0.0002

Green Tea Extract Banana Leaves Extract 0.0004

Banana Leaves Extract Ampicillin 0.8812

Note: In red are p-values that are <0.05, i.e. difference observed in terms of antibacterial 120

efficacy of the test compounds is significant (and not due to chance). 121 122 123 124 125 126 127 Table 4: One-way ANOVA (analysis of variance) for average number of M. luteus colonies 128

counted after mixing bacterial broth with 2-7 (or 1/128) full concentration of the various 129

compounds tested and after 24-h incubation 130

Sum of Squares df Mean Square F P-value F-critical

Between Groups 75.6 2 37.8 3.287 0.07272 3.885

Within Groups 138 12 11.5

Total 213.6 14 15.257

131

Table 5: Tukey’s all pairs comparison for average number of M. luteus colonies counted after 132

mixing bacterial broth with 2-7 (or 1/128) full concentration of the various compounds tested 133

and after 24-h incubation 134

Pair for Comparison

P-value Test Compound 1 Test Compound 2

Green Tea Extract Ampicillin 0.0651

Green Tea Extract Banana Leaves Extract 0.2529

Banana Leaves Extract Ampicillin 0.6868

135

136

137

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RESULTS AND DISCUSSION 138

(all graphs and statistical analyses are done using MS Excel 2007® and KaleidaGraph® v3.6) 139

Table 6: Total Polyphenol Content in 20 µl of Green Tea or Banana Leaves Extract (n=5) 140

20 µl of Extract (±0.1µl) ABS at 765 nm (±0.005A) Total Phenol Concentration (mg/L)

Green Tea Leaves 1.312 495.1

Banana Leaves 0.601 221.6

141

The “Total Phenol Concentration” was estimated using the “Folin-Ciocalteau Gallic Acid 142

Standard Calibration Curve”, by interpolating the ABS values obtained for the extracts after 143

quantification procedure. 144

145

Table 7: Summary of MBC findings for the various test compounds 146

(MBC value is highlighted in orange) 147

E. coli: 148

1 2 4 8 16 32 64 128

Green Tea Extract - - - - - + + +

Banana Leaves Extract - - - + + + + +

Ampicillin - - + + + + + +

Ethyl acetate + + + + + + + + + + + + + + + +

Note: + indicates growth, – indicates inhibition (no growth) 149

150

M. luteus: 151

1 2 4 8 16 32 64 128

Green Tea Extract - - - - - + + +

Banana Leaves Extract - - - - + + + +

Ampicillin - - - - + + + +

Ethyl acetate + + + + + + + + + + + + + + + +

Note: + indicates growth, – indicates inhibition (no growth) 152

153

It was encouraging that on the average, for the same dry mass of banana leaves as green tea 154

leaves, the prepared banana leaves extract possessed nearly half the total polyphenol content 155

of the green tea leaves extract. This implies that banana leaves could therefore prove to be a 156

cheap and abundant source of naturally-occurring polyphenols. 157

158

Compound

Dilution Factor

Concentrations:

100µl of undiluted

green tea extract (21)

has a total phenol

concentration of

2476mg/L

100µl of undiluted

banana leaves extract

(21) has a total

phenol concentration

of 1108mg/L

100µl of undiluted

ampicillin (21) has a

concentration of

30mcg/mL

Dilution Factor

Compound

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From the antimicrobial assay done, we can infer that apart from ethyl acetate (the solvent used 159

to extract the polyphenolic compounds from green tea and banana leaves), the other compounds 160

tested all showed antimicrobial activity (to varying degrees) towards E. coli and M. luteus. 161

162

The total number of visible bacteria colonies of each of the dilution factors for each trial was 163

counted using a colony counter which electronically keeps track of the number of colony 164

counted, and a black marker was also used to mark every colony counted. In order to better 165

gauge the antimicrobial efficacy of these test extracts and combinations, Tukey’s Honestly 166

Significant Difference (HSD) analysis was done to conduct an all pairs comparison for the 167

average number of bacterial colonies counted for the lowest concentration of the various 168

compounds and combinations (1/128 or 2-7) tested. The produced p-value (shown in Graphs 1 169

and 2) can then be used to determine which test extract or combination is a significantly more 170

effective treatment method (at the lowest concentration tested) against E. coli and M. luteus. 171

The rationale behind this is to determine the antimicrobial compound which is the most 172

effective at the lowest concentration tested; so as to enable a lower concentration/ dosage 173

(yet being effective at the same time) to be prescribed to combat bacterial infections by E. coli 174

or M. luteus. 175

176

Graph 1: Graph of average number of E. coli colonies counted against 2-7 concentration of the 177

different compounds tested (Error bars showing ±1 std dev, n=5) 178

179

180 181

182

0

10

20

30

40

50

60

70

80

90

21

Av

era

ge

nu

mb

er o

f E

. co

li c

olo

nie

s

cou

nte

d

Compounds Tested

A, Ampicillin (30mcg/ml)

B, Green Tea Extract

C, Banana Leaves Extract

D, Ampicillin + Green Tea

E, Ampicillin + Banana Leaves

A B C D E

Note:

implies p < 0.005

implies p < 0.001

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Graph 2: Graph of average number of M. luteus colonies counted against 2-7 concentration of 183

the different compounds tested (Error bars showing ±1 std dev, n=5) 184

185

186

To further assess the antimicrobial efficacy of applying ampicillin in close association with the 187

polyphenol-rich extract – fractional bactericidal concentration (FBC) indices were calculated 188

as follows: FBC = FBCA + FBCB. 189

For example, FBCA = 𝐌𝐁𝐂 𝐨𝐟 𝐜𝐨𝐦𝐩𝐨𝐮𝐧𝐝 𝐀 𝐰𝐡𝐞𝐧 𝐚𝐩𝐩𝐥𝐢𝐞𝐝 𝐢𝐧 𝐜𝐨𝐦𝐛𝐢𝐧𝐚𝐭𝐢𝐨𝐧

𝐌𝐁𝐂 𝐨𝐟 𝐜𝐨𝐦𝐩𝐨𝐮𝐧𝐝 𝐀 𝐚𝐥𝐨𝐧𝐞 190

The results (calculated FBC value) were then interpreted as either synergistic (if ≤ 0.5), additive 191

(if 0.5-1), indifferent (if >1), or antagonistic (if >4) [10]. 192

Table 7: FBC indices of Ampicillin when applied in close association with polyphenol 193

extracts-“enhanced”-agar against E. coli 194

Combined treatment MBC value FBC index (to 2 d.p.) Type of interaction

Ampicillin + Green Tea Extract 2-7

4

7

1

7

2

2

2

2 0.14 Synergistic

Ampicillin + Banana Leaves Extract 2-4

2

4

1

4

2

2

2

2 0.38 Synergistic

195 Table 8: FBC indices of Ampicillin when applied in close association with polyphenol 196

extracts-“enhanced”-agar against M. luteus 197

Combined treatment MBC value FBC index (to 2 d.p.) Type of interaction

Ampicillin + Green Tea Extract 2-7

4

7

3

7

2

2

2

2 0.19 Synergistic

Ampicillin + Banana Leaves Extract 2-5

3

5

3

5

2

2

2

2 0.50 Synergistic

198

0

10

20

30

40

50

60

70

80

21

Av

era

ge

nu

mb

er o

f M

. lu

teu

s co

lon

ies

cou

nte

d

Compounds Tested

A, Ampicillin (30mcg/ml)

B, Green Tea Extract

C, Banana Leaves Extract

D, Ampicillin + Green Tea

E, Ampicillin + Banana Leaves

A B C D E

Note:

implies p < 0.005

implies p < 0.001

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Clear synergistic interactions were observed in all cases, except when antibiotic ampicillin 199

was applied in close association with a sub-bactericidal concentration of banana leaves extract 200

against M. luteus as the interaction tended towards additive. This was further supported by the 201

results of Tukey’s HSD test, as the antimicrobial efficacy of applying ampicillin in close 202

association with extract-“enhanced”-agar was significantly better than applying the different 203

test compounds individually (as p <0.0001 in all cases). The combined application of ampicillin 204

and green tea extract “enhanced” agar was also observed to be significantly better than the 205

combined application of ampicillin and banana leaves extract “enhanced” agar (as p <0.0001). 206

207

The results obtained can be explained by the fact that green tea and banana leaves contain large 208

amounts of polyphenols [11] which are partially hydrophobic (due to the presence of non-polar 209

groups e.g. alkyl and aryl groups) and hydrophilic (due to the presence of polar groups e.g. 210

hydroxyl group) [12]. The polar groups on the polyphenols are able to attract the ionised 211

phosphate head of the phospholipids at the surface of the bacterial membrane, while the non-212

polar groups tend to repel the membrane. This combination of opposing forces thus gives rise 213

to instability of the bacterial membrane. This could have not only explained for the 214

antimicrobial effects of the polyphenol-rich extracts but also the decrease in MBC values 215

observed in the synergy studies done as the polyphenol-rich green tea and banana leaves 216

extracts render the bacterial cell more susceptible to ampicillin via destabilisation of bacterial 217

membranes. Other possible synergistic effects due to the application of the polyphenol-rich 218

extracts include (i) impediment of receptor or active site modification to allow enhanced 219

binding of the antibiotic [13], (ii) inhibition of mycobacterial enzymes which can degrade the 220

antibiotic [14], and (iii) inhibition of efflux pumps thus allowing accumulation of the antibiotic 221

within the bacterial cell [15]. 222

223

224

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CONCLUSION 225

The polyphenol-rich extracts of green tea and banana leaves are potential antimicrobial agents 226

for treatment of bacterial infections. These extracts also possess synergistic interactions when 227

applied in close association with ampicillin, a commonly-prescribed moderate-spectrum 228

antibiotic, effectively increasing the antimicrobial potency of ampicillin against E. coli and M. 229

luteus. This holds great promise for alleviating the impact of prevailing bacterial infections and 230

the cost of ineffective antibiotics – as such synergistic inhibitory actions can not only help to 231

reduce application dosage – they can also serve as an adjuvant to “enhance” our current 232

antibiotics so as to better combat against multiple drug-resistant ‘superbugs’ prevalent in 233

society today [16]. 234

Future work can be done to study the possible cytotoxic and genotoxic effects of the proposed 235

treatment methods as they should be selectively toxic i.e. target only the disease-causing 236

bacteria [17]. Lastly, some bioactive substances extracted from botanical materials tend to be 237

much more poorly absorbed as compared to the standard antibiotics [18]. Thus, the 238

bioavailability of the green tea and bananas leaves extracts should be evaluated. 239

240

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ACKNOWLEGEMENTS 241

Research was conducted with assistance from the National University of Singapore. 242

No conflict of interest to declare. 243

244

245

246

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REFERENCES 247

[1] Mead, Paul (1999). “Food related illness and deaths in the US”. Emerging Infectious 248

Diseases 5, 1999. 249

[2] “Drug-resistant NDM-1 'superbug' Found in London and Nottingham Hospitals | Mail 250

Online”. Home | Mail Online. Web. 22 Oct. 2010. URL: 251

<http://www.dailymail.co.uk/health/article-1302432/Drug-resistant-NDM-1-superbug-252

London-Nottingham-hospitals.html>. 253

[3] Hanberger H, Nilson LE. (1996) “Increased Antibiotic Resistance of Common Microbe 254

Strains”. Lakartidningen. 93, No. 3, 148 – 51, 154. 255

[4] Havsteen B (1983). “Flavonoids, A Class of Natural Products of High Pharmacological 256

Potency”. Biochem Pharmacol 1983; 32:1141–8. 257

[5] Lotito SB, Frei B (2006). “Consumption of Flavonoid-rich Foods and Increased Plasma 258

Antioxidant Capacity in Humans: Cause, Consequence, or Epiphenomenon?” Free Radic. 259

Biol. Med. 41 (12): 1727–46 260

[6] Cristobol, Miranda (2008). “Antioxidant Activities of Flavonoids”. Department of 261

Environmental and Molecular Technology. Oregon State University. 262

[7] Cushnie TPT, Lamb AJ (2005). “Antimicrobial Activity of Flavonoids”. International 263

Journal of Antimicrobial Agents 26 (5): 343–356. 264

[8] Singleton VL, Orthofer R, Lamuela-Raventos RM (1999). “Analysis of total phenols and 265

other oxidation subtrates and antioxidants by means of Folin-Ciocalteu reagent”. Meth 266

Enzymol 1999; 299: 152-178. 267

[9] “Medical microbiology”, Mims and Playfair, Mosby Europe, 1993, 35.31 268

[10] Odds FC (2003). “Synergy, antagonism, and what the chequerboard puts between them”. 269

J Antimicrob Chemother 2003, 52:1. 270

[11] Hsu, S.D., Singh, B.B., Lewis, J.B., Borke, J.L., Dickinson, D.P., Drake, L., et al. 271

(2002). “Chemoprevention of oral cancer by green tea”. Gen. Dent. 50(2): 140–146. 272

PMID:12004708. 273

[12] Cabrera C, Artacho R, Giménez R (April 2006). “Beneficial effects of green tea--a 274

review”. Journal of the American College of Nutrition, Vol. 25, No. 2, 79-99 (2006). 275

[13] Takashi Kuzuhara, Yuma Iwai, Hironobu Takahashi, Dai Hatakeyama, and Noriko 276

Echigo (2009). “Green tea catechins inhibit the endonuclease activity of influenza A virus 277

RNA polymerase”. PLoS Curr. 2009 October 13; 1: RRN1052. 278

PeerJ PrePrints | https://doi.org/10.7287/peerj.preprints.1580v1 | CC-BY 4.0 Open Access | rec: 10 Dec 2015, publ: 10 Dec 2015

Page 16: (Camellia sinensis) and banana

[14] Lambert, P., & Hammond, S. (1973). “Potassium fluxes, first indications of membrane 279

damage in micro-organisms”. Biochemical and Biophysical Research Communications , 796-280

799. 281

[15] Judis, J. (1962). “Studies on the mechanism of action of phenolic disinfectants. I. 282

Release of radioactivity from carbon-14-labeled Escherichia coli”. Journal of Pharmaceutical 283

Sciences , 261-265. 284

[16] F. Tenover (2006). “Mechanisms of Antimicrobial Resistance in Bacteria”. American 285

Journal of Infection Control, Volume 34, Issue 5, Pages S3-S10. 286

[17] “Antimicrobial Agents in the Treatment of Infectious Disease” (2009). Online Textbook 287

of Bacteriology. Web. 20 Nov. 2010. URL: 288

<http://www.textbookofbacteriology.net/antimicrobial_2.html>. 289

[18] Rosato, A., Vitali, C., De Laurentis, N., Armenise, D. and Antonietta Milillo, M. (2007). 290

“Antibacterial effect of some essential oils administered alone or in combination with 291

Norfloxacin”. Phytomed., 14, 727-732. 292

PeerJ PrePrints | https://doi.org/10.7287/peerj.preprints.1580v1 | CC-BY 4.0 Open Access | rec: 10 Dec 2015, publ: 10 Dec 2015