(camellia sinensis) and banana
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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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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 ng.qin.xiang@u.nus.edu 17
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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
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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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