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Easa et al. Int J Gastroenterol Hepatol Transpl Nutr 2017;2(iii):1-10 ISSN 2455–9393
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Original Article
Effect of different extracts of four Egyptian mango leaves on some food
borne bacteria Saadia Mohamed Easa1, AfafAli Amin2, Hala Mohamed Refaat3, Al-shimaa Mahfouz Attala2
ABSTRACT This study is to investigate the antimicrobial activity of leaves of four varieties of
Mangiferaindica L., Anacardiaceae, by 4 different solvents (Ethanol 70%, acetone,
boiling water and water at 37C°). These extracts were tested for their inhibitory effects
using the agar diffusion method at different concentrations (10% and 20%) on nine
bacterial strains, five of them are gram-negative (S. typhimurium, S. cerro, Sh.
dysenteriae, E.coli and P. mirabilis) and the remaining four are gram-positive (B.
cereus, B. lichiniformis, Staph. aureus and L. monocytogenes). The study demonstrated
that gram-positive bacteria are more susceptible than gram-negative. It was found that
Staph. aureus is the most sensitive pathogen compared to the others, it exhibited the
largest inhibition zones in all extracts which reached to maximum with ethanolic
extract to give 28mm. Shigelladysenteriae, Echerichia coli and Proteus mirabilis gave
a weak response with water 37°C and boiled water extracts of Alphonso leaves at 20%
(w⁄v) concentration, acetone extracts at 20% (w⁄v) concentration affected Sh.
dysenteriae, E.coli and P. mirabilis with zones of inhibition (13, 16 and 12 mm)
respectively, also Ethanol extracts at 20% (w⁄v) concentration affected these strains
with zones of inhibition which were 14, 9, and 13mm respectively. Salmonella
typhimurium showed no inhibition effects by water 37°C, but with boiled water
extracts of Kiett leaves, showed that inhibition zone of 12 mm was formed at 20%
(w⁄v) concentration ,while acetone extracts of Alphonso leaves at 10 and 20% (w⁄v)
concentrations inhibition zones of 9 and 12 mm respectively were formed and ethanol
extracts of Alphonso leaves at 10 and 20% (w⁄v) concentrations afforded inhibition
zones of 8 and 11 mm respectively were formed .L. monocytogenes inhibited by all
Alphonso leave extractes with maximum inhibition zone 25 mm with 10% (w⁄v)
concentrations acetone and 20% (w⁄v) concentrations ethanol extracts. It was found
that, Alphonso leaves were a source of natural antimicrobial agent in boiled water,
acetone and ethanol extracts at 20% (w⁄v) concentrations and as additive of processed
food as natural preservative, these extracts showed inhibition zone diameters near to
the antibiotic ciprofloxacin.
INTRODUCTION
Despite the availability of a range of synthetic antibiotics, the
infectious diseases continue to be the major health problem
worldwide. The development of widespread antibiotic resistance
among the pathogens (Al-Sokari and El-Sheikha, 2015) and
undesirable side effects associated with the continued use of
synthetic drugs has stimulated a renewed interest in the
alternative therapeutics (Kauret al., 2010). Because of serious
threats of synthetic drugs people turning towards the use of
medicinal plants as cure for infectious disease (Rajan et al.,
2012).
Medicinal plants contain biologically active components which
over the years have been exploited in the traditional medical
practice for the treatment of various ailments. Many plants have
been used because of antimicrobial properties which are due to
compounds produced in the secondary metabolism of the plant,
for instance the phenolic compounds (Mustapha et al., 2014).
Medicinal plants are valuable natural sources effective against
various infectious agents and are rich in bioactive compounds
International Journal of Gastroenterology, Hepatology,
Transplant & Nutrition
1 Ain Shams University, Microbiology Department, Science faculty, Egypt 2 National Nutrition Institute, Food Hygiene
Department, Microbiology Unit, Egypt 3 National Research Center, Microbial
Chemistry Department, Egypt
Address for Correspondence:
Saadia Mohamed Easa
E-mail: af_amin@hotmail.com
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Key words: Mangiferaindica, ethanolic extract, pathogenic bacteria
Easa et al. Int J Gastroenterol Hepatol Transpl Nutr 2017;2(iii):1-10 ISSN 2455–9393
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which can resist health hazards. Plant extracts has been used
traditionally to treat a number of infectious diseases including
those caused by bacteria and fungi. New antimicrobial agents
are needed to treat diseases in humans and animals caused by
drug resistant microorganisms (Kumar, 2015).
Medicinal uses of these plants range from the administration of
the plant’s roots, bark, stem, leaves, fruits and seeds, to the use
of extracts from the whole plant (Akujobiet al., 2004).
Antimicrobials of plant origin have enormous therapeutic
potential. Many of these medicine plants are source of
phytochemicals such as polyphenols (Albuquerque et al.,
2013).They are effective in the treatment of infectious diseases
while simultaneously minimizing many of the side effects that
are often associated with synthetic antimicrobials (Shinde and
Mulay, 2015). Polyphenols are well documented to have
microbicide activities against a huge number of pathogenic
bacteria.
Rajanet al. (2011) proved that mango is one such medicinal
plant used as cure among the traditional and indigenous people
of India since ages. Mango (Mangiferaindica L.) is one of the
most popular and economically important fruit marketed
worldwide and is often referred to as "the king of fruits" (Engels
et al., 2009). It is a member of the Anacardiaceae family which
comprises more than 70 genera and historical records suggest
that its cultivation as a fruit tree originated in India around 4000
years ago (Riberoand Schieber ,2010). All parts of mango trees
have been used in traditional south Asian medicine: kernels,
flowers, leaves, gum, bark and peel. Diseases commonly treated
with herbal remedies obtained from parts of the mango tree
include dysentery, diarrhea, urinary tract inflammation,
rheumatism and diphtheria and a number of these uses are
supported by scientific evidence (Ross, 2003). Mango
(Mangiferaindica) fruit is rich in polyphenolic compounds
(Luoet al., 2014). Mango leaves (MLs) are rich source of
phenolic compounds and their extract can be used as natural
preservative in food applications (Morsi et al., 2010; Teresa et
al., 2013). Severi et al. (2009) found that the phytochemical
study of the aqueous decoction (AD) from MLs leads to
isolation of two phenolic compounds (mangiferin "xanthone"
and benzophenone glycoside) and the percentage of total
phenolic content obtained from AD leaves was greater than
57.3%. Bakana et al, (1987) deduced that group of
benzophenone display many biological activities including
antimicrobial properties, in addition , some studies have shown
the activity of benzophenone against Helicobacter
pyloricontributing to the healing effects of AD from mango to
gastric ulcers (Ma et al., 2004). Hannan et al. (2013), found that
acetone mango leaf extract has antibacterial activity against
multi-drugresistant S.typhias it wasinhibited in all dilutions
tested (50, 100, 150, 200, 250 mg/ml). However, Bharti (2013)
proved that hexane-ethyle acetate leaf extract of M.indica
exhibited pronounced activity against (S.typhi, Proteus vulgaris,
Klesiella pneumonia, Enterobacteraerogens, Mycobacterium
tuberclosis, S.aureus, Streptococcus pyogens, P. aeuroginsa).
On the other hand Masibo and He. (2009) proved that Chinese
mango leaf extracts have mild antimicrobial activity against
E.coli, S. typhi, Staph. aureus and B. cereus.
MATERIAL AND METHODS
1. Media and chemical reagents
Nutrient agar (Oxoid): Commonly used for the routine
cultivation of bacteria (Lapageet al., 1970).
Nutrient broth (Oxoid): for the cultivation of a wide variety of
microorganisms ( Boltonet al.,1984).
Peptone water (Himedia): It used for bacteriological analysis.
Ethyl alcohol 70%: (El-Nasr Pharmaceutical Chemical
Company)
Sterile distilled water
Pure acetone 99%: (El-Nasr Pharmaceutical Chemical
Company)
Ciprofloxacin (Ciprofloxacin susceptibility discs 5 mc,
bioanalyse): as antibiotic
0.5 McFarland standard (Biomerieux)
Sodium carbonate, methanol, gallic acid and Folin-Ciocalteu
reagents.
2. Plant materials
Fresh leaves of different four mango varieties (Alphoso, Awis,
Zebda and Kiett) were collected from the farm of The
Agriculture Research Center, Ministry of Agriculture, Giza,
Egypt
3. Identified bacterial strains
Staphylococcus aureus ATCC29213, Listeria monocytogenes
ATCC 7644, Salmonella typhimorium ATCC 14028, Proteus
mirabilis ATCC 43071, E.coli ATCC 10536, Bacillus
licheniformis ATCC 14580, Bacillus cereus ATCC 10876, were
obtained from TCS bioscience LTD, Botolph Clydon,
Buckingham, MK 18 2LR, England.
Salmonella cerro (6, 14), 18: Z4, Z23, Shigelladysenteriae (Sub
group A), Proved by Hygiene Institute/National Salmonella
Center, (Fedral German Republic).
All strains were stored in nutrient agar stabs, sealed and kept in
refrigerator and sub-cultured every 3 months.
4. Methods
Preparation of ethanol and acetone extracts of leaves from
different varieties of mango
According to (Akerele et al., 2008; Doughari and Manzara,
2008) with slight modifications, samples of mango leaves of
each kind were air dried separately then milled using electrical
blender to obtain fine powder. One hundred milliliter (100 ml)
from ethanol 70% and acetone were added to 10 g of each kind
of leaf powder (Alphoso, Awis, Zebda and Kiett) in separate
conical flasks and allow to soak at ambient temperature for 72h,
for 1-3 times till exhaustion with frequent shaking from time to
time. The extracts were then filtered using Whatman no.1 and
then the filtrates were concentrated at 40˚C using oven till
getting rid of all solvent and having solid residue, which stored
in freezer at -20˚C till use and rehydrated by water during
screening of antimicrobial activity.
Easa et al. Int J Gastroenterol Hepatol Transpl Nutr 2017;2(iii):1-10 ISSN 2455–9393
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Preparation of water 37˚C and boiled water extracts of
leaves from different varieties of mango
According to (Masibo and He, 2009) with slight modifications,
samples of mango leaves of each kind were air dried separately
then milled using electrical blender to obtain fine powder. Then
in 2 flasks add 30 g of leaves powder and 500 mL of ionized
water and incubate the first flask at 37˚C for 48 hours, but the
other flask at boiled temperature for 3 hours using water bath.
The extracts were then filtered by Whatman no.1 and the
filtrates were concentrated at 40˚C using oven till getting rid of
all water and having solid residue, which stored in freezer at -
20˚C till use and rehydrated by water during screening of
antimicrobial activity.
Preparation and standardization of inoculum
The tested bacteria were first inoculated to test tubes of nutrient
broth separately and incubated at 37˚C for 18-24 hours and each
of the cultures turbidity were then adjusted to 0.5 McFarland
turbidity standard (Lino and Deogracios, 2006; Doughari and
Manzara, 2008).
Comparison between the two opacities of bacterial suspension
tube and 0.5McFaland tube did by placing the tubes against a
black background (Washington et al., 1972).
Preparation of extract concentration
One gram of extract added to 10 ml sterile distilled water and
shake well until complete dissolve under aseptic condition. Use
this extract concentration (10%) in antibacterial activity tests.
The above steps repeated by 2 grams of extract in 10 ml sterile
distilled water to make concentration (20%).
Screening of antibacterial activity for the mango leave
extracts
Well diffusion technique
The well diffusion technique was employed to observe the
inhibitory spectra of different four varieties (Alfonso, Zebda,
Awis and kit) of (Mangiferaindica) leave extracts on nine
pathogenic bacteria. By using sterile cotton swabs from
standardized streaked onto nutrient agar plates. 50 µl of extract
(from different variety at concentration of 10% and 20%) were
soaked in plate well. Ciprofloxacin discs were used as positive
control, and distilled water as negative control. The plates were
incubated at 37˚C for 18-24 hours. The screening of
antibacterial activity was assessed based on the diameter of the
clear zone surrounding the well (including the well diameter)in
millimeter (mm). The tests were conducted triplicates (Aboaba
et al., 2006; Nurmahani et al., 2012). The transparently clear
zones showed bactericidal activity while the clear zones
containing micro colonies showed bacteriostatic activity
(Aboaba et al., 2006).
The statistical analysis
The statistical analysis includes:
A. Descriptive Statistics: arithmetic mean or average and
standard deviation.
B. The results were analyzed by SPSS (Statistical Package for
Social Science) statistical package version 15 and the
results were tabulated by Harvard graphics packages
version 4 (1998) were used for representing the results
graphically.
C. Quantitative variables from normal distribution were
expressed as (mean ±SD).
Independent t-test was used to compare between the two groups
10% and 20%and F-test (One way ANOVA) and (Two ways
ANOVA) were used for comparing between groups, there are
two assumptions underlying the analysis of variance and
corresponding F test. The first is that the variable is normally
distributed. The second is that the standard deviation between
individuals is the same in each group. Data were analyzed by
SPSS statistical package version 15 (1994).
If the F ratio is significant, then SPSS conduct post hoc tests as
LSD test (Least Significant Difference) (Armitage et al., 2002).
A significant P-value was considered when P is less than 0.05
(Jayawardana et al., 2015).
RESULTS
Screening of antimicrobial activity for different types of
mango leaves (MLs) with different solvents
Table 1: Antibacterial activities of water 37°C extract of different varieties of MLs expressed as inhibition zone diameter (mm)
at different concentration (10 and 20%) w/v
Bacterial species
Different varieties of water 37°C extract of MLs
Alphonso Awis Keitt Zebda 10% 20%
10% 20% 10% 20% 10% 20% 10% 20% Mean SD Mean SD t-value p-value
1. S. typhimurium - - - - - - - - 0.0 0.0 0.0 0.0 0.0 0.0
2. S. cerro 11 16 - 11 - 13 - 6 2.75 5.5 11.5 4.2 2.53 0.04*
3. Sh. dysenteriae - 10 - - - - - - 0.0 0.0 2.5 5.0 1.0 NS
4. E.coli - - - - - 12 - - 0.0 0.0 3.0 6.0 1.0 NS
5. P. mirabills - - - - - 12 - - 0.0 0.0 3.0 6.0 1.0 NS
6. B. cereus 11 12 - 9 - 13 - 8 2.75 5.5 10.5 2.38 2.59 0.04*
7. B. Licheniformis 11 13 - 10 10 13 - 8 5.25 6.08 11.0 2.45 1.76 NS
8. Staph. aureus 15 19 - 13 11 15 12 15 9.5 6.56 15.5 2.52 1.71 NS
9. L. monocytogenes 12 13 - - - 16 - - 3.0 6.0 7.25 8.46 0.82 NS
Mean 6.67 9.22 0.0 4.78 2.33 10.4 1.33 4.11
SD 6.44 7.37 0.0 5.76 4.64 6.06 4.0 5.44
t-value 0.78 2.49 3.19 1.23
p-value NS 0.02* 0.006** NS
*p < 0.05 (Significant), **p < 0.01 (Highly significant), NS = Not Significant, (-) no inhibition
Easa et al. Int J Gastroenterol Hepatol Transpl Nutr 2017;2(iii):1-10 ISSN 2455–9393
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Figure 1a: Mean values of inhibition zones of each bacterial
species with different varieties at the two concentrations 10
and 20% with water 37°C extract of MLs
Figure 1b: Mean values of bacterial species with each
variety at both concentrations 10 and 20% with water 37°C
extract of MLs
Table 1a: Multiple comparisons of dependent variable
(organism) at concentration 10% with water 37°C extract of
MLs
Multiple Comparisons
Dependent Variable: Organism
LSD
(I) Organism (J) Organism Mean
Difference (I-J) Sig.
Staphylococcus
aureus
1 9.50 0.001**
2 6.75 0.013*
3 9.50 0.001**
4 9.50 0.001**
5 9.50 0.001**
6 6.75 0.013*
9 6.50 0.016*
*p<0.05 (significant), **p<0.01 (high significant), I: The
pathogen that has the higher mean value, J: The other pathogens
that have lower mean values.
Table 1b: Multiple comparisons of dependent variable
(organism) at concentration 20% with water 37°C extract of
MLs
Multiple Comparisons
Dependent Variable: Organism
LSD
(I) Organism (J) Organism Mean
Difference (I-J) Sig.
Staphylococcus
aureus
1 15.50 0.000***
3 13.00 0.000***
4 12.50 0.000***
5 12.50 0.000***
9 8.25 0.005**
LSD: The least significance difference, ***p<0.001 (high
significant)
Figure 2a: Mean values of inhibition zones of each bacterial
species with different varieties at the two concentrations 10
and 20% (w/v) with boiled water extracts of MLs
Figure 2b: Mean values of all bacterial species with each
variety at both concentrations 10 and 20% (w/v) with boiled
water extracts of MLs
Easa et al. Int J Gastroenterol Hepatol Transpl Nutr 2017;2(iii):1-10 ISSN 2455–9393
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Table 2: Antibacterial activities of boiling water extract of different varieties of MLs expressed as inhibition zone diameter
(mm) at different concentrations (10 and 20%) w/v
Bacterial species
Different varieties of MLs (Boiling water extracts)
Alphonso Awis Keitt Zebda 10% 20%
10% 20% 10% 20% 10% 20% 10% 20% Mean SD Mean SD t-value p-value
S. typhimurium - - - - - 12 - - 0.0 0.0 3.0 6.0 1.0 NS
S. cerro 13 18 12 13 - 12 - 7 6.25 4.23 12.5 4.5 1.5 NS
Sh. dysenteriae - 10 - - - - - - 0.0 0.0 2.5 5.0 1.0 NS
E.coli - - - - - - - - 0.0 0.0 0.0 0.0 0.0 NS
P. mirabills - - - - - - - - 0.0 0.0 0.0 0.0 0.0 NS
B. cereus 12 16 11 14 - 13 - 9 5.75 6.65 13.0 2.94 1.99 NS
B. Licheniformis 11 17 11 13 - 10 - 7 5.5 6.35 11.75 4.27 1.6 NS
Staph. aureus 15 22 11 14 14 18 8 15 12.0 3.16 17.25 3.59 2.19 NS
L. monocytogenes 13 18 - - - 16 - - 3.25 6.5 8.5 9.85 0.89 NS
Mean 7.11 11.2 5.0 6.0 1.56 9.0 0.89 4.22
SD 6.83 8.96 5.94 7.12 4.67 7.14 2.67 5.52
t-value 1.09 0.32 2.6 1.63
p-value NS NS 0.019* NS
*p < 0.05 (significant), NS = Not Significant, (-) no inhibition
Table 2a: Multiple comparisons of dependent variable
(organism) at concentration 10% with boiled water extracts
of MLs
Multiple Comparisons
Variable dependiente: organism
LSD
(I) Organism (J) Organism Mean
Difference (I-J) Sig.
Staphylococcus
aureus
1 12.00 0.000***
2 5.75 0.040*
3 12.00 0.000***
4 12.00 0.000***
5 12.00 0.000***
6 6.25 0.026*
7 6.50 0.021*
9 8.75 0.003**
*p < 0.05 (significant), **p < 0.01 or ***p < 0.001 (high
significant)
Table 2b: Multiple comparisons of dependent variable
(organism) at concentration 20% with boiled water extracts
of MLs
Multiple Comparisons
Variable dependiente: organism
LSD
(I) Organism (J) Organism Mean
Difference (I-J) Sig.
Staphylococcus
aureus
1 14.25 0.000***
3 14.75 0.000***
4 17.25 0.000***
5 17.25 0.000***
9 8.75 0.005**
LSD: The least significance difference, NS = Not Significant
Table 3: Antibacterial activities of acetone extract of different varieties of MLs expressed as inhibition zone diameter (mm) at
different concentrations (10 and 20%) w/v
Bacterial species
Different varieties of MLs (Boiling water extracts)
Alphonso Awis Keitt Zebda 10% 20%
10% 20% 10% 20% 10% 20% 10% 20% Mean SD Mean SD t-value p-value
S. typhimurium 9 12 - - - - - - 2.25 4.5 3.0 6.0 0.20 NS
S. cerro 19 20 18 18 8 13 11 13 14.0 5.35 16.0 3.56 0.10 NS
Sh. dysenteriae 9 13 - - - - - - 2.25 4.5 3.25 6.5 0.25 NS
E.coli 13 16 - - - - - - 3.25 6.5 4.0 8.0 0.15 NS
P. mirabills - 12 - - - - - - 0.0 0.0 3.0 6.0 1.0 NS
B. cereus 18 19 17 18 12 14 11 14 14.5 3.51 16.25 2.63 0.80 NS
B. Licheniformis 18 20 18 16 10 12 11 12 13.75 3.86 15.0 3.83 0.46 NS
Staph. aureus 25 28 18 20 16 21 13 16 18.0 5.09 21.25 4.99 0.91 NS
L. monocytogenes 25 24 19 22 13 16 - - 13.5 9.75 15.5 10.9 0.27 NS
Mean 14.8 18.2 9.78 10.4 6.56 8.44 5.11 6.11
SD 7.78 5.54 9.31 10.0 6.58 8.4 6.09 7.32
t-value 1.1 0.15 0.53 0.32
p-value NS NS NS NS
NS = Not Significant, (-) no inhibition
Easa et al. Int J Gastroenterol Hepatol Transpl Nutr 2017;2(iii):1-10 ISSN 2455–9393
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Figure 3a: Mean values of inhibition zones of each bacterial
species with different varieties at the two concentrations 10
and 20% with acetone extract of MLs
Figure3b: Mean values of all bacterial species with each
variety at both concentrations 10 and 20% with acetone
extract of MLs
Table 3a: Multiple comparisons of dependent variable
(organism) at concentration 10% with acetone extract of
MLs
Multiple Comparisons
Dependent Variable: Organism
LSD
(I) Organism (J) Organism Mean
Difference (I-J) Sig.
Staphylococcus
aureus
1 15.75 0.000***
2 14.00 0.000***
5 14.50 0.000***
6 13.75 0.000***
7 18.00 0.000***
9 13.50 0.000***
***p < 0.001 (high significant)
Figure 3c: Some plates showing antimicrobial activity with
acetone extract of MLs
Table 3b: Multiple comparisons of dependent variable
(organism) at concentration 20 %with acetone extract of
MLs
Multiple Comparisons
Dependent Variable: Organism
LSD
(I) Organism (J) Organism Mean
Difference (I-J) Sig.
Staphylococcus
aureus
1 18.25 0.000***
3 18.00 0.000***
4 17.25 0.000***
5 18.25 0.000***
7 6.25 0.024*
9 5.75 0.036*
LSD: The least significance difference, *p < 0.05 (significant)
Easa et al. Int J Gastroenterol Hepatol Transpl Nutr 2017;2(iii):1-10 ISSN 2455–9393
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Table 4: Antibacterial activities of ethanol extract of different varieties of MLs expressed as inhibition zone diameter (mm) at
different concentrations (10 and 20%) w/v
Bacterial species
Different varieties of MLs (Boiling water extracts)
Alphonso Awis Keitt Zebda 10% 20%
10% 20% 10% 20% 10% 20% 10% 20% Mean SD Mean SD t-value p-value
S. typhimurium 8 11 - - - - - - 2 4.0 2.75 5.5 0.22 NS
S. cerro 18 21 15 18 11 14 10 11 13.5 3.7 16.0 4.4 0.87 NS
Sh. dysenteriae 9 14 - - - - - - 2.25 4.5 3.5 7.0 0.30 NS
E.coli 7 9 - - - - - - 1.75 3.5 2.25 4.5 0.18 NS
P. mirabills - 13 - - - - - - 0.0 0.0 3.25 6.5 1.0 NS
B. cereus 17 20 16 17 12 13 11 12 14.0 2.9 15.0 3.7 0.64 NS
B. Licheniformis 16 19 15 17 11 14 11 13 13.25 2.63 15.75 2.8 0.13 NS
Staph. aureus 23 28 18 20 17 20 14 17 18.0 3.7 21.25 4.7 1.1 NS
L. monocytogenes 21 25 18 22 15 17 13 17 16.75 3.9 20.25 3.9 1.3 NS
Mean 13.2 17.8 9.11 10.4 7.3 8.7 6.6 7.8
SD 7.58 6.5 8.7 10.0 7.2 8.5 6.3 7.6
t-value 1.4 0.3 0.36 0.37
p-value NS NS NS NS
NS = Not Significant, (-) no inhibition
Figure 4a: Mean values of inhibition zones of each bacterial
species with different varieties at the two concentrations 10
and 20% (w/v) with ethanolic extract of MLs
Figure 4b: Mean values of all bacterial species with each
variety at both concentrations 10 and 20% (w/v) with
ethanolic extract of MLs
Table 4a: Multiple comparisons of dependent variable
(organism) at concentration 10% (w/v) with ethanolic
extract of MLs
Multiple Comparisons
Dependent Variable: Organism
LSD
(I) Organism (J) Organism Mean
Difference (I-J) Sig.
Staphylococcus
aureus
1 12.00 0.000***
2 5.75 0.000***
3 12.00 0.000***
4 12.00 0.000***
5 12.00 0.000***
6 6.25 0.026*
7 6.50 0.021*
9 8.75 0.003**
*p < 0.05 (significant), **p < 0.01 or ***p < 0.001 (high
significant)
Table 4b: Multiple comparisons of dependent variable
(organism) at concentration 20% (w/v) with ethanolic
extract of MLs
Multiple Comparisons
Dependent Variable: Organism
LSD
(I) Organism (J) Organism Mean
Difference (I-J) Sig.
Staphylococcus
aureus
1 14.25 0.000***
3 14.75 0.000***
4 17.25 0.000***
5 17.25 0.000***
9 8.75 0.005**
LSD: The least significance difference
Easa et al. Int J Gastroenterol Hepatol Transpl Nutr 2017;2(iii):1-10 ISSN 2455–9393
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Table 5: Antibacterial activities of standard synthetic antibiotic
(Ciprofloxacin) on the tested strains
Bacterial strains Ciprofloxacin
Salmonella typhimurium 30
Salmonella cerro 31
Shigella dysenteriae 30
E.coli 29
Proteus mirabilis 28
Bacillus cereus 25
Bacillus licheniformis 25
Staphylococcus aureus 24
Listeria monocytogenes 29
mean + SD 27.9 + 2.6
p-value ---
DISCUSSION
To inhibit food-borne pathogens and to extend shelf life,
synthetic chemicals with antimicrobial properties are often used
as preservative in food processing and storage. Concerns over
the potential risks of synthetic food additives for human health
and consumer awareness have directed the interest in using
naturally occurring alternatives. The market of health and herbal
neutraceuticals are addressing their attention to rich plants
sources offering functional efficacy (Duaet al., 2013).
Regarding the antibacterial activity of mango leaf extracts
(MLEs) showed in generalthe bacterial species (S. typhimurium,
Shigella dysenteriae, Echerichia coli and Proteus mirabilis)
gave a weak response with water 37°C and boiled water extracts
of Alphonso leaves at 20% (w⁄v) concentration which affected
Sh. dysenteriae with a zone of inhibition 10 mm, but with
acetone extracts at 20% (w⁄v) concentration which affected Sh.
dysenteriae, E.coli, and P. mirabilis with zones of inhibition
(13, 16 and 12 mm) respectively, while at 10% (w⁄v)
concentration affected Sh. dysenteriae and E.coli with 9 and 13
mm respectively (Tables 1,2,3). However ethanol extracts at
20% (w⁄v) concentration affected Sh. dysenteriae, E.coli and P.
mirabilis with zones of inhibition which were 14, 9, and 13mm
respectively (Table 4),while 10% (w⁄v) concentration affected
Sh. dysenteriaeand E.coli with zones of inhibition which were 9
and 7 mm respectively. The present results showed that Sh.
dysenteriae and P. mirabilis have a weak inhibition zone (12
mm) with water 37°C extracts of Kiett leaves at 20% (w⁄v)
concentration. This weak response of the above pathogens for
most extracts is followed by the resistance of Salmonella
typhimurium which showed no inhibition effects by water 37°C,
concerning with boiled water extracts of Kiett leaves, results
showed that inhibition zone of 12 mm was formed at 20% (w⁄v)
concentration, applying acetone extracts of Alphonso leaves at
10 and 20% (w⁄v) concentrations inhibition zones of 9 and 12
mm respectively were formed, application of ethanol extracts of
Alphonso leaves at 10 and 20% (w⁄v) concentrations afforded
inhibition zones of 8 and 12 mm respectively were formed.
These results were in agreement with Islam et al. (2010), who
reported that mango leaf extract has no effect on S. typhi and P.
mirabilis. On the other hand, Salmonella cerro showed a good
response especially with ethanol and acetone extracts of
Alphonso leaves at 20% (w⁄v) concentration, the inhibition zone
diameters were reached to 20 and 21 mm respectively.
Doughariand Manzara (2008) reported that the mango leaf
extract (MLE) has a potential effect on E. faecalis and P.
mirabilis ,buthas a low effect on S. typhi.The data in this study
clarified that the four pathogenic bacteria (B. cereus, B.
licheniformis, Staph. aureus and L. monocytogenes), have high
sensitivity to the MLE. They were partly sensitive to most
varieties in both extract concentrations and with all solvents.
Alphonso leaves were a source for antimicrobial in boiled water,
acetone and ethanol extracts as cleared from mean values of
different leaf varieties in (Table 2) 11.2 mm, (Table 3) 18.2 mm
and (Table 4) 17.8 mm. On the other hand, it was found that
Kiett leaves were the best source with water 37°Cextract; it was
10.4 mm in (Table 1). From these results it was concluded that
the most of mango leaf extracts (MLEs) have an effect on gram-
positive as Staph. aureus than gram-negative species. Similar
results were reported by Hannan et al. (2013). This may be due
to the presence of lipopolysaccharides (LPS) in the cell wall of
gram-negative bacteria (Zakaria et al., 2006). The results
indicated that the MLEs may possessed certain phyto chemicals,
which had moderate activity on gram- positive and low activity
on gram-negative bacteria .The results obtained revealed that the
MLEs exhibited relatively weak antimicrobial potential against
the tested pathogens (Tables 1, 2,3and 4) this agreed with
Masibo and He (2009) especially with Water extracts. Doughari
and Manzara (2008) demonstrated that the elevating of the
temperature to 60 -100°C enhanced the activity of MLE against
the tested pathogens, but the water extract at 30 °C gave less
activity. These results are accorded to the present results, which
indicated that the boiled water extract was more effective on the
tested species than in water 37°C extract, this was cleared from
the mean values especially with Alphonso and Awis varieties
(Tables 1 and 2), so it could be concluded that the bioactive
component in the MLE may be heat stable. Marjorie (1999)
reported that different solvents have diverse solubility capacities
for different phyto constituents. This research showed that
acetone and ethanol extracts have more efficacy on bacterial
pathogens than aqueous extracts especially on gram-positive
bacteria. So, acetone and ethanol were suitable solvents for
active components extraction of mango leaves, moreover the
acetone extract was slightly higher than that of ethanol, Tables
(1, 2, 3 and 4), in agreement with those found by Basha et al.
(2015) who reported that ethanol extract has the highest
inhibitory activity against all tested organisms compared to
water extract.
Gram-positive pathogenic bacterium, Staph. aureus, is the most
commonly isolated human bacterial pathogen, which
persistently and asymptomatically colonizes up to 20%–30%of
humans and intermittently colonizes up to 50% – 60%
(Wertheim et al., 2005). Staph. aureus colonizes skin and
mucosa of human, it also causes many infectious diseases
ranging from minor skin infections and abscesses to life-
threatening ailments (such as necrotizing pneumonia,
Easa et al. Int J Gastroenterol Hepatol Transpl Nutr 2017;2(iii):1-10 ISSN 2455–9393
9
endocarditis, and septicemia) associated with high morbidity
and mortality (Liu et al., 2015).Statistical analysis showed that
Staph. aureus is the most sensitive pathogen compared to the
others, it exhibited the largest inhibition zones in all extracts
which reached to maximum with ethanolic extract to give 28mm
(Tables 1a, 1b, 2a,2b, 3a, 3b, 4a, 4b). This was not in agreement
with Islam et al. (2010), who approved that Staph. aureus is the
least sensitive pathogen for MLE compared to other gram-
positive (B. cereus, B. subtilis) and gram-negative (Sh. flexneri,
Sh. sonni). The statistical analysis results of the present extracts
showed that there was no significant relation between 10and
20% (w⁄v) concentrations with Alphonso and Zebda varieties,
but with Awis and Kiett varieties the mean of 20%
concentration exceed that of 10% concentration and the
difference was significant with Awis P= 0.02 and highly
significant with Kiett P=0.006. However, there were no
significant difference between 10 and 20% (w⁄v) concentrations
with all tested species except with S. cerro and B. cereus and
found that the mean of 20% concentration exceed the mean of
10% concentration and the difference was significant P=0.04
(Table 1). In this part of the study there were no significant
relation between 10 and 20% (w⁄v) concentrations with all tested
bacteria and all varieties unless the significant difference of
Kiett variety P=0.019 ( Table 2). On the other hand, Tables 3and
4, showed no significant difference between 10 and 20% (w⁄v)
concentration, this was due to the mean of 10% concentration
was nearly same as the mean of 20% concentration with all
varieties. This non- significance resulted from the similar effect
of 10% and 20% with similar inhibition zones or by having no
effect on the pathogens. Basha et al. (2015) reported that
different concentrations might have significant because of the
effect of these concentrations.
Natural ethanolic mango leave extract of alphonso type and
standard synthetic antibiotic ciprofloxacin (Table 5), showed
nearly equal efficiency on the tested bacterial strains, this was in
agreement with (Aloket al., 2013) who found that mango leave
extracts of bangnapalli variety has a better antibacterial activity .
The results of our previous study in Mohamed (2016) revealed
that ethanolic extract of alphonso kernel was the most potent
extract on S. typhimurium, S. cerro, Staph. aureus and L.
monocytogenes, the polyphenol content of alphonso kernel was
(365.75 mg/ml) higher than the other types, the ethanolic extract
of alphonso type was a promising source of natural
antimicrobial agent and as additive of processed food as natural
preservative.
In conclusion, because of the side effects resulted from using
antibiotics and hazards caused to humans by using chemical
preservatives can be avoided by replacing them by natural
ethanolic extract of mango leaves or kernelespecially alphonso
type. So, further investigations on large scale were
recommended.
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