effect of different extracts of four egyptian mango leaves...

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: [email protected]

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Key words: Mangiferaindica, ethanolic extract, pathogenic bacteria


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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.


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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


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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



LSD



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)



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


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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)



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


(organism) at concentration 10% with boiled water extracts

of MLs


Variable dependiente: organism

LSD



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)


(organism) at concentration 20% with boiled water extracts

of MLs


Variable dependiente: organism

LSD



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




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


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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


(organism) at concentration 10% with acetone extract of

MLs



LSD



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


(organism) at concentration 20 %with acetone extract of

MLs



LSD



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)


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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




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



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


(organism) at concentration 10% (w/v) with ethanolic

extract of MLs



LSD



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)


(organism) at concentration 20% (w/v) with ethanolic

extract of MLs



LSD



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


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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,


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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