African Journal of Microbiology Research Vol. 4(22), pp. 3664-3668, 16 October, 2011
Available online http://www.academicjournals.org/ajmr
ISSN 1996-0808 ©2011 Academic Journals
Full Length Research Paper
Antimicrobial activity of pomegranate rind peel extracts
Hany M. Yehia1*, Manal F. Elkhadragy2 and Ahmed E. Abdel Moneim2
1Department of Food Science and Nutrition, College of Food and Agriculture Sciences, King Saud University, Al-Riyadh,
KSA, Saudi Arabia.
2Department of Zoology and Entomology, Faculty of Science, Helwan University, Egypt.
Accepted 19 September, 2011
The pomegranate, Punica granatum L., is an ancient, mystical, unique fruit borne on a small, long-living
tree cultivated throughout the Mediterranean region. Pomegranate is used in several systems of
medicine for a variety of ailments. The synergistic action of the pomegranate constituents appears to
be superior to that of single constituents. P. garantum, have been reported to have antimicrobial
activity against a range of Gram positive and negative bacteria. Pomegranate formulations containing
ferrous salts have enhanced although on short-term. The aim of this experiment is to determine the
antimicrobial activities of combinations of pomegranate rind extract with range of metal salts with the
addition of vitamin C. Phytochemical analyses was made to determine the active inhibitors in rind
extract, including phenolics and flavonoids.
Key words: Antimicrobial activity, pomegranate rind extract, phenolics, flavonoids.
Punica granatum L. (Punicaceae), commonly called
pomegranate, recently described as nature’s power fruit,
is a plant used in folkloric medicine for the treatment of
various diseases (Abdel Moneim et al., 2011; Ajaikumar
et al., 2005) widely cultivated in the Mediterranean
region. Pomegranate has strong antioxidant and anti-
inflammatory properties, recent studies have
demonstrated its anti-cancer activity in several human
cancers (Adhami and Mukhtar, 2007; Longtin, 2003). In
addition, pomegranate peel extract with an abundance of
flavonoids and tannins has been shown to have a high
antioxidant activity (Abdel Moneim et al., 2011).
Antimicrobial drug resistance in human bacterial
pathogens is a worldwide issue and as a consequence,
effective treatment and control of such organisms remain
an important challenge. Bacterial resistance has
appeared for every major class of antibiotic (Lambert,
2005). Since their introduction the emergence of resistant
is evident, particularly for important pathogens such as
Escherichia coli, Salmonella spp., Campylobacter spp.,
Enterococcus spp. and Staphylococcus spp. (Musgrove
*Corresponding author. E-mail: email@example.com.
Tel: (+2) 01001013346.
et al., 2006).
Over the last decade research into the antimicrobial
properties of traditional plant based medicines has been
revisited (Melendez and Capriles, 2006; Navarro et al.,
1996). Numerous plants have been screened for
antimicrobial properties, for example Holetz et al. (2002)
tested 13 plants used in Brazilian traditional medicine and
they demonstrated activity against bacteria such as
Staphylococcus aureus and E. coli. Melendez and
Capriles (2006) tested 172 plant species used in Puerto
Rico and they demonstrated that 14 of these showed
activity against bacteria including S. aureus and E. coli.
Prashanth et al. (2001) tested a number of extracts of
pomegranates against a range of bacteria (S. aureus, E.
coli, Klebsiella pneumoniae, Proteus vulgaris, Bacillus
subtilis and Salmonella typhi), and they found activity
against all isolates. Braga et al. (2005) observed that
pomegranate extracts were able to inhibit not only the
growth of S. aureus but also the production of
enterotoxin. The methanolic extract derived from 200 g of
dried pomegranate produced bactericidal effects at 1%
(v/v) over an extended incubation period (50 h),
demonstrating longevity of action.
Many bacteria have advanced protective mechanisms
for the detoxification of heavy metal ions (Silver, 1996).
Despite this, numerous literature reports address the
development of metal compounds as antimicrobial
agents. Many low molecular-mass metal compounds
exhibit bactericidal and/or bacteriostatic activities. In one
study the susceptibilities of Staphylococcus strains to
solutions of metal salts (in the range of 50 µmol to 80
mmol) were determined and frequencies of resistance
were found to be CuSO4 and NiCl2, 36.2%; ZnSO4,
13.6% and CoCl2, 4.5%, respectively (Ug and Ceylan,
MATERIALS AND METHODS
Preparation of pomegranate rind extracts
Pomegranate rind extracts (PRE) were prepared by blending 50 g
of the seed with 100 mls of distilled water for 10 min. The crude
extract was filtered through muslin followed by Whatman No. 1 filter
paper prior to autoclaving (121°C for 15 mins) before storage at -
20° C (Stewart et al., 1998).
Test for tannins
The aqueous extract (1 ml) was mixed with 10 ml of distilled water
and filtered. Ferric chloride reagent (3 drops) was added to the
filtrate. A blue-black or green precipitate confirmed the presence of
gallic tannins or catechol tannins, respectively.
The Fe3+ reducing power of the extract was determined according
to the method of Oyaizu (1986). The extract (2 ml) was mixed with
0.2 M phosphate buffer, pH 6.6 (2 ml) and 1% potassium
ferricyanide (2 ml). The mixture was then incubated at 50°C for 20
min. Afterwards, the mixture was stopped by adding 10%
trichloroacetic acid (2 ml) and then centrifuged at 3,000 rpm for 10
min. The upper layer of supernatant (2 ml) was mixed with distilled
water (2 ml) and 0.1% FeCl3 solution (0.5 ml). The absorbance was
measured at 700 nm against a blank with a spectrophotometer, and
ascorbic acid was used as a standard. Higher absorbance of the
reaction mixture indicated greater reducing power. The percents of
reducing power were presented as ascorbic acid equivalents using
a calibration curve between the absorbance of the reaction and the
percent of the reducing power ability of ascorbic acid:
OD = (0.0146 x [percent]) + 0.0016, R2 = 0.9999
Determination of total flavonoids
For the assessment of flavonoids, colorimetric method introduced
by Dewanto et al. (2002) was adapted. To determine the amount of
flavonoids by the above mentioned method, 1.50 ml of the
deionized water was added to 0.25 ml of the sample extract and
then 90 µl of 5% Sodium nitrite (NaNO2). Six minute later, after
addition of 180 µl of 10% AlCl3, mixture was allowed to stand for
another 5 min before mixing 0.6 ml of 1 M NaOH. By adding
deionized water and mixing well, final volume was made upto 3 ml.
Using blank, absorbance was measured at 510 nm. Calibration
curve was prepared using querestin acid as standard for total
flavonoids which was measured as milligram querestin equivalents
(QE) per gram of the sample (mg/g).
Determination of total phenolics
To analyze the total phenolic content (TPC), Kim et al. (2003)
Yehia et al. 3665
method was followed to make the use of Folin Ciocalteu reagent.
To 0.4 ml of the extract (prepared in methanol with a concentration
of 1.0 mg/ml), 1.0 ml of (10%) Folin-Ciocalteu reagent was mixed
and solution was allowed to stand at 25°C for 5 to 8 min before
adding 0.8 ml of 7.5% sodium carbonate solution and using
deionized water, final volume was made to 10.0 ml. After two hours,
absorbance was measured at 765 nm. Calibration curve was
prepared using gallic acid as standard for TPC which was
measured as mg gallic acid equivalents (GAE) per gram of the
Antimicrobial activity of PRE/metal salt combinations
A sample of the PRE was preserved in refrigerator at 4°C until
used. An aliquot of PRE (330 µl) was added to (700 µl) solutions
(4.8 mM) of each metal salts used (CuSO4, MnSO4 and ZnSO4) and
the final solution was protected from light (Stewart et al., 1998).
Antimicrobial activity of PRE/metal salt combinations plus
The assay was carried out as described above with the following
addition: A sample of the PRE was added to vitamin C (1:1),
sample of the PRE (330 µl) was added to (330 µl) of vitamin C and
to the metal salt 330 µl of (CuSO4, MnSO4 and ZnSO4) solution
immediately prior to mixing.
Overnight cultures of the Gram positive strains Staphylococcus
spp., B. subtilis, Bacillus indicus and the Gram negative strains E.
coli, Enterobacter aerogenes, Serratia marcescens and Brucella
spp. were prepared on nutrient agar plates (Oxoid Ltd, UK). All
bacterial isolates were suspended in Ringer's solution (Oxoid Ltd,
UK) to a turbidity equivalent to 0.5 McFarland (1.5 × 108 CFU/ml)
and 100 µl were spread onto Mueller-Hinton agar plates (Oxoid Ltd,
UK). Also yeasts as Saccharomyces cerevisiae and Rhodotorula
glutinis and Geotrichum spp. The PRE (10 µl) was then spotted
onto sterile Whatman no 1 filter paper discs (5 mm diameter) placed
centrally on the plates which were incubated at 37°C for 24 h prior
to recording zones of inhibition (McCarrell et al., 2008).
RESULTS AND DISCUSSION
Pomegranate is an important source of anthocyanins,
hydrolysable tannins punicalagin and punicalin (Afaq et
al., 2005), ellagic and gallic acids (Lansky and Newman,
2007) and also contains vitamin C (Turk et al., 2008). The
antioxidant and free radical scavenging activity of
phenolic compounds derived
(Rosenblat et al., 2006) and vitamin C (Sonmez et al.,
2005) have been reported (Abdel Moneim, 2011). The
PRE gave positive tests for gallic tannins while the
extract gave negative result for catechol tannins as
shown in Table 1. The reducing power (RP) and the total
phenolic (TPC) and flavonoids (TFC) contents in
pomegranate rind extracts are shown in Table 2. The
direct correlation between antioxidant activity and
reducing power of certain plant extracts were reported.
The presence of reductants (antioxidants) in the extracts
3666 Afr. J. Microbiol. Res.
Table 1. Quantitative analysis of tannins and its type in the rind
extract of pomegranate.
Table 2. Reducing power (%/ g sample), total phenolic (mg GAE/g
sample) and flavonoids (mg QE/ g sample) contents in the rind
extract of pomegranate.
Reducing power (RP)
Total phenolic content (TPC)
Total flavonoids contents (TFC)
a Values expressed are the mean of three replications.
would result in the reduction of iron (III) to iron (II). PRE
showed 14.65% of activity at 1 g sample/ml. The result
indicates that PRE contained electron donors and
possessed the ability to reduce iron (III) to iron (II).
It was well-known that plant phenolics and flavonoids
are highly effective free radical scavengers and antioxi-
dants. P. granatum contained high amounts of phenolic
and flavonoids compounds. It was shown that PRE
contained phenolic and flavonoids compounds at 104.68
mg GAE/g and 47.27 mg QE/g, respectively (Table 2).
This result indicates that the potent antioxidant activity of
PRE may be related to the phenolic and flavonoids
compounds in the extract.
It has been reported that oxidizing biological material
leads to a rapid burst of ROS, such as superoxide (O2– •),
hydrogen peroxide (H2O2) and hydroxyl (•OH) generated
primarily because of the ionizing of water molecules
(Agrawal et al., 2001), which then interact with biological
target molecules, causing lipid peroxidation and DNA
damage, and subsequently resulting in cell killing and
mutations (Abdel Moneim et al., 2011).
Plants, vegetables, herbs and spices used in folk and
traditional medicine have been accepted currently as one
of the main sources of chemo preventive drug discovery
and development (Aruoma, 2003). It has been observed
that many plant polyphenols, such as ellagic acid,
catechins, and chlorogenic, caffeic and ferulic acids act
as potent antioxidant, antimutagenic and anticarcinogenic
agents (Ayrton et al., 1992).
Data in Table 3 indicates antimicrobial activity of the
different mixture of PRE, vitamin C and metals after
autoclaving as separated and mixed components where
the PRE/ vitamin C (1/1), exhibited antimicrobial activity
against Gram positive (B. indicus) and Gram negative (E.
coli) organisms reached 18 and 20 mm respectively.
While mixing PRE/ZnSO4 showed antimicrobial activity
against B. subtilis, Staphylococcus spp. and Brucella spp.
and were 15, 15 and 15 mm respectively. PRE/MnSO4
affected only on the yeast Saccharomyces cerevisiae.
Moderate antimicrobial activity was seen with the mixing
of PRE/vitamin C /salts metals on the different micro-
organisms. Results indicated that PRE/vitamin C/ MnSO4,
just effected on S. cerevisaie and the zone of inhibition
was 10 mm whilst PRE/vitamin C/ CuSO4 effected on
Brucella spp. and gave 10 mm zone of inhibition.
PRE/vitamin C/ ZnSO4 were not effected on any of the
tested strains. The PRE alone did not exhibit anti-
microbial activity against any of the isolates. Variations in
results between studies on pomegranate extracts are not
only seen in disc diffusion assays, but have also been
recorded with minimum inhibition concentration (MIC)
No detectable effect of any of the extraction mixture on
the growth of Enterobacter aerogenes and the yeast
Rhodotorula glutinis. McCarrell et al. (2008) prepared
ethanol extractions of a number of plants and tested
these against a range of laboratory and clinical isolates.
Interestingly, this group only reported antimicrobial
activity for pomegranate extracts against laboratory
strains of P. aeruginosa and B. subtilis. In their test, B.
subtilis produced a zone of clearing equal to or greater
than 7 mm. These differences may in part be due to the
different extraction methods employed, potentially the
freshness of the fruit used, and variations in the season
and region of growth.
Melendez and Capriles (2006) tested the antimicrobial
properties of a number of tropical plants from Puerto Rico
using the disc diffusion method against E. coli and S.
aureus. They demonstrated that pomegranate extract
produced inhibition zone sizes of 11 and 20 mm, for E.
coli and S. aureus respectively. Thus, their results
contrast to the present study in that a smaller zone of
inhibition for S. aureus was observed along with
antimicrobial activity against E. coli. In the present work
the value of MIC was 0.5 mg/l against all positive results
that was mentioned in Table 2. Values for MIC have been
reported in a number of studies, ranging from 0.62 to 10
mg/ml against S. aureus, E. coli and P. aeruginosa
(Navarro et al., 1996) and up to 250 mg/L against S.
aureus (Machado et al., 2003). These differences could
be due to the extraction method, freshness of the fruit,
season and region of growth (McCarrell et al., 2008).
Combinations of PRE with metal salt ZnSO4 exhibit
enhanced antimicrobial effects against Bacillus subtilis,
Staphylococcus spp. and Brucella spp. Mixture of PRE/
Vitamin C (1:1) exhibited higher zone of inhibition against
E. coli and B. indicus in comparison to the three mixtures.
Further work is underway to establish the mode of action,
along with the mechanism of enhancement by metal salts
Yehia et al. 3667
Table 3. Diameter of the zones of inhibition (mm) of the autoclaved pomegranate rind extract compared to penicillin and the results.
PRE/Vitamin C and PRE/Vitamin C /metal salts
0: No effect on the growth of the microorganisms.
and vitamin C.
This project was supported by King Saud
University, Deanship of Scientific Research,
College of Food and Agriculture Sciences,
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