Industrial Crops and Products 34 (2011) 1523–1527

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Industrial Crops and Products

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High potential of agro-industrial by-products of pomegranate (Punica granatum

L.) as the powerful antifungal and antioxidant substances
Ali Tehranifar a,∗ , Yahya Selahvarzi a , Mahdiyeh Kharrazi a , Vahid Jahan Bakhsh b
a
Center of Pomegranate Research, Department of Horticultural Science, Faculty of Agriculture, Ferdowsi University of Mashhad, Mashhad, Iran
b
Department of Plant Protection, Faculty of Agriculture, Ferdowsi University of Mashhad, Mashhad, Iran

a r t i c l e i n f o a b s t r a c t

Article history: Microorganisms such as fungi are one of the most important factors that cause oxidative processes during
Received 7 January 2011 postharvest stage and consequently deterioration of agriculture products would not be unexpected. On
Received in revised form 26 April 2011 the other hand, high antioxidant properties of industrial by-products of pomegranate propose them
Accepted 10 May 2011
as powerful antioxidant and antifungal substances. So to investigate the antioxidant and antifungal
Available online 8 June 2011
properties of pomegranate, two independent factorial experiments based on randomized design with
5 replications were conducted. In the first experiment the effect of 3 different parts of pomegranate
Keywords:
(peel, seed and leaf) and 2 different kinds of extracts (aqueous and methanolic) with 4 concentrations
Pomegranate
Methanolic extract
(0, 500, 1000 and 1500 ppm) were investigated on 3 postharvest fungi (Penicillium italicum, Rhizopus
Antifungal activity stolonifer and Botrytis cinerea). In the second experiment antioxidant capacity and phenolic content were
Phenolic content measured for two different extracts from different parts. Based on the results the methanolic extract
Antioxidant activity showed the highest inhibitory effects on the mycelia growth (IMG) and spore germination (ISG) with
49.82 and 41.25% respectively. On the other hand, peel and seed extracts had more inhibitory effect (IMG
and ISG) than leaf extract. The phenolic content of peel extract were also measured 2.8 fold higher than
pomegranate leaf extract and antioxidant capacity of peel, seed and leaf extracts of pomegranate were
55.3%, 35.7% and 16.4% respectively. Therefore, it seems that the high percentage of phenolic content
in the peel and seed of pomegranate could cause the high antifungal and antioxidant activity of their
extracts.
© 2011 Elsevier B.V. All rights reserved.

1. Introduction Antioxidants have an important role in body’s health. Recently,

Pomegranate (Punica granatum L.) is a deciduous shrub that
is native to Iran (Sarkhosh et al., 2007). By producing around
670,000 tons annually, Iran is the largest producer of this edible
fruit (Anonymous, 2005). Because of the high antimicrobial activ-
ity against many pathogens, pomegranate has been widely used for
the treatment of different types of human disease.
On the other hand, during the industrial processing of
pomegranate, large volumes of industrial wastes are produced,
which have a wide range of nutritional values. Therefore, in the
recent years, the attention has been focused on the industrial by-
products of pomegranate that have a high potential of antioxidant
and antifungal properties (Orzuaa et al., 2009). Furthermore, the
presence of abundant effective compounds has been reported in
many parts of this plant such as leaves, barks, roots, peels, juice
and seeds that cause high antimicrobial and antioxidant activity of
them (Seeram et al., 2006).
∗ Corresponding author. Tel.: +98 915 1107389; fax: +98 511 8787430.
E-mail address: tehranifar2009@yahoo.com (A. Tehranifar).
a series of studies had demonstrated that there is a positive cor-
relation between prevention of many cancers and the amounts of
absorption and consumption of vegetables and fruits that contain
natural antioxidants (Casanova et al., 2008).
Ellagic acid is one of the most important compounds in peel of
pomegranate. The phenolic structure of this compound causes its
drastic antioxidant activity. Although pomegranate is used as an
edible fruit, in recent years, medicinal properties of this fruit have
been noticed by many investigators (Sarkhosh et al., 2007). Pheno-
lic compounds especially punicalagin extracted from the peels of
pomegranate have antimicrobial activity against Candida albicans
(Burapadaja and Bunchoo, 1995). According to the type of tested
microorganism, the reports about antifungal properties of the
pomegranate peel extract are various. For example, this extract can
prevent the growth of Penicillum citrinum and Aspergillus ochraceus
for 8 and 3 days respectively. In some cases in China the peel extract
of pomegranate is used as a fungicide (Seeram et al., 2006).
Nowadays, many people in Asia and Africa who are under the
poverty line have to tolerate the malefic effects of fungi (Majumder
et al., 1997). On the other hand, for controlling fungi, the most
popular way is using synthetic chemicals and fungicides. But their

0926-6690/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.indcrop.2011.05.007
1524 A. Tehranifar et al. / Industrial Crops and Products 34 (2011) 1523–1527
high accumulation capacity in bodies of consumers, long durabil-
ity period and many environmental disadvantages of them lead
to hesitation for using of them (Barnard et al., 1997). Chang et al.
(2008) mentioned that application of synthetic chemicals leads to
creation of resistant strains. So during two recent decades, applica-
tions of some natural compounds such as essential oils and extracts
for biological control of pathogens have been noticed.
Despite several previous studies on the antimicrobial proper-
ties of pomegranate against many human’s disease, to date there
is no any research about its antifungal activity against postharvest
fungi, while it has been determined that the huge amounts of dete-
rioration of agricultural products occur during storage because of
oxidative processes and microorganisms attack (Casanova et al.,
2008). So, the aim of the present study is to investigate the anti-
fungal properties of aqueous and methanolic extracts of industrial
wastes of pomegranate and to determine the phenolic content and
antioxidant activity of this extracts for introducing them as a safe
products.
2. Material and methods
2.1. Preparation of plant materials and fungi isolates
Pomegranate cultivar “shishe kab” that is cultured in Iran as
a commercial cultivar, was selected for this investigation. Fresh
peels, leaves and seeds of this cultivar were collected from Ferdows,
Khorasan, Iran. The parts were dried in the shade and after-
ward powdered. Three common plant pathogenic fungi (Penicillium
italicum, Botrytis cinerea and Rhizopus stolonifer) were selected from
Center of Pomegranate Research of Ferdowsi University of Mash-
had, Iran in 2009.
2.2. Preparation of the extracts
In this research aqueous and methanolic extracts were used.
Powdered plant materials (2 g) of peel, seed and leaves of
pomegranate were macerated with 20 ml solvent (distilled water
or methanol) for 24 h at room temperature. The macerated was
first filtered through double layered muslin cloth and then spun
at 3000 rpm for 15 min. The extracts were concentrated by using
Rotary Evaporator at 40 ◦ C and afterward dried in oven at 50 ◦ C for
48 h. Finally, by using these dried extracts, the different concentra-
tions of aqueous and methanolic extracts (500, 1000, 1500 ppm)
were prepared (Fernandes et al., 1997).
2.3. Antifungal activity against the mycelia growth
After sterilization the PDA (potato dextrose agar) medium, it
was cooled in a water bath to 40 ◦ C. The aqueous and methano-
lic extracts were mixed with sterile molten PDA for obtaining the
final concentrations (0, 500, 1000 and 1500 ppm). 15 ml of each
medium (contains different concentrations of extracts) was poured
into 90 mm Petri plates and then were inoculated with 5 mm plugs
from 7-day-old cultures. Five replicates were used per treatment.
Plates were incubated at 28 ± 1 ◦ C in 12 h light/12 h dark cycle.
Growth inhibition of treatment against control was calculated by
using the following formula:
a − b
Inhibitory of mycelia growth (%) = × 100
a (30.92%) (Table 1). As shown in Fig. 1, the lowest amount of ISG
where a and b represented the average increase in mycelium
growth of control and average increase in mycelium growth of
treatment, respectively (Satish et al., 2007).
2.4. Antifungal activity against the spore germination
Spore suspension of plant pathogens were obtained from their
respective 7-day-old cultures and mixed with sterile distilled water
to obtain the homogenous spore suspension of 2 × 106 spore/ml.
500 ␮l of spore suspension of each fungi was added to Petri plates
that contained different concentrations of extracts (0, 500, 1000
and 1500 ppm) and spread with sterile lob. After 20 h, spore germi-
nation was counted around a limited area (2 cm diameter) under
the microscope (Bajpai et al., 2007).
a − b
Inhibitory of spore germination (%) = × 100
a
where a and b represented the average of spore germination in
control and treatments, respectively.
2.5. Total phenols and antioxidant activity evaluation
Determination of the total phenols was done by applying
the Folin–ciocalteu regent using a UV–vis spectrophotometer at
660 nm (Singleton and Rossi, 1965).Gallic acid used as standard
absorbance read. Results were expressed as milligram (mg) gallic
acid equivalents per gram dry weight (Mousavinejad et al., 2009).
DPPH method was used to measure the antioxidant activity of
pomegranate extracts based on the evaluation of the free radi-
cal scavenging capacities of the extracts (Gil and Tomas, 2000).
In this method 0.1 ml of methanolic extract of each plant sample
was combined with DPPH (500 ␮mol in methanol). The mixture
was strongly shaken and after 30 min the change in absorbance at
517 nm was measured. Finally, calculation of antioxidant activity
was done by using the following formula:
 
Asample (517 nm)
Antioxidant activity = 1− × 100
Acontrol (517 nm)
Antioxidant activity of extracts was compared with the activity
of butylated hydroxyanisole (BHA) and l-ascorbic acid.
2.6. Experimental design and statistical analysis
In this study the effect of aqueous and methanolic extracts of
3 different parts of pomegranate (peel, seed and leaf) and with 4
concentrations (0, 500, 1000 and 1500 ppm) were investigated on 3
postharvest fungi (P. italicum, R. stolonifer and B. cinerea) in one fac-
torial experiment and the antioxidant activity and phenolic content
of these two extract methods from different parts of pomegranate
(one concentration) were evaluated in another experiment. Both
experiments conducted in five replications. The analysis of the data
was conducted by Statistical Analysis System (SAS) software Ver-
sion 9.1 and Mean values and standard error (SE) were calculated
for all tests.
3. Results
Results showed that the effect of plant material type, extraction
method, type of fungi and extract concentration on the inhibitory
of mycelia growth (IMG) and inhibitory of spore germination (ISG)
were significant (Table 1). Leaf extract of pomegranate had the least
effect on ISG and there were no significant differences between
seed and peel extract. Among the fungi, P. italicum has the least
ISG (17.86%) compared with R. stolonifer (30.29%) and B. cinerea
was observed in the treatment of leaf extract for P. italicum.
The effect of methanolic extract on IMG was 90.8% higher than
the aqueous extract (Table 1). In addition, there was no significant
difference between R. stolonifer and B. cinerea. The percentage of
 A. Tehranifar et al. / Industrial Crops and Products 34 (2011) 1523–1527 1525

Methanolic extract

49.82 ± 2.43
41.25 ± 2.19
Extraction method

Aqueous extract

26.10 ± 1.55
11.73 ± 1.10
61.78 ± 2.22
45.00 ± 2.84

Fig. 1. Interaction effect of extracts from different parts of pomegranate and fungi
1500

species on inhibitory spore germination (ISG).

49.20 ± 2.26
33.86 ± 2.62

IMG in P. italicum was the lowest amount (28.46%) in comparison

Concentration of extract (ppm)

to two other types of fungi (Table 1). According to the results given
in Fig. 2, the highest amount of IMG was observed in methanolic
1000
Extraction method, concentration, plant material type and fungi spices effect on inhibitory mycelia growth (IMG) and inhibitory spore germination (ISG).

extract for R. stolonifer and B. cinerea (55%), whereas the lowest

amount of it was obtained in aqueous extract for P. italicum (18.5%).
27.05 ± 2.55
40.99 ± 2.30

By investigating the methanolic and aqueous extracts of differ-

ent parts of pomegranate in various concentrations, we concluded
that 1500 ppm methanolic extract of pomegranate seed and peel
500

can better prevent the mycelia growth. On the other hand, the main
inhibitory effects of extracts, especially the methanolic extract,
0

0
0

were observed at 0–500 ppm whereas at higher concentrations

32.16 ± 2.18
17.13 ± 1.57

(1000–1500 ppm), the slop of their inhibitory effect reduced (Fig. 3).
As shown in Table 2, the antioxidant activity of peel extract was
54% and 237% higher than seed and leaf extracts. In addition, the
Leaf

phenolic content of peel, seed and leaf extracts was 302, 325 and
116 mg/g respectively (Table 2).
43.00 ± 2.91
32.25 ± 2.74

The interaction between plant material type and solvent type on

antioxidant activity and phenolic content was significant (p < 0.01).
Peel
Plant material type

Aqueous extract of pomegranate leaves was the lowest amount

of this trait, while methanolic extract of pomegranate peel had
29.93 ± 2.81
38.60 ± 2.91

the highest antioxidant activity (85.4%) and phenolic content

(423.5 mg/g) in comparison with other extracts (Table 2).
Seed

4. Discussion
Rhizopus stolonifer

Consistent with our results, many researchers have confirmed

42.29 ± 2.85
30.29 ± 2.58

the superiority of antifungal properties of methanolic extracts

Botrytis cinerea

42.77 ± 2.85
30.92 ± 2.64

Values are the mean of five replicates ± SE.

Penicillium italicum
a
28.46 ± 2.14
17.86 ± 2.09
Fungi
Inhibitory effect
Table 1

IMG
ISG
a

Fig. 2. Interaction effect of extraction method and fungi species on inhibitory

mycelia growth (IMG).
1526 A. Tehranifar et al. / Industrial Crops and Products 34 (2011) 1523–1527
Table 2
Antioxidant activity and phenolic content of aqueous and methanolic extracts of different parts of pomegranate.
Seed Peel
Methanolic Aqueous Methanolic
Antioxidant 51.7 ± 2.4a 19.6 ± 1.3 85.4 ± 3.1
activity (%)
Mean 35.7 ± 3.4 55.3 ± 4.2
Phenolic content 384.7 ± 24.2 219.8 ± 16.6 423.5 ± 31.8
(mg/g)
Mean 3023.3 ± 15.1 325.23 ± 20.0
a
Values are the mean of five replicates ± SE.
of different plants compared with other solvents of them (Choi
et al., 2008; Marjorie, 1999). Alcoholic extract of pomegranate
peel has the drastic antimicrobial properties against Escherichia coli
and it has been mentioned that the most important contents of
this extract are flavonoides, steroles, triterpens, phenols and tan-
nins (Supayang et al., 2005). Doughari and Obidah (2008) found
that the bark methanolic extract of Leptadenia lancifollia can com-
pletely prevent the spore germination of many fungi, while aqueous
extract of this plant had no significant effects on spore germina-
tion of fungi. In fact, the differences between solvents are related
to the polarity and solubility of them. Consequently, it seems that
methanolic extract can draw out more compounds from plant cells
compared with other extracts. So this method has more effective
role in prevention of microbial growth.
Studies show that there are some compounds in alcoholic
extract of pomegranate that have antifungal, antibacterial and even
antiviral activity (Ahmad and Beg, 2001). In addition, in vivo studies
show the inhibitory effects of pomegranate extracts on the growth
of Candida albicans (Vasconcelos et al., 2003). In another research,
it has been demonstrated that the different parts of pomegranate
including fresh fruit and sterile juice can have the inhibitory effect
on another species of this fungi (Candida mycoderma) (Seeram et al.,
2006). So the results of previous reports along with our findings
can confirm the inhibitory effects of different parts of pomegranate
extracts on growth and development of fungi.
An antimicrobial activity of pomegranate extract is probably
due to the presence of large scale of antibiotic compounds (Seeram
et al., 2006). In most investigated plants, phenols and tannins are
Fig. 3. Interaction effect of extraction method, concentration and plant part of
pomegranate on IMG.
Leaf Standard
Aqueous Methanolic Aqueous Butylated l-Ascorbic acid
hydroxyanisole
25.2 ± 2.0 22.1 ± 1.9 10.7 ± 1.4 57.6 ± 1.9 22.7 ± .9
16.4 ± 2.4 –
226.9 ± 13.5 133.3 ± 8.7 98.6 ± 12.7 – –
116.1 ± 9.7 –
the most important compounds in this field (Seeram et al., 2006).
Nasr et al. (1996) reported that different parts of pomegranate plant
such as peels and leaves contain phenolic compounds that lead to
antimicrobial activity of their extracts. In fact, phenolic materials
along with high-molecular weight proteins constitute a complex.
Thus they can react with the cellular enzymes (oxidoreductase)
that exist in cytoplasm and cell wall. Furthermore, these materials
can inhibit the access of cellular receptors against microorganisms
(Cowan, 1999).
By investigating the phenolic content and antioxidant capacity
of extracts shown in Table 2, it is clearly obvious that the pres-
ence of the phenolic compounds in this extract is an important
factor in creating the antioxidant activity of them. In other words,
the more phenolic content causes the more antioxidant activity.
Kim et al. (2002) introduced the peel of pomegranate as a rich
source of antioxidants and phenolic materials. Undoubtedly, the
antioxidant capacity of pomegranate is related to the presence of
phenolic materials, especially elagic acid and punicalagin (Sarkhosh
et al., 2007). These compounds that are mostly found in the fruit
peels cause the anti-mutation, antiviral, antimicrobial and antiox-
idant activity of different parts of pomegranate extracts (Zargari,
1996). However, according to the results of this study, it is dis-
tinguished that the highest and lowest antioxidant activity was
obtained in the methanolic and aqueous extracts of pomegranate
peel, respectively. This phenomenon is due to the presence of phe-
nols and the capacity of them in freeing radicals scavenging. These
results are consistent with the data reported by Negi et al. (2003).
They compared the antioxidant activity of aqueous, methanolic,
acetone and ethyl acetate extracts of pomegranate peel and found
that methanolic extract has the highest antioxidant capacity, while
Li et al. (2006) reported that the mixture extract of aqueous and
methanolic solvent is more effective than either of them alone.
5. Conclusion
From the present study, it can be concluded that the level of phe-
nolic content of different parts of pomegranate extracts, especially
peel and seed extracts are so high. These compounds can cause anti-
fungal activity of these extracts. In addition, by their free radical
scavenging capacities, they can lead to a high antioxidant activ-
ity. As a consequence, we can consider peel and seed extracts of
the pomegranate, which are the agro-industrial waste of this fruit,
as a postharvest fungi controlling, because of their antifungal and
antioxidant properties.
Acknowledgment
The authors are grateful to Ferdowsi University of Mashhad, Iran
for providing financial support and Center of Pomegranate Research
of Ferdowsi University of Mashhad for supplying facilities.
 A. Tehranifar et al. / Industrial Crops and Products 34 (2011) 1523–1527
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