Phytochemical screening and antibacterial activity

of different parts of the Prosopis farcta extracts against

methicillin-resistant Staphylococcus aureus (MRSA)

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J. SHARIFI-RAD 1, 2, S. M. HOSEINI-ALFATEMI 3*, M. SHARIFI-RAD 4, A. MIRI 1, 2, 5

The present study investigated the phytochemical

screening and antibacterial potentiality of different
parts (roots, leaf, pods and seeds) of the Prosopis farcta extracts against Methicillin-resistant Staphylococcus aureus (MRSA). Phytochemical screening of the
P. farcta methanolic extract was carried out for estimation of saponins, glycosides, alkaloids, tannins and
flavonoids by using standard phytochemical screening
methods. The antibacterial activity was determined by
both the disc diffusion method and the microbroth dilution method. Antibacterial screening of P. farcta extracts showed that inhibited zones for roots, leaf, pods
and seeds extract were 50.1, 60.4, 806 and 120.1
mm, respectively. MIC for roots, leaf, pods and seeds
extract were 450.4, 35.50.8, 150.1 and 50.4 g/
mL, respectively. MBC for roots, leaf, pods and seeds
extract were 1000.4, 750.3, 250.5, and 50.2 g/
mL, respectively. In this study, MIC range for Vancomycin was between 0.4-4 g/mL for all strains isolated
from various clinical samples. All P. farcta extracts exhibited different degrees of antibacterial activity on
MRSA; this difference is mainly due to the presence of
different components in these parts. The seeds extract
showed more effect than other parts of the plant. This
effect may be related to high concentrations of alkaloids, tannins or glycosides in this part. These components are known to high antibacterial activity. P. farcta extracts possess potent antibacterial against MRSA,
and may be used as an antibacterial to treat diseases.
Key words: Anti-bacterial agents - Prosopis - Methicillin-resistant Staphylococcus aureus - Alkaloids - Tannins - Glycosides.

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means which may allow access to the Article. The use of all or any part of the Article for any Commercial Use is not permitted. The creation of derivative works from the Article is not permitted. The production of reprints for personal or commercial use is
not permitted. It is not permitted to remove, cover, overlay, obscure, block, or change any copyright notices or terms of use which the Publisher may post on the Article. It is not permitted to frame or use framing techniques to enclose any trademark, logo,
or other proprietary information of the Publisher.

MINERVA BIOTEC 2014;26:287-93

he increasing use of chemical drugs causing bacterial resistance to the drugs caused side effects

Corresponding author: S. M. Hoseini-Alfatemi, Pediatric Infections

Research Center, Mofid Children Hospital, Shahid Beheshti University
of Medical Sciences, Tehran, Iran. E-mail: m.hoseinialfatemi@gmail.com

Vol. 26 - No. 4

1ZabolMedicinalPlantsResearchCenter
ZabolUniversityofMedicalSciences, Zabol, Iran
2DepartmentofPharmacognosy, FacultyofPharmacy
ZabolUniversityofMedicalSciences, Zabol, Iran
3Pediatric Infections Research Center
Mofid Children Hospital, Shahid Beheshti
University of Medical Sciences, Tehran, Iran
4DepartmentofRangeandWatershedManagement
FacultyofNaturalResources, UniversityofZabol, Iran
5Zabol University of Medical Sciences, Zabol, Iran

more dangerous than the disease itself. Nowadays,

due to the deformation resistance of bacteria becoming resistant to commonly used antibiotics, tendency
to replace them with new antibiotics has increased.1
In spite of the progress of science and the development of synthetic drugs, medicinal plants are still
used in large-scale and traditional medicine.2-7 In recent years, extensive study was carried out to evaluate the antimicrobial effect of plant essential oils and
extracts. These results showed the potency of these
compounds in inhibiting the growth of a wide range
of pathogenic microorganisms.
The genus Prosopis belongs to the Leguminosea
family (subfamily Mimosoidae). It includes almost
50 species, whose 25 are on the list of federal harmful weeds. The Prosopis farcta is a little prickly spiny
shrub or tree. Its native of range land, it is widespread and a weed of cotton fields and wheat, infringed by root suckers. It is native of United States,
northern Africa, south western Asia, Kuwait, Turkey,
Iraq and Iran. It grows in southern Iran in the Sistan
and Baluchestan, Hormozgan, Bushehr, Khuzestan
and southern Fars provinces.8 The antimicrobial ac-

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tivity of extracts from Prosopis spp. was examined

and the results showed antibacterial activity against
some gram-positive and negative bacteria, antidiarrheic, antiparasitic and antifungal properties.9, 10
The bacterial resistance to drugs has become a
major problem worldwide. One of these bacteria
that have been resistant to drugs is Staphylococcus
aureus (S. aureus). S. aureus is one of the most consequential human pathogen responsible for nosocomial, hospital- and community-acquired infections.
It can bring about a range of infectious diseases
from moderate conditions, such as soft tissue infections, to serious life-threatening debilitation like endocarditis, to death.11, 12 Methicillin-resistant strains
of staphylococci were recognized shortly upon the
preface of methicillin into clinical train. The first outbreak caused by Methicillin-resistant Staphylococcus
aureus (MRSA) happened in an English hospital in
the early 1960s and appeared in the United States
in the 1980s.12 In recently years, MRSA strains have
often become resistant to multiple other antimicrobial agents and many researcher have reported an
intensification in the incidence of MRSA. On the
basis of some investigations on phytochemical and
antibacterial properties of different parts extracts of
the P. farcta against MRSA, it can be asserted that the
importance of traditional medicine and medicinal
plants with very small side effects on human can be
useful for discovering natural extracts to that reduce
the growth of these bacteria.

Figure 1.Prosopis farcta plants used in this study.

These working solutions were used for all the tests

in this study.
Preliminary phytochemical analysis

Phytochemical screening for major constituents

include saponins, glycoside, flavonoids, tannins, alkaloids, resin and phenols was undertaken by using
standard qualitative methods as described by Fadeyi
et al.,14 Odebiyi and Sofowora,15 Harborne,16 and
Abulude.17
Bacterial isolates and antibiotic susceptibility assay

Materials and methods

Plant preparation and extracts

The Prosopis farcta were collected during the mature period, May 2013, from the area surrounding
Hamun Lake, Zabol (Coordinates: 31 1 43 N, 61
30 4 E), in Sistan and Baluchestan Province of Iran
(Figure 1). The plant was taxonomically identified
by the Ferdowsi University Mashhad Herbarium,
Iran (Voucher No: 25685). Twenty grams of each
parts viz. roots, leaf, pods and seeds were powdered
separately and then dissolved in 200 mL methanol
85% using a shaker water bath for 24 hours at 25
C. After filtration with Whatman No. 1 filter paper,
the resulting solutions were concentrated by a rotary
evaporator at 40 C for 35 min to remove solvents
from the extracts. Solid extracts (residues of plant
extracts) were dissolved in 20 mL of distilled water.

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(either sporadically or systematically, either printed or electronic) of the Article for any purpose. It is not permitted to distribute the electronic copy of the article through online internet and/or intranet file sharing systems, electronic mailing or any other
means which may allow access to the Article. The use of all or any part of the Article for any Commercial Use is not permitted. The creation of derivative works from the Article is not permitted. The production of reprints for personal or commercial use is
not permitted. It is not permitted to remove, cover, overlay, obscure, block, or change any copyright notices or terms of use which the Publisher may post on the Article. It is not permitted to frame or use framing techniques to enclose any trademark, logo,
or other proprietary information of the Publisher.

PROSOPIS FARCTA AGAINST STAPHYLOCOCCUS AUREUS

SHARIFI-RAD

288

Four hundred and seventy-seven clinical specimens such as burn, wound, urine, pus and throat
swab were collected from patients attended in emergency Hospital and Internal Laboratory of Hospital
and Central Laboratory in Zabol city (Iran) for different bacteriological examinations (Table I). Standard
isolation protocols were used for all the samples.
Identification of S. aureus was approved by standard techniques accord on colonial and microscopic
morphology, coagulase test and biochemical activities.17 Detection of MRSA was done by the use of
Cefoxitin disc (30 g) diffusion test. The strains of
S. aureus with a zone diameter of <19 mm based
on Clinical and Laboratory Standard Institute (CLSI)
were taken to show methicillin-resistance.19 All
strains of S. aureus were confirmed as methicillin
resistant by oxacillin agar dilution using Muller Hinton agar (beef extract, acid hydrolysate of casein
17.5g/L, starch 1.5 g/L, agar 17.0 g/L, final 7.30.1

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Table I.The number and percentage of isolated MRSA from various clinical specimens.
Type of specimens

Number of specimens

Burn
Wound
Urine
Pus
Throat
Total

104
45
254
14
60
477

S. aureus
N.

53
38
11
4
1
107

MRSA
%

N.

50.69
84.44
4.33
28.57
1.66
22.43

9
4
4
0
1
18

16.98
10.52
36.36
0.00
100
16.82

terial growth. Bacterial cells from the extracts MIC

test plate were sub-cultured on solid nutrient agar
by making streaks on the surface of the agar. The
plates were incubated overnight at 37 C and the
minimum bactericidal concentration (MBCs) were
determined after 24 h. The plates that did not show
growth were measured to be the MBC for the extract
used. The experiment was performed in triplicate.
All strains were assayed for MIC by vancomycin agar
dilution method using Muller Hinton agar. The concentrations assayed ranged from 1 g/mL to 33 g/
mL of vancomycin.

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at 25 C) supplemented with 2% NaCl. The concentrations tested ranged from 1 g/mL to 16 g/mL
of oxacillin. The strains having minimum inhibitory
concentration (MIC) 4 g/mL were taken as resistant. Among 477 clinical specimens, of the 107 specimens (22.43%) were S. aureus that from these 18
specimens (16.82%) were MRSA.
Disc diffusion method

Antimicrobial activity was based on the disc diffusion method 20 using a cell suspension of microorganisms. The concentration of the cell suspension
was equilibrated to a 0.5 McFarland standard and 50
L of each microorganisms suspension was spread
on a Mueller-Hinton agar plate. In addition, 50 L of
diluted methanolic extracts was pipetted onto sterile
paper discs (6 mm in diameter), which were dried
in an open sterile Petri dish in a biological laminar
flow bench. Discs were placed on the surface of
inoculated plates and incubated at 37 C for 24 h.
Diameters (mm) of the zones of bacterial inhibition
minus the discs diameter were recorded.21

Statistical analysis

The extracts were prepared in triplicate for

chemical characterization and antibacterial assays.
Data was subjected to analysis of variance following a completely random design to determine the
least significant difference (LSD) at P<0.05 using
SPSS vs. 11.5 (IBM SPSS, New York, USA). In this
study values for antibacterial assays were based on
meanSE.

Determination of MIC and minimum bactericidal

concentration

The MIC values for each plant extracts were determined by microbroth method. Residues of plant extracts were dissolved in 20 mL of distilled water. All
extracts are tested at 1000 g/mL 22 and serially diluted two-fold to 1.95 g/mL in a 96-multiwell polystyrene flat-bottomed microplate (Sigma-Aldrich, St.
Louis, MO, USA) after which 100 L (1106 CFU/mL)
of bacteria are added to each of them. Preincubation
absorbance values were read from an ELISA reader.
The microplates were then incubated overnight at
37 C and absorbance values were read after 24 h.
The MIC values are recorded as the lowest concentration of the extract that completely inhibited bac-

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(either sporadically or systematically, either printed or electronic) of the Article for any purpose. It is not permitted to distribute the electronic copy of the article through online internet and/or intranet file sharing systems, electronic mailing or any other
means which may allow access to the Article. The use of all or any part of the Article for any Commercial Use is not permitted. The creation of derivative works from the Article is not permitted. The production of reprints for personal or commercial use is
not permitted. It is not permitted to remove, cover, overlay, obscure, block, or change any copyright notices or terms of use which the Publisher may post on the Article. It is not permitted to frame or use framing techniques to enclose any trademark, logo,
or other proprietary information of the Publisher.

PROSOPIS FARCTA AGAINST STAPHYLOCOCCUS AUREUS

Vol. 26 - No. 4

Results

Preliminary phytochemical analysis

The results of preliminary phytochemical analysis
of different parts (roots, leaf, pods and seeds) of the
P. farcta showed that roots methanolic extract demonstrated high concentrations of flavonoids, saponins,
phenols and moderate concentrations of alkaloids,
tannins, glycosides and resins. The leaf and pods
methanolic extract showed a high concentration of
saponins and flavonoids and moderate concentrations
of alkaloids, resins, glycosides, tannins and phenols.
Seeds methanolic extract presented high concentrations of alkaloids, tannins, glycosides and moderate

MINERVA BIOTECNOLOGICA

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Table II.Results of phytochemical analysis of extracts from root, leaf, pod and seed of the P. farcta.
Methanolic extracts
Chemical Compounds

Saponins
Tannins
Flavanoids
Glycoside
Alkaloids
Phenols
Resin

Root

Leaf

Pod

Seed

++
+
++
+
+
++
+

++
+
++
+
+
+
+

++
+
++
+
+
+
+

+
++
+
++
++
+
+

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The values: High (++), Moderate: (+)

concentrations of flavonoids, saponins, phenols and

resins (Table II). The results demonstrated that all
parts of the plant included same compounds.
Disc diffusion method

The results of disc diffusion method showed that

all parts of the methanolic extract of the P. farcta
recorded different degrees of antibacterial activity

on MRSA bacteria as evidenced by the inhibited

zones (50.1, 60.4, 806 and 120.1 mm, respectively). The inhibited zones of all part of the plant
on plate are shown in Figure 2A-D. Results showed
that seeds extract were significantly more efficacious
on the MRSA bacteria than other extracts (P<0.05).
The roots extract was inhibited for MRSA bacteria
with 50.1 mm, which is significantly different with
other extracts at P<0.05.

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(either sporadically or systematically, either printed or electronic) of the Article for any purpose. It is not permitted to distribute the electronic copy of the article through online internet and/or intranet file sharing systems, electronic mailing or any other
means which may allow access to the Article. The use of all or any part of the Article for any Commercial Use is not permitted. The creation of derivative works from the Article is not permitted. The production of reprints for personal or commercial use is
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PROSOPIS FARCTA AGAINST STAPHYLOCOCCUS AUREUS

SHARIFI-RAD

Figure 2.Inhibition zones of all parts of the Prosopis farcta. A) roots; B) leaf; C) pods; D) seeds.

290

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SHARIFI-RAD

MIC and MBC

nium strictipes and Polygonum multiflorum plants

was 1.43 mg/mL.
Chomnawang et al.36 investigated antibacterial activity of Thai Medicinal plants against MRSA. They
illustrated that Garcinia mangostana extract was
identified as the most potent plant against MRSA
(MIC and MBC values of 1.95 and 3.91 g/mL, respectively).
Radji et al.37 carried out a study on antimicrobial
activity of green tea extract against isolates of MRSA.
The results of this study showed that the inhibited
zone diameter of green tea extract for MRSA was
19.130.25 mm and MIC of green tea extract was
400 g/mL. The MIC and MBC values obtained in all
mentioned studies demonstrated that these plants
have effective impact against MRSA.
Rad et al.38 investigated in vitro antibacterial activity of Xanthium strumarium L. extracts on methicillin-susceptible and methicillin-resistant Staphylococcus aureus. They reported that X. strumarium
extract affected both methicillin-susceptible S. aureus and MRSA, though antibacterial activity was
more effective on methicillin-susceptible S. aureus
spp. Also they illustrated that the antibacterial activity exhibited by the methanol extract may justify
the traditional use of this plant as a folk remedy
worldwide.
Darogha 39 analyzed phytochemical and antibacterial activity of some medicinal plants against methicillin-resistant S. aureus. The results showed that the
MIC/MBC of pods aqueous and ethanolic extracts of
P. farcta against MRSA isolates were 100/25 mg/mL
and 25/12.5 mg/mL, respectively. In our study we
observed that MIC and MBC for pods methanolic
extract were 150.1 and 250.5 g/mL, respectively.
In addition, Darogha 39 reported that pods aqueous
and ethanolic extracts of P. farcta was included alkaloids, tannins, glycosides, flavonoids, saponins, phenols and resins. The result of our study also showed
that these compounds were available in pods extract
but the values of these compounds were various.
Al-Ameri 40 illustrated that MIC for pods extract
of P. farcta from Iraq was 1.5 g/mL. In comparison with our results (MIC 150.1 g/mL), this value
was modest. This difference between values can be
related to geographic location and ecosystems and
different climate.
All P. farcta extracts demonstrated various degrees of antibacterial activity on MRSA as evidenced
by the inhibited zones, MIC and MBC. The basis of
varying degree of sensitivity of tested MRSA to plant

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The values of MIC for roots, leaf, pods and seeds

extract were 450.4, 35.50.8, 150.1 and 50.4 g/
mL, respectively. MBC for roots, leaf, pods and seeds
extract were 1000.4, 750.3, 250.5 and 50.2 g/
mL, respectively. The plant seeds extract showed
lowest MIC (50.4 g/mL) and MBC (50.2 g/mL)
against MRSA while highest MIC and MBC observed
in roots extract compared with other extracts. These
results illustrated that seeds extract was more effective against MRSA bacteria than other extracts of P.
faracta at P<0.05. These results also confirmed the
result of disc diffusion method. In this study, MIC of
vancomycin for all strains isolated from various clinical samples ranged between 0.4 to 4 g/mL. Among
MIC of all extracts, MIC of seeds extract (50.4 g/
mL) was near the highest vancomycin MIC (4 g/
mL).
Discussion

In this study, all isolated MRSA were entirely sensitive to vancomycin. Similar results were achieved
for vancomycin assay in previous studies.23, 24 Vancomycin has been the most effective therapeutic
agent against MRSA. However, increased use of vancomycin has set a basis of selection for vancomycinresistance in MRSA.
S. aureus is a versatile human pathogen; it was
powerfully considered as a major reason of nosocomial infection. In recent years, the prevalence of
MRSA has enhanced universal as it is evident from
numerous supervision studies.25-33 However, infection with MRSA changes greatly from one geographic location to another, from hospital to hospital.34
Today, some antimicrobial agent such as vancomycin and teicoplanin are still exclusively efficient
against MRSA; thus, this pathogen can cause critical
infection in different body systems in patients. The
emergences of expanding in antibiotic resistance
due to many researchers were examined optional
accesses to treat staphylococcal infection. Plant extracts have been extensively studied as natural products.
Zuo et al.35 performed a study on potential antibacterial activity of aerial parts of the Chinese medicinal plants against clinical isolates of MRSA. They
reported that the MIC for Dendrobenthamia capitata, Elsholtzia rugulosa, Elsholtzia blanda, Gera-

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(either sporadically or systematically, either printed or electronic) of the Article for any purpose. It is not permitted to distribute the electronic copy of the article through online internet and/or intranet file sharing systems, electronic mailing or any other
means which may allow access to the Article. The use of all or any part of the Article for any Commercial Use is not permitted. The creation of derivative works from the Article is not permitted. The production of reprints for personal or commercial use is
not permitted. It is not permitted to remove, cover, overlay, obscure, block, or change any copyright notices or terms of use which the Publisher may post on the Article. It is not permitted to frame or use framing techniques to enclose any trademark, logo,
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extract may be due to the natural tolerance of MRSA

and the nature and combinations of phytochemical
present in the P. farcta extracts. Seed extract of P.
farcta among all part of the plant was more efficient
against MRSA. This effect can be related to highest
concentration of alkaloids, tannins and glycosides.
In previous studies it was reported that these phytochemicals are known to have antimicrobial activity.38, 41

Kahoor and effectiveness for compost production in Hormozgan

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activity of certain Indian medicinal plants used in folkloric medicine. J Ethnopharmacol 2001;74:211-20.
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Chemical composition, antioxidant activity and in vitro antibacterial activity of Achillea wilhelmsii C. Koch essential oil on
methicillin-susceptible and methicillin-resistant Staphylococcus
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standard for antimicrobial susceptibility testing. 12th edition. Information supplement 2010;M100-520.
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and antibacterial activities of Nephelium lappaceum L. extracts.
LWT-Food Sci Technol 2008;41:2029-35.
21. Ilim A, Dgrak M, Bagci E. The investigation of antimicrobial
effect of some plant extract. Turk J Bio1998;22:119-25.
22. Al-Fatimi M, Wurster M, Schwoder G, Lindequist U. Antioxidant antimicrobial and cytotoxic activities of selected medicinal
plants from Yemen. J Ethnopharmacol 2007;111:657-66.
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24. Kumari N, Mohapatra TM, Singh YL. Prevalence of methicillinresistant Staphylococcus aureus (MRSA) in a Tertiary-Care Hospital in Eastern Nepal. J Nepal Med Assoc 2008;47:53-6.
25. Simor AE, Ofiner-Agostini M, Bryce E, Green K, McGeer A, Mulvey M et al. The evolution of methicillin-resistant Staphylococcus
aureus in Canadian Hospitais: 5 years of national surveillance.
Canadian Med Assoc 2001;165:21-6.
26. Tiemersma EW, Bronzwaer LAM, Lyltikalinen O, Degener JE,
Schrijnemakers P, Bruinsma N, Monen J, Witte W, Grundmann
H. Methicillin-resistant Staphylococcus aureus in Europe, 19992002. Emerg Infect Dis 2004;10:1627-34.
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screening. J Hosp Infect 2008;69:315-20.
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HA, Tacconelli E. Meticillin-resistant Staphylococcus aureus
(MRSA): screening and decolonization. Int J Antimicrob Agents
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Conclusions

The mechanism concept to be responsible for the

activity of these phytochemicals against MRSA may
include enzyme inhibition by the oxidized compounds which act as an origin of stable free radical
and often lead to deactivation of the protein and
loss of function. They have the ability to compound
not only with extracellular and soluble proteins
but also bacterial cell walls and interrupt microbial
membranes. However, the search for new antibacterial factors should be continued by screening many
other plant families. The antimicrobial and phytochemical studies would provide valuable knowledge to the understanding media universal.
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Acknowledgments.This research was self-funded by Authors. The
authors are very grateful to Department of Range and Watershed Management, Faculty of Natural Resources, University of Zabol, Iran and
Zabol Medicinal Plants Research Center, Zabol University of Medical
Sciences, Iran for excellent cooperation in this study.
Conflicts of interest.The authors certify that there is no conflict of
interest with any financial organization regarding the material discussed
in the manuscript.
Received on September 13, 2013.
Accepted for publication on November 1, 2014.

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This document is protected by international copyright laws. No additional reproduction is authorized. It is permitted for personal use to download and save only one file and print only one copy of this Article. It is not permitted to make additional copies
(either sporadically or systematically, either printed or electronic) of the Article for any purpose. It is not permitted to distribute the electronic copy of the article through online internet and/or intranet file sharing systems, electronic mailing or any other
means which may allow access to the Article. The use of all or any part of the Article for any Commercial Use is not permitted. The creation of derivative works from the Article is not permitted. The production of reprints for personal or commercial use is
not permitted. It is not permitted to remove, cover, overlay, obscure, block, or change any copyright notices or terms of use which the Publisher may post on the Article. It is not permitted to frame or use framing techniques to enclose any trademark, logo,
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