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,
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
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

MINERVA BIOTEC 2014;26:287-93

Phytochemical screening and antibacterial activity

of different parts of the Prosopis farcta extracts against
methicillin-resistant Staphylococcus aureus (MRSA)
J. SHARIFI-RAD 1, 2, S. M. HOSEINI-ALFATEMI 3*, M. SHARIFI-RAD 4, A. MIRI 1, 2, 5, M. SHARIFI-RAD 5

® A
T C
H DI
The present study investigated the phytochemical 1Zabol Medicinal Plants Research Center
screening and antibacterial potentiality of different Zabol University of Medical Sciences, Zabol, Iran
parts (roots, leaf, pods and seeds) of the Prosopis farc- 2Department of Pharmacognosy, Faculty of Pharmacy

ta extracts against Methicillin-resistant Staphylococ- Zabol University of Medical Sciences, Zabol, Iran

IG E
cus aureus (MRSA). Phytochemical screening of the
P. farcta methanolic extract was carried out for esti-
3Pediatric Infections Research Center
Mofid Children Hospital, Shahid Beheshti
R M
mation of saponins, glycosides, alkaloids, tannins and University of Medical Sciences, Tehran, Iran
4Department of Range and Watershed Management
flavonoids by using standard phytochemical screening
methods. The antibacterial activity was determined by Faculty of Natural Resources, University of Zabol, Iran
5Zabol University of Medical Sciences, Zabol, Iran
both the disc diffusion method and the microbroth di-
lution method. Antibacterial screening of P. farcta ex-
P A

tracts showed that inhibited zones for roots, leaf, pods

and seeds extract were 5±0.1, 6±0.4, 8±06 and 12±0.1
O V

mm, respectively. MIC for roots, leaf, pods and seeds more dangerous than the disease itself. Nowadays,
extract were 45±0.4, 35.5±0.8, 15±0.1 and 5±0.4 µg/
mL, respectively. MBC for roots, leaf, pods and seeds due to the deformation resistance of bacteria becom-
C ER

extract were 100±0.4, 75±0.3, 25±0.5, and 5±0.2 µg/ ing resistant to commonly used antibiotics, tendency
mL, respectively. In this study, MIC range for Vanco- to replace them with new antibiotics has increased.1
Y

mycin was between 0.4-4 μg/mL for all strains isolated In spite of the progress of science and the develop-
from various clinical samples. All P. farcta extracts ex- ment of synthetic drugs, medicinal plants are still
hibited different degrees of antibacterial activity on used in large-scale and traditional medicine.2-7 In re-
IN

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 alka-
M
cent years, extensive study was carried out to evalu-
ate the antimicrobial effect of plant essential oils and
extracts. These results showed the potency of these

loids, tannins or glycosides in this part. These compo-
nents are known to high antibacterial activity. P. farc-
ta extracts possess potent antibacterial against MRSA,
and may be used as an antibacterial to treat diseases.
Key words: Anti-bacterial agents - Prosopis - Methicillin-resist-
ant Staphylococcus aureus - Alkaloids - Tannins - Glycosides.
or other proprietary information of the Publisher.
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 harm-
ful weeds. The Prosopis farcta is a little prickly spiny
shrub or tree. Its native of range land, it is wide-

spread and a weed of cotton fields and wheat, in-

T he increasing use of chemical drugs causing bac-
terial 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
fringed 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-

Vol. 26 - No. 4 MINERVA BIOTECNOLOGICA 287

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,
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
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

SHARIFI-RAD PROSOPIS FARCTA AGAINST STAPHYLOCOCCUS AUREUS

tivity of extracts from Prosopis spp. was examined

and the results showed antibacterial activity against
some gram-positive and negative bacteria, antidi-
arrheic, 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 con-
sequential human pathogen responsible for nosoco-
mial, hospital- and community-acquired infections.
It can bring about a range of infectious diseases
from moderate conditions, such as soft tissue infec-
tions, to serious life-threatening debilitation like en-

® A
docarditis, to death.11, 12 Methicillin-resistant strains
of staphylococci were recognized shortly upon the

T C
preface of methicillin into clinical train. The first out- Figure 1.—Prosopis farcta plants used in this study.
break caused by Methicillin-resistant Staphylococcus

H DI
aureus (MRSA) happened in an English hospital in
the early 1960s and appeared in the United States These working solutions were used for all the tests
in the 1980s.12 In recently years, MRSA strains have in this study.
often become resistant to multiple other antimicro-

IG E
bial agents and many researcher have reported an
intensification in the incidence of MRSA. On the
Preliminary phytochemical analysis
R M
basis of some investigations on phytochemical and Phytochemical screening for major constituents
antibacterial properties of different parts extracts of include saponins, glycoside, flavonoids, tannins, al-
the P. farcta against MRSA, it can be asserted that the kaloids, resin and phenols was undertaken by using
importance of traditional medicine and medicinal standard qualitative methods as described by Fadeyi
P A

plants with very small side effects on human can be et al.,14 Odebiyi and Sofowora,15 Harborne,16 and
useful for discovering natural extracts to that reduce Abulude.17
O V

the growth of these bacteria.

Bacterial isolates and antibiotic susceptibility assay
C ER

Materials and methods Four hundred and seventy-seven clinical speci-

Y

mens such as burn, wound, urine, pus and throat

Plant preparation and extracts swab were collected from patients attended in emer-
gency Hospital and Internal Laboratory of Hospital
IN

The Prosopis farcta were collected during the ma- and Central Laboratory in Zabol city (Iran) for differ-
ture period, May 2013, from the area surrounding ent bacteriological examinations (Table I). Standard
Hamun Lake, Zabol (Coordinates: 31° 1′ 43″ N, 61° isolation protocols were used for all the samples.
M

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
or other proprietary information of the Publisher.
Identification of S. aureus was approved by stand-
ard techniques accord on colonial and microscopic
morphology, coagulase test and biochemical activi-
ties.17 Detection of MRSA was done by the use of
Cefoxitin disc (30 µg) diffusion test. The strains of
S. aureus with a zone diameter of <19 mm based
on Clinical and Laboratory Standard Institute (CLSI)

°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.
were taken to show methicillin-resistance.19 All
strains of S. aureus were confirmed as methicillin
resistant by oxacillin agar dilution using Muller Hin-
ton agar (beef extract, acid hydrolysate of casein
17.5g/L, starch 1.5 g/L, agar 17.0 g/L, final 7.3±0.1

288 MINERVA BIOTECNOLOGICA December 2014

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,
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
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
PROSOPIS FARCTA AGAINST STAPHYLOCOCCUS AUREUS SHARIFI-RAD
Table I.—The number and percentage of isolated MRSA from various clinical specimens.
S. aureus
Type of specimens Number of specimens
N.
Burn 104 53
Wound 45 38
Urine 254 11
Pus 14 4 28.57
Throat 60 1 1.66
Total 477 107
at 25 °C) supplemented with 2% NaCl. The concen-
trations tested ranged from 1 μg/mL to 16 μg/mL
® A
of oxacillin. The strains having minimum inhibitory
concentration (MIC) ≥4 μg/mL were taken as resist-
T C
ant. Among 477 clinical specimens, of the 107 speci-
mens (22.43%) were S. aureus that from these 18
H DI
specimens (16.82%) were MRSA.
Disc diffusion method
IG E
Antimicrobial activity was based on the disc dif-
fusion method 20 using a cell suspension of micro-
R M
organisms. The concentration of the cell suspension
was equilibrated to a 0.5 McFarland standard and 50
μL of each microorganism’s suspension was spread
on a Mueller-Hinton agar plate. In addition, 50 μL of
P A
diluted methanolic extracts was pipetted onto sterile
paper discs (6 mm in diameter), which were dried
O V
in an open sterile Petri dish in a biological laminar
flow bench. Discs were placed on the surface of
C ER
inoculated plates and incubated at 37 °C for 24 h.
Diameters (mm) of the zones of bacterial inhibition
Y
minus the discs diameter were recorded.21
IN
Determination of MIC and minimum bactericidal
concentration
The MIC values for each plant extracts were deter-
M
mined by microbroth method. Residues of plant ex-
tracts were dissolved in 20 mL of distilled water. All
extracts are tested at 1000 μg/mL 22 and serially di-
luted two-fold to 1.95 μg/mL in a 96-multiwell poly-
styrene flat-bottomed microplate (Sigma-Aldrich, St.
Louis, MO, USA) after which 100 μL (1×106 CFU/mL)
of bacteria are added to each of them. Preincubation
or other proprietary information of the Publisher.
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 concen-
tration of the extract that completely inhibited bac-
Vol. 26 - No. 4 MINERVA BIOTECNOLOGICA 289
MRSA
% N. %
50.69 9 16.98
84.44 4 10.52
4.33 4 36.36
0 0.00
1 100
22.43 18 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 con-
centrations assayed ranged from 1 μg/mL to 33 μg/
mL of vancomycin.
Statistical analysis
The extracts were prepared in triplicate for
chemical characterization and antibacterial assays.
Data was subjected to analysis of variance follow-
ing 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
mean±SE.
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 dem-
onstrated 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 concentra-
tions of alkaloids, tannins, glycosides and moderate
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,
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
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

SHARIFI-RAD PROSOPIS FARCTA AGAINST STAPHYLOCOCCUS AUREUS

Table II.—Results of phytochemical analysis of extracts from root, leaf, pod and seed of the P. farcta.
Methanolic extracts
Chemical Compounds Root Leaf Pod Seed
Saponins ++ ++ ++ +
Tannins + + + ++
Flavanoids ++ ++ ++ +
Glycoside + + + ++
Alkaloids + + + ++
Phenols ++ + + +
Resin + + + +
The values: High (++), Moderate: (+)

® A
concentrations of flavonoids, saponins, phenols and on MRSA bacteria as evidenced by the inhibited
resins (Table II). The results demonstrated that all zones (5±0.1, 6±0.4, 8±06 and 12±0.1 mm, respec-

T C
parts of the plant included same compounds. tively). The inhibited zones of all part of the plant
on plate are shown in Figure 2A-D. Results showed

H DI
Disc diffusion method that seeds extract were significantly more efficacious
on the MRSA bacteria than other extracts (P<0.05).
The results of disc diffusion method showed that The roots extract was inhibited for MRSA bacteria
all parts of the methanolic extract of the P. farcta with 5±0.1 mm, which is significantly different with

IG E
recorded different degrees of antibacterial activity other extracts at P<0.05.
R M
P A
O V
C ER
Y
IN

A B
M
or other proprietary information of the Publisher.

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

290 MINERVA BIOTECNOLOGICA December 2014

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,
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
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
PROSOPIS FARCTA AGAINST STAPHYLOCOCCUS AUREUS SHARIFI-RAD
MIC and MBC
The values of MIC for roots, leaf, pods and seeds
extract were 45±0.4, 35.5±0.8, 15±0.1 and 5±0.4 µg/
mL, respectively. MBC for roots, leaf, pods and seeds
extract were 100±0.4, 75±0.3, 25±0.5 and 5±0.2 µg/
mL, respectively. The plant seeds extract showed
lowest MIC (5±0.4 µg/mL) and MBC (5±0.2 µg/mL)
against MRSA while highest MIC and MBC observed
in roots extract compared with other extracts. These
results illustrated that seeds extract was more effec-
tive against MRSA bacteria than other extracts of P.
faracta at P<0.05. These results also confirmed the
® A
result of disc diffusion method. In this study, MIC of
vancomycin for all strains isolated from various clin-
T C
ical samples ranged between 0.4 to 4 μg/mL. Among
MIC of all extracts, MIC of seeds extract (5±0.4 µg/
mL) was near the highest vancomycin MIC (4 μg/
H DI
mL).
IG E
Discussion
R M
In this study, all isolated MRSA were entirely sen-
sitive to vancomycin. Similar results were achieved
for vancomycin assay in previous studies.23, 24 Van-
comycin has been the most effective therapeutic
P A
agent against MRSA. However, increased use of van-
comycin has set a basis of selection for vancomycin-
O V
resistance in MRSA.
S. aureus is a versatile human pathogen; it was
C ER
powerfully considered as a major reason of noso-
comial infection. In recent years, the prevalence of
Y
MRSA has enhanced universal as it is evident from
numerous supervision studies.25-33 However, infec-
tion with MRSA changes greatly from one geograph-
IN
ic location to another, from hospital to hospital.34
Today, some antimicrobial agent such as vanco-
mycin and teicoplanin are still exclusively efficient
M
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 ex-
tracts have been extensively studied as natural prod-
ucts.
or other proprietary information of the Publisher.
Zuo et al.35 performed a study on potential anti-
bacterial activity of aerial parts of the Chinese me-
dicinal plants against clinical isolates of MRSA. They
reported that the MIC for Dendrobenthamia capi-
tata, Elsholtzia rugulosa, Elsholtzia blanda, Gera-
Vol. 26 - No. 4 MINERVA BIOTECNOLOGICA 291
nium strictipes and Polygonum multiflorum plants
was ≤1.43 mg/mL.
Chomnawang et al.36 investigated antibacterial ac-
tivity 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 µg/mL, re-
spectively).
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
19.13±0.25 mm and MIC of green tea extract was
400 μg/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 activ-
ity of Xanthium strumarium L. extracts on methi-
cillin-susceptible and methicillin-resistant Staphylo-
coccus aureus. They reported that X. strumarium
extract affected both methicillin-susceptible S. au-
reus and MRSA, though antibacterial activity was
more effective on methicillin-susceptible S. aureus
spp. Also they illustrated that the antibacterial ac-
tivity exhibited by the methanol extract may justify
the traditional use of this plant as a folk remedy
worldwide.
Darogha 39 analyzed phytochemical and antibacte-
rial activity of some medicinal plants against methi-
cillin-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 15±0.1 and 25±0.5 µg/mL, respectively.
In addition, Darogha 39 reported that pods aqueous
and ethanolic extracts of P. farcta was included alka-
loids, tannins, glycosides, flavonoids, saponins, phe-
nols 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 µg/mL. In compari-
son with our results (MIC 15±0.1 µg/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 de-
grees 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
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,
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
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
SHARIFI-RAD PROSOPIS FARCTA AGAINST STAPHYLOCOCCUS AUREUS
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 phy-
tochemicals are known to have antimicrobial activ-
ity.38, 41
Conclusions
® A
The mechanism concept to be responsible for the
activity of these phytochemicals against MRSA may
T C
include enzyme inhibition by the oxidized com-
pounds which act as an origin of stable free radical
H DI
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
IG E
membranes. However, the search for new antibacte-
rial factors should be continued by screening many
R M
other plant families. The antimicrobial and phyto-
chemical studies would provide valuable knowl-
edge to the understanding media universal.
P A
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O V
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interest with any financial organization regarding the material discussed

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