Antibacterial, Antifungal and Toxicity of Rare Iranian Plants
As a part of our drug discovery program, an effort to
introduce new effective medicinal plants, with antibacterial, antifungal
and cytotoxic properties was made. The extracts of aerial part of 8 plants,
collected in southeastern Iran, were investigated against standard strains
of Bacillus subtilis, Enterococcus faecalis, Pseudomonas
aeruginosa, Salmonella entrica subsp. entrica ser. Typhi,
Escherichia coli, meticillin resistant Staphylococcus aureus (isolated
from patients), Fusarium oxyosporum, Aspergilus niger, and
Aspergilus fumigatus. We used brine shrimp (Artemia salina)
cytotoxicity bioassay in order to provide a better base for introducing
the extracts as the new therapeutic candidates. Capparis deciduas (Forsk.)
Edjw. and Cleome oxypetala Boiss. (both from Capparidaceae family)
were recognized as having the potential for development of new antibiotics.On
the other hand, Cistanche tubulosa (Schrenk) R.Wight, could be
worthy of attention for finding anticancer phytochemicals.
The emergence of pathogens resistant to antibiotics as a result of excessive
use of them in clinical and veterinary applications, represent a serious
problem for public health. Despite the existence of efficient antibiotics,
drug resistant or multi-drug resistant strains are steadily appearing
and require longer and more expensive treatments (Alanis, 2005). These
facts play a driving force for scientists to explore new anti-infective
agents for developing new drugs. Among several options, plants are the
most known source of healing compounds. Plants have limitless ability
to synthesize substances, many of them with anti-infective activity. Nowadays,
scientists all over the world are engaged to explore bioactive properties
of plants with the hope of adding new therapeutic agents (Gurib-Fakim,
2006). Iran, in addition to having a very honorable tradition of plant-healing
remedies (Gorji and Khaleghi Ghaderi, 2002), is one of the richest countries
in terms of its plant biodiversity. In an attempt to introduce plants
with bioactive compounds, we selected and gathered several plants for
their desired bioactivity and collected them. After identification, the
plant extracts were evaluated through various bioassays to assess the
anti-microbial potency as well as their cytotoxic property. Such study
is part of our expanded bioactive lead discovery program from natural
products and their introduction for potential anti-infective or anticancer
The potential and rational for collections of plants stemmed from their
potential of previous usage and novelty in the report of investigated
bioactivity. Capparidaceae is a vast family with several genera. Many
of plants which have been used in traditional medicine belong to this
family particularly to Cappris and Cleome genera (Jeruto
et al., 2008; González-Tejero et al., 2008). Capparis
deciduas is a well known plant in Saudi Arabia with a broad range
of medical usage (Rahman et al., 2004). In addition to C. deciduas
we have investigated C. oxypetala from Capparidaceae. Cleome
genus includes many species with vast applications in herbal remedies.
Cleome gynandra has been used against malaria as well as stomach
congestion in Kenya (Jeruto et al., 2008). Cleome arabica
is mentioned as a plant with versatile medical uses in Saudi Arabia (Rahman
et al., 2004). There are many other plants of Cleome genus
with documented traditional medical application. Tamarix dioica from
Tamaricaceae family was among the collected plants. Actually, T. dioica
is known as an important medicinal plant in India and has been investigated
due to richness of bioactive phytochemicals (Pamar et al., 1994).
Other species from Tamarix genus have reputable therapeutic applications
too. For example Tamarix aphylla and Tamarix gallica are
used to alleviate several physiological distresses and infectious diseases
in the Central Sahara region in Africa (Hammiche and Maiza, 2006). Plants
from genus Taverniera have been studied for search of valuable
phytochemicals particularly saponins. Many pharmacological activities
have been reported about saponins such as antibiotic, antifungal and antiviral
activities. Taverniera aegyptiaca is indicated to have various
kinds of this bioactive phytochemical (Ibraheim et al., 2003).
Taverniera cuneifolia (Roth) Arn. exhibited promising anti-inflammatory,
anti-tumor, anti germ tube formation (Zore et al., 2008). In this
context, T. glabra was investigated for assessment of antimicrobial
activity in this study. Rumex genus belongs to Polygonaceae family
and several species of this genus has indicated noteworthy therapeutic
potentials. Rumex cyprius, the plant from this genus which we assessed
its antimicrobial activity has shown antiviral activity against HIV through
inhibition of reverse transcriptase (Vermani and Garg, 2002). Blepharis
edulis is an Indian medicinal plant which is source of several bioactive
phytochemicals such as blepharin (Pratt et al., 1995). Extracts
of Blepharis ciliaris has indicated significant antimicrobial activity
and is used in folk medicine of Saudi Arabia (Harraz et al., 1996).
Blepharis panduriformis Linda, another member of blepharis genus
is applied in form of decoction for dysentery treatment in Tanzanian traditional
medicine (Maregesi et al., 2007). Convolvulus sericeus is
collected and assessed for anti infective activity. Some members of Convolvulus
genus are among traditional medicinal plants of some cultures. For example,
Convolvulus arvensis is known as carminative, antiseptic and stimulant
in Egypt (Atta and Mouneir, 2004). The Orobanchaceae parasitic plant,
Cistanche tubulosa (Schrenk) R. Wight, is widely distributed in
North Africa, Arabia and Asian countries and has been traditionally used
as a promoting agent of blood circulation and treatment of impotence,
sterility, lumbago, body weakness and tonic (Yoshikawa et al.,
2006). Another member of Cistanche genus is Cistanche deserticola
which is a Chinese traditional herb. The dried whole plant is used for
the treatment of kidney pain, gynecological diseases, intestinal infection
and constipation (Dong et al., 2007).
MATERIALS AND METHODS
Plant material: Plant materials were collected in March 2005,
from Chabahar, in Sistan and Baluchestan, the southeastern province of
Iran (Table 1). The identification was carried out by
our team members and confirmed by Dr. S. Sardari. The voucher specimens
were deposited in our herbarium. After identification, aerial parts of
each specimen were dried in ambient temperature for 4 days and mill ground
to a fine powder using an electric grinder.
Preparation of the plant extracts: About 10 g of each plant sample
was extracted three times with 100 mL of 80% ethanol in water each time.
The plant extracts were filtered through Whatman No. 1 filter paper. The
filtrates were concentrated to total dryness in a rotary evaporator at
40°C until a constant dry weight for each extract was obtained, all
in dark brown color. The residues were stored at 4°C. The extraction
yield was between 25-32%. For Capparis deciduas the alcohol extract
was further fractionated by dichloromethane. After concentration, the
extract was dissolved in ethanol and the remaining undissolved extract
was further dispersed in dichloromethane.
Antibacterial and antifungal screening: Bacillus subtilis ATCC
6633, Enterococcus faecalis NCTC 8213, Pseudomonas aeruginosa
ATCC 9027, Salmonella entrica subsp. entrica ser. Typhi
NCTC 5761, Escherichia coli ATCC 8739, methicillin resistant Staphylococcus
aureus (isolated from patients), Aspergillus niger N 402, Aspergillus
fumigatus PTCC 5009 and Fusarium oxyosporum CBS 620.87 were
used as test strains. The plant extracts were dissolved in dimethyl sulfoxide
(DMSO) to reach a concentration of 10 mg mL-1. The broth microdilution
method was performed for antibacterial and antifungal activity tests.
The absorbance was read at 530 nm for fungi and 600 nm for bacteria inoculums
to reach the suitable density of pathogen or test microorganisms. From
the prepared stock fungal culture, a 1:1000 dilution with broth (e.g.,
10 μL stock fungal culture: 10 mL broth) was prepared, which is called
working fungal culture. Modified antimicrobial susceptibility testing
based on NCCLS M27-A method was performed. From the prepared stock culture,
a 1:1000 dilution with broth medium (10 μL stock culture: 10 mL broth)
was prepared, which is called working microorganism culture. Mueller-Hinton
broth was used for the screening of antimicrobial activity against the
bacterial test strains. Tween 20 (0.1% w/v) was added to facilitate the
preparation of fungal spores suspensions of the fungi (Cuenca-Estrella
et al., 2002; Gomez-Lopez et al., 2005). Mueller-Hinton
broth and Sabourod Dextrose broth were used for the screening of antimicrobial
and antifungal activities, respectively. Broth medium (100 μL) was
added to each well of a 96-well microplate and then 40 μL of plant
extract and 60 μL broth were added to well (A), then a solution (100
μL) serially diluted from well (A) by taking 100 μL into (B)
was obtained. This two-fold dilution was continued down the plate and
100 μL from the last well (H) was discarded. Then all the wells were
filled with 100 μL of working microorganism suspension. Fluconazole
was used as a reference in fungal test whereas Penicillin, Kanamycin and
Ciprofloxacin were references for bacterial tests.
||Scientific names of collected plants, their family and
their abbreviations used in this study
For this experiment the following controls were prepared: no inoculum
was added to a number of wells were used for control of sterility; control
of inoculum viability, no plant extract was added to wells containing
inoculum and nutrient medium and control of the DMSO inhibitory effect,
DMSO was added to some wells containing inoculum and nutrient medium.
The plates were covered and incubated at 37°C for 24 h. The MIC values
were obtained by reading the concentration of the well with no growth.
Toxicity screening: Extracts of all plants were screened for toxicity
with larvae (nauplii) of Artemia salina (brine shrimp). In order
to improve the exposure of nauplii with the extract; we made some changes
to conventional method of McLaughin et al. (1998). The test was
performed in triplicate in vials with extract concentrations of 10, 100
and 1000 μg mL-1. The eggs were placed in an aerated bottle,
using electric pump with a flow of 3-4 L min-1, containing
33 g L-1 NaCl saline and natural lighting. After two days of
hatching period at room temperature, the nauplii were ready for the experiment.
To obtain a concentration of 5 mg mL-1, relative mass of each
extract was accurately measured and dissolved in appropriate volume of
solvent (acetone or ethanol). From the stock solutions 10, 100 and 1000
μL were placed in vials (three vials for each concentration). A mixture
of 33 g L-1 saline and polyethylene glycol (PEG) 6000 (3% w/v)
was prepared to have a better dispersion of the plant extract. After evaporation
of solvent, the above mixture was added to vials making the volume up
to 5 mL to make the final concentrations of 10, 100 and 1000 μg mL-1.
The mixture of saline and PEG 6000 was used as control solution. Ten shrimps
were added to each vial. For better aeration, the vials were put horizontally
on a shaker to be moved slowly. After 24 h, the survivors were counted
under microscope and recorded. The data were analyzed by SPSS version
10 for probit analysis to determine LC50 values.
RESULTS AND DISCUSSION
The hydro-alcoholic extract of ten Iranian plants were screened for their
antimicrobial and toxicity properties. The effect of extracts was compared
to control agents. At the same time, the solvent of the extracts was tested
to identify the level of inhibition that is attributable to the solvent
and the results are shown in Table 2 and 3.
There was no inhibition by the DMSO solvent for microorganisms tested.
Between the plants extracts tested, C. tubulosa, R. cyprius
and C. deciduas (extracted by CH2Cl2) had
maximum toxicity against A. salina (Table 4).
The extracts of these plants may not be used as antimicrobial agents for
further therapeutic research in spite of their good antimicrobial activities,
unless we indicate that the toxicity is originated from different materials
present in the extract than the antimicrobials. However, the extracts
of such plants are potential cases for finding anticancer phytochemicals.
T. glabra, T. dioica, B. edulis and C. oxypetala
and ethanolic phaseof C. deciduas extracts had milder toxicity.
It is useful to remember that caffeine showed LC50 300 μg
mL-1 against A. salina (Meyer et al., 1982).
However, B. edulis, T. dioica and C. sericeus do
not show acceptable antimicrobial activity. Cleome oxypetala and
ethanolic phase of C. deciduas extract had potent antibacterial
activity against methicillin resistant S. aureus (11 times more
than penicillin and kanamycin and 47 times more than ciprofloxacin). It
should be remembered that methicillin resistant Staphylococcus aureus
(MRSA) has shown relatively high resistance when exposed to reference
antibiotics. These plant extracts can be useful for further researches.
Compared to fluconazole, C. oxypetala is more effective against
F. oxyosporum and A. niger. Between the two Aspergillus
species tested, A. niger N 402showed more susceptibility to plant
extracts than, A. fumigatus PTCC 5009. R. cyprius and T.
dioica represent the least MIC among the extracts against A. niger
N 402 and A. fumigatus PTCC 5009, respectively. Due to A.
fumigatus PTCC 5009 strikethrough susceptibility to T. dioica,
it could be an option for more studies.Thoughall the extracts showed considerable
inhibitory effect against F. oxyosporum CBS 620.87, C. deciduas,
C. sericeus and B. edulis act in 8 times less concentration
in comparison with the reference antibiotic fluconazole. The extracts
show weak effects against bacteria as compared to conventional antibiotics,
but we should not neglect the least MICs that guide us to know extracts
with potential ingredients against bacterial pathogens. In a general view,
B. subtilis is the most susceptible bacterial organism among the
others. C. deciduas (the ethanolic phase), T. glabra and
C. oxypetala show the strongest effect against B. subtilis.
Another considerable result is the effect of R. cyprius extract
against meticillin resistant Staphylococcus aureus,which has shown
relatively high resistance when exposed to reference antibiotics. Among
the extracts, the ethanolic phase of C. deciduas, with the least
MICs against two pathogens, F. oxyosporum and B. subtilis
and relatively, low toxicity in comparison with other extracts is considered
as a promising material that merit more detailed studies. In the same
manner, C. oxypetala extract with low MIC against the two above
mentioned microorganisms and the least cytotoxicity among the extracts,
could be another option for antibiotic development. C. tubulosa
extract, has the highest cytoxicity among the others, a trait which introduce
C. tubulosa as a potential case for finding anticancer phytochemicals.
We intend to continue the research by further study on the promising plants
towards the active ingredients and the bioactivity profile.
||Antimicrobial activity of extracts expressed as Minimum
Inhibitory Concentration (MIC)
|CD1: Capparis deciduas
ethanolic phase, CD2: Capparis deciduas dichloromethane
phase, SA: Staphylococcus aureus, BS: Bacillus subtilis,
EF: Enterococcus faecalis, PA: Pseudomonas aeruginosa,
SE: Salmonella entrica subsp. entrica ser. Typhi, EC:
Escherichia coli, P: Penicillin, K: Kanamycin, C: Ciprofloxacin
|| Antifungal activity of extracts expressed as Minimum
Inhibitory Concentration (MIC)
|AN: Aspergillus niger, AF: Aspergillus
fumigatus, FO: Fusarium oxyosporum, F: Fluconazole
|| The results of cytotoxic effect of plant extracts on
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