Antimicrobial activities from extracts of seven medicinal plant species against multidrug-resistant bacteria and fungi

This is the first to report the antimicrobial effects of extracts of seven endemic medicinal plants. Flower extracts of Onopordum jordanicolum exhibited antibacterial activity against Staphylococcus aureus, MRSA, Klebsiella pneumonia, and Proteus mirabillis. Ethanol extract displayed significant antibacterial activity against S. aureus with the best MIC and MMC values. Onopordum blancheanum flower extracts produced antibacterial activity against S. aureus, Escherichia coli, K. pneumonia, and P. mirabillis. Ethanol extract of Aethionema carneum leaves exhibited antibacterial activity against all test bacteria except MRSA and produced significant antibacterial activity against E. coli with the best MIC and MMC values. Methanol and acetone extracts of Delphinium ithaburense leaves showed significant antibacterial activity against K. pneumonia. Aqueous extract of Lathyrus hirticarpus leaves revealed a broad spectrum of antibacterial activity. Aqueous and acetone extracts of Orchis sancta flowers showed significant antibacterial activity against MRSA and Pseudomonas aeruginosa, respectively. Aqueous and methanol extracts from Papaver umbonatum flowers exhibited significant antibacterial activity against Streptococcus pyogenes with the best MIC and MMC values. For antifungal activity, it was found that Aspergillus brasiliensis and Candida albicans were inhibited by aqueous extracts of A. carneum and P. umbonatum, acetone extract of D. ithaburense, ethanol extract of L. hirticarpus, methanol extracts of O. blancheanum and O. sancta. Interestingly, acetone extract of O. jordanicolum displayed significant antifungal activities against A. brasiliensis and C. albicans with the best MIC and MMC values. Phytochemical screening of promising extracts revealed the presence of alkaloids, flavonoids, saponins, and/or tannins which might be responsible for their antimicrobial activity.


INTRODUCTION
Traditional medicines that are based on medicinal plants still play an essential role in health care.Medicinal plants are an important source of drugs and a huge number of bioactive medicines against different diseases are developed from plants origin (Sahoo et al., 2010).Secondary metabolites derived from medicinal plants E-mail: cherfiarr@yahoo.fr,obeidat@bau.edu.jo.Tel: 00962775609846.
Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License Recently, more than one hundred secondary plant metabolites derived from plants are used worldwide as drugs to treat different diseases (Jagatheeswari et al., 2013).
Increasing antibiotic resistance by pathogenic bacteria is developing at an alarming rate, as the rate of discovery for new antibiotics has been on the decline (Hopwood, 2007).Therefore, medicinal plants shift the attention of scientists and researchers to herbal medicine to find effective antimicrobial agents for treatment of diseases caused by multidrug resistant pathogens like bacteria and fungi (Parungao et al., 2007).
Although natural products derived from medicinal plants are used for thousands of years to treat different diseases, most medicinal plants around the world are not yet explored for their medicinal activities (Hassan, 2012).Therefore, seven endemic medicinal plants in Jordan (Aethionema carneum, Delphinium ithaburense, Lathyrus hirticarpus, Onopordum blancheanum, Onopordum jordanicolum, Orchis sancta, and Papaver umbonatum) were selected in the present study and evaluated for their antimicrobial activities against human pathogenic bacteria and fungi (Table 1).Few phytochemical investigations about A. carneum have been described in the literature.This annual plant has a unique character, which produce glucosinolate (mustard oil) compounds (Adigizel, 2000).
Nevertheless, the significant antimicrobial activity of this plant is not known.The plant D. ithaburense has been utilized for the treatment of various problems (Table 1).Many species of Delphinium have numerous toxic alkaloids (Gardner and Pfister, 2007).However, few phytochemical investigations about D. ithaburense have been described in the literatures and the antimicrobial effects were not identified.The medicinal plant L. hirticarpus is grown in the northern part of Jordan.This plant is commonly called Indian vetch.Seeds of members of the genus Lathyrus are toxic if ingested in high quantity (Grela et al., 2000).Until today, there is no antimicrobial study related to this plant.Two species of Onopordum from Asteraceae family were investigated in this study, O. blancheanum and O. jordanicolum.The O. blancheanum distributed throughout the Mediterranean areas (Ronel et al., 2009) and it is commonly known as cotton thistle.The O. jordanicolum is a Jordanian local plant and it is commonly called camel thistle.This plant species is found in Jordan Eastern desert.There are no previous studies that assessed the antimicrobial activity of O. blancheanum and O. jordanicolum.The orchid O. sancta belong to the family Orchidaceae.This family is the largest family of the plant kingdom, comprising more than 30,000 species (Jin-Ming et al., 2003).
In Jordan, there is a unique species of orchids called O. sancta commonly known as holy orchid and considered as an elegant perennial plant.The existence of alkaloids in orchid and considered as an elegant perennial plant.The existence of alkaloids in orchid constituents suggests that orchids possess some biological activity against disease and cancer (Bulpitt et al., 2007).Even so, the antimicrobial activity of O. sancta was not evaluated.The native plant to Jordan, P. umbonatum, is grown wildly in many parts of the world.Flowers of P. umbonatum are used for treating various mild pains (Table 1).Recently, it was revealed that active phytochemicals found in P. umbonatum are antioxidants and contain various phenolic compounds (Bernáth, 2006).Nonetheless, no previous antimicrobial investigations of P. umbonatum have been described in the literature.Therefore, no previous studies were performed concerning the use of those medicinal plants selected in this study as sources of antibacterial and antifungal agents.Thus, this study is established to determine the antibacterial and the antifungal activities of those selected medicinal plants.Collected plant materials (roots, leaves, fruits, and flowers) from each plant species were washed and dried in the shade in an aerated place at room temperature until complete drying.The dried plant material was ground into a fine powder for initiating the extraction process.

Preparation of plant extracts
Each powdered plant material was soaked in each of acetone, Ethanol, methanol, and hot water solvents (plant material to solvent ratio was 1:10, w/v) and extracted for two weeks at room temperature with shaking at 150 rpm.All extracts were filtered through White canvas and filter paper.The collected filtrates of the extracts were dried by evaporation until dryness.The dried extracts were weighed and dissolved in 0.05% dimethyl sulphoxide (DMSO) to a final stock concentration of 200 mg/ml.All crude extracts were purified by filtration through 0.22 m filter units and kept at -20°C until use.

Preparation of inoculums
Test bacteria and test fungi were grown in nutrient broth (NB) at 37°C for 24 h and in sabouraund dextrose broth (SDB) at 28°C for 48 h, respectively.Bacterial and fungal cultures were serially diluted and adjusted to achieve 2×10 6 colony forming units (CFU/ml) for bacteria and 2×10 5 spore/ml for fungi (Ceylan et al., 2008).

Antimicrobial activity
The antibacterial activity of plant extracts was screened in triplicates against test bacteria by using the agar well diffusion method (Perez et al., 1990).An inoculum of 100 μl bacterial suspension was swapped uniformly to solidified nutrient agar (NA) plates and the plates were allowed to dry for 5 min.Holes of 5 millimeters (mm) in diameter were made in the seeded agar using sterile cork borer.Aliquot of 50 μl from each plant extract was added into each hole on the seeded medium and allowed to stand for one hour for proper diffusion and incubated at 37°C for 24 h.The antibacterial activity was evaluated by measuring the inhibition zone diameter in mm around the wells.The antifungal activity of plant extracts was performed in triplicates against test fungi in the same manner as described for bacteria but by using sabouraund dextrose agar (SDA) plates and incubation at 28°C for 48 h.

Determination of the minimum inhibitory concentration and minimum microbial concentration
The minimum inhibitory concentration (MIC) and the minimum microbial concentration (MMC) were determined for plant extracts that showed the most significant antimicrobial activity according to the modified methods previously described by Obeidat (2011).All samples were examined in triplicates.

Phytochemical screening
The phytochemical components of promising extracts (extracts that exhibited broad spectrum antibacterial activity and extracts that showed the most significant antifungal activity) from the selected medicinal plants in this study were performed according to the standard protocols; Mayer's test for alkaloids (Siddiqui and Ali, 1997), sodium hydroxide test for flavonoids (Roopashree et al., 2008), foam test for saponins (Roopashree et al., 2008), and ferric chloride test for tannins (Iyengar, 1995).

Statistical analysis
All measured inhibition zones were expressed as the mean ± standard error (SE).For statistical evaluation of data for generated inhibition zones, one-way ANOVA (Tukey's studentized range) was applied using the program IBM SPSS statistics 19.0 for Windows.Significant differences were considered significant at P < 0.05.

RESULTS
Antimicrobial activities were estimated by measuring the average diameters of the formed inhibition zones around wells.The inhibitory effects of plant extracts prepared by different solvent (water, Ethanol, methanol, and acetone) from A. carneum, D. ithaburense, L. hirticarpus, O. blancheanum, O. jordanicolum, O. sancta, and P. umbonatum were investigated against 10 antibioticresistant test microorganisms including eight bacteria and two fungi.In general, most plant extracts of selected plants in this study showed a broad spectrum of antimicrobial activity.Although all plant parts of selected  plants were screened for the potential antimicrobial activity, it was found that antimicrobial activity was produced from leaves of A. carneum and D. ithaburense from flowers of L. hirticarpus, O. blancheanum, O. jordanicolum, O. sancta, and P. umbonatum (Table 2).It was found that flower extracts of O. jordanicolum were the best source for antimicrobial agents.All test microorganisms were affected by at least one extract.It was found that all flower extracts of O. jordanicolum exhibited antibacterial activity against Gram-positive bacteria except acetone extract which did not show antibacterial activity against S. pyogenes (Table 2).Methanol and water extracts were significantly the most active extracts against S. aureus and MRSA, respectively (Table 2).
Ethanol extract of O. jordanicolum exhibited antibacterial activity against Gram-negative bacteria (Table 3).On the other hand, water and ethanol extract of O. blancheanum, which belongs to the same plant genus, showed a broad spectrum of antimicrobial activity with significant antibacterial activity against S. aureus.
As shown in Table 2, the aqueous and methanol extracts of P. umbonatum flowers exhibited significant antibacterial activity against S. pyogenes.Remarkably, it was found that leaf extracts of D. ithaburense (methanol and acetone extracts) and A. carneum (water and ethanol extract) produced significant antibacterial activity against K. pneumonia (Table 3).
It was observed that S. typhimurium (Gram-negative bacterium) followed by MRSA (Gram-positive bacterium) and P. aeruginosa (Gram-negative bacterium) are the least sensitive among all test microorganisms used in this study (Table 2 and 3).The bacterium S. typhimurium was affected by only three extracts (water and ethanol extract of A. carneum and ethanol extract of O. jordanicolum); ethanol extract of A. carneum gave the most significant antibacterial activity (Table 3), whereas eight extracts inhibited MRSA growth (Table 2).Interestingly, all extracts of O. jordanicolum exhibited anti-MRSA activity.The highest significant anti-MRSA activity was produced from aqueous extracts of O. jordanicolum and O. sancta flowers.It was found that only 13 extracts (out of 32) exhibited antibacterial activity against P. aeruginosa (Table 3).The most significant antibacterial activity against P. aeruginosa was obtained from aqueous extract of A. carneum and acetone extract of O. sancta.However, the most sensitive bacteria were P. mirabillis and E. coli, which were affected by most plant extracts (Table 3).The growth of P. mirabillis was inhibited by all plant extracts except acetone extract of D. ithaburense.The bacterium E. coli was insensitive to only three plant extracts; ethanol and acetone extracts of L. hirticarpus and aqueous extract of O. jordanicolum.
For antifungal activity, it was observed that the test fungi was inhibited by few plant extracts (Table 4).For instance, it was observed that only water extract of A. carneum leaves, ethanol extract of L. hirticarpus flowers, and methanol extract of O. sancta flowers exhibited antifungal activity against A. brasiliensis and C. albicans (Table 4).However, it was found that all flower  extracts of O. jordanicolum exhibited antifungal activity.Furthermore, acetone extract of O. jordanicolum flowers produced the highest significant antifungal activity against A. brasiliensis and C. albicans.It was interesting to notice that acetone extract of D. ithaburense leaves also exhibited significant antifungal activity against both test fungi (Table 4).It was found that methanol extract of O. blancheanum and aqueous extract of P. umbonatum exhibited high antifungal activities.
Significant antibacterial activities, expressed as MIC and MMC, of promising plant extracts against test bacteria are shown in Table 5.Extracts of selected plants were among the most active with MIC values ranging from 4 to 32 mg/ml and MMC values ranging from 8 to 64 mg/ml.The antibacterial activities with the best MIC and MMC values were produced significantly by ethanolic extracts of A. carneum against E. coli and O. blancheanum against S. aureus and by methanolic extracts of O. jordanicolum against S. aureus and P. umbonatum against S. pyogenes (Table 5).Whereas, MIC and MMC values of the most active antifungal plant extracts ranged from 32 to 64 mg/ml and from 64 to 128 mg/ml, respectively.
The antifungal activities with the best MIC and MMC values were produced significantly by acetone extracts of O. jordanicolum followed by D. ithaburense against A.  The efficacy of plant extracts evaluated as antimicrobial agents was dependent on the solvent of extraction.In general, ethanol was the best extracting solvent from A. carneum leaves and from flowers of O. blancheanum and O. jordanicolum, produced broad-spectrum and/or significant antibacterial activity (Tables 2 and 3).Whereas, extraction of D. ithaburense leaves, L. hirticarpus flowers, and O. sancta flowers by hot water was found to be the best method for extraction of antibacterial active compounds while methanol appeared to be the appropriate solvent for extraction of antibacterial agents from P. umbonatum flowers.For production of broad spectrum antifungal activity, it was observed that acetone is the best extracting solvent from most selected medicinal plants.Obeidat et al. (2012) reported that ethanol is the best extracting solvent for plant leaves.Alzoreky and Nakahara (2003) found that both methanol and acetone proved to be suitable solvents for extraction of bioactive inhibitory effects from medicinal plants.Eloff (1998) and Cowan (1999) revealed that methanol was more efficient than acetone in extracting phytochemicals from plant materials.In accordance with those contradictory findings and in agreement with Obeidat et al. (2012), the results of this study indicate that the antimicrobial efficacy depends on selected plant species, plant part, solvent type, test microorganism, and phytochemical components.
The obtained results show that most plant extracts displayed antibacterial activity against all test bacteria except S. typhimurium which was affected only by three extracts.In contrast to previous findings (Ripa et al., 2009;Obeidat et al., 2012), this study showed in general no significant differences in susceptibility of Grampositives and Gram-negative bacteria to plant extracts but unfortunately most extracts produced no or even limited antibacterial activity against S. typhimurium.
diameters are expressed as Means±SE.The means±SE within column followed by the same letter are not significantly different (Tukey's studentized range test: α = 0.05).
diameters are expressed as Means±SE.The means±SE within column followed by the same letter are not significantly different (Tukey's studentized range test: α = 0.05).

Table 1 .
Selected plant species for this study and their uses in folk medicine.

Table 2 .
Antibacterial activity of extracts from selected medicinal plants against pathogenic Gram-positive bacteria.

Table 3 .
Antibacterial activity of extracts from selected medicinal plants against pathogenic Gram-negative bacteria *de *Inhibition zone diameters are expressed as Means±SE.The means±SE within column followed by the same letter are not significantly different (Tukey's studentized range test: α = 0.05).

Table 4 .
Antifungal activity of extracts from selected medicinal plants against pathogenic fungi.

Table 5 .
Minimum inhibitory concentration and minimum microbial concentration of the most significant active extracts of selected medicinal plants

Table 6 .
Screening of phytochemical components of selected medicinal plants' extracts which displayed broad-spectrum antibacterial activity.

Table 7 .
Screening of phytochemical components of selected medicinal plants' extracts which displayed the most significant antifungal activity.Generally, it was revealed that most prepared extracts from leaves of A. carneum and D. ithaburense and from flowers of L. hirticarpus, O. blancheanum, O. jordanicolum, O. sancta, and P. umbonatum exhibited promising antibacterial and antifungal activities.Thus, this study provides valuable information on the abilities of the extracts of selected medicinal plants to yield bioactive compounds that could be potentially used to treat diseases caused by multidrug-resistant bacteria and fungi.