Assessment of the antibacterial effects of Moringa peregrina extracts

Al-Ghad International College for Applied Medical Sciences, Tabuk, Saudia Arabia. Department of Biology, University of Jordan, Amman, Jordan. Department of Biology, Mutah University, Karak, Jordan. Department of Medical Support, Al-karak University College, Al-Balqa' Applied University, Jordan. Faculty of Medicine, University of Hail, Hail, KSA. Department of Laboratory Medical Sciences, Princess Aisha Bent Al-Hussein Faculty of Nursing & Health Sciences, Al Hussein bin Talal University, Jordan.


INTRODUCTION
Since the early stages for human society's evolution, the usage of medicinal plants in folk medicine is highly a potential regiment for several disorders therapy.A small percentage of plants is used by humans as food source, eventually the majority are used for medicinal purposes.Medicinal plants are considered as one of the major constituents for disease therapy in rural areas.Most of the populations in the world depend on herbal medicine for their health care needs (Manandhar, 1994).
Phytochemicals in fruits, vegetables, spices and traditional herbs have been found to play a protective role against human diseases (Schippmann et al., 2002).Different plant extracts as crude, fractionated and sometime whole plant compounds are used for their antibacterial, anti-inflammatory, and antioxidant activities (Lev and Amar, 2002).
In addition, a high sensibility is paid for the usage of medicinal plants in pharmaceutical industries not only due to the toxicity of new synthetic drugs, but also to their high cost.Thus, plants possess medicinal properties play an important role in the discovery of new therapeutic agents (Verma and Singh, 2008).Jordan has Mediterranean like climate and shares different demographical regions as mountains, land scabs and deserts.In Jordan, it is estimated that more than 2000 species belongs to about 700 genera of wild plants grown over the Kingdom.From these species, about 485 (99 families) are used as medicinal plants (Oran and Al-Eisawi, 1998).Ethnopharmacological examination of traditional drugs showed that there are 236 local and imported plants used in Jordan traditionally for treatment of different diseases (Lev and Amar, 2002).The genus Moringa which is called miracle tree belongs to "Moringaceae" family with 13 known species (Olson, 2002).Moringa peregrina is known in Arabic as "Habb El Yasar, Habb El Pan", the seeds are known as "Habba Ghalia".It is widely grown in dry or semiarid countries neighboring the Red Sea, from Somalia and Yemen to Palestine and to Syria (Somali et al., 1984).The development and increase of antibiotic resistance, as well as the continuously evolving new strains of disease causing bacteria, have pointed to the need for new and safe antibacterial agents discovery.Medicinal plants are an attractive source for new discoveries in antibacterial agents (Cowan, 1999).Various parts of M. peregrina possess antibacterial activity (Lockett et al., 2000;Anwar and Rashid, 2007).Majorly, the mechanism of action for these antibacterial compounds is either to confer a death of the microorganism (bactericidal) or by preventing their growth (bacteriostatic).This is just a listing of antibiotic effects in general.More recently, M. perigrina aerial parts were fractionated using n-hexane and e --amyrin -sitosterol-3-O--D-glucoside and apigenin were tested against various bacterial and fungal species.Results demonstrated that each of these constituents had significant antibacterial inhibitory effect as compared with standard antibiotics (Abdel-Rahman et al., 2010).
Quinones naturally occurring phenol compounds are found in Moringa oleifera (genus Moringa "Moringaceae" family with 13 known species: mentioned in the introduction) with laxative effects.M. oleifera leaves content such as terpenoids and steroids have antimicrobial effect against S. aureus, alkaloids (nitrogencontaining compound) have antimicrobial activity due to their ability to disrupt microorganisms DNA (Bennett et al., 2003).Moringa flavonoids, which are many in numbers, have a strong antibacterial activity against many microorganisms by inhibiting the enzyme bound membrane activity (Li-Weber, 2009).M. peregrina are taxa of the natural flora in Jordan especially in Wad-Bin-Hammad and Wade-Arabah.This is the first assessment of antibacterial effect of the M. peregrina plant ethanolic crude extracts in Jordan.

Plant
M. peregrina wild trees were collected on June from the Wadi Bin-Hammad Valley in the South of Jordan in Karak.Root, seed and leaf each part were collected separately (exposed root on the surface, seed from large dried pods from under of the tree, and semi dried leaf).

Preparation of ethanolic extract
After collection of the plant material, the leaves, roots and seeds were dried in shade at room temperature (27 to 30°C) to constant weight (to calculate the %yield of the plant extract = dry weight of extract (g)/initial weight of plant sample × 100%).Different parts of the plant were powdered by mortar and pestle and further reduced to a fine powder using blender.Powdered sample were transferred to closed flasks.Each flask sample was extracted with 95% ethanol.Each sample (50 g) mixed with 500 ml of 95% ethanol, kept for shaking at 120 rpm for half an hour and allowed to precipitate at room temperature for 72 h.After 72 h, each extract was filtered by Whatman filter paper.The resulting filtrates were then concentrated in rotary evaporator and left to completely dry.

Bacterial strains and antibacterial activity
Three different bacterial species were used Escherichia coli (E. coli ATCC25922), Staphylococcus aureus (S. aureus ATCC 43300) and Klebsiella pneumonia (K.pneumonia ATCC 13883).These species were kindly provided by Dr. Wael Zereini, Department of Biological Sciences at Mut' ah University, Karak, Jordan.
Antimicrobial activity of M. peregrina ethanolic extracts was evaluated by disc diffusion method (Kirby, 1996).Briefly, inoculum containing 106 colony forming unit (CFU)/ml was spread on nutrients agar plates.Sterile filter disc (diameter 6 mm) emerging with 17 μl of ethanolic extract in minimum amount of solvent (17 μl which is the maximum capacity of the disc) at 10, 50 and 100 mg/ml concentrations.One disc paper emerging with 17 μl of ethanol was used as negative control.
Author(s) agree that this article remains permanently open access under the terms of the Creative Commons Attribution License 4.0 International License was repeated with 30 μg/disc of chloramphenicol, tetracycline and with ampicillin at 10 μg/disc.The plates were incubated at 37°C for 24 h.The antibacterial activity was detected by the measurement of the diameter of the zone of inhibitions around each filter disc.Each sample was repeated three times (Romero et al., 2005).

Determination of minimum inhibitory concentration (MIC)
The MICs of the M. peregrina ethanolic extracts were identified in triplicates.0.5 ml of different concentrations of the extracts (from 20 to 0.5 mg/ml) and 2 ml of the nutrient broth were added and first inoculum containing 106 CFU/ml of each organism was added to each tube.A tube without any extracts was used as negative control (media and bacteria), stranded antibiotics (antibiotics disc chloramphenicol has known effect), chloramphenicol was used as positive control.All broth samples were incubated at 37°C for 24 h.The antimicrobial results were measured by observing turbidity at 600 nm wavelength (Bibitha et al., 2002).

Antibacterial effect of M. peregrina ethanolic extracts
The antibacterial activities of M. peregrine leaves, roots, and seeds ethanolic extracts against E. coli, S. aureus and K. pneumonia were tested and compared to known antibiotics (Tables 1, 2, and 3).The results showed that increasing the concentration of the ethanolic extracts increased the zone of inhibition.
In addition, the ethanolic roots' extracts have significantly higher antibacterial activity against E. coli, S. aureus and K. pneumonia than the leaves or seeds' extracts (Tables 1, 2 and 3).
Furthermore, the antibacterial activity of the M. peregrina extracts against gram positive and negative bacteria seemed to be similar.Regarding MIC results, similar results were obtained.The roots' ethanolic extracts yielded lower MIC values, which agreed with the zone of inhibition studies (Table 4).In comparison to known antibiotics, M. peregrina ethanolic roots' extracts of 850 µg have higher antibacterial activities than 30 μg/disc of chloramphenicol, tetracycline and ampicillin at 10 μg/disc (Tables 1, 2 and 3). 100 µl of the extracts prepared at three different concentrations (10, 50, and 100 mg/ml) were pipetted onto filtered paper disc and allow to dry overnight at 37°C).

DISCUSSION
Traditionally plants with medicinal value have been widely used throughout human history and still are used in many regions around the world (Heinrich, 2000).
There is an increasing interest in medicinal plants and homeopathic contemporary medicine especially with the raised awareness of toxic or unwanted side effects of classical drugs (Uprety et al., 2012).The current study, aimed to assess the antibacterial effect of the endemic Jordanian M. peregrina plant.There is plethora of studies assessing the role of the Moringa plant in folk medicine in many countries (Elbatran et al., 2005;Gupta et al., 2010;Dehshahri et al., 2012).However, there are neither actual uses of M. peregrina in Jordan, nor studies done to assess its antibacterial activity of the Jordanian M. peregrina ethanolic extracts.Therefore, the present study examined the effect of short-term application of the ethanolic extracts from different parts of M. peregrina on the antibacterial activity against three different bacterial species.Previous studies demonstrated high prevalence of active antibacterial substances in plants.Worldwide, plants and herbs are used differently as remedy for certain diseases (Brantner et al., 1996).In the present study, M. peregrina ethanolic extracts exhibited antibacterial activity against broad spectrum of gram negative and gram positive bacteria.The roots ethanolic extract of the plant showed the highest antibacterial effects.Seeds and leaves of the plant also showed antibacterial activity against three bacterial strains, but less than roots extract.
Furthermore, ethanolic extracts showed a dose dependent activity against different bacterial species, but interestingly MIC values were significantly higher against three bacterial species.Afolayan and Meyer (1995) showed low susceptibility of gram negative bacteria toward plant extracts.In this study, M. peregrina ethanolic extracts exhibited antibacterial activity against both gram positive and negative bacteria, which indicates that the seed of M. peregrina contains compounds with antibacterial activity against both gram negative and positive bacteria; this implicates an activity against protein structure or even on a transcription level, but not a specific disturbance among cell wall structure.
In conclusion, the activity of M. peregrina ethanolic extracts showed antibacterial activity against both gram positive and gram negative bacteria, which indicates that M. peregrina contains putative compounds with potential antibacterial activity against protein or DNA structures.The traditional methods of treating bacterial infection are decoction or boiling the plant in water (Nair et al., 2005).In the present study M. peregrina ethanolic extracts showed better antibacterial activity than that obtained by Nair et al. (2005).
A further advanced study should be pursued that specifically focuses on the antibacterial agent dosages of these plant parts, which may be used by the pharmaceutical industry.

Table 1 .
Zone of inhibition of ethanolic extracts of Moringa peregrina plant at different concentrations on the bacterial growth of E. coli.

Table 2 .
Zone of inhibition of ethanolic extracts of M. peregrina plant at different concentrations on the bacterial growth of Staphylococcus aureus.
*Mean of three replicates ± SD.

Table 3 .
Zone of inhibition of ethanolic extracts of Moringa peregrina plant at different concentrations on the bacterial growth of Klebsiella pneumoniae.
*Mean of three replicates ± SD.

Table 4 .
Minimum inhibitory concentration (MIC) of Moringa peregrina ethanolic extracts.The antimicrobial results were measured by observing turbidity at 600 nm wavelength.