Antimicrobial activities of six plants used in Traditional Arabic Palestinian Herbal Medicine

Ethanolic extracts of six plants: Arum palaestinum Bioss, Urtica pilulifera L., Thymbra capitata (L.) Cav., Origanum syriacum L., Teucrium creticum L., and Teucrium polium L., used in Traditional Arabic Palestinian Herbal Medicine were evaluated for their antibacterial, anti-candida, and antidermatophyte activities using well diffusion, micro-dilution and food poisoned techniques. The extracts were tested against: six bacterial strains including Staphylococcus aureus, Proteus vulgaris, Pseudomonas aeruginosa, Salmonella typhi, Escherichia coli and Klebsiella pneumoniae; five Candida albicans isolates, and two dermatophytes: Microsporum canis, and Trichophyton rubrum. The most active plants extracts were T. capitata and O. syriacum against the tested bacteria, while the remaining plant extracts did not express any activity or exhibited only very low activity against tested bacteria species and candida isolates. O. syriacum was also the most active plant against all Candida strains with inhibition zones that ranged from 22.5 to 29.5 mm. On the other hand, T. capitata extract showed the highest activity against the test dermatophytes (producing a complete inhibition at ≤ 45 μg/mL).


INTRODUCTION
In Palestine, the screening of flora for pharmacological active compounds started in the late nineties (Ali-Shtayeh et al., 1998) and continue to provide useful means for treating ailments (Ali-Shtayeh and Jamous, 2008).Little scientific research has been carried out to investigate the plants used in Traditional Arabic Palestinian Herbal Medicine (TAPHM) (Ali-Shtayeh et al., 1998;2003).In the course of our investigations we found that several plants of the Palestinian ethno-medicine possess interesting biological activities, which could be of interest for all parts of the world.The activities have been selected because of their strong medicinal relevance (Saad et al., 2005;Abu-Lafi et al., 2007).
Arum palaestinum Bioss (Araceae) is considered edible after being soaked in salty water or dried.The plant is also used in folk medicine to treat several diseases such as stomach acidity, atherosclerosis, cancer, diabetes and food toxicity (Ali-Shtayeh et al., 1998).Phytochemical and biological investigations carried out on the plant have shown that it possesses an inhibitory effect on smooth muscle contraction in rats (Afifi et al., 1999), anticancer activity against lymphoblastic leukemia due to the presence of a pyrrole alkaloid (El-Desouky et al., 2007), and antioxidant activity (Husein et al., 2014).Urtica pilulifera L. (Urticaceae), has long been used in traditional medicine in many countries around the world to treat various ailments including: sore joints, rheumatism, hemorrhage, renal stones and inflammation of the bladder, diabetes mellitus and other ailments (Kavalali et al., 2003;Lopatkin et al., 2005;Irshid and Mansi, 2009).
Thymbra capitata (L.) Cav.(Lamiaceae) is an aromatic plant found in Palestine and locally known under the common name Zaa'tman.The essential oil of the plant was reported to have antimicrobial activities most of which are mediated by thymol and carvacrol (Bhaskara et al., 1998).The plant is also important as a source of perfume, cosmetics, flavoring and pharmaceutical industries (Tabata et al, 1988).In traditional food the plant has been used for its flavors as refreshing drink or in cooking.It is used in folk medicine against cold, influenza and throat infection (Ali-Shtayeh and Jamous, 2008).Later on the plant was found to contain antiseptic and antimicrobial agents (Bremnes, 2002).
Origanum syriacum L. (Lamiaceae) is one of the most popular herbs among Palestinians (Ali-Shtayeh and Jamous, 2008).The green leaves of the herb are rich in essential oil, which is responsible for its characteristics of flavor and fragrance.Oil of cultivated O. syriacum is an important commercial product and is obtained mainly by steam distillation of the fresh leaves.Plant extract is also found to have strong biological activity, and this may be due to the presence of phenols, thymol and carvacrol as major constituents of thyme oil in the plant (Abu-Lafi et al., 2007).
Teucrium creticum L. (Lamiaceae) is found in Palestine and locally known under the common name Ja'adh.The plant is used traditionally to cure diabetes in Palestine (Ali-Shtayeh et al., 2012).No phytochemical studies were found on this plant, and this may be attributed to its limited distribution in Palestine (Saad et al., 2005).
In this study, the ethanolic extracts prepared from six Palestinian medicinal plants have been investigated for their antibacterial and antifungal activities by means of the agar diffusion, micro-dilution and food-poisoned methods.In addition, the current study reports values of minimum inhibitory concentration (MIC) for active plants extracts.

Plants extracts preparation
Fifty grams of each dried plant was ground using a Molenix (Mooele Depose type 241) for a minute and the resulting powder was extracted by continuous stirring with 200 ml 70% ethanol at 24°C for 72 h.Extracts were filtered through Whatman No. 4 filter paper, and the residue was then washed with additional 50 ml ethanol.The combined ethanol extracts were dried using rotary evaporator followed by freeze drying and stored at -20°C for future use.
Five Candida albicans strains were used, three of which were clinical isolates from cutaneous candidiasis patients (BERC-N43, BERC-N72, and BERC-N66), and the other two from CBS type cultures: CBS6589, and CBS9120.

Disk diffusion method
Disc diffusion method was made according to Zongo et al. (2009).
Dried plant extracts were dissolved in DMSO to a final concentration (100 mg/mL) and sterilized by filtration through a 0.45 µm membrane filter.Inoculums (10 6 bacterial cells/mL) were spread on Muller-Hinton agar plates (1 mL inoculum/ plate).Filter paper discs (6 mm in diameter) were individually impregnated with 40 µL of each plant extract, and controls, and placed onto the surface of inoculated Petri dishes.Before incubation, all Petri dishes were kept in the refrigerator (4°C) for 2 h and incubated after at 37°C for 24 h for bacteria growth.After incubation, the diameters (mm) of inhibition zones were measured including diameter of discs.The antimicrobial potentials were estimated according to the index reported by Rodriguez et al. (2007).All the experiments were done in triplicates.Gentamicin (10 mg/mL) and DMSO served as a positive and negative control, respectively.

Well diffusion method
Anti-Candida susceptibility testing was done using well-diffusion method to detect the antifungal activities of plant samples (Perez et al., 1990).A sterile swab was used to evenly distribute fungal culture over Muller-Hinton agar plates supplemented with glucose-methylene blue.The plates were allowed to dry for 15 min before use in the test.Wells were then created and 50 µL of the crude extract (100 mg/mL) of each plant extract were pippeted into each well.The same extract was used in each plate; with a total of two plates used for each extract including two wells for the positive and negative controls.The plates were incubated at 37ºC for 24 h after which they were examined for inhibition zones (Delahaye et al., 2009).All the experiments were done in triplicates.Amphotericin B (32 µg/mL) and DMSO were used as positive and negative controls, respectively.

Micro-dilution Test
Broth micro-dilution was performed following the CLSI M27-A2 method (NCCLS, 2002) with little modifications.Muller-Hinton media (pH 7.2) was used for bacteria, while Muller-Hinton supplemented with glucose-methylene blue was used for Candida.Plant extracts were dissolved in DMSO and the c o r r e c t v o l u m e w a s pippeted in the first micro-well plate with Muller-Hinton, for the concentration of each plant extract to be 25 mg/mL in that well.The cell suspension was prepared in 0.85% saline, with an optical density equivalent to 0.5 McFarland standards, and diluted 1:100 in the media to obtain a final concentration of 1 × 1 0 4 to 5 × 1 0 4 colony-forming units per milliliter (CFU/mL).This suspension was inoculated in each well of a micro-dilution plate pr evious ly p r e p a r e d w it h t he plant extracts to give concentrations from 25 mg/mL down to 0.012 mg/mL (Scorzoni et al., 2007).The plates were incubated with agitation at 37°C for 24 h for all species.The control drugs were gentamicin for bacteria strains, and amphotericin B for Candida, respectively.Concentrations of controls were ranged from 250-1 µg/mL for gentamicin, and from 16.0-0.125µg/mL for amphotericin B. Value of minimum inhibitory concentration ( MIC), d et er m i n ed by broth m acr o-dilution, and defined as the lowest concentration of the drug completely inhibited the growth of the isolate.For plant extracts, MIC value was defined as the lowest concentration able to inhibit any visible bacterial or candidal growth.Results were read visually and spectrophotometerically.

Antidermatophytes testing
Plants extracts were tested at different concentrations for their antidermatophytes activity against the test pathogens using a modified poisoned food technique (Dikshit and Husain, 1984).Different amounts of each extract were incorporated in pre-sterilized SDA medium to prepare a series of concentrations of the extract (15, 30, 45, and 60 µg/mL).A mycelial agar disk of 5 mm diameter was cut out of 12 days old culture of the test dermatophytes and inoculated onto the freshly prepared agar pl at es .In controls, sterile distilled water was used in place of the tested sample as a n e g a t i v e c o n t r o l , w h i l e econazole (5 µg/mL) was used as the positive control.Three replicate plates were used for each treatment (concentration).The inoculated plates were incubated in the dark at 24C and the results were recorded after 10 days.
Percentage of mycelial inhibition was calculated using the following formula: Where, dc is the colony diameter of the negative control and ds is colony diameter of the sample.

RESULTS
Six medicinal plant species belonging to 3 families were selected based on their uses in Traditional Arabic Palestinian Herbal Medicine (TAPHM) for the treatment of various ailments.The antibacterial, anti-Candida and antidermatophytes activities of ethanolic extracts of the selected plants were screened in this study.

Antibacterial activity
The ethanolic extracts of six Palestinian plants were subjected to a preliminary screening for antimicrobial activity against six human pathogenic bacteria S. aureus, E. coli, K. pneumoniae, P. vulgaris, P. aeruginosa and S. typhi.The inhibition zones of the plant extracts against different types of bacteria strains and MIC values of plants extracts are presented in Table 1.Gentamicin was used as a positive control.Two plant extracts (T.capitata, and O. syriacum) inhibited the growth of all tested bacteria strains.The MIC values for T .capit at a were 390 µg/mL for Pr=.vulgaris, E. coli, and K. pneumonia while for O. syriacum it was 390 µg/mL for K. pneumonia.Other plants extracts revealed very low or no activity against different types of bacteria (Table 1).

Anti-candida activity
The anticandida activity of tested plants extracts was evaluated according to their mean of inhibition zone (100 mg/mL) against various candida isolates, and t h e MIC values of active plants extracts against the five isolates of Candida.Results o f inhibition zone were compared with the activity of standard amphotericin B (32 µg/mL).Origanum syriacum and T. capitata exhibited strong and moderate inhibition activity against candida isolates, while the other 4 plant extracts show no activity (Table 2).The MIC value for O. syriacum is 150 µg/mL   against the type culture isolate, while it was 390 µg/mL against the clinical isolates.

Antidermatophyte activity
The antidermatophyte activity of the tested plants extracts was expressed as the means of % inhibition values of dermatophytes growth.Four different concentrations of plants extracts concentrations were tested.The percent of inhibition of the two dermatophytes ranged between 49.6-100%, with T. capitata exhibiting a significant % of inhibition (100%) against the two dermatophyte species at a concentration of 45 µg/mL comparable to the positive control (Table 3).

DISCUSSION
Substances derived from plants have recently attracted much attention with regard to human health, due to their low cost, broad availability and limited toxicity.Thus, plant based antimicrobial compounds have vast therapeutically potential as they can serve the purpose without any side effects that are often associated with synthetic drugs.

Antimicrobial activity
The primary results of bioassays for plants extracts open the possibility of finding new clinically effective antimicrobial compounds, such as the extract of Micromeria nervosa which is found in different locations of Palestine and are well known plant used for various medical purposes ( Ali-Shtayeh et al., 1997).
Antimicrobial activity of plant materials can be classified according to their MIC values.MIC values lower than 500 indicate the strong antimicrobial activity.Furthermore, MIC values equal to 600-1500 µg/mL and higher than 1600 µg/mL indicate intermediate and weak antimicrobial activities respectively (Durate et al., 2007;Fabri et al., 2009;Michielin et al., 2009).Therefore, it was clear from the results of inhibition zone that the most pronounced activity against all test bacteria with inhibition zones ≥12 mm at a concentration of 100 mg/mL was shown by the ethanolic extracts of O. syriacum and T. capitata.The majority of the remaining pl ant extracts d i d not express any activity (Table 1).This tends to show that the biological activities of the plant extracts are different from one plant to another.The absence of antibacterial activity of ethanolic extracts of those plants indicates ei t her the absence of active chemical compounds or the insolubility of the active ingredients in the solvent used.
The activity of T. capitata and O. syriacum might be attributed to the active compounds in their essential oils.O. syriacum is an aromatic, perenial, herbaceous plant.The plant has been reported to pose antibacterial activity (Leja and Thopil, 2007).The major volatiles and semivolatiles of Palestinian wild O. syriacum are αphelandrene, α -pinene, b-myrecene, o-cymene, pcymene, c-terpinene, thymol, and carvacrol (Abu-Lafi et al., 2008).The plant has a long history of medicinal use among Palestinian (Ali-Shtayeh and Jamous, 2008) as well as by the Creks as an antidote to poisoning and snake venom, by the Romans for stomach disorders and more recently for digestive, antispasmodic and sedative properties (Evans, 2002).
Continued further exploration of plant-derived antimicrobials is needed today.Further research is necessary to determine the identity of antibacterial compounds from these plants and also to determine their full spectrum of efficacy.However the present study of in vitro antibacterial evaluation of some extracts forms a primary platform for further phytochemical and pharmacological studies to discover new antibiotic drugs.

Anti-Candida activity
The results reveal that extracts of T. capitata and O. syriacum are potent antimicrobials against all the microorganisms studied.O. syriacum showed inhibition zone o f 29.5±0.5 mm against BERC N66, (94% of amphotericin B activity) and T. capitata (inhibition zone 20.0 mm) against the same genotype of Candida.Other extracts did not show significant activity against the studied microorganisms (Table 3).It is noteworthy to mention that plants belonging to the same family sometimes exhibited comparable anticandidal activity since these plants are expected to possess similar active constituents (T.capitata, and O. syriacum of the Family Labiateae), while some plants of the same family exhibited different effects (T.creticum and T. polium of the Family Labiateae) indicating that there might be some constituents found in one member of the family but not in the other.

Antidermatophyte activity
Only a few antifungal substances are known or available in the market as compared to antibacterial substances.Antimycotic substances are also relatively unsatisfactory in the control of dermatophytes.The discovery of active components exhibiting a broad spectrum antifungal activity may prove useful for the development of antifungal agents.Laboratory assessment showed the nature of fungi static activity encountered in plant extracts.Table 3 sh o w s percentage of inhibition due to plant type at (45 µg/mL) concentration.Values of % inhibition increase with increasing concentration of each plant extract (Figure 1).
All tested plants revealed antimycotic activity against all t w o tested dermatophytes, with T. capitata exhibiting the highest activity; a complete inhibition by the ethanolic extract of th i s p l a n t was at a concentration lower than 45 µg/mL.O. syriacum extract have also showed considerable antimycotic activity against the same tested dermatophytes, complete inhibition was at a concentration ˂60 µg/mL.The oil of T. capitata have been shown to exhibit antidermatophytic activity, with MIC values ranging from 0.08 to 0.32 μL/mL (Salgueiro  , 2004).In addition to these plants, A. palaestinum showed 50% inhibition against M. canis at a concentrationof ~45µg/mL.
The present work has shown that most of the studied plants are potentially good source of antidermatophytes and demonstrates the importance of such plants in medicine and in assisting primary health care.However, the screened plants are among the wild edible plants consumed by Palestinian (Ali-Shtayeh et al., 2008).From those results we can suggest that plants extracts investigated here could find practical application in the introduction of highly active and safe antimicrobial agents including antidermatophytes.These results justify the usage of those plants in folk medicine (Ali-Shtayeh et al., 2008, Ali-Shtayeh andJamous, 2008).

Conclusion
Overall, some crude extracts prepared from plants commonly grown in Palestine or commonly used by Palestinian were found to exert, in-vitro some antimicrobial effect.However, further work is needed to isolate and identify the active compound(s) from active plant species.

Table 1 .
Inhibition zones (mm) of plant extract against different types of bacteria strains using the disc diffusion method.
* Values of inhibition zone diameter in mm ± SD.

Table 2 .
Inhibition zone (mm) and MIC values of tested extracts against Candida albicans i s ol at es compared to amphotericin B.

capitata O. syriacum Amphotericin B T. capitata O. syriacum Amphotericin B
* Values of inhibition zone diameter in mm ± SD.