Biological activities of Peganum harmala leaves

1 Laboratoire des maladies transmissibles et des substances biologiquement actives Faculté de Pharmacie-5000Monastir, Tunisie. 2 Laboratoire de Microbiologie C H U Fattouma Bouguiba -5000Monastir, Tunisie. 3 Laboratoire de chimie des substances naturelles et de synthèse organique 99/UR/12-26 Faculté des sciences de Monastir, 5000 Monastir, Tunisie. 4 Laboratoire de Microbiologie et de Virologie, Hôpital Européen George Pompidou, APHP, 20, Rue Leblanc 75905 Paris cedex 15, France.


INTRODUCTION
Herbal medicine represents one of the most important fields of traditional medicine all over the world.Medicinal plants are traditionally used for the treatment of pain.Formation of free radicals may play an important role in the origin of life and biological evolution, implying their beneficial effects on aging of organisms and cancer promotion (Ashok and Ali, 1999).The interest in natural antioxidants, especially of plant origin has greatly increased in recent years (Jayaprakasha et al., 2000).Natural antioxidants can protect the human body against free radicals that may cause some chronic diseases (Kinsella et al., 1993;Lai et al., 2001).The researches conducted on the antioxidant activities of some plants as natural antioxidants generally focused on the herbs and aromatic plants (Miliauskas et al., 2004;Pizzale et al., 2002).
Peganum harmala L. (Syrian rue) is a wild growing flowering plant belonging to the Zygophylaceae family and is considered an important medicinal plant.The seeds are known to possess hypothermic and hallucinogenic properties (Kuhn and Winston, 2000;Lamchouri et al., 1999).
P. haramala was shown to possess antihelmitic, lactogogue, antispasmodic, antipyretic, abortifient, emetic and emmenagogue properties (Chopra et al., 1958).Thus, the objective of this study was to determine the antimicrobial, antiviral and antioxidant properties in extracts obtained from the leaves of P. harmala grown in Tunisia.

Plant materials
Plants materials were collected in 2008 in Kerker (Tunisian centre).The plant was identified by Pr.Mohamed Chaieb, a botanist in the University of Science (Sfax, Tunisia).A voucher specimen was deposited in the herbarium of our laboratory.

Extraction
Five hundred gram (500 g) of dried P. harmala leaves were extracted with methanol (MeOH) through maceration for five days.The resulting extract was evaporated at reduced pressure to obtain a green residue (231 g) which was successively fractionated with petroleum ether, chloroform and ethyl acetate, and these fractions were used for biological tests.

Total phenolic content
The polyphenol content of the extracts were determined spectrophotometrically according to the Folin-Ciocalteu colorimetric method (Singleton and Rosi, 1965), calibrating against catechin standards and expressing the results as mg catechin equivalents (CAE) g _ 1 extract.Data presented are the average of three measurements.

Microorganisms
The microorganism strains used in the biological assays are listed in Table 1.Different American-Type Cell Culture (ATCC) reference bacteria and fungi as well as clinical isolates including methicillin susceptible (MSSA) and methicillin resistant Staphylococcus aureus (MRSA) strains were used.

Determination of minimal inhibitory concentration
Various concentrations (from 1 to 10 mgml -1 ) of extracts of P. harmala leaves were used to determine the antimicrobial activity.Overnight, broth cultures were adjusted to yield approximately 1 x 10 6 CFUml -1 of bacteria or yeast.The minimum inhibitory concentrations (MICs) were determined on brain heart infusion (BHI) agar plates (Bio-Rad, Marne la Coquette, France) by a standard method (NCCLS, 1997) with a Steers-type replicator device that delivered approximately 10 4 bacteria per spot.One milliliter (1 ml) of each of the extracts previously dissolved in 10% dimethyl sulfoxide (DMSO) were mixed for each concentration with 19 ml of BHI agar at 40°C and poured over Petri dishes.The resulting DMSO concentration was approximately 0.5%.Plates containing only medium or medium with 0.5% DMSO were used as controls to ensure that DMSO did not affect growth standard antibiotics (oxacilin, amoxicillin, ticarcillin and cefotaxim were used in order to control the sensitivity of the tested microorganism).After 18 h incubation at 37°C, the MIC was defined as the lowest concentration of the extract inhibiting the visible growth of each microorganism.Each test was carried out in triplicate.The microorganisms tested in this study were provided from the European Hospital of George Pompidou (HEGP) (France).

Cell toxicity assay
The cytotoxic activity was determined by the MTT assay (Polydoro et al., 2004).

Antiviral activity test
The test used in this paper is a modification of the generally used 'shell-vial' culture method for the early detection of the CMV infection (Alpert et al., 1985).The diagnostic method is sensitive (Pepin et al., 1991) and quantitative (Mazeron et al., 1991).The antiviral activity of the extracts of the plants was diluted in MEM-D, then the virus was added and centrifuged for 1 h at 1000 g.After centrifugation, the wells were incubated for 48 h at 37°C, and the viral antigens were revealed by immunofluorescence.The culture medium was aspirated and the cells were infected with 600 ml of the virus dilution followed by centrifugation at 1000 g for 1 h.After centrifugation, the mixture was incubated for 48 h at 37°C, then the viral antigens were revealed by immunofluorescence.
The immunofluorescence detection of the viral antigens was common for the above procedures and was as follows: at the end of incubation, the content of the wells were aspirated and the cells were fixed with cold methanol at 20°C for 10 min.The methanol was aspirated and 250 ml of anti immediate-early antibody was added and incubated for 45 min at 37°C in a humidified chamber.At the end of this time the wells were washed three times for 5 min with phosphate buffered saline (PBS).Then the FITC labeled antimouse antibody was incubated for 40 min and finally washed under the same conditions three times as previously done.After that, the cells were observed under an immunofluorescence microscope.The number of infected cells were compared with a known number of viral particles (average of 1000 particles) in the presence or absence of the plant extract.Because the extracts were dissolved in dimethylsulphoxide (DMSO) which positively affects the infectivity of the viruses (Li and Fong, 1990), DMSO was also included in the wells without any extract.The antiviral activity was determined by counting the number of cells infected by the virus (presenting fluorescent nuclei) in comparison with the number of infected cells in the reference well (no presence of any antiviral agent).The procedure was selected to compare the antiviral activity of the extracts and references ganciclovir and ribavirine.The reference well contained cells infected with the virus without any antiviral agent.The above described experiments were done three to four times.

Antioxidant activity by ammonium thiocyanate method
The lipid peroxidation inhibition activity was measured in a linoleic acid emulsion system according to the methods of Osawa and Namiki (1985).Briefly, a sample (1.3 mg) was dissolved in 10 ml of 50 mM phosphate buffer (pH 7), and a solution of 0.13 ml of linoleic acid and 10 ml of 99.5% ethanol was added.Then the total volume was adjusted to 25 ml with distilled water.The mixture was incubated at 40°C for 5 days in a dark room, and the degree of oxidation was evaluated by measuring the ferric thiocyanate (FTC), according to Mitsuda et al. (1966).A total of 100 ml of the oxidized linoleic acid solution described above was mixed with 4.7 ml of 75% ethanol, 0.1 ml of 30% ammonium thiocyanate and 0.1 ml of 0.02 M

Total phenolic content
The results indicate that among the tested extracts, methanolic and butanolic extracts had the highest total phenolic content (112.5;63 mgCEg -1 ) Figure 1.The ethyl acetate extract had the lowest total phenolic content (25.7 mg CEg -1 ).The results indicate that the efficiency of methanol for the extraction of total phenolic compounds was the highest.

Antibacterial activity
The results of the antibacterial and antifungal activities of P. harmala leaves extracts are presented in Table 1.The chloroformic, ethyl acetate, butanolic and methanolic extracts of P. harmala leaves all displayed good antifungal activity with MIC values of 2.5 mgml -1 .Chloroformic and methanolic extracts showed important antibacterial activity against gram positive bacteria than gram negative bacteria with MIC values ranging between 0.251 and 2.5 mgml -1 (Table 1).Butanolic and ethyl acetate extracts showed antibacterial activity against gram positive bacteria with MICs values ranging between 0.512 and 5 mgml -1 but this activity is lower than the other extracts.

Antiviral activity
The antiviral activity of the extracts of leaves of P. harmala in different concentrations in comparison with that of ganciclovir are summarized in Table 2.According to this table, all extracts had anti-HCMV activity in different concentrations except petroleum ether extract which did not have any antiviral activity.The most active extract seemed to be methanol extract.The active concentrations of the different extracts ranged from 25 mgml -1 (80% activity) to 100 mgml -1 (95% activity).As shown in Table 2, all extracts did not exhibit important anti-Cox B3 viruses, only methanol extract manifested moderate antiviral activity against HCMV.

Antioxidant activity by ammonium thiocyanate method
The antioxidant effects of the extracts from the leaves of P. harmala and α-tocopherol on the peroxidation of linoleic acid were investigated and the results are presented in Table 3.In the ammonium thiocyanate system, oxidation of linoleic acid was effectively inhibited by the methanol extract (75.9 ± 0.3), butanol and ethyl acetate extracts (67.8 ± 0.48 and 60.8 ± 0. 3, respectively) after incubation for 120 h (5 days).As seen in Figure 2, methanol extract displayed higher antioxidant activity when compared with positive control α-tocopherol (80.12 ± 0.4).

DISCUSSION
To our knowledge, this study represents the first demonstration of antibacterial antiviral and antioxidant activity of P. harmala leaves extracts.
In the present study, we determined the phenolic composition of P. harmala leaves extracts from an arid region in Tunisia.Differences in polarity of the antioxidative components may explain why extraction yield and antioxidant activity of the extracts differ.The solubility of phenolic compounds are governed by the type of solvent used and the degree of polymerization of phenolics.In previous work done by Edziri et al. (2009), the phenolic content in aerial part of P. harmala is higher than the phenolic content in the leaves.In the present work, the methanol extract showed the highest amount of the polyphenols.For this purpose, methanol was recommended and frequently used for the extraction of phenolics (Abreu et al., 2005;Galvez, et al., 2005).
In comparison with other works done by Marzouk et al. ( 2009), we found that the methanol and chloroform extracts of P. haramala had good antibacterial activity against gram positive bacteria than the extracts of Citrullus colocynthis which have weaker antibacterial activity that ranged from 0.41 to 3.25 mgml -1 .In addition, the methanol extract of Bacillus subtilis showed antibacterial activity against S. aureus at MIC 512 mgml -1 (Basile et al., 1998).Same result was found in our work with methanolic and chloroformic extract of P. haramala leaves.
Antibiotic resistance is increasing worldwide in both outpatients as well as hospitalized patients.In the last two decades, the problem has escalated as the prevalence of antibiotic-resistant bacteria has increased and multi-drug-resistant strains have emerged in many species that cause disease in humans.There are no treatments available for infections caused by many of the antibiotic-resistant bacteria, and resistance to commonly used antibiotics is steadily increasing.Our finding shows that methanolic and chloroformic extract displayed higher antibacterial activity against gram-positive than gramnegative bacteria which are greater than other extracts.The reason for higher sensitivity of the gram-positive than gram-negative bacteria may be due to the differences between their cell wall compositions.The gram-positive bacteria contain an outer peptidoglycan layer, which is an ineffective permeability barrier (Nikaido and Vaara, 1985).
The observed antibacterial activity of the extracts of aerial part of P. harmala might also be attributed to the high quantity of polyphenols, which are known to possess efficient antibacterial activity (Scherrer and Gerhar, 1971).Cowan (1999) showed that several classes of polyphenol such as phenolic acids, flavonoids and tannins serve as plant defence mechanism against pathogenic microorganisms.
The antiviral activity of methanolic extract is probably due to the high phenolic content and the presence of polar substances such as flavonoids and tannins which are known to possess antiviral activity (Abidi and Ali, 1999;Fukuchi et al., 1989;Guillen and Manzanos, 1998;Kujumgiev et al., 1999).
Recently, numerous reports described antioxidants and compounds with radical scavenging activity present in fruit, vegetable, herb and cereal extracts (Cotelle et al., 1996;Hou et al., 2005).It seems that the antioxidant activity seen with the ammonium thiocyanate method for methanol, butanol, ethyl acetate and chloroform extracts may be predominantly related to the presence of the phenolic compounds such as flavonoids and tannins present in these polar extract.
The correlation level between the phenolic content and antioxidant activity between the plant organs is an interesting aspect, which supports the hypothesis that the former compounds contribute directly to antioxidant activity.Thus our findings show that leaves extracts of P. harmala also possess antibacterial, antiviral and antioxidant properties, which may be of beneficial when used therapeutically in humans.

Table 1 .
Antibacterial activity of leaves extracts of P. harmala.

Table 2 .
Antiviral activity of leaves extracts of P. harmala.
b: Ganciclovir, which is clinically used anti-HCMV drug, was used as positive controls in the antiviral activity.

Table 3 .
Antioxidant activity of the leaves of P. harmala extracts by the ammonium thiocyanate method.