Antimicrobial Effects of Aqueous and Alcoholic Extract of Peganum Harmala L. Seeds on Two Types of Salivary Isolated Microorganisms in Al-Ramadi City

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1 JKAU: Med. Sci., Vol. 17 No. 4, pp: 3-17 (2010 A.D. / 1431 A.H.) DOI: /Med Antimicrobial Effects of Aqueous and Alcoholic Extract of Peganum Harmala L. Seeds on Two Types of Salivary Isolated Microorganisms in Al-Ramadi City Minan Y. H. Al-Izzy, MSc Department of Preventive Dentistry, Faculty of Dentistry, Al-Anbar University, Baghdad, Iraq minanyasseen@yahoo.com Abstract. In the present study Lactobacilli and Candida were isolated from saliva of twenty dental students at Al-Ramady City, aged between years at the period between March and May These microorganisms were isolated, purified and diagnosed according to cultural and microscopical characteristics, and confirmed by biochemical tests. Dried seeds of Peganum harmala L. were extracted. Different concentrations of the extract were prepared and estimated in g/100ml distilled water. Chlorhexidene was used as an antimicrobial agent for comparison. Agar diffusion technique was employed in this research. The study concluded that harmala extract was effective in the inhibition of the growth of these microorganisms and being more potent than chlorhexidene. Keywords: Peganum harmala L., Harmaline, Lactobacilli, Candida Albicans. Introduction Recently, searching for drugs and dietary supplements derived from plants have been accelerated. Plants are rich in a wide variety of secondary metabolites, such as tannins, terpenoids, alkaloids and flavonoids which have been found in vitro to have antimicrobial properties [1,2]. From ancient times, Peganum harmala L. was claimed to Correspondence & reprint request to: Minan Al-Izzy Twenty-Fourth Street, Al-Yarmouk 616, Baghdad, Iraq. Accepted for publication: 13 June Received: 27 February

2 4 M.Y.H. Al-Izzy be an important medicinal plant and it was used in global folk medicine. This plant was called Assyrian Rue or Latin Peganum harmala L. Its seeds were known to posses hypothermic, and essentially hallucinogenic properties [3-5]. Peganum is a small genus belonging to the family Zygophylaceae and mainly distributed in the Mediterranean region, also found in central Asia, North Africa and has been also distributed in America and Australia [6,7]. Peganum harmala L. is the only species found growing wild in the middle and northern parts of Iraq. The plant is rich in alkaloids and contains up to 4% total alkaloids [8-10]. There are several reports which indicate the variety of pharm zacological and biological activities of Peganum harmala L. such as antibacterial, antifungal and MAO inhibition (Monoamine oxidases). In addition to be effective in the treatment of dermatosis vasorelaxant, antihemosporidian, antitumour, antinociceptive, anti-inflammatory, and antiprotozoal effects [11-13]. The smoke of its seeds is traditionally used as a disinfectant [14]. The coactions of Lactobacilli with Candida albicans have probably received more attention than those of any other pair of microorganisms commonly found in the mouth. When Lactobacilli are grown in the presence of C. albicans, there is an increased yield of acid. Since the same effect occurs with heat-killed Candida cells, the yeast probably provides some nutritional needs for Lactobacilli [15]. The main objective of the present study is to find the antimicrobial activity of Peganum harmala L. against Lactobacilli and Candida isolated from the oral cavity. Aqueous Extract Preparation Materials and Method The crude extract was obtained according to Al-Mizrakchi [16]. The dry seeds of Peganum harmala L. were purified washed and dried under fresh air, then ground in electrical grinder to get fine powder of the seeds. Then, 100 gm of the ground seeds were infused in 500 ml. of distilled water for 24 hours at room temperature. Agitation of the infusion using magnetic stirrer had been done alternatively. Then the infusion was filtered by filter paper (Wattman No.1) and the residue was discarded. The extract left to dry in a Petri dish at room temperature and the resulted

3 Antimicrobial Effects of Aqueous and Alcoholic Extract of Peganum Harmala 5 powder was kept in a tightly closed glass container in refrigerator until it s used to prepare different concentrations. Alcoholic Extract Preparation The crude extract was obtained according to Subhani et al. [17]. The dry seeds of Peganum harmala were purified washed and dried under fresh air, then ground in electrical grinder to get fine powder of the seeds. Then 100 g of the ground seeds were infused in 500 ml. of 96% of ethanol for 48 hours at room temperature. Agitation of the infusion using magnetic stirrer had been done alternatively. Then the infusion was filtered by filter paper (Wattman No.1) and the residue discarded. The extract left to dry in a Petri dish at room temperature, the resulted powder kept in tightly closed glass container in refrigerator until used to prepare different concentrations. Isolation of Microorganisms Stimulated salivary samples were collected under standard conditions according to Dasanayake et al. [18]. Twenty dental students with no medical history, aged years were selected to participate in this project (ten isolates of both Lactobacilli and Candida and ten isolates were excluded from being participated in the research as there was no growth of each type of the tested microorganisms). Each individual was asked to chew a piece of Arabic chewing gum ( g) for five minutes to stimulate salivary collection as much as possible, then saliva was collected in sterilized screw capped bottles. Afterwards, saliva was homogenized by vortex mixer for two minutes. Ten fold serial dilutions were prepared using normal saline. Two dilutions were selected for each microbial type; inoculated on Glucose-yeast extract-acetic acid agar (SLA) (selective Lactobacilli agar) and Sabouraud dextrose agar (SDA). The plates were incubated aerobically for 48 h at 37 C. The identification of Lactobacilli and Candida colonies on SLA and (SDA) had been done directly and under dissecting microscope (magnification 15), respectively. Morphological and biochemical tests for bacterial cells were done according to Koneman et al. [19]. Purification and Maintenance of Microorganism Isolates The purity of both types of cultures was checked by re-inoculation of 0.1 ml. of Lactobacilli culture on SLA agar and 0.1 ml of Candida

4 6 M.Y.H. Al-Izzy culture on Sabouraud dextrose agar plates. Plates were incubated aerobically for h at 37 C. Then a selective colony from both types of microorganisms separately was transferred to 10 ml. of a sterile brain heart infusion broth with glycerol at ratio of 15% which was added for microorganism cells preservation and incubated for 24 h aerobically at 37 C. These broths were stored in the refrigerator until used. Different concentrations (20%, 30% and 50%) from aqueous and alcoholic extract were prepared by using distilled water. Each concentration prepared was sterilized by filtration using millipore filter size 0.22 µm (Inlet Lot, 1824). Chlorhexidene gluconate solution (CHX) 0.2% (Corsodyl, GlaxoSmithKline PLC, UK) was used as a positive antimicrobial control. Sensitivity Tests In this experiment, agar diffusion technique was applied to study the antimicrobial effect of the previously mentioned agents on Mueller Hinton agar (MHA) (Difco, Voigt Global Distribution Inc, Lawrence, KS USA). Twenty isolates of Lactobacilli and the same for Candida were used. A well method was employed by making holes (5 mm in diameter and 4 mm in depth). Each well was filled with 40µl of selected agents. Inhibition zones were across the diameter of each well. Complete resistance of microorganisms to the tested agent was indicated when there were no zones of inhibition [20]. Ethical A written consent signed by volunteers, including the purpose of the study. Statistical Analysis Data were analyzed using SPSS version 13 computer software. An expert statistical advice was performed. A difference in mean inhibition zone diameter between CHX control and the 3 different concentrations of aqueous or alcoholic extract was assessed for statistical significance by ANOVA. When ANOVA model showed a statistically significant difference, further exploration for the site of statistical significance between all possible paired combinations of tested groups was assessed by LSD. P value less than 0.05 was considered statistically significant.

5 Antimicrobial Effects of Aqueous and Alcoholic Extract of Peganum Harmala 7 Such a difference was attributed to the effect of extract at the tested concentration compared to CHX control. The percent change in mean inhibition zone diameter attributed to the effect of extract was computed by dividing the difference in mean by the mean of control group and then multiplied by 100. The difference in mean between aqueous and alcoholic extract was assessed by independent samples t-test. The statistical significance strength and direction of linear correlation between the concentration of extract (which is a discrete quantitative variable) and inhibition zone diameter was assessed by Spearman s rank correlation coefficient. P value less than the 0.05 level of significance was considered statistically significant. Results As shown in Table 1, the mean diameter of inhibition zone of Lactobacilli after the addition of 20% aqueous extract solution was (18.7 mm) which was significantly lower than that of 0.2% CHX. The 20% extract decrease the zone diameter by % compared to CHX. The mean diameter of inhibition zone of 30% aqueous extract solution in comparison with that of CHX was significant, and the zone diameter of it was decreased by - 5.2% compared with CHX solution. The action of the highest concentration of the aqueous extract (50%) was better than that of CHX with significant difference between their mean diameters of inhibition zones (Fig. 1 and 2) The 50% increase the mean inhibition zone by 6.6% compared with CHX. The concentration of aqueous extract showed a statistically significant moderately strong positive linear correlation with inhibition zone diameter of Lactobacilli culture. The mean diameter of inhibition zone for Lactobacilli after adding 20% alcoholic extract solution (25.4 mm) was significantly higher than that of CHX control. The 20% extract increase the zone diameter by 19.2% compared to CHX control. The 30% alcoholic extract significantly increase the mean diameter of inhibition zone by 26.8% compared to CHX control. The highest concentration (50%) of alcoholic extract significantly increases the mean diameter of inhibition zone by 34.7% compared to CHX control. All concentrations of alcoholic extract showed a statistically significant

6 8 M.Y.H. Al-Izzy moderately strong positive linear correlation with inhibition zone diameter of Lactobacilli culture (Table 2). Table 1. The difference in mean inhibition zone diameter in Lactobacilli culture plates between chlorhexidine (control) and 3 selected concentrations of the tested aqueous and alcoholic extract. Inhibition Zone Diameter CHX (control) Concentration (%) 0.2% 20% 30% 50% Aqueous extract Range (18-26) (17-23) ( ) (20-30) Mean+/-SE (21.3 +/- 0.7) (18.7 +/- 0.58) (20.2 +/- 0.71) (22.7 +/- 1.24) N Change ratio compared to CHX -12.2% -5.2% 6.6% P (ANOVA) = r = 0.61 P < CHX (control) x 20% solution = CHX (control) x 30% solution = 0.36[NS] CHX (control) x 50% solution = 0.25[NS] Alcoholic extract Range (18-26) (21-30) (24-33) (24-32) Mean+/-SE (21.3 +/- 0.7) (25.4 +/- 0.9) (27 +/- 1.02) (28.7 +/- 0.79) N Change ratio compared to CHX 19.2% 26.8% 34.7% P (ANOVA) = < r = 0.49 P = CHX (control) x 20% solution = CHX (control) x 30% solution < CHX (control) x 50% solution < A C B Fig. 1. Sensitivity of Lactobacilli to different concentrations of Peganum harmala L. aqueous extract on MHA: (A) 50%; (B) 30%; (C) 20%.

7 Antimicrobial Effects of Aqueous and Alcoholic Extract of Peganum Harmala 9 Fig. 2. The error bar chart showing the mean (with its 95% confidence interval) inhibition zone diameter of Lactobacilli colonies after adding aqueous or alcoholic extract compared to CHX. Table 2. The difference in mean inhibition zone diameter in Lactobacilli culture plate between aqueous and alcoholic extract at 3 selected concentrations of the tested extract. Inhibition Zone Diameter Concentration (%) 20% 30% 50% Aqueous extract Range (17-23) ( ) (20-30) Mean+/-SE (18.7 +/- 0.58) (20.2 +/- 0.71) (22.7 +/- 1.24) N Alcoholic extract Range (21-30) (24-33) (24-32) Mean+/-SE (25.4 +/- 0.9) (27 +/- 1.02) (28.7 +/- 0.79) N Change ratio with alcoholic extraction compared to aqueous 35.8% 33.7% 26.4% extraction P (t-test) < < < 0.001

8 10 M.Y.H. Al-Izzy For Candida, the mean diameter of inhibition zone after the addition of 20% of aqueous extract of Peganum harmala was 20.5 mm, which is slightly better than CHX (20 mm) by the ratio of 2.5% and considered to be insignificant compared to it. The 30% concentration, also has similar results and was slightly better than CHX by only 4%. The higher concentration of Peganum harmala, 50% only has better results than CHX by the ratio 7%, but still not significant (Table 3, Fig. 3 and 4). Table 3. The difference in mean inhibition zone diameter in Candida culture plates between chlorhexidine (control) and 3 selected concentrations of the tested aqueous and alcoholic extract. Inhibition Zone Diameter CHX (Control) Concentration (%) 0.2% 20% 30% 50% Aqueous extract Range (18-26) (19-22) ( ) (20-23) Mean+/-SE (20+/-0.79) (20.5+/-0.4) (20.8+/-0.19) (21.4+/-0.45) N Change ratio compared to CHX 2.5% 4% 7% P (ANOVA) = 0.28[NS] r = 0.23 p = 0.22 [NS] r-0.23 P=0.22[NS] Alcoholic extract Range (18-26) (25-28) (28-30) (29-35) Mean +/- SE (20 +/- 0.79) (26 +/- 0.39) (29 +/- 0.24) (31.2 +/- 0.59) N Change ratio compared to CHX 30% 45% 56% P (ANOVA) = < r = 0.90 p < CHX (control) x 20% solution < CHX (control) x 30% solution < CHX (control) x 50% solution < The linear correlation was only 0.23 for the aqueous extract. In general, alcoholic extract had the better results than the aqueous extract in relation to the tested bacteria. The 20% concentration was significantly better than CHX by the ratio of 30%, and the mean diameter of inhibition zone was (26 mm) which was better than the diameter of inhibition zone of CHX (20 mm). The 30 % concentration was also better than CHX by the ratio of 45% and the mean diameter of inhibition zone was (29 mm). The 50% concentration had the strongest activity than CHX by the ratio of 56% and the mean diameter of inhibition zone was (31.2 mm). The linear correlation coefficient was statistically strong and represented by 0.9 (Table 4).

9 Antimicrobial Effects of Aqueous and Alcoholic Extract of Peganum Harmala 11 A C B Fig. 3. Sensitivity of Candida to different concentrations of Peganum harmala L. alcoholic extract on MHA (A):50% (B):30% (C):20%. Fig. 4. The error bar chart showing the mean (with its 95% confidence interval) inhibition zone diameter of Candida colonies after adding aqueous or alcoholic extract compared to CHX.

10 12 M.Y.H. Al-Izzy Table 4. The difference in mean inhibition zone diameter in Candida culture plate between aqueous and alcoholic extract at 3 selected concentrations of the tested extract. Inhibition Zone Diameter Concentration (%) 20% 30% 50% Aqueous extract Range (19-22) ( ) (20-23) Mean+/-SE (20.5 +/- 0.4) (20.8 +/- 0.19) (21.4+/-0.45) N Alcoholic extract Range (25-28) (28-30) (29-35) Mean+/-SE (26 +/- 0.39) (29 +/- 0.24) (31.2 +/- 0.59) N Change ratio with alcoholic extraction compared to 26.8% 39.4% 45.8% aqueous extraction P ( t -test) < < < Discussion Initial screening of plants for possible antimicrobial activities typically begin by using crude aqueous or alcohol extractions and can be followed by various organic extraction methods [2]. The amateur herbalists are advised to prepare healing compounds from plants and herbs using water extraction methods, since water is universally the solvent used in extract activity even at home. The best way to sterilize the extract, whether it was aqueous or alcoholic, is by filtration using millipore filter size (0.2 µm) to keep the integrity of the antimicrobial components of the plant extract. Since nearly all of the identified components from plants active against microorganisms are aromatic or saturated organic compounds, they are most often obtained through initial ethanol or methanol extraction. Both of these solvents are used as initial extractants in approximately 35% of recent studies as they may not demonstrate the greatest sensitivity in yielding antimicrobial chemicals on an initial screening. One hundred grams from the ground seeds were used; extracted in 500 ml. of either hot water in the aqueous extract or ethanol alcohol in alcoholic extract, the reason for using this ratio was to obtain as much extract as possible. The sensitivity of Lactobacilli to different concentrations of Peganum harmala L. aqueous extract in comparison to 0.2% CHX was tested using

11 Antimicrobial Effects of Aqueous and Alcoholic Extract of Peganum Harmala 13 Agar Diffusion Technique. Results of this experiment were affected by agar thickness, molecular weight and concentration of antimicrobial agent. It was found that Peganum harmala L. aqueous and alcoholic extracts have the ability to inhibit the growth of these bacteria. These findings were in coincidence with Cowan [2], who discovered that Peganum harmala L. extract (aqueous and alcoholic) is very effective against all gram positive bacteria including Lactobacilli. Al-Mizrakchi [16] also studied the antibacterial activity in vitro of different concentrations of aqueous extract of Peganum harmala L. seeds against S. mutans which is also gram positive bacteria and he discovered that the mentioned extract has a great antibacterial effect against S. mutans. For Candida, it was found that it was more sensitive against both aqueous and alcoholic extract than Lactobacilli. These findings were in coincidence with Shahverdi et al. [14] who found that Fungi in general, appeared to be more sensitive than bacteria against Peganum harmala L. smoke extract generated from the burned seeds. This smoke extract contained the vapor of alkaloids and other vapors of cytotoxic components against microorganisms. The zones of inhibition were found to increase when the concentrations of Peganum harmala L. extract increase. This may be attributed to the increased levels of the principle alkaloids, while increasing the extract concentrations, and these alkaloids (which are heterocyclic nitrogen compounds) have the ability to intercalate with DNA of the microorganisms [22]. Some of these alkaloids are present in Peganum harmala L. seeds extracts are: harmaline, harmine, harmalol peganine, β-carboline alkaloids, anthroquinons and fixed oils [3]. The effect of 50% concentration of aqueous and alcoholic extract had better results than 0.2% CHX in this research for both tested microorganisms. In general the alcoholic extract of Peganum harmala L. was found to be more potent than the aqueous extract. This may have been contributed to the fact that alcoholic soluble substances are more potent against tested microorganisms than aqueous soluble substances. As far as we know, no studies have been conducted on the antimicrobial activity of this seeds extract against Lactobacilli or Candida. This is the first study of the cytotoxicity of the crude aqueous or alcoholic extract of such plant seeds on these two types of microorganisms.

12 14 M.Y.H. Al-Izzy However, further studies need to be performed using the crude extract of Peganum harmala L. seeds (aqueous or alcoholic) or its other chemical constituents to be tested in vivo, as a mouthwash or in the dental products vehicles. Plus, make this extract obtainable for everyone to prevent dental caries progression in the future. These studies should be employed after excluding the extract toxicity to the human being. In regards to the sample size remark, the present study, being an explorative one, was trying to find a probable role for a novel plant extract in causing inhibition of Candida and lactobacilli aimed to look for a difference in inhibition zone of 20% or larger compared to its baseline value of 20 mm diameter. Such an effect was considered by the researcher to be clinically relevant difference from the CHX effect. The above difference in mean would be equivalent to an effect size of at least 2 (measured according to Cohen's effect size and knowing that the SD is on average equal to 2). Therefore, the sample size formula calculated a sample size of 7 for each of the 2 comparisons group, using considering the following parameters: Alpha = 0.05, study power (1-Beta) = 0.95, effect size (Cohen's d) = 2. Furthermore, a decision to use a sample size of 10 was considered sufficient to reach a conclusion about the present study objectives. It is obvious that the main conclusion that both types of plant extract (aqueous or alcoholic was at least as comparable to CHX effect) holds true even in what seems to be a small sample size. A larger scale study might indeed be needed to conclude that aqueous extract at certain larger concentrations might have a significantly stronger effect than CHX. Such an effect, even if proved to be statistically significant is of much smaller magnitude than the proven superior effect of alcoholic extract in the present study, which can be safely generalized to the population level even with what seems to be a small sample size. Conclusion and Recommendations In conclusion, the present study provides evidence that the aqueous and alcoholic extract derived from seeds of Peganum harmala L. possess some antimicrobial properties against Lactobacilli and Candida in vitro. However, further studies need to be performed in vitro and in vivo with specified chemical constituents of Peganum harmala L. extract, especially with antimicrobial action and be tested separately for the

13 Antimicrobial Effects of Aqueous and Alcoholic Extract of Peganum Harmala 15 antimicrobial activity. The possibility to mix these extracts with other chemical constituents or other plants extracts, plus test their effects with the same tested microorganisms or other cariogenic bacteria, and larger samples are needed to confirm the results of this research. Acknowledgments The author is very grateful to Professor Dr. Abbas Sabry Al- Mizrakchi and Asst. Prof. Dr. Mushtak Talib and Dr. Ahmed Sameer for their unlimited assistance and support during this research. References [1] Adaay MH, Rashan LJ, Sulayman KD, Al-Abaar M, Ayoub T. Antimicrobial activity of different extracts from the seeds of Peganum harmala. Fitoterapia 1989; 60: [2] Cowan M. Plant products as antimicrobial agents. Clin Microbiol Rev 1999; 12(4): [3] Sharaf M, el-ansari A, Matlin SA, Saleh NA. Four flavonoid glycosides from Peganum harmala. Phytochemistry 1997; 44(3): [4] Lamchouri F, Settaf A, Cherrah Y, Zemzami M, Lyoussi B, Zaid A, Atif N, Hassar M. Antitumour principles from Peganum harmala seeds. Therapie 1999; 54(6): [5] Fan B, Liang J, Men J, Gao F, Li S, Zhao S, Hu T, Dang P, Zhang L. Effect of total alkaloid of Peganum harmala L. in the treatment of experimental haemosporidian infections in cattle. Trop Anim Health Prod 1997; 29(4 Suppl): 77S-83S. [6] Juma'a GM. Peganum harmala production. Eur J Sci Res 2005; 11(1): [7] Shi CC, Chen SY, Wang GJ, Liano JF, Chen CF. Vasorelaxant effect of harmaline. Eur J Pharmacol 2000; 390(3): [8] Lala S, Pramanick S, Mukhopadhyay S, Bandyopadhyay S, Basu MK. Harmine: evaluation of its antileishmanial properties in various vesicular delivery systems. J Drug Target 2004; 12(3): [9] Monsef HR, Ghobadi A, Iranshahi M, Abdollahi M. Antinociceptive effects of Peganum harmala L. alkaloid extract on mouse formalin test. J Pharm Pharm Sci 2004; 7(1): [10] Muhi-eldeen Z, Al-Shamma KJ, Al-Hussainy TM, Al-Kaissi EN, Al-Daraji AM, Ibrahim H. Acute toxicological studies on extract of Iraqi Peganum harmala in rats. Eur J Sci Res 2008; 22(4): [11] Arshad N, Neubauer C, Hasnian S, Hess M. Peganum harmala can minimize Escherichia coli infection in poultry, but long-term feeding may induce side effects. Poult Sci 2008; 87(2): [12] Berrougui H, Martin C, Khalil A, Hamamouchi M, Ettaib A, Marhuenda E, Herrara MD. Vasorelaxant effects of harmine and harmaline extracted from Peganum harmala L. seeds in isolated rat aorta. Pharmacol Res 2006; 54(2): [13] Asghari G, Lockwood GB. Stereospecific biotransformation of (±) phenelethyl propionate by cell cultures of Pganum harmala L. Iranian Biomedical J 2002; 6(3):

14 16 M.Y.H. Al-Izzy [14] Shahverdi AR, Ostad SN, Khodaee S, Bitarafan L, Monsef-Esfahani HR, Jamalifar H, Nickavar B, Mohseni M. Antimicrobial and cytotoxicity potential of Peganum harmala smoke. Phcog Mag 2008; 4(15): [15] Nolte W. (1) The microbial parasite and the host (2) pathogenic microorganisms and the host. In: Oral Microbiology with Basic Microbiology and Immunology. 4 th ed. CV Mosby Co , 287. [16] Al-Mizrakchi A. Adherence of mutans Streptococci on teeth surfaces: microbiological and biochemical studies. PhD Thesis. 1998; University of Al-Mustansiriya. [17] Subhani AM, Ebrahimi SA, Mahmoudian M. An in vitro evaluation of human DNA topoisomerase 1 inhibition by Peganum harmala L. seeds extract and its β-carboline alkaloids. J Pharm Pharm Sci 2002; 5(1): [18] Dasanayake AP, Caufield PW, Cutter GR, Roseman JM, Köhler B. Differences in the detection and enumeration of Mutans Streptocicci due to differences in methods. Archs Oral Biol 1995; 40(4): [19] Koneman EW, Schreckenberge PC, Allens SD, Jr, WCW, Janada WM. Textbook of Diagnostic microbiology; 4 th ed. Philadelphia, USA: JB Lippincott Co, [20] Al-Izzy M. Antibacterial effects of black and green tea extract on Mutans Streptococci and Lactobacilli (in vitro and in vivo study). MSc. Thesis 2005; College of Dentistry, Baghdad University. [21] Prashanth D, John S. Antibacterial activity of Peganum harmala. Fitoterapia 1999; 70(4): [22] Phillipson JD, O Neill MJ. New leads to the treatment of protozoal infections based on natural product molecules. Acta Pharm Nord 1987; 1:

15 Antimicrobial Effects of Aqueous and Alcoholic Extract of Peganum Harmala 17 - (Lactobacilli). (Candida) - ( ).( ). /...