In our study, it was aimed to determine antifungal activity of Peganum harmala on medically important yeasts and molds in vitro. Fungal suspensions of Candida spp and aspergillus spp isolated from the clinical specimens were treated with serial dilutions of P. harmala alcoholic extract in Sabouraud Broth. The titers included: 1/20, 1/40, 1/80, 1/160, 1/320, 1/640, and incubated for 24 h at 30Â°C. Anti fungal activity of the extract was determined as MIC and MFC. For the determination of MFC, a small volume of above serial dilutions was cultured on SGA 4% medium. After 24 to 72 h of incubation at 30Â°C, the cultures were looked for fungal growth. The highest and lowest inhibitory effects of P. harmala extract were determined on C. glabrata (MIC: 0.312 mg/ml) and C. albicans (MIC: 1.25 mg/ml), respectively. The MFC for Candida species was differed from 0.62 to 2.5 mg/ml. The highest fungicidal effect was seen on C. glabrata and C. tropicalis (MFC: 0.62 and 0.125 mg/ml, respectively) and the lowest was for C. albicans (MFC: 2.5 mg/ml). The treatment of Aspergillus suspensions with Perganum extract showed a decreasing of growth and sporolation without definite inhibition. The alcoholic extract of P. harmala showed a fungicidal activity on opportunistic yeasts, Candida spp, and a decreasing sporolation for aspergillus most importamt species. Â© 2011 Academic Journals

Diba, K.

Khorshivand, Z.

Shabatkhori, M.

Shoar, M.G.

English

Golestan University of Medical Sciences Repository

  Journal of Medicinal Plants Research Vol. 5(23), pp. 5550-5554, 23 October, 2011 Available online at http://www.academicjournals.org/JMPR  ISSN 1996-0875 ©2011 Academic Journals     Full Length Research Paper  Anti fungal activity of alcoholic extract of  Peganum harmala seeds  K. Diba1*, M. Gerami Shoar2, M. Shabatkhori3 and Z. Khorshivand1  1Department of Medical Parasitology and Mycology, School of Medicine, Urmia University of Medical Sciences,  Uromia, Iran. 2Department of Medical Parasitology and Mycology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran. 3Infectious Diseases Research Center, Department of Medical Parasitology and Mycology, Faculty of Medicine, Golestan University of Medical Sciences, Gorgan, Iran.  Accepted 8 June, 2011   In our study, it was aimed to determine antifungal activity of Peganum harmala on medically important yeasts and molds in vitro. Fungal suspensions of Candida spp and aspergillus spp isolated from the clinical specimens were treated with serial dilutions of P. harmala alcoholic extract in Sabouraud Broth. The titers included: 1/20, 1/40, 1/80, 1/160, 1/320, 1/640, and incubated for 24 h at 30°C. Anti fungal activity of the extract was determined as MIC and MFC. For the determination of MFC, a small volume of above serial dilutions was cultured on SGA 4% medium. After 24 to 72 h of incubation at 30°C, the cultures were looked for fungal growth. The highest and lowest inhibitory effects of P. harmala extract were determined on C. glabrata (MIC: 0.312 mg/ml) and C. albicans (MIC:  1.25 mg/ml), respectively. The MFC for Candida species was differed from 0.62 to 2.5 mg/ml. The highest fungicidal effect was seen on C. glabrata and C. tropicalis (MFC: 0.62 and 0.125 mg/ml, respectively) and the lowest was for C. albicans (MFC: 2.5 mg/ml). The treatment of Aspergillus suspensions with Perganum extract showed a decreasing of growth and sporolation without definite inhibition. The alcoholic extract of P. harmala showed a fungicidal activity on opportunistic yeasts, Candida spp, and a decreasing sporolation for aspergillus most importamt species.  Key words: Growth inhibitory, Peganum harmala, fungi.   INTRODUCTION  Peganum harmala (Zygophyllaceae) is an intriguing herb with a long history of medicinal use. It is widely grow in the most parts of the world including North and South America, Mexico, Africa and Asia. P. harmala is widely distributed in the Southern part of Iran too. It is known as “espand” in Persian and is famous for its medical uses as analgesic and antiseptic in folk medicine. P. harmala has significant usages in industrial and medical aspects (Berrougui et al., 2006). It has been medically used by the people of ancient countries such as India, Iran, Egypt and Spain. Some people use it typically for enhancing limbs. People in the west Asia burn  the  seeds   to  make     *Corresponding author.  E-mail: kambiz37diba@gmail.com. Tel: +98 441 367 4886, +98 912 446 4972 (Mobile). Fax: +98 441 278 0800-1. smoke for keeping safe against voodoo (Rojhan, 2002). Levchenko (1987), Kang (1994) and Rashan (1989) carried out first studies on anti parasitic and anti viral activities of P. harmala. Also anti parasitic effect of the plant seeds have been recently reported by Iranian researchers (Mahdavi and Masoud, 2003; Nateghpour et al., 2006). Also anti tumor activity of P. harmala was shown by Lamchouri et al. (1999). Peganum seeds contain carbohydrates, lipids, proteins, minerals, amino acids and alcoholic compounds as well as saturated fatty acids.  On the other hand, during last decades, fungi have emerged as new life threatening agents, considering increased opportunistic infections in human and animals. In fact, many medical centers in the world are challenge with the event. Furthermore some of these organisms have earned a resistance to anti fungal drugs. Regarding   the    high   frequency   of  fungal  infections   among   the       immunocompromised patients, and also the desirable effects of the P. harmala on the growth of other micro organisms, such as bacteria and parasites, it is necessary to study the antifungal usage of P. harmala seeds which seem to be practical. Present research is one of the first studies in Iran which carried out on antifungal activities of P. harmala in vitro. The goal was to measure absolute minimum concentration of alcoholic extract of P. harmala for inhibiting and killing of most important fungi causing human infection.   MATERIALS AND METHODS  Collection and identification of the plant   About 2000 g of the plant seeds were prepared from traditional drug stores as the seeds of Espand (in Persian) and then confirmed as Iranian P. harmala by the Plants Systematic laboratory, UM School of Botany, Iran.      Preparation of alcoholic extract  Amount of 1000 g of P. harmala seeds was powdered in an electrical grinder and then added to 3200 ml of ethanol 96°, shaken for 2 h and incubated over night at room temperature to remove alcoholic solvent from the crude extract. Again, the same amount of solvent was added to the crude extract and the process was repeated five times. The solvent was separated completely by a vacuum filtering system. The final product was 110 g of a brown color extract with biting odor.   Micro organisms  Main subjects included 111 isolates of Candida species and Aspergillus species obtained from clinical specimens of patients in department of Medical Mycology, School of Public Health and Institute of Public Health Research. For the improvement of accuracy in the results MIC and minimum fungicidal concentration (MFC) tests, all of the 111 clinical isolates were used. Candida isolates were identified in the level of species using differential medium CHROM agar Candida included C. albicans, C. dubliniensis, C. glabrata, C. tropicalis, C. kefyre and C. parapsilosis. Identification of Aspergillus species by using morphological characteristics resulted three species of A. fumigatus, A. flavus and A. niger. Each fungal species was confirmed by Molecular Mycology laboratory of TUMS.    Preparation of fungal suspensions  Fungal suspensions including 105 Candida cells and 103 Aspergillus spores in aqueous Sabouraud medium were made. For avoiding spore propagations a little volume of Tween 80 were added into the suspensions. Standard Mc farland 0.5 containing barium chloride and sulphoric acid was used as turbidity control.    Serial dilution of P. harmala extract  Serial dilutions in tubes  Small volume (1 ml) of sabouraud broth in capped tubes was used as substrate.  1 ml  of  10%  solution  of  the  crude  extract in sterile  Diba et al.          5551    water was added into the substrate and mixed thoroughly. Then 1 ml of the solution was transferred into the next tube containing 1 ml substrate, after which it was mixed and transferred into the next tube with the same substrate. This was done sequentially till it got to the 6th tube, so that six decreasing titers of P. harmala alcoholic extract in substrate including:  1/20, 1/40, 1/80, 1/160, 1/320, 1/640 were made. Then 1 ml of each Candida suspension was inoculated into dilution tubes and incubated at 30°C for 24 h. Negative and positive controls were prepared by adding 1 ml of 1/20 diluted extract and Candida suspension, respectively, into the substrate.   Serial dilutions in agar media  Sabouraud glucose agar 4% (SGA 4%) was measured, mixed with distilled water, autoclaved and dispensed in medium size (8 cm diameter) plates, 10 ml in each after cooling up to 60 to 55°C. While it was melted, the alcoholic extract was added into media plates in a decreasing pattern to make titers: 1/10, 1/100, 1/200, 1/400, 1/800, respectively. After making solid agar media, 1 ml of Aspergillus suspensions was inoculated into the dilution plates and left at 30°C for 72 h.    MIC (minimum inhibitory concentration) determination  After incubation time at 30°C, the tubes and plates of reactions were investigated for fungal growth. It was performed for Candida isolates, comparing turbidity of test tubes with that of Mc Farland standard 0.5. Positive tubes were found without any turbidity of growth.    MFC (minimum fungicidal concentration) determination  All tubes without turbidity (negative for growth) in serial dilution test were used for the determination of MFC. A small volume from the negative tubes was transferred into plates of Sabouraud agar and the incubated at 30°C for 72 h. MFC of peganum extract on Candida isolates was determined finding the media plates without any growth of Candida colonies. For the Aspergillus isolates, the first dilution plate without fungal growth was considered as MFC point.     RESULTS  Totally 111 Candida isolates including C. albicans, C. parapsilosis, C. keiffir, C. glabrata tested for MIC and MFC. Inhibitory test on 6 Candida species and 3 Aspergillus species resulted a range of MIC: 0.312 to 1.25 mg/ml. Alcoholic extract of P. harmala showed; MIC: 0.312 mg/ml on C. glabrata and MIC: 1.25 mg/ml on C. albicans as the highest and lowest inhibitory effects, respectively (Table 1). Moreover, minimum fungicidal concentration of the extract on Candida isolates was determined in a range of 0.625 to 2.5 mg/ml. As our results, the highest fungicidal effect of the extract (MFC: 0.625 mg/ml) was found on C. glabrata, and the lowest on C. albicans (MFC: 2.5 mg/ml). Our assay on Aspergilli showed different results in the level of species. The extract was effective on A. niger and A. fumigatus as growth inhibitory but for A. flavus the result  was  restricted   to  some  changes  in morphology    5552          J. Med. Plants Res.    Table 1. MIC (minimum inhibition concentration) and MFC (minimum fungicidal concentration) in Candida species.  Candida species No of isolates MIC  (mg/ml) MFC (mg/ml) C. albicans 102 1.25 2.5 C. parapsilosis 11 0.625 1.25 C. Keiffir 3 0.625 1.25 C. glabrata 2 0.312 0.625 C. tropicalis  2 0.312 1.25 C. dubliensis 1 0.625 1.25      Figure 1. Effect of PAE on Aspergillus fumigatus (left) and A. niger (right) in the dilution of 0.3.    characteristics, macroscopic features and sporolation rate, color and size of colonies (Figures 1 and 2). Microscopic variations were not included for Aspergillus species. Time effectiveness in MIC assay was shown in Table 2. It is clear that 24 h treatment for fungal suspension with P. harmala is much more effective than 2 and 6 h treatment.   DISCUSSION  P. harmala seeds have been considered from ancient time to date as a plant with drug usages regarding to some alkaloids compounds such as harmalin and harmalol. The compounds extracted from this plant have shown different medical characteristics such as anti inflammation effects of pitolin were reported by El-Saad El Rifaei (1980). He confirmed anti bacterial/anti fungal, anti fever and anti parasite effects on P. harmala. Most studies on medical effects of P. harmala are on parasite infections. Levchenko was one of the first people (Levchenko, 1987) who reported the medical effects of alkaloids of P. harmala on bovine Theileriosis et al. (1994) indicated protoscolocidal effects on P. harmala in hydatid  cysts  (Rashan,  1989). Some compounds of this plant have anti parasite and anti microbial effects such as alkaloids that have drug usage on Babesia infected cattle (Fan et al., 1997). Anti bacteria and anti fungal activity of harmaline have reported by Abdel-Fattah et al, (1995). Most relevant researches are in vivo studies, but they are rare in vitro studies such as Shahverdi et al. on anti microbial effects of smoking of P. harmala seeds, also chemical effects of the plant on Staphylococcus    epidermidis    and Cryptococcus neoformans (Shahverdi et al., 2005).  The plant on Staphylococcus epidermidis and In this study anti fungal activity of P. harmala on 6 and 3 species of Candida and Aspergillus, (respectively) was evaluated in vitro. The inhibitory activity of alcoholic extract of P. harmala has been measured by the determination of MIC and MFC parameters. The lowest MIC was shown on Candina albicans the highest MIC on C. glabrata. Low activity of Peganum extract on C. albicans can be explained by the resistance of most of C. albicans clinical strains to anti fungal drugs. Shafiee and Gilani tested anti fungal activity of P. harmala seeds on Microsporom, Trichophyton and Fonescae pedrosoei with serial dilutions and they reported 0.125 mg/ml as lowest MIC. In the present study MIC differs in different species of Candida. MICs  were  decreased  from  0.25 to 0.031 mg/   Diba et al.          5553      Figure 2. Modified Morphology of A. flavus in colony coloration and sporolation in the dilutions 0.3 to 0.02.    Table 2. Effect of incubation time on fungicidal activity of P. harmala extract.  Species No. of isolates Growth after 2 h Growth after 6 h Growth after 24 h C. albicans 16 G G NG C. parapsilosis             1 NG NG NG C. keffyre                        1 NG NG NG C. glabrata                    2 G NG NG C. tropicalis                  1 NG NG NG  G: growth, NG: no growth.    ml on Candida species:  C. albicans, to C. tropicalis and C. glabrata, respectively. By the consideration of dense and compact cell wall of yeasts comparing filamentous fungi cell walls, it seems to be reasonable that Candida spp. were resistant to P. harmala extract more than Aspergillus spp. Although P. harmala alcoholic extract inhibited mildly A. niger and A. fumigatus but it was not considerable. The alcoholic extract of Iranian P. harmala showed inhibitory and fungicidal activities on opportunistic pathogenic Candida spp, but it caused very low inhibition of growth and sporolation of tested Aspergillus spp. ACKNOWLEDGEMENTS  We thank Dr. Jamal Hashemi for helping us to access the clinical isolates, also Leila Hosseinpur for assisting us in the lab. This project was supported by the deputy head of Research, Institute of Health Research, TUMS.   REFERENCES  Abdel-Fattah AFM, Matsumoto K, Gammaz  HAK, Watanabe  H (1995). Hypothermic effect of harmala alkaloid in rats: Involvement of serotonergic mechanism. Pharmacol. Biochem., 52: 421-426.   5554          J. Med. Plants Res.    Berrougui H, Isabelle M, Cloutier M, Hmamouchi M, Khalil  A (2006). Protective effects of Peganum harmala extract harmine and harmaline against human low-density lipoprotein oxidation. J. Pharmacol., 58: 967-974. El-Saad EM (1980). Peganum harmala: its use in certain dermatoses. Int. J. Dermatol., 19: 221-222. Fan B, Liang J, Men J, Gao F, Li G, Zhao S (1997). Effect of total alkaloid of Peganum harmala in the treatment of experimental haemosporidian infections in cattle. Trop. Anim. Health. Prod., 29: 77S-83S. Kang J (1994). In vitro cidal effect of 10 chinese traditional herbs against Echinococcus granulosus protoscoleces. End. Dis. Bull., 94: 22-24. Lamchouri F, Settaf A, Cherrah Y, Zemzami M, Lyoussi B, Zaid  A (1999). Antitumour principles from Peganum harmala seeds. J. Ther., 54(6): 753-758. Levchenko F (1978). Comparative study of three chemotherapeutic preparations against natural theileriosis in cattle. Trud. Nauch. Vetr. Inst. Tadzh, 8: 117-119. Mahdavi M, Masoud J (2003). Protoscolocidal effects of aquatic and alcoholic extracts and alkaloids of Peganum harmala seeds (In persian). J. Sch. Med. TUMS, 3: 215-226.                                                         Rashan LJ, Adaay MH, Al-Khazraji  ALT (1989). In vitro antiviral activity of the aqueous extract from the seeds of Peganum harmala. J. Fitoterapia, 60: 365-367. Rojhan M (1982). Herbal treatment (In persian). Khayyam ed. p. 28. Shahverdi AR, Monsef-Esfahani HR, Nickavar  B, Bitarafan  L, Khodaee  S, Khoshakhlagh  N (2005). Antimicrobial activity and main chemical composition of two smoke condensates from Peganum harmala seeds. Z. Naturforsch, 60: 707-710. Nateghpour M, Sharbatkhori M, Edrissian GH, Souri E, Mohebali M, Akbarzadeh K, Motevalli Haghi A, Satvat M, Rahimi A (2006). Assessment of in vitro activity of Peganum harmala extract on Plasmodium falciparum growth compared with chloroquine. Pak. J. Bio Sci., 9: 214-216. 

Anti fungal activity of alcoholic extract of Peganum harmala seeds

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Anti fungal activity of alcoholic extract of Peganum harmala seeds

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