Skip Navigation
Skip to contents

PHRP : Osong Public Health and Research Perspectives



Page Path
HOME > Osong Public Health Res Perspect > Volume 7(2); 2016 > Article
Original Article
Anti-biofilm Properties of Peganum harmala against Candida albicans
Elham Aboualigalehdaria, Nourkhoda Sadeghifarda, Morovat Taherikalanib, Zaynab Zargousha, Zahra Tahmasebia, Behzad Badakhshc, Arman Rostamzadd, Sobhan Ghafouriana, Iraj Pakzada
Osong Public Health and Research Perspectives 2016;7(2):116-118.
Published online: January 8, 2016

aClinical Microbiology Research Center, Ilam University of Medical Sciences, Ilam, Iran

bResearch Center & Department of Microbiology, School of Medicine, Lorestan University of Medical Sciences, Khorramabad, Iran

cDepartment of Gastroenterology, Faculty of Medicine, Ilam University of Medical Sciences, Ilam, Iran

dDepartment of Biology, Faculty of Sciences, Ilam University of Medical Sciences, Ilam, Iran

∗Corresponding authors.
∗Corresponding authors.
• Received: November 29, 2015   • Revised: December 6, 2015   • Accepted: December 20, 2015

Copyright © 2016 Korea Centers for Disease Control and Prevention. Published by Elsevier Korea LLC.

This is an open access article under the CC BY-NC-ND license (

  • 21 Download
  • 12 Crossref
  • 18 Scopus
  • Objectives
    Vaginitis still remains as a health issue in women. It is notable that Candida albicans producing biofilm is considered a microorganism responsible for vaginitis with hard to treat. Also, Peganum harmala was applied as an anti fungal in treatment for many infections in Iran. Therefore, this study goal to investigate the role of P. harmala in inhibition of biofilm formation in C. albicans.
  • Methods
    So, 27 C. albicans collected from women with Vaginitis, then subjected for biofilm formation assay. P. harmala was applied as antibiofilm formation in C. albicans.
  • Results
    Our results demonstrated that P. harmala in concentration of 12 μg/ml easily inhibited strong biofilm formation; while the concentrations of 10 and 6 μg/ml inhibited biofilm formation in moderate and weak biofilm formation C. albicans strains, respectively.
  • Conclusion
    Hence, the current study presented P. harmala as antibiofilm herbal medicine for C. albicans; but in vivo study suggested to be performed to confirm its effectiveness.
Unfortunately, vaginitis remains a health issue in women. It has been estimated that there are around 10 million physician visits for vaginitis annually [1]. However, biofilm formation is known as a major worldwide concern [2]. Despite this, there are a vast majority of microbiology studies focusing on bacterial biofilm, but there is less consideration to medically important fungal biofilms. Among fungi, the genus Candida, especially Candida albicans, is responsible for around 15–20% of vaginitis cases in women [3]. Studies have demonstrated that the majority of biofilm formation by C. albicans occurred in the oral cavities, environment, and vaginas of patients [4].
New drug discovery could also be useful for the eradication of potent fungi in biofilm formation. Herbal medicines are defined as natural products, which are used in traditional medicine. The use of medicinal plants goes back 60,000 years ago [5]. Peganum harmala, usually called Esfand, is a plant of the family of Nitrariaceae. It is applied as a traditional medicine for so many infections in Iran [6]. Hence, the current study aimed to investigate the role of P. harmala in the inhibition of biofilm formation produced by C. albicans.
2.1 Organisms and identification
C. albicans were collected from vulvovaginal candidacies women in the Ilam province in the west of Iran. The isolates were cultured on Sabouraud dextrose agar and then re-identified with a germ tube test [7].
2.2 Cell culture
The P. harmala extract was tested for its cytotoxicity effect on a Vero cell line. The percentage viability of the cell line was carried out by using the MTT assay (Sigma, United States).
2.3 Toxicity assay
The cells were coated in a 96-well flat bottom plates at a density of 5,000–10,000 cells per well. Following this, the cells were treated with different concentrations of P. harmala ethanolic extract (1–100 μg/mL). After 24 hours, the MTT assay was applied to determine the toxicity concentration of P. harmala. The absorbance of the converted dye was measured at a wavelength of 570 nm with a background subtraction at 600 nm.
2.4 Biofilm formation assay
A 0.5 McFarland solution of C. albicans inoculated to 200 uL lurian broth (LB broth) in 96 micro plates for evaluation of biofilm formation. One colony from each C. albicans was applied to inoculate 5 mL LB broth. Then, the culture incubated for 24 hours at 35°C with aeration at 4 ×g. Then, 0.5 McFarland from each C. albicans was prepared. Following this, 200 μL of each C. albicans was transferred to a 96-well micro plate. The experiment was performed in duplicate. LB broth without C. albicans was considered as a negative control. The plates were incubated for 48 hours at 35oC.
2.5 Semiquantification of biofilm biomass
Biofilm biomass was quantified using a methodology described by Mowat et al [8].
2.6 Analysis of biofilm formation
The capacity of each strain to form a biofilm was compared with that of the confluent biofilm-forming C. albicans control by analyzing the absorbance of the crystal violet stain obtained for each biofilm. This allowed each isolate to be assigned a percentage value depending on the proportion of biofilm biomass it was able to establish after 48 hours in comparison with the control (taken as 100%).
Isolates were also divided into three groups depending on whether they formed fully established biofilms with 75% of the biomass of the positive control, moderately adherent biofilms with 25–75% biomass, or weak biofilms with 25% of the biomass of the positive control.
2.7 Antibiofilm formation activity determination of P. harmala
Different concentrations (1–15 μg/mL) of P. harmala were applied on the positive biofilm formation strains. Then, the biofilm formation assay, as described above, was performed to evaluate the efficacy of P. harmala on biofilm formation by C. albicans isolates.
3.1 Biofilm formation in C. albicans
Our results demonstrated that among 100 yeasts responsible for vaginitis, 27% (n = 27) of them were C. albicans. Our analysis demonstrated that all types of biofilm formation were observed; while the most biofilm formation was observed as weak biofilms (37%, n = 10). The lowest biofilm formation was observed for both moderate and strong biofilm formation (each, 7.4%, n = 2). The remaining strains (48.2%, n = 13) showed no biofilm formation (Figure 1).
3.2 P. harmala as a candidate for inhibition of biofilm formation in C. albicans
The IC50 for P. harmala was 15 μg/mL. P. harmala, in a concentration of 12 μg/mL, easily inhibited biofilm formation on strong biofilm formation strains; while concentrations of 6 μg/mL and 10 μg/mL inhibited biofilm formation in weak and moderate biofilm formation C. albicans strains, respectively.
C. albicans is considered as a human pathogen and causes many infections ranging from severe to mild candidacies. Many factors are involved in the pathogenicity of C. albicans, including the production of extracellular enzymes, biofilm formation, and surface adherence 9, 10. Also, C. albicans is settled in the vaginal cavity as a microbiota that causes vulvovaginal candidiasis. Our finding demonstrated that there is variation in biofilm formation in C. albicans, which is consistent with obtained results by Sherry et al [11]. It seems to be necessary to investigate traditional herbs against pathogenic microorganisms. Our findings demonstrated the antibiofilm effectiveness of P. harmala against C. albicans. However, more investigations, including in vivo studies, are needed to study P. harmala as an antibiofilm in C. albicans.
The author has no conflicts of interest to declare.
  • 1. Kent H.L.. Epidemiology of vaginitis. Am J Obstet Gynecol 165(4 Pt 2). 1991 Oct;1168−1176. PMID: 1951572.ArticlePubMed
  • 2. Naparstek L., Carmeli Y., Navon-Venezia S.H.. Biofilm formation and susceptibility to gentamicin and colistin of extremely drug-resistant KPC-producing Klebsiella pneumoniae. J Antimicrob Chemother 69(4). 2014 Apr;1027−1034. PMID: 24408988.ArticlePubMed
  • 3. Sobel J.D., Faro S., Force R.W.. Vulvovaginal candidiasis: epidemiologic, diagnostic, and therapeutic considerations. Am J Obstet Gynecol 178(2). 1998 Feb;203−211. PMID: 9500475.ArticlePubMed
  • 4. Li X., Yan Z., Xu J.. Quantitative variation of biofilms among strains in natural populations of Candida albicans. Microbiology 149(Pt 2). 2003 Feb;353−362. PMID: 12624197.ArticlePubMed
  • 5. Thompson J.D., Manicacci D., Tarayre M.. Thirty-five years of thyme: a tale of two polymorphisms. Why so many females? Why so many chemotypes? Bioscience 48(10). 1998 Oct;805−815.Article
  • 6. Moloudizargari M., Mikaili P., Aghajanshakeri S.H.. Pharmacological and therapeutic effects of Peganum harmala and its main alkaloids. Pharmacogn Rev 7(14). 2013 Jul–Dec;199−212. PMID: 24347928.ArticlePubMed
  • 7. Ahmadzadeh A., Valavi E., Shamsizadeh A.. Fungal urinary tract infection in an infant with posterior urethral valves. Jundishapur J Microbiol 4(Suppl. 1). 2011;S71−S76.
  • 8. Mowat E., Rajendran R., Williams C.. Pseudomonas aeruginosa and their small diffusible extracellular molecules inhibit Aspergillus fumigatus biofilm formation. FEMS Microbiol Lett 313(2). 2010 Dec;96−102. PMID: 20964704.ArticlePubMed
  • 9. Calderone R.A., Fonzi W.A.. Virulence factors of Candida albicans. Trends Microbiol 9(7). 2001 Jul;327−335. PMID: 11435107.ArticlePubMed
  • 10. Gokce G., Cerikcioglu N., Yagci A.. Acid proteinase, phospholipase, and biofim production of Candida species isolated from blood cultures. Mycopathologia 164(6). 2007 Dec;265−269. PMID: 17874282.ArticlePubMed
  • 11. Sherry L., Rajendran R., Lappin D.F.. Biofilm formed by Candida albicans bloodstream isolates display phenotypic and transcriptional heterogeneity that are associated with resistance and pathogenicity. BMC Microbiol 14:2014 Jul;182PMID: 24996549.ArticlePubMed
Figure 1
Biofilm formation among Candida albicans collected from patients with vaginitis.

Figure & Data



    Citations to this article as recorded by  
    • Effect of Peganum harmala Extract on Biofilm and Involved Gene Expression in Biofilm Production of Candida albicans
      Maryam Erfaninejad, Elham Aboualigalehdari, Mahnaz Fatahinia
      Jundishapur Journal of Microbiology.2023;[Epub]     CrossRef
    • Antifungal Mechanisms of a Chinese Herbal Medicine, Cao Huang Gui Xiang, Against Candida Species
      Huizhen Yue, Xiaolong Xu, Shasha He, Xuran Cui, Yuhong Guo, Jingxia Zhao, Bing Peng, Qingquan Liu
      Frontiers in Pharmacology.2022;[Epub]     CrossRef
    • An overview on ethnobotanico-pharmacological studies carried out in Morocco, from 1991 to 2015: Systematic review (part 1)
      Jamila Fakchich, Mostafa Elachouri
      Journal of Ethnopharmacology.2021; 267: 113200.     CrossRef
    • Improving health benefits with considering traditional and modern health benefits of Peganum harmala
      Mohamad Hesam Shahrajabian, Wenli Sun, Qi Cheng
      Clinical Phytoscience.2021;[Epub]     CrossRef
    • Phytomedicine from Middle Eastern Countries: An Alternative Remedy to Modern Medicine against Candida spp Infection
      Mohammad Zubair Alam, Mohd Sajjad Ahmad Khan, Jenny Wilkinson
      Evidence-Based Complementary and Alternative Medic.2021; 2021: 1.     CrossRef
    • In vitro and in vivo activity of Peganum harmala L. alkaloids against phytopathogenic bacteria
      Hanan A. Shaheen, Marwa Y. Issa
      Scientia Horticulturae.2020; 264: 108940.     CrossRef
    • Molecules and Metabolites from Natural Products as Inhibitors of Biofilm in Candida spp. pathogens
      Rajeev K. Singla, Ashok K. Dubey
      Current Topics in Medicinal Chemistry.2019; 19(28): 2567.     CrossRef
    • Assessment of the Antimicrobial and Antioxidant Activities of Ziziphus lotus and Peganum harmala
      Leila Ait Abderrahim, Khaled Taïbi, Chahinez Ait Abderrahim
      Iranian Journal of Science and Technology, Transac.2019; 43(2): 409.     CrossRef
    • Evaluation of Biofilm Formation and Anti-biofilm Properties of Peganum Harmala and Crocus Sativus in Shigella Flexneri Clinical Isolates
      Mahsa Jalili, Mansour Amraei, Nourkhoda Sadeghifard, Sobhan Ghafourian
      The Open Microbiology Journal.2019; 13(1): 297.     CrossRef
    • PgTeL, the lectin found in Punica granatum juice, is an antifungal agent against Candida albicans and Candida krusei
      Pollyanna Michelle da Silva, Maiara Celine de Moura, Francis Soares Gomes, Danielle da Silva Trentin, Ana Patrícia Silva de Oliveira, Gabriela Souto Vieira de Mello, Maira Galdino da Rocha Pitta, Moacyr Jesus Barreto de Melo Rego, Luana Cassandra Breitenb
      International Journal of Biological Macromolecules.2018; 108: 391.     CrossRef
    • Antibiofilm activities of norharmane and its derivatives against Escherichia coli O157:H7 and other bacteria
      Jin-Hyung Lee, Yong-Guy Kim, Sang Hee Shim, Jintae Lee
      Phytomedicine.2017; 36: 254.     CrossRef
    • Synthesis of silver nanoparticles using Peganum harmala extract as a green route
      Matin Azizi, Sajjad Sedaghat, Kambiz Tahvildari, Pirouz Derakhshi, Ahad Ghaemi
      Green Chemistry Letters and Reviews.2017; 10(4): 420.     CrossRef

    • PubReader PubReader
    • Cite
      export Copy
    • XML DownloadXML Download

    PHRP : Osong Public Health and Research Perspectives