This document reports on a study that evaluated the in vitro antibacterial activity of methanolic and aqueous extracts of Anacyclus pyrethrum, a plant used in Moroccan traditional medicine. The extracts were tested against 6 bacterial strains using the disc diffusion method to determine zones of inhibition and the MTT assay to determine minimum inhibitory concentrations (MICs). The results showed that both extracts inhibited the growth of all bacterial strains, with MICs as low as 3.125 mg/ml. This supports the traditional use of this plant and suggests it may be a potential source of new antibiotics.
This document reports on a study that evaluated the in vitro antibacterial activity of methanolic and aqueous extracts of Anacyclus pyrethrum, a plant used in Moroccan traditional medicine. The extracts were tested against 6 bacterial strains using the disc diffusion method to determine zones of inhibition and the MTT assay to determine minimum inhibitory concentrations (MICs). The results showed that both extracts inhibited the growth of all bacterial strains, with MICs as low as 3.125 mg/ml. This supports the traditional use of this plant and suggests it may be a potential source of new antibiotics.
This document reports on a study that evaluated the in vitro antibacterial activity of methanolic and aqueous extracts of Anacyclus pyrethrum, a plant used in Moroccan traditional medicine. The extracts were tested against 6 bacterial strains using the disc diffusion method to determine zones of inhibition and the MTT assay to determine minimum inhibitory concentrations (MICs). The results showed that both extracts inhibited the growth of all bacterial strains, with MICs as low as 3.125 mg/ml. This supports the traditional use of this plant and suggests it may be a potential source of new antibiotics.
This document reports on a study that evaluated the in vitro antibacterial activity of methanolic and aqueous extracts of Anacyclus pyrethrum, a plant used in Moroccan traditional medicine. The extracts were tested against 6 bacterial strains using the disc diffusion method to determine zones of inhibition and the MTT assay to determine minimum inhibitory concentrations (MICs). The results showed that both extracts inhibited the growth of all bacterial strains, with MICs as low as 3.125 mg/ml. This supports the traditional use of this plant and suggests it may be a potential source of new antibiotics.
1 University Mohammed V Souissi, Faculty of Medicine and Pharmacy, Department of Drugs Sciences, Laboratory of Pharmacology and Toxicology, Research Team 10000 Rabat, Morocco, 2 Received: 03 Mar 2012, Revised and Accepted: 06 Apr 2012 Laboratory of Microbiology, Hospital Ibn Baja, 35000 Taza, Morocco. Email: [email protected] ABSTRACT The antibacterial activity against six standard bacterial strains evaluated with the disc diffusion assay and the MTT assay to determinate the Minimum Inhibitory Concentration (MIC). The result of the antimicrobial activity indicated by zone of inhibition of growth ranged from 13.0 to 22.2mm. The antibacterial activity demonstrated that all extracts have an inhibitory activity against bacteria with the lowest MIC at 3.125mg/ml. The extract contains some major bioactive compounds that inhibit the growth of microorganisms thereby proving very effective as alternative source of antibiotics. Our results support the ethno-pharmacological uses of this plant in folk medicine and could provide useful data for the utilization of this extracts in pharmaceutical, cosmetic and food industries. Keywords: Anacyclus perythrum, Root extract, Medicinal plants, Antibacterial activity, Minimum inhibitory concentration
INTRODUCTION All over the world, infectious disease is the number one cause of death accounting for approximately one-half of all the deaths in tropical countries. This perhaps may be attributed to the increasing incidence of multiple resistances in human pathogenic microorganisms in recent years, largely due to the indiscriminate use of commercial antimicrobial drugs commonly employed in the treatment of infectious diseases 1 . Medicinal plants have been used for centuries as remedies for human diseases as they contain components of therapeutic value 2- 4 . Very few antimicrobial studies have focused on in vivo models 5, 6 . Recently much attention has been paid to extracts and biologically active compounds isolated from plant species used in herbal medicine. However, there have been some efforts to mimic the traditional use of plant material as noted by the addition of aqueous extracts in screening assays 7-12 . Several methods are currently available to detect the antibacterial activity of plant extracts using different principles to assess bacterial growth or its inhibition. Some reports have focused on the antimicrobial screening together with other pharmacological investigations including toxicity 13, 14 . In Morocco, there is more than 42,000 species of plants; divided into 150 families and 940 genus used in traditional medicine 2, 15 . Anacyclus Perythrum (AP) locally known as Aud el-attas, Akkar Karha and Agargarha is a species commonly used in Moroccan traditional medicine. The powder of the root is well known as sternutatory, diaphoretic and used for many ailments. Showing the root is considered to be sialagogue, and to relieve toothache. In liver diseases is recommended, mixed with olive oil it is used in rheumatism, sciatic, colds, neuralgic, and paralysis 16 . In addition, the AP is also used as cardiotonic to treat typhus fever 17 . The chemical analysis of the roots shows that they contain three fatty acids, one sterol and ten unsaturated amides, more specifically: pellitorine, anacycline, phenylethylamide, enetriyne alcohol, inulin, polyacetylenic amides I-IV, and sesamin. The plant contains also tannins, gum and essential volatile oil. Pyrethrine, an alkaloid, yielding pyrethric acid, is stated to be one of the active principles 32 . However the antibacterial activity has not yet been studied. Thus, the aim of this study is to evaluate the acute toxicity and the antibacterial activity of the methanolic and aqueous extracts of Anacyclus perythrum against different bacterial strains which may be involved in such diseases, using a prelimary bioassay screening and to quantify the minimum inhibition concentration (MIC) using colorimetric method 18 MATERIALS AND METHODS . Plant material Anacyclus perythrum, from Asteraceae family, was collected in 28 March 2009, 5km east of Morocco with collection number 77765. The plant was identified by a botanist M. Fennane from the Scientific Institute of Rabat (Morocco), where a voucher specimen has been deposited with number 3949. Extraction procedure The roots were air dried for 20 days, and then washed free from soil, powdered and stored in airtight containers at room temperature until extraction. 1kg of the powdered roots was extracted with methanol through a maceration process within a period of 48h. The dark residues were obtained after the evaporation of the solvent under reduced pressure at 40C; the percentage of extracts yield was 12% (w/w). Simultaneously, water extract was prepared by adding 1.5L of distilled water to 1Kg of powdered plants roots and macerated within a period of 48h. It was then filtered and concentrated the extract under reduced pressure and percentage of extracts yield was 10% (w/w) for plant material. Acute toxicity The toxicological study was conducted according to the method of OECD Guideline 423. Swiss mice (20-30g) were kept at 23C of temperature, 50% humidity, on a 12h light/dark cycle and were fed at libitum with standard feed and water in the course of the study. The animals were obtained from the elevage of animal centre of Faculty of Medicine and Pharmacy of Mohammed-V Souissi University, Rabat. The extracts of plant were administered by oral route in a single dose. The mice of both sexes received the initial dose of methanolic and aqueous extracts of AP that was chosen 2000mg/kg of body weight. The control group received only the water. The animals must be individually observed, the symptoms and weight variation was recorded during the first 30min and regularly during the first 24h after treatment and daily for 14 days. Care and treatment of the mice were in compliance with the guidelines of the guide for the care and use of laboratory animals (commission on life science, national research council, 1996). Antibacterial Activity Test Bacterial strains Six bacterial strains, namely Bacillus subtilis ATCC 6633, Pseudomonas aeruginosa ATCC 15442, Klebsiella pneumoniae ATCC 53153, Escherichia coli ATCC 54127, Staphylococcus aureus ATCC 6538, Micrococcus luteus ATCC 9341, were used for antibacterial testing; The cultures of bacteria were maintained in their appropriate agar slants at +4C throughout the study and used as stock cultures. Disk diffusion method and Minimum inhibitory concentration Antibacterial activity of aqueous and methanolic extracts diluted in sterile distilled water, was evaluated by paper disc diffusion method. International Journal of Pharmacy and Pharmaceutical Sciences ISSN- 0975-1491 Vol 4, Issue 3, 2012 A Ac ca ad de em mi ic c S Sc ci ie en nc ce es s Doudach et al. Int J Pharm Pharm Sci, Vol 4, Issue 3, 402-405 403 Disks impregnated with sterile distilled water served as negative controls, Cephaxitin and Amikacin are used as positive control 19, 30, 31 . The viability indicator MTT (3-(4, 5 dimethylthiazol-2-yl)-2, 5 diphenyltetrazolium bromide, Sigma, Aldrich) and the quick microplate metho 20 were used for the determination of the minimum inhibitory concentration (MIC) 21 . For the inoculum, turbidity was adjusted to 1.5 x 108 CFU/ml with the reference to Mc Farland turbidometer 22 The mean values and standard deviations of all replicates described in the above tests were calculated using Delta Graph (USA). The data of the extracts were statistically analysed by ANOVA. A students t- test was computed for the statistical significance of the results. Differences were considered significant with p < 0.05. . Serial dilutions of extracts were dissolved in distilled water and made in a concentration range from 25mg/ml to 200mg/ml in sterile test tubes. Each well of micro-titer plate was inoculated with 20L of the bacterial suspension (15l of Mueller Hinton broth and 5l of the bacterial Inoculum) and 20l of sample of various concentrations of the extracts. The plate was incubated under optimal conditions at 37C for 24h. After incubation, as an indicator of bacterial growth 1 mg/ml of MTT was added to each well, after incubation periods ranging from 3 to 5h at 37C, control without plant extracts with MTT and bacterial inoculums were used as negative control. Optical densities were measured at 570nm by using a microplate reader. The bacterial suspension changed to blue when bacterial growth occurred. The MIC is defined as the lowest concentration of antibiotic or extract at which there is no visible growth. All tests were performed in triplicate. Statistical analysis RESULTS AND DISCUSSION Acute toxicity of Anacyclus pyrethrum During the 14 days, the evolution of the weight was established. Abdominal contraction was observed by 20-25min after the oral administration of aqueous extract of Anacyclus pyrethrum and no mortality was recorded. Generally, no toxicity was demonstrated at the dose of 2000mg/kg and the extracts of the plant does not lead to mortality neither by oral route. Under the system of global harmonization of chemicals (GHS), this product is classified category 5, which the LD50 was higher than 2000mg/kg. Antibacterial potential of plant extracts The antibacterial activity of methanol and aqueous extracts of AP were assayed in vitro by a disk diffusion method (Table1) using Cephaxitin and Amikacin as positif control against Gram positive and Gram negative pathogenic bacteria. All the assayed extracts significantly inhibited the growth of the bacterial strains, the methanol extract showed the highest antibacterial activity (P<0.05), the diameter of the zone inhibition ranging from 16 - 40mm (Fig.1).
Table 1: Diameters of the inhibition zones of the growth of the bacterial strains in mm induced by Antibiotics of third generation.
Fig. 1: Graph of the inhibition diameters in mm of methanolic extract tested on bacterial strains. The significance was evaluated by means values S.E. of Student test in triplicate experiments: *p < 0.05 **p <0.01 ***p<0.001). 25 mg/ml 50 mg/ml 200 mg/ml 100mg/ml
Doudach et al. Int J Pharm Pharm Sci, Vol 4, Issue 3, 402-405 404
Fig. 2: Graph of the inhibition diameters in mm of aqueous extract tested on bacterial strains. The significance was evaluated by means values S.E. of Student test in triplicate experiments: *p < 0.05 **p <0.01 ***p<0.001).
According to the results, methanolic and aqueous extracts (Figure 2) were found to be active against all pathogenic bacteria and the best results were observed with methanolic extract. The strongest antibacterial activity was seen against Bacillus subtilis, Staphylococcus aureus, Escherichia coli and Micrococcus luteus with a MIC value of 3.125mg/ml followed by MIC 6.25mg/ml while Klebsiella pneumoniae methanolic extracts and Pseudomonas aeruginosa aqueous extracts of AP. Overall, the reduction in bacterial viability was dose dependent and it was higher (p<0.05) (Table 2). Most of the researchers have evaluated first the antibacterial spectra of plant extracts; secondly they have determined the chemical composition of the extracts, and thirdly they have retroactively attributed such activity to such molecule 23-27
. In this study the extracts of AP showed higher antibacterial potential against different bacterial strains, this antibacterial activity is may be related to their secondary metabolites; alkaloids, phenolics and terpenoids reported in this plant (Guide to Medicinal Plants in North Africa).
Table 2: The minimum inhibitory concentration (MIC) of the extracts of the plant tested against different bacteria. Data represent the MICs (mg/mL). Standard reference bacterial strains Gram-negative organisms Gram-positive organisms Extracts EC KB PA SA ML BS AE. AP 3.125 3.125 6.25 3.125 3.125 3.125 ME. A P 3.125 6.25 3.125 3.125 3.125 3.125 BS: Bacillus subtilis ATCC 6633, PA: Pseudomonas aeruginosa ATCC 15442, KB: Klebsiella pneumoniae CIP 53153, EC: Escherichia coli ATCC 54127, SA: Staphylococcus aureus ATCC 6538 and ML: Micrococcus luteus ATCC 9341, AE: aqeous extract; ME: methanolic extract; AP: Anacyclus perythrum.
Gram-negative bacteria which are responsible for a large number of infectious diseases have a unique outer membrane that contains lipopolysaccharides which render them impermeable to certain antibacterial compounds 28 . All extracts (methanolic and aqueous extracts of AP) showed antibacterial activity against both Gram- positive and Gram-negative bacteria. This gives an indication of the presence of promising antibacterial compounds. The selection of bacterial strains was based on their pathogenicity lead to infectious diseases, poor sanitary conditions, unclean unsafe drinking water and inappropriate food storage conditions are common in poor families that frequently suffer from diarrhea and vomiting. In most cases, they treat themselves by using local herbs 29 CONCLUSION . Our results against these bacterial strains suggest the efficacy of plant claimed by traditional healers. This plant may be a source which could yield drug that could improve the treatment of infections caused by this organism. The data indicate that the extracts of the Anacyclus pyrethrum species inhibit efficaciously some resistant bacterial strains which make serious sanitary problems worldwide and confirm that there is a good correlation between the reported traditional uses of the plant for infectious diseases. This indicates that this plant may be useful for developing alternative compounds to treat infections caused by these antibiotic resistant pathogens. According to these results, it is possible to conclude that Anacyclus pyrethrum had a strong and a broad spectrum of antibacterial activity. To the best of our knowledge, this is the first study to provide data that the methanolic 50mg/ml 25mg/ml 100mg/ml 200mg/ml Doudach et al. Int J Pharm Pharm Sci, Vol 4, Issue 3, 402-405 405 and aqueous extracts of Anacyclus pyrethrum evaluated against a wide range of bacteria. ACKNOWLEDGEMENTS The authors wish to thank all the individuals and institutions who made this survey possible. REFERENCES 1. Aliyue AB, Musa A, Abdullahi MA, Ibrahim MS, Tijjani MB, Aliyu MS, Oyewale AO. Activity of saponin fraction of Anisopus mannii against some pathogenic microorganisms. J Med Plants Res 2011; 5: 6709-6713. 2. Alnamer R, Alaoui K, Bouidida El H, Benjouad A, Cherrah Y. Psychostimulant activity of Rosmarinus officinalis essential oils. J Nat Prod 2012a; 5: 83-92. 3. Alnamer R, Alaoui K, Bouidida El H, Benjouad A, Cherrah Y. Sedative and Hypnotic Activities of the Methanolic and Aqueous Extracts of Lavandula officinalis from Morocco. Adv Pharmacol Sci 2012b; 2012: 5 Pages. 4. Al-Bakri AG, Afifi FU. Evaluation of antimicrobial activity of selected plant extracts by rapid XTT colorimetry and bacterial enumeration. J Microbiol Methods 2007; 68(1): 19-25 5. Dube A, Manthata LN, Syce JA. The design and evaluation of placebo material for crude herbals: Artemisia afra herb as amodel. J Ethnopharmacol 2007; 112: 138144. 6. Mukinda JT, Syce JA. Acute and chronic toxicity of the aqueous extract of Artemisia afra in rodents. J Ethnopharmacol 2007; 112: 138144. 7. Rabe T, Van Staden J. Antibacterial activity of South African plants used for medicinal purposes. J Ethnopharmacol 1997; 56: 8187. 8. Kelmanson JE, Jger AK, Van Staden J. Zulu medicinal plants with antibacterial activity. J Ethnopharmacol 2000; 69: 241 246. 9. McGaw LJ, Jger AK, Van Staden J. Antibacterial, anthelmintic and anti-amoebic activity in South African medicinal plants. J Ethnopharmacol 2000; 72: 247263. 10. Scott G, Springfield EP, Coldrey N. A pharmacognostical study of 26 South African plant species used as traditional medicines. Pharm Biol 2004; 43: 186213. 11. Eldeen MS, Elgorashi EE, Van Staden J. Antibacterial, anti- inflammatory, anti-cholinesterase and mutagenic effects of extracts obtained from some trees used in South African traditional medicine. J Ethnopharmacol 2005; 102: 457464. 12. Reid KA, Jger A, Light ME, Mulholland DA, Van Staden J. Phytochemical and pharmacological screening of Sterculiaceae species and isolation of antibacterial compounds. J Ethnopharmacol 2005; 97: 285291. 13. Kamatou GPP, Viljoen AM, Figueiredo AC, Tilney PM, Van Zyl RL, Barroso JG, Pedro LG, Van Vuuren SF. Trichomes, essential oil composition and biological activities of Salvia albicaulis Benth and S. dolomitica Codd, two species from the Cape region of South Africa. South African J Bota 2007; 73: 102108. 14. Viljoen AM, Moolla A, VanVuuren SF, VanZyl RL, Bas KHC, Demirci B, zek Trinder-Smith T. The biological activity and essential oil composition of 17 Agathosma (Rutaceae) species. J Essen Oil Res 2006; 18: 216. 15. Alnamer R, Alaoui K, Bouidida El H, Benjouad A, Cherrah Y. Toxicity and Psychotropic Activity of Essential Oils of Rosmarinus officinalis and Lavandula officinalis from Morocco. J Biol Act Prod Nat 2011; 1(4): 262-272. 16. Bellakhdar J. Anacyclus pyrethrum L.: La Pharmacope Marocaine Traditionnelle. Ed Le Fennec, Ibis Press; 1997: pp. 177. 17. Alluri V, Tayi V, Sundararajua D, Vanisreeb M, Tsayb H, Gottumukkala VS. Assessment of Bioactivity of Indian Medicinal Plants Using Brine Shrimp (Artemia salina) Lethality Assay. Int J Appl Sci Eng 2005; 3(2): 125-134. 18. Grare M, Fontanay S, Cornil C, Finance C, Duval E.R. Tetrazolium salts for MIC determination in microplates: Why? Which salt to select? How?. J Microbiol Methods 2008; 75: 156 159. 19. Zampini IC, Vattuone M., Isla MI. Antibacterial activity of Zuccagnia punctata Cav ethanolic extracts. J Ethnopharmacol 2005; 102(3): 450-456. 20. Eloff JN. A sensitive and quick method to determine the minimal inhibitory concentration of plant extracts for bacteria. Planta Med 1998; 64: 711-713. 21. Oumzil H, Ghoulami S, Rhajaoui M, Ilidrissi A, Fkih-Tetouani S, Faid M, Benjouad A. Antibacterial and antifungal activity of essential oils of Mentha suaveolens. Phytother Res 2002; 16: 727-731. 22. Andrews W. Manual of food quality control. 4. Rev. 1. Microbiological analysis. Washington: Food and Drug Administration; 1992. 23. Cowan MM. Plant products as antimicrobial agents. Clin Microbiol Rev 1999; 12: 564582. 24. Chattopadhyay D, Arunachalam G, Mandal AB, Sur TK, Mandal SC, Bhattacharya SK. Antimicrobial and anti-inflammatory activity of folklore: Mallotus peltatus leaf extract. J Ethnopharmacol 2002; 82: 229 237. 25. Chandramu C, Manohar RD, Krupadanam DG, Dashavantha RV. Isolation, characterization and biological activity of betulinic acid and ursolic acid from Vitex negundo L. Phytother Res 2003; 17: 129 134. 26. Horiuchi K, Shiota S, Hatano T, Yoshida T, Kuroda T, Tsuchiya T. (): Antimicrobial activity of oleanolic acid from Salvia officinalis and related compounds on vancomycin-resistant enterococci (VRE). Biol Pharm Bull 2007; 30: 11471149. 27. Zenasni, L, Bouidida H, Hancali A, Boudhane A, Amzal H, Il Idrissi A, El Aouad R, Bakri Y, Benjouad A. The essentials oils and antimicrobial activity of four nepeta species from Morocco. J Med Plants Res 2008; 2(5): 111-114. 28. Clements JM, Coignard F, Johnson I, Chandler S, Palan S, Waller A, Wijkmans J, Hunter MG. Antibacterial activities and characterization of novel inhibitors of LpxC. Antimicrob Agen Chemother 2002; 46: 17931799. 29. Maregesi SM, Pieters L, David Ngassapa O, Apers S, Vingerhoets R, Cos P, Vanden Berghe DA, Vlietinck AJ. Screening of some Tanzanian medicinal plants from Bunda district for antibacterial, antifungal and antiviral activities. J Ethnopharmacol 2008; 119(1): 58 66. 30. Pratibha N, Sushma D, Rajinder G. In vitro antibacterial and antioxidant potential of medicinal plants used in the treatment of Acne. Int J Pharm Pharm Sci 2012; 4(1): 185-190. 31. Geeta S , Padma K. Antibacterial potential of alkaloids of Withana Somnifera L and Euphorbia Hirta L. Int J Pharm Pharm Sci 2012; 4(1): 78-81. 32. Guide to Medicinal Plants in North Africa. Centre for Mediterranean Cooperation, International Union for Conservation of Nature and Natural Resources: Anacyclus pyrethrum L in, Ed Kamal Batanouny 2005; pp. 35.