Biological properties of Algerian Cyclamen africanum extracts

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Biological properties of Algerian Cyclamen africanum extracts
  Advances in Environmental Biology , 8(4) March 2014, Pages: 900-903 AENSI Journals Advances in Environmental Biology ISSN-1995-0756 EISSN-1998-1066 Journal home page: Corresponding Author:  Dr. Samah Djeddi, Department of Biology, Faculty of Science, Badji Mokhtar University, PO Box 12, Annaba 23000. Algeria. Tel: +213 697 19 21 43, Fax +213 38 87 54 00 Biological Properties of lgerian yclamen africanum  Extracts 1 Wissam Mazouz   and 2 Samah Djeddi   1  Laboratory of Biomolecules and Plant Breeding, Life Science and Nature Department, Faculty of Exact Science and Life Science and  Nature, Larbi Ben Mhidi University , PO Box 358, Oum El Bouaghi 04000, Algeria. 2  Department of Biology, Faculty of Science, Badji Mokhtar University, PO Box 12, Annaba 23000. Algeria. ARTICLE INFO ABSTRACT Article history:  Received 14 January 2014  Received in revised form 19  April 2014  Accepted 23 April 2014  Available online 5 May 2014  Key words: Cyclamen africanum, antioxidant activity, antibacterial activity. Cyclamen africanum  is an endemic species to the North of Africa, in Algeria it has never been subject to any previous biological studies, for this reason we choose to investigate the antioxidant as well as the antibacterial activities of its extracts using the DPPH free radical scavenging and the disc diffusion methods. The results obtained showed that all extracts (polar and non-polar) showed a significant antioxidant activity while we noticed that there is no significant antibacterial activity observed for all extracts tested. © 2014 AENSI Publisher All rights reserved . To Cite This Article:  Wissam Mazouz and Samah Djeddi., Biological Properties of Algerian Cyclamen africanum  Extracts. Adv. Environ. Biol., 8(4),  900-903, 2014   INTRODUCTION It is widely known that plants are a major source of drugs, by using the secondary metabolics they control their environment. It should also be noted that these molecules: tannins, glycosides, mucilage, flavonoids, saponins, resins, gum, provide significant healing properties that no synthesis and combinatorial chemistry can offer [9][14][15]. Many previous studies have proved that the phenol compounds, such as flavonoids, exhibit antioxidant  properties due to their high redox potential. They also exhibit a wide range of biological activities like antimicrobial, anticarcinogenicity and antiproliferation [19]. In continuation on our investigation on the Algerian flora [5,6]. We report here the biological investigation of Cyclamen africanum Boiss & Reut which is a perennial plant [2] endemic to the North of Africa [4], belongs to the family of  primulaceae [12]. Since the biological activities of C. africanum have not previously been reported, the main goal of this study was to evaluated antioxidant and antibacterial activities of three crude extracts of C. africanum ; namely, dichloromethane, methanol and methanol-water. MATERIALS AND METHODS  Plant material: The aerial parts of Cyclamen africanum Boiss & Reut growing wild in Maouna Mountain located at Guelma city (North East of Algeria), were collected during the pre-flowering stage in May 2013. The samples were cleaned and dried at room temperature.  Preliminary phytochemical tests: The preliminary phytochemical tests are required the use of 20 g of powdered plant material to prepare: ether, methanol and aqueous extracts obtained by successive extraction with solvents of increasing polarity (petroleum ether, methanol and distilled water). Detection of chemical groups is performed according the  protocol described by Zellagui et al [20] on these three types of extracts:    Identification of volatile oils  : the ether extract has been evaporated to dryness. The presence of a pleasant odor indicating the presence of volatile oils;    Identification of sterols and triterpenes  : the residue of the ether extraction has been dissolved in 0.5ml of acetic anhydride, then in 0.5ml of chloroform, later in 1 ml of concentrated sulphuric acid (Lieberman-Burchards reaction). The appearance at the interface, a brownish red ring indicates a positive reaction;  901 Wissam Mazouz and Samah Djeddi, 2014 Advances in Environmental Biology , 8(4) March 2014, Pages: 900-903    Identification of flavone aglycones  : the residue of the ether extract was dissolved in 2ml methanol at 50 Cº. Metallic magnesium and 5 drops of concentrated HCl were added. A red or orange colour indicates the presence of flavones aglycones (Shibata’s reaction);      Identification of anthracenoside aglycones (emodols)  :1 ml of 25% NH 4 OH was added to the etheric extract (Bortrager reaction). After agitation the appearance of a red colour indicates the presence of emodols;    Identification of coumarins: the residue of the etheric extract was dissolved after the drought in hot water. The solution is divided into two equal volumes: one containing the reference and the second is made alkaline with 0.5ml of 10% ammonia solution. The appearance of an intense fluorescence under UV light shows the  presence of coumarin and its derivatives;    Identification of tannins  : 1 ml of the water extract was diluted with 2 ml of water and 3 drops of the diluted solution of ferric chloride was added. The appearance of light blue or light green colour indicates the  presence of tannins;    Identification of anthracenosides  : in alcoholic extract (method Bortrager reaction using the ether extract);    Identification of polyuronides  : 2 ml of the extract were added dropwise in a test tube, where 10 ml of acetone have been placed. The observation of a wholesale flake precipitates indicating the presence of  polyuronides. Samples extraction: Dried materiel (200g) was ground finely and extracted following the Dall’Acqua et al [3] modified method. We used four solvents with increasing polarity: petroleum ether, dichloromethane, methanol and methanol-water (5:1), successively. The extractions were performed at room temperature for 72 h for each sample.  Antioxidant potential: Antioxidant activities of the crude extracts obtained (DCM, ME, AME) were investigated by the evaluation of the free radical scavenging capacity using the DPPH modified method described by Bounatirou et al   [1]. Methanol solution (100µl) containing different concentrations (100, 250, 500, 750 and 1000 mg l -1 ) of each crude extracts or standard antioxidants (BHT and Gallic acid) was added to 2ml of freshly prepared DPPH methanol solution (24µg/ml). An equal amount of DPPH methanol solution was used as a control. The measurements were performed in triplicate. After incubation for 30min at room temperature, scavenging capacity was read spectrophotometrically by monitoring the decrease in absorbance at 517 nm. Activity of scavenging was calculated using the following formula: DPPH radical scavenging (%) = [(Abs control    –   Abs sample ) / Abs control ] x 100  Antibacterial activity: Antibacterial activities of all crude extracts as well as the positive control Gentamicin were tested against seven human pathogenic bacterial strains, including Gram positive ( Staphylococcus aureus ATCC 252923, Sterptococcus sp ) and Gram negative (  Pseudomonas aeruginosa ATCC 27853,  Escherichia coli ATCC 25922,  Klebsiella oxytoca ,  Klebsiella pneumoniae , Salmonella sp ) by using disc diffusion method described by Djeddi et al   [5] with slight modifications. The bacterial suspension concentration was equilibrated to 0.5 McFarland standard, and was inoculated over the surface of a Petri dishes containing Muller-Hinton agar. Whatman paper disc injected by 20µl of each sample (1mg/ml) obtained by sterile distilled water, while Gentamicin disc were placed on the surface of Petri dishes. The bacteria were incubated at 37°C for 24h. The inhibition zones were calculated by measuring the diameter of inhibition in mm (including disc). Experiments were performed in duplicate and the inhibition zones were compared with those of the positive control. RESULTS AND DISCUSSION  Preliminary phytochemical tests: The results indicate the presence of volatile oils, tannins, polyuronides, traces of sterols and triterpenes; while we observe the absence of flavones aglycones, anthracenosides aglycones and anthracenosides. To the  best of our knowledge, there is no phytochemical investigation reported on the aerial parts of this species.  Extractions yield: The extractions yield of the four solvents used are represented in Table 1. The highest extract yield was obtained by methanol-water one, so we conclude that the highest polar solvent was more efficient in extracting than the less polar solvents, this observation is in agreement with the results reported by some previous research [3,13].  902 Wissam Mazouz and Samah Djeddi, 2014 Advances in Environmental Biology , 8(4) March 2014, Pages: 900-903 Table 1:  Extractions yield of C. africanum  using various solvents. Solvents Yield (%)   Petroleum ether 1.56 Dichloromethane 1.61 Methanol 6.88 Methanol-water (5:1) 7.25  Antioxidant activity : As summarized in Table 2, crude extracts of C. africanum and the standard antioxidants tested showed a significant antioxidant activity. The methanol extract (ME) possessed the highest antioxidant activity followed by the methanol-water (MW) extract and finally dichloromethane extract (DCM) which showed the lowest potential. This result is compared with other previous research, where it was seen that the methanol extract was the most efficient solvent for extracting the antioxidant natural compounds [13.17]. Table 2:  DPPH scavenging activity (%) of C. africanum  crude extracts and standard antioxidant. Concentrations (mg l -1 ) C. africanum crude extracts Standard antioxidants DCM ME MW BHT Gallic acid 100 250 500 750 1000 16.92 ± 0.837 17.943 ± 0.109 17.753 ± 0.290 19.096 ± 2.297 22.046 ± 4.30 29.516 ± 1.579 36.533 ± 0.385 47.430 ± 0.773 61.210 ± 2.818 70.440 ± 1.656 21.033 ± 5.060 39.546 ± 3.466 46.276 ± 10.380 55.126 ± 2.324 64.286 ± 12.718 59.293 ± 5.771 82.046 ± 2.938 86.790 ± 0.913 87.560 ± 0.294 88.006 ± 0.109 87.746 ± 0.721 88.200 ± 0.294 88.776 ± 0.109 87.946 ± 1.057 89.096 ± 0.295 Each value represents the mean of three replicates ± standard deviation Previous study have proved that the petroleum ether, acetone, methanol and water extracts of Cyclamen mirabile leaves showed higher antioxidant activities copared with α -tocophérol and BHT (Mazouz and Djeddi, 2013).  Antibacterial activity: For the assessment of the antibacterial potential of the extracts studied, we choose to test them against many  bacterial species, because each one has a particular cellular structures and metabolism. The results presented in Table 3 showed that the ME extract don’t have any antibacterial activity against all  bacteria tested, DCM extract and MW extract, showed a slight activity against  Pseudomonas aeruginosa . All these extract are considered inactive compared to the positive control which have a strong activity against all bacterial strains tested. Many researchers demonstrate that Cyclamen mirabile  is used as an antifungal agent against Candida  species and Cryptococcus neoformans  [9]. The period of collection of the plant can influence the antibacterial activity of the crude extracts. Bounatirou et al   [1] have proved that Thymus capitatus   Hoff & Link essential oil don’t hav e antibacterial activity in pre-flowering period, while it exhibited a higher antibacterial activity during flowering period. The method used to evaluate the antibacterial activity also can affects the results; Natarajan et    al   [10] and Fazeli et al   [8] found that the diffusion method from wells on agar is more appropriate to study the activity of aqueous and organic extracts of  Euphorbia fusiformis and hydro-ethanolic extracts of  Rhus coriaria and  Zataria multifora , than the method of agar diffusion. Table 3:  Antibacterial activity of C. africanum  crude extracts expressed as the diameter of the inhibition zone in mm in the disk sensitivity assay. Bacterial strains C. africanum ’s  crude extracts Gentamicin DCM ME MW S. aureus ATCC 252923 Streptococcus sp  P. aeruginosa ATCC 27853  E. coli ATCC 25922  K. oxytoca    K. pneumonie Salmonella sp - - 9.5 ± 0.707 - - - - - - - - - - - - - 6.5 ± 0.707 - - - - 10.5 ± 0.707 32 ± 2.828 39 ± 1.414 39.5 ± 0.707 14 ± 1.414 15 ± 0.000 34.5 ± 0.707 Each value represents the mean of two replicates ± standard deviation *The sensitivity to the different strains was classified by the diameter of the inhibition zone as follows (Ponce et al  , 2003): -: diameter less than 8 mm, not sensitive; +: sensitive, diameter 9-14 mm; ++: very sensitive, diameter 15-19 mm; +++: extremely sensitive for diameter larger than 20 mm. Conclusion: From the present study we concluded that the crude extracts of C. africanum  possessed only antioxidant activity, the result indicate that the extraction yield and the antioxidant potential depend on the polarity of the solvent. However, the main components responsible of the antioxidant activity of the extracts of C. africanum    903 Wissam Mazouz and Samah Djeddi, 2014 Advances in Environmental Biology , 8(4) March 2014, Pages: 900-903 still unknown. Future studies will be performed to clarify the antioxidant mechanisms of C. africanum  compounds. ACKNOWLEDGMENT Authors warmly thanks Miss Amina Beljazia (Department of Ecology, Faculty of Biology, University of Setif, Algeria) for plant authentication and kind help, they are grateful for the financial support provided by the DRSDT (Direction Générale de la Recherche Scientifique et du Développement Technologique, Algeria) under the code: National Research Project n° 237/ANDRS/2011. REFERENCES [1] Bounatirou, S., S. Smiti, M.G. Miguel, L. Faleiro, M.N. Rejeb, M. Neffati, M.M. Costa, A.C. Figueiredo, J.G. Barroso and L.G. Pedro, 2007. 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