Advances in Natural and Applied Sciences, 8(8) July 2014, Pages: 131-140
AENSI Journals Advances in Natural and Applied Sciences
ISSN:1995-0772 EISSN: 1998-1090
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Phytochemical study of Anacyclus pyrethrum (L.) of Middle Atlas (Morocco), and in vitro study of antibacterial activity of pyrethrum
1,2Hanane Elazzouzi, 1Aminata Soro, 1Fatima Elhilali, 1Amar Bentayeb, 2Mohamed Alaoui El Belghiti, 1Touriya Zair
1Laboratory of Chemistry of Bioactive Molecules and Environment, University of Sciences Moulay Ismail, BP 11201. Zitoune, Meknès, Morocco. 2Laboratory of Chemistry – General Physics – University of Sciences- Agdal, 4- Avenue Ibn Battouta. B.P. 1014 RP, Rabat, Morocco.
ARTICLE INFO ABSTRACT Article history: Exploitation of plants used in traditional medicine is important to identify substances Received 2 April 2014 belonging to different secondary metabolites classes. These metabolites have efficient Received in revised form properties and their study and exploitation is important to rationalize their utilisation. 13 May 2014 The aim of this study is to determine the chemical composition of the spontaneous and Accepted 28 May 2014 endemic Anacyclus pyrethrum Link (Asteraceae) essential oils, to perform a Available online 27 June 2014 phytochemical screening and to assess in vitro antibacterial activity of Pyrethrum extracts. Essential oils obtained from hydrodistillation of pyrethrum samples collected Keywords: in April and June (2012) from Timahdite (Morrocan middle Atlas region) were Ancyclus Pyrethrum L., Essential oils, analysed by CG/MS. Phytochemical screening of Anacyclus pyrethrum roots was Chemical composition, Phytochemical carried out through precipitation and color reaction. Antibacterial test of volatiles and Screening, Antibacterial activity non volatiles extracts of Anacyclus pyrethrum were done against pathogen germs isolated from hospital. Disc-diffusion method in solid medium and macrodilution method in liquid medium were respectively used to determine inhibition diameters and of the antibacterial parameters to knowing, minimal inhibitory and bactericide concentrations (MIC and MBC). Essential oils yielded 0,051% and 0,07% (ml/100g) respectively in full-bloom (April) and post-bloom (June) periods. For these periods, essential oils analyses revealed spathulenol as the most abundant compound in the plant with 13,31% and 16,9% respectively in April and June. Oxygenated sesquiterpenes is the most important group among identified compounds. Phytochemical screening proved the presence of alkaloids, gallic and cathechic tanins, flavonoids, and coumarins. For antibacterial activity, results proved that aqueous macerate extract was the most active. Its inhibition diameters are 9±0,81 mm and 9,66±0,47 mm respectively against susceptible Escherichia coli and Staphylococcus aureus. Both strains have the same inhibition concentration (MIC=1/200 v/v). MBC was assessed to be superior (CMB>1/100 v/v).
© 2014 AENSI Publisher All rights reserved. To Cite This Article: Hanane Elazzouzi, Aminata Soro, Fatima Elhilali, Amar Bentayeb, Mohamed Alaoui El Belghiti, Touriya Zair, Phytochemical study of Anacyclus pyrethrum (L.) of Middle Atlas (Morocco), and in vitro study of antibacterial activity of pyrethrum. Adv. in Nat. Appl. Sci., 8(8): 131-140, 2014
INTRODUCTION
These last years, aromatic and medicinal plants exploitation and culture are in constant growth in developed and developing countries (Benjilali et al., 2005). Due to their frequency and gravity, infectious diseases are considered as a problem of great importance in public health (Traoré et al., 2012). Various types of fungi and bacteria are responsible of these infections. In the constant concern of preservation from diseases, poor populations have always sought in their biotope brut or transformed substances for their needs. Thus plants constitute a real alternative for health care’s (Bencheqroun et al., 2012). The exploitation of the plants used in traditional medicine is searched to identify substances having of the effective properties for a more rational use. Nowadays, we know that medicinal plants active principles are related to secondary metabolism products. Numerous of active principles that have considerable benefits in traditional and modern medicine have been discovered and listed (Bourgaud et al., 2001; Kar, 2007). North Africa possesses almost 1700 endemic species and subspecies and half of them are specific to Morocco (EL Oualid et al., 2012). To Anacyclus genus, belong 13 annual and perennial species mostly encountered in North -West Africa and also in other Mediterranean countries (Harald, 1978). Many Anacyclus species such as A. pyrethrum, A. radiatus, A. valentinus, A. cyrtolepodioide and A. Clavatus are used in
Corresponding Author: Hanane Elazzouzi, Laboratory of Chemistry -General Physics- Department of chemistry, University of Sciences, Rabat, Morocco. Tel: +212658330106 E-mail: [email protected] 132 Hanane Elazzouzi et al, 2014 Advances in Natural and Applied Sciences, 8(8) July 2014, Pages: 131-140 traditional medicine. Their medicinal properties are due to the presence of flavonoids and terpenoids (Harald, 1978; Efraim et al., 2008; Benitez et al. 2010). In the Mediterranean wide flora, Anacyclus pyrethrum L. (Asteraceae), commonly named “African pyrethrum” or " Tigenthast" by Moroccan people (Batanouny, 2005) was chosen. It is an endemic herbaceous and perennial species (EL Oualid et al., 2012) present in sunny medium. In North Africa, the species is encountered in wild on slimy and well-drained soils (Batanouny, 2005). Previous chemical studies show that Anacyclus pyrethrum has immunostimuling properties (Bendjeddou, 2010). Its roots appreciated as a nervous system tonic, are also used in the treatment of paralysis and epilepsy. They have anti-inflammatory (Annalakshmi et al., 2012; Rimbau, 1999), antibacterial, and insecticidal properties (Zaidi et al., 2013). They are also considered as aphrodisiac (Vicas, 2009), antidiabetic (Satyanand et al., 2011) and antioxidant (Kalim et al., 2010). Roots powder of Anacyclus pyrethrum is well known as sternutatory, sudorific and anti infectious (Doudach et al., 2012). It is considered as sialagogue and is frequently used for toothache (Selles et al., 2013). It’s also used against rheumatism, sciatic, cold and neuralgia (Doudach et al., 2012). Chemical analysis of roots shows the presence of three fatty acids, a sterol and ten unsaturated amides. The most important compounds discovered in roots are pellitorin, anacyclin, phenylethylamid, inulin, polyacetylenic amides I-IV, and sesamin. The species contains also tannins, gum and essential oil traces (Selles et al. (a), 2012; Zaidi et al., 2013, Sujith, 2012). In the aim to valorize A. Pyrethrum from Timahdite (Moroccan middle atlas), we focused on the plant potentialities through essential oil study including extraction, yield and chemical composition. Then a phytochemical screening and antibacterial study of the species were performed.
MATERIALS AND METHODS
Plant material: Wild specimens of A. Pyrethrum were manually harvested in Timahdite region in April and June (2012) corresponding to full-bloom and post-bloom period. Biomass was dried in the shade for ten days before extraction process. Botanical identification of the species was done at scientific institute of Rabat.
Microbial strains: Microbial material consists in four susceptible and resistant bacteria strains responsible of human pathology isolated from neonatology department of University Healthcare Centre (CHU) Hassan II of Fès : Staphylococcus aureus (positive gram), susceptible and resistant Escherichia coli (negative gram), Klebsiella pneumoniae resistant (negative gram), and susceptible and resistant Pseudomonas aeruginosa (negative gram). They are stored at 4°C in test tubes containing Mueller Hinton solid medium.
Phytochemical study of A. pyrethrum: Essential oils extraction: Essential oils (EOs) extraction was performed by hydrodistillation for three hours using a Clevenger-type apparatus with 100g biomass of A. pyrethrum roots, leaves/stems and flowers. EOs were then dried over anhydrous sodium sulfate (Afnor, 2000), protected from light and stored at 4°C until use.
Analyses and identification of EO’s chemical composition: Chromatographic analyses of EOs samples were performed with gas chromatograph Thermo Electron type (Trace GC Ultra) coupled to a mass spectrometer Thermo Electron Trace MS system (Thermo Electron: Trace GC ultra; Polaris Q MS), fragmentation is performed by electronic impact with 70 eV intensity. The chromatograph is equipped with a column DB-5 (5% phenyl-methyl-siloxane) (30m x 0.25 mm x 0.25 microns film thickness), a flame ionization detector (FID) supplied by H2/Air gas mixture. The column temperature rises at a gradient rate of 4 °C/min from 50 to 200°C for 5 min. The chromatograph is equipped with a column DB-5 (5% phenyl-methyl-siloxane) (30m x 0.25 mm x 0.25 microns film thickness), a flame ionization detector (FID) supplied by H2/Air gas mixture. The injection mode is split (split ratio: 1/70 ml/min flow rate), the carrier gas was nitrogen with a flow rate of 1ml/min. Determination of chemical composition of A. Pyrethrum oils has been performed based on the comparison of their Kovats’ indices (IK). Theses indices were calculated based on the relation between the compounds and linear alkanes (C7-C40) injected in the experimental condition and compared to those in the literature (Kovàts, 1965; Adams, 2007). The mass spectra were also compared to different references (Adams, 2007; National Institute of Standards and Technology, 2014).
Phytochemical Screening: Phytochemical study was achieved only with Anacyclus pyrethrum L. roots extracts. 133 Hanane Elazzouzi et al, 2014 Advances in Natural and Applied Sciences, 8(8) July 2014, Pages: 131-140
Dry drug was first crushed in a mortar and then with a brand Moulinex mill until fine powder with brownish color. Selective extractions were made specifically for each family of compounds studied. Extracts were obtained with several solvents (petroleum ether, methanol, ethanol, chloroform and distilled water). Various phytochemical tests were performed using the methodology described by Harborne (Harborne, 1998). These qualitative tests were based on color and/or precipitation reactions.
Alkaloids Extraction: Alkaloids extraction, described by Jilani et al. was made with 10 g of root powder and 300 ml of ethyl acetate in a soxhlet apparatus for 18 hours. The filtrate was concentrated in vacuo. The residue obtained was dissolved in water and then acidified with sulphuric acid until pH=3-4. Then, it was extracted with 50 ml of petroleum ether and 50 ml of diethyl ether. After alkalinisation of the aqueous phase to pH 9-10 with ammonia (25%), this solution was extracted with 100 ml of chloroform. The extract was washed with distilled water to neutral pH, dried over sodium sulphate and concentrated to dryness at reduced pressure to give crude alkaloids (Djilani et al., 2006).
Preparation of total macerate extracts : aqueous macerate and ethanolic macerate: Pyrethrum roots extracts were prepared according to Motamed et al. method. Fifty grams of plant powder were brought to maceration in 300 ml solvent (distilled water or ethanol). The macerate was homogenized for 48 hours under stirring at room temperature. After filtration and evaporation at 40°C using a rotary evaporator system, the residue was stored at 4°C until use (Motamed et al., 2010).
Preparation of total soxhlet extracts: water and ethanolic extracts: Aqueous and ethanolic extracts were prepared according to Masturah et al. method. Fifty grams of roots powder was extract with 300 ml of solvents (distilled water or ethanol) in soxhlet apparatus (16 cycles). After filtration and evaporation using rotary evaporator at 40°C, the residue was stored at 4°C until use (Masturah et al., 2007).
Antibacterial tests: Disc- diffusion method on solid medium: Essential oils, alkaloïds, aqueous and ethanolic extracts from the plant were tested against bacterial strains through disc-diffusion method (Sacchetti et al., 2005; Celiktas et al., 2007). Inocula of 108 CFU/ml were prepared in isotonic sterile water from 24 hours-bacterial pure culture. The inocula were spread on 90mm- diameter Petri dishes containing Tryptone Soya Agar (TSA). Petri dishes were allowed to dry. Then, 6 mm- diameter sterile discs of wathman paper filter loaded with 2µl of the extract were placed in the centre of the plate. Tests were done in triplicate. Inhibition diameters were reported after 18 to 24 hours of incubation at 37˚C. Gentamicin (GM10)10µg and Cefalotin (CF30) 30µg were used as positive controls.
Macrodilution method in liquid medium: The aim is to determine minimal inhibitory and bactericide concentrations MIC and MBC (Benbelaid et al., 2012). Tests were performed in tubes containing 4ml of Brain heart infusion broth (BHIB). Fresh bacterial inocula of 107 UFC/ml were first prepared in BHIB. Only extracts with significant inhibition diameters (D ≥ 8 mm) were selected. Extracts were emulsified in Dimethylsulfoxid (20% DMSO) solution. In 4ml- BHIB test tubes, 40µl of initial inoculum (107UFC/ml) were added. Emulsified extracts were then added to obtain a spectrum ranged from C1 to C5 corresponding to 1/1000, 1/500, 1/250, 1/200, 1/100 v/v final concentrations of extract and 105 UFC/ml final bacterial concentration. Tests were performed in triplicate. Tubes without extract were used as negative controls and test tubes containing Amoxicilline (4ug/ml) were considered as positive controls. MIC was determined after 18-24 hours of incubation at 37°C. MBC is determined after plating 100µl of all tubes without any visible bacterial growth on TSA medium. Petri dishes were incubated at 37°C for 24 hours. RESULTS AND DISCUSSION
Variation in the content of essential oils and chemical composition: Essential oil content: The yields of A. Pyrethrum’s essential oils obtained during the two harvest periods are summarized in table 1. We found that the yield during June (0.07%) is higher than the one during April (0.05%). These rates are relatively high compared to those obtained in Algeria by Selles et al. (2013) (0.019%). Intraspecific variations of the yields can be depend to harvest period. Several authors confirmed that the best yield occurs at the flowering stage (Selles et al, 2013. Ghanmi et al, 2010. Simonnet et al, 2006. , Bourkhiss et al, 2011).
134 Hanane Elazzouzi et al, 2014 Advances in Natural and Applied Sciences, 8(8) July 2014, Pages: 131-140
Table 1: A. pyrethrum essential oils yields for both harvest periods. Harvest period April (2012) June (2012) EOs volumes (ml) 0,03 0,05 Humidity rates (%) 39,10 28,57 Essential oils yields (%) 0,05 0,07 Color Red- orange Red- orange Aspect Liquid Liquid
Chemical composition of essential oils: Analyses of A. Pyrethrum EO from Timahdite area (Morocco) revealed the presence of 42 compounds for April sample and 36 compounds for June sample. These compounds represent about 91,32 % and 91,82 % of the total of these Eos. Figure 1 and table 2, show respectively chromatograms and chemical compounds of EOs. Oxygenated sesquiterpenes are the most abundant group among the identified compounds. Their level rises from 89,17 % (April) to 90,58 % (June) during maturation step. Similarly, this group is the most abundant in the Algerian species as showed by Selles et al. (2013). In his study percentage of sesquiterpenes rises from 37,1% to 58,6 % respectively before and after flowering stage. Comparison of essential oils’ chemical composition showed quantitative and qualitative changes. The percentage of the major constituent spathulenol increases significantly from April (13,31%) to June (16,9%). Germacra -4 (15),5, 10 (14) - trien -1-a –ol percentage also increases from April (2,07%) to June (12,89%). We also note that selina -3 ,11- dien- 6-a -ol has its highest proportion in the first period (9,24% ) while acetate cedryl highest percentage is obtained during the second period ( 8,10% ). The percentage of caryophyllene oxide falls from April to June (9,65 to 7,11%). Finally, it is important to note the high rates of -biotol and salvial -4 (14) -en-1-one during the first period of harvest (5,16% and 4,66% respectively). Eudesma -4 (15),7- diene-1- ol and β – himachalol have their high rates during the second period (5,85% and 5,67% respectively). In our plant essential oils, spathulenol is the most important compound at both stages (April and June). So whatever the time of harvest, the plant EO can be classified as spathulenol chemotype. However, in other studies the results are quite different. Anacyclus Pyrethrum EO from Algeria is dominated by germacrene-D and defined by the germacrene-D chemotype (Selles, 2012; Selles et al, 2013). Since in both harvest periods, essential oils have other major constituents like germacra-4 (15), 5, 10 (14)- trien -1-a-ol, caryophyllene oxide , etc. Then, we can define intermediate chemotypes such as chemotype of April with spathulenol (13,31%) / caryphylene oxide (9,65 %) /cedryl acetate ( 8,10% )/ and eudesma -4 (15) ,7- diene-1-β-ol (5,85%). And the chemotype of June with spathulenol (16,9%)/ germacra -4( 15) , 5, 10(14)- trien-1-a-ol (12,89 %)/ and selina -3, 11-dien-6-a-ol (9,24%). Indeed, the difference observed in compounds content between these two collection dates can be explained by the biosynthesis process of these main constituents (Ghanmi et al., 2010). Therefore, Asteraceae family is particularly characterized by the chemical polymorphism. This chemical variation can depend on the harvest period of the plant. This period constitute a parameter which influences both chemical yield and quality of the essential oil (Garneau, 2001).
(a) 135 Hanane Elazzouzi et al, 2014 Advances in Natural and Applied Sciences, 8(8) July 2014, Pages: 131-140
(b)
Fig. 1: GC-MS chromatograms of A. pyrethrum essential oils collected in April 2012 (a), and June 2012 (b).
Table 2: Chemical composition of A. pyrethrum essential oils according to harvest periods. Compounds Adams IR Area (%) April June α-Neocallitropsene 1476 - 0.20 Germacrene D 1481 0.17 - Trans-β-Ionone 1488 0.20 - Cubebol 1515 0.10 0.15 δ-Cadinene 1523 0.18 - Ar-Macrocarpene 1526 - 0.40 Italicence epoxide 1548 0.58 - occidentalol 1552 1.31 2 .20 1α,10α-Epoxy-amorph-4-ene 1572 0.78 2.83 Spathulenol 1578 13.31 16.90 Caryphylene oxide 1583 9.65 7.11 β-copaen-4-α-ol 1590 0.28 - Salvial-4(14)-en-1-one 1594 4.39. 4.66 Mayurone < cis – dihydro> 1595 4.96 - -Atlantol 1608 - 2.90 -Biotol 1613 2.76 5.16 E-Isoeugenol acetate 1615 1,35 0.36 Trans-Isolongifolanone 1626 1.05 0.45 Muurola-4,10(14)-dien-1--ol 1631 4.34 1.81 -Acorenol 1637 - 2.58 Caryophylla-4(18), 8(13)-dién-5α-ol 1640 0.54 - 3-iso-Tujopsanone 1642 - 1.54 Selina-3,11-dien-6α-ol 1644 0.91 9.24 cis-guai-3.9-dien-11-ol 1649 1.71 - Cedr-8(15)-en-9-α-ol 1651 1.44 - Himachalol 1653 5.67 - 3-Thujopsanone 1654 - 0.42 E-Caryophylene-14-hydroxy-9-epi 1669 1.88 - Z--Santalol 1675 - 1.86 Khusinol 1680 0.95 3.29 Germacra-4(15),5,10(14)-trien-1-α-ol 1686 2.07 12.89 Eudesma-4(15),7-diene-1-β-ol 1688 5.85 - Nootkatol
Hinesol acetate 1784 0.56 - 8-Cedren-13-ol acetate 1788 0.32 0.29 Isovalencenol 1793 - 0.14 -Eudesmol acetate 1795 - 0.68 14-Hydroxy-δ-cadinene 1803 1.74 1.26 Vetivenic acid 1811 0.46 2.18 Khusinol acétate 1823 0.08 - 8-hydroxy-eremphilone 1847 - 0.59 Chenopodiol<α> 1856 0.19 - Murolan-3,9(11)-diene-10-peroxy 1876 - 0.21 Carissone 1927 0.16 - Selorene 1974 0.25 - Kaurene 2043 - 0.28 Total % 91,32 91,82 Oxygenated monoterpenes (%) 0,2 - Sesquiterpenes hydrocarbons (%) 0,35 0,6 Oxygenated Sesquiterpenes (%) 89,17 90,58 Diterpenes hydrocarbons (%) 0,25 0,28 Phenylpropanoid (%) 1,35 0,36 IR : Adams Retention Indices
Phytochemical Screening: The results of the phytochemical screening showed that A. pyrethrum roots are rich in alkaloids, reducing compounds and cathechic tannins. Similar results were obtained by Selles et al. (2012) in the roots, leaves and flowers of Algerian pyrethrum. In India, the species lacks tannins while alkaloids are present in the roots (Amrita et al., 2011). Phytochemical tests also revealed the presence of flavonoids while the roots of Algerian and Indian species are devoid of flavonoids (Selles, 2012; Selles et al. (b), 2012; Amrita et al., 2011). Our plant contains other chemicals such as gallic tannins, triterpenes, sterols, mucilage, coumarins, saccharids and holosids. However, tests for saponins and anthracenic compounds gave negative results. The absence of saponins in this study is in agreement with the work already done by Amrita et al. (2011) and in contrast with those of Selles et al. ( Selles, 2012; Selles et al.(b), 2012). Anacyclus pyrethrum therefore appears to be a plant rich in secondary metabolites. This fact could justify their extraction and use especially in the prevention of diseases and the management of many infections.
Table 3. Phytochemical screening reactions Chemical groups Reagents or Reaction name Results Tannins Total FeCl3 ++ (Dark green color) Cathechic Stiasny reagent + (red precipitate) Gallic Reaction with sodium acetate ++ (bleue precipitate) Flavonoids Anthocyans Acido-basic reaction - Flavons Cyanidin reaction with Mg + (Pink-orange color) shavings Leucoanthocyans Cyanidin reaction without Mg - shavings Catechols Cyanidin reaction without Mg ++ (Red-brown color) shavings Alkaloids Valser - Mayer Reagent ++ (orange precipitate) Dragendorff Reagent ++ (red precipitate) Saponosids Foam Index (FI) - (Positif Test if FI<100) Sterols and Triterpenes Liebermann Buchard Reaction + (Red ring and brownish violet color of the supernatant layer) Anthracenic Free anthraquinones Börntragger Reaction - compounds Combined o-heterosids Color Reaction - anthraquinones c-heterosids Color Reaction - Narcotics Color Reaction - Reducing compounds Fehling Reaction ++ (Brick-red precipitate) Mucilage precipitation Reaction ++ (Flake) Coumarins fluorescence Reaction + (Intense fluorescence) Oses and holosids Color Reaction ++ (red color) Highly positive reaction: + +; positive Reaction : + Moderately positive reaction: + / -; Negative test: -
Yields extracts: Each extract was characterized by its color, appearance and the dry matter-based yield. These elements are shown in table 4. 137 Hanane Elazzouzi et al, 2014 Advances in Natural and Applied Sciences, 8(8) July 2014, Pages: 131-140
In general, the yields not only vary within a single species but also according to solid-liquid extraction parameters: temperature, extraction solvent, particle size and diffusion coefficient of solvent. Non volatile and volatile extracts from the plant may contain a variety of biologically active molecules. In this context, we attempted to evaluate the antibacterial activity of these A. pyrethrum extracts.
Table 4: Extracts yields, aspects and color. Extracts Yields (%) Color Aspect Aqueous macerate 22,02 Brownish Viscous ethanolic macerate 6,36 Greenish Viscous Soxhlet with water 36,88 Brownish Viscous Soxhlet with ethanol 13,90 Greenish Viscous Alkaloids 3,60 Brownish Viscous
Antibacterial activity: Aromatogram performed by disc-diffusion method: The results of susceptibility testing are shown in table 5. Antibacterial activity of pyrethrum extracts against germs was qualitatively and quantitatively assessed by the presence or absence of inhibition zones. For this method, an extract is considered active when it induces an inhibition zone greater or equal to 9 mm (Celikel et al., 2008). Thus, the analysis shows that the inhibition zones are more pronounced for the aqueous macerate than other extracts (sensitive and resistant E. coli: 9±0,81 mm, S. aureus: 9,66±0,47 mm , resistant P. aeruoginosa 7,6±0,47 mm , sensitive P. aeruoginosa : 6,5±0,5 mm and resistant K. pneumoniae: 7,33±0,47 mm). We note that the aqueous macerated exerts an antibacterial effect on S. aureus strains and on susceptible and resistant E. coli strains, these strains are susceptible to the extract. This effect is low compared to that of Gentamicin and Cephalothin used as antibiotics reference. These results are consistent with those reported by Douddach et al., (2012) who tested the antibacterial effect of aqueous extract of A. pyrethrum from Eastern Morocco. Inhibition diameters obtained in that studies are larger compared to our results (13mm and 22mm respectively for the E. coli and S. aureus). Moreover, the species of Algeria develops 6mm and 16mm as inhibition zones (Selles et al. (a), 2012). The activity of the aqueous extract prepared by soxhlet ranks second (sensitive E. coli: 8 ± 0 mm, resistant E. coli: 7±0,81 mm, S. aureus : 6±0 mm, resistant P. aeruoginosa : 6±0 mm, and resistant K. pneumoniae: 7±0 mm). We note that the inhibition area are ≤ 8 mm and are small compared to the positive control, the bacterial strains are resistant (Celikel and Kavas, 2008). Moreover, the inhibitory activity of essential oils and pyrethrum alkaloids were also low (≤ 7mm) and even no inhibition were observed for certain strains. Alkaloid from pyrethrum roots extract (pellitorin) proved to endow antibacterial activities by several authors (Chaaib, 2004; Molina, 1999; Crombia, 1954). However, in other works alkaloids are less potent to inhibit microbial growth of S. aureus and E. coli (Perumalsamy et al., 2013). However, the inhibitory action of pyrethrum essential oil against S. aureus remains low compared to that described by Selles, (2012). The essential oils of the roots and leaves/stems of Algerian pyrethrum develop respectively 11 and 10mm inhibition areas against S. aureus (Selles, 2012). In fact, in other works by the same author, EO from aerial parts exerts a significant inhibitory effect against the same strain (14mm) (Selles et al., 2013). Despite the high content of oxygenated compounds in essential oils, it had not the expected effect on the different microorganisms. Activity of ethanol extract (macerate and soxhlet) shows a weak inhibition diameter ≤ 7mm, no significant inhibition was observed for certain strains (Table 5). In other works, Sqalli et al. (2007) show that the ethanolic extract of pyrethrum has an interesting antimycobacterial effect. Other authors showed the inhibitory effect of the extract against the tested strains (Jalayer et al., 2012; Selle et al. (a), 2012; Doudach et al, 2012; Annalakshmi et al., 2012). The antibacterial activity appears to correlate well with the total phenolic values, previous studies indicate that water and methanol are the most used solvents for a high recovery of phenolic compounds which are responsible for many biological activities, including antimicrobial activity (Xia et al., 2010; Bouzid et al., 2011; Cushnie et al., 2005).
Table 5: Inhibition diameters of A. Pyrethrum extracts on bacterial strains presented as Means (mm) ± standard deviation. E. coli E. coli S. aureus P. aeruginosa P. aeruoginosa K. pneumoniae sensitive résistant sensitive résistant résistant Essential Oil 0 0 7±0 0 0 0 Aqueous macerate 9±0,81 9±0,81 9,66±0,47 6,5±0,5 7,6±0,47 7,33±0,47 Ethanol macerate 0 7±0,81 7±1 6,33±0,47 7±0 0 Soxhlet with 8±0 7±0,81 6±0 0 6±0 7±0 water Soxhlet with - - 7±1,41 - 6±0 - ethanol Alkaloids - 7±0,81 6±1,41 6±0 - 7±0 138 Hanane Elazzouzi et al, 2014 Advances in Natural and Applied Sciences, 8(8) July 2014, Pages: 131-140
Determination of the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of tested samples: MIC and MBC of A. pyrethrum extracts have been determined only for those that have previously exhibited significant antibacterial effects. From aromatogramme results, resistant and susceptible strains of E. coli and S. aureus strains were all susceptible to the aqueous macerate of pyrethrum. Despite the small inhibition diameter registered for the soxhlet aqueous extract against resistant E. coli, the result is still important against the resistance of the bacteria to antibacterial effects. In fact, the results of our work show that pyrethrum aqueous macerate is endowed with inhibitory activity against susceptible E .coli and S. aureus at a concentration of 1/200 v/v (Table 6 and 7). Doudach et al. (2012), reported that of the aqueous extract of A. Pyrethrum from East Morocco exhibited similar MIC against E. coli and S. aureus. They have registered a minimum concentration of 3.125 mg/ml. In fact, the bactericidal effect of the extract may be at a concentration greater than the concentration range of our study (˃ C5=1/100 v/v). Thus, the inhibitory and bactericidal activity of the same extract against resistant E. coli can be at a higher concentration ˃ 1/100 v/v. However, the soxhlet aqueous extract can demonstrate inhibition and bactericidal power against susceptible E. coli at a MIC and MBC greater than ˃ 1/100 v/v.
Table 6: Susceptibility of tested germs with A. pyrethrum extracts and MIC determination. Extracts/ Strains Concentrations (v/v) 1 /1000 1/500 1/250 1/200 1/100 negative control T(-) (without extracts) + + + + + positive control T(+) (Amoxicillin, 4ug/ml) - - - - - aqueous macerate / S. aureus + + + - - aqueous macerate / resistant E. coli + + + + + aqueous macerate / susceptible E. coli + + + - - aqueous extract (soxhlet) / susceptible E. coli + + + + + (-) antibacterial action. (+) no antibacterial action.
Table 7: Antibacterial parameters of A. Pyrethrum (MIC and MBC). Resistant E. coli Sensitive E. coli S. aureus MIC (v/v) MBC (v/v) MIC (v/v) MBC (v/v) MIC (v/v) MBC (v/v) Aqueous macerate ˃1/100 ˃1/100 1/200 ˃1/100 1/200 ˃1/100 Soxhlet equeous n. d n. d ˃1/100 ˃1/100 n. d n. d extract n. d : not determined
Conclusion: In this work, we have attempted to contribute to the valorization of Moroccan A. pyrethrum by establishing a chemical and biological characterization of the plant. The chemical composition of essential oils extracted from A. pyrethrum harvested in two different flowering periods, allowed us to identify two intermediate chemotypes with spathulenol as a major compound. In fact, its presence as a major constituent in A. pyrethrum’s EOs is an important indicator for their potential use in biological activities. Phytochemical screening has identified various secondary metabolites (alkaloids, reducing compounds, tannins, flavonoids and coumarins). Moreover, only the aqueous macerate of A. Pyrethrum has shown a relatively high antibacterial activity against two strains namely, S. aureus and susceptible E. coli. This activity could be explained by the nature of the compounds present in this plant which may be used for other interesting biological activities.
ACKNOWLEDGMENTS
We are grateful to Mr M. Ibn Tattou, Professor at the Scientific Institute of Rabat, for the species identification.
REFERENCES
Adams, R.P., 2007. Identification of Essential Oil Components by Gas Chromatography/ Mass Spectrometry, 4th edition Allured Publishing Corporation, Carol Stream. Afnor, 2000. Huiles essentielles, Échantillonnage et méthodes d’analyse (tome 1) – Monographies relatives aux huiles essentielles (tome 2. volumes 1 et 2). Amrita, M., K.M. Arun, K.G. Ashoke and J. Shivesh, 2011. Pharmacognostical, Physicochemical and Phytochemical Studies of Some Marketed Samples of Roots used in Ayurvedic Medicines. Pharmacognosy Journal, 3(24): 55-61. DOI: 10.5530/ pj.2011.24.11. Annalakshmi, R., R. Uma, G.C. Subash and A. Muneeswaran, 2012. A treasure of medicinal herb - Anacyclus pyrethrum. A review Indian Journal of Drugs and Diseases, 1(3): 59-67. ISSN: 2278- 2958. 139 Hanane Elazzouzi et al, 2014 Advances in Natural and Applied Sciences, 8(8) July 2014, Pages: 131-140
Batanouny, K., 2005. Guide to Medicinal Plants in North Africa. Centre for Mediterranean Cooperation, International Union for Conservation of Nature and Natural Resources: Anacyclus pyrethrum L. ISBN 2-8317- 0893-1. 35-37. Bellakhdar, J., 1997. La Pharmacopée Marocaine Traditionnelle: Médecine arabe ancienne et savoirs populaires - Saint –Etienne, Edit. Ibis Press. . Benbelaid, F., M. Bendahou, A. Khadir, M.A. Abdoune, C. Bellahsene, F. Zenati, W. Bouali, and D.E. Abdelouahid, 2012. Antimicrobial activity of essential oil of Lavandula multifida L. Journal of Microbiology and Biotechnology Research Scholars Research Library J. Microbiol. Biotech. Res., 2(2): 244-247. ISSN: 2231 –3168 CODEN (USA): JMBRB4244. Bencheqroun, H.K., M. Hhanmi, B. Satrani, A. Aafi, and A. chaouch, 2012. Activité antimicrobienne des huiles essentielles d’Artemisia mesatlantica, plante endémique du Maroc. Bulletin de la Société Royale des Sciences de Liège, 81: 4-21. Bendjeddou, D., K. Lalaoui and D. Satta, 2010. Immunologically Active Polysaccharides Isolated from Anacyclus pyrethrum. Libyan Agric Res Cen J Intl, 1(3): 128–33. Benjilali, B., 2005. Plantes aromatique et médecinales : atouts du secteur et exigences pour une valorisation durable. Editions Actes. Benitez, G., M.R. Gonzalez-Tejero, and J. Molero-Mesa, 2010. Pharmaceutical ethnobotany in the western part of Granada province (southern Spain). Ethnopharmacological synthesis. J Ethnopharma, 129: 87–105. Bourgaud, F., A. Gravot, S. Milesi, and E. Gontier, 2001. Production of plant secondary metabolites: a historical perspective. Plant Science, 161: 839-851. Bourkhiss, M ., M. Hnach, T. Lakhlifi, A. Boughdad, A. Farah, and B. Satrani, 2011. Effet de l’Age et du Stade Vegetatif sur la Teneur et la Composition Chimique des Huiles Essentielles de Thuya de Berbere, Les Technologies De Laboratoire -, Volume 6, N°23. Article original, pp : 64-68. Bouzid, W., M. Yahia, M. Abdeddaim, M.C. Aberkane, and A. Ayachi, 2011. Evaluation de l’activité antioxydante et antimicrobienne des extraits de L’Aubepine Monogyne. Lebanese Science Journal, 12(1): 59-69. Celikel, N. and G. Kavas, 2008. Antimicrobial properties of some essential oils against some pathogenic microorganisms. Food Science, 26: 174-181. Celiktas, O.Y., E.E. Hames Kocabas, E. Bedir, O.S. Verdar, and K.H.C. Baser, 2007. Antimicrobial activities of methanolic extract and essential oils of Rosmarinus officinalis, depending on location and seasonal variations. Food Chemistry, 100: 553-559. Chaaib, K.F., 2004. Investigation phytochimique d’une brosse à dents africaine Zanthoxylum zanthoxyloides (Lam.) Zepernick et Timler (Syn. Fagara zanthoxyloides L.) (Rutaceae), Thèse, Genève. Crombia, L., 1954. Isolation and Structure of an N-iso Butyldienediynamide from Pellitory (Anacyclus pyrethrum DC.). Nature, 174: 832-833. Cushnie, T.P.T. and A.J. Lamb, 2005. International. Journal. Antimicrobial, 26: 343-356. Djilani, A., B. Legseir, R. Soulimani, A. Dicko, and C. Younos, 2006. New technique for alkaloids. J.Braz. Chem. Soc., 17: 518-520. Doudach, L., B. Meddah, R. Alnamer, F. Chibani, and Y. Cherrah, 2012. In vitro antibacterial activity of the methanolic and aqueous extracts of Anacyclus pyrethrum used in moroccan traditional medicine. Academic science International Journal of Pharmacy and Pharmaceutical Sciences, 4(3): 402-405. ISSN- 0975-1491. Efraim, L. and A. Zohar, 2008. “Fossils” of practical medical knowledge from medieval Cairo. J Ethnopharma, 119: 24–40. El Oualidi, J., H. Khamar, M. Fennane, M. Ibn Tattou, S. Chauvet, M.S. Taleb, 2012. Checklist des endémiques et spécimens types de la flore vasculaire de l'Afrique du Nord. Document De L’institut Scientifique, N°25, Universite Rabat. ISBN : 978-9954-20-667-6. Publication présentant les résultats de projets réalisés dans le cadre du programme GPI « Global Plants Initiative » & API « African Plants initiative » en partenariat avec Tela Botanica & SANBI avec l’appui financier de la Fondation Andrew W. Mellon. 187. Garneau François-Xavier, 2001. Le matériel végétal et les huiles essentielles. Corporation :LASEVE- UQAC, Chicoutimi (Québec) G7H 2B1 Huiles essentielles : de la plante à la commercialisation. Ghanmi, M., B. Satrani, A. Aafi, M.R. Isamili, H. Houti, H. El Monfalouti, K.H. Benchakroun, M. Aberchane, L. Harki, A. Boukir, A. Chaouch, and Z. Charrouf, 2010. Effet de la date de récolte sur le rendement, la composition chimique et la bioactivité des huiles essentielles de l’armoise blanche (Artemisia herba-alba) de la région de Guerçif (Maroc oriental). Phytothérapie , 8(5): 295-301. DOI 10.1007/s10298-010- 0578-1. Print ISSN 1624-8597. Harborne, J. B., 1998. Phytochemical Methods. A guide to modern techniques of plant analysis, Chapman &Hall, London. Harald, G., 1978. Comparative phytochemistry and systematics of Anacyclus. Biochem Syst Ecol, 6: 11– 17. Jalayer, N.N., M. Niakan and E. Khodadadi, 2012. Determination of Antibacterial Activity of Anacyclus Pyrethrum Extract against Some of the Oral Bacteria: An In Vitro Study. Shiraz Univ Dent J, 13(2): 59-6. 140 Hanane Elazzouzi et al, 2014 Advances in Natural and Applied Sciences, 8(8) July 2014, Pages: 131-140
Kalim, M.D., D. Bhattacharyya, A. Banerjee, and S. Chattopadhyay, 2010. Oxidative DNA damage preventive activity and antioxidant potential of plants used in Unani system of medicine. BMC Complement Altern. Med., 10: 77-87. Kar, A., 2007. Pharmacognosy and Pharmabiotechnologie. Ed 2: New Age International Publishers, pp: 1- 30. Kováts, E., 1965. Gas chromatographic characterization of organic substances inthe retention index system. Advances in Chromatography, 1: 229-247. Masturah, M., H. Masitah, W.D. Wan Ramli, S. Harcharan, and M.J. Jamaliah, 2007. Extraction of hydrolysable tannins from Phyllanthus niruri Linn.: Effects of solvents and extraction methods. Separation and Purification Technology, 52: 487–496. Molina, T.J., 1999. Antimicrobial properties of alkamides present in flavouring plants traditionally used In Mesoamerica: affinin and capsaicin. Journal of Ethnopharmacology, 64: 241-248. Motamed, S. M. and F. Naghibi, 2010. Antioxidant activity of some edible plants of the Turkmen Sahra region in northern Iran. Food Chemistry, 119: 1637-1642. Perumalsamy, H., M.Y. Jung, S.M. Hong, and Y.J. Ahn, 2013. Growth-Inhibiting and morphostructural effects of constituents identified in Asarum heterotropoides root on human intestinal bacteria. BMC Complementary and Alternative Medicine, 13(245): 11. Rimbau, V., Cerdan, R. Vila, J. Iglesias and 1999. Anti-inflammatory activity of some extracts from plants used in the traditional medicine of North-African countries. Phytother Res, 10(5): 421–3. Sacchetti, G., S. Maietti, M. Muzzoli, M. Scaglianti, S. Mansredini, M. Radice, and R. Irimi, 2005. Comparative evaluation of 11 essentials oils of different origin as functional antioxydants, antiradicals and antimicrobial in food. Food Chemistry, 91: 621-632. Satyanand, T., H.M. Mohd, K.S. Narendra, K.S. M anoj, B. Poonam, K.S. Rahul, 2011. Antidiabetic Effect of Anacyclus pyrethrum DC in Alloxan Induced Diabetic Rats. European Journal of Biological Sciences, 3(4): 117-120. ISSN 2079-2085. Selles, Ch., 2012. Valorisation d’une plante médicinale à activité antidiabétique de la région de Tlemcen : Anacyclus pyrethrum L. Application de l’extrait aqueux à l’inhibition de corrosion d’un acier doux dans H2SO4 0.5M, Universite Abou Bekr Belkaid. Algerie, 175. Selles, Ch., D.M. El Amine, H. Allali, and B. Tabti, 2012(a). Evaluation of antimicrobial and antioxidant activities of solvent extracts of Anacyclus pyrethrum L., from Algeria. Mediterranean Journal of Chemistry, 2(2): 408-415. Selles, Ch., O. Benali, B. Tabti, L. Larabi, Y. Harek, 2012(b). Green corrosion inhibitor: inhibitive action of aqueous extractof Anacyclus pyrethrum L. for the corrosion of mild steel in 0.5 M H2SO4. J. Mater. Environ. Sci. 3 (1): 206-219. ISSN : 2028-2508 CODEN: JMESCN. Selles, Ch., D.M. ElAmine, N. Djabou, F. Beddou, A. Muselli, B. Tabti, J. Costa, and B. Hammouti, 2013. L'activité antimicrobienne et l'évolution de la composition de l'huile essentielle d’Anacyclus pyrèthrum de l'Algérie L. à travers le cycle végétatif. Natural Product Research: Lettres du produit Autrefois naturelles, 2231- 2234. DOI:10.1080/14786419.2013.811409 Simonnet, X., M. Gaudin, P. Jacquemettaz, and U. Piantini, 2006. Stade phénologique et qualité des hampes florales du genépi blanc. Médiplant Centre de recherches sur les plantes médicinales et aromatiques. Revue suisse Vitic. Arboric. Hortic, 38(3):189-193. Sqalli, H., A. El Ouarti, A. Ennabili, S. Ibnsouda, A. Farah, A. Haggoud, A. Houari, and M. Iraqui, 2007. Évaluation De L’effet Antimycobactérien De Plantes Du Centre-Nord Du Maroc. Bull. Soc. Pharm. Bordeaux, 146: 271-288. Sujith, K., R. Darwin, and V. Suba, 2012. Toxicological evaluation of ethanolic extract of Anacyclus pyrethrum in albino wistar rats. Asian Pacific Journal of Tropical Disease, pp: 437-441. Traoré, Y., K. Ouattara, D. Yéo, I. Doumbia, and A. Coulibaly, 2012. Activités antifongique et antibactérienne des feuilles d’Annona senegalensis Pers. Recherche des activités antifongique et antibactérienne des feuilles d’Annona senegalensis Pers. (Annonaceae). J. Appl. Biosci. 58: 4234-4242. ISSN 1997–5902. Vikas, S., T. Mayank, S.C. Nagendra, and K.D. Vinod, 2009. Evaluation of the anabolic, aphrodisiac and reproductive activity of Anacyclus pyrethrum DC. in male rats. Organization for Economic Cooperation and Development. Sci Pharm, 77: 97-110. Zaidi, S.M.A., A.P. Shadab, S. Surender, J. Shakir, J.A. Farhan and K.K. Roop, 2013. Anticonvulsant, Anxiolytic and Neurotoxicity Profile of Aqarqarha (Anacyclus pyrethrum) DC. (Compositae) Root Ethanolic Extract. Pharmacology & Pharmacy, 4: 535-541. Xia, E.Q., G.F. Deng, Y.J. Guo, and H.B. Li, 2010. Biological activities of polyphenols from grapes. International Journal of Molecular Sciences, 11: 622-646.