Advances in Natural and Applied Sciences Anacyclus pyrethrum vitro

Advances in Natural and Applied Sciences, 8(8) July 2014, Pages: 131-140
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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
1,2
Hanane Elazzouzi, 1Aminata Soro, 1Fatima Elhilali, 1Amar Bentayeb, 2Mohamed Alaoui El Belghiti, 1Touriya
Zair
1
Laboratory of Chemistry of Bioactive Molecules and Environment, University of Sciences Moulay Ismail, BP 11201. Zitoune, Meknès,
Morocco.
2
Laboratory of Chemistry – General Physics – University of Sciences- Agdal, 4- Avenue Ibn Battouta. B.P. 1014 RP, Rabat, Morocco.
ARTICLE INFO
Article history:
Received 2 April 2014
Received in revised form
13 May 2014
Accepted 28 May 2014
Available online 27 June 2014
Keywords:
Ancyclus Pyrethrum L., Essential oils,
Chemical composition, Phytochemical
Screening, Antibacterial activity
ABSTRACT
Exploitation of plants used in traditional medicine is important to identify substances
belonging to different secondary metabolites classes. These metabolites have efficient
properties and their study and exploitation is important to rationalize their utilisation.
The aim of this study is to determine the chemical composition of the spontaneous and
endemic Anacyclus pyrethrum Link (Asteraceae) essential oils, to perform a
phytochemical screening and to assess in vitro antibacterial activity of Pyrethrum
extracts. Essential oils obtained from hydrodistillation of pyrethrum samples collected
in April and June (2012) from Timahdite (Morrocan middle Atlas region) were
analysed by CG/MS. Phytochemical screening of Anacyclus pyrethrum roots was
carried out through precipitation and color reaction. Antibacterial test of volatiles and
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]
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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.
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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 90mmdiameter Petri dishes containing Tryptone Soya Agar (TSA). Petri dishes were allowed to dry. Then, 6 mmdiameter 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).
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Table 1: A. pyrethrum essential oils yields for both harvest periods.
Harvest period
EOs volumes (ml)
Humidity rates (%)
Essential oils yields (%)
Color
Aspect
April (2012)
0,03
39,10
0,05
Red- orange
Liquid
June (2012)
0,05
28,57
0,07
Red- orange
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)
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(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
α-Neocallitropsene
Germacrene D
Trans-β-Ionone
Cubebol
δ-Cadinene
Ar-Macrocarpene
Italicence epoxide
occidentalol
1α,10α-Epoxy-amorph-4-ene
Spathulenol
Caryphylene oxide
β-copaen-4-α-ol
Salvial-4(14)-en-1-one
Mayurone < cis – dihydro>
-Atlantol
-Biotol
E-Isoeugenol acetate
Trans-Isolongifolanone
Muurola-4,10(14)-dien-1--ol
-Acorenol
Caryophylla-4(18), 8(13)-dién-5α-ol
3-iso-Tujopsanone
Selina-3,11-dien-6α-ol
cis-guai-3.9-dien-11-ol
Cedr-8(15)-en-9-α-ol
Himachalol
3-Thujopsanone
E-Caryophylene-14-hydroxy-9-epi
Z--Santalol
Khusinol
Germacra-4(15),5,10(14)-trien-1-α-ol
Eudesma-4(15),7-diene-1-β-ol
Nootkatol<epi>
Eudesm−7(11)−en−4−ol
Amorpha-4,9-dien-2-ol
γ-Gurjunenepoxide
(+)-Trans-Nootkatol
Vetiselinenol
Isobicyclogermacrenal
Khusimol
-acoradienol
Cedryl acétate
14-oxy--Muurolenee
Amorpha-4,11-diene<2-α-hydroxy>
14- hydroxy--muurolene
1476
1481
1488
1515
1523
1526
1548
1552
1572
1578
1583
1590
1594
1595
1608
1613
1615
1626
1631
1637
1640
1642
1644
1649
1651
1653
1654
1669
1675
1680
1686
1688
1699
1700
1700
1704
1715
1731
1734
1742
1763
1767
1768
1775
1780
Area (%)
April
0.17
0.20
0.10
0.18
0.58
1.31
0.78
13.31
9.65
0.28
4.39.
4.96
2.76
1,35
1.05
4.34
0.54
0.91
1.71
1.44
5.67
1.88
0.95
2.07
5.85
0.15
0.79
0.15
0.55
2.35
3.72
8.10
4.03
1.44
-
June
0.20
0.15
0.40
2 .20
2.83
16.90
7.11
4.66
2.90
5.16
0.36
0.45
1.81
2.58
1.54
9.24
0.42
1.86
3.29
12.89
0.12
0.62
0.28
1.41
2.88
1.58
1.49
0.86
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Hinesol acetate
8-Cedren-13-ol acetate
Isovalencenol
-Eudesmol acetate
14-Hydroxy-δ-cadinene
Vetivenic acid
Khusinol acétate
8-hydroxy-eremphilone
Chenopodiol<α>
Murolan-3,9(11)-diene-10-peroxy
Carissone
Selorene
Kaurene
Total %
Oxygenated monoterpenes (%)
Sesquiterpenes hydrocarbons (%)
Oxygenated Sesquiterpenes (%)
Diterpenes hydrocarbons (%)
Phenylpropanoid (%)
IR : Adams Retention Indices
1784
1788
1793
1795
1803
1811
1823
1847
1856
1876
1927
1974
2043
0.56
0.32
1.74
0.46
0.08
0.19
0.16
0.25
91,32
0,2
0,35
89,17
0,25
1,35
0.29
0.14
0.68
1.26
2.18
0.59
0.21
0.28
91,82
0,6
90,58
0,28
0,36
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
Tannins
Total
Cathechic
Gallic
Flavonoids
Anthocyans
Flavons
Reagents or Reaction name
FeCl3
Stiasny reagent
Reaction with sodium acetate
Acido-basic reaction
Cyanidin reaction with Mg
shavings
Cyanidin reaction without Mg
shavings
Cyanidin reaction without Mg
shavings
Valser - Mayer Reagent
Dragendorff Reagent
Foam Index (FI)
Liebermann Buchard Reaction
Leucoanthocyans
Catechols
Alkaloids
Saponosids
Sterols and Triterpenes
Anthracenic
compounds
Free anthraquinones
Combined
anthraquinones
Narcotics
Reducing compounds
Mucilage
Coumarins
Oses and holosids
Highly positive reaction: + +; positive Reaction : +
Moderately positive reaction: + / -; Negative test: -
o-heterosids
c-heterosids
Börntragger Reaction
Color Reaction
Color Reaction
Color Reaction
Fehling Reaction
precipitation Reaction
fluorescence Reaction
Color Reaction
Results
++ (Dark green color)
+ (red precipitate)
++ (bleue precipitate)
+ (Pink-orange color)
++ (Red-brown color)
++ (orange precipitate)
++ (red precipitate)
- (Positif Test if FI<100)
+ (Red ring and brownish
violet color of the supernatant
layer)
++ (Brick-red precipitate)
++ (Flake)
+ (Intense fluorescence)
++ (red color)
Yields extracts:
Each extract was characterized by its color, appearance and the dry matter-based yield. These elements are
shown in table 4.
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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
Aqueous macerate
ethanolic macerate
Soxhlet with water
Soxhlet with ethanol
Alkaloids
Yields (%)
22,02
6,36
36,88
13,90
3,60
Color
Brownish
Greenish
Brownish
Greenish
Brownish
Aspect
Viscous
Viscous
Viscous
Viscous
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
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.
1/250
+
+
+
+
+
Table 7: Antibacterial parameters of A. Pyrethrum (MIC and MBC).
Resistant E. coli
Sensitive E. coli
MIC (v/v)
MBC (v/v)
MIC (v/v)
MBC (v/v)
Aqueous macerate
˃1/100
˃1/100
1/200
˃1/100
Soxhlet equeous
n. d
n. d
˃1/100
˃1/100
extract
n. d : not determined
1/200
+
+
+
MIC (v/v)
1/200
n. d
1/100
+
+
+
S. aureus
MBC (v/v)
˃1/100
n. d
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.
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