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Phytochemical study of Anacyclus pyrethrum (L.) of middle atlas (morocco), and in vitro study of antibacterial activity of pyrethrum.

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 (Traore 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 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 Fes : 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[degrees]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[degrees]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 [degrees]C/min from 50 to 200[degrees]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 (Kovats, 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. 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[degrees]C using a rotary evaporator system, the residue was stored at 4[degrees]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[degrees]C, the residue was stored at 4[degrees]C until use (Masturah et al., 2007).

Antibacterial tests:

Disc--diffusion method on solid medium:

Essential oils, alkaloids, 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 90 mm-diameter Petri dishes containing Tryptone Soya Agar (TSA). Petri dishes were allowed to dry. Then, 6 mmdiameter sterile discs of wathman paper filter loaded with 2pl 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[degrees]C. Gentamicin (GM10)10[micro]g and Cefalotin (CF30) 30pg 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 [greater than or equal to] 8 mm) were selected. Extracts were emulsified in Dimethylsulfoxid (20% DMSO) solution. In 4ml--BHIB test tubes, 40pl of initial inoculum ([10.sup.7]UFC/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 [10.sup.5] 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[degrees]C. MBC is determined after plating 100pl of all tubes without any visible bacterial growth on TSA medium. Petri dishes were incubated at 37[degrees]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).

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 p-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 P--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-p-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).

[FIGURE 1 OMITTED]

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.

Yields extracts:

Each extract was characterized by its color, appearance and the dry matter-based yield. These elements are shown in table 4.

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.

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 [+ or -] 0,81 mm, S. aureus: 9,66 [+ or -] 0,47 mm, resistant P. aeruoginosa 7,6 [+ or -] 0,47 mm, sensitive P. aeruoginosa : 6,5 [+ or -] 0,5 mm and resistant K. pneumoniae: 7,33 [+ or -] 0,47mm). 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 22 mm 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 [+ or -] 0 mm, resistant E. coli: 7 [+ or -] 0,81 mm, S. aureus : 6 [+ or -] 0 mm, resistant P. aeruoginosa : 6 [+ or -] 0 mm, and resistant K. pneumoniae: 7 [+ or -] 0mm). 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 ([less than or equal to] 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 [less than or equal to] 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).

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.

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.

ARTICLE INFO

Article history:

Received 2 April 2014

Received in revised form 13 May 2014

Accepted 28 May 2014

Available online 27 June 2014

ACKNOWLEDGMENTS

We are grateful to Mr M. Ibn Tattou, Professor at the Scientific Institute of Rabat, for the species identification.

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(1,2) Hanane Elazzouzi, (1) Aminata Soro, 1Fatima Elhilali, (1) Amar Bentayeb, (2) Mohamed Alaoui El Belghiti, (1) Touriya Zair

(1) Laboratory of Chemistry of Bioactive Molecules and Environment, University of Sciences Moulay Ismail, BP 11201. Zitoune, Meknes, Morocco.

(2) Laboratory of Chemistry--General Physics--University of Sciences--Agdal, 4--Avenue Ibn Battouta. B.P. 1014 RP, Rabat, Morocco.

Corresponding Author: Hanane Elazzouzi, Laboratory of Chemistry -General Physics--Department of chemistry, University of Sciences, Rabat, Morocco. Tel: +212658330106 E-mail: ezzhanane@hotmail.com
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

Table 2: Chemical composition of A. pyrethrum essential oils according
to harvest periods.

Compounds                                     Adams         Area (%)
                                              IR

                                                       April   June

[alpha]-Neocallitropsene                      1476     --      0.20
Germacrene D                                  1481     0.17    --
Trans-[beta]-Ionone                           1488     0.20    --
Cubebol                                       1515     0.10    0.15
[delta]-Cadinene                              1523     0.18    --
Ar-Macrocarpene                               1526     --      0.40
Italicence epoxide                            1548     0.58    --
occidentalol                                  1552     1.31    2 .20
1 [alpha],10[alpha]-Epoxy-amorph-4-ene        1572     0.78    2.83
Spathulenol                                   1578     13.31   16.90
Caryphylene oxide                             1583     9.65    7.11
[beta]-copaen-4-[alpha]-ol                    1590     0.28    --
Salvial-4(14)-en-1-one                        1594     4.39.   4.66
Mayurone < cis--dihydro>                      1595     4.96    --
[beta]-Atlantol                               1608     --      2.90
[beta]-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-P-ol                  1631     4.34    1.81
[beta]-Acorenol                               1637     --      2.58
Caryophylla-4(18), 8(13)-dien-5a[alpha]-ol    1640     0.54    --
3-iso-Tujopsanone                             1642     --      1.54
Selina-3,11-dien-6[alpha]-ol                  1644     0.91    9.24
cis-guai-3.9-dien-11 -ol                      1649     1.71    --
Cedr-8(15)-en-9-[alpha]-ol                    1651     1.44    --
Himachalol                                    1653     5.67    --
3-Thujopsanone                                1654     --      0.42
E-Caryophylene- 14-hydroxy-9-epi              1669     1.88    --
Z-[alpha]-Santalol                            1675     --      1.86
Khusinol                                      1680     0.95    3.29
Germacra-4(15),5,10(14)-trien-1-[alpha]-ol    1686     2.07    12.89
Eudesma-4(15),7-diene-1-[beta]-ol             1688     5.85    --
Nootkatol<epi>                                1699     0.15    --
Eudesm-7(11)-en-4-ol                          1700     0.79    --
Amorpha-4,9-dien-2-ol                         1700     --      0.12
[gamma]-Gurjunenepoxide                       1704     --      0.62
(+)-Trans-Nootkatol                           1715     0.15    0.28
Vetiselinenol                                 1731     0.55    --
Isobicyclogermacrenal                         1734     2.35    1.41
Khusimol                                      1742     --      2.88
[beta]-acoradienol                            1763     3.72    1.58
Cedryl acetate                                1767     8.10    --
14-oxy-[alpha]-Muurolenee                     1768     4.03    1.49
Amorpha-4,11-diene<2-[alpha]-hydroxy>         1775     1.44    --
14-hydroxy-[alpha]-muurolene                  1780     --      0.86
Hinesol acetate                               1784     0.56    --
8-Cedren-13-ol acetate                        1788     0.32    0.29
Isovalencenol                                 1793     --      0.14
[alpha]-Eudesmol acetate                      1795     --      0.68
14-Hydroxy-S-cadinene                         1803     1.74    1.26
Vetivenic acid                                1811     0.46    2.18
Khusinol acetate                              1823     0.08    --
8-hydroxy-eremphilone                         1847     --      0.59
Chenopodiol<a>                                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

Table 3. Phytochemical screening reactions

Chemical                                       Reagents or
groups                                         Reaction name

Tannins        Total                           Fe[Cl.sub.3]

               Cathechic                       Stiasny reagent

               Gallic                          Reaction with sodium
                                               acetate

Flavonoids     Anthocyans                      Acido-basic reaction

               Flavons                         Cyanidin reaction
                                               with Mg  shavings

               Leucoanthocyans                 Cyanidin reaction
                                               without Mg  shavings

               Catechols                       Cyanidin reaction
                                               without Mg  shavings

Alkaloids                                      Valser--Mayer
                                               Reagent  Dragendorff
                                               Reagent

Saponosids                                     Foam Index (FI)

Sterols and                                    Liebermann Buchard
Triterpenes                                    Reaction

Anthracenic    Free anthraquinones             Borntragger Reaction
  compounds    Combined         o-heterosids   Color Reaction
               anthraquinones   c-heterosids   Color Reaction
Narcotics                                      Color Reaction

Reducing                                       Fehling Reaction
compounds

Mucilage                                       precipitation
                                               Reaction

Coumarins                                      fluorescence
                                               Reaction

Oses and                                       Color Reaction
holosids

Chemical                                       Results
groups

Tannins        Total                           (++) (Dark green
                                               color)

               Cathechic                       (+) (red
                                               precipitate)

               Gallic                          (++) (bleue
                                               precipitate)

Flavonoids     Anthocyans                      --

               Flavons                         (+) (Pink--orange
                                               color)

               Leucoanthocyans                 -

               Catechols                       (++) (Red--brown
                                               color)

Alkaloids                                      (++) (orange
                                               precipitate)

                                               (++) (red
                                               precipitate)

Saponosids                                     (-) (Positif Test if
                                               FI<100)

Sterols and                                    (+) (Red ring and
Triterpenes                                    brownish  violet
                                               color of the
                                               supernatant layer)

Anthracenic    Free anthraquinones             -
  compounds    Combined         o-heterosids   -
               anthraquinones   c-heterosids   -
Narcotics                                      -

Reducing                                       (++) (Brick--red
compounds                                      precipitate)

Mucilage                                       (++) (Flake)

Coumarins                                      (+) (Intense
                                               fluorescence)

Oses and                                       (++) (red color)
holosids

Highly positive reaction: (++) ; positive Reaction : (+)
Moderately positive reaction: (+ / -); Negative test: (-)

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

Table 5: Inhibition diameters of A. Pyrethrum extracts on
bacterial strains presented as Means (mm)  [+ or -]  standard
deviation.

                      E. coli           E. coli
                      sensitive         resistant

Essential Oil         0                 0
Aqueous macerate      9 [+ or -] 0,81   9 [+ or -] 0,81
Ethanol macerate             0          7 [+ or -] 0,81
Soxhlet with water    8 [+ or -] 0      7 [+ or -] 0,81
Soxhlet with ethanol        --                --
Alkaloids                   --          7 [+ or -] 0,81

                      S. aureus            P. aeruginosa
                                           sensitive

Essential Oil         7 [+ or -] 0         0
Aqueous macerate      9,66 [+ or -] 0,47   6,5 [+ or -] 0,5
Ethanol macerate         7 [+ or -] 1      6,33 [+ or -] 0,47
Soxhlet with water    6 [+ or -] 0         0
Soxhlet with ethanol  7 [+ or -] 1,41              --
Alkaloids             6 [+ or -] 1,41         6 [+ or -] 0

                      P. aeruoginosa      K. pneumoniae
                      resistant           resistant

Essential Oil         0                   0
Aqueous macerate      7,6 [+ or -] 0,47   7,33 [+ or -] 0,47
Ethanol macerate        7 [+ or -] 0              0
Soxhlet with water    6 [+ or -] 0        7 [+ or -] 0
Soxhlet with ethanol  6 [+ or -] 0                -
Alkaloids                    --           7 [+ or -] 0

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

                   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

                          S. aureus

                   MIC (v/v)   MBC (v/v)

Aqueous macerate   1/200       >1/100
Soxhlet equeous      n. d        n. d
extract

n. d : not determined
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Author:Elazzouzi, Hanane; Soro, Aminata; Elhilali, Fatima; Bentayeb, Amar; El Belghiti, Mohamed Alaoui; Zai
Publication:Advances in Natural and Applied Sciences
Article Type:Report
Geographic Code:6MORO
Date:Jul 1, 2014
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