Partial chemical composition and antimicrobial activity of Daucus crinitus Desf

The carrot is a plant belonging to the Apiaceae family and is grown throughout the temperate regions for its roots that are consumed raw and cooked. The carrot is reported to be endowed with medicinal properties, i.e. hypotensive, diuretic, carminative, stomachic and antilipemic (Gilani et al., 2000; Kumarasamy et al., 2005; Nicolle et al., 2004). In Algerian folk medicine, the leaves are used raw or in an infusion as depurative and diuretic agents. In Algeria, the Daucus genus is represented by some species living in dry and uncultivated areas and, among them, D. carota is widespread together with its many subspecies. In this group D. crinitus is different from the other species. The morphological features observed in the leaves of the Daucus genus are alternately arranged, except in D. crinitus, in which they appear to be pseudoverticillate. Also, D. crinitus is characterized by the presence of many subspecies that colonize sands and cliffs (Quezel and Santa, 1963; Sáenz Laín, 1981). The species studied is known by two botanical classifications that are considered synonyms: D. crinitus Desf. and D. meifolius Brot. This species grows along the Central-Western Mediterranean coasts; in Algeria it can be found along the Western coast from Tlemcen to Mascara (Quezel and Santa, 1963; Sáenz Laín, 1981). In folk medicine, it is also used to expel the placenta after childbirth or to give some stoutness. The mixture is also galactogene and depurative. RESUMEN


INTRODUCTION
The carrot is a plant belonging to the Apiaceae family and is grown throughout the temperate regions for its roots that are consumed raw and cooked.The carrot is reported to be endowed with medicinal properties, i.e. hypotensive, diuretic, carminative, stomachic and antilipemic (Gilani et al., 2000;Kumarasamy et al., 2005;Nicolle et al., 2004).In Algerian folk medicine, the leaves are used raw or in an infusion as depurative and diuretic agents.
In Algeria, the Daucus genus is represented by some species living in dry and uncultivated areas and, among them, D. carota is widespread together with its many subspecies.In this group D. crinitus is different from the other species.The morphological features observed in the leaves of the Daucus genus are alternately arranged, except in D. crinitus, in which they appear to be pseudoverticillate.Also, D. crinitus is characterized by the presence of many subspecies that colonize sands and cliffs (Quezel and Santa, 1963;Sáenz Laín, 1981).
The species studied is known by two botanical classifications that are considered synonyms: D. crinitus Desf.and D. meifolius Brot.This species grows along the Central-Western Mediterranean coasts; in Algeria it can be found along the Western coast from Tlemcen to Mascara (Quezel and Santa, 1963;Sáenz Laín, 1981).In folk medicine, it is also used to expel the placenta after childbirth or to give some stoutness.The mixture is also galactogene and depurative.

Partial chemical composition and antimicrobial activity of Daucus crinitus Desf. extracts.
The chemical composition of fatty acids and the unsaponifiable fraction of the roots, leaves and stems from Daucus crinitus Desf.were, determined using gas chromatography (GC) and gas chromatography-Mass Spectrometry (GC-MS).The fatty acid fractions of different organs (leaves, stems and roots) were characterized by lauric acid (17.9, 17.5 and 18.1 % respectively) and other long chain fatty acids (until C 22 ).Qualitative and quantitative differences were reported between the unsaponifiable fractions of different organs from D. crinitus.The unsaponifiable fractions of the leaves, roots and stem showed high amounts of aliphatic components (83.4%, 87.2% and 91.4%, respectively).The monoterpen, diterpen and sesquiterpen components were only

Sample preparation
Samples were air-dried and crushed.Obtained powders were sieved in size grading 0.355 and 0.600 mm for roots, leaves and stems, respectively.50 g of each organ were extracted with 2x200 mL of methanol and petroleum ether under reflux (Soxhlet apparatus) separetely.After evaporation of the solvent under reduced pressure, extracts were gathered and dried on MgSO 4 .The extraction was performed in triplicate and the yields (average of three extractions) in methanol were 2.5% for stems, 6.1% for leaves and 1.8% for roots and in petroleum ether were 1.2% for stems, 5.8% for leaves and 1.5% for roots.For aqueous extracts, 5 g of each organ were extracted with 100 mL of wáter and were lyophilized.

Extraction of unsaponifiable compounds
1 g of petroleum ether extract was saponified with 50 mL of methanolic potassium hydroxide solution (2 mol/L) for 1 h under reflux; the unsaponifiable components were then extracted three times with 100 mL of ether.The pooled extracts were washed three times with 50 mL of deionized water.The solvent was subsequently removed at 35°C under reduced pressure with a rotary evaporator.The unsaponifiable fractions of the leaves, stems and roots represent 54.1%, 69.1% and 55.3% (w/w) of petroleum ether extract, respectively.

Extraction of fatty acids
The soapy aqueous solution obtained previously was acidified (HCl 1N) until precipitation of fatty acids (pH 5-6).The freed fatty acids were extracted with ether (3x 50 mL) and dried on MgSO 4 and then weighed.Fatty acids were transformed into their methyl ester derivatives by the addition of a methanolic solution of BF 3 10% (Metcalfe and Schmitz, 1961).Thereafter, fatty acids were extracted three times with 50 mL of hexane at room temperature.The organic layer was evaporated and dried on Na 2 SO 4 .The acid fractions of the leaves, stems and roots represent 21.2%, 29.4% and 11.4% (w/w) of diethyl ether extract, respectively.
In Algeria, many patients used medicinal plants, aside from conventional treatments, as an alternative therapy for many diseases.Recently, special attention has been paid to the bioactive elements extracted from plants in order to discover new drugs for the treatment of several pathologies (Kivçak and Mert, 2001).Therefore, the aim of the present work was: (i) to establish the chemical composition of solvent extracts (fatty acids and unsaponifiable fractions) of the stems, leaves and roots from D.crinitus for the first time.For this purpose, the analysis of extract composition was carried out by Gas Chromatography (GC) and Gas Chromatography-Mass Spectrometry (GC-MS); (ii) to test the antibacterial and antifungal activities of unsaponifiable, methanolic and aqueous fractions against four species of micro-organisms using a microdilution method.The assessment of the D. crinitus potential as a source of natural compounds for pharmaceutical application is expected as a possible outcome of this study.

Plant material
Samples from each organ (stems, leaves and roots) of D. crinitus were collected in the Bensekrane PARTIAL CHEMICAL COMPOSITION AND ANTIMICROBIAL ACTIVITY OF DAUCUS CRINITUS DESF.EXTRACTS and incubated at 30±1°C for 24 to 48 h.0.1 mL of each culture was added to 10 mL sterile physiological water.
For the antimicrobial assay, bacterial strains were grown on Mueller-Hinton Agar (MHA, Pronadisa Hispanalab) while C. albicans was grown on Sabouraud Dextrose Agar + Chloramphenicol (SDA, Merck).Bacterial and yeast inocula reached microbial densities in the range of 10 6 to 10 7 cfu/mL.

Antimicrobial activity
Two different techniques were used to test the anti-microbial activity: the paper disc diffusion and the dilution agar method.The minimum inhibitory concentration (MIC) was determined by the later method.

Paper-disc diffusion method
Paper discs (6 mm in diameter) saturated with a 40 mg/mL solution of plant extract were applied to the surface of agar plates that were previously seeded by spreading 0.1 mL of culture overnight.The plates were incubated overnight at the appropriate temperature (see above), and the diameter of the resulting zone of inhibition was measured in millimeters.The results indicated in Table 3 and in the text represent the net zone of inhibition including the diameter (6 mm) of the paper disk.The scale of measurement was the following (disk diameter included) : > 20 mm zone of inhibition is strongly inhibitory; < 20-12 mm zone of inhibition is moderately/mildly inhibitory; and <12 mm is not inhibitory.All the data collected for each assay are the averages of three determinations.

Dilution-agar method
A dilution agar method was used to determine the Minimum Inhibitory Concentrations (MIC).Stock solutions were obtained by dissolving extracts in dimethylsulfoxide (DMSO 1%).Serial dilutions were made to obtain concentrations ranging from 0 to 4000 µg/mL of unsaponifiable, methanolic and aqueous extracts.Each mixture was added to Mueller-Hinton agar for bacteria and Sabouraud dextrose agar with Chloramphenicol for yeast (cooled to 45-50°C) (Cowan, 1999;Lennette and Balows, 1985).Two controls were included in this test.The Petri dishes contained a sterile solution of DMSO and the culture medium, respectively.The experiments were performed in triplicate.After incubation at 37°C for 24h for bacteria and at 30°C for 48h for the yeast.

Fatty acids of the leaves, stems and roots from D. crinitus
Fatty acids from the leaves, stems and roots, their retention indexes and their relative abundance 1 (polydimethylsiloxane).The oven temperature was programmed from 60°C to 230°C at 2°C/min and then held isothermally at 230°C for 35 min.Injector and detector temperatures were maintained at 280°C.Samples were injected in the split mode (1/50), using helium as the carrier gas (1 ml/min); the injection volume was 0.2 µL.Retention indexes (I) of the compounds were determined relative to the retention times of the series of n-alkanes (C5-C30) with linear interpolation, using the Van den Dool and Kratz equation (Van Den Dool and Kratz, 1963) and software from Perkin-Elmer.Component relative concentrations were calculated based on GC peak areas without using correction factors.

Gas chromatography-Mass spectrometry analysis (GC-MS)
Samples were analyzed with a Perkin-Elmer Turbo mass detector (quadrupole), coupled to a Perkin-Elmer Autosystem XL, equipped with the fused-silica capillary columns Rtx-1 and Rtx-Wax (ion source temperature 150°C; energy ionization 70 eV).EI mass spectra were acquired over the mass range 35-350 Da (scan time: 1 s).Other GC conditions were the same as described under GC except split 1/80.

Component identification
As previously reported (Paolini et al., 2005), the methodology used for the identification of individual components was based on: (a) a comparison of calculated retention indices I a on an apolar column with those of authentic compounds or literature data I l (Jennings and Shibamoto, 1980; National Institute of Standards and Technology, 2005; National Institute of Standards and Technology, 2005) and (b) computer matching with commercial mass spectral libraries (König et al., 2001;Adams, 2001;Mc Lafferty and Stauffer,1994;Mc Lafferty and Stauffer,1988;National Institute of Standards and Technology, 1999) and a comparison of mass spectra with those of our own library of authentic compounds or literature data (Jennings and Shibamoto,1980;Joulain and König,1998).

Bacterial and yeast strains and media
The bacterial strains used in this study, i.e.Bacillus cereus, Staphylococus aureus and Escherichia coli, were isolated at the Medical Reanimation Department of the Hospital University Center of Tlemcen in Algeria.The yeast Candida albicans was isolated at the Dermatology Department of the same hospital.

Unsaponifiable fraction of leaves, stems and roots from D.crinitus
The combined analysis (GC and GC-MS) of D. crinitus unsaponifiable fractions revealed the presence of 33 components: 33 in leaf fraction, 29 in stem fraction and 15 in root fraction accounting for 91.5%, 92.8% and 91.4% of the total composition, respectively (Table 2).Even if aliphatic compounds are given in Table 1; the components are listed in the order of elution on the Rtx-1 column.25 acid constituents were identified from leaf, stem and root extracts amounting to 84.1%, 85.7% and 86.6% of the total acid fraction, respectively (Table 1).The three fractions were qualitatively similar.However, propanoic acid (0.9%) was reported only in the stem extract and 15 methyl hexadecanoic acid (0.6%) in the root extract.The leaf, stem and root extracts were characterized by high contents of saturated fatty acids with lauric acid (17.9%, 17.5% and 18.1%, respectively) as the major compound followed by palmitic acid (16.8%, 14.3% and 10.7%, respectively), myristic acid (10.2%, 14.5% and 16.1%, respectively), behenic acid (7.2%, 9.1% and 6.5%, respectively), and stearic a Order of elution are given on a polar column (Rtx-1).b Retention indexes of literature of corresponding esters on the apolar column (I l ) (Jennings and Shibamoto. 1980;König and al.. 2001).c Retention indexes of corresponding esters on the apolar Rtx-1 column (I a ).d Relative percentages (%) on the a polar column (Rtx-1); tr = trace (< 0.1%).
PARTIAL CHEMICAL COMPOSITION AND ANTIMICROBIAL ACTIVITY OF DAUCUS CRINITUS DESF.EXTRACTS diterpenes (2.4% and 1.9%) and sesquiterpenes (0.7% and 06%) respectively.Moreover, the monoterpene hydrocarbon family exhibited qualitative and quantitative differences in their composition.The γ-terpinene was identified as the major components of were in high abundance in the three fractions, these were qualitatively and quantitatively different.Indeed, the leaf and stem fractions were strongly dominated by aliphatic compounds (83.4% and 87.2% respectively), monoterpene (5% and 3.1%), Order of elution are given on a polar column (Rtx-1).b Retention indexes of literature on the apolar column (I l ) (Jennings and Shibamoto. 1980;König and al.. 2001).c Retention indexes on the apolar Rtx-1 column (I a ).d Relative percentages (%) on the apolar column (Rtx-1); tr = trace (<0.1%).

Antibacterial activity
The in vitro antibacterial activities of root, leaf and stem unsaponifiable, methanolic and aqueous extracts of D.crinitus against the employed bacteria were qualitatively and quantitatively assessed by the presence or absence of inhibition zones.As shown in Table 3, the root, leaf and stem unsaponifiable extracts exhibited inhibitory effects against the tested bacterial pathogens.C. albicans, S. aureus and B. cereus were found to be the most inhibited pathogens by the unsaponifiable extracts with their respective diameter zones of inhibition of 30 (roots, leaves and stems), 18 (leaves) and 14 (roots) mm.The rest of the bacterial strain (E.coli) showed no To the best of our knowledge, the composition of fatty acid and unsaponifiable extracts and antimicrobial activity of D. crinitus have not been reported before and therefore our results can be evaluated as the first report about the antimicrobial properties and the chemical compositions of fatty acids and unsaponifiable factions.inhibition, with diameter of zones of inhibition ranging from 8 to 9 mm (Table 3).
Also the methanolic extracts exerted potential effects of antibacterial activity against the tested bacteria.However, the roots, leaves and stems exhibited inhibitory effects against C.albicans with diameters of zones of inhibition from 13, 16 and 15 mm respectively, E. coli (14, 13 and 15 mm respectively), B. cereus (14, 15 and 10 mm respectively) and S. aureus (13, 11 and 11 mm respectively).However, aqueous extracts exhibited no effect of antibacterial activity against of the bacterial pathogens tested.

Minimum inhibitory concentrations (MIC)
As shown in Table 4, the most promising results were obtained from the unsaponifiable extract which not only had the lowest MIC value (46.5, 56.2and 76.2 µg/mL for leaf, stem and root extracts respectively) against C.albicans, but also inhibited the growth of other micro-organisms.The unsaponifiable extract of leaves, stems and roots also shows a strong antimicrobial effect against E. coli with an MIC of 111.5, 111.1 and 126 µg/mL respectively and S. aureus with a MIC of 122.1, 122.1 and 136 µg/mL respectively and average activity on the B. cereus with a MIC of 255, 255 and 250 µg/mL respectively (Table 4).Using the dilution agar method technique, root, leaf and stem methanolic extracts of D. crinitus showed antimicrobial properties with MIC values ranging from 500 to 1800 µg/mL for bacteria and 400 µg/mL for C. albicans.The root, leaf and stem aqueous extracts showed no inhibition towards any of the microorganisms assayed up to the value of 3000 µg/mL for bacteria and 2000 µg/mL for C. albicans.
The unsaponifiable extracts of roots, leaves and stems evaluated in this work have different varieties of phytochemicals that could be considered responsible for their antimicrobial activity.Although they usually occur as complex mixtures, their activity can generally be accounted for in terms of their major components.The antimicrobial activity of the unsaponifiable extracts of roots, leaves and stems could be due to the investigated strain sensitivity to α-pinene, sabinene, limonene and the other lighter components present in unsaponifiable extracts (Magiatis et al., 1999;Oussou et al., 2008;Oussou et al., 2004).

CONCLUSIONS
The leaves, stems and roots of D. crinitus were characterized by the presence of fatty acids.It appears that all parts of the plant were rich in saturated fatty acids with lauric acid (17.9% for leaves, 17.5% for stems and 18% for roots) as the major compound.In all extracts, lauric acid is accompanied by fatty acids with long chain (from C 3 to C 22 ).The unsaponifiable fraction of leaves and stems were characterized by aliphatic constituent products and monoterpene . A. DIB, M. BENDAHOU, A. BENDIABDELLAH, N. DJABOU, H. ALLALI, B. TABTI, J. PAOLINI AND J. COSTA forest area (at about 30 km north west of Tlemcen -Algeria) [287 m, 35°07'N 1°22'O] in September, 2009.Voucher specimens were deposited in the herbarium of the Tlemcen University Botanical Laboratory (Voucher number: UBL 05.09).A portion of each organ was stored at 4°C for further analysis . M

Table 4 Minimum Inhibitory Concentrations MIC (µg/mL) of unsaponifiable, methanol and water extracts from D. crinitus.
CHEMICAL COMPOSITION AND ANTIMICROBIAL ACTIVITY OF DAUCUS CRINITUS DESF.EXTRACTS hydrocarbons.Sesquiterpenic compounds were also present but in small amounts.The antimicrobial properties of the unsaponifiable, methanolic and aqueous extracts (leaves, stems and roots) were tested on four microorganism species.The root, leaf and stem unsaponifiable and methanol extracts were active against B. cereus, S. aureus, E. coli and C. albicans bacteria strain which are deemed very dangerous and very difficult to eliminate. PARTIAL