INTRODUCTION
As apart of our investigation into some medicinal plants known in Tunisia
(Bouaziz et al., 2002; Ben Salah et al., 2002; Koubaa and
Damak, 2003; Louati et al., 2003; Fakhfakh and Damak, 2007) and
in order to continue present research on potential biological natural
compounds, we report below the study of oil composition and antioxidant
activity of Daucus maritimus.
Oil yielding crop plants are very important for economic growth of agricultural
sector. The oilseeds containing unusual fatty acids are very important
for industry; they can be used in pharmaceuticals, cosmetics, detergents,
soaps, textiles, surfactants (Hosamani and Sattigeri, 2000). On the other
hand, no oil from a single source has been found to be suitable for all
purposes because oils from different sources generally differ in their
composition. This requires the search for new sources of novel oils. Several
plants are now growing, not only for food and fodder, but also for a striking
range of products with an industrial application.
The genus Daucus, consisting of 14 species, belongs to the Umbelliferae
family (Apiaceae). Daucus maritimus (Lam.) Batt. is a subspecies
of Daucus carota. It is a biennial plant, with fleshy leaves. It
is widespread in the Mediterranean region (L’abbé, 1983; Tutin
et al., 1981). No report indicates its uses in traditional medicine,
but relatively few phytochemical analyses have been published by Baranska
et al. (2005) and Miladi et al. (2005). Furthermore, insufficient
information is available regarding to the seeds content in this species.
The seeds were undertaken and extracted in a soxhlet apparatus successively
with solvents of different polarities (hexane, dichloromethane, ethyl
acetate and ethanol) to provide four oily extracts: HE, DM, EA and ET.
The aim of the present investigation is to determine the composition
of Daucus maritimus seed oils and to evaluate their antioxidant
activities by 2,2-diphenyl-1-picryl hydrazyl andβ-carotene bleaching
methods.
MATERIALS AND METHODS
Plant material: Daucus maritimus (Lam.) Batt. was collected
in June 2005 at Sfax, Tunisia. It was identified by Pr. M. Chaieb (Département
de Biologie, Faculté des Sciences, Université de Sfax, Tunisie).
Voucher specimens (N° LCSN109) were deposited at the Laboratoire de
Chimie des Substances Naturelles, Faculté des Sciences, Université
de Sfax, Tunisie.
Extraction and physicochemical properties: Seed powder of Daucus
maritimus (Lam.) Batt. (300 g) was extracted in a Soxhlet apparatus
with hexane, dichloromethane, ethyl acetate and ethanol to give successively
four oily extracts: HE, DM, EA and ET.
Some physicochemical indices of the four oils were determined. The following
were evaluated according to the methods listed in the ISO (International
Organization for Standardization) and in IOC (International Oil Council)
: acid value (ISO 660, 1996); saponification value (ISO 3657, 1988); Iodine
value (ISO 3961, 1996); unsaponifiable value (ISO 3596, 1996); peroxide
value (IOC 3960, 1998).
Fatty acid analysis: The fatty acids methyl esters were prepared
by adding 10 mL of sodium methoxide solution (0.25 M) to 1 g of oil. The
mixtures were refluxed for 15 min, followed by adding 15 mL of acid methanolic
solution (1M) and refluxed again for 10 min according to the procedure
reported by ISO 5509. The solutions were cooled by adding 20 mL of water
and the fatty acids methyl esters were extracted three times with 20 mL
of hexane and concentrated under reduced pressure. Then, the fatty acid
composition of the four oils was determined on a Hewlett-Packard GC/MS
(HP6890-HP5973 MSD-Agilent Technology, Wiling, USA) equipped with a HP-5
column (30 mx 0.25 mm i.d.) under the following operating conditions:
oven temperature: 150°C for 4 min, rising to 290°C at 3°C
min-1 and held for 10 min; injector temperature: 250°C;
scanning scope: 50-600 amu; ionization voltage: 70 eV; ionization electric
current: 30 μA.
Sterol composition: Unsaponifiable matter of Daucus maritimus
seed (DMS) oils was extracted and determined in oils samples according
to IOC (COI/T.20/Doc.No. 10, 2001). One gram of each oil was refluxed
with 10 mL of 10% ethanolic potassium hydroxide for 1 h. The reaction
mixture was diluted with 10 mL of distilled water and the unsaponifiable
matter was extracted three times with 20 mL portion of ether. The ether
extracts were combined and washed three times with distilled water until
they became alkali-free. The ether extract was dried over anhydrous sodium
sulphate and evaporated. The sterol fractions obtained by TLC separation
from the unsaponifiables of the oils were derivitised with silanizing
mixture (pyridine-hexamethyl disilazane-trimethyl chlorosilane 9:3:1,
v/v/v) in the ratio of 50 μL for every mg of sterols.
Further determination of the sterols was carried out on a Hewlett-Packard
GC/MS (HP6890-HP5973 MSD-Agilent Technology, Wiling, USA) equipped with
a HP-5 column (30 m x 0.25 mm i.d.) under the following operating conditions:
oven temperature: 150°C for 1 min, rising to 260°C at 10°C
min-1 and held for 10 min, then to 270°C at 2°C min-1
and held for 10 min, then to 300°C at 5°C min-1; injector
temperature: 280°C; carrier gas: helium (1 mL min-1); scanning
scope: 50-600 amu; ionization voltage: 70 eV; ionization electric current:
30 μA.
Scavenging activity on DPPH radical: The antioxidant activity
of the DMS extracts was assessed by the scavenging effect on DPPH radical
(2,2-diphenyl-1-picrylhydrazyl) (Brand-Williams et al., 1995; Chen
et al., 1999; Naik et al., 2003). Briefly, 1.5 mL of DPPH
solution (10-5 M, in 95% Ethanol) was incubated with 1.5 mL of DMS extract
at varying concentrations (0.1-5 mg). The reaction mixture was shaken
well and incubated in the dark for 30 min at room temperature. The absorbance
of each sample was measured at the spectrophotometer (Bibby Anadéo)
at 517 nm against a blank of EtOH.
The radical scavenging activity (RSA) was measured as a decrease in the
absorbance of DPPH and was calculated using the following equation:
The extract concentration providing 50% inhibition (EC50%)
was calculated from the graph of Scavenging effect percentage against
the extract concentration. BHT was used as a reference compound. Tests
were carried out in triplicate.
β-carotene bleaching test: The oxidative losses ofβ-carotene/linoleic
acid emulsion were used to assess the anti-oxidation ability of the DMS
extracts (Chevolleau et al., 1992; Moure et al., 2000).
Ten milligram ofβ-carotene (type 1 synthetic, Sigma, St. Louis,
MO, USA) was dissolved in 10 mL of CHCl3 and 0.2 mL of the
solution was placed into a boiling flask containing 20 mg linoleic acid
and 200 mg Tween-40 (Sigma). After removal of CHCl3, 50 mL
of oxygenated distilled water was added to the flask and vigorously shaken
to form an emulsion. An absorbance at 470 nm was immediately recorded
after adding 2 mL of the sample to the emulsion, which was regarded as
t = 0 min. The round-bottomed flasks were stoppered and placed in an incubator
at 50°C. The absorbance at 470 nm was determined every 15 min for
120 min. A second emulsion, consisting of 20 mg of linoleic acid and 200
mg of Tween 40 and 50 mL of oxygenated water, was also prepared and served
as blank to zero the spectrophotometer. The antioxidant activity coefficient
(AAC) was calculated according to the following equation:
AAC = [(A A(120)-A C(120)
) / (A C(0)-A C(120) )] x 1000 |
where, A A(120) is the absorbance of the antioxidant after
120 min, A C(120) is the absorbance of the control after 120
min and A C(0) is the absorbance of the control at 0 min. The
AAC was calculated from the graph of absorbance against time. BHT was
used as a reference compound. Tests were carried out in triplicate.
RESULTS AND DISCUSSION
Physicochemical properties: Table 1 presents
the yields and some physico-chemical characteristics of DMS oils.
| Table 1: |
Physicochemical properties of Daucus maritimus seed (DMS)
oils |
 |
| Values are Means±SD of three determination |
| Table 2: |
Fatty Acid Compositions of Daucus maritimus oils |
 |
| -: Not detected |
All oils show high saponification values suggesting their use in production
of liquid soap and shampoos. The high levels of acid value indicate that
these oils could only be recommended for industrial use. The four oils
samples have high iodine value, thus reflecting a high degree of unsaturation
and placing them in the drying group oils similar to Linseed and Conophor
oils (Akpuaka and Nwankwor, 2000). These iodine values were confirmed
by the fatty acid composition of the oils presented in Table
2.
Fatty acids: Fatty acid composition of the oils and their percentages
are presented in order of their elution on the column in Table
2. All samples were characterized by high amounts of unsaturated fatty
acids (76.12-90.28%). Oleic acid was the main unsaturated fatty acids
in HE (62.07%), DM (73.16%) and EA (80.48%) oils whereas petroselinic
acid prevails in ET oil (77.31%). The major saturated fatty acid in these
oils was palmitic acid (8.48-13.26%).
The presence of a very high level of unusual petroselinic acid in ET
oil is of interest as this compound could be used as a plastic lubricant,
in the manufacture of nylons and for cosmetics. Moreover, petroselinic
acid is used in treatment of inflammations.
The results obtained in this study showed that the fixed oils from DMS
are rich in polyunsaturated fatty acids which play an important role in
industry. Therefore, it would be interesting to know more about the possible
pharmacological effects of this specie.
Sterol composition: In almost all plant fats and oils, sterols
are the most quantitatively important unsaponifiable components. Cholesterol
was detected in DM, EA and ET oils in a small amount (0.33-1.28%). We
have observed the presence of campesterol in the DM, EA and ET oils. The
main components for all oils wereβ-sitosterol (54.91-67.90%) and
stigmasterol (26.43-38.84%). Two unknown peaks were detected, one eluted
after cholesterol in HE oil (3.26%) and the other one eluted after Δ7-sitosterol
in DM oil (0.82%). Further work is in progress to identify these peaks
(Table 3).
Scavenging activity on DPPH radical: The RSA of the DMS extracts
was assessed using an ethanolic solution of the stable free radical, DPPH.
A freshly prepared DPPH solution exhibited a deep purple color with a
maximum absorption at 517 nm. This purple color disappears when an antioxidant
is present in the medium. Thus, antioxidant molecules can quench DPPH
free radicals and convert them into a colorless product, resulting in
a decrease in absorbance at 517 nm. The RSA values of hexane, dichloromethane,
ethyl acetate and ethanol extracts of DMS are presented in Fig.
1. The results are expressed as the ratio percentage of sample absorbance
decrease and the absorbance of DPPH solution in the absence of an extract
at 517 nm. RSA% was proportional to the concentration of the extract.
EC50 values (concentration of sample required to scavenge 50%
of free radicals) of DMS extracts and BHT are indicated in Table
4.
| Table 3: |
Relative retention times and mass spectra of the sterols in DMS
oily extracts |
 |
| -: Not detected. RRt : Relative Retention
time toβ-sitosterol (26.80 min) using a HP-5 30 m column |
|
| Fig. 1: |
Scavenging activity (%) on DPPH radicals of DMS oils |
| Table 4: |
Comparison of antioxidant properties of DMS oily extracts and BHT |
 |
The EA extract has the greater scavenging effect followed by ET extract.
From the analyses of Fig. 1, we can conclude that the
scavenging effects of seeds extract increase as the concentration increases.
Antioxidant assay using theβ-carotene bleaching method:
The antioxidant activities of the DMS extracts were evaluated by theβ-carotene
bleaching method, in which the oxidation of linoleic acid takes place.
Linoleic acid hydroperoxides attack theβ-carotene molecule and as
a result, it undergoes a rapid decolorization. The corresponding decrease
in absorbance can be monitored spectrophotometrically. The presence of
antioxidant extracts can hinder the extent ofβ-carotene bleaching
by acting on the linoleate-free radical and other free radicals formed
in the system (IENICA, 2000).
|
| Fig. 2: |
Change of absorbance at 470 nm with time for DMS oils (2 mg mL)
inβ-carotene/linoleic acid emulsion |
Accordingly, the absorbance decreased rapidly in samples without antioxidant
whereas, in the presence of an antioxidant, they retained their color and thus
their absorbance, for a longer time. The absorbance of the emulsion decreased
with time (Fig. 2). DMS extracts, BHT showed a variant anti-oxidation
activity. The decreasing rate of absorbance for an emulsion sample with the
added of hexane and ethyl acetate extracts was significantly lower than the
samples with the addition of other extracts. The AAC are summarized in Table
4. The order of AAC at 2 mg mL-1 among the four extracts of DMS
was as follows: ET extract > EA extract > HE extract > DM extract.
CONCLUSION
This study shows that Daucus maritimus seeds provide different
oils containing a large amount of unsaponifiable matter. The physico-chemical
characteristics and fatty acid composition of these oils suggest that
they have some industrial potential. Furthermore, the ethanol extract
could be used as alternative resource of petroselinic acid.
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