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Antioxidant Activity in Leaves of Some Mediterranean Plants

S. Chevolleau a, J.F. Mallet a, E. Ucciani a,*, J. Gamisans b and M. Gruber b
aLaboratoire de Chimie Organique Appliqude, URA CNRS 1409 and bLaboratoire de Botanique et Ecologie, URA CNRS 1152,
Facultd des Sciences et Techniques, 13397 Marseille Cedex 13, France

Hexane and m e t h a n o l leaf extracts of sixteen Mediter- light spectrophotometer (Sequoia Turner Corporation,
ranean plant species were obtained by percolation. Higher Mountain View, CA). Chemical reagents and products were
yields were observed for m e t h a n o l (16.4-47.8%} than for obtained from Fluka (Mulhouse~ France). An a-tocopherol
hexane extracts (1.3-13%}. Antioxidant activity of these solution (15 mg/L) was used to establish, from 20 ex-
extracts w a s determined by a rapid spectrophotometric periments, the relative dispersion of the results (4.8%}.
method involving the combined oxidation of beta-carotene
and linoleic acid. Leaf extracts were thus characterized
RESULTS AND DISCUSSION
by an Antioxidant A c t i v i t y Coefficient (AAC) ranging
from 0 to 1000. Hexane extracts gave m u c h higher A A C Several preliminary experiments were performed in our
values than the corresponding methanol extracts. In both laboratory concerning plant extraction with hexane and
cases, myrtle (Myrtuscommunis) showed the best antiox- methanol (Chevolleau, M., unpublished results}. Leaves
idant effectiveness. were dried by various methods, such as heating (40°C)
under vacuum, azeotropic distillation and lyophilization.
KEY WORDS:Antioxidant activity, hexane extracts, Mediterranean Best results were obtained from air-dried leaves at room
plant leaves, methanol extracts. temperature, protected from light, humidity and dust.
Choice of solvent and extraction procedures were also
considered. Among all the common solvents, hexane was
Lipid protection against autoxidative degradation is en- preferred to alkyl chlorides, aromatic hydrocarbons and
sured by antioxidants. Among the naturally occurring an- ethers, owing to their disadvantages. On the other hand,
tioxidants, the liposoluble tocopherols have been studied we found that methanol, in spite of its toxicity, was con-
extensively. The search for new natural antioxidants venient for solubilization of the more polar compounds.
began in the fifties (1-3) and has received increasing in- Efficiencies of some extraction methods (Soxhlet,
terest recently (4-10). Most of the isolated compounds are Kumagawa, maceration, percolation) were compared, and
found in plant leaves and have been characterized as it was observed that percolation was less time-consuming
polyphenols. and gave rise to higher antioxidant activity. However, the
Food, cosmetic and pharmaceutic industries are in- yield was less than that obtained by the other methods.
terested in new sources of natural antioxidants. The plant Sixteen plant species were studied, and the yields
kingdom is rich in phenolic compounds (11), which are par- resulting from hexane followed by methanol extractions
ticularly found in spices and aromatic plants of warm and of the leaves are given in part A of Table 1.
dry regions (12,13). It must be noted that in all cases, low amounts of hex-
This paper describes a method for obtaining plant ex- ane extracts were obtained. The yield varied from 1.3%
tracts as well as determination of their antioxidant ac- (R. alaternus) to 13% (L. angustifolia) and was rarely above
tivity. The species we have examined occur frequently near 5%. Higher yields for some species (E. globulus, L.
the Mediterranean coast. Our results constitute the first angustifolia, P. halepensis) could be explained by the
report on these plants. presence of essential oils and oleoresins. Hexane extracts
were all viscous oils with a brownish green color.
Presence of chlorophyll in the methanol extracts was
EXPERIMENTAL PROCEDURES
responsible for the deep-green coloration. Extraction with
Air-dried leaves were ground, and 10 g of the resulting methanol gave yields ranging from 16.4% (F. comrnunis)
powder was introduced into a glass column (55 X 2 cm). to 47.8% (G. alypum), and more than half of them were
Hexane (300 mL) was allowed to percolate through at over 20%.
room temperature and protected from light. Percolation Determination of antioxidant activity of a pure com-
was complete after about 2 h. The crude extract solution pound or a mixture is always achieved through measure-
was vacuum evaporated to dryness at 40°C and stored ment of lipid oxidation. The methods currently utilized
under argon at - 2 0 °C. A second extraction process was have been reviewed recently (17), and all of them give com-
carried out with 300 mL methanol. Flow rate was com- parable results, although the compounds involved are
paratively slower with this solvent. After vacuum evapora- different.
tion, the dry methanol extract was stored as described We used a simple and rapid method to evaluate the an-
above. tioxidant activity of the plant extracts. Inhibition rate
Hexane and methanol extracts were prepared as of the combined oxidation of beta-carotene and linoleic
methanol solutions (100 mg/L), and their antioxidant ac- acid was monitored by optical density readings at 470 nm
tivity was determined by a spectrophotometric method of an oxygenated aqueous emulsion containing these two
(14-16). The experimental procedure has been described substrates and the extracts. Antioxidant effectiveness was
previously by Taga et al. (15). Optical density measure- calculated in the following way. At time t = 0, the absor-
ment at 470 nm was made on a Sequoia Turner 690 visible- bance of beta-carotene at 470 nm is maximum and a coef-
ficient of 1000 was thus attributed. At time t = 120 min,
*Towhom correspondenceshould be addressed at LCOA, boite 412, the minimum absorbance was observed for the control,
Facult~ des Sciences et Techniques, 13397 Marseille Cedex 13, to which we attributed a 0 coefficient. For each extract,
France. an antioxidant activity coefficient (AAC), ranging from

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TABLE 1

Percentage of Leaf Extracts and Antioxidant Activity of Various Plant Species

A B
Leaf extract (%) Antioxidant activity
(dry weight basis) coefficient (AAC)
Plant species Hexane Methanol Hexane Methanol

Centranthus tuber DC. 2.5 24.0 29 (0.7)a 0 (0)

(Valerianaceae)
Cistus albidus L. 3.3 26.9 5 (0.2) 20 (5.4)
(Cistaceae)
Conium maculatum L. 3.5 20.8 102 (3.6) 14 (2.9)
(Apiaceae)
CoronillajunceaL. 1.8 31.6 7 (0.1) 10 (3.2)
(Fabaceae)
Eucalyptus globulus Labil. 11.1 31.2 18 (2.0) 2 (0.6)
(Myrtaceae)
Ferula communis L. 2.5 16.4 2 (0.1) 14 (2.3)
(Apiaceae)
Globularia alypum L. 1.7 47.8 464 (7.9) 6 (2.9)
(Globulariaceae)
Hedera helix L. 1.5 32.8 214 (3.2) 8 (2.6}
(Araliaceae)
Lavandula angustifolia L. 13.0 18.4 2 (0.3) 14 (2.6)
(Lamiaceae)
Myrtus communis L. 1.9 19.8 641 (12.2) 260 (51.5)
(Myrtaceae)
Phillyrea angustifolia L. 2.0 26.2 243 (4.9) 7 (1.8
(Oleaceae)
Pinus halepensis Mill. 10.5 16.8 4 (0.4) 10 (1.7
tPinaceae)
Quercus ilex L. 1.8 23.7 515 (9.3) 150 (35.6
(Fagaceae)
Rhamnus alaternus L. 1.3 22.3 585 (7.6) 13 (2.9
(Rhamnaceae)
Smilax aspera L. 1.7 17.3 292 (5.0) 27 (4.7
(Liliaceae)
Staehelina dubia L. 4.6 16.6 11 (0.5) 8 (1.3
{Asteraceae)

aValues in parentheses correspond to AAC of dry leaves.

0 to 1000, was d e t e r m i n e d b y t h e following e x p r e s s i o n (16): We h a v e t h u s , in d e c r e a s i n g order, for t h e h e x a n e ex-

t r a c t s , M. c o m m u n i s > Q. ilex > G. a l y p u m > R. alater ~
AAC -- 1000 [(Am20 - AC120)/(Aco -- AC120) [1] nus . . . . a n d for t h e m e t h a n o l e x t r a c t s , M. c o r n m u n i s >
Q. ilex > C albidus > S. aspera . . . .
w h e r e AE120 c o r r e s p o n d s t o a b s o r b a n c e of t h e e x t r a c t a t M. c o m m u n i s (myrtle) s h o w e d t h e b e s t a n t i o x i d a n t ef-
t = 120 min, Ac0 a n d Ac120 c o r r e s p o n d to a b s o r b a n c e of fectiveness whatever the solvent used. In a future paper,
t h e c o n t r o l a t t -- 0 a n d t -- 120 min, r e s p e c t i v e l y . r e s u l t s c o n c e r n i n g o t h e r p l a n t species a n d s t r u c t u r a l d a t a
T h e c a l c u l a t e d A A C v a l u e s a r e g i v e n in p a r t B of on s o m e a n t i o x i d a n t s p r e s e n t t h e r e i n will be d i s c u s s e d .
Table 1. H e x a n e e x t r a c t s h a v e m u c h h i g h e r A A C (up t o
641) t h a n t h e c o r r e s p o n d i n g m e t h a n o l e x t r a c t s , for w h i c h
A A C r a n g e s f r o m 0 t o 260. B y c o m p a r i s o n of t h e A A C REFERENCES
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