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Antioxidative activities of algal extracts, synergistic effect with vitamin E
Phytochemistry, Vol. 29, No. 12, pp. 3759-3765, 1990 Printed in Great Britain.
ANTIOXIDATIVE
0
ACTIVITIES OF ALGAL EXTRACTS, EFFECT WITH VITAMIN E BERNARD
003 l-9422/90 $3.00 + 0.00 1990 Pergamon Press plc
SYNERGISTIC
LE TUTOUR
Laboratoire de GEnie des Pro&d&s, IUT de Saint-Nazaire, Le Heinlex, BP 420, 44606 Saint-Nazaire Cedex, France (Received 25 January 1990)
Key Word Index-Algae; docosahexaenoate;
antioxidant; vitamin E; BHT; tocopherol; ethyl eicosapentaenoate.
methyl linoleate; sunflower oil; ethyl
Abstract-The antioxidant effect of seven species of marine algae on the preservation of sunflower oil were investigated by storage experiments at 75”. Laminaria digitata (Kombu) and Himanthalia elongata were the most effective in extending the induction period. Extracts of these two seaweeds were then compared with vitamin E and BHT as regards to their antioxidative activities, by kinetic studies in a model system. The thermal initiated oxidation of methyl linoleate was performed at 60” in heptanol, with or without antioxidants. When they reached 1% of the substrate, seaweed extracts exhibited antioxidant activities by extending the induction period but they were not actually effective. Effects of the addition of algal extracts together with vitamin Eon the preservation of methyl linoleate, sunflower oil or mixed ethyl eicosapentaenoate and docosahexaenoate were investigated at 60” by the same kinetic studies. Algal extracts synergistically enhance the antioxidant effect of vitamin E when the latter is present at a level higher than 1 x 10e4 M. On mechanistic grounds, the understanding of this synergism is outlined. Seaweed extracts should find applications in the cosmetic and food industries without any further purification.
INTRODUCTION
The natural occurrence of substituted phenols and polyphenols in seaweeds has been known about for a long time [l-7]. Although this class of compounds includes effective antioxidants [4,8-l 11, very little is known about antioxygenic activities of seaweed extracts [4, 12-171. Out of 21 species of marine algae which were examined by Fujimoto et al. [13] in 1980, about 60% ofthem exhibited antioxidant activity to some extent. The active principles identified were either phospholipids (Porphyra tenera [ 123 and Eisenia bicyclis [ 131) or substituted phenols and o-diphenols (Polysiphonia urceolata [4, 141). The research workers failed to determine the chemical structures of the antioxygenic components of Undaria pinnatijda but found synergistic effects between several fractions [13]. In the present study, seven species of marine algae collected on French coasts and used as food were examined for antioxidant activity. In order to attempt in a preliminary way to select active species, peroxide formation in sunflower oil with added extracts was measured during storage experiments at 75”. As a result, two species, Laminaria digitata and Himanthalia elongata, which showed the strongest effect on extending the induction periods of the substrate were chosen for further investigation in a model system. Oxidation of methyl iinoleate in heptanol, initiated with 2,2’-azo-bis-isobutyronitrile (AIBN), was carried out in the presence of both algal extracts, BHT or vitamin E respectively. The inhibiting effect of each compound or extract was characterized by the induction period and effectiveness. Experiments were also performed with mixtures of seaweed extracts and vitamin E in order to check for synergistic action between these products. Methyl
linoleate, sunflower oil and a mixture of ethyl eicosapentaenoate and docosahexaenoate were then tested as substrates.
RESULTS AND DISCUSSION
Seven species of seaweeds were used in the first screening test: the brown algae (3), Himanthalia elongata, Laminaria digitata and Undaria pinnatifda; the green algae (2), Ulva lactuca and Enteromorpha; and the red algae(2), Porphyra and Rhodymenia palmata. Yields of extracts are given in Table 1. The extracts of H. elongata, Enteromorpha, U. pinnatijda and L. digitata were very viscous, owing to their high lipid content. The results of the storage experiments at 75” are shown in Fig. 1 and the antioxidant activities of the extracts and BHT at peroxide values of 20, 70 and 120 meq kg-’ compared in Table 1. All extracts added at a 1% level improved the stability of the sunflower oil and extracts of L. digitata and H. elongata were the most effective at extending the induction periods. The protection factor (FP2,,) reached 4.2 and 4.4 respectively for L. digitata and H. elongata, while it only reached 2 for BHT added at a 0.05% level. Therefore, it seems that effective antioxidants could be isolated from these two seaweeds. The rates of oxidation of methyl linoleate in heptanol at 60”, initiated by AIBN, in the presence of L. digitata or H. elongata are shown in Figs 2 and 3 respectively. Some representative data for these oxidations are given in Table 2. At this temperature, the autoxidation of methyl linoleate in homogeneous solution, initiated by AIBN, is a free radical chain process which can be represented by the
3760
B. LE TUTOUR
Table
1. Relative antioxidant
activities
of seaweeds extracts
and BHT in sunflower
oil at 75”
Algae
Yield (% )*
Added(%)?
FP,,,:
FP,,
FP,,,
Himanthalia elongata Enteromorphu Undaria pinnat$du Uica iuctuca Lam. digitata (Kombu) Porph,yra (Nori) Rhodymeniu palmarcr (Dulce) BHT
7.4 3.9 3 2 1.8 0.8 0.7
1 1 I 1 1
4.2 1.7 1.4 1.8 4.4 1.X 0.9 2
2.1 I.5 1.2 1.9 2.2 2
I.6
0.45 0.24 0.05
1 1.5 ______
1.3 1.1 1.5 1.9 1.5 1.3 1.2
*Yield of extract from alga: % dry weight. tpercentage of sunflower oil (w;w). SProtection Factor: PF,=- T,/7,, T,, and T; are the times for peroxide value to reach n(meq kg- r) in presence and absence of antioxidant. rj’, = 40, T;‘, = 140, T,~20= 220 hr.
Tame (mlnl
120 ‘P 100 1 3 ^ ?!
80 60
Reference
0
0 BHT 0.05% u p,nnat/fida. I% + R polmoto 0 24% x Enteromorpha I% n U Ioctuca I% Pwphyro0 45 % - H elongoto I% L dig&to I%
I
l
l
Time (hr)
Fig. 1. Effects of algal extracts or BHT on peroxide formation in sunflower oil at 75 [concentrations in O/Osubstrate (w/w)].
Fig. 3. Inhibition of oxidation of methyl linoleate (RH) at 60” in heptanol by H. rlongata (HE) or vitamin E (TH). (RH): 0.5 M; (AI RN): 0.0063 M.
Time (mlnl 20
40
60
80
120
RH RH RH RH RH RH
AO,‘+RH-+AO,H+R’
(1)
A’+RH-AH+R’
(1’) (2)(-2)
RO;+RHARO,H+R.
Fig. 2. Inhibition of oxidation of methyl linoleate (RH) at 60 in heptanol by L. digitata (LD) or vitamin E (TH). (RH): 0.5 M; (AIBN): 0.0063 M.
A’+ 0, -
R’-
2RO;
-’
RO;+InH&K&H i _
following scheme [8, 11, 18-221: A-N=N-AL2eA’+(l
R‘+R’ RO;+
-e)A, AO,’
+ N,
(3)
inactive products
+In’
(4) (9 (6)
(7)(-7)
(i) R’+InH&RH+In. (x)
k
ii
@I(-@
Algal extracts as antioxidants RO,‘+In’--+ R’+In’
-
In’+In’
-
chain initiation e and hence the rate of initiation Ri. Under conditions employed in heptanol, the computed efficiency of chain initiation by AIBN was e = 0.45 at 60”. The kinetic chain lengths during inhibition period vinh and after inhibition period v were given by eqns VI and VII:
(9)
inactive
(10)
products
(11)
In,‘+In,H-----+In,
H+In,‘.
(12)
R,,,=k,
(2 k6)- 1’2R;‘* [RH]
v =
(I)
where Ri is the rate of chain initiation, k, and k, are the rate constants for the key-reactions (3) and (6) (propagation and termination respectively). Upon introduction of InH, the inhibited rate of ouidation Rinh given by eqn II, provided the key-reactions are steps (3), (7) and (9) [ll, 221:
R. =k,Ri CRHI
(11)
Inh f k, [InH] wherefis
the stoichiometric 1 fcCInH,,
coefficient of inhibition flnh o Ri dt* s
[8]:
(III)
exp (-k,
. t)
CBHTI,
=e[l-exp(-k,.tinh)]_
WI)
L g-l see-’
for vitamin
E
f k, = 37 250
L g-’ set-’
for BHT
fk7=
440
L g-l set-’
for L. digitutu
300
L g-’ set-’
for H. elongutu.
Another characteristic of the antioxidant effect is the length of inhibition. Increasing the concentration of seaweed extracts extended the induction period. Furthermore, algal extracts added at a 3% level still inhibited the rate of oxidation of methyl linoleate after the induction period, suggesting that all the effective principles were not consumed at this time. Usually when the induction period is over, oxidation proceeds at the same rate as an uninhibited oxidation (i.e. R, = Rref. as for TH curve in Figs 2 and 3). When seaweed extracts amounts reached 3% of the substrate, values of the ratio RJR,,, were 0.6
where [AIBN], is the initial concentration of the azo initiator. Assuming f= 2 for the chain-breaking antioxidant BHT [l&22,24], eqns III and IV give eqn V: (V)
CAIBNI,
R, JR,
fk,=182000
fk,=
(IV)
WI)
where R, is the rate of oxidation after the induction period. Laminaria digituta and H. elongutu extracts clearly inhibited oxidation of methyl linoleate when they were added at a level equal to or higher than 1% of the substrate. However, these crude extracts did not suppress the rate of oxygen uptake at I, as did vitamin E. The effectiveness of the inhibition was calculated as the ratio of Rinh to the uninhibited rate of oxidation Rref of the reference run. A plot of this ratio as a function of [InH],/[RH], for algal extracts, vitamin E and BHT is shown in Fig. 4. Concentration units are g I-‘. Crude extracts of L. digitutu and H. elonguta were much less effective than either vitamin E or BHT. According to k,= 189 M-‘see-’ at 60” for methyl linoleate [26, 271, eqn II gives:
The induction period tinh was determined graphically on kinetic curves as described by Tsepalov et al. [23], and the rate of chain initiation Ri was determined by the conventional inhibitor method developed by Boozer et al. [24, and also 18, 223: Ri = 2e k, [AIBN],
= &h/R,
‘inh
In this scheme A - N=N - A is the azo initiator AIBN, RH is the organic substrate and InH is an inhibitor. In the absence of an antioxidant, the rate of oxidation Rrel is given by eqn I [8, 111: dCO,l -p= dt
3761
According to k, = 1.58 x 1015 exp (- 308OO/RT) set- ’ for AIBN [22,25], a plot of the ratio [BHT]J[AIBN],as a function of [ 1 -exp (-k, tinh)] yields the efficiency of
Table 2. Effects of algal extracts on the initiated oxidation of methyl linoleate RH in heptanol at 60
Algal
CRHI,
CAIBNI, x lo3
species
(M)
(W
L. digitata
0.50 0.51 0.51 0.53 0.50 0.51 0.50 0.52 0.51 0.50
6.3 6.3 6.4 6.4 6.3 6.4 6.3 6.4 6.4 6.3
H. elongata
[extracts], (% RH) (gl-‘) 0 0.01 0.10
0.50 1 3.10 0.10
0.50 1.00 3.00
(0.015) (0.16) (0.78) (1.50)
:,
180 6:
5.4 5.5
840
5.5 5.5 5.5
(4.70)
3060
5.5
(0.15) (0.76) (1.54) (4.48)
0 0 390 1500
5.5 5.5 5.5 5.5
*The uninhibited oxidation rate Rrsr as described in text. PHYTO 29:12-F
Ri X 10s (M set-‘)
RP
Rin,,
(M set-‘)
(M set-‘)
x 106
4.9* 4.7 5.3 5.0 4.4 3.1 5.0 4.9 4.2 2.5
x 106
4.0 4.4 2.4 1.5
3.4 1.6
v
Vi”h
90 86 95 90 80 57 92 88 77 47
72 80 44 28 62 29
B. LETUTOUR
3162
Time (hr)
+ TH (v1t.E) 0 L. d/g/tot0 0 H. eh-tgata I 001
0
(HE): 0 (HE) : 0 01 I (HE) 0.031 I 002
I 003
Fig. 4. Initiated oxidation of methyl linoleate (RH) at 60”. Effect of the ratio (InH)JRH), on the effectiveness of antioxidants (InH). (RH): 0.5 M; (AIBN): 0.0063 M; (P,J,: 500 torr; solv: heptanol.
Fig. 6. Initiated oxidation of a commercial sunflower oil (OIL) at 60” in the presence of H. elonyata extract (HE). (OIL): 270 g I ‘; (AIBN): 0.0063 M; solvent: heptanol.
0
P
50 I
Time (mm) 100 I
150 I
200 I
Time (hr) 4
0
(LD): (LDI: (LD): (LD):
a I
12 I
0 0.005 0.010 0029
Fig. 5. Initiated oxidation of a commercial sunflower oil (OIL) at 60” in the presence of L. digitata extract (LD). (OIL): 270 g l- 1; (AIBN): 0.0063 M; solvent: heptanol.
for L. digitata and 0.5 for H. elongata respectively. Thus these two extracts were only ca 0.2% as reactive as vitamin E (ratio of thefk, values) but Figs 2 and 3 clearly show that added at a 3% level, they were as good inhibitors as vitamin E added at 0.06%. So, these crude extracts could be estimated to ensure at least 2% of the antioxidative activity of vitamin E, but obviously, these results did not emphasize the oven test conclusions. Commercial sunflower oil and Activepa 50E were then used as a substrate in the oxygen uptake method. The rates of oxidation of these substrates in heptanol at 60”, initiated by AIBN, in the presence of L. digitata or H. elongata are shown in Figs 5-8. Additions of algal extracts resulted in an extension of the inhibition period. This extension occurred when amount of extract reached 1% of the sunflower oil or 0.5% of the Activepa 50E. The extracts did not affect the effectiveness of vitamin E. The inhibited oxidation curves
Fig. 7. Initiated oxidation of activepa 50 E (ACT) at 60” in the presence of vitamin E (TH) and L. digitatu extracts (LD). (ACT): 2OOglI’; (AIBN): 0.0063 M; (TH): 0.00018 M; solvent: heptano1.
showed a sharp break when the inhibition period was over. Furthermore, the rate of oxidation was lessened after tinh when extracts were added at a 3% level. The values of the ratio RJR,,, were 0.6 for L. digitata and 0.5 for H. elongate added to sunflower oil and 0.7 for both extracts added to Activepa 50E. Synergistic effects of algal extracts together with vitamin E on the preservation of methyl linoleate were investigated by kinetic studies at 60” in heptanol. At a defined initial vitamin E concentration of 1 x 10m4 M, the addition of algal extracts extended the induction period to the sum of the induction periods observed when either vitamin E or the algal extracts were used alone. The results are summarized in Table 3. During the whole induction period, the effectiveness of the combined antioxidants was vitamin E effectiveness (Figs 9 and 10). For an added extract amount of 3% of the substrate, the rate of oxidation R, did not return to its uninhibited value Rref after vitamin E was consumed. Values of the ratio R,/RrCf were still found to be 0.6 for L. digitata and
Algal extracts as antioxidants
3763
Table 3. Initiated oxidation of methyl linoleate RH at 60” in heptanol, effects of the addition of algal extracts together with vitamin E (TH) on the induction periods
Algal species
x lo4 (M)
CBXTI:
tTnYf
tZY$
TH.EXT tinll §
t;:. ExT -(t;t+t:y)
(% RH)
(s)
(s)
(s)
(s)
None
1 2 4 1 1 1 1 2 4 1 1 1 1 2 4
0 0 0 0.5 1 3 1 1 1 0.5 1 3 1 1 1
3120 5300 9970 3120 3120 3120 3120 5300 9970 3120 3120 3120 3120 5300 9970
60 840 3060 840 840 840 0 390 1500 390 390 390
3660 4140 6480 4140 7620 14820 3420 4020 4440 4020 1320 14700
480 180 300 180 148011 401011 300 510 -180 510 163011
C-W,
L. digitata
H. elongata
434011
[RH],=0.5 M; [AIBN], =0.0063 M, initial oxygen pressure: 500 torr. *Initial concentration of algal extracts. tbrduction period with vitamin E alone. $ Induction period with algal extract alone. 0 Antioxidants (vitamin E and algal extracts) combined. IlSignificant synergistic effect.
Time 0
50
(min) loo
Time 150
ZCXJ
r 3
(min)
‘i X’
” ”
-5
I
E 1: 0
-10
+ (HE): x (HE): 0 (HE):
0.005 0.011 0.029
(ACT) (ACT) (ACT)
Fig. 8. Initiated oxidation of activepa 50 E (ACT) at 60” in the presence of vitamin E (TH) and H. elongata extract (HE). (ACT):
A (LD): x (LD): 0 (LD):
0.005 0.01 0.03
(RH) (RHJ (RH)
200 g I- ‘; (AIBN): 0.0063 M; (TH): 0.00018M, solvent: heptanol.
Fig. 9. Inhibition of oxidation of methyl linoleate (RH) at 60” in heptanol by a mixture of vitamin E (TH) and L. digitata extract (LD). (RH): 0.5 M; (AIBN): 0.0063 M; (TH): 0.0001 M; solvent: heptanol.
0.5 for H. elongata. Thus, the vitamin E content did not seem to affect the ratio RJR,,,. Experiments were also run at a defined initial amount of algal extracts in the reaction mixture (1% of the substrate), while the initial concentration of vitamin E varied from 1 x 10m4 M to 4 x 10e4 M. Algal extracts synergistically enhanced the antioxidant effect of vitamin E when the latter was present at a concentration higher than 1 x 10m4 M (Table 3). The difference between the induction period observed in the presence of combined antioxidants and the sum of the induction periods of each antioxidant used alone markedly increased as the initial
concentration of vitamin E was increased. These results account for the very effective antioxidant activity exhibited by these two extracts in the preservation of sunflower oil during storage experiments as the vitamin E concentration in the oil was 9 x low4 M. Vitamin E is known to be a highly efficient chainbreaking antioxidant in vitro, especially at low concentrations [9,22,28,29], whereas it can exhibit a prooxidant effect when used at high concentrations [30,31]. This takes place when the a-T’ radicals formed from TH participate in chain propagation via steps (- 7) and (- 8), i.e. when the amount of a-T’ radicals in the reaction
B. LE TUTOUR
3764
Table
4. Fatty
LD HE
acid composition of L. digitata (LD) and H. elongata extracts, expressed as percentage of total fatty acids.
1
16:0
18:
19.1 17.1
15.6 8.2
*Percentage of extract internal standard.
18:2
18:4’“_3’
20 : 4’” --6,
20:
4.9 5.2
10.2 (2)* 11.1(1.7)*
24.3 (4.9)* 33.4 (5.2)$
9.6 (1.8)* 3.9 (0.6)*
(w/w) determined
Time (min)
‘t
(HE): (HE): (HE). (HE) :
0
0.005 0.01 003
IRH)‘, \ (RH) * (RH)
(HE)
’
Fig. 10. Inhibition of oxidation of methyl linoleate (RH) at 60 by a mixture of vitamin E (TH) and H. elongata extract (HE). (RH): 0.5 M; (AIBN): 0.0063 M; (TH): 0.0001 M; solvent: heptanol.
becomes high enough. As described elsewhere [l 1,221, it is safe to think that the effective principles of algal extracts react with sr-T’ radicals via steps (12) to regenerate vitamin E. Thereby, the rates of key-reactions (- 7) and (- 8) are lessened and seaweed extracts also become involved in the chain termination process. Vitamin C, phenols, amines and phospholipids are known to synergistically enhance the antioxidative activity of vitamin E [l 1,22,28, 29, 32, 331. As mentioned in the experimental section, vitamin C amounts were very low in the extracts. We plan to extend this study to the identification of the effective components of L. digitata and H. elongata with the aim of clarifying the mechanism of this synergism. Taking into account their polyunsaturated fatty acids composition (Table 4) these crude extracts would find applications in both the cosmetic and the food industry without any further purification. mixture
EXPERIMENTAL
Materinls. All chemicals were analytical grade reagents unless mentioned. They were supplied by Fluka and used as received. Dried algae (Hz0 < 10%) packed by Nature Algues were kindly donated by the Centre d’Etudes et de Valorisation des Algues CEVA, 22610 Pleubian, France. The fatty acid composition of the commercial sunflower oil was as follows: palmitate 6%; stearate 4%; oleate 20%; linoleate 70%. Its initial peroxide value was 2 meq kg-i and its tocopher01s content [34]: a-: 415 meq kg- i, p-: 16 meq kg- *.
by using methyl
ytr- 31
nonadecanoate
as
Activepa 50E was purchased from Jomar (Bergen, Norway). It was mostly a mixture of ethyl esters of the following fatty acids: 16:l 1.9%; 18:l 6.1%; 18:4 (n-3) 2.2%; 2O:l 6%; 20:4 (n-6) 2.7%; 20:5 (n-3) 33%; 2215 5.1%; 2216 (n-31 23%. Its tocopherol content was Z- 385 mg kg- ’ [34]. Autoxidation procedure. Oxidations were carried out at 60 under 500 torr 0, in an automatic recording gas absorption apparatus using a Leybold CI 1000 absolute pressure sensor connected to a Leybold Membranovac control unit supplying a chart recorder output voltage of 10 mV torr- *. The reaction mixture was prepared by dissolving an appropriate amount of thermal initiator in heptanol followed by the addition of methyl linoleate and antioxidants. Total liquid volume in all runs was maintained at 5 ml. The reaction vessel, of total vol. ca 55 ml, was connected to the pressure transducer and agitation was provided by a magnetic stirrer during the course of the experiment. The continuously recorded pressure decreases were converted to rates of oxidation of the substrate in M sec. ’ units. GC. In the early stages of this work, it was ascertained that the rates of substrate disappearance and oxygen uptake agreed with each other. The consumption of methyl linoleate was measured by GC during experiments in the presence of algal extracts. Methyl heptadecanoate was used as int. standard. In a typical run, 0.25 ml of the reaction mixture was cleaned up on a SepPack silica cartridge using 25 ml of heptane as a mobile phase. This soln (1 ~1) was directly injected into the gas chromatograph fitted with a FID and a 2 m x 1’8 in. stainless steel column packed with 10% DEGS on 100-120 mesh chromosorb WAWDMCS. Column temp.: 150’ for 1 min, prog. 4’ min- ’ to 180‘. Run time was 17 i 2 min. Storage test. Ahquots of each seaweed extract or BHT were added as a methanohc soln to 100 g of sunflower oil in a beaker. Initial methanol content was 2 ml per 100 g oil. Samples were stored in the dark at 75”. Peroxide values were determined by the AOCS method [35]. Preparation qf algal extracts. Samples were extracted ( x 2) from dried algae (100 g) with 500 ml of CHCI,~-MeOH (2: 1) [4, 13,141 under reflux 5 hr. The pooled fractions were evapd to dryness in ~‘acuo. The dry residues were then transferred to a separatory funnel with EtOAC and washed with H,O. The organic layer was recovered. dried over Na,SO,, filtered, evapd to dryness under red. pres. Extracts were stored under N, atmosphere in the dark at -4’. The fatty acid composition of L. digitata and H. elongata extracts are summarized in Table 4. The vitamin E and C contents were: L. digitatu extract: E 9.2 pg g _ ’ : C 27 pg g- I ; H. elongata extract: E 90 pgg i ; C 33 pgg- i. AcknowledgementsThis study was in part supported by grants from the Institut Francais de Recherche et d’Exploitation de la Mer IFREMER, Nantes, France. The author thanks Drs Patrick Durand and Jean-Paul Gouigou (Ifremer, Nantes) for the fatty acid analyses of algal extracts and Activepa 50E, and Dr Gerard
Algal extracts as antioxidants Briantais (Laboratoire Interregional, Rennes, France) for the tocopherol determinations in Activepa 50E and sunflower oil. Vitamins E and C were determined in algal extracts by the Institut Pasteur, Lyon, France.
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France. 23. Tsepalov, V. F., Kharitonova, A. A., Gladyshev, G. P. and Emanuel’, N. M. (1977) Kinet. Catal. 18, 1034. 24. Boozer, C. E., Hammond, G. S., Hamilton, C. E. and Sen, J. N. (1955) J. Am. Chem. Sot. 77, 3233. 25. Van Hook, J. P. and Tobolsky, A. V. (1958) J. Am. Chem. Sot. 80, 779. 26. Howard, J. A. and Ingold, K. U. (1967) Can. J. Chem. 45,793. 27. Korseck, S., Chenier, J. H. B., Howard, J. A. and Ingold, K. U. (1972) Can. J. Chem. 50,2285. 28. Niki, E., Saito, T., Kawakami, A. and Kamiya, Y. (1984) J. Biol. Chem. 259,4177.
29, Aoyama, M., Maruyama, T., Kanematsu, H., Niiya, I., Tsukamoto, M., Tokairin, S. and Matsumoto, T. (1989) Yukagaku 38, 72. 30. Cillard, J. and Cillard, P. (1980) J. Am. Oil Chem. Sot. 57, 255. 31. Koskas, J. P., Cillard, J. and Cillard, P. (1984) J. Am. Oil
Chem. Sot. 61, 1466. 32. Barclay, L. R. C., Locke, S. J. and Macneil, J. M. (1983) Can. J. Chem. 61, 1288. 33. Niki, E. (1988) Vitamin 62, 601. 34. Speek, A. J., Schrijver, J. and Schreurs, W. H. P. (1985) J. Food Sci. 50, 121. 35. AOCS (1973) Ojicial and Tentative Methods, 3rd Edn. American Oil Chemists’ Society, Chicago.
ANTIOXIDATIVE
0
ACTIVITIES OF ALGAL EXTRACTS, EFFECT WITH VITAMIN E BERNARD
003 l-9422/90 $3.00 + 0.00 1990 Pergamon Press plc
SYNERGISTIC
LE TUTOUR
Laboratoire de GEnie des Pro&d&s, IUT de Saint-Nazaire, Le Heinlex, BP 420, 44606 Saint-Nazaire Cedex, France (Received 25 January 1990)
Key Word Index-Algae; docosahexaenoate;
antioxidant; vitamin E; BHT; tocopherol; ethyl eicosapentaenoate.
methyl linoleate; sunflower oil; ethyl
Abstract-The antioxidant effect of seven species of marine algae on the preservation of sunflower oil were investigated by storage experiments at 75”. Laminaria digitata (Kombu) and Himanthalia elongata were the most effective in extending the induction period. Extracts of these two seaweeds were then compared with vitamin E and BHT as regards to their antioxidative activities, by kinetic studies in a model system. The thermal initiated oxidation of methyl linoleate was performed at 60” in heptanol, with or without antioxidants. When they reached 1% of the substrate, seaweed extracts exhibited antioxidant activities by extending the induction period but they were not actually effective. Effects of the addition of algal extracts together with vitamin Eon the preservation of methyl linoleate, sunflower oil or mixed ethyl eicosapentaenoate and docosahexaenoate were investigated at 60” by the same kinetic studies. Algal extracts synergistically enhance the antioxidant effect of vitamin E when the latter is present at a level higher than 1 x 10e4 M. On mechanistic grounds, the understanding of this synergism is outlined. Seaweed extracts should find applications in the cosmetic and food industries without any further purification.
INTRODUCTION
The natural occurrence of substituted phenols and polyphenols in seaweeds has been known about for a long time [l-7]. Although this class of compounds includes effective antioxidants [4,8-l 11, very little is known about antioxygenic activities of seaweed extracts [4, 12-171. Out of 21 species of marine algae which were examined by Fujimoto et al. [13] in 1980, about 60% ofthem exhibited antioxidant activity to some extent. The active principles identified were either phospholipids (Porphyra tenera [ 123 and Eisenia bicyclis [ 131) or substituted phenols and o-diphenols (Polysiphonia urceolata [4, 141). The research workers failed to determine the chemical structures of the antioxygenic components of Undaria pinnatijda but found synergistic effects between several fractions [13]. In the present study, seven species of marine algae collected on French coasts and used as food were examined for antioxidant activity. In order to attempt in a preliminary way to select active species, peroxide formation in sunflower oil with added extracts was measured during storage experiments at 75”. As a result, two species, Laminaria digitata and Himanthalia elongata, which showed the strongest effect on extending the induction periods of the substrate were chosen for further investigation in a model system. Oxidation of methyl iinoleate in heptanol, initiated with 2,2’-azo-bis-isobutyronitrile (AIBN), was carried out in the presence of both algal extracts, BHT or vitamin E respectively. The inhibiting effect of each compound or extract was characterized by the induction period and effectiveness. Experiments were also performed with mixtures of seaweed extracts and vitamin E in order to check for synergistic action between these products. Methyl
linoleate, sunflower oil and a mixture of ethyl eicosapentaenoate and docosahexaenoate were then tested as substrates.
RESULTS AND DISCUSSION
Seven species of seaweeds were used in the first screening test: the brown algae (3), Himanthalia elongata, Laminaria digitata and Undaria pinnatifda; the green algae (2), Ulva lactuca and Enteromorpha; and the red algae(2), Porphyra and Rhodymenia palmata. Yields of extracts are given in Table 1. The extracts of H. elongata, Enteromorpha, U. pinnatijda and L. digitata were very viscous, owing to their high lipid content. The results of the storage experiments at 75” are shown in Fig. 1 and the antioxidant activities of the extracts and BHT at peroxide values of 20, 70 and 120 meq kg-’ compared in Table 1. All extracts added at a 1% level improved the stability of the sunflower oil and extracts of L. digitata and H. elongata were the most effective at extending the induction periods. The protection factor (FP2,,) reached 4.2 and 4.4 respectively for L. digitata and H. elongata, while it only reached 2 for BHT added at a 0.05% level. Therefore, it seems that effective antioxidants could be isolated from these two seaweeds. The rates of oxidation of methyl linoleate in heptanol at 60”, initiated by AIBN, in the presence of L. digitata or H. elongata are shown in Figs 2 and 3 respectively. Some representative data for these oxidations are given in Table 2. At this temperature, the autoxidation of methyl linoleate in homogeneous solution, initiated by AIBN, is a free radical chain process which can be represented by the
3760
B. LE TUTOUR
Table
1. Relative antioxidant
activities
of seaweeds extracts
and BHT in sunflower
oil at 75”
Algae
Yield (% )*
Added(%)?
FP,,,:
FP,,
FP,,,
Himanthalia elongata Enteromorphu Undaria pinnat$du Uica iuctuca Lam. digitata (Kombu) Porph,yra (Nori) Rhodymeniu palmarcr (Dulce) BHT
7.4 3.9 3 2 1.8 0.8 0.7
1 1 I 1 1
4.2 1.7 1.4 1.8 4.4 1.X 0.9 2
2.1 I.5 1.2 1.9 2.2 2
I.6
0.45 0.24 0.05
1 1.5 ______
1.3 1.1 1.5 1.9 1.5 1.3 1.2
*Yield of extract from alga: % dry weight. tpercentage of sunflower oil (w;w). SProtection Factor: PF,=- T,/7,, T,, and T; are the times for peroxide value to reach n(meq kg- r) in presence and absence of antioxidant. rj’, = 40, T;‘, = 140, T,~20= 220 hr.
Tame (mlnl
120 ‘P 100 1 3 ^ ?!
80 60
Reference
0
0 BHT 0.05% u p,nnat/fida. I% + R polmoto 0 24% x Enteromorpha I% n U Ioctuca I% Pwphyro0 45 % - H elongoto I% L dig&to I%
I
l
l
Time (hr)
Fig. 1. Effects of algal extracts or BHT on peroxide formation in sunflower oil at 75 [concentrations in O/Osubstrate (w/w)].
Fig. 3. Inhibition of oxidation of methyl linoleate (RH) at 60” in heptanol by H. rlongata (HE) or vitamin E (TH). (RH): 0.5 M; (AI RN): 0.0063 M.
Time (mlnl 20
40
60
80
120
RH RH RH RH RH RH
AO,‘+RH-+AO,H+R’
(1)
A’+RH-AH+R’
(1’) (2)(-2)
RO;+RHARO,H+R.
Fig. 2. Inhibition of oxidation of methyl linoleate (RH) at 60 in heptanol by L. digitata (LD) or vitamin E (TH). (RH): 0.5 M; (AIBN): 0.0063 M.
A’+ 0, -
R’-
2RO;
-’
RO;+InH&K&H i _
following scheme [8, 11, 18-221: A-N=N-AL2eA’+(l
R‘+R’ RO;+
-e)A, AO,’
+ N,
(3)
inactive products
+In’
(4) (9 (6)
(7)(-7)
(i) R’+InH&RH+In. (x)
k
ii
@I(-@
Algal extracts as antioxidants RO,‘+In’--+ R’+In’
-
In’+In’
-
chain initiation e and hence the rate of initiation Ri. Under conditions employed in heptanol, the computed efficiency of chain initiation by AIBN was e = 0.45 at 60”. The kinetic chain lengths during inhibition period vinh and after inhibition period v were given by eqns VI and VII:
(9)
inactive
(10)
products
(11)
In,‘+In,H-----+In,
H+In,‘.
(12)
R,,,=k,
(2 k6)- 1’2R;‘* [RH]
v =
(I)
where Ri is the rate of chain initiation, k, and k, are the rate constants for the key-reactions (3) and (6) (propagation and termination respectively). Upon introduction of InH, the inhibited rate of ouidation Rinh given by eqn II, provided the key-reactions are steps (3), (7) and (9) [ll, 221:
R. =k,Ri CRHI
(11)
Inh f k, [InH] wherefis
the stoichiometric 1 fcCInH,,
coefficient of inhibition flnh o Ri dt* s
[8]:
(III)
exp (-k,
. t)
CBHTI,
=e[l-exp(-k,.tinh)]_
WI)
L g-l see-’
for vitamin
E
f k, = 37 250
L g-’ set-’
for BHT
fk7=
440
L g-l set-’
for L. digitutu
300
L g-’ set-’
for H. elongutu.
Another characteristic of the antioxidant effect is the length of inhibition. Increasing the concentration of seaweed extracts extended the induction period. Furthermore, algal extracts added at a 3% level still inhibited the rate of oxidation of methyl linoleate after the induction period, suggesting that all the effective principles were not consumed at this time. Usually when the induction period is over, oxidation proceeds at the same rate as an uninhibited oxidation (i.e. R, = Rref. as for TH curve in Figs 2 and 3). When seaweed extracts amounts reached 3% of the substrate, values of the ratio RJR,,, were 0.6
where [AIBN], is the initial concentration of the azo initiator. Assuming f= 2 for the chain-breaking antioxidant BHT [l&22,24], eqns III and IV give eqn V: (V)
CAIBNI,
R, JR,
fk,=182000
fk,=
(IV)
WI)
where R, is the rate of oxidation after the induction period. Laminaria digituta and H. elongutu extracts clearly inhibited oxidation of methyl linoleate when they were added at a level equal to or higher than 1% of the substrate. However, these crude extracts did not suppress the rate of oxygen uptake at I, as did vitamin E. The effectiveness of the inhibition was calculated as the ratio of Rinh to the uninhibited rate of oxidation Rref of the reference run. A plot of this ratio as a function of [InH],/[RH], for algal extracts, vitamin E and BHT is shown in Fig. 4. Concentration units are g I-‘. Crude extracts of L. digitutu and H. elonguta were much less effective than either vitamin E or BHT. According to k,= 189 M-‘see-’ at 60” for methyl linoleate [26, 271, eqn II gives:
The induction period tinh was determined graphically on kinetic curves as described by Tsepalov et al. [23], and the rate of chain initiation Ri was determined by the conventional inhibitor method developed by Boozer et al. [24, and also 18, 223: Ri = 2e k, [AIBN],
= &h/R,
‘inh
In this scheme A - N=N - A is the azo initiator AIBN, RH is the organic substrate and InH is an inhibitor. In the absence of an antioxidant, the rate of oxidation Rrel is given by eqn I [8, 111: dCO,l -p= dt
3761
According to k, = 1.58 x 1015 exp (- 308OO/RT) set- ’ for AIBN [22,25], a plot of the ratio [BHT]J[AIBN],as a function of [ 1 -exp (-k, tinh)] yields the efficiency of
Table 2. Effects of algal extracts on the initiated oxidation of methyl linoleate RH in heptanol at 60
Algal
CRHI,
CAIBNI, x lo3
species
(M)
(W
L. digitata
0.50 0.51 0.51 0.53 0.50 0.51 0.50 0.52 0.51 0.50
6.3 6.3 6.4 6.4 6.3 6.4 6.3 6.4 6.4 6.3
H. elongata
[extracts], (% RH) (gl-‘) 0 0.01 0.10
0.50 1 3.10 0.10
0.50 1.00 3.00
(0.015) (0.16) (0.78) (1.50)
:,
180 6:
5.4 5.5
840
5.5 5.5 5.5
(4.70)
3060
5.5
(0.15) (0.76) (1.54) (4.48)
0 0 390 1500
5.5 5.5 5.5 5.5
*The uninhibited oxidation rate Rrsr as described in text. PHYTO 29:12-F
Ri X 10s (M set-‘)
RP
Rin,,
(M set-‘)
(M set-‘)
x 106
4.9* 4.7 5.3 5.0 4.4 3.1 5.0 4.9 4.2 2.5
x 106
4.0 4.4 2.4 1.5
3.4 1.6
v
Vi”h
90 86 95 90 80 57 92 88 77 47
72 80 44 28 62 29
B. LETUTOUR
3162
Time (hr)
+ TH (v1t.E) 0 L. d/g/tot0 0 H. eh-tgata I 001
0
(HE): 0 (HE) : 0 01 I (HE) 0.031 I 002
I 003
Fig. 4. Initiated oxidation of methyl linoleate (RH) at 60”. Effect of the ratio (InH)JRH), on the effectiveness of antioxidants (InH). (RH): 0.5 M; (AIBN): 0.0063 M; (P,J,: 500 torr; solv: heptanol.
Fig. 6. Initiated oxidation of a commercial sunflower oil (OIL) at 60” in the presence of H. elonyata extract (HE). (OIL): 270 g I ‘; (AIBN): 0.0063 M; solvent: heptanol.
0
P
50 I
Time (mm) 100 I
150 I
200 I
Time (hr) 4
0
(LD): (LDI: (LD): (LD):
a I
12 I
0 0.005 0.010 0029
Fig. 5. Initiated oxidation of a commercial sunflower oil (OIL) at 60” in the presence of L. digitata extract (LD). (OIL): 270 g l- 1; (AIBN): 0.0063 M; solvent: heptanol.
for L. digitata and 0.5 for H. elongata respectively. Thus these two extracts were only ca 0.2% as reactive as vitamin E (ratio of thefk, values) but Figs 2 and 3 clearly show that added at a 3% level, they were as good inhibitors as vitamin E added at 0.06%. So, these crude extracts could be estimated to ensure at least 2% of the antioxidative activity of vitamin E, but obviously, these results did not emphasize the oven test conclusions. Commercial sunflower oil and Activepa 50E were then used as a substrate in the oxygen uptake method. The rates of oxidation of these substrates in heptanol at 60”, initiated by AIBN, in the presence of L. digitata or H. elongata are shown in Figs 5-8. Additions of algal extracts resulted in an extension of the inhibition period. This extension occurred when amount of extract reached 1% of the sunflower oil or 0.5% of the Activepa 50E. The extracts did not affect the effectiveness of vitamin E. The inhibited oxidation curves
Fig. 7. Initiated oxidation of activepa 50 E (ACT) at 60” in the presence of vitamin E (TH) and L. digitatu extracts (LD). (ACT): 2OOglI’; (AIBN): 0.0063 M; (TH): 0.00018 M; solvent: heptano1.
showed a sharp break when the inhibition period was over. Furthermore, the rate of oxidation was lessened after tinh when extracts were added at a 3% level. The values of the ratio RJR,,, were 0.6 for L. digitata and 0.5 for H. elongate added to sunflower oil and 0.7 for both extracts added to Activepa 50E. Synergistic effects of algal extracts together with vitamin E on the preservation of methyl linoleate were investigated by kinetic studies at 60” in heptanol. At a defined initial vitamin E concentration of 1 x 10m4 M, the addition of algal extracts extended the induction period to the sum of the induction periods observed when either vitamin E or the algal extracts were used alone. The results are summarized in Table 3. During the whole induction period, the effectiveness of the combined antioxidants was vitamin E effectiveness (Figs 9 and 10). For an added extract amount of 3% of the substrate, the rate of oxidation R, did not return to its uninhibited value Rref after vitamin E was consumed. Values of the ratio R,/RrCf were still found to be 0.6 for L. digitata and
Algal extracts as antioxidants
3763
Table 3. Initiated oxidation of methyl linoleate RH at 60” in heptanol, effects of the addition of algal extracts together with vitamin E (TH) on the induction periods
Algal species
x lo4 (M)
CBXTI:
tTnYf
tZY$
TH.EXT tinll §
t;:. ExT -(t;t+t:y)
(% RH)
(s)
(s)
(s)
(s)
None
1 2 4 1 1 1 1 2 4 1 1 1 1 2 4
0 0 0 0.5 1 3 1 1 1 0.5 1 3 1 1 1
3120 5300 9970 3120 3120 3120 3120 5300 9970 3120 3120 3120 3120 5300 9970
60 840 3060 840 840 840 0 390 1500 390 390 390
3660 4140 6480 4140 7620 14820 3420 4020 4440 4020 1320 14700
480 180 300 180 148011 401011 300 510 -180 510 163011
C-W,
L. digitata
H. elongata
434011
[RH],=0.5 M; [AIBN], =0.0063 M, initial oxygen pressure: 500 torr. *Initial concentration of algal extracts. tbrduction period with vitamin E alone. $ Induction period with algal extract alone. 0 Antioxidants (vitamin E and algal extracts) combined. IlSignificant synergistic effect.
Time 0
50
(min) loo
Time 150
ZCXJ
r 3
(min)
‘i X’
” ”
-5
I
E 1: 0
-10
+ (HE): x (HE): 0 (HE):
0.005 0.011 0.029
(ACT) (ACT) (ACT)
Fig. 8. Initiated oxidation of activepa 50 E (ACT) at 60” in the presence of vitamin E (TH) and H. elongata extract (HE). (ACT):
A (LD): x (LD): 0 (LD):
0.005 0.01 0.03
(RH) (RHJ (RH)
200 g I- ‘; (AIBN): 0.0063 M; (TH): 0.00018M, solvent: heptanol.
Fig. 9. Inhibition of oxidation of methyl linoleate (RH) at 60” in heptanol by a mixture of vitamin E (TH) and L. digitata extract (LD). (RH): 0.5 M; (AIBN): 0.0063 M; (TH): 0.0001 M; solvent: heptanol.
0.5 for H. elongata. Thus, the vitamin E content did not seem to affect the ratio RJR,,,. Experiments were also run at a defined initial amount of algal extracts in the reaction mixture (1% of the substrate), while the initial concentration of vitamin E varied from 1 x 10m4 M to 4 x 10e4 M. Algal extracts synergistically enhanced the antioxidant effect of vitamin E when the latter was present at a concentration higher than 1 x 10m4 M (Table 3). The difference between the induction period observed in the presence of combined antioxidants and the sum of the induction periods of each antioxidant used alone markedly increased as the initial
concentration of vitamin E was increased. These results account for the very effective antioxidant activity exhibited by these two extracts in the preservation of sunflower oil during storage experiments as the vitamin E concentration in the oil was 9 x low4 M. Vitamin E is known to be a highly efficient chainbreaking antioxidant in vitro, especially at low concentrations [9,22,28,29], whereas it can exhibit a prooxidant effect when used at high concentrations [30,31]. This takes place when the a-T’ radicals formed from TH participate in chain propagation via steps (- 7) and (- 8), i.e. when the amount of a-T’ radicals in the reaction
B. LE TUTOUR
3764
Table
4. Fatty
LD HE
acid composition of L. digitata (LD) and H. elongata extracts, expressed as percentage of total fatty acids.
1
16:0
18:
19.1 17.1
15.6 8.2
*Percentage of extract internal standard.
18:2
18:4’“_3’
20 : 4’” --6,
20:
4.9 5.2
10.2 (2)* 11.1(1.7)*
24.3 (4.9)* 33.4 (5.2)$
9.6 (1.8)* 3.9 (0.6)*
(w/w) determined
Time (min)
‘t
(HE): (HE): (HE). (HE) :
0
0.005 0.01 003
IRH)‘, \ (RH) * (RH)
(HE)
’
Fig. 10. Inhibition of oxidation of methyl linoleate (RH) at 60 by a mixture of vitamin E (TH) and H. elongata extract (HE). (RH): 0.5 M; (AIBN): 0.0063 M; (TH): 0.0001 M; solvent: heptanol.
becomes high enough. As described elsewhere [l 1,221, it is safe to think that the effective principles of algal extracts react with sr-T’ radicals via steps (12) to regenerate vitamin E. Thereby, the rates of key-reactions (- 7) and (- 8) are lessened and seaweed extracts also become involved in the chain termination process. Vitamin C, phenols, amines and phospholipids are known to synergistically enhance the antioxidative activity of vitamin E [l 1,22,28, 29, 32, 331. As mentioned in the experimental section, vitamin C amounts were very low in the extracts. We plan to extend this study to the identification of the effective components of L. digitata and H. elongata with the aim of clarifying the mechanism of this synergism. Taking into account their polyunsaturated fatty acids composition (Table 4) these crude extracts would find applications in both the cosmetic and the food industry without any further purification. mixture
EXPERIMENTAL
Materinls. All chemicals were analytical grade reagents unless mentioned. They were supplied by Fluka and used as received. Dried algae (Hz0 < 10%) packed by Nature Algues were kindly donated by the Centre d’Etudes et de Valorisation des Algues CEVA, 22610 Pleubian, France. The fatty acid composition of the commercial sunflower oil was as follows: palmitate 6%; stearate 4%; oleate 20%; linoleate 70%. Its initial peroxide value was 2 meq kg-i and its tocopher01s content [34]: a-: 415 meq kg- i, p-: 16 meq kg- *.
by using methyl
ytr- 31
nonadecanoate
as
Activepa 50E was purchased from Jomar (Bergen, Norway). It was mostly a mixture of ethyl esters of the following fatty acids: 16:l 1.9%; 18:l 6.1%; 18:4 (n-3) 2.2%; 2O:l 6%; 20:4 (n-6) 2.7%; 20:5 (n-3) 33%; 2215 5.1%; 2216 (n-31 23%. Its tocopherol content was Z- 385 mg kg- ’ [34]. Autoxidation procedure. Oxidations were carried out at 60 under 500 torr 0, in an automatic recording gas absorption apparatus using a Leybold CI 1000 absolute pressure sensor connected to a Leybold Membranovac control unit supplying a chart recorder output voltage of 10 mV torr- *. The reaction mixture was prepared by dissolving an appropriate amount of thermal initiator in heptanol followed by the addition of methyl linoleate and antioxidants. Total liquid volume in all runs was maintained at 5 ml. The reaction vessel, of total vol. ca 55 ml, was connected to the pressure transducer and agitation was provided by a magnetic stirrer during the course of the experiment. The continuously recorded pressure decreases were converted to rates of oxidation of the substrate in M sec. ’ units. GC. In the early stages of this work, it was ascertained that the rates of substrate disappearance and oxygen uptake agreed with each other. The consumption of methyl linoleate was measured by GC during experiments in the presence of algal extracts. Methyl heptadecanoate was used as int. standard. In a typical run, 0.25 ml of the reaction mixture was cleaned up on a SepPack silica cartridge using 25 ml of heptane as a mobile phase. This soln (1 ~1) was directly injected into the gas chromatograph fitted with a FID and a 2 m x 1’8 in. stainless steel column packed with 10% DEGS on 100-120 mesh chromosorb WAWDMCS. Column temp.: 150’ for 1 min, prog. 4’ min- ’ to 180‘. Run time was 17 i 2 min. Storage test. Ahquots of each seaweed extract or BHT were added as a methanohc soln to 100 g of sunflower oil in a beaker. Initial methanol content was 2 ml per 100 g oil. Samples were stored in the dark at 75”. Peroxide values were determined by the AOCS method [35]. Preparation qf algal extracts. Samples were extracted ( x 2) from dried algae (100 g) with 500 ml of CHCI,~-MeOH (2: 1) [4, 13,141 under reflux 5 hr. The pooled fractions were evapd to dryness in ~‘acuo. The dry residues were then transferred to a separatory funnel with EtOAC and washed with H,O. The organic layer was recovered. dried over Na,SO,, filtered, evapd to dryness under red. pres. Extracts were stored under N, atmosphere in the dark at -4’. The fatty acid composition of L. digitata and H. elongata extracts are summarized in Table 4. The vitamin E and C contents were: L. digitatu extract: E 9.2 pg g _ ’ : C 27 pg g- I ; H. elongata extract: E 90 pgg i ; C 33 pgg- i. AcknowledgementsThis study was in part supported by grants from the Institut Francais de Recherche et d’Exploitation de la Mer IFREMER, Nantes, France. The author thanks Drs Patrick Durand and Jean-Paul Gouigou (Ifremer, Nantes) for the fatty acid analyses of algal extracts and Activepa 50E, and Dr Gerard
Algal extracts as antioxidants Briantais (Laboratoire Interregional, Rennes, France) for the tocopherol determinations in Activepa 50E and sunflower oil. Vitamins E and C were determined in algal extracts by the Institut Pasteur, Lyon, France.
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