Synthesis of new azulene derivatives and study of their effect on lipid peroxidation and lipoxygenase activity.

Article abstract of DOI:10.1248/cpb.50.904

The relationship between free radicals and acute or chronic inflammation has been well established. We have previously reported the significant antioxidant activity of the natural azulene derivatives chamazulene and guaiazulene. Furthermore, some syntheti

Full text of DOI:10.1248/cpb.50.904

904
Chem. Pharm. Bull. 50(7) 904—907 (2002)
Vol. 50, No. 7
Synthesis of New Azulene Derivatives and Study of Their Effect on Lipid
Peroxidation and Lipoxygenase Activity
Eleni REKKA,* Michael CHRYSSELIS, Ioanna SISKOU, and Angeliki KOUROUNAKIS
Department of Pharmaceutical Chemistry, School of Pharmacy, Aristotelian University of Thessaloniki; Thessaloniki
54124, Greece. Received January 21, 2002; accepted April 10, 2002
The relationship between free radicals and acute or chronic inflammation has been well established. We
have previously reported the significant antioxidant activity of the natural azulene derivatives chamazulene and
guaiazulene. Furthermore, some synthetic azulene analogues have been found to possess anti-inflammatory ac-
tivity. In this investigation we report the synthesis of five 3-alkyl or 3-(hydroxy)alkylazulene-1-carboxylic acids
and esters, from tropolone, via the corresponding furanone. The synthesised compounds were tested for their ef-
fect on the peroxidation of rat hepatic microsomal membrane lipids, applying the 2-thiobarbituric acid test.
Their anti-inflammatory activity was evaluated in vitro by the offered inhibition of soybean lipoxygenase. All the
tested molecules were found to inhibit lipid peroxidation by 100% at 1 mM. They were also found to considerably
inhibit lipoxygenase activity. The above results are discussed in relation to the structure and physicochemical
properties of the examined azulene derivatives.
Key words antioxidant activity; lipoxygenase inhibition; azulene derivative; lipid peroxidation; inflammation
In aerobic life various molecular and cellular events in- tributing to the anti-inflammatory activity in a series of pro-
volve oxygen activation and related radical reactions. A bal- piophenone derivatives.7)
ance between the production and elimination of oxygen radi-
Chemistry The synthesis of the azulene derivatives is
cals is critical in human health and disease. When more ac- shown in Chart 2. The structures of the intermediate and final
1
tive oxygen species are generated than can be counteracted products are confirmed spectroscopically (IR, H-NMR) and
by the defence system of the organism, the external applica- by elemental analysis.
tion of synthetic or natural antioxidants appears to be a ratio-
nal approach to the management of conditions associated
with oxidative stress.1)
For the synthesis of the azulene derivatives, tropolone (1,
The radical-mediated oxidation of biomolecules, espe-
cially of lipids, can result into organ and tissue injury. The
modification of biomolecules by lipid peroxidation products
has a central role in most of the major health disorders in the
industrialised world.2) In particular, during the inflammation
process, active oxygen species are generated at inflammatory
sites and aggravate tissue damage. Thus, oxidative stress is
considered to be one of the pathogenic factors in acute and
chronic inflammation, while agents combining anti-inflam-
matory and antioxidant activities would be useful in the treat-
ment of inflammation. Chamazulene and guaiazulene (Chart
1) have been found to possess anti-allergic, anti-inflamma-
tory and anti-ulcer properties.3) We have reported4,5) that
these natural, non toxic products are potent antioxidants. Fur-
thermore, some synthetic azulenes have been reported to af-
fect mediators of inflammation.6)
Chart 1. Structures of Chamazulene and Guaiazulene
In this investigation, we report the synthesis of five azu-
lene derivatives, namely, 3-ethyl-, 3-n-butylazulene-1-car-
boxylic acids and their (2-hydroxy)ethyl esters, as well as 3-
(2-hydroxyethyl)azulene-1-carboxylic acid. In addition, we
study their effect on lipid peroxidation and on lipoxygenase
activity in vitro.
The rationale for the design of the above compounds is the
preservation of the main azulene structure, in combination
with a polar substituent at position 1 which would lower the
high lipophilicity of the natural derivatives. Furthermore, we
have taken into account that an essential structural element
for the activity of azulenes affecting some inflammatory me-
diators is a carboxylic group or sulfonic acid moiety at posi-
tion 1 of the azulene ring.6) Finally, we have found that the
(2-hydroxy)ethyl group is a useful structural component con-
Chart 2. Synthetic Route for the Preparation of the Azulene Derivatives
© 2002 Pharmaceutical Society of Japan
To whom correspondence should be addressed. e-mail: rekka@pharm.auth.gr

July 2002
905
Chart 2), via its tosyl ester (2), reacted with dimethyl mal-
onate, an active methylene compound, in the presence of a
strong base, to give the furanone 3. The subsequent reaction
of the furanone with the in situ generated morpholino enam-
ines of aldehydes resulted in the formation of the 3-alkylazu-
lene-1-carboxylic acids 4b—6b, after the hydrolysis of their
methyl esters 4a—6a. The final products 7 and 8 were ob-
tained after the reaction with the appropriate alcohol, using
both 1,3-dicyclohexylcarbodiimide (DCC) and a catalytic
amount of 4-dimethylaminopyridine (DMAP), at room tem-
perature. Applying the above method for the preparation of
the hydroxyethyl ester 8, a by-product was obtained in a sub-
stantial yield, which was identified to be the diester 9, having
both hydroxylic groups of ethylene glycol esterified with the
carboxylic acid 6b.
The products were obtained in good yields. Spectral data
and elemental analyses confirmed their structures.
Results and Discussion
The natural derivatives chamazulene and guaiazulene are
potent antioxidants, nontoxic compounds, found to possess
anti-inflammatory activity. However, a limitation to their
broader therapeutic use is their high lipophilicity (clogP
about 6). We calculated the clogP values of the synthesised
compounds, and found them to be noticeably lower, i.e. 4.09,
5.14, 2.10 for the acids 4b, 5b, 6b, and 3.43, 4.48 for the es-
ters 7 and 8.
The time course of lipid peroxidation, as affected by com-
pounds 4b, 5b, 6b, 7 and 8, at 0.5 mM, is shown in Figs. 1A,
B. All the tested compounds were found to completely in-
hibit the peroxidation reaction at 1 mM, throughout the incu-
bation period (2.5—45 min), while their antioxidant activity
was practically lost at 0.1 mM (data not shown). Compound
5b, shown in Fig. 1A to be the most potent antioxidant at
0.5 mM, was found to inhibit this reaction by 82% and 60%
after 30 and 45 min of incubation, respectively, at 0.2 mM
(data not shown).
Fig. 1. Time Course of Lipid Peroxidation, as Affected by Compounds 4b,
5b (Panel A), 6b, 7 and 8 (Panel B), at 0.5 mM (C: Control)
We propose that the mechanism by which these derivatives
exhibit their antioxidant activity is connected with the pres-
ence of an allylic-type of hydrogen on the substituent at posi-
tion 3, which could be abstracted by the attacking radical.
The resulting radical of the azulenic molecule could be stabi-
lized by resonance, while it may be further affected by the in-
ductive effect of the methyl or hydroxylic group of this sub-
stituent. Moreover, our results show that the antioxidant ac-
tivity of the compounds increases with lipophilicity. It can be
suggested that this increase in lipophilicity facilitates their
access, retention, as well as interaction with biological mem-
branes, the site of lipid peroxidation.
The effect of compounds 4b, 5b, 6b, 7 and 8 on lipoxyge-
nase activity at 100 mM is shown in Figs. 2A, B. It was found
that the most active was compound 6b (Fig. 2B), which
demonstrated a 61% inhibition, followed by compound 5b,
which inhibited the enzyme by 40%. For compounds 4b, 7
and 8 approximately the same degree of inhibition (20—
25%) was observed.
It is known that the 5-lipoxygenase pathway is the
source of a potent group of inflammatory mediators, the
leukotrienes, implicated in neutrophil activation, which is as-
sociated with the induction of inflammation. Neutrophils
may contribute to local tissue damage through the production
Fig. 2. Effect of 0.1 mM of Compounds 4b, 5b, 7, 8 (Panel A) and 6b
(Panel B) on Lipoxygenase Activity (C: Control)

906
Vol. 50, No. 7
the residue recrystallised from ethyl acetate–petroleum ether. Yield 75%, mp
174—175 °C. 1H-NMR (CDCl3) d 3.9 (s, 3H, COOCH3), 7.5 (m, 5H, Harom).
Anal. Calcd for C11H8O4: C, 64.71; H, 3.95. Found: C, 64.40; H, 3.73.
General Procedure for the Preparation of 3-Alkylazulene-1-car-
boxylic Acids 4b—6b To a solution of 3 (1 mmol) in absolute ethanol, the
of destructive proteinases and reactive oxygen or nitrogen
metabolites. Several non-steroidal-anti-inflammatory drugs
are lipoxygenase inhibitors, most of them having the ability
to participate in redox processes. Since they inhibit lipid
peroxidation, the synthesised compounds intervene in such corresponding aldehyde (3.5 mmol) and morpholine (0.8 mmol) were added.
The mixture was refluxed for 8—10 h, then left at room temperature for 12 h
under stirring, and finally ethanol was evaporated and the methyl esters 4a—
6a were isolated from the residue by flash chromatography and identified.
The esters were suspended in aqueous sodium hydroxide solution (10%
reactions. Therefore, their effect on soybean lipoxygenase
activity was examined, as an indication of potential anti-in-
flammatory activity. It is established that, even when the
quantitative results of this assay cannot be extrapolated to
the inhibition of mammalian lipoxygenase, the inhibition of
plant lipoxygenase by known non-steroidal-anti-inflamma-
tory drugs is at least qualitatively similar to their inhibition
of the rat mast cell lipoxygenase and can be used as a simple
screen for such activity.8) In our study, the offered inhibition
of lipoxygenase did not seem to correlate completely with
the antioxidant activity of the studied compounds, and the
presence of a free carboxylic group appeared important for
this enzyme inhibition. Compound 6b was a better lipoxyge-
nase inhibitor than the other two carboxylic acids 4b and 5b.
This is considered to be due to the lower lipophilicity of 6b,
compared to 4b and 5b. The above is in accordance with pre-
vious work reporting increased anti-inflammatory activity of
azulene-sulphonic acids of comparable lipophilic character
with compound 6b.9)
w/v), the mixture was refluxed for 12 h, cooled, acidified with hydrochloric
acid and extracted with ether. The organic layer was dried (Na2SO4), ether
was evaporated, the carboxylic acids 4b—6b were purified by flash chro-
matography and/or recrystallisation and identified.
3-Ethylazulene-1-carboxylic Acid 4b Prepared according to the gen-
eral method, starting from the furanone 3 and butanal. The methyl ester 4a
was isolated by flash chromatography using petroleum ether/ethyl acetate
1
(50/1), as a dark blue oil. Yield 90%. H-NMR (CDCl3) d: 1.4 (t, 3H, Ar-
CH2CH3), 3.1 (q, 2H, Ar-CH2CH3), 3.9 (s, 3H, Ar-COOCH3), 7.2—7.3 (m,
3H, C5-H, C6-H, C7-H), 8.1 (s, 1H, C2-H), 8.3 (d, 1H, C4-H), 9.5 (d, 1H,
C8-H). Anal. Calcd for C14H14O2: C, 78.48; H, 6.59. Found: C, 78.55; H,
6.67. The final product 4b was obtained after recrystallisation from ether-pe-
troleum ether. Yield 80%, mp 186—187 °C. 1H-NMR as for 4a, without the
peak at d 3.9. Anal. Calcd for C13H12O2: C, 77.98; H, 6.04. Found: C, 77.63;
H, 5.94.
3-n-Butylazulene-1-carboxylic Acid 5b According to the general pro-
cedure, starting from 3 and hexanal. The methyl ester 5a was isolated (dark
blue oil, yield 85%) and identified as described for 4a. 1H-NMR (CDCl3) d:
1.2 [t, 3H, Ar-(CH2)3CH3] 1.6 [m, 4H, Ar-CH2(CH2)2CH3], 3.0 [t, 2H, Ar-
CH2(CH2)2CH3], 3.9 (s, 3H, Ar-COOCH3), 7.2—7.3 (m, 3H, C5-H, C6-H,
C7-H), 8.1 (s, 1H, C2-H), 8.3 (d, 1H, C4-H), 9.5 (d, 1H, C8-H). Anal. Calcd
for C16H18O2: C, 79.30; H, 7.49. Found: C, 79.08; H, 7.61. The final acid 5b
was isolated by flash chromatography (petroleum ether/ethyl acetate 3/1) as
an oil, yield 57%. 1H-NMR as for 5a, without the peak at d: 3.9. Anal. Calcd
for C15H16O2: C, 78.92; H, 7.06. Found: C, 78.56; H, 6.98.
3-(2-Hydroxyethyl)azulene-1-carboxylic Acid 6b According to the
general procedure, starting from 3 and 4-hydroxybutanal. The methyl
ester 6a was isolated (dark blue oil, yield 60%) by flash chromatography
(petroleum ether/ethyl acetate 20/1). 1H-NMR (CDCl3) d: 3.0 (t, 2H, Ar-
In conclusion, this study indicates that new azulene deriva-
tives with anti-inflammatory and antioxidant activities can be
designed, and proposes structural and physicochemical fea-
tures that contribute to this combination of actions. Further
work is in progress, to extend the structural modifications, as
well as the evaluation of activity in this series of compounds.
Experimental
Tropolone, dimethyl malonate, butanal, hexanal, 1,2-ethylenediol, 2,3-di- CH2CH2OH), 3.6—3.9 (m, 5H, Ar-CH2CH2OH, COOCH3), 5.0 [s, 1H, Ar-
hydrofuran, DMAP, DCC and L(ϩ) ascorbic acid were purchased from
(CH2)2OH], 7.2—7.3 (m, 3H, C5-H, C6-H, C7-H), 8.1 (s, 1H, C2-H), 8.3 (d,
Merck Chemical Company (Germany). 2-Thiobarbituric acid, soybean 1H, C4-H), 9.5 (d, 1H, C8-H). Anal. Calcd for C14H14O3: C, 73.04; H, 6.09.
lipoxygenase (lipoxidase Type I-B) and sodium linoleate were from Sigma Found: C, 72.98; H, 6.11. The acid 6b was obtained in a yield of 20% by
Chemical Company (U.S.A.). TLC was performed on silica gel 60F254 alu- flash chromatography (petroleum ether/ethyl acetate 1/1) as a solid, mp
minium sheets (Merck). For flash chromatography, silica gel 60 (40—63 173—174 °C. 1H-NMR (CDCl3) d: 3.0 (t, 2H, Ar-CH2CH2OH), 3.7 (m, 2H,
mesh, Merck) was used. When petroleum ether is mentioned, it corresponds Ar-CH2CH2OH), 5.0 [s, 1H, Ar-(CH2)2OH], 7.2—7.3 (m, 3H, C5-H, C6-H,
to the fraction with bp 40—60 °C. All other common chemicals and solvents C7-H), 8.1 (s, 1H, C2-H), 8.3 (d, 1H, C4-H), 9.5 (d, 1H, C8-H). Anal. Calcd
of the appropriate purity were from various commercial sources.
for C13H12O3: C, 72.21; H, 5.59. Found: C, 71.81; H, 5.80.
Melting points were obtained on a MEL-TEMPII (Laboratory Devices,
The starting 4-hydroxybutanal was obtained from 2,3-dihydrofuran, ac-
U.S.A.) apparatus and are uncorrected. IR spectra were recorded on a cording to the literature.11) Briefly, 2,3-dihydrofuran was added to an aque-
Perkin-Elmer 597 infrared spectrophotometer. 1H-NMR spectra were ob-
tained with a Brucker AW 80 MHz spectrometer. Chemical shifts (d) are re- was left at room temperature for 2 h, then neutralised with sodium hydroxide
ported in ppm relative to tetramethylsilane (TMS), and signals are given as
(1 M) and extracted with ether. After drying (CaCl2) and evaporation of the
follows: s, singlet; d, doublet; t, triplet; q, quadruplet; m, multiplet. Elemen- solvent, 4-hydroxybutanal was obtained by distillation under reduced pres-
ous hydrochloric acid solution (0.8 M) with stirring and cooling. The mixture
tal analyses were performed with a Perkin-Elmer 2400 CHN analyzer.
Chemistry. 2-Tolylsulfonyloxytropone 2 Tropolone (1, 6.1 g, 50 mmol)
was dissolved in freshly distilled pyridine (15 ml) and to this a solution of 4-
sure (bp3 46—48 °C).
General Method for the Preparation of (2-Hydroxy)ethyl Esters 7 and
8
To a stirred solution of the appropriate carboxylic acid 5b or 6b
toluenesulfonyl (tosyl) chloride (10.5 g, 55 mmol) in dry chloroform (15 ml) (1 mmol) in dry dichloromethane (70 ml), DCC (1.5 mmol), 1,2-ethylenediol
was added under stirring and cooling. The mixture was left at room tempera- (8 mmol) and DMAP (0.15 mmol) were successively added and the mixture
ture for 48 h, while an additional amount (1 g) of tosyl chloride was added
after 24 h. Then, the precipitated solid was filtered off, the filtrate was tered off, the filtrate was washed with an aqueous CH3COOH solution (5%
washed with water, hydrochloric acid solution (2 M), sodium hydrogen
v/v) and with water, dried (MgSO4) and the final products were isolated by
carbonate solution (5%) and again with water. The organic layer was dried flash chromatography and identified.
(MgSO4), the solvent evaporated and the residue recrystallised from
(2-Hydroxy)ethyl 3-Ethylazulene-1-carboxylate
was stirred at room temperature for 8 h. Then, the formed precipitate was fil-
7
Prepared as de-
methanol. Yield 85%, mp 151—154 °C. 1H-NMR (CDCl3) d 2.4 (s, 3H, scribed above, starting from 5b. The final product (oil) was obtained in 67%
C6H4CH3), 7.5 (m, 9H, Harom). Anal. Calcd for C14H12O4S: C, 60.86; H, 4.38.
yield by flash chromatography, using petroleum ether/ethyl acetate (5/1). 1H-
Found: C, 61.02; H, 4.14.
NMR (CDCl3) d: 1.4 (t, 3H, Ar-CH2CH3), 3.1 (q, 2H, Ar-CH2CH3), 4.0 (m,
3-Methoxycarbonyl-2H-cyclohepta[b]furan-2-one 310) In anhydrous 4H, Ar-COOCH2CH2OH), 4.5 (t, 1H, Ar-COOCH2CH2OH), 7.2—7.3 (m,
methanol (30 ml), sodium (300 mg) was added under stirring and cooling,
3H, C5-H, C6-H, C7-H), 8.1 (s, 1H, C2-H), 8.3 (d, 1H, C4-H), 9.5 (d, 1H,
followed by the successive addition of dimethyl malonate (1.5 ml, 13 mmol) C8-H). Anal. Calcd for C15H16O3: C, 73.75; H, 6.60. Found: C, 73.40; H,
in methanol (30 ml), and 2-tolylsulfonyloxytropone (1.8 g, 6.5 mmol) sus- 6.78.
pended in methanol (100 ml). The mixture was stirred at room temperature
(2-Hydroxy)ethyl 3-n-Butylazulene-1-carboxylate 8 Starting from
for 12 h. Then, methanol was evaporated, ethyl acetate was added and the 6b, compound 8 (oil) was prepared, in a 50% yield, according to the general
mixture was washed with water, dried (MgSO4), the solvent evaporated and procedure, flash chromatographed with petroleum ether/ethyl acetate

July 2002
907
(50/1) and identified. 1H-NMR (CDCl3) d: 1.4 [t, 3H, Ar-(CH2)3CH3], 1.6
[m, 4H, Ar-CH2(CH2)2CH3], 3.0 [t, 2H, Ar-CH2(CH2)2CH3], 4.0 (m, 4H,
Ar-COOCH2CH2OH), 4.5 (t, 1H, Ar-COOCH2CH2OH), 7.2—7.3 (m, 3H,
C5-H, C6-H, C7-H), 8.1 (s, 1H, C2-H), 8.3 (d, 1H, C4-H), 9.5 (d, 1H, C8-
H). Anal. Calcd for C17H20O3: C, 74.97; H, 7.40. Found: C, 74.40; H, 7.50.
(3-n-Butylazulene-1-carboxy)ethyl 3-n-Butylazulene-1-carboxylate 9
During the preparation of the hydroxyethyl ester 8, a second product
was formed (Chart 2), which was isolated as dark blue crystals by flash
chromatography and identified to be the ester of 8 with the carboxylic
acid 5b. Yield 35%, mp 90—93 °C. 1H-NMR (CDCl3) d: 1.1 [t, 6H
2ϫAr-(CH2)3CH3], 1.6 [m, 8H, 2ϫAr-CH2(CH2)2CH3], 4.7 (s, 4H, Ar-
COOCH2CH2OCO-Ar), 7.2—7.3 [m, 6H, 2ϫ(C5-H, C6-H, C7-H)], 8.1 [s,
4H, 2ϫ(C2-H)], 8.3 [d, 2H, 2ϫ(C4-H)], 9.5 [d, 2H, 2ϫ(C8-H)]. Anal. Calcd
for C32H34O4ϫ0.3H2O: C, 78.78; H, 7.09. Found: C, 78.37; H, 7.30.
The lipophilicity of the synthesized compounds 4b—8, as well as of
chamazulene and guaiazulene was calculated according to the method of
Hansch and Leo12) and expressed as clogP values.
Lipid Peroxidation Heat inactivated hepatic microsomal fraction from
untreated male Fischer-344 rats (180—220 g) was prepared.13) The incuba-
tion mixture contained the microsomal fraction, corresponding to 2.5 mg of
hepatic protein/ml (final concentration), or 4 mM fatty acid residues,14) ascor-
bate (0.2 mM) in Tris–HCl/KCl buffer (50 mM/150 mM, pH 7.4) and the stud-
ied compounds, dissolved in dimethyl sulfoxide, at various concentrations
(0.1—1.0 mM), or the solvent (control). The reaction of peroxidation was ini-
tiated by the addition of a freshly prepared FeSO4 solution (10 mM) and the
mixture was incubated at 37 °C for 45 min. Aliquots were taken at various
time intervals, and lipid peroxidation was assessed by spectrophotometric
(535 against 600 nm) determination of the 2-thiobarbituric acid reactive ma-
terial.4,13) Under the same experimental conditions, DL-(a)-tocopherol ac-
etate, used as a reference, offered 100% and 10% inhibition of this reaction
at 1 mM and 0.5 mM respectively.15)
same experimental conditions.
Acknowledgements E.R. and A.K. wish to acknowledge support for
this work by the European Commission Financed Programme QLK6-CT-
1999-51170.
References
1) Paya M., Halliwell B., Hoult J. R. S., Free Rad. Res. Commun., 17,
293—298 (1992).
2) Rice-Evans C., Burdon R., Prog. Lipid Res., 32, 71—110 (1993).
3) Yanagisawa T., Kosakai K., Tomiyama T., Yasunami M., Takase K.,
Chem. Pharm. Bull., 38, 3355—3358 (1990).
4) Rekka E. A., Kourounakis A. P., Kourounakis P. N., Res. Commun.
Molec. Pathol. Pharmacol., 92, 361—364 (1996).
5) Kourounakis A. P., Rekka E. A., Kourounakis P. N., J. Pharm. Phar-
macol., 49, 938—942 (1997).
6) Yokota M., Uchibori S., Hayashi H., Koyama R., Kosakai K., Waka-
bayashi S., Tomiyama T., Bioorg. Med. Chem., 4, 575—591 (1996).
7) Gavalas A., Hadjipetrou L., Kourounakis P., J. Pharm. Pharmacol., 50,
583—591 (1998).
8) Taraporewala I. B., Kauffman J. M., J. Pharmaceut. Sci., 79, 173—178
(1990).
9) Yanagisawa T., Wakabayashi S., Tomiyama T., Yasunami M., Takase
K., Chem. Pharm. Bull., 36, 641—647 (1988).
10) Nozoe T., Takase K., Kato M., Nogi T., Tetrahedron, 27, 6023—6035
(1971).
11) Bergman N.-A., Jansson M., Chiang Y., Kresge A. J., Yin Y., J. Org.
Chem., 52, 4449—4453 (1987).
12) Hansch C., Leo A. (ed.), “Substituent Constants for Correlation
Analysis in Chemistry and Biology,” John Wiley & Sons, New York,
1991.
13) Rekka E., Kolstee J., Timmerman H., Bast A., Eur. J. Med. Chem., 24,
43—54 (1989).
14) Eichenberger K., Bohni P., Winterhalter K. H., Kawato S., Richter C.,
FEBS Lett., 142, 59—62 (1982).
15) Andreadou I., Tasouli A., Bofilis E., Chrysselis M., Rekka E., Tsantili-
Kakoulidou A., Iliodromitis E., Siatra T., Kremastinos D., Chem.
Pharm. Bull., 50, 165—168 (2002).
Effect on Lipoxygenase Activity The reaction mixture contained (final
concentrations) the test compounds, dissolved in 60% ethanol (100 mM), or
the solvent (control), soybean lipoxygenase, dissolved in 2% ethanol in
0.9% NaCl solution (250 u/ml) and sodium linoleate (100 mM), in Tris/HCl
buffer, pH 9.0. The reaction was monitored for 7—8 min at 28 °C, by record-
ing absorbance at 234 nm.8) The performance of the assay was checked
using nordihydroguaiaretic acid (10 mM) as a reference under exactly the