Synthesis and biological activity of α-methylene-γ-lactones as new aroma chemicals

Article abstract of DOI:10.1021/jf000346t

Seven kinds of α-methylene-γ-lactones with an alkyl group at the C-4 position were synthesized according to a previously described method, with yields of 28-34%. These α-methylene-γ-lactones had characteristic and unique odors. All α-methylene-γ-lactones added a roast-like odor to materials. The antimicrobial effects of α-methylene-γ-lactones were investigated by using a paper disk diffusion method. The results showed the α-methylene-γ-lactones inhibited the growth of three bacteria (Staphylococcus aureus, Escherichia coli, and Pseudomonas fluorescens) and two fungi (Saccharomyces cerevisiae and Aspergillus niger). In particular, α-methylene-γ-undecalactone and α-methyleneγ-dodecalactone exhibited potent inhibition of the growth of these microorganisms compared to butyl p-hydroxybenzoate as standard antibiotic. The umu test revealed that the α-methylene-γ-lactones suppressed the SOS-inducing activity of three mutagens, furylfuramide, UV irradiation, and TrpP-1, respectively. The antimicrobial effects and the suppressive effects of SOS induction by α-methylene-γ-lactones had a tendency to intensify as the number of carbons in the side chain increased.

Full text of DOI:10.1021/jf000346t

5406
J. Agric. Food Chem. 2000, 48, 5406−5410
Syn th esis a n d Biologica l Activity of r-Meth ylen e-γ-la cton es a s New
Ar om a Ch em ica ls
Mitsuo Miyazawa,*,† Hiroshi Shimabayashi,† Shuichi Hayashi,‡ Seiji Hashimoto,‡
Sei-ichi Nakamura,§ Hiroshi Kosaka,§ and Hiromu Kameoka†
Department of Applied Chemistry, Faculty of Science and Engineering, Kinki University, Kowakae,
Higashiosaka-shi, Osaka 577-8502, J apan; Nagaoka Perfumery Company, Ltd., Itsukaichi, Ibaraki-shi,
Osaka 567-0005, J apan; and Osaka Prefectural Institute of Public Health, Nakamichi-1, Higashinari-ku,
Osaka-shi, Osaka 537-0025, J apan
Seven kinds of R-methylene-γ-lactones with an alkyl group at the C-4 position were synthesized
according to a previously described method, with yields of 28-34%. These R-methylene-γ-lactones
had characteristic and unique odors. All R-methylene-γ-lactones added a roast-like odor to materials.
The antimicrobial effects of R-methylene-γ-lactones were investigated by using a paper disk diffusion
method. The results showed the R-methylene-γ-lactones inhibited the growth of three bacteria
(Staphylococcus aureus, Escherichia coli, and Pseudomonas fluorescens) and two fungi (Saccharo-
myces cerevisiae and Aspergillus niger). In particular, R-methylene-γ-undecalactone and R-methylene-
γ-dodecalactone exhibited potent inhibition of the growth of these microorganisms compared to butyl
p-hydroxybenzoate as standard antibiotic. The umu test revealed that the R-methylene-γ-lactones
suppressed the SOS-inducing activity of three mutagens, furylfuramide, UV irradiation, and Trp-
P-1, respectively. The antimicrobial effects and the suppressive effects of SOS induction by
R-methylene-γ-lactones had a tendency to intensify as the number of carbons in the side chain
increased.
Keyw or d s: R-Methylene-γ-lactones; odor; antimicrobial activity; SOD-like activity; umu test
INTRODUCTION
to a previously described method (Minato and Horibe,
1967), and their aromas were examined. In addition,
R-methylene-γ-lactones were investigated for several
biological activities, including antimicrobial activity,
SOD-like activity, and suppression of SOS-inducing
activity. Structure-property relationships for each bio-
logical activity were examined.
The R-methylene-γ-lactone skeleton is a common
grouping in various naturally occurring terpenoid lac-
tones. Their antibiotic (Park et al., 1987; Goren et al.,
1990), antitumor (Hall et al., 1978), and insecticidal
(Kim et al., 1998) activities and other biological func-
tions have been studied. As a result of various investi-
gations, it was found that one of the structural require-
ments for significant cytotoxicity was an exocyclic
double-bond system as part of an ester, ketone, or
lactone (Lee et al., 1975). In addition, γ-lactones are
used frequently as fragrance or flavor compounds.
However, there are no studies about aroma properties
and other biological activities of R-methylene-γ-lactones
with an alkyl group at the C-4 position.
MATERIALS AND METHODS
Gen er a l P r oced u r es. GC-MS was performed on a Hewlett-
Packard 5972 series mass spectrometer interfaced with a
Hewlett-Packard 5890 gas chromatograph fitted with an HP-
5MS column (30 m × 0.25 mm i. d.). High-resolution MS was
carried out with a J EOL-HX100 (with a J EOL J MA-DA 5000
mass data system) apparatus. IR spectra were determined
In recent years, the occurrence of food poisoning
caused by microorganisms, such as enteropathogenic
Escherichia coli O-157, has increased. Inhibition of
microorganisms for food safety is therefore an important
issue. Furthermore, ordinary human diets contain
several mutagens such as products of heat degradation,
food ingredients, and various chemicals. For these
reasons, γ-lactones with several functions (e.g., antimi-
crobial activity, antitumor activity, and so on) maybe
valuable as aroma compounds.
with a Perkin-Elmer 1760-X infrared Fourier transform
spectrometer. Nuclear magnetic resonance (NMR) spectra (δ,
J in hertz) were recorded on a J EOL GSX 270 NMR spectrom-
eter. Tetramethylsilane (TMS) was used as the internal
1
reference (δ 0.00) for H NMR spectra measured in CDCl3. This
solvent was also used for 13C NMR spectra.
Ma ter ia ls. Butyl p-hydroxybenzoate was purchased from
Tokyo Kasei Kohgyo Co. Ltd. (Tokyo, J apan). Lactose broth
and potato dextrose were purchased from Nissui Pharmaceuti-
cal Co., Ltd. (Tokyo, J apan). Agar powder was purchased from
Nacalai Tesque Inc. (Kyoto, J apan). Furylfuramide, Trp-P-1,
nitro blue tetrazolium (NBT), xanthine oxidase (XOD), and
xanthine were purchased from Wako Pure Chemicals Co.
(Osaka, J apan). Bovine serum albumin (BSA) was purchased
from Sigma Chemical Co. (St. Louis, MO). S9 (supernatant of
9000 g) and coenzymes NADPH, NADH, and G-6-P were
purchased from Oriental Yeast Co. (Osaka, J apan). All other
reagents were of analytical grade.
In this study, R-methylene-γ-lactones with an alkyl
group at the C-4 position were synthesized according
* Author to whom correspondence should be addressed
(telephone +81-6-6721-2332, ext. 4153; fax +81-6-6727-4301;
e-mail miyazawa@apch.kindai.ac.jp).
† Kinki University.
‡ Nagaoka Perfumery Co., Ltd.
Micr oor ga n ism s. The synthetic R-methylene-γ-lactones
were tested for antimicrobial activity against the Gram-
§ Osaka Prefectural Institute of Public Health.
10.1021/jf000346t CCC: $19.00 © 2000 American Chemical Society
Published on Web 10/14/2000

Synthesis and Biological Activity of R-Methylene-γ-lactones
J. Agric. Food Chem., Vol. 48, No. 11, 2000 5407
positive bacteria Staphylococcus aureus IFO 14462, the Gram-
negative bacteria Escherichia coli IFO 12734 and Pseudomo-
nas fluorescens IFO 3081, and the fungi Saccharomyces
cerevisiae SW-33 and Aspergillus niger IFO 4414.
[M+] (tr), 153 (2), 140 (14), 111 (3), 97 (100), 69 (26), 41 (32);
IR vmax (KBr) cm-1 2930, 1762, 1667, 1276, 1118, 1010; 1H
NMR (CDCl3) δH 6.21 (1H, dd, J ) 3.0 and 2.5, H-11a), 5.62
(1H, dd, J ) 3.0 and 2.5, H-11b), 4.51 (1H, tt, J ) 7.5 and 6.0,
H-4), 3.06 (1H, dddd, J ) 17.0, 7.5, 3.0, and 2.5, H-3a), 2.58
(1H, dddd, J ) 17.0, 6.0, 3.0, and 2.5, H-3b), 1.78-1.68 (2H,
m, H-5), 1.36-1.22 (8H, m, H-6, 7, 8, 9), 0.89 (3H, t, J ) 7.0,
H-10); 13C NMR (CDCl3) δC 170.3 (s, C-1), 134.8 (s, C-2), 121.8
(t, C-11), 76.5 (d, C-4), 36.2 (t, C-3), 33.5 (t, C-5), 31.6 (t, C-6),
28.9 (t, C-7), 24.7 (t, C-8), 22.5 (t, C-9), 14.0 (q, C-10).
R-Methylene-γ-undecalactone (13). The lactone was obtained
as a colorless oil (917 mg, 29%): HRMS m/z 196.1466 ([M+],
calcd for C12H20O2, 196.1464); El-MS m/z (rel intensity) 196
[M+] (tr), 178 (tr), 163 (tr), 140 (13), 111 (7), 97 (100), 69 (27),
41 (28); IR vmax (KBr) cm-1 2931, 1767, 12667, 1278, 1118,
1000; 1H NMR (CDCl3) δH 6.22 (1H, dd, J ) 3.0 and 2.5,
H-12a), 5.62 (1H, dd, J ) 3.0 and 2.5, H-12b), 4.51 (1H, tt, J
) 7.5 and 6.0, H-4), 3.05 (1H, dddd, J ) 17.0, 7.5, 3.0, and
2.5, H-12a), 2.58 (1H, dddd, J ) 17.0, 6.0, 3.0, and 2.5, H-12b),
1.36-1.20 (12H, m, H-5, 6, 7, 8, 9, 10), 0.88 (3H, t, J ) 7.0,
H-11); 13C NMR (CDCl3) δC 170.0 (s, C-1), 136.0 (s, C-2), 122.0
(t, C-12), 83.8 (d, C-4), 41.3 (t, C-3), 26.5 (t, C-5), 39.4 (t, C-6),
31.6 (t, C-7), 29.4 (t, C-8), 23.6 (t, C-9), 22.5 (t, C-10), 14.0 (q,
C-11).
Syn th esis (Min a to a n d Hor ibe, 1967). A solution of
γ-lactone (20 mmol) and ethyl formate (25 mmol) in dry ether
(30 mL) was added dropwise to a suspension of sodium hydride
(20 mmol) in dry ether (15 mL) with stirring during 15 min in
an ice bath. The mixture was stirred for 3 h at room
temperature and set aside overnight. The sodium salt of the
R-formyl-γ-lactone was separated and added to 2 N hydro-
chloric acid (30 mL) with stirring in an ice bath. The solution
was extracted with ether, washed with saturated sodium
chloride solution, dried (Na2SO4), and evaporated to give a light
yellow oil. The crude product was dissolved in methanol (45
mL) and reduced with sodium borohydride (10 mmol) with
stirring for 1 h at room temperature to give a viscous oil. This
was chromatographed on silica gel (eluent: CH2Cl2/acetone)
to give an R-hydroxymethyl-γ-lactone. The R-hydroxymethyl-
γ-lactone was tosylated with toluene-p-sulfonyl chloride (8
mmol) in pyridine (15 mL). The tosylate was dissolved in
pyridine and refluxed for 4-6 h to give crude product. This
was chromatographed on silica gel (eluent: CH2Cl2/acetone)
to give R-methylene-γ-lactone.
R-Methylene-γ-hexalactone (8). The lactone was obtained as
a colorless oil (781 mg, 31%): HRMS m/z 126.0683 ([M+], calcd
for C7H10O2, 126.0681); El-MS m/z (rel intensity) 126 [M+] (12),
111 (tr), 97 (100), 69 (35), 41 (32); IR vmax (KBr) cm-1 2971,
1762, 1666, 1278, 1119, 963; 1H NMR (CDCl3) δH 6.22 (1H,
dd, J ) 3.0 and 2.5, H-7a), 5.63 (1H, dd, J ) 3.0 and 2.5, H-7b),
4.47 (1H, tt, J ) 7.5 and 6.0, H-4), 3.06 (1H, dddd, J ) 17.0,
7.5, 3.0, and 2.5, H-3a), 2.59 (1H, dddd, J ) 17.0, 6.0, 3.0,
and 2.5, H-3b), 1.81-1.63 (2H, m, H-5), 1.01 (3H, t, J ) 7.5,
H-6); 13C NMR (CDCl3) δC 170.3 (s, C-1), 134.7 (s, C-2), 121.8
(t, C-7), 78.6 (d, C-4), 33.0 (t, C-3), 29.1 (t, C-5), 9.0 (q, C-6).
R-Methylene-γ-heptalactone (9). The lactone was obtained
as a colorless oil (952 mg, 34%): HRMS m/z 140.0836 ([M+],
calcd for C8H12O2, 140.0837); El-MS m/z (rel intensity) 140 [M+]
(3), 125 (tr), 111 (4), 97 (100), 69 (27), 41 (30); IR vmax (KBr)
cm-1 2963, 1762, 1667, 1274, 1118, 1003; 1H NMR (CDCl3) δH
6.22 (1H, dd, J ) 3.0 and 2.5, H-8a), 5.62 (1H, dd, J ) 3.0 and
2.5, H-8b), 4.53 (1H, tt, J ) 7.5 and 6.0, H-4), 3.06 (1H, dddd,
J ) 17.0, 7.5, 3.0, and 2.5, H-3a), 2.58 (1H, dddd, J ) 17.0,
6.0, 3.0, and 2.5, H-3b), 1.77-1.55 (2H, m, H-5), 1.55-1.39 (2H,
m, H-6), 0.97 (3H, t, J ) 7.5, H-7); 13C NMR (CDCl3) δC 170.3
(s, C-1), 134.8 (s, C-2), 121.8 (t, C-8), 77.3 (d, C-4), 38.3 (t, C-3),
33.5 (t, C-5), 18.1 (t, C-6), 13.7 (q, C-7).
R-Methylene-γ-octalactone (10). The lactone was obtained as
a colorless oil (864 mg, 28%): HRMS m/z 154.0995 ([M+], calcd
for C9H14O2, 154.0994); El-MS m/z (rel intensity) 154 [M+] (tr),
139 (tr), 125 (3), 111 (2), 97 (100), 69 (27), 41 (26); IR vmax (KBr)
cm-1 2931, 1763, 1666, 1278, 1117, 1005; 1H NMR (CDCl3) δH
6.22 (1H, dd, J ) 3.0 and 2.5, H-9a), 5.63 (1H, dd, J ) 3.0 and
2.5, H-9b), 4.52 (1H, tt, J ) 7.5 and 6.0, H-4), 3.06 (1H, dddd,
J ) 17.0, 7.5, 3.0, and 2.5, H-3a), 2.58 (1H, dddd, J ) 17.0,
6.0, 3.0, and 2.5, H-3b), 1.82-1.54 (2H, m, H-5), 1.52-1.28 (4H,
m, H-6, 7), 0.92 (3H, t, J ) 7.0, H-8); 13C NMR (CDCl3) δC 170.3
(s, C-1), 134.7 (s, C-2), 121.8 (t, C-9), 77.5 (d, C-4), 35.9 (t, C-3),
33.5 (t, C-5), 26.9 (t, C-6), 22.3 (t, C-7), 13.8 (q, C-8).
R-Methylene-γ-dodecalactone (14). The lactone was obtained
as a colorless oil (882 mg, 31%): HRMS m/z 210.1619 ([M+],
calcd for C13H22O2, 210.1620); El-MS m/z (rel intensity) 210
[M+] (tr), 192 (tr), 153 (1), 140 (12), 111 (6), 97 (100), 69 (31),
41 (35); IR vmax (KBr) cm-1 2927, 1767, 1667, 1278, 1118, 1003;
1H NMR (CDCl3) δH 6.22 (1H, dd, J ) 3.0 and 2.5, H-13a),
5.62 (1H, dd, J ) 3.0 and 2.5, H-13b), 4.52 (1H, tt, J ) 7.5
and 6.0, H-4), 3.06 (1H, dddd, J ) 17.0, 7.5, 3.0, and 2.5, H-3a),
2.58 (1H, dddd, J ) 17.0, 6.0, 3.0, and 2.5, H-3b), 1.36-1.18
(14H, m, H-5, 6, 7, 8, 9, 10, 11), 0.88 (3H, t, J ) 7.0, H-12); 13
C
NMR (CDCl3) δC 170.3 (s, C-1), 134.8 (s, C-2), 121.8 (t, C-13),
76.5 (d, C-4), 36.3 (t, C-3), 33.5 (t, C-5), 31.7 (t, C-6), 29.4 (t,
C-7), 29.3 (t, C-8), 29.1 (t, C-9), 24.8 (t, C-10), 22.6 (t, C-11),
14.1 (q, C-12).
Eva lu a tion of An tim icr obia l Activity. The paper disk
diffusion method was used to determine the activity of
synthetic compounds on test microorganisms. The overnight
cultures of microorganisms were spread over (106 cfu/plate)
the appropriate media (bacteria, lactose broth; fungi, potato
dextrose) in Petri plates (90 mm). Paper disks (8 mm)
impregnated with DMSO solutions of the test compounds were
placed on the air-dried surface of the media seeded with the
respective microorganisms. After incubation (bacteria, 37 °C
for 24 h; fungi, 30 °C for 48 h), the zones of inhibition around
the disks were measured. The antimicrobial effects of com-
pounds that produced zones of inhibition >2 mm against
bacteria were quantified using the broth dilution method
(Gotou et al., 1983) and minimum inhibitory concentration
(MIC) values were determined. Broths of serial 2-fold dilution
of the compound dissolved in DMSO (maximum concentration
) 800 µg/mL) were made, and the tubes were inoculated with
microorganisms at a density of 105 cfu/mL. After incubation
(37 °C for 18 h), the tubes were visually examined for growth
of bacteria. In both methods, DMSO was used as a control.
SOD-lik e Activity Test. The method for the SOD-like
activity assay of test compounds was carried out according to
the NBT (IV) improved method of Beauchamp et al. (1971). A
buffer of 0.3 M Na2CO3-NaHCO3 (pH 10.2) containing 0.6 mM
EDTA‚2Na, 0. 15 mM NBT (nitro blue tetrazolium), 0.3 mM
xanthine, and 0.15 w/v % BSA was mixed. The solution was
subdivided into 2.5 mL portions in each test tube. The test
compounds (0. 1 mL in DMSO, end concentration ) 0.33 mg/
mL), ethanol (0.1 mL), and water (0.2 mL) were added to each
test tube. In the tube without xanthine oxidase (XOD), 0.3 mL
of water was added. After 10 min of incubation at 25 °C, the
enzyme reaction was initiated by the addition of XOD (0. 1
mL). After 20 min of incubation at 25 °C, the absorbance at
560 nm was measured. The intensity of the SOD-like activity
was calculated as
R-Methylene-γ-nonalactone (11). The lactone was obtained
as a colorless oil (1042 mg, 31%): HRMS m/z 168.1150 ([M+],
calcd for C10H16O2, 168.1151); El-MS m/z (rel intensity) 168
[M+] (tr), 153 (tr), 139 (3), 111 (3), 97 (100), 69 (25), 41 (25);
IR vmax (KBr) cm-1 2922, 1734, 1667, 1457, 937, 814; 1H NMR
(CDCl3) δH 6.22 (1H, dd, J ) 3.0 and 2.5, H-lOa), 5.63 (1H,
dd, J ) 3.0 and 2.5, H-lOb), 4.52 (1H, tt, J ) 7.5 and 6.0, H-4),
3.06 (1H, dddd, J ) 17.0, 7.5, 3.0, and 2.5, H-3a), 2.58 (1H,
dddd, J ) 17.0, 6.0, 3.0, and 2.5, H-3b), 1.50-1.41 (2H, m,
H-5), 1.36-1.28 (6H, m, H-6, 7, 8), 0.90 (3H, t, J ) 7.0, H-9);
13C NMR (CDCl3) δC 170.3 (s, C-1), 134.8 (s, C-2), 121.8 (t,
C-10), 76.6 (d, C-4), 36.2 (t, C-3), 33.5 (t, C-5), 31.4 (t, C-6),
24.5 (t, C-7), 22.4 (t, C-8), 13.9 (q, C-9).
R-Methylene-γ-decalactone (12). The lactone was obtained
as a colorless oil (1201 mg, 33%): HRMS m/z 182.1305 ([M+],
calcd for C11H18O2, 182.1307); EI-MS m/z (rel intensity) 182
intensity of activity ) [B - (A - C)]/B × 100 (%)

5408 J. Agric. Food Chem., Vol. 48, No. 11, 2000
Miyazawa et al.
Sch em e 1. Syn th esis of r-Meth ylen e-γ-la cton esa
Ta ble 1. Ar om a Ch a r a cter istics of
r-Meth ylen e-γ-la cton es
R-methylene-γ-
aroma characteristics
hexalactone (8)
roast; coconut, coumarin, herbal
roast; nut, coumarin
roast; coconut, herbal
roast; peach, coconut, cream
roast; mild, peach, cream
roast; peach, fatty
heptalactone (9)
octalactone (10)
nonalactone (11)
decalactone (12)
undecalactone (13)
dodecalactone (14)
roast; peach, fatty
Ta ble 2. An tim icr obia l Activity of
r-Meth ylen e-γ-la cton es
microorganisma
compd
1
2
3
4
5
a
1, NaH, HCO2Et; 2, NaBH4; 3, TsCl.
8
9
-
++
++
++
++
+++
++
-
++
-
+
+++
+++
+++
+++
++
++++
++++
++++
++++
++++
++
++++
++++
++++
++++
++++
++++
+++
where A is the absorbance at 560 nm of sample with XOD, B
is the absorbance at 560 nm of DMSO with XOD, and C is the
absorbance at 560 nm of sample without XOD.
10
11
12
13
14
+++
+++
++
++
++
Um u Test. The method for the umu test for detecting SOS-
inducing activity of chemicals was carried out according to that
of Oda et al. (1985) using Salmonella typhimurium TA1535/
pSK1002, having plasmid pSK1002 carrying a umuC′-′lacZ
fused gene. The overnight culture of bacterial strain was
diluted 50-fold into TGA medium (1% Bactotryptone, 0.5%
NaCl, and 0.2% glucose supplemented with 50 mg/L of
ampicillin) and incubated at 37 °C until the bacterial density
reached 0.25-0.30 at 600 nm. The bacterial culture was
subdivided into 2.1 mL portions in test tubes, and the test
compound (50 µL), 0.1 M phosphate buffer (300 µL, pH 7.4),
and mutagens (50 µL in DMSO) were added to each tube. In
the case of Trp-P-1, S9 mix was added in each tube instead of
phosphate buffer. After 2 h of incubation at 37 °C with
shaking, the culture was centrifuged (3000 rpm) to collect cells,
which were resuspended in 2.5 mL of PBS. The level of
â-galactosidase activity was measured by using a slight
modification of Miller’s method (Miller, 1972). Fractions (0.25
mL) of the culture were diluted with 2.25 mL of Z buffer and
0. 1% SDS solution (50 µL) and chloroform (10 µL) were added
to each fraction. The enzyme reaction was initiated by the
addition of 0.25 mL of 2-nitrophenyl â-D-galactopyranoside
solution (ONPG, 4 mg/mL in 0.1 M phosphate buffer, pH 7.4)
at 28 °C. After 15 min, 0.1 M Na2CO3 was added, and the
absorbance at 420 and 500 nm was measured. Using the
remainder of culture, the bacterial density was measured at
600 nm. The unit of â-galactosidase activity was calculated
according to the method of Miller (1972).
+
BHBb
+++
+++
++
+++
++++
a
Microorganisms: 1, S. aureus; 2, E. coli; 3, P. fluorescens; 4,
S. cerevisiae; 5, A. niger. The results are the average of three
readings. Inhibition zones: ++++, >15 mm; +++, 11-15 mm;
++, 6-10 mm; +, 2-5 mm; -, <2 mm. Dose of the compound: 1
b
mg/disk. Standard antibiotic: butyl p-hydroxybenzoate.
UV Ir r a d ia tion a s Mu ta gen . The overnight cultured cells
(S. typhimunuium TA1535/pSK1002) were diluted 50-fold with
fresh TGA medium and incubated at 37 °C until the bacterial
density at 600 nm reached 0.25-0.30 with 5 mL of 0. 1 M
phosphate buffer. The cells were transferred to a Petri plate
(40 mm) and UV-irradiated for 5 s (2 J /m2) with a germicidal
lamp at room temperature.
F igu r e 1. Inhibition zone of R-methylene-γ-lactones against
fungi.
p-hydroxybenzoate as a standard antibiotic. The MIC
values of compounds 9)14 against S. aureus were 400,
400, 200, 100, 50, and 50 µg/mL, respectively. The MIC
values of compounds 9)14 against E. coli were 400,
200, 200, 100, 50, and 25 µg/mL, respectively. The MIC
values of compounds 9)14 against P. fluorescens were
800, 800, 400, 200, 100, and 50 µg/mL, respectively.
According to the MIC value, the antimicrobial effects
of R-methylene-γ-lactones with respect to bacteria had
a tendency to intensify as the number of carbons in the
side chain increased. From the diameter of the inhibi-
tion zones (Figure 1), the antimicrobial effects of R-me-
thylene-γ-lactones on fungi were greatest when the
number of carbons in the side chain was five (R-
methylene-γ-nonalactone).
RESULTS AND DISCUSSION
R-Methylene-γ-lactones were easily synthesized ac-
cording to the method of Minato and Horibe (1967)
(Scheme 1). The synthetic R-methylene-γ-lactones had
unique odors, which were roast-like in character (de-
scribed in Table 1). From the results of the antimicrobial
test (Table 2), compounds 9)14 were observed to inhibit
the growth of bacteria (S. aureus, E. coli, and P.
fluorescens) and fungi (S. cerevisiae and A. niger).
R-Methylene-γ-hexalactone (8) inhibited the growth of
only fungi. Butyl p-hydroxybenzoate as a standard
antibiotic inhibited the growth of all microorganisms.
In particular, R-methylene-γ-undecalactone (13) and
R-methylene-γ-dodecalactone (14) exhibited potent in-
hibition to the growth of bacteria similar to butyl
The results of the superoxide dismutase (SOD-like)
activity test showed (Table 3) that compounds 9, 10, 11,
and 12 exhibited 1, 2, 4, and 1% of SOD-like activity at
a concentration of 0.33 mg/mL, respectively, whereas

Synthesis and Biological Activity of R-Methylene-γ-lactones
J. Agric. Food Chem., Vol. 48, No. 11, 2000 5409
Ta ble 3. SOD-lik e Activity of r-Meth ylen e-γ-la cton es by
pounds 8)14, except for 9, showed suppression of the
SOS-inducing activity of UV irradiation. Compounds 8
and 11)13 suppressed 20, 65, 88, and 97% of the SOS-
inducing activity of UV irradiation at a concentration
of 0.04 mg/mL, respectively. Compounds 10 and 14
suppressed 21 and 65% of the SOS-inducing activity of
UV irradiation at a concentration of 0.01 mg/mL,
respectively. Furthermore, compounds 8)14 also showed
suppression of the SOS-inducing activity of Trp-P-1,
which requires metabolic activation. Compounds 8, 9,
and 11)13 suppressed 14, 66, 83, 43, and 72% of the
SOS-inducing activity of Trp-P-1 at a concentration of
0.04 mg/mL, respectively, whereas compounds 10 and
14 suppressed 10 and 14% of the SOS-inducing activity
of Trp-P-1 at a concentration of 0.01 mg/mL. This was
the maximum concentration of compounds 10 and 14
that could be tested because of their cytotoxicity toward
S. typhimurium. From these results, compounds 8)14
showed similar suppressive effects on furylfuramide-,
UV irradiation-, and Trp-P-1-induced SOS response, and
these effects intensified as the number of carbons in the
side chain increased. The increase in antibiotic effects
and suppressive effects on SOS induction with the
increase in the side chain length may reflect the ability
of R-methylene-γ-lactones to pass through the phospho-
lipid bilayer as a result of the increasing hydrophobicity.
NBT(IV) Meth od
compda
activity (%)
compda
activity (%)
8
9
10
11
inactive
12
13
14
1
1
2
4
inactive
inactive
TPb
75
a
b
Dose of the compound: 0.33 mg/tube. Standard antioxidant:
dl-R-tocopherol.
compounds 8, 13, and 14 did not exhibit SOD-like
activity. dl-R-Tocopherol, as a control, exhibited 75% of
SOD-like activity at the same concentration. On the
basis of these results, R-methylene-γ-lactones had little
SOD-like activity. Thus, those compounds did not
quench or scavenge free radicals (superoxide anion).
From the results of the umu test, the suppressive effects
of compounds 8)14 were determined (Figure 2). All
compounds inhibited the SOS induction of furylfura-
mide. Compounds 8, 9, and 11)13 suppressed 39, 38,
98, 85, and 97% of the SOS-inducing activity of furyl-
furamide at a concentration of 0.04 mg/mL, respectively.
Compounds 10 and 14 suppressed 41 and 92% of the
SOS-inducing activity of furylfuramide at a concentra-
tion of 0.01 mg/mL, respectively. Additionally, com-
F igu r e 2. Suppression of furylfuramide-, Trp-P-1-, and UV-induced SOS responses by R-methylene-γ-lactones in S. typhimurium
TA 1535/pSK1002. Furylfuramide (1 µg/mL in DMSO) was added at 50 µL. Trp-P-1 (40 µg/mL in DMSO) was added at 50 µL. The
cells were exposed to UV light (2.0 J /m2) with a germicidal lamp at room temperature.

5410 J. Agric. Food Chem., Vol. 48, No. 11, 2000
Miyazawa et al.
Lee, H. K.; Ibuka, T.; Kim, H. S.; Vestal, R. B.; Hall, H. I.;
Huang, S. E. Antitumoragents. 16. Steroidal R-methylene-
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Miller, J . W. Experiments in Molecular Genetics; Cold Spring
Harbor Laboratory: Cold Spring Harbor, NY, 1972; pp 352-
355.
Further studies will be needed to clarify the mecha-
nisms of antimicrobial effects and suppressive effects
of SOS induction by R-methylene-γ-lactones.
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Received for review March 16, 2000. Revised manuscript
received August 1, 2000. Accepted August 3, 2000.
J F000346T