Lytic activity of l-menthol derivatives against the snow blight disease fungus, Micronectriella nivalis

Article abstract of DOI:10.1021/jf0210831

Lytic activity of l-menthol (1) derivatives [(-)-(1S,3R,4S,6S)-6-hydroxymenthol (2), (-)-(1S,3R,4S)-1-hydroxymenthol (3), and (+)-(1S,3R,4R,6S)-6,8-dihydroxymenthol (4)] against the snow blight disease fungus, Micronectriella nivalis was investigated. Compounds 2, 3, and 4 had 85.0, 63.9, and 81.9% lytic activity, respectively, at a concentration of 0.2 mg/mL. The activity of each of the three compounds increased in a dose-response manner. To study the structure-activity relationship, acetyl esters of 1-4 [(-)-menthyl acetate (1Ac), (-)-6-hydroxymenthyl diacetate (2Ac), (-)-1-hydroxymenthyl 3-monoacetate (3Ac), and (-)-6,8-dihydroxymenthyl 3,6-diacetate (4Ac)] were synthesized with yields of 80.2-99.8% and were also assayed. The acetyl esters of 1Ac, 2Ac, 3Ac, and 4Ac had 51.2, 91.5, 66.0, and 95.2% lytic activity, respectively, at a concentration of 0.2 mg/mL, and these compounds showed further high lytic activity compared with the alcohols of 1-4. These acetyl esters also showed higher lytic activity as their concentration was increased. Of particular interest is the fact that 2Ac and 4Ac both had higher lytic activity at 0.05-0.2 mg/mL compared with copper 8-hydroxyquinolate, a standard chemical widely used to control snow blight. This is the first report on lytic activity of I-menthol derivatives.

Full text of DOI:10.1021/jf0210831

1880 J. Agric. Food Chem. 2003, 51, 1880−1883
Lytic Activity of l-Menthol Derivatives against the Snow Blight
Disease Fungus, Micronectriella nivalis
MITSUO MIYAZAWA,*† HIDEKI KAWAZOE,† YUJI SUMI,§ AND
§
MITSURO HYAKUMACHI
Department of Applied Chemistry, Faculty of Science and Engineering, Kinki University,
Kowakae, Higashiosaka-shi, Osaka 577-8502, Japan, and Laboratory of Plant Disease Science,
Faculty of Agriculture, Gifu University, 1-1 Yanagido, Gifu 501-1112, Japan
Lytic activity of l-menthol (1) derivatives [(-)-(1S,3R,4S,6S)-6-hydroxymenthol (2), (-)-(1S,3R,4S)-
1-hydroxymenthol (3), and (+)-(1S,3R,4R,6S)-6,8-dihydroxymenthol (4)] against the snow blight
disease fungus, Micronectriella nivalis was investigated. Compounds 2, 3, and 4 had 85.0, 63.9, and
81.9% lytic activity, respectively, at a concentration of 0.2 mg/mL. The activity of each of the three
compounds increased in a dose-response manner. To study the structure-activity relationship, acetyl
esters of 1-4 [(-)-menthyl acetate (1Ac), (-)-6-hydroxymenthyl diacetate (2Ac), (-)-1-hydroxymenthyl
3-monoacetate (3Ac), and (-)-6,8-dihydroxymenthyl 3,6-diacetate (4Ac)] were synthesized with yields
of 80.2-99.8% and were also assayed. The acetyl esters of 1Ac, 2Ac, 3Ac, and 4Ac had 51.2,
91.5, 66.0, and 95.2% lytic activity, respectively, at a concentration of 0.2 mg/mL, and these
compounds showed further high lytic activity compared with the alcohols of 1-4. These acetyl esters
also showed higher lytic activity as their concentration was increased. Of particular interest is the
fact that 2Ac and 4Ac both had higher lytic activity at 0.05-0.2 mg/mL compared with copper
8-hydroxyquinolate, a standard chemical widely used to control snow blight. This is the first report on
lytic activity of l-menthol derivatives.
KEYWORDS: Rhizoctonia solani; l-menthol derivatives; Micronectriella nivalis MAFF 305031; lytic activity;
copper 8-hydoxyquinolate
INTRODUCTION
Our laboratory has studied the biotransformation (4) and
biological activity (5) of terpenoids. Terpenoids are known as
raw materials for biologically active substances and are easily
available in large amounts from plants or chemical synthesis.
A great majority of biologically active terpenoids are produced
as plant secondary metabolites, and these terpenoids have been
shown to have biological activity against plants, microorganisms,
and insects. On the other hand, they serve as flavors and fra-
grances or can be useful as chiral synthons for chemical syn-
thesis. As the properties of these compounds depend on specific
configurations, modifications demand reactions with high stereo-
and enantioselectivity. Therefore, biotransformation of terpe-
noids presents a helpful support in this field. Various attempts
have been made to search for new biologically active terpenoids.
Biotransformation is one way to produce biologically active ter-
penoids. For example, biotransformation of terpenoids has been
investigated not only in many kinds of microorganisms such as
bacteria, yeasts, and fungi (6) but also in mammals (7) and
insects (8, 9).
Snow blight brings about significant disease damage on
gramineous plants such as wheat, barley, and oat and on
leguminous plants in cold snowy districts. This disease is
characterized by damage on the plants under the fallen snow,
whereby the damaged plant may, after the thawing of the snow,
be dead from rotting of the foliage and blighted upon drying or
may suffer from retarded growth. Such damage due to snow
blight disease is well-known for lawn grass. Because disease
occurs beneath the fallen snow, a prophylaxis or therapy for
the disease is difficult. To control the disease, organic copper
compounds, such as copper 8-hydroxyquinolate, have hitherto
been used as an effective component. However, these com-
pounds provoke the problem (1, 2) of environmental pollution
in watering canals by rainfall or an accidental flooding.
Therefore, it is necessary to find safe control methods alternative
to the organic copper compounds. Using a lytic activity of
beneficial microorganisms against the pathogen is one such
method (3).
We previously reported the isolation and structure elucida-
tion of (-)-(1S,3R,4S,6S)-6-hydroxymenthol (2) as a metab-
olite of l-menthol, in incubation with the soil-borne plant
pathogenic fungus Rhizoctonia solani (10). From the same
source, two other compounds, (-)-(1S,3R,4S)-1-hydroxymenthol
* Author to whom correspondence should be addressed (telephone
+81-6-6721-2332; fax +81-6-6727-4301; e-mail miyazawa@
apch.kindai.ac.jp).
† Kinki University.
§ Gifu University.
10.1021/jf0210831 CCC: $25.00 © 2003 American Chemical Society
Published on Web 02/28/2003

Lytic Activity of l-Menthol
J. Agric. Food Chem., Vol. 51, No. 7, 2003 1881
at 25 °C for 5 days. Mycelial plugs (diameter ) 3 mm) from colonies
growing on PDA medium were placed on autoclaved cellophane (7
mm × 7 mm) on water agar media [2% (w/v) agar and 1% (w/v)
glucose] and then incubated at 25 °C for 48 h. After 48 h of incubation
at 25 °C, the cellophanes were removed from the media and placed on
the 300 µL solution [sterilized distilled water and test compound
dissolved in DMSO (concentration ) 0.025, 0.05, 0.1, and 0.2 mg/
mL)] in the well (inside diameter ) 10 mm; depth ) 15 mm) of 24
well multidish cases and then incubated at 3 °C for 48 h. After 48 h of
incubation at 3 °C, the cellophanes were removed from the case and
placed on microscopic slides. The hyphal area was stained with 5%
cotton blue. The existence ratio of stained and nonstained hyphe was
counted using the microscope (×40). The existence ratio was necessary
for evaluation.
(-)-(1S,3R,4S,6S)-6-Hydroxymenthol (2): colorless needlelike crys-
tal; mp 143 °C; [R]25D -42.0° (CHCl3; c 0.4); HR-MS, m/z 172.1465;
EI-MS, m/z (rel int) 154 (15), 139 (48), 121 (13), 111 (15), 97 (40), 95
(17), 83 (33), 69 (37), 67 (10), 55 (67), 43 (100); IR, Vmax cm-1 3264,
1450, 1343, 1029; 13C NMR δ 15.9 (C-9), 18.1 (C-10), 20.9 (C-8),
25.7 (C-7), 32.4 (C-5), 38.3 (C-1), 42.5 (C-2), 48.5 (C-4), 70.6 (C-3),
75.9 (C-6); 1H NMR (500.00 MHz, in CDCl3, TMS as internal standard)
δ 0.82 (3H, d, J ) 7.0 Hz, H-7), 0.99 (3H, d, J ) 7.1 Hz, H-9), 1.03
(3H, d, J ) 7.2 Hz, H-10), 2.15 (1H, dd, J ) 7.2, 7.1 Hz, H-8), 3.19
(1H, ddd, J ) 11.2, 11.0, 3.9 Hz, H-6), 3.76 (1H, m, H-3).
Figure 1. Compounds tested for lytic activity.
(3) and (+)-(1S,3R,4R,6S)-6,8-dihydroxymenthol (4), were
isolated. However, there are no reports of the lytic activity of
these metabolites.
In this study, the lytic activity of l-menthol (1) derivatives
[(-)-(1S,3R,4S,6S)-6-hydroxymenthol (2), (-)-(1S,3R,4S)-1-
hydroxymenthol (3), and (+)-(1S,3R,4R,6S)-6,8-dihydroxym-
enthol (4)] against the snow blight disease fungus, Micronec-
triella niValis was investigated at concentrations of 0.025, 0.05,
0.1, and 0.2 mg/mL, respectively. To study the structure-
activity relationship, acetyl esters of 1-4 (Figure 1) were
synthesized and were also assayed. Finally, to compare levels
of lytic activity, copper 8-hydroxyquinolate (as a standard
chemical used to control snow blight) was also tested, and we
discuss the possibility of whether these compounds could be
used alternative to the organic compound, copper 8-hydroxy-
quinolate.
(-)-(1S,3R,4S)-1-Hydroxymenthol (3): colorless needlelike crystal;
mp 164 °C; [R]25D -38.6° (CHCl3; c 1.0); HR-MS, m/z 172.1463; EI-
MS, m/z (rel int) 154 (8), 139 (62), 121 (12), 111 (20), 97 (13), 87
(76), 81 (15), 71 (48), 67 (8), 55 (34), 43 (100); IR, Vmax cm-1 3342,
1463, 1348, 1051; 13C NMR δ 16.4 (C-9), 19.3 (C-5), 20.9 (C-10),
26.1 (C-8), 31.6 (C-8), 38.6 (C-6), 48.4 (C-4), 50.2 (C-2), 68.6 (C-3),
71.4 (C-1); 1H NMR (500.00 MHz, in CDCl3, TMS as internal standard)
δ 0.85 (3H, d, J ) 7.0 Hz, H-9), 0.95 (3H, d, J ) 7.0 Hz, H-10), 1.24
(3H, s, H-7), 3.76 (1H, ddd, J ) 11.0, 11.1, 4.0 Hz, H-3).
(+)-(1S,3R,4R,6S)-6,8-Dihydroxymenthol (4): colorless oil; [R]25
D
+5.2° (CHCl3; c 1.0); HR-MS, m/z 188.1413; EI-MS, m/z (rel int) 173
(1), 155 (1), 137 (3), 116 (22), 94 (80), 83 (10), 79 (30), 70 (11), 58
(100), 53 (4), 43 (47); IR, Vmax cm-1 3332, 1369, 1159, 1022; 13C NMR
δ 17.9 (C-7), 23.7 (C-9), 30.1 (C-10), 36.1 (C-5), 38.1 (C-1), 42.2 (C-
MATERIALS AND METHODS
1
2), 51.8 (C-4), 72.0 (C-3), 74.8 (C-8), 75.6 (C-6); H NMR (500.00
Materials. l-Menthol was purchased from Wako Pure Chemical
Industries, Ltd. Acetyl chloride and copper 8-hydroxyquinolate were
purchased from Tokyo Kasei Kohgyo Co., Ltd.
Microorganisms. Micronectriella niValis MAFF 305031 isolated
from diseased bent grass was used.
MHz, in CDCl3, TMS as internal standard) δ 1.02 (3H, d, J ) 7.0 Hz,
H-7), 1.22 (3H, s, H-9), 1.24 (3H, s, H-10), 3.21 (1H, ddd, J ) 10.5,
10.2, 4.3 Hz, H-6), 3.77 (1H, ddd, J ) 10.3, 10.1, 4.1 Hz, H-3).
Synthesis of Acetyl Esters. Acetyl chloride (1.2 equiv mol) was
added dropwise with stirring to l-menthol (1) (20.0 mg, 1.0 equiv mol),
(-)-6-hydroxymenthol (2) (20.0 mg, 0.5 equiv mol), (-)-1-hydroxy-
menthol (3) (20.0 mg, 1.0 equiv mol), and (+)-6,8-dihydroxymenthol
(4) (20.0 mg, 0.5 equiv mol), respectively, and pyridine (0.6-1.2 equiv
mol) in an organic solvent (CHCl3) (30 mL). Then, the solution was
heated at 25 °C for 1 h. After cooling, the mixture was poured into
water (100 mL) and the aqueous phase was extracted thoroughly with
CHCl3. The combined CHCl3 extracts were washed successively three
times with 5% HCl (100 mL each time), three times with 5% NaHCO3
(100 mL each time), and then with water (100 mL), dried with Na2-
SO4, and evaporated to give a colorless oil. This oil was chromato-
graphed on silica gel (eluent: hexane/EtOAc) to give each acetate (1Ac,
2Ac, 3Ac, and 4Ac) of compounds 1-4.
General Procedure. Thin-layer chromatography (TLC) was carried
out on a 0.25 mm thick silica gel plate (Merck silica gel 60 GF254) in
EtOAc/hexane (7:3). For gas chromatography (GC), a Hewlett-Packard
5890A gas chromatograph equipped with a flame ionization detector,
a DB-5 capillary column (30 m length, 0.25 mm i.d.), and a split
injection of 20:1 were used. Helium at a flow rate of 0.6 cm3 min-1
was used as a carrier gas. The oven temperature was programmed from
80 to 240 °C at 4 °C min-1. The injection temperature was 270 °C,
and the detector temperature was 280 °C. The peak area was integrated
with a Hewlett-Packard HP3396 series 2 integrator. For gas chroma-
tography-mass spectrometry (GC-MS), a Hewlett-Packard 5890A gas
chromatograph equipped with a split injector was combined by direct
coupling to a Hewlett-Packard 5972A mass spectrometer; an HP-5MS
capillary column (30 m length, 0.25 mm i.d.), and a split injection of
20:1 were used. Helium at 0.6 cm3 min-1 was used as a carrier gas.
The temperature of the ion source was 180 °C, and the electron energy
was 70 eV. The electron impact (EI) mode was used.
High-resolution mass spectrometry (HR-MS) was carried out with
a JEOL-HX100 (with a JEOL JAM-DA 5000 mass data system)
instrument. The infrared (IR) spectra were obtained with a Perkin-Elmer
1760X spectrometer. Chloroform was used as a solvent. Nuclear
magnetic resonance (NMR) spectra were obtained with a JEOL FX-
500 [500 MHz (1H) or 125.65 MHz (13C), chloroform/deuterium with
tetramethylsilane (1H) or chloroform (13C) as internal standard]
spectrometer.
(-)-(1S,3R,4S)-Menthyl monoacetate (1Ac): colorless oil; [R]25
D
-118.9° (CHCl3; c 1.0); HR-MS, m/z 198.1624; EI-MS, m/z (rel int)
183 (1), 165 (1), 138 (40), 123 (31), 109 (8), 95 (87), 82 (24), 81 (56),
67 (19), 55 (24), 43 (100); IR, Vmax cm-1 2956, 1732, 1462, 1372, 1241,
1051; 13C NMR δ 16.3 (C-9), 20.7 (C-10), 21.3 (CH3CO), 22.0 (C-7),
23.5 (C-5), 26.3 (C-8), 31.4 (C-1), 34.3 (C-6), 40.9 (C-2), 47.2 (C-4),
1
74.2 (C-3), 170.7 (CH3CO); H NMR (500.00 MHz, in CDCl3, TMS
as internal standard) δ 4.68 (1H, ddd, J ) 11.0, 10.9, 4.0 Hz, H-3).
(-)-(1S,3R,4S,6S)-6-Hydroxymenthyl diacetate (2Ac): colorless oil;
[R]25D -88.2° (CHCl3; c 1.0); HR-MS, m/z 256.1678; EI-MS, m/z (rel
int) 154 (3), 136 (30), 121 (21), 107 (5), 93 (40), 81 (8), 69 (8), 55
(10), 43 (100); IR, Vmax cm-1 2960, 1735, 1456, 1370, 1241, 1025; 13
C
NMR δ 16.2 (C-9), 17.8 (C-10), 20.5 (C-7), 21.1 and 21.2 (CH3CO),
26.2 (C-8), 29.0 (C-5), 35.4 (C-1), 38.1 (C-2), 45.2 (C-4), 72.4 (C-3),
77.1 (C-6), 170.6 and 170.8 (CH3CO); 1H NMR (500.00 MHz, in
Lytic Activity Test. M. niValis MAFF 305031 isolated form diseased
bent grass was previously grown on potato dextrose agar (PDA) medium

1882 J. Agric. Food Chem., Vol. 51, No. 7, 2003
Miyazawa et al.
Figure 2. Typical photographs of hyphal lysis stained with 5% cotton blue.
Figure 3. Lytic activity of 1−4 against M. nivalis MAFF 305031.
Figure 5. Lytic activity of 2, 4, 2Ac, 4Ac, and copper 8-hydroxyquinolate
against M. nivalis MAFF 305031.
(-)-(1S,3R,4R,6S)-6,8-Dihydroxymenthyl 3,6-diacetate (4Ac): color-
less oil; [R]25D -35.8° (CHCl3; c 1.0); HR-MS, m/z 272.1621; EI-MS,
m/z (rel int) 197 (1), 152 (2), 138 (1), 134 (6), 109 (3), 100 (2), 94
(77), 79 (29), 69 (3), 59 (21), 55 (7), 43 (100); IR, Vmax cm-1 3472,
2928, 1736, 1456, 1371, 1241, 1025; 13C NMR δ 17.7 (C-7), 21.1 and
21.5 (CH3CO), 22.7 (C-9), 28.3 (C-10), 35.3 (C-1), 36.1 (C-5), 38.1
(C-2), 49.6 (C-4), 72.5 (C-3), 74.1 (C-8), 76.5 (C-6), 170.0 and 170.7
(CH3CO); 1H NMR (500.00 MHz, in CDCl3, TMS as internal standard)
δ 4.45 (1H, ddd, J ) 10.2, 10.3, 4.1 Hz, H-6), 4.80 (1H, ddd, J )
10.0, 10.1, 4.0 Hz, H-3).
RESULTS AND DISCUSSION
The existence ratio of stained hyphae and no stain was
counted using the microscope (Figure 2). Compounds 2, 3, and
4 had 85.0, 63.9, and 81.9% lytic activity, respectively, at the
concentration of 0.2 mg/mL. On the other hand, the lytic activity
of 1 was 45.7% at 0.2 mg/mL. These compounds each showed
higher lytic activity as their concentration was increased (Figure
3). Especially, compounds 2 and 4 had 57.1 and 68.1% lytic
activities at 0.05 mg/mL.
Acetyl esters 1Ac, 2Ac, 3Ac, and 4Ac showed high lytic
activity against M. niValis. Compounds 1Ac, 2Ac, 3Ac, and 4Ac
had 51.2, 91.5, 66.0, and 95.2% lytic activities at a concentration
of 0.2 mg/mL and showed higher lytic activity compared with
compounds 1, 2, 3, and 4. Each of these acetyl esters showed
higher lytic activity as their concentration was increased (Figure
4). In addition, copper 8-hydroxyquinolate (as a standard snow
blight control agent) was tested and also compared with the
high lytic activity compounds 2, 2Ac, 4, and 4Ac (Figure 5).
Copper 8-hydroxyquinolate had 87.5% activity at 0.2 mg/mL.
However, 2Ac had a higher lytic activity compared with copper
8-hydroxyquinolate at 0.05-0.2 mg/mL, and 4Ac had a higher
activity at 0.1-0.2 mg/mL.
Figure 4. Lytic activity of 1Ac, 2Ac, 3Ac, and 4Ac against M. nivalis
MAFF 305031.
CDCl3, TMS as internal standard) δ 4.45(1H, ddd, J ) 11.0, 10.7, 3.9
Hz, H-6), 4.68 (1H, ddd, J ) 11.2, 10.9, 3.9 Hz, H-3).
(-)-(1S,3R,4S)-1-Hydroxymenthyl 3-monoacetate (3Ac): colorless
oil; [R]25D -97.8° (CHCl3; c 1.0); HR-MS, m/z 214.1562; EI-MS, m/z
(rel int) 154 (7), 139 (13), 136 (13), 121 (9), 111 (17), 97 (10), 87
(10), 81 (9), 69 (14), 55 (13), 43 (100); IR, Vmax cm-1 3450, 2959,
1714, 1460, 1371, 1244, 1026; 13C NMR δ 16.5 (C-9), 19.4 (C-5),
21.0 (C-10), 26.3 (C-8), 31.4 (C-7), 37.8 (C-6), 44.6 (C-4), 47.0 (C-
2), 71.0 (C-3), 71.9 (C-1); 13C NMR δ 16.5 (C-9), 19.4 (C-5), 20.7
(C-10), 21.3 (CH3CO), 26.3 (C-8), 31.4 (C-7), 37.8 (C-6), 44.6 (C-4),
1
47.0 (C-2), 71.0 (C-3), 77.9 (C-1), 170.6 (CH3CO); H NMR (500.00
MHz, in CDCl3, TMS as internal standard) δ 5.00 (1H, ddd, J ) 10.0,
10.1, 4.0 Hz, H-3).

Lytic Activity of l-Menthol
J. Agric. Food Chem., Vol. 51, No. 7, 2003 1883
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From the above results, compounds 2-4 showed high lytic
activity compared with 1, and 2 and 4 showed high lytic activity
compared with 3. These results indicated that the increasing
hydroxyl group at 1 and hydroxylation at the C-6 position are
important factors for lytic activity against M. niValis. On the
other hand, the acetyl esters 1Ac, 2Ac, 3Ac, and 4Ac showed
further high lytic activity compared with the alcohols 1-4.
These results also indicated that the acetylation of the hydroxyl
group is an important factor for lytic activity. We considered
that hydroxylation at the C-6 position and acetylation of the
C-6 position hydroxyl group are the most important factors for
lytic activity. Finally, 2Ac and 4Ac had equal levels of lytic
activity compared with copper 8-hydroxyquinolate. On the basis
of these findings, 2Ac and 4Ac could be potential alternative
methods to the organic copper compound (copper 8-hydroxy-
quinolate) for the control of snow blight disease.
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Received for review October 29, 2002. Revised manuscript received
January 7, 2003. Accepted January 11, 2003.
JF0210831