Biotransformation of (+)- and (-)-menthol by the larvae of common cutworm (Spodoptera litura)

Article abstract of DOI:10.1021/jf980924u

(+)-Menthol was mixed in an artificial diet at a concentration of 1 mg/g of diet, and the diet was fed to the last instar larvae of common cutworm (Spodoptera litura). Metabolites were recovered from grass and analyzed spectroscopically. (+)-Menthol was transformed mainly to (+)-7- hydroxymenthol. Similarly, (-)-menthol was transformed mainly to (-)-7- hydroxymenthol. The C-7 position of (+)and (-)-menthol was preferentially oxidized.

Full text of DOI:10.1021/jf980924u

3938
J. Agric. Food Chem. 1999, 47, 3938−3940
Biotr a n sfor m a tion of (+)- a n d (-)-Men th ol by th e La r va e of Com m on
Cu tw or m (Spod opter a litu r a )
Mitsuo Miyazawa,* Shingo Kumagae, and Hiromu Kameoka
Department of Applied Chemistry, Faculty of Science and Engineering, Kinki University, Kowakae,
Higashiosaka-shi, Osaka 577-8502, J apan
(+)-Menthol was mixed in an artificial diet at a concentration of 1 mg/g of diet, and the diet was fed
to the last instar larvae of common cutworm (Spodoptera litura). Metabolites were recovered from
frass and analyzed spectroscopically. (+)-Menthol was transformed mainly to (+)-7-hydroxymenthol.
Similarly, (-)-menthol was transformed mainly to (-)-7-hydroxymenthol. The C-7 position of (+)-
and (-)-menthol was preferentially oxidized.
Keyw or d s: Common cutworm; Spodoptera litura; biotransformation; menthol; (+)-7-hydroxy-
menthol; (-)-7-hydroxymenthol
INTRODUCTION
Ga s Ch r om a togr a p h y/Ma ss Sp ectr om etr y (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. The same type of column
and the same temperature program as just described for GC
were used. Helium at 1 mL/min was used as a carrier gas.
The temperature of the ion source was 280 °C, and the electron
energy was 70 eV. The electron impact (EI) mode was used.
In fr a r ed (IR) Sp ectr oscop y. The IR spectra were ob-
tained with a Perkin-Elmer 1760X spectrometer. CHCl3 was
used as a solvent.
Terpenoids are known as not only raw materials for
flavor and fragrance but also biologically active sub-
stances. A great majority of biologically active terpe-
noids are produced as plant secondary metabolites, and
these terpenoids have been shown to have biological
activity against plants, microorganisms, and insects.
Various attempts have been made to search for new
biologically active terpenoids. Biotransformation is one
of way to produce biologically active terpenoids.
Previously we reported biotransformation of R-ter-
pinene and (+)- and (-)-limonene by the larvae of
common cutworm (Spodoptera litura) (Miyazawa et al.,
1996, 1998). Consequently, we revealed that the C-7
position (allylic methyl group) of R-terpinene was pref-
erentially oxidized. The results indicated that the
intestinal bacteria probably participated in the metabo-
lism of R-terpinene. (+)- and (-)-limonene were oxidized
at the 8,9-double bond and the C-7 position (allylic
methyl group). In the present paper, the biotransfor-
mation of (+)- and (-)-menthol (1) by the larvae of S.
litura was investigated for the purpose of estimating
possible metabolic pathways in insects. Compound 1 is
a major component of various mint oils. It has a pleasant
odor and taste and is widely used to flavor foods and
oral pharmaceutical preparations. This paper deals with
the metabolism from frass and the metabolic pathways.
Nu clea r Ma gn et ic R eson a n ce (NMR ) Sp ect r oscop y.
The NMR spectra were obtained with a J EOL GSX-270 (270.05
1
MHz, H; 67.80 MHz, 13C) spectrometer.
Rea r in g of La r va e. The larvae of S. litura were reared in
plastic cases (200 × 300 mm wide, 100 mm high, 100 larvae/
case) covered with a nylon mesh screen. The rearing conditions
were as follows: 25 °C, 70% relative humidity, and constant
light. A commercial diet (Insecta LF; Nihon Nosan Kogyo Co.,
Ltd.) was given to the larvae from the first instar. From the
fourth instar, the diet was changed to an artificial diet
composed of kidney beans (100 g), brewer’s dried yeast (40 g),
ascorbic acid (4 g), agar (12 g), and water (600 mL; Yushima
et al., 1991).
Ad m in istr a tion of (+)-Men th ol (1). The artificial diet
without the agar was mixed with a blender. Five hundred
milligrams of (+)-1 was then added directly into the blender
at 1 mg/g of diet. Agar was dissolved in water and boiled and
then added into the blender. The diet was then mixed and
cooled in a tray (220 × 310 mm wide, 30 mm high). The diet
containing (+)-1 was stored in a refrigerator until the time of
administration. The last instar larvae (average weight ) 0.5
g) were moved into new cases (100 larvae/case), and the diet
was fed to the larvae in limited amounts. Groups of 500 larvae
were fed the diet containing (+)-1 (actually 200-300 mg, 0.4-
0.6 mg for a body) for 2 days, and then the artificial diet not
containing (+)-1 was fed to the larvae for an additional 2 days.
Frass was collected daily (total of 4 days) and stored in a
solution of CH2Cl2 (500 mL). (-)-1 was administered to 500
larvae in the same manner. For diet and frass separation, the
fresh frass was extracted as soon as the last instar larvae
excreted.
MATERIALS AND METHODS
Ch em ica ls. The (+)- and (-)-forms of menthol (1) were
purchased from Taiyo Perfume Co., Ltd. (Osaka, J apan).
Ga s Ch r om a togr a p h y (GC). A Hewlett-Packard 5890A
gas chromatograph equipped with a flame ionization detector,
an HP-5MS 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 1
mL/min was used as a carrier gas. The oven temperature was
programmed from 80 to 240 °C at 4 °C/min. The injector and
detector temperatures were 250 °C. The peak area was
integrated with a Hewlett-Packard HP3396 Series 2 integra-
tor.
Isolation an d Iden tification of Metabolites fr om Fr ass.
The frass were extracted three times with CH2Cl2 each time.
The extract solution was evaporated under reduced pressure,
and 2401 mg of extract was obtained. The extract was
distributed between 5% NaHCO3 (aq) and CH2Cl2, the CH2-
Cl2 phase was evaporated, and the neutral fraction (1454 mg)
* Author to whom correspondence should be addressed
(telephone +81-6-6721-2332; fax +81-6-6727-4301).
10.1021/jf980924u CCC: $18.00 © 1999 American Chemical Society
Published on Web 08/18/1999

Biotransformation of Menthol by Cutworm Larvae
J. Agric. Food Chem., Vol. 47, No. 9, 1999 3939
was obtained. The neutral fraction was analyzed by GC/MS;
metabolite (+)-2 occurred in this fraction. The alkali phase
was acidified with 1 N HCl and distributed between water and
CH2Cl2. The CH2Cl2 phase was evaporated, and the acidic
fraction (716 mg) was obtained. The neutral fraction was
subjected to silica gel open-column chromatography (silica gel
60, 230-400 mesh, Merck) with a 9:1 n-hexane/EtOAc solvent
system, and (+)-2 (213 mg) was isolated. Metabolite (+)-2 was
identified by a comparison of established MS, IR, and NMR
data.
Sch em e 1. Meta bolites of (+)- a n d (-)-Men th ol (1) by
th e La r va e of S. litu r a a
(+)-7-Hyd r oxym en th ol (2) was obtained as a crystal: mp
110-101 °C; [R]D +28.4° (CHCl3, c 0.84); EIMS, m/ z (rel
intensity) 172 (9), 93 (19), 71 (21), 43 (100); IR (vmax, cm-1
)
1
3318, 1464, 1386, 1020; H NMR (CDCl3) δ 0.82 (3H, d, Me-
9), 0.94 (3H, d, Me-10), 3.48 (3H, m, H-3 and H-7); 13C NMR
(CDCl3) δ 16.0 (t, C-9), 20.9 (t, C-10), 22.7 (q, C-5), 25.8 (d,
C-8), 28.7 (q, C-6), 39.1 (q, C-2), 39.4 (d, C-1), 50.3 (d, C-4),
67.8 (q, C-7), 71.2 (d, C-3).
Biotr a n sfor m a tion of (-)-Men th ol (1). The same proce-
dure as described for (+)-1 was used. Substrate (-)-1 was
transformed to metabolite (-)-2 (167 mg).
(-)-7-Hyd r oxym en th ol (2) was obtained as a crystal: [R]D
-38.7° (CHCl3, c 0.16); spectral data of the enantiomer (-)-2
were identical to those of (+)-2.
In cu ba tion of In testin a l Ba cter ia w ith (+)-Men th ol
(1). This experiment was intentionally carried out under sterile
conditions. Petri dishes, pipets, and solutions were autoclaved.
A GAM broth (Nissui Pharmaceutical Co., Ltd.) was adjusted
to pH 9.0 and placed in Petri dishes at 10 mL/Petri dish. The
fresh frass (5 g) of the last instar larvae were suspended in
physiological saline (100 mL), and the suspension (1 mL) was
pipetted in the medium. The medium without frass was also
prepared for a blank experiment. These media were incubated
(25 °C, darkness, 2 days) under aerobic and anaerobic condi-
tions. After growth of bacteria, 1 (10 mg/Petri dish) was added
to the medium and the incubation was continued. The per-
centage of metabolites in the medium was determined 12, 24,
and 48 h after addition of 1. The medium was acidified with 1
N HCl and distributed between Et2O and saturated solution
of salt. The Et2O phase was evaporated, and the extract was
obtained. For the quantitative analysis of metabolites, the GC
analysis was used as an internal standard with 1. (-)-1 was
tested as well as (+)-1.
a
Percentage was calculated from the peak area in the GC
spectra of the extract of frass. 100% was defined as total
metabolites of each 1.
of metabolite was (+)-2 (90%). Percentage was calcu-
lated from the peak area in the GC spectra of the extract
of frass. 100% was defined as total metabolites of (+)-
1. Substrate (+)-1 and intermediary metabolites (alco-
hol, aldehyde, and epoxide) were not detected in the
frass by GC analysis. Metabolite (+)-2 was produced by
oxidation at the C-7 position (+)-1.
In the biotransformation of (-)-1, similarly, the one
metabolite isolated from the frass was identified as (-)-
7-hydroxymenthol (2). The percentage conversion of
metabolite was (-)-2 (86%). These results were similar
to those for (+)-1.
In testin a l Ba cter ia . A previous paper described the
participation of intestinal bacteria in the metabolism
of R-terpinene (Miyazawa et al., 1996). The aerobically
active intestinal bacteria transformed R-terpinene to
p-mentha-1,3-dien-7-ol, and the anaerobically active
intestinal bacteria transformed R-terpinene to p-cymene.
In the present study, the in vitro metabolism of (+)- and
(-)-1 by intestinal bacteria was also examined in a
manner similar to that of the previous paper. However,
(+)- and (-)-1 were not metabolized at all (no reaction).
These results suggested that the intestinal bacteria did
not participate in the metabolism of (+)- and (-)-1. The
difference of reaction between (+)- and (-)-1 and R-ter-
pinene was suggested to be due to the difference of
substrate.
Met a b olic P a t h w a ys. In the present study of
biotransformation of (+)- and (-)-1, the larvae trans-
formed (+)-1 to (+)-2; similarly, the larvae transformed
(-)-1 to (-)-2 (Scheme 1). The C-7 position of (+)- and
(-)-1 were preferentially oxidized like the biotransfor-
mation of R-terpinene. These results indicate C-7 is
rather the preferred position for oxidation.
RESULTS AND DISCUSSION
Meta bolites fr om F r a ss. Biotransformation by the
larvae of S. litura was observed as follows: substrate
was administered to the larvae through their diet;
metabolite was then detected and isolated from the frass
of larvae. In a previous paper, R-terpinene was mixed
in the diet of larvae at a high concentration (10 mg/g of
diet) to increase the production of potential metabolites
(Miyazawa et al., 1996). Although alcohols were detected
by GC analysis, intermediary metabolites (alcohols and
aldehydes) were not isolated. This suggested that
intermediary metabolites were rarely excreted into the
frass. In the present study, a concentration of 1 mg/g of
diet was therefore chosen as optimum for administra-
tion. “Optimum” means the concentration results in
complete consumption of substrate. The larvae that
were fed the diet without substrate were used as control,
and the extract of frass was analyzed by GC. The result
was that terpenoids in the frass were not observed. For
the consumption of substrate in the diet observed, we
varied the quantity of substrate in the diet by the
internal standard method. The result was that con-
sumption of (+)-menthol (1) was 10%. The result for
(-)-1 was 14%.
Compound 1 is the most important and widespread
terpene known; however, there are a few reports on the
biotransformation of 1 by other organisms. It seems
natural to obtain different metabolites with different
species of organisms: (+)- and (-)-1 were barely con-
verted to their corresponding ketones by the cultured
cells of Nicotiana tabacum (Suga et al., 1987); glycosyl-
In the biotransformation of (+)-1, the one metabolite
isolated from the frass was identified as (+)-7-hydroxy-
menthol (2) (see Scheme 1). The percentage conversion

3940 J. Agric. Food Chem., Vol. 47, No. 9, 1999
Miyazawa et al.
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ation at the hydroxyl is the main metabolic pathway in
the biotransformation of (-)-1 by Eucalyptus perriniana
cultured cells (Furuya et al., 1989); hydroxylation at the
C-7 and C-9 positions is the main metabolic pathway
in the biotransformation of 1 by Aspergillus species
(Asakawa et al., 1991); and oxidation of the methyl and
isopropyl groups as major urinary metabolites occurs
after the daily administration of menthol to rats (Madha-
va Madyastha et al., 1988). The present study is the
first report of the C-7 position of compound 1 being
hydroxylated to a high degree of efficiency.
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Received for review August 19, 1998. Revised manuscript
received J une 3, 1999. Accepted J une 23, 1999.
J F980924U