Transformation of a monoterpene ketone, (R)-(+)-pulegone, a potent hepatotoxin, in Mucor piriformis

Article abstract of DOI:10.1021/jf980164n

Biotransformation of a monoterpene ketone, (R)-(+)-pulegone (I), a potent hepatotoxin, was studied using a fungal strain, Mucor piriformis. Eight metabolites, namely, 5-hydroxypulegone (II), piperitenone (III), 6- hydroxypulegone (IV), 3-hydroxypulegone (V), 5-methyl-2-(1-hydroxy-1- methylethyl)-2-cyclohexene-1-one (VI), 3-hydroxyisopulegone (VII), 7- hydroxypiperitenone (VIII), and 7-hydroxypulegone (IX), have been isolated from the fermentation medium and identified. GC analysis of the metabolites indicated that II was the major metabolite formed. The organism initiates transformation either by hydroxylation at the C-5 position or by hydroxylation of the ring methylenes, the former being the major activity. On the basis of the identification of the metabolites, pathways for the biotransformation of (R)-(+)-pulegone have been proposed. The mode of transformation of (S)-(-)-pulegone by this organism was shown to be similar to that of its (R)-(+)-enantiomer. When isopulegone (X) was used as the substrate, the organism isomerized it to pulegone (I), which was then transformed to metabolites II-IX.

Full text of DOI:10.1021/jf980164n

J. Agric. Food Chem. 1999, 47, 1203−1207
1203
Tr a n sfor m a tion of a Mon oter p en e Keton e, (R)-(+)-P u legon e, a
P oten t Hep a totoxin , in Mu cor pir ifor m is
K. M. Madyastha*,†,‡ and H. V. Thulasiram†
Department of Organic Chemistry, Indian Institute of Science, Bangalore 560012, India, and Chemical
Biology Unit, J awaharlal Nehru Centre for Advanced Scientific Research, Bangalore 560064, India
Biotransformation of a monoterpene ketone, (R)-(+)-pulegone (I), a potent hepatotoxin, was studied
using a fungal strain, Mucor piriformis. Eight metabolites, namely, 5-hydroxypulegone (II),
piperitenone (III), 6-hydroxypulegone (IV), 3-hydroxypulegone (V), 5-methyl-2-(1-hydroxy-1-meth-
ylethyl)-2-cyclohexene-1-one (VI), 3-hydroxyisopulegone (VII), 7-hydroxypiperitenone (VIII), and
7-hydroxypulegone (IX), have been isolated from the fermentation medium and identified. GC
analysis of the metabolites indicated that II was the major metabolite formed. The organism initiates
transformation either by hydroxylation at the C-5 position or by hydroxylation of the ring methylenes,
the former being the major activity. On the basis of the identification of the metabolites, pathways
for the biotransformation of (R)-(+)-pulegone have been proposed. The mode of transformation of
(S)-(-)-pulegone by this organism was shown to be similar to that of its (R)-(+)-enantiomer. When
isopulegone (X) was used as the substrate, the organism isomerized it to pulegone (I), which was
then transformed to metabolites II-IX.
Keyw or d s: (R)-(+)- and (S)-(-)-pulegone; monoterpene ketone; biotransformation; fungal system;
hepatotoxin; metabolites
INTRODUCTION
the mammalian system. Earlier, Mucor piriformis was
shown to carry out novel and preparatively useful
transformations of steroids (Madyastha and J oseph,
1994, 1995) and alkaloids (Madyastha and Reddy, 1994).
This fungal strain very efficiently transforms (R)-(+)-
and (S)-(-)-pulegone to various metabolites. In fact, M.
piriformis is more versatile in its ability to transform
(R)-(+)-pulegone than some of the fungi tested earlier
for carrying out the biotransformation of (R)-(+)-pule-
gone (Mustapha et al., 1992).
We report here the isolation and identification of eight
metabolites, of which five of them have not been
reported earlier. The mode of transformation of (R)-
(+)-pulegone (I), its (S)-enantiomer, and isopulegone by
M. piriformis are also compared.
(R)-(+)-Pulegone (I), a monoterpene ketone, is the
major constituent of pennyroyal oil from Mentha
pulegium. The oil is used as a fragrance component and
a flavoring agent. Studies carried out with animals
have shown that (R)-(+)-pulegone is both hepatotoxic
and pneumotoxic (Gordon et al., 1982; Thorup et al.,
1983; Moorthy et al., 1991). Surprisingly, (S)-(-)-
pulegone is far less toxic than its (R)-enantiomer (Gor-
don et al., 1982). The metabolic fate of (R)-(+)-pulegone
(I) in rats has been studied in great detail, and these
studies have indicated the existence of two major
pathways for its biotransformation. One of the path-
ways is initiated through regiospecific hydroxylation of
I to 9-hydroxypulegone, which is further converted to
menthofuran (Gordon et al., 1987; Madyastha and Paul
Raj, 1990). The other major pathway involves stereo-
selective hydroxylation of I to 5-hydroxypulegone, which
upon dehydration yields piperitenone (Moorthy et al.,
1989; Madyastha and Paul Raj, 1993).
Whereas considerable work has been carried out on
the mammalian degradation of (R)-(+)-pulegone, very
little is known regarding the microbial metabolism of
this hepatotoxin. It is of interest to know whether
microbes bring about transformation in (R)-(+)-pulegone
in a manner similar to what has been observed in the
mammalian system and, if so, then it would be easier
to prepare some of the reactive metabolites in large
quantities that can be used for their detailed study in
MATERIALS AND METHODS
Ma ter ia ls. (R)-(+)-Pulegone (I) and isopulegol were ob-
tained from Aldrich Chemical Co. (Milwaukee, WI). (S)-(-)-
Pulegone was synthesized as reported earlier (Corey et al.,
1976). Isopulegone was prepared by the pyridinium chloro-
chromate oxidation of isopulegol (Corey and Suggs, 1975).
Isopulegone obtained was purified by column chromatography
over silica gel using 5% ethyl acetate in hexane. 1H NMR
(CDCl3) spectrum showed signals at δ 5.14 (s, 2H, olefinic
protons), 2.18-1.8 (m, 8H, ring protons), 1.78 (s, 3H, allylic
methyl), and 0.98 (d, 3H, J ) 7.2 Hz, ring methyl). Mass
spectrum gave the following: m/z 152 (M+), 137 (M+ - CH3)
109 (M+ - C3H7), 93 (M+ - C3H7O), 81 (M+ - C4H7O), and 67
(M+ - C5H9O).
Micr oor ga n ism . The organism used in the present study
was isolated from soil and identified as Mucor piriformis
(Madyastha and Srivatsan, 1987). It was maintained and
propagated as reported earlier (Madyastha and Srivatsan
1987).
* Author to whom correspondence should be addressed
(telephone 091-080-3092541; fax 091-080-3341683; e-mail
kmm@orgchem.iisc.ernet.in).
† Indian Institute of Science.
‡ J awaharlal Nehru Centre for Advanced Scientific Re-
search.
Con d ition s for F er m en ta tion . Fermentations were car-
ried out in modified Czapek Dox medium (Prema and Bhat-
10.1021/jf980164n CCC: $18.00 © 1999 American Chemical Society
Published on Web 02/10/1999

1204 J. Agric. Food Chem., Vol. 47, No. 3, 1999
Madyastha and Thulasiram
tacharyya, 1962). The pH of the medium was adjusted to 6.8-
7.0 with 1 N NaOH. Flasks (500 mL) containing 100 mL of
sterile medium were inoculated with 1 mL of a spore suspen-
sion from a 5-day-old culture grown on potato dextrose agar
(PDA) slants and were incubated at 29-30 °C on a rotary
shaker (220 rpm) for 36 h. After this growth period, the pH
of the medium was again adjusted to 7.0 by the addition of
sterile 1 N NaOH, 60 mg of (R)-(+)-pulegone in acetone (0.2
mL) was added to each flask, and the incubation was continued
for an additional period of 48 h (increase in the substrate
concentration considerably decreased the yields of the me-
tabolites). A control experiment was also run with the
substrate but without the organism. In a similar way fer-
mentation was carried out using (S)-(-)-pulegone and iso-
pulegone as substrates.
In time course experiments, incubations were carried out
for 24, 48, 72, and 96 h and the metabolites formed at the end
of each incubation period were monitored by GC analysis.
Quantification was made by measuring the area under the
peak. The levels of different metabolites formed were deter-
mined on the basis of the area under the respective peaks and
compared with the standard graphs obtained for each metabo-
lite by injecting known amounts of these metabolites. All
analyses were carried out under identical conditions.
Extr a ction of Meta bolites. At the end of the incubation
period, the contents from all of the flasks were pooled, the pH
was adjusted to 5.5-6.0, and the mixture was filtered. The
filtrate (broth) and mycelia were separately extracted with
CHCl3. The two CHCl3 extracts were combined, concentrated,
and separated into acidic and neutral fractions by treatment
with 5% sodium bicarbonate solution. The bicarbonate phase
was acidified and re-extracted with CHCl3. The acidic fraction
contained compounds derived from the organism and hence
was not processed further. The neutral fraction was subjected
to column chromatography and preparative TLC to isolate the
metabolites in the pure form.
F igu r e 1. Transformation of (R)-(+)-pulegone by M. pirifor-
mis.
the presence of at least eight compounds that were
absent in the control experiment. This fraction was
subjected to column chromatography on silica gel (80
g), and the metabolites were eluted with hexane/ethyl
acetate mixtures. Elution of the column with hexane
yielded a fraction containing the least polar compound
(Rf 0.79, system I). The GC analysis of this fraction
showed the presence of one major peak (tR ) 3.7 min)
and one minor peak (tR ) 2.4 min). The major and
minor peaks were enhanced when mixed with pulegone
(I) and isopulegone (X), respectively. The mass spectral
analysis of these two peaks (tR ) 3.7 and 2.4 min)
corresponded well with those of authentic pulegone (I)
and isopulegone (X).
Ch r om a togr a p h y. Thin-layer chromatography (TLC) was
carried out on silica gel G-coated plates (0.25 mm for analyti-
cal; 0.75 mm for preparative) developed with either hexanes/
ethyl acetate (8:2, v/v, system I) or chloroform/methanol (9.7:
0.3, v/v, system II). Compounds were visualized by spraying
with 3% vanillin in 1% methanolic H2SO4, followed by heating
at 100 °C for 5-10 min. GC analyses were carried out on a
Shimadzu model 14A instrument equipped with a hydrogen
flame ionization detector and a Shimadzu HR-1 wide bore
capillary column (15 cm × 0.5 mm). N2 was used as the carrier
The fraction eluted with ethyl acetate (0.5%) in
hexane contained a mixture of two compounds with Rf
values of 0.62 (IV) and 0.59 (III) (system I), which were
separated and purified by repeated preparative TLC
(system I). Compound IV showed an optical rotation
of +66 (c 1.0, CHCl3) and UV absorption maximum at
254 nm. The IR, 1H NMR, and MS data for this
compound are given in Table 1. From the spectral
characteristics, the compound was identified as 6-hy-
droxypulegone (IV, Figure 1). The spectral data agreed
with an earlier report for this compound (Imamura et
al., 1975). Earlier studies also suggested that the C-5
methyl group and the C-6 hydroxyl group in IV are cis
to each other (Imamura et al., 1975). Compound III (Rf
0.59, system I; tR ) 6.8 min) showed UV absorption
maxima at 276 and 244 nm. From the spectral char-
acteristics (Table 1), the compound was identified as
piperitenone (III, Figure 1). The identity of the me-
tabolite was further confirmed by comparing the NMR
and MS data with those of an authentic compound
prepared as reported earlier (Nakanishi et al., 1980).
Elution of the column with 3% ethyl acetate in hexane
gave fractions containing mainly one compound (Rf 0.33,
system I; tR ) 4.9 min), which was further purified by
preparative TLC (system I). The IR, 1H NMR, and MS
gas at
a flow rate of 30 mL/min. Initially the column
temperature was maintained at 80 °C for 10 min, after which
time the temperature was raised at 5 °C/min to 150 °C and
maintained at 150 °C for 5 min.
Hyd r ogen a tion of Com p ou n d s VIII a n d IX. The com-
pound (5.0 mg, VIII/IX) was dissolved in dry methanol (0.5
mL), and a catalytic amount of palladium charcoal was added.
The mixture was stirred for 2 h at room temperature under
hydrogen atmosphere. The reaction mixture was then diluted
with chloroform, passed through a Celite bed, concentrated,
and subjected to column chromatography over silica gel, and
the compound was eluted using 5% ethyl acetate in hexane.
Sp ectr a l Stu d ies. Infrared (IR) spectra were recorded
either on a Perkin-Elmer model 781 or on a Hitachi 270-50
spectrophotometer. 1H nuclear magnetic resonance (NMR)
spectra were recorded on a J EOL FT-90 MHz spectrometer.
Chemical shifts are reported in parts per million, with respect
to tetramethylsilane as the internal standard. Mass spectra
(MS) were recorded on a J EOL-J MS-DX 303 instrument with
J MA-DA 5000 data system.
RESULTS
A batch of 50 flasks was inoculated with M. piriformis,
and at the end of 2 days after the addition of substrate,
the contents of all the flasks were pooled and processed
as described under Materials and Methods. The neutral
fraction (2.6 g) upon TLC analysis (system I) showed
26
data for this compound {VI, [R]D ) -32° (c 1.0, in
CHCl3)} are shown in Table 1. On the basis of the
spectral characteristics, the compound was assigned the
structure 5-methyl-2-(1-hydroxy-1-methylethyl)-2-cyclo-

Transformation of (R)-(+)-Pulegone in Mucor piriformis
Ta ble 1. Sp ectr a l Da ta for Meta bolites II-IXa
J. Agric. Food Chem., Vol. 47, No. 3, 1999 1205
compd
IR (neat) γmax
1H NMR (δ) CDCl3
MS (LR and HRMS)
II
3400 cm-1 (-OH)
2.48 (4H, m, H-3 and H-6), 1.95 (3H, s,
H-9), 1.8-2.0 (2H, m, H-4), 1.8 (3H, s,
m/z: 168 (M+, 31%), 150 (M+ - H2O, 20%), 135 (M+
-
1665 and 1600 cm-1
CH3 - H2O, 30%), 107 (M+ - C2H5O, 39%), 67 (M+
-
(conjugated carbonyl) H-10), 1.25 (3H, s, H-7)
C5H9O2, 48%)
HRMS: C10H16O2 requires 168.1150, found 168.1154
III
IV
1640 and 1595 cm-1
(conjugated carbonyl) (2H, t, H-3), 2.0 (3H, s, H-7), 1.85 (3H,
s, H-9), 1.8 (3H, s, H-10)
5.8 (1H, br s, H-6), 2.6 (2H, t, H-4), 2.2
m/z: 150 (M+, 100%), 135 (M+ - CH3, 50%), 122 (M+
CO, 12%), 91 (M+ - C3H7O, 17%)
-
3460 cm-1 (-OH)
4.2 (1H, d, J ) 6.4 Hz, H-6), 3.98 (1H,
br s, 6-OH), 2.2-2.6 (4H, m, H-3 and
m/z: 168 (M+, 50%), 150 (M+ - H2O, 13%), 125 (M+
C3H7, 100%)
-
1675 and 1610 cm-1
(conjugated carbonyl) H-4), 1.98 (3H, s, H-9), 1.83 (3H, s,
HRMS: C10H16O2 requires 168.1150, found 168.1140
H-10), 0.88 (3H, d, J ) 7.7 Hz, H-7)
V
3400 cm-1 (-OH)
4.95 (1H, t, H-3), 1.92-2.6 (5H, m,
m/z: 168 (M+, 9%), 150 (M+ - H2O, 100%), 135 (M+
CH3 - H2O, 49%)
-
1670 and 1600 cm-1
H-4, H-5, and H-6), 1.9 (3H, s, H-9),
(conjugated carbonyl) 1.85 (3H, s, H-10), 0.95 (3H, d, J ) 6.4
HRMS: C10H16O2 requires 168.1150, found 168.1144
Hz, H-7)
VI
VII
3420 cm-1 (-OH)
6.88 (1H, dd, J ) 5.6 and 2.4 Hz, H-3),
4.38 (1H, br s 8-OH), 2.1-2.5 (5H, m,
m/z: 168 (M+, 4%), 153 (M+ - CH3, 100%), 150 (M+
H2O, 26%), 135 (M+ - CH3 - H2O, 29%), 109 (M+
-
655 cm-1
-
(conjugated carbonyl) H-4, H-5, and H-6), 1.4 (6H, s, H-9 and C3H7O, 19%)
H-10), 1.07 (3H, d, J ) 5.98 Hz, H-7)
HRMS: C10H16O2 requires 168.1150, found 168.1157
3400 cm-1 (-OH)
700 cm-1
(carbonyl)
5.28 and 4.98 (2H, 2s, H-9), 3.8 (1H,
m, H-3), 2.9 (1H, d, J ) 11.6 Hz, H-2),
2.48-1.7 (5H, m, H-4, H-5 and H-6),
1.88 (3H, s, H-10), 1.1 (3H, d, J ) 7.7
Hz, H-7)
m/z: 168 (M+, 20%), 153 (M+ - CH3, 30%), 150 (M+
-
H2O, 43%), 135 (M+ - CH3 - H2O, 30%), 109 (M+
C3H7O, 58%)
-
HRMS: C10H16O2 requires 168.1150, found 168.1152
VIII
3400 cm-1 (-OH)
6.2 (1H, s, H-6), 4.3 (2H, s, H-7), 2.68
(2H, t, H-4), 2.28 (2H, t, H-3), 2.15
m/z: 166 (M+, 100%), 135 (M+ - CH2OH, 31%), 123 (M+
C3H7, 29%)
-
1650 and 1590 cm-1
(conjugated carbonyl) (3H, s, H-9), 1.85 (3H, s, H-10)
HRMS: C10H14O2 requires 166.0994, found 166.0998
IX
3400 cm-1 (-OH)
3.55 (2H, d, J ) 5.1 Hz, H-7), 2.6-2.05
(7H, m, ring protons), 2.0 (3H, s, H-9),
m/z: 168 (M+, 29%), 137 (M+ - CH2OH, 12%), 84 (M+
C5H8O, 100%)
-
1660 and 1590 cm1
(conjugated carbonyl) 1.8 (3H, s, H-10), 1.68 (1H, s, 7-OH)
HRMS: C10H16O2 requires 168.1150, found 168.1142
a
Details are as mentioned under methods.
hexene-1-one (VI, Figure 1). The spectral data agreed
with those given in an earlier paper on this compound
(Nagasawa et al., 1975).
of 0.30 and 0.26 (system II). These two compounds were
separated and purified by preparative TLC (system II).
The IR, 1H NMR, and mass spectral data for these
compounds (VIII, IX) are given in Table 1. From the
spectral characteristics, the compounds with Rf 0.30 and
0.26 (system II) were identified as 7-hydroxypulegone
(VIII, Figure 1) and 7-hydroxypiperitenone (IX, Figure
1), respectively. The structures assigned (VIII and IX)
were further confirmed by hydrogenation of VIII and
IX using palladium charcoal as a catalyst. Both VIII
and IX upon hydrogenation yielded the same product,
which had the following spectral characteristics: IR
spectrum (neat) γmax, 3400 cm-1 (hydroxyl) and 1695
Elution of the column with 7% ethyl acetate in hexane
yielded fractions containing a compound (Rf 0.22, system
I; tR ) 5.2 min) that was further purified by preparative
TLC (system I). The spectral data of this optically active
26
compound {[R]D ) +20° (c 1.0, in CHCl3)} are pre-
sented in Table 1. From the spectral characteristics,
the compound was identified as 3-hydroxyisopulegone
(VII, Figure 1).
Further elution of the column with 9% ethyl acetate
in hexane gave a fraction which upon TLC analysis
revealed the presence of one major compound and one
minor compound (Rf 0.36 and 0.43, system II). In GC
analysis, these two compounds did not separate (condi-
tions as mentioned under Materials and Methods) and
gave a single peak (tR ) 10.7min). It was also observed
from GC analysis of this mixture that the peak corre-
sponding to tR ) 10.7 min decreased upon storage at
room temperature, with the appearance of a new peak
(tR ) 6.8 min) that was enhanced when mixed with
piperitenone (III, Figure 1). These two compounds were
separated by preparative TLC (system II). The minor
1
cm-1 (carbonyl); H NMR (CDCl3) δ 3.49 (2H, d, J )
5.42 Hz, H-7), 2.3-1.62 (9H, m, ring protons), and 0.86,
0.79 (6H, 2d, J ) 6.58, 6.54 Hz, H-9 and H-10); MS,
m/z 170 (M+, 52%), 155 (M+ - CH3, 40%), 152 (M+
-
H2O, 10%), 139 (M+ - CH2OH, 33%), 128 (M+ - C3H6,
65%), and 69 (100%); HRMS, C10H18O2 requires 170.1307,
found 170.1302. The hydrogenated product was identi-
fied as 2-isopropyl-5-(hydroxymethyl)cyclohexanone.
The time course experiment carried out with (R)-(+)-
pulegone (I) revealed that during the early stages of
incubation (24 h) nearly 40% of I was transformed into
various metabolites (II-IX, Figure 1). By prolonging
the incubation period to 48 and 72 h, the transformation
was increased to 62% and 70%, respectively. However,
incubation carried out beyond 72 h did not result in any
appreciable improvement in the yields of the metabo-
lites. GC profiles of the total metabolites formed at
different time intervals (24, 48, and 72 h) clearly
indicated that 5-hydroxypulegone (II) was the major
metabolite formed (Figure 2).
26
compound {Rf 0.43, system II; {[R]D ) +12° (c 1.0, in
CHCl3)} was identified as 3-hydroxypulegone (V, Figure
1) on the basis of various spectral characteristics (Table
1). The major compound {Rf 0.36, system II; [R]D26 -31°
(c 1.0, in CHCl3)} was identified as 5-hydroxypulegone
(II, Figure 1) by comparing the spectral characteristics
with the earlier study on this compound (Mustapha et
al., 1992; Madyastha and Paul Raj, 1993).
The fraction eluted with 12% ethyl acetate in hexane
contained a mixture of two compounds with Rf values

1206 J. Agric. Food Chem., Vol. 47, No. 3, 1999
Madyastha and Thulasiram
droxypulegone, in which the hydroxy and keto groups
are syn to each other. This allylic alcohol readily
undergoes intramolecular cyclization followed by dehy-
dration to menthofuran, which is considered as the
proximate toxin responsible for at least 50% of the
toxicity elicited by (R)-(+)-pulegone (Thomassen et al.,
1988). Menthofuran is further metabolized to an R,â-
unsaturated-γ-keto aldehyde and p-cresol (McClanahan
et al., 1989; Madyastha and Paul Raj, 1992), and these
two metabolites substantially contribute toward (R)-(+)-
pulegone-mediated toxicity. In contrast, the present
studies have clearly demonstrated that M. piriformis
does not carry out 9-hydroxylation of both (R)-(+) and
(S)-(-)-pulegone, suggesting that these enantiomers are
not transformed to menthofuran. However, the organ-
ism very efficiently transformed both (R)-(+) and (S)-
(-)-pulegone to various metabolites, and the mode of
metabolism seems to be operating either through hy-
droxylation at the C-5 position or through ring meth-
ylene hydroxylation. The 5-hydroxypulegone (II) upon
dehydration yields piperitenone (III). Piperitenone (III)
has also been shown to be formed in the mammalian
system as one of the metabolites of both (R)-(+)- and
(S)-(-)-pulegone, although its formation is significantly
greater from (R)-(+)-pulegone than from its (S)-enan-
tiomer (Madyastha and Paul Raj, 1993; Madyastha and
Gaikwad, 1998), suggesting hydroxylation at C-5 posi-
tion is stereoselective. On the contrary, M. piriformis
transformed both of the enantiomers of pulegone to III
to the same extent, indicating that hydroxylation at the
C-5 position is not stereoselective. It is interesting to
note that mammals convert piperitenone (III) to 9-hy-
droxypiperitenone, which upon cyclization yields 6,7-
dehydromenthofuran (unpublished observation), a furan
compound that has the potential to generate toxicity.
However, such a transformation is not possible in M.
piriformis because the organism is not capable of
carrying out 9-hydroxylation of piperitenone (III). In
the mammalian system, further metabolism of III leads
to the formation of p-cresol, a known toxin (Sax, 1951).
It is believed that part of the toxicity mediated by
(R)-(+)-pulegone is due to the formation of p-cresol
(Madyastha and Paul Raj, 1991; Thompson et al., 1994).
Present studies have also noted that M. piriformis does
not transform III to p-cresol.
Although C-5 hydroxylation seems to be the major
activity, the organism also carries out hydroxylation at
C-3 and C-6 positions to form 3-hydroxy- (V) and
6-hydroxypulegone (IV), respectively. It is possible to
envisage the formation of VI and VII from V through
an allylic alcohol rearrangement and double-bond isomer-
ization. 7-Hydroxypiperitenone (VIII) could have been
formed through oxidation of the allylic methyl group in
III. In fact, hydroxylation of the allylic methyl group
in some of the monoterpenes has been observed earlier
in microbial (Ranganathan and Madyastha, 1983; Mad-
yastha and Murthy, 1988) and mammalian systems
(Madyastha and Chadha, 1982). In the present studies,
it was observed that significantly higher levels of
6-hydroxypulegone (IV) were formed from (R)-(+)-pule-
gone than from its (S)-enantiomer (Figure 2). It is quite
possible that inversion of configuration of the C-5
methyl group would affect the hydroxylation reaction
at the C-6 position. From the present and earlier
studies (Mustapha et al., 1992) it can be inferred that
fungal species tested so far do not produce reactive
metabolites which have been shown to be formed during
F igu r e 2. GC separation of the neutral metabolites obtained
by incubating (R)-(+)- and (S)-(-)-pulegone with M. piriformis
for 48 h. Experimental conditions are as reported under
Materials and Methods. I, pulegone; II, 5-hydroxypulegone;
III, piperitenone; IV, 6-hydroxypulegone; V, 3-hydroxypule-
gone; VI, 5-methyl-2-(1-hydroxy-1-mthylethyl)-2-cyclohexene-
1-one; VII, 3-hydroxyisopulegone; VIII, 7-hydroxypiperitenone;
IX, 7-hydroxypulegone; X, isopulegone. *5 indicates that
particular area is magnified 5 times.
Biotr a n sfor m a tion of (S)-(-)-P u legon e. The or-
ganism M. piriformis also accepts (S)-(-)-pulegone as
substrate. The fermentation of (S)-(-)-pulegone was
carried out under the same experimental conditions as
those used for its (R)-enantiomer (I). Nearly 60% of (S)-
(-)-pulegone was biotransformed at the end of 48 h of
incubation. The GC analysis of the total neutral frac-
tion from (S)-(-)-pulegone clearly indicated that all of
the metabolites isolated and identified (II-IX) from (R)-
(+)-pulegone (I) were also shown to be formed from its
(S)-enantiomer (Figure 2) as judged by their retention
time and peak enhancement, which occurred when
mixed with the metabolites (II-IX) isolated and identi-
fied from (R)-(+)-pulegone. There was no significant
difference in the levels of various metabolites formed
from both the enantiomers except that the level of
6-hydroxypulegone (IV) formed was appreciably higher
(70-80% more) in the case of (R)-(+)-pulegone than (S)-
(-)-pulegone (Figure 2). When isopulegone was used
as the substrate, it was observed that the organism
initiated the biotransformation by converting it to
pulegone, which was further transformed into various
metabolites (II-IX, Figure 1).
DISCUSSION
Earlier studies have clearly demonstrated that the
hepatotoxin (R)-(+)-pulegone is extensively metabolized
in the rat system (Madyastha and Paul Raj, 1993). One
of the major pathways and perhaps the most significant
pathway involved in the biotransformation is the re-
giospecific oxidation of (R)-(+)-pulegone (I) to 9-hy-

Transformation of (R)-(+)-Pulegone in Mucor piriformis
J. Agric. Food Chem., Vol. 47, No. 3, 1999 1207
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Received for review February 19, 1998. Revised manuscript
received J uly 7, 1998. Accepted J uly 8, 1998. Financial
assistance from the Department of Biotechnology, New Delhi,
India, CSIR (New Delhi) India, and J NCASR (Bangalore) is
gratefully acknowledged. H.V.T is thankful to Indian Institute
of Science, Bangalore, for the award of a research fellowship.
J F980164N