New terpenoids from Mentha pulegium L. and their antimicrobial activity

Article abstract of DOI:10.1080/14786419.2012.691488

In addition to one previously reported compound: two new terpenoidal compounds 1α, 6βdimethyl-5β-hydroxy-4β-(prop-1-en-2-yl)- decahydronaphthalen-2-one (1) and 1-(O-β-D-glucopyranosyl)-2,7-dimethyloct- 5-en-3-one (2) were isolated from the chloroformic ex

Full text of DOI:10.1080/14786419.2012.691488

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New terpenoids from Mentha pulegium
L. and their antimicrobial activity
a
Amany K. Ibrahim
a
Department of Pharmacognosy, Faculty of Pharmacy , Suez Canal
University , Ismailia 41522 , Egypt
Published online: 24 May 2012.
To cite this article: Amany K. Ibrahim (2013): New terpenoids from Mentha pulegium L. and their
antimicrobial activity, Natural Product Research: Formerly Natural Product Letters, 27:8, 691-696
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Natural Product Research, 2013
Vol. 27, No. 8, 691–696, http://dx.doi.org/10.1080/14786419.2012.691488
New terpenoids from Mentha pulegium L. and their antimicrobial activity
Amany K. Ibrahim*
Department of Pharmacognosy, Faculty of Pharmacy, Suez Canal University,
Ismailia 41522, Egypt
(
Received 1 October 2011; final version received 26 March 2012)
In addition to one previously reported compound: two new terpenoidal
compounds 1ꢀ, 6ꢁdimethyl-5ꢁ-hydroxy-4ꢁ-(prop-1-en-2-yl)-decahydronaphtha-
len-2-one (1) and 1-(O-ꢁ-D-glucopyranosyl)-2,7-dimethyloct-5-en-3-one (2) were
isolated from the chloroformic extract of Mentha pulegium L. The structure
elucidation of these compounds was based primarily on 1D and 2D-nuclear
magnetic resonance analyses. Compound 1 displayed moderate anti-MRSa
ꢀ
1
(
IC50 8.5 mg mL ).
Keywords: Mentha pulegium; Lamiaceae; terpenoids; isolation; spectroscopic
identification
1
. Introduction
Terpenoids are a diverse class of natural products that have many functions in the plant
kingdom and in human health and nutrition. Their chemical diversity has led to the
discovery of over 40,000 different structures, with several classes serving as important
pharmaceutical agents, including the anticancer agent paclitaxel (Taxol) and terpenoid-
derived indole alkaloids (Roberts, 2007).
Mentha pulegium L. Family Lamiacea (pennyroyal) is one of the four most
commercially important Mentha species, although it is not a cultivated plant. It can be
abundantly located in the Iberian Peninsula and North African countries (Kanakis,
Petrakis, Kimbaris, Pappas, & Polissiou, 2011).
The essential oil of Mentha pulegium known as Pennyroyal is generally considered to be
a rich source of pulegone, menthone, isomenthone as well as many other important
terpenoidal compounds (Kokkini, Hanlidou, Karousou, & Lanaras, 2002). The infusion is
widely reputed as safe to ingest in restricted quantities. It has been traditionally employed
and was reported successfully as an emmenagogue (menstrual flow stimulant) or as an
abortifacient. Pennyroyal is also used to settle an upset stomach and to relieve flatulence
(
Kowalchik and William, 1998; Ritchason, 1995). Pennyroyal leaves, both fresh and dried,
are especially noted for repelling insects (Kowalchik and William, 1998). However, when
treating infestations such as fleas, using the plant’s essential oil should be avoided due to
its toxicity to both humans and animals, even at extremely low levels (Anderson et al.,
1996). In this report, the isolation and structure elucidation of three compounds from
Mentha pulegium including two new 1 and 2 and one known compound 3 as well as their
biological evaluation were performed.
*
Email: am_kamal66@yahoo.com
©
2013 Taylor & Francis

6
92
A.K. Ibrahim
2
. Results and discussion
Following collection, extraction and further chromatographic processing of the plant
under investigation; three compounds were purely isolated and spectroscopically
analysed. The structure elucidation procedure of the three compounds is stated in the
following.
2
.1. Compound 1
2
D
5
Compound 1 was obtained as whitish oily matter. {½ꢀꢁ ꢀ35.19 (c 0.5, CHCl )} and its
3
molecular formula was determined to be C H O by high resolution electrospray
1
5
24
2
þ,
ionisation mass spectrometric (HRESIMS) [m/z Calcd for C H O : 237.1885 (MþH)
2
0
25
2
found 237.1855] representing four degrees of unsaturation. The IR spectrum exhibited
ꢀ1
hydroxyl and carbonyl absorption bands at 3646 and 1640 cm , respectively.
1
The H-nuclear magnetic resonance (NMR) spectrum showed two methyl connected to
3
sp carbons ꢂ 0.97 (d, 6.8) and 0.92 (d, 6.8), One vinylic methyl singlet ꢂ 1.61 (s) attached
H
H
2
to sp olefinic carbon atom, two methylene protons ꢂ 4.61 (d, 8.0) for a methylene double
bond, four methine protons ꢂH 2.52 (m), 2.54 (t, 4.4), 1.87 (m) and 2.47 (m) in addition
H
3
to eight protons attached to sp carbons at ꢂH 1.87, 1.32, 1.32 and 1.39 corresponding to
1
3
2
H in positions 3, 7, 8 and 9 respectively. The C-NMR spectrum showed 15 carbon
resonances, which were identified by DEPT and HMQC experiments as three methyl ꢂC
2
1
methylene ꢂC 112.1 (C-12)], three sp methines and three quaternary carbon atoms
7.9, 14.5and 13.6 (C-13, C-14 and C-15), five methylene [one of them is olefinic sp
3
3
[
carbon sp ꢂ 146.1 (C-11)]. This observation suggested the absence of an angular methyl
one carbonyl carbon ꢂ 215.1 (C-2), one oxygenated sp ꢂ 78.5 (C-5) and one quaternary
C C
2
C
group at C-10 (a signal for quaternary carbon was not seen around ꢂ 37 ppm). In the
C
1
H –H COSY experiment, correlations between H-7/H-8 and H-8/H-9 were observed.
1
The HMBC spectrum showed correlations from ꢂ 0.92 (d, 6.8 H-15) to ꢂ 215.1(C-2),58.3
H
C
3
2
(
2
(
1
C-10) ( JCH) and ꢂH 0.92 (d, 6.8 H-15) to ꢂ 40.2 (C-1) ( JCH), ꢂH 2.54(t, 4.4 H-4) to ꢂC
C
3
15.1(C-2), 38.7 (C-6), 112.1 (C-12), (17.9) C-13 ( JCH), ꢂH 2,54 (t, 4.4, H-4) to ꢂ 78.5
C
2
3
C-5), 146.1 (C-11) ( JCH), ꢂH 4.61 (d, 8.0 H-12) to ꢂ 17.8 (C-13), 49.0 (C-4) ( J ), ꢂH
C CH
3
2
.61 (s, H-13) to 49.0 (C-4), 112.1(C-12) ( J ), ꢂ 1.61 (s, H-13) to ꢂ 146.1 (C-11) ( JCH),
CH H C
HMQC, COSY and HMBC experiments established its chemical structure and enabled
also the assignment of all resonances in the NMR spectra which indicated that 1
possessed a sesquiterpene skeleton (4-isopropyl-1,6 dimethyldecahydronaphthalen)
(
Figure 1).
A ꢁ-orientation was suggested for the isopropylidine group at C-4, based on the
downfield shift of C-4 (ꢂC 49.0), compared to that of the epi-isomers (ꢂC 41–44)
Raharivelomanana et al., 1998; Schmeda-Hirschmann and Papastergiou, 1994). The
(
stereoorientation of the OH at C-5 was further confirmed as ꢁ-, based on the fact that
the vicinal isopropylidine group, which is ꢁ-oriented, must assume an equatorial
position to avoid steric interaction with the axial H-10, in the trans-decalin skeleton
(
De Rosa, De Giulio, Iodice, & Zavodink, 1994; Queiroga, Ferracini, & Marsaioli, 1996;
Raharivelomanana et al., 1998; Schmeda-Hirschmann and Papastergiou, 1994).
The relative stereochemistry of 1 was deduced from 2D NOESY experiments
which indicated that Me-15, H-4 and H-10, were on one side of the molecule, while
Me-14 and H-1 were on the opposite side of the molecule. From the aforementioned data,
1 can be formulated as 1ꢀ,6ꢁdimethyl-5ꢁ-hydroxy-4ꢁ-(prop-1-en-2-yl)-decahydronaphtha-
len-2-one.

Natural Product Research
693
1
5
10
3
H
HO
O
2
9
1
1
4
O
H
H
2
3
4
HO
OH
1
0
8
7
H
1`
5
O H 6`
5
9
O
6
H
OH
6
OH
14
7
11
8
1
2
13
2
1
H
HO
H
O
O
H
HO
OH
H
H
O
O H
H
OH
OH
Figure 1. Important HMBC (blue), COSY(red) and Roesy (violet) correlations for 1 and 2.
2
.2. Compound 2
2
D
5
Compound 2 was obtained as whitish oil. {½ꢀꢁ þ23.49 (c 0.5, CHCl )}and its molecular
3
formula was determined to be C H O based on positive mode HR-ESI-MS data of [m/z
1
6
28
7
þ,
calcd for C H O : 333. 1957 (MþH) found 333.1913].
1
6
29
7
1
3
H-NMR spectrum showed three methyl connected to sp carbons ꢂ 2.52 (m) and 2.26
H
(
their chemical shifts of ꢂH 4.39 and ꢂH 3.70–4.12. Nine methinic protons; two of them
d, 8.4). Three methylinic protons; two of them were proposed to be oxygenated based on
2
ꢂH 5.56 (m) attached to sp olefinic carbon atoms, five of them were proposed to be
1
oxygenated based on their chemical shifts ranging from ꢂH 3.93–4.85. The C-NMR
3
spectrum showed 16 carbon resonances, which were identified by DEPT and HMQC
experiments as three methyl groups ꢂ 27.1, 27.1 and 24.4 (C-8, C-9 and 10) respectively,
C
0
3
three methylene [two of them are oxygenated ꢂC 67.7 (C-1) and 61.0 (C-6 )], nine sp
methines [five of them were resonating from (ꢂC 69.9–103.0), suggesting a conjugation
of a glycosyl moiety in the molecule] and one quaternary carbonyl carbon atom ꢂC
2
19.1 (C-3).
1
1
In the H –H COSY experiment, correlations between H-6/H-7 and H-7/H-8 were
observed. The HMBC spectrum showed correlations from ꢂ 3.70 (d, 7.2 H -1) to ꢂ 52.7
H
2
C
2
3
(
C-2) ( J ), ꢂ 3.70 (d, 7.2 H -1) to ꢂ 219.1 (C-3), 24,4 (C-10) ( J ), ꢂ 2.26 (d, 8.4 H -
CH H 2 C CH H 3
3
3
1
H -10) to ꢂ 52.7 (C-2) ( JCH), ꢂH 5.56 (m, H-5) to ꢂ 219.1 (C-3) ( JCH) and ꢂH 2.5 (m,
0) to ꢂ 67.6 (C-1) ( J ), ꢂ 2.26 (d, 8.4 H -10) to ꢂ 219.1 (C-3) ( J ), ꢂ 2.26 (d, 8.4
C CH H 3 C CH H
2
3
3
C
C
3
H-7)to ꢂ 126.6 (C-5) ( JCH) (Figure 1). The presence of a glycosyl moiety in the molecule
C

6
94
A.K. Ibrahim
was confirmed by the acid hydrolysis of 2, which afforded 2a and a sugar component,
which accounted for the last degree of unsaturation. The sugar was identified to be
D-glucose by co-thin-layer chromatography (TLC) (EtOAc-MeOH-H O ¼ 9:3:1, R value:
2
f
1
13
0
.17) with a D-glucose standard. The H- and C-NMR spectrum also confirmed the
presence of D-glucose (Yang, Kim, Lee, Kim, & Lee, 2007). Based on the previously
mentioned data, compound (2) was identified to be 1-(ꢁ-glucosyl)-2,7-dimethyloct-5-en-
3
-one.
2
.3. Compound 3
Based on the spectroscopic data and by comparing with previous reports, compound 3 was
identified to be ꢁ-sitosterol-3-O-ꢁ -D-glucopyranoside (Chang, Yun, & Yamasaki, 1981).
2
.4. Biological evaluation of the isolated compounds
Compound 1 displayed moderate anti-MRSa [Methicillin Resistant Staphylococcus
ꢀ
1
aureus ATCC 43300] with (IC50 8.5 mg mL ). The other compounds have shown no
activity. All compounds have shown no antioxidant activity.
3
3
1
. General experimental procedures
.1. Material and methods
D and 2D NMR spectra were recorded in C D N and CDCl on a Varian AS 400
3
5
5
spectrometer using tetra methyl silane (TMS) as the internal standard with chemical shifts
expressed in ꢂ and coupling constants (J) in Hz. Infra red (IR) spectra were recorded
on a Bruker Tensor 27 spectrophotometer. Optical rotations were measured at ambient
temperature using a Rudolph Research Analytical Autopol IV automatic polarimeter.
HRESIMS was obtained using a Bruker Bioapex FTMS in ESI mode.
TLC was carried out on aluminum-backed plates pre-coated with silica gel F254
˚
(
F
20 cmꢂ 20 cm, 200 mm, 60 A, Merck) and on glass-backed plates pre-coated with silica gel
˚
254
(10 cmꢂ 20 cm, 200 mm, 60 A, Analtech). Visualisation was accomplished by spraying
˚
with p-anisaldehyde spray reagent followed by heating. Flash silica gel (40–63 mm, 60 A,
Silicycle) and Sephadex LH20 were used for column chromatography.
3
.2. Plant material
Mentha Pulegium L. was collected from Faculty of Agriculture farm (SCU), Ismailia,
Egypt. Taxonomically authenticated voucher specimens were deposited in the Herbarium
Section of Pharmacognosy Department, Faculty of Pharmacy (SCU) sample no. SCPG 27.
3
.3. Extraction and isolation procedures
The dried plant leaves (1.0 kg) was sequentially extracted with chloroform CHCl3
(
evaporated under reduced pressure at 40 C to afford chloroformic (78.1 g) and methanolic
3 L ꢂ 6 L), Methanol MeOH (3 L ꢂ 6 L) overnight at room temperature. The extracts were
ꢃ
(
20 g) extracts. The resulting chloroformic extract was fractionated using vacuum liquid
chromatography over silica gel 40 mm column (200 g, column ꢀ 15 cm) using hexanes
gradually enriched with CHCl till reaching pure chloroform to start enriching with
MeOH to reach pure MeOH. The obtained 17 fractions were vacuum dried and accurately
weighed. The fraction eluted with [15% MeOH/CHCl (80 mg)] was subjected to further
3
3
purification using Sephadex LH-20 packed open glass columns (100 g, column ꢀ 2 cm)
with pure MeOH as an eluent to yield two metabolites 1 (13.2 mg) and 3 (5.5 mg).
Purification of metabolite 2 (15.3 mg) was achieved through repeated open glass columns

Natural Product Research
695
˚
packed with Flash silica gel (40–63 mm, 60 A, Silicycle) and preparative thin layer
chromatography for the [25% MeOH/CHCl ] fraction (117 mg).
3
3
.4. Acid hydrolysis for identification of the sugar
Compound 2 (2 mg) was refluxed with 0.5 N HCl (3 mL) for 2 h. The reaction mixture was
diluted with water and extracted with CHCl . The water layer was dried under reduced
3
pressure as well as under N to give the monosaccharide.
2
3
.5. 1a,6bdimethyl-5b-hydroxy-4b-(prop-1-en-2-yl)-decahydronaphthalen-2-one (1)
1
25
Whitish oily matter. {½ꢀꢁ ꢀ35.19 (c 0.5, CHCl )}; H NMR (CDCl , 400 MHz): ꢂ 0.92
D
d, 6.8, H -15), 0.97 (d, 6.8, H -14), 1.32 (m, H -7), 1.32 (m, H -8), 1.39 (m, H -9), 1.61
3
3
H
(
(
(
(
3 3 2 2 2
s, H -13), 1.87 (m, H-6), 2.38 (dd, 5.2, H-2), 2.41 (dd, 5.2, H -3), 2.47 (m, H-10), 2.52
3
2
1
m, H-1), 2.54 (t, 4.4, H-4), 4.61 (d, 8.0, H -12); C NMR (CDCl , 100 MHz): ꢂ 13.6
3
2
3
C
C-15), 14.5 (C-14), 17.9 (C-13), 25.1 (C-8), 29.7 (C-7), 31.1 (C-3), 32.5 (C-9), 38.7 (C-6),
0.2 (C-1), 49.0 (C-4), 58.3 (C-10), 78.5 (C-5), 112.1 (C-12), 146.1 (C-11), 215.1 (C-2).
4
þ,
HR-ESI-MS data of [m/z Calcd for C H O : 332. 1957 (MþH) found 332.1913].
1
6
29
7
3
.6. 1-(b-glucosyl)-2,7-dimethyloct-5-en-3-one (2)
1
2
5
Whitish oil. {½ꢀꢁ þ23.49 (c 0.5, CHCl )}; H NMR (C D N, 400 MHz): ꢂ 2.0 (m, H-3),
D
3
5
5
H
2
.26 (d, 8.4, H -10), 2.32 (m, H-14), 2.5 (m, H-7), 2.52 (m, H -8), 2.52 (m, H -9), 3.70
3 3 3
0
0
0
(
(
d, 7.2, H-1), 3.93 (br d, H-3 ), 4.05 (d,0.76, H-2 ), 4.12 (d, 7.2, H-1), 4.22(m, H-5 ), 4.39
13
m, H-4 ), 4.39 (m, H-6 ), 4.85 (d,0.76, H-1 ), 5.56 (m, H-5), 5.56 (m, H-6); C NMR
0
0
0
(
C D N, 100 MHz): ꢂ 24.4 (C-10), 27.1 (C-8), 27.1 (C-9), 36.5(C-4), 37.1 (C-7), 52.7 (C-3),
C
5
5
0 0 0 0 0 0
6
(
(
1.0 (C-6 ), 67.7 (C-1), 69.9 (C-4 ), 73.4 (C-2 ), 76.7 (C- 3 ), 76.8 (C-5 ), 103.0 (C-1 ), 126.6
C-5), 127.2 (C-6), 219.1(C-2). HR-ESI-MS data of [m/z Calcd for C H O : 333. 1957
1
6
29
7
þ
MþH) , found 333.1913].
3
.7. b-Sitosteryl-3-O-b-D-glucopyranoside (3)
1
White amorphous powder; H NMR (CDCl , 400 MHz): ꢂ 0.63 (9H, m, H -26, 27, 29),
3
H
3
0
H -2-H -4-H -7-H -8-H -9-H -11-H -12-H -14-H -15-H -16-H -17-H -20-H -22-
.75 (3H, s, H -18), 0.90 (3H, d, 6.8 Hz, H -21), 0.99 (3H, s, H -19),1.15–2.75 (29H, H -1-
3 3 3 2
2
2
2
1
1
2
2
1
2
2
1
1
2
0
0
H -23-H -24-H -25-H -28), 3.49 (1H, m, H-3), 3.35-4.05 (4H, H-2 -H-5 ), 4.33 (1H, d,
2
2
1
1
0
0
13
J ¼ 7.6 Hz, H-1 ), 4.1-4.2 (2H, m, H-6 ) 5.33 (1H, bs, H-6); C NMR (CDCl , 100 MHz):
3
ꢂ 37.6 (C-1), 29.6 (C-2), 78.3 (C-3), 39.4 (C-4), 141.0 (C-5), 122.0 (C-6), 32.2 (C-7), 32.1
C
(
(
C-8), 50.5 (C-9), 36.4 (C-10), 21.3 (C-11), 40.0 (C-12), 42.5 (C-13), 57.0 (C-14), 24.5
C-15), 28.3 (C-16), 56.4 (C-17), 12.0 (C-18), 19.6 (C-19), 36.4 (C-20), 19.0 (C-21), 34.2 (C-
2
1
5
2), 26.4 (C-23), 46.0 (C-24), 29.4 (C-25), 19.2 (C-26), 20.0 (C-27), 23.3 (C-28), 12.2 (C-29),
0
0
0
0
0
0
02.6 (C-1 ), 75.8 (C-2 ), 76.6 (C-3 ), 70.8 (C-4 ), 73.8 (C-5 ), 63.0 (C-6 ); HRESIMS m/z
-
75.4326 [M-H] (Calcd for C H O , 575.4312).
3
5
59
6
3
.8. Antimicrobial, antileishmanial and antimalarial bioassay
The isolated compounds were evaluated for antimicrobial (Candida albicans ATCC 90028,
Escherichia coli ATCC 35218, Pseudomonas aeruginosa ATCC 27853, Mycobacterium
intracellulare ATCC 23068, Aspergillus fumigat ATCC 90906 and Methicillin Resistant
Staphylococcus aureus ATCC 43300). Susceptibility testing was performed for all
organisms, except M. intracellulare and A. fumigatus, using modified versions of the
CLSI/NCCLS methods (NCCLS, 2002), while optical density was used to monitor
growth. Media supplemented with 5% Alamar Blue was utilized for growth detection of

6
96
A.K. Ibrahim
M. intracellulare (NCCLS, 2000), and A. fumigatus (NCCLS, 1998). The in vitro
antileishmanial activity was evaluated against a culture of L. donovani promastigotes
ꢃ
grown in RPMI 1640 medium supplemented with 10% GIBCO fetal calf serum at 26 C
(Bharate et al., 2007) and antimalarial activity [Plasmodium falciparum (D6 clone) and
P. falciparum (W2 clone)]. The assay is based on the determination of plasmodial LDH
activity (Bharate et al., 2007).
3
.9. Anti-oxidant activity
A TLC autographic assay for DPPH (1,1-diphenyl-2-picrylhydrazyl) radical-scavenging
effect was used to determine anti-oxidant activity (Takamatsu et al., 2003).
Acknowledgements
The Author is greatly thankful to Prof. Dr Ikhlas Khan (National Centre of Natural Products
Research, University of Mississippi, USA) for his help and worthy advice through this work until its
full accomplishment.
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