Evaluation of antiparasitic activity of mentha crispa essential oil, its major constituent rotundifolone and analogues against trypanosoma brucei

Article abstract of DOI:10.1055/s-0042-107082

Considering the pressing need for new drugs to treat sleeping sickness and Nagana disease, Mentha crispa essential oil, its principal constituent rotundifolone, and four related p-menthane-Type monoterpenes (two stereoisomers of limonene epoxide, perillyl alcohol, and perillyl aldehyde) were investigated for their activity against bloodstream forms of Trypanosoma brucei. The general cytotoxicity of the compounds was determined with human myeloid HL-60 cells. The effect of the M. crispa essential oil and the monoterpenes on the growth of parasite and human cells was evaluated in cell cultures with the resazurin viability assay. Of all of the compounds tested, M. crispa essential oil, rotundifolone, and perillyl aldehyde showed the highest trypanocidal activities with 50% growth inhibition (GI₅₀) and minimum inhibitory concentration values of 0.3 μg/ mL and 1 μg/mL, respectively. In contrast, HL-60 cells were considerably less sensitive to the compounds with minimum inhibitory concentration values of 100 μg/mL and GI₅₀ values ranging between 3.4 to 13.8 μg/mL. As a consequence of this, GI50 and minimum inhibitory concentration ratios of cytotoxic to trypanocidal activity (selectivity index) of these three compounds were promising with values of 11-45 and 100, respectively. These results indicate that the p-menthane-Type monoterpenes rotundifolone and perillyl aldehyde are interesting lead candidates for further rational antitrypanosomal drug development.

Full text of DOI:10.1055/s-0042-107082

Original Papers
Evaluation of Antiparasitc Activity of Mentha crispa
Essential Oil, Its Major Constituent Rotundifolone and
Analogues against Trypanosoma brucei
Authors
Damião Pergentino de Sousa1, Tamires Cardoso Lima2, Dietmar Steverding3
1
Affiliations
Department of Pharmaceutical Sciences, Federal University of Paraíba, João Pessoa, Paraíba, Brazil
Department of Pharmacy, Federal University of Sergipe, São Cristóvão, Sergipe, Brazil
Bob Champion Research and Education Building, Norwich Medical School, University of East Anglia, Norwich,
2
3
United Kingdom
Key words
Abstract
aldehyde showed the highest trypanocidal activ-
ities with 50% growth inhibition (GI50) and mini-
mum inhibitory concentration values of 0.3 µg/
mL and 1 µg/mL, respectively. In contrast, HL-60
cells were considerably less sensitive to the com-
pounds with minimum inhibitory concentration
values of 100 µg/mL and GI50 values ranging be-
tween 3.4 to 13.8 µg/mL. As a consequence of this,
GI50 and minimum inhibitory concentration ra-
tios of cytotoxic to trypanocidal activity (selectiv-
ity index) of these three compounds were prom-
ising with values of 11–45 and 100, respectively.
These results indicate that the p-menthane-type
monoterpenes rotundifolone and perillyl alde-
hyde are interesting lead candidates for further
rational antitrypanosomal drug development.
"
l Mentha crispa essential oil
!
"
l monoterpenes
Considering the pressing need for new drugs to
treat sleeping sickness and Nagana disease, Men-
tha crispa essential oil, its principal constituent
rotundifolone, and four related p-menthane-type
monoterpenes (two stereoisomers of limonene
epoxide, perillyl alcohol, and perillyl aldehyde)
were investigated for their activity against blood-
stream forms of Trypanosoma brucei. The general
cytotoxicity of the compounds was determined
with human myeloid HL-60 cells. The effect of
the M. crispa essential oil and the monoterpenes
on the growth of parasite and human cells was
evaluated in cell cultures with the resazurin via-
bility assay. Of all of the compounds tested,
M. crispa essential oil, rotundifolone, and perillyl
"
l rotundifolone
"
l Trypanosoma brucei
"
l HL‑60 cells
"
l trypanocidal activity
Introduction
cial formulation Giamebil® that displays amoebi-
cidal and giardicidal activities [6]. This remedy
has also been shown to be effective in women
with Trichomonas vaginalis infection [7].
!
The genus Mentha (commonly known as mint)
comprises approximately 19 species and 13 natu-
ral hybrids and is one of the most popular essen-
tial oil crops due to the remarkable chemical di-
versity and bioactivity of its secondary com-
pounds [1–3]. The plants are commonly known
for their culinary use as spices and seasonings
and as teas and infusions, while their essential
oils and isolated compounds are used as flavour-
ings in toothpaste, antiseptic mouth rinses,
breath fresheners, chewing gum, drinks, desserts,
and candies. However, mint is also used as a me-
dicinal herb in traditional medicine. In addition,
extracts and essential oils of mint plants have
been shown to display activity against parasites.
For example, the essential oil of the hybrid species
Mentha crispa L. (syn. Mentha x villosa Huds.) has
been reported to exhibit larvidical activity against
Aedes aegypti [4] and Schistosoma mansoni [5].
A dry extract from the leaves and stems of
M. crispa is the active ingredient of the commer-
One of the predominant compounds found in
many Mentha species is the monoterpene rotun-
difolone (aka piperitenone oxide) [4,8–11]. For
instance, the essential oil of M. crispa contains
generally around 70% of this secondary plant
compound [4,10]. Rotundifolone is a p-men-
thane-type epoxide and has been reported to ex-
hibit strong trypanocidal activity against epimas-
tigote and trypomastigote forms of Trypanosoma
cruzi, the causative agent of Chagas disease in Lat-
in America, with IC50 values of < 10 µg/mL [12].
In the search for new compounds with activity
against African trypanosomes that cause sleeping
sickness in humans and Nagana disease in cattle,
we investigated the trypanocidal activity of
M. crispa essential oil (MCEO) and its major con-
stituent rotundifolone as well as some structur-
ally related monoterpenes against bloodstream
forms of Trypanosoma brucei. In this respect, it is
received
revised
February 16, 2016
April 7, 2016
accepted
April 13, 2016
Bibliography
DOI http://dx.doi.org/
10.1055/s-0042-107082
Published online
Planta Med © Georg Thieme
Verlag KG Stuttgart · New York ·
ISSN 0032‑0943
Correspondence
D. P. de Sousa
Department of Pharmaceutical
Sciences
Federal University of Paraíba
Cidade Universitária
58051–970 João Pessoa – PB
Brazil
Phone: + 558332167347
Fax: + 558332167156
damiao_desousa@
yahoo.com.br
De Sousa DP et al. Evaluation of Antiparasitc… Planta Med

Original Papers
noteworthy to mention that monoterpenes have previously been
shown to display promising trypanocidal activities [13,14].
Results and Discussion
!
The present work investigated the in vitro antitrypanosomal and
cytotoxic activities of MCEO, its major constituent rotundifolone,
and four analogous monoterpenes [(+)-limonene epoxide, (−)-
limonene epoxide, (−)-perillyl alcohol, and (−)-perillyl aldehyde]
"
(l Fig. 1) using the resazurin (Alamar blue) assay described pre-
viously [15]. The trypanocidal activity of the compounds was de-
termined with T. brucei bloodstream forms 427–221 a [16], while
the general cytotoxicity was evaluated with human myeloid leu-
kaemia HL-60 cells [17]. MCEO and the other compounds all
showed a dose-dependent effect on the growth of trypanosomes
with MIC (minimum inhibitory concentration; i.e., the concen-
tration of the compounds at which all cells were killed) values
varying between 1 and 100 µg/mL and GI50 (50% growth inhibi-
tion; i.e., the concentration of a compound necessary to reduce
the growth rate of cells by 50% to that of controls) values ranging
Fig. 1 Chemical structures of p-menthane-type monoterpenes tested for
trypanocidal activity.
"
from 0.3 to 13.3 µg/mL (l Table 1). MCEO, rotundifolone, and
(−)-perillyl aldehyde were the most trypanocidal agents with
identical MIC and GI50 values. The antitrypanosomal activity of
MCEO is most likely due to its major constituent rotundifolone
(the MCEO used in this study contained 58.11% rotundifolone,
oxide display the same antitrypanosomal activity. Likewise, no
difference in larvicidal activity against A. aegypti has been re-
cently reported for the enantiomers (+)-limonene epoxide and
(−)-limonene epoxide [4]. However, enantioselectivity was ob-
served for the cytotoxic activity of (+)-limonene epoxide and
(−)-limonene epoxide against HL-60 cells. This example shows
that determination of the activity of stereoisomers can be worth-
while as one enantiomer may be active while the other one in-
active. In this context it would have been interesting to see
whether (+)-perillyl aldehyde would have a reduced cytotoxicity
but the same trypanocidal activity as its stereoisomer (−)-perillyl
aldehyde. Unfortunately, the required parent compound (+)-
perillyl alcohol for synthesis of (+)-perillyl aldehyde is not com-
mercially available at an affordable price.
Over the past years, essential oils and their constituents have
been proven to be profitable sources for compounds with anti-
parasitic activity. This study has shown that rotundifolone, the
major constituent of the essential oil of M. crispa, and the related
monoterpene perillyl aldehyde display promising trypanocidal
activity towards bloodstream forms of T. brucei. Both compounds
almost fulfil the activity criteria for drug candidates for African
trypanosomiasis (GI50 < 0.2 µg/mL; selectivity > 100) [22]. How-
ever, one should bear in mind that in the present study a cancer
cell line was used for determining the selectivity. Therefore, com-
pared with nonmalignant cells, it is likely that the cytotoxicity of
the compounds is overestimated. Further optimisation of the
compounds through structural modification may lead to mole-
cules with improved trypanocidal activity and reduced cytotoxic-
ity.
"
l Table 2), as the essential oil and the monoterpene have similar
trypanocidal activities. When compared with the MIC value and
the GI50 value of suramin (reference control), one of the drugs
used in the treatment of sleeping sickness, the three compounds
"
were 10 and 6 times less trypanocidal, respectively (l Table 1).
The cytotoxicity of the compounds towards human HL-60 cells
was generally lower with MIC values of 100 or > 100 µg/mL and
"
GI50 values ranging between 3.4 and > 100 µg/mL (l Table 1).
As a result, the MIC and GI50 ratios of cytotoxic to trypanocidal
activities (selectivity indices) were found to be in a modest range
"
for most compounds (l Table 1). Only MCEO, rotundifolone, and
(−)-perillyl aldehyde had a substantial selectivity index for the
MIC ratio of 100. For comparison, the reference drug suramin
had an MIC ratio and a GI50 ratio of > 1000 and > 2000, respective-
"
ly (l Table 1).
Structure-activity relationship analysis revealed some structural
characteristics that can be correlated with the antitrypanosomal
effect of the compounds tested. It appears that the trypanocidal
activity increases with the presence on an α,β-unsaturated car-
bonyl, since rotundifolone and perillyl aldehyde containing this
functional group were the most potent compounds. This sugges-
tion is supported by previous findings that the presence of a car-
bonyl group (aldehyde or ketone) conjugated to a C‑C double
bond seems to be important for the biological activity of many
compounds [4,18–20]. In line with this is the finding that perillyl
alcohol, which contains a hydroxyl group, was 40 to 100 times
less trypanocidal than its analogue perillyl aldehyde. In addition,
the increased hydrophilicity of perillyl alcohol due to the pres-
ence of a hydroxyl group may also contribute to its lower anti-
trypanosomal activity. It is well established that lipophilicity is a
critical parameter for the membrane and cell permeation of
drugs [21]. This is corroborated by the finding that the limonene
epoxide stereoisomers, which do not contain a carbonyl or a
hydroxyl group, exhibited trypanocidal activities that ranged
between those of perillyl alcohol and perillyl aldehyde. Another
interesting observation is that both enantiomers of limonene ep-
Materials and Methods
!
Reagents
MCEO was obtained from Hebron® company (Recife, Brazil).
(−)-Perillyl alcohol (purity 96%, GC), (+)-limonene (purity 97%,
GC), (−)-limonene (purity 96%, GC), and suramin sodium salt
(purity ≥ 99%, TLC) were purchased from Sigma-Aldrich.
De Sousa DP et al. Evaluation of Antiparasitc… Planta Med

Original Papers
Table 1 Bioactivity of MCEO and p-menthane-type monoterpenes against T. brucei bloodstream forms and HL-60 cells.
Compound
T. brucei
HL-60
Selectivity
MIC (µg/mL)a
GI50 (µg/mL)b
MIC (µg/mL)a
GI50 (g/mL)b
MIC ratioc
GI50 ratiod
MCEO
1
0.33 0.03
0.32 0.05
(1.93)
100
8.31 2.10
3.40 1.21
(20.5)
100
100
25
Rotundifolone
1
100
10.6
(6.0)e
10–100
(66–655)
10
(602)
(+)-Limonene epoxide
(−)-Limonene epoxide
(−)-Perillyl alcohol
(−)-Perillyl aldehyde
Suraminf
3.03 0.47
(19.9)
> 100
> 100
> 1–10
10
> 33
(> 655)
100
(> 655)
34.6 0.7
(227)
2.94 0.15
(19.3)
11.7
2.6
(66)
100
(655)
100
13.3 1.6
(87)
34.6 0.6
(227)
1
(655)
1
(655)
100
0.31 0.02
(2.06)
13.8 4.1
(92)
100
45
(6.7)
0.1
(666)
> 100
(> 70)
0.050 0.003
(0.035)
> 100
> 1000
> 2000
(0.07)
(> 70)
a Data shown are the mean values of three independent experiments; b data shown are the mean values SD of three independent experiments; c defined as MIC(HL-60)/MIC(T. brucei)
d defined as GI50(HL-60)/GI50(T. brucei) e values in brackets are concentrations in µM; f reference control.
;
;
tion with CH2Cl2 followed by filtration and evaporation under re-
duced pressure to obtain a yellowish oil. The oil was confirmed as
Table 2 Composition of the essential oil from leaves of M. crispa determined
by GC/MS.
1
rotundifolone by H and 13C NMR analysis, IR spectroscopy, and
b
RIa
934
Constituent
%
comparison with published data [25].
Rotundifolone: 1H NMR (CDCl3, 200 MHz): δH 1.41 (s, 3 H), 1.74
(s, 3 H), 1.92–1.78 (m, 2 H), 2.04 (s, 3 H), 2.40–2.24 (m, 2 H), 3.17
(s, 1 H); 13C NMR DEPT (CDCl3, 50 MHz): δC 21.7, 23.0, 23.0, 24.0,
27.7, 63.2, 63.4, 127.5, 149.2, 198.4; IR (cm−1): ν 3050, 2990, 1700,
1640 and 880; purity: > 93% (1H NMR and TLC).
α-Pinene
2.00
972
976
Sabinene
1.08
4.43
7.79
10.58
5.01
58.11
0.17
2.00
0.22
0.37
6.55
98.31
β-Pinene
Myrcene
990
1029
1035
1363
1386
1420
1454
1457
1481
Total
Limonene
cis-β-Ocimene
Rotundifolone
β-Bourbonene
trans-Caryophyllene
α-Humulene
trans-β-Farnesene
Germacrene
Synthesis of (+)-limonene epoxide,
(−)-limonene epoxide and (−)-perillyl aldehyde
(+)-Limonene epoxide and (−)-limonene epoxide were synthes-
ised from (+)-limonene and (−)-limonene, respectively, by endo-
cyclic epoxidation of the double bond using meta-chloroperben-
zoic acid (m-CPBA) as the oxidising agent and CH2Cl2 as the sol-
vent system, as previously described [26]. In brief, to a solution of
(−)-limonene or (+)-limonene (7.35 mmol) in 40 mL of dry
CH2Cl2, a 70% solution of m-CPBA (7.35 mmol) in CH2CL2 was
added dropwise. The mixture was stirred at 0°C (ice bath) for
4 h, followed by washing 4 times with 50 mL of an aqueous solu-
tion of 10% NaHSO3. The aqueous layer was extracted with
CH2Cl2 (2 × 50 mL) and the organic layers were washed with an
aqueous solution of 5% NaHCO3 (2 × 50 mL). The combined organ-
ic solutions were dried over anhydrous Na2SO4. After evaporation
of the solvent under reduced pressure, the product was purified
by column chromatography on silica gel.
(−)-Perillyl aldehyde was prepared from (−)-perillyl alcohol by
selective oxidation using pyridinium chlorochromate (PCC) as
the oxidising agent and dry CH2Cl2 as the solvent, according to
the method described in [27]. In brief, to a solution of (−)-perillyl
alcohol (59.21 mmol) in 462 mL of dry CH2Cl2, a solution of PCC
(177.63 mmol) in CH2Cl2 was slowly added. The mixture was
stirred at room temperature for 4 h and filtered through a Büch-
ner funnel containing silica gel. The filtrate was concentrated on a
rotavapor and the product was purified by silica gel column chro-
matography.
a Retention index determined on an OV-5 column; b constituentʼs percentage
GC/MS analysis of MCEO
MCEO was analysed by GC/MS using a Hewlett Packard system
(gas chromatograph model 5890 equipped with a mass spec-
trometer model 5988 A) and an OV-5 capillary column
(30 m × 0.25 mm, bonded 0.25 µm). The following analytical con-
dition were used: electron impact, 70 eV; carrier gas, helium;
flow rate, 1 mL/min; temperature, programmed from 60–240°C
at 3°C/min; injection temperature, 240°C; detection tempera-
ture, 230°C; split ratio, 1/20. The injected volume was 1 µL of a
solution containing approximately 0.1 µL of MCEO in 1 mL ethyl
acetate. The identification of each component was determined
by comparing their mass spectra with a GC/MS database (Nist 62
library) and Kovats retention indices [23]. The results of the
"
GC/MS analysis of MCEO are shown in l Table 2.
Isolation of rotundifolone
The terpenic ketone rotundifolone was isolated from MCEO as
previously described [24]. MCEO was subjected to preparative
silica gel thin-layer chromatography using hexane as the mobile
phase. The plates were exposed to UV light (254 nm) and rotun-
difolone was identified as the major component of MCEO. Rotun-
difolone was removed from the plates and recovered by extrac-
The identity of all three monoterpenes was confirmed by 1H and
13C NMR analysis, IR spectroscopy, and comparison with pub-
lished data [27,28].
(+)-Limonene epoxide: 1H NMR (CDCl3, 200 MHz): δH 1.30–0.90
(m, 2 H), 1.22 (s, 3 H), 1.72–1.63 (m, 2 H), 1.68 (s, 3 H), 2.05–1.74
De Sousa DP et al. Evaluation of Antiparasitc… Planta Med

Original Papers
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(m, 2 H), 2.18–2.10 (m, 1 H), 2.97 (t, J = 4.2 Hz, 1 H), 4.62
(d, J = 0.8 Hz, 1 H), 4.66 (d, J = 0.8 Hz, 1 H); 13C NMR DEPT (CDCl3,
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148.9; IR (cm−1): ν 3040, 3000, 1670, 1250, and 860; purity:
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(−)-Limonene epoxide: 1H NMR (CDCl3, 200 MHz): δH 1.40–1.00
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ν 3050, 3000, 1690, 1260, and 890; purity: > 93% (1H NMR and
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(−)-Perillaldehyde: 1H NMR (CDCl3, 200 MHz): δH 1.46–1.36
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2.48–2.42 (m, 1 H), 4.71 (s, 2 H), 6.01–5.77 (m, 1 H), 9.40 (s, 1 H);
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109.4, 141.1, 148.2, 150.5, 193.8; IR (cm−1): ν 3080, 2980, 1700,
1680, and 880; purity: > 93% (1H NMR and TLC).
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In brief, cells were seeded in 96-well plates in a final volume of
0.2 mL of appropriate culture medium (T. brucei: Baltz medium
[29]; HL-60 cells: RPMI 1640 medium [30]) supplemented with
16.7% heat-inactivated foetal calf serum and containing 10-fold
serial dilutions of test compounds (10−1 to 10− 6 mg/mL) dissolved
in 100% DMSO. Controls contained DMSO alone. In all experi-
ments, the final DMSO concentration was 1%. The seeding den-
sities were 104 trypanosomes/mL and 105/HL-60 cells/mL. After
24 h incubation at 37°C in a humidified atmosphere containing
5% CO2, 20 µL of a 0.44-mM resazurin solution prepared in PBS
were added and the cells were incubated for a further 48 h so that
the total incubation time was 72 h. It should also be noted that
the addition of 20 µl resazusin resulted in a 9% dilution of the test
compounds, which, however, does not have a significant effect on
the determination of MIC and GI50 values. This was previously
shown when the resazurin assay was compared with direct cell
counting [15]. Thereafter, the absorbance was read on a micro-
plate reader using a test wavelength of 570 nm and a reference
wavelength of 630 nm. GI50 values were determined by linear in-
terpolation according to the method described in [31]. MIC values
were determined microscopically.
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Acknowledgements
!
The authors are thankful to CAPES and CNPq for financial
support.
Conflict of Interest
!
The authors have declared that there is no conflict of interest.
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