In vitro anticancer activity of extracts of Mentha Spp. against human cancer cells

Abstract

In vitro anticancer potential of methanolic and aqueous extracts of whole plants of Mentha arvensis, M. longifolia, M. spicata and M. viridis at concentration of 100 μg/ml was evaluated against eight human cancer cell lines--A-549, COLO-205, HCT-116, MCF-7, NCI-H322, PC-3, THP-1 and U-87MG from six different origins (breast, colon, glioblastoma, lung, leukemia and prostate) using sulphorhodamine blue (SRB) assay. Methanolic extracts of above-mentioned Mentha Spp. displayed anti-proliferative effect in the range of 70-97% against four human cancer cell lines, namely COLO-205, MCF-7, NCI-H322 and THP-1; however, aqueous extracts were found to be active against HCT-116 and PC-3. The results indicate that Mentha Spp. contain certain constituents with cytotoxic properties which may find use in developing anticancer agents.

Full text

Indian Journal of Biochemistry & Biophysics
Vol. 51, October 2014, pp. 416-419
NOTE
In vitro anticancer activity of extracts
of Mentha Spp. against human cancer cells
Vikas Sharma*, Shabir Hussain, Moni Gupta and
Ajit Kumar Saxena#
Division of Biochemistry, Faculty of Basic Sciences,
Sher-e-Kashmir University of Agricultural Sciences
and Technology of Jammu, Main Campus Chatha,
Jammu-180009, J&K, India
#Cancer Pharmacology Division, Indian Institute of Integrative
Medicine, Canal Road Jammu-180001, J&K, India
Received 12 December 2013; revised 10 September 2014
In vitro anticancer potential of methanolic and aqueous
extracts of whole plants of Mentha arvensis, M. longifolia,
M. spicata and M. viridis at concentration of 100 µg/ml
was evaluated against eight human cancer cell lines — A-549,
COLO-205, HCT-116, MCF-7, NCI-H322, PC-3, THP-1 and
U-87MG from six different origins (breast, colon, glioblastoma,
lung, leukemia and prostate) using sulphorhodamine blue (SRB)
assay. Methanolic extracts of above-mentioned Mentha Spp.
displayed anti-proliferative effect in the range of 70-97%
against four human cancer cell lines, namely COLO-205, MCF-7,
NCI-H322 and THP-1; however, aqueous extracts were found
to be active against HCT-116 and PC-3. The results indicate
that Mentha Spp. contain certain constituents with cytotoxic
properties which may find use in developing anticancer agents.
Keywords: Mentha Spp., Cancer cells, SRB assay
Mentha is a plant with worldwide distribution and
contains several species which are used in traditional
medicine, mainly for gastrointestinal disturbances.
Cytotoxic and other pharmacological activities have
also been reported from Mentha Spp.1-3. Methanolic
extract and essential oils from six Mentha species viz,
M. piperita, M. spicata, M. pulegium, M. longifolia,
M. aquatica and M. crispa have shown cytotoxicity
against HeLa and HEP-2 cancer cell lines4. Aqueous
extract of M. spicata (pahari pudina), an important
aromatic spice has also shown cytotoxic effect in
mouse fibrosarcoma Wehi-64 and human monocytic
U937 cells5. The cytotoxic effect of essential oil from
M. spicata leaves on some cancer cell lines is also
reported in vitro6,7. Chloroform and ethylacetate
extracts of leaves of M. piperita (Gamathi pudina)
has shown significant dose and time-dependent
anticarcinogenic activity against HeLa, MCF-7,
Jurkat, T24, HT-29 and MIAPaCa-2 cancer cell lines8.
M. arvensis, commonly known as pudina is used in
various symptoms of diseases, such as abdominal
pain, vomiting, cough, loss of appetite, menstrual
disorders, joint pain and in diseases of liver, spleen
and asthma9.
In the present study, anticancer potential of four
Mentha Spp. Viz., M. arvensis, M. longifolia, M. spicata
and M. viridis has been investigated against eight
human cancer cell lines (A-549, COLO-205,
HCT-116, MCF-7, NCI-H322, PC-3, THP-1 and
U-87MG) of six different origins (breast, colon,
glioblastoma, lung, leukemia and prostate).
Materials and Methods
Chemicals
RPMI-1640 medium, Dulbecco’s minimum essential
medium (DMEM), dimethyl sulfoxide (DMSO),
EDTA, fetal calf serum (FCS), sulphorhodamine blue
(SRB) dye, phosphate buffer saline (PBS), trypsin,
gentamycin, penicillin and 5-flurouracil were purchased
from Sigma Chemical Co., USA. All other chemicals
were of high purity and obtained locally with the brand
Sigma-Aldrich Chemicals Pvt. Ltd. and S.D. Fine
Chemicals Pvt. Ltd.
Plant material and preparation of extracts
The whole plants of four Mentha Spp. viz,
M. arvensis, M. longifolia, M. spicata and M. viridis
were authenticated at site by Prof. M Saleem,
Division of Agroforestry, SKUAST-Jammu and then
collected in the month of May from Herbal Garden,
Sher-e-Kashmir University of Agricultural Sciences
and Technology of Jammu (SKUAST-Jammu),
J&K, India. The freshly collected plant material
was chopped, shade-dried and ground into powdered
form and extracted with different solvents at room
temperature to obtain extracts for bioevaluation.
The methanolic extract was prepared by percolating
the dried ground plant material (100 g) with 95%
methanol and then concentrating it to dryness under
reduced pressure The aqueous extract was obtained by
boiling dried ground plant material (100 g) for 30 min
in distilled water (300 ml) and freeze-dried. Stock
——————
*Author for correspondence
E-mail: vikas.skuast@gmail.com
Mobile: 09469752697, 09419634588

NOTES
417
solutions of 20 mg/ml were prepared by dissolving
95% methanolic extract in DMSO and aqueous
extract in sterile water. Stock solutions were prepared
at least one day in advance and were not filtered and
the microbial contamination was controlled by
addition of 1% gentamycin in complete growth
medium i.e. used for dilution of stock solutions to
make working test solutions of 200 µg/ml.
Cell lines and cultures
The human cancer cells — A-549, COLO-205,
HCT-116, MCF-7, NCI-H322, PC-3, THP-1 and
U-87MG were obtained from National Centre for
Cell Science, Pune, India and National Cancer
Institute, Frederick, USA. These human cancer cells
were further grown and maintained in RPMI-1640
medium and DMEM. The media was supplemented
with FCS (10%), penicillin (100 units/ml), streptomycin
(100 µg/ml) and glutamine (2 mM).
Preparation of positive controls
Positive controls like adriamycin and 5-fluorouracil
were prepared in distilled water, while paclitaxel was
prepared in DMSO. These were further diluted in
gentamycin medium to obtain desired concentrations
of 2 × 10-5 M and 2 × 10-6 M.
In vitro assay for cytotoxic activity
Extracts were subjected to in vitro anticancer
activity against various human cancer cell lines10.
In brief, the cells were grown in tissue culture flasks
in growth medium at 37oC in an atmosphere of
5% CO2 and 90% relative humidity in a CO2
incubator (Hera Cell; Heraeus; Asheville, NCI, USA).
The cells at sub-confluent stage were harvested from
the flask by treatment with trypsin (0.05% trypsin in
PBS containing 0.02% EDTA) and suspended in
growth medium. Cells with more than 97% viability
(trypan blue exclusion) were used for determination
of cytotoxicity. An aliquot of 100 µl of cells
(105 cells/ml) was transferred to a well of 96-well
tissue culture plate. The cells were allowed to grow
for 24 h. Extracts (100 µl/well) were then added to
the wells and cells were further allowed to grow for
another 48 h.
The anti-proliferative SRB assay which estimates
cell number indirectly by staining total cellular
protein with the dye SRB was performed to assess
growth inhibition. The SRB staining method is
simpler, faster and provides better linearity with
cell number. It is less sensitive to environmental
fluctuations and does not require a time sensitive
measurement of initial reaction velocity11. In brief, the
cell growth was stopped by gently layering 50 µl of
50% (ice cold) trichloroacetic acid on the top of
growth medium in all the wells. The plates were
incubated at 4oC for 1 h to fix the cells attached to the
bottom of the wells. Liquid of all the wells was then
gently pipetted out and discarded. The plates were
washed five-times with distilled water and air-dried.
SRB 100 µl (0.4% in 1% acetic acid) was added to
each well and the plates were incubated at room
temperature for 30 min.
The unbound SRB was quickly removed by
washing the cells five-times with 1% acetic acid.
Plates were air-dried, tris buffer (100 µl, 0.01 M,
pH 10.5) was added to all the wells to solubilize
the dye and then plates were gently stirred for 5 min
on a mechanical stirrer. The optical density (OD) was
recorded on ELSIA reader at 540 nm. Suitable
blanks (growth medium and DMSO) and positive
controls (prepared in DMSO and distilled water) were
also included. Each test was done in triplicate and
the values reported were mean values of three
experiments.
The cell growth was determined by subtracting
average absorbance value of respective blank from the
average absorbance value of experimental set. Percent
growth in presence of test material was calculated as
under:
• OD Change in presence of control = Mean OD of
control – Mean OD of blank
• OD Change in presence of test sample = Mean OD
of test sample – Mean OD of blank
• % Growth in presence of control = 100/OD change
in presence of control
• % Growth in presence of test sample = % Growth
in presence of control × OD change in presence of
test sample
• % Inhibition by test sample = 100 – % Growth in
presence of test sample
The growth inhibition of 70% or above was
considered active while testing extracts, but in testing
of active ingredients at different molar concentrations,
the growth inhibition of 50% or above was the criteria
of activity.
Results and Discussion
The in vitro cytotoxic activity of whole plants of
Mentha Spp. is summarized in Table 1. The methanolic
extract of M. arvensis showed in vitro cytotoxic effect
against four human cancer cell lines from four different
tissues. Maximum growth inhibition (92%) was observed

INDIAN J. BIOCHEM. BIOPHYS., VOL. 51, OCTOBER 2014
418
against THP-1 (leukemia). The extract showed 86%,
85% and 76% growth inhibition against colon cancer
cells (COLO-205), lung cancer cells (NCI-H322) and
breast cancer cells (MCF-7), respectively. Whereas
the aqueous extract of M. arvensis suppressed
97% and 75% proliferation of HCT-116 (colon) and
PC-3 (prostate), respectively.
The methanolic extract from M. longifolia suppressed
the proliferation in the range of 75-92% against four
human cancer cell lines from colon, breast, lung
and leukemia origin. Its aqueous extract showed
maximum growth inhibitory effect (81%) against
colon cancer cells (HCT-116), which was considered
significant. The methanolic extract of M. spicata
displayed in vitro anticancer efficacy the range of
71-97% against THP-1, COLO-205, MCF-7 and
NCI-H322. However, its aqueous extract showed
in vitro cytotoxicity only against PC-3 (85%).
Similarly, methanolic extract from M. viridis
exhibited in vitro cytotoxic efficiency against COLO-
205, MCF-7, NCI-H322, THP-1 in the range of 71-
94%, but its aqueous extract was found active against
only HCT-116 (70%) and PC-3 (71%).
Most of the drugs used in cancer chemotherapy
exhibit cell toxicity and can induce genotoxic,
carcinogenic and teratogenic effects in non-tumor
cells. Therefore, there is a need for alternative drugs
of natural origin that are less toxic, endowed with
fewer side effects and more potent in their mechanism
of action. Recently, we have reported that methanolic
extract from the leaves of Nardostachys jatamansi
(commonly known as muskroot) exhibits in vitro
anticancer effect against five human cancer cell
lines viz., NCI-H23, HeLa, SK-N-MC, SW-620 and
COLO-205 in the range of 70-93%12.
Similarly, the methanolic extract from the fruit
part of ‘Kamala tree’ (Mallotus philippinensis) has
displayed significant cytotoxic effect against fourteen
human cancer cell lines — A-549, COLO-205,
DU-145, HEP-2, HeLa, IMR-32, KB, MCF-7,
NCI-H23, OVCAR-5, SiHa, SK-N-MC, SW-620 and
ZR-75-113. Also, the methanolic extract from the
stem-leaves of Calotropis procera has shown 70%
growth inhibition of colon cancer cells (HCT-15)14
and the seed part of Apium graveolens is observed to
be active against COLO-205, HeLa, KB, SK-N-MC15.
The strong anti-proliferative effect on a range of
human cancer cell lines is also displayed by the
methanolic extract from the fruit part of Momordica
charantia; this particular extract has been observed to
be cytotoxic to a wide spectrum of cancer cells
(A-549, COLO-205, MCF-7, NCI-H322, PC-3,
THP-1 and U-87MG) with the growth inhibition
ranging between 75-100%. The extract has shown a
Table 1—Growth inhibitory effect of extracts of whole plants from Mentha Spp. with appropriate positive controls against human
cancer cell lines
Plant Extract Conc.
(µg/ml) Lung Colon Colon
Breast Lung Prostate Leukemia Glioblastoma
A-549 COLO-
205 HCT-
116 MCF-7
NCI-
H322 PC-3 THP-1 U-87MG
Growth inhibition (%)
Methanolic 100 58 86 23 76 85 0 92 36
M. arvensis
Aqueous 100 46 21 97 63 42 75 3 9
Methanolic 100 58 92 20 84 75 0 92 58 M. longifolia Aqueous 100 56 14 81 57 31 3 7 2
Methanolic 100 53 81 53 75 71 0 97 38 M. spicata Aqueous 100 42 22 68 61 34 85 18 16
Methanolic 100 61 88 7 70 71 0 94 54 M. viridis
Aqueous 100 48 24 70 59 39 71 9 12
Positive controls Conc.
(Molar)
5-Flurouracil 2×10–5 - 51 68 - - - 73 60
Paclitaxel 1×10–6 79 - - - 52 - - -
Adriamycin 1×10–6 - - - 60 - 59 - -
Growth inhibition of 70% or above is indicated in bold.
The mark (-) indicates that particular human cancer cell line was not treated with that particular positive control.

NOTES
419
high degree of growth inhibition against NCI-H322
(lung, 100%), MCF-7 (breast, 99%) and COLO-205
(colon, 97%). Interestingly, the extract has exhibited
the cytotoxic effect significantly higher than 5-flurouracil,
adriamycin and paclitaxel used as positive controls16.
Moreover, potent cytotoxic effect of 80% methanolic
extract and chloroform fractions of Mentha spicata
has been reported against HeLa, HEP-2 and PC-3
cancer cell lines1,4. Aqueous extract from M. spicata
has also shown cytotoxic effect on U-937 human
monocytic leukemia cells5. M. longifolia has also
shown cytotoxicity of IC50 at a concentration of
119 µg/ml and 89.9 µg/ml against HeLa and HEP-2
human cancer cells4. The oral administration of
aqueous extract from M. piperita leaves has exhibited
a significant reduction in number of lung tumors
from an incidence of 67.92% in animals given only
benzo[a]pyrene to 26.31%17. M. piperita has a
chemopreventive effect against the tumorigenicity of
shamma which could be due to anti-mutagenic
properties18. Essential oil from M. pulegium is also
found to be a cytotoxic agent against human ovary
adenocarcinoma SK-OV-3, human malignant cervical
adenocarcinoma HeLa and human lung carcinoma
A-549 cell lines19.
To conclude, Mentha Spp. possess in vitro
cytotoxic effect against COLO-205, HCT-116,
MCF-7, NCI-H322, PC-3 and THP-1 cancer cells.
However, further studies are required for the isolation
of active ingredient(s) which may serve as lead
molecule(s) in the development of anticancer agents,
especially for colon, breast, lung cancer and leukemia
carcinoma.
References
1 Arumugam P, Ramamurthy P & Ramesh A (2010) Int
J Food Prop 13, 23-31
2 Manosroi J, Dhumtanom P & Manosroi A (2006) Cancer
Lett 235, 114-120
3 Londonkar R L & Poddar P V (2009) World J Gastrointes
Oncol 1, 82-88
4 Rahimifard N, Hajimehdipoor H, Hedayati M H, Bagheri O,
Peshehvar H & Ajani Y (2010) J Med Plants 9, 88-92
5 Hajighasemi F Hashemi V & Khoshzaban F (2011) J Med
Plant Res 5, 5142-5147
6 Zu Y, Yu H, Liang L, Fu Y, Efferth T, Liu X & Wu N (2010)
Molecules 15, 3200-3210
7 Hussain A I, Anwar F, Nigam P S, Ashraf M & Gilani A H
(2010) J Sci Food Agric 90, 1827-1836
8 Jain D, Pathak N, Khan S, Raghuram G V, Bhargava A,
Samarth R & Mishra P K (2011) Int J Toxicol 30, 225-236
9 Paranjape P (2001) Indian Medicinal Plants: A Guide to
Ayurvedic Herbal Medicine, pp 1-31, Chaukhamba Sanskrit
Pratishthan, New Delhi.
10 Monks A, Scudiero D, Skehan P, Shoemaker R, Paull K,
Vistica D, Hose C, Langley J, Cronise P,Vaigro– Wolff A,
Gray–Goodrich M, Campbell H, Mayo J & Boyd M (1991)
J Natl Cancer Inst 83, 757-766
11 Skehan P, Storeng R, Scudiero D, Monks A, McMohan J,
Vistica D, Warren J T, Bokesh H, Kenny S & Boyd M
(1990) J Natl Cancer Inst 82, 1107-1112
12 Sharma V & Hussain S (2013) Bioinfolet 10, 567-571
13 Sharma V (2011) J Plant Biochem Biotechnol 20, 190-195
14 Bhagat M & Sharma V (2010) Int J Med Sci 2, 152-154
15 Sharma V & Tiku A K (2008) Asian J Exp Chem 3, 41-45
16 Hussain S, Sharma V & Saxena A K (2013) Int J Plant Sci 8,
140-143
17 Samarth R M, Panwar M, Kumar M & Kumar A (2006)
Mutagenesis 21, 61-66
18 Samman M A, Bowen I D, Taiba K, Antonius J & Hannan M
A (1998) Carcinogenesis 19, 1795-1801
19 Shirazi F H, Ahmadi N & Kamalinejad M (2004)
J Pharm Sci 12, 106-110.