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Antioxidative effects of the spice cardamom against non-melanoma skin cancer by modulating nuclear factor erythroid-2-related factor 2 and NF-κB signalling pathways

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Antioxidative effects of the spice cardamom against non-melanoma skin cancer by modulating nuclear factor erythroid-2-related factor 2 and NF-κB signalling pathways

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The role of dietary factors in inhibiting or delaying the development of non-melanoma skin cancer (NMSC) has been investigated for many years. Cardamom, which is a dietary phytoproduct, has been commonly used in cuisines for flavour and has numerous health benefits, such as improving digestion and stimulating metabolism and having antitumorigenic effects. We have investigated the efficacy of dietary cardamom against 7,12-dimethylbenz[a]anthracene (DMBA)-induced skin papillomatogenesis in Swiss albino mice that closely resembles human NMSC. Mice were grouped into normal wild type (untreated), vehicle-treated (acetone), carcinogen-treated (DMBA), and DMBA and cardamom-treated (DMBA+CARD) to delineate the role of cardamom against DMBA-induced papillomatogenesis. Oral administration of cardamom to DMBA-treated mice up-regulated the phase II detoxification enzymes, such as glutathione-S-transferase and glutathione peroxidase, probably via activation of nuclear factor erythroid-2-related factor 2 transcription factor in 'DMBA+CARD' mice. Furthermore, reduced glutathione, glutathione reductase, superoxide dismutase and catalase were also up-regulated by cardamom in the same 'DMBA+CARD' group of mice compared with DMBA-treated mice. Cardamom ingestion in DMBA-treated mice blocked NF-κB activation and down-regulated cyclo-oxygenase-2 expression. As a consequence, both the size and the number of skin papillomas generated on the skin due to the DMBA treatment were reduced in the 'DMBA+CARD' group. Thus, the results from the present study suggest that cardamom has a potential to become a pivotal chemopreventive agent to prevent papillomagenesis on the skin.
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Antioxidative effects of the spice cardamom against non-melanoma skin
cancer by modulating nuclear factor erythroid-2-related factor 2 and NF-kB
signalling pathways
Ila Das
1
, Asha Acharya
2
, Deborah L. Berry
3
, Supti Sen
3
, Elizabeth Williams
3
, Eva Permaul
3
,
Archana Sengupta
1
, Sudin Bhattacharya
1
and Tapas Saha
3
*
1
Department of Cancer Chemoprevention, Chittaranjan National Cancer Institute, Kolkata, India
2
Genetarn Corporation, Rockville, MD, USA
3
Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University, 3970 Reservoir Road, NW,
PCS Building, Room GD3, Washington, DC 20057, USA
(Submitted 9 May 2011 – Final revision received 28 September 2011 – Accepted 17 October 2011 – First published online 19 December 2011)
Abstract
The role of dietary factors in inhibiting or delaying the development of non-melanoma skin cancer (NMSC) has been investigated for many
years. Cardamom, which is a dietary phytoproduct, has been commonly used in cuisines for flavour and has numerous health benefits,
such as improving digestion and stimulating metabolism and having antitumorigenic effects. We have investigated the efficacy of dietary
cardamom against 7,12-dimethylbenz[a]anthracene (DMBA)-induced skin papillomatogenesis in Swiss albino mice that closely resembles
human NMSC. Mice were grouped into normal wild type (untreated), vehicle-treated (acetone), carcinogen-treated (DMBA), and DMBA
and cardamom-treated (DMBA þCARD) to delineate the role of cardamom against DMBA-induced papillomatogenesis. Oral adminis-
tration of cardamom to DMBA-treated mice up-regulated the phase II detoxification enzymes, such as glutathione-S-transferase and glu-
tathione peroxidase, probably via activation of nuclear factor erythroid-2-related factor 2 transcription factor in ‘DMBA þCARD’ mice.
Furthermore, reduced glutathione, glutathione reductase, superoxide dismutase and catalase were also up-regulated by cardamom in
the same ‘DMBA þCARD’ group of mice compared with DMBA-treated mice. Cardamom ingestion in DMBA-treated mice blocked
NF-kB activation and down-regulated cyclo-oxygenase-2 expression. As a consequence, both the size and the number of skin papillomas
generated on the skin due to the DMBA treatment were reduced in the ‘DMBA þCARD’ group. Thus, the results from the present study
suggest that cardamom has a potential to become a pivotal chemopreventive agent to prevent papillomagenesis on the skin.
Key words: Chemoprevention: Cyclo-oxygenase-2: Superoxide dismutase: Catalase: Glutathione: 7,12-
Dimethylbenz[a]anthracene: Detoxification enzymes
Non-melanoma skin cancer (NMSC) is a common form of skin
cancer that occurs either in basal or squamous cells. These
cells are located at the epidermal layer of the skin and protect
the internal organs and the outer surface of the body. Skin
cancer cells vary in their proliferation rate depending upon
the type of skin cancer, but rarely spread to the other parts
of the body. More than 1 000 000 new cases and less than
1000 deaths from NMSC have been reported in the USA in
2010
(1)
. UVA and UVB (UV radiation) exposure to exposed
skin is the leading cause of NMSC
(2)
. Outdoor workers,
people of fair skin, large number of moles in the body,
large dark-coloured birth mark known as congenital melano-
cytic nevus, pre-cancerous skin lesion such as actinic kerato-
sis, family history of sunburn and skin cancer, and aged
people are at a serious risk of developing skin cancer.
Oxidative stress, which is defined as an excess of toxic reac-
tive oxygen species (ROS), and concomitant inadequate
expression levels of various ROS detoxification enzymes,
such as phase II detoxification and antioxidation enzymes,
play a crucial role in developing several cancers including
*Corresponding author: Dr T. Saha, fax þ1 202 687 5324, email ts283@georgetown.edu; tapassaha2000@gmail.com
Abbreviations: 4-HNE, 4-hydroxynonenal; CARD, cardamom-ingested; CAT, catalase; CDNB, 1-chloro-2-4-dinitrobenzene; COX-2, cyclo-oxygenase-2;
DMBA, 7,12-dimethylbenz[a]anthracene; GPx, glutathione peroxidase; GR, glutathione reductase; GSH, reduced glutathione; GST, glutathione-S-
transferase; IHC, immunohistochemistry; Keap1, kelch-like ECH-associated protein 1; LPO, lipid peroxides; MDA, malondialdehyde; NMSC,
non-melanoma skin cancer; Nrf2, nuclear factor erythroid-2-related factor 2; ROS, reactive oxygen species; SOD, superoxide dismutase; TPA,
12-O-tetradecanoylphorbol-13-acetate.
British Journal of Nutrition (2012), 108, 984–997 doi:10.1017/S0007114511006283
qThe Authors 2011
British Journal of Nutrition
skin carcinogenesis
(3)
. ROS is produced during distinct intra-
cellular pathways and has been found to be elevated during
infection, inflammation, and exposure to environmental carci-
nogens, radiation and sunlight
(4)
, which in part might lead to
the development of NMSC. Cancer chemoprevention, as first
defined by Sporn in 1976
(5)
, utilises naturally occurring phyto-
chemicals, biological compounds and/or synthetically syn-
thesised chemical agents to reverse, repress or prevent the
development of several cancers including skin cancer. Spices
and condiments are naturally occurring dried herbs that are
known for their high therapeutic potential. Numerous epide-
miological studies have shown that a diet rich in vegetables,
fruits, herbs and spices is associated with a reduced risk of
most cancers, and thus serve as potent chemopreventive
agents
(5,6)
.
Cardamom is a well-known aromatic spice that is regularly
used in Eastern, Arab and Scandinavian cuisines for its aroma.
From ancient times, it has also been used as herbal medicine
especially for the common cold. Cardamom has also been
used for teeth, gum and throat infection, as well as against
lung congestion, pulmonary tuberculosis and digestive dis-
orders. Cardamom is one of the common ingredients of
Indian Ayurveda and Chinese traditional medicine
(7)
. Carda-
mom belongs to Elettaria and Amomum genera of the ginger
family Zingiberaceae. Both forms have small spindle-like seed-
pod but Elettaria is smaller and light green in colour, whereas
Amomum is big and blackish grey in appearance. T. John
Zachariah in his chapter titled ‘Chemistry of cardamom’ from
the book Cardamom, The Genus Elettaria
(8)
had compiled
different components of cardamom that have been investigated
and published earlier. The main chemical ingredients of carda-
mom is the oil component that is composed of a- and b-pinene,
a-phellandrene, sabinene, limonene, myrcene, 1,8-cineole,
g-terpinene, p-cymene, terpinolene, linalool, terpinen-4-oil,
a-terpineol, a-terpineol acetate, linalyl acetate, citronellol,
geraniol, nerol, methyl eugenol and trans-nerolidol
(8,9)
.A
couple of bioactive compounds of cardamom have been
studied for anti-cancer, anti-inflammatory and chemopreven-
tive properties. Monoterpene compounds of cardamom oil
were shown to enhance the activity of indomethacin on skin.
A lower dose of cardamom suppressed oedema to a lesser
extent, whereas a higher dose of cardamom reveals a more
potent anti-inflammatory effect on the skin in the presence of
indomethacin
(10)
. Chemopreventive function of cardamom
has also been demonstrated earlier to regulate colorectal
cancer
(11)
. The present study was designed to evaluate the
chemopreventive efficacy of nutritional cardamom on
7,12-dimethylbenz[a]anthracene (DMBA)-induced skin carcino-
genesis in a well-defined mouse model
(12)
that highly mimics
human NMSC.
Experimental methods
Chemicals
DMBA, croton oil, acetone, NADPH (nicotinamide adenine
dinucleotide phosphate, reduced form), pyrogallol, thiobarbi-
turic acid, diethylene triamine penta-acetic acid and pyridine
were purchased from Sigma Chemicals Company.
Animals used and dose selection for 7,12-
dimethylbenz[a]anthracene and cardamom
Female Swiss albino mice aged 56 weeks, and weighing
20 –22 g were housed in plastic cages with stainless-steel
wire lids (four mice per cage). The housing facility was main-
tained at 308C temperature under alternating light and dark
conditions. Standard food pellets and drinking water were
provided ad libitum to all the treated/untreated mice.
7,12-Dimethylbenz[a]anthracene treatment
DMBA was purchased from Sigma Chemicals Company. In the
first week, three topical applications of 100 nmol DMBA in
100 ml acetone were applied on the shaved dorsal skin. This
was followed by 1 % (w/v) croton oil in acetone at the same
site twice per week up to 8 weeks for the initiation and devel-
opment of skin papillomatogenesis.
Cardamom treatment
Small-pod cardamom was bought from the local market (avail-
able to common people) in bulk in anticipation to complete
one set of experiments (3 months of supply) so that the
batch of cardamom does not differ between feedings to the
animals. Just before the oral administration of cardamom to
DMBA-treated mice, the required amount of spice was
ground in a mixer and an aqueous suspension of cardamom
solution was prepared fresh that contained all the oil com-
ponents of cardamom as emulsion. Initially, a pilot study
was carried out using different doses, namely 250, 500, 750
and 1000 mg cardamom/kg body weight per d, in order to
standardise the most effective dose against papilloma for-
mation during DMBA-induced skin carcinogenesis. Treatment
with 500 mg cardamom/kg body weight per mouse per d was
found to be most effective without any toxic manifestation.
Experimental design and treatment groups
The experiments were divided into three sets such that mice
can be killed after 2nd, 8th and 12th week of first DMBA appli-
cation. Each set was again divided into five groups with eight
mice in each group. Each set comprised forty mice that
were divided into ‘untreated’ (normal control), ‘acetone’
(vehicle control), ‘DMBA’ (carcinogen control), ‘DMBA þ
CARD’ (DMBA-treated and cardamom-ingested) and ‘CARD’
(cardamom-ingested) groups. The protocol used for DMBA
application on the shaved dorsal skin of Swiss albino mice,
and oral administration of cardamom to wild-type and
DMBA-treated mice are given in Table 1. Acetone treatment
was done exactly similar to the DMBA treatment and con-
sidered as the vehicle control because DMBA was dissolved
in acetone. All animal protocols have been approved by the
Institutional Animal Care and Use Committee of the institution.
The animals were carefully examined once per week for
recording the body weight and papilloma growth.
Chemopreventive effects of dietary cardamom 985
British Journal of Nutrition
Table 1. Correlation between the different groups of mice, treatments and body weight
(Mean values and standard deviations)
Initial average
weight (g),
week 0
Average weight
(g), week 8
Final average
weight (g), week
12
Experimental groups Treatments
No. of animals/
duration
of study (weeks) Mean SD Mean SD Mean SD
Untreated (normal control) Water and standard food pellets during treatments
and serve as a untreated control
24/02
24/08
24/12
20·31 1·17 28·5 1·28 32·42 1·25
Acetone (vehicle-treated) Three topical application of acetone, followed by
100 ml of 1 % (w/v) croton oil in acetone at the
same site twice per week up to 8th week
and treated as ‘normal’
24/02
24/08
24/12
20·18 0·96 28·05 1·44 30·98 1·29
DMBA (carcinogen-treated) Three topical application of 100 nmol DMBA in
100 ml acetone, followed by 100 ml of 1 % (w/v)
croton oil in acetone at the same site twice
per week up to 8th week and treated as ‘normal’
24/02
24/08
24/12
20·19 1·22 22·92 0·89 25·79 1·131
DMBA þCARD (carcinogen and cardamom-treated) DMBA treatment was performed same as above.
In parallel, cardamom suspension was administered
orally to the mice simultaneously with DMBA
application and continued until the end of the
experiment (12 weeks)
24/02
24/08
24/12
20·15 1·01 26·01 1·19 29·46* 1·15
CARD (cardamom-treated) Cardamom suspension was administered to the mice
without DMBA application and continued
until the end of the experiment (12 weeks)
24/02
24/08
24/12
19·57 1·15 27·4 0·95 31·45 1·19
DMBA, 7,12-dimethylbenz[a]anthracene; CARD, cardamom ingested.
* Mean value was significantly different from that of the DMBA group (P,0·001).
I. Das et al.986
British Journal of Nutrition
Tissue preparation for histology with haematoxylin and
eosin and immunohistochemistry
Collected skin tissues were fixed in 10 % neutral formalin for
24 h and then dehydrated by graded ethyl alcohol, starting
from 50 to 95 %. The tissues were then soaked in xylene
and paraffin blocks were prepared. Serial microtome sections,
in the form of a paraffin ribbon, were made at a thickness of
5mm. The tissues were soaked in lukewarm water (588C) for
stretching, and the desired portion of the paraffin ribbon
was placed on a clear glass slide previously coated with egg
albumin and gently stretched using forceps or needles. Any
excess water on the slide was drained off, and the slide was
placed on a hot plate to allow the paraffin to dry and
adhere to the slide.
Haematoxylin and eosin staining was performed with the
sectioned tissues. The slides were rehydrated by graded
ethyl alcohol series (from 95 to 50 % ethyl alcohol) and were
stained with 1 % haematoxylin for 3 min. The slides were
washed slowly in running tap water for 8– 10 min to develop
the colour of haematoxylin that stains DNA-rich nuclei. The
stained slides were then dipped in an acidalcohol solution
(1:1 HCl–C
2
H
5
OH) to remove extra stain and washed under
running water. The haematoxylin-stained slides were again
passed through ascending grades of ethyl alcohol. Counter-
staining was done with eosin (0·5 g in 100 ml of 95 % absolute
alcohol). The slides were then treated with xylene for 15 min
each. Mounting was performed with a small amount of DPX
mounting solution over a thin 22 –50 mm coverslip.
Immunohistochemical staining of mouse skin samples was
performed with the following antibodies: nuclear factor ery-
throid-2-related factor 2 (Nrf2; Santa Cruz SC-722, 1:100);
NF-kB (Santa Cruz SC-109, 1:75); IkBa(Santa Cruz SC-371,
1:600); kelch-like ECH-associated protein 1 (Keap1; Sigma
AV38981, 1:4000). A 5 mm section from formalin-fixed paraf-
fin-embedded tissues was deparaffinised with xylene and
rehydrated through a graded alcohol series (from 95 to 50 %
ethyl alcohol). Antigen retrieval was performed by immersing
the tissue sections at 988C for 20 min in 10 mM-citrate buffer
(pH 6·0) with 0·05 % Tween. Immunohistochemical staining
was performed using the Vectastain Kit (Vector Labs), accord-
ing to the manufacturer’s instructions. Briefly, slides were pre-
treated with 3 % H
2
O
2
, avidin/biotin blocking and 10 % normal
goat serum, and then exposed to the stated dilution of primary
antibody for 1 h at room temperature. The slides were
exposed to appropriate biotin-conjugated secondary anti-
bodies (Vector Labs), Vectastain ABC reagent (Vector Labs)
and DAB chromagen (Dako) to detect horseradish peroxidase.
The slides were then counterstained with haematoxylin
(Fisher, Harris Modified Haematoxylin) at a 1:17 dilution for
2 min at room temperature, blued in 1 % ammonium hydrox-
ide for 1 min at room temperature, dehydrated and mounted
with Acrymount. Consecutive sections with the omitted pri-
mary antibody were used as negative controls. Images were
captured using an Olympus DP70 microscope (Olympus) at
200 £magnification.
Preparation of tissue lysates for immunoblotting
Treated/untreated skin tissues from the dorsal sides were
excised from each group of mice for lysate preparation. In
the case of the ‘DMBA’ and ‘DMBA þCARD’ groups, skins
with three to four papillomas per mice were excised to pre-
pare the tissue lysates. This whole procedure had been
repeated three times for reproducibility and statistical signifi-
cance. Approximately 50 mg of skin tissues from treated and
untreated mice were excised from the different groups of
mice and subsequently soaked in tissue lysis buffer (Roche
Biochemicals) containing protease inhibitor cocktail (Roche
Biochemicals) at 48C. The tissues were then homogenised
for 30 s and then centrifuged at 14 000 gfor 20 min at 48C.
The supernatants (tissue lysates) were collected into a fresh
tube and stored in 2808C until further use in subsequent Wes-
tern blotting. Concentrations of the total proteins were
measured by the Lowry method using bovine serum albumin
as a standard.
Methods for biochemical assays
Skin tissues were sampled for biochemical assays from all
groups of mice for the measurement of lipid peroxides
(LPO), glutathione-S-transferase (GST), glutathione peroxidase
(GPx), catalase (CAT), superoxide dismutase (SOD), gluta-
thione reductase (GR) and reduced glutathione (GSH) as pub-
lished protocols
(12)
. For the 4-hydroxynonenal (4-HNE) assay,
generation of 4-HNE was measured by following the manufac-
turer’s instructions using OxiSelect HNE-His Adduct Elisa Kit
from Cell BioLabs.
LPO generation in mice was obtained from 400 mg of the
treated/untreated skin tissue lysates by measuring thiobarbitu-
ric acid-reactive substance, formed/mg protein using an extinc-
tion coefficient of 1·56 £10
5
mol/l per cm
(13)
. Thiobarbituric
acid-reactive substance assay is a tool for the direct measure-
ment of malondialdehyde (MDA) in the treated/untreated
skin tissue lysates. In brief, skin tissues were washed in 0·9 %
NaCl and homogenised with nine volumes (3·6 ml) of 1·15 %
KCl, followed by centrifugation at 12 000 gfor 10 min at 48C.
The supernatant was further centrifuged for 10 min at 10 000 g
at 48C. Finally, the supernatant was centrifuged at 25 000 gat
48C for 1 h and the precipitate was retained. The precipitate
was then resuspended in KCl and aliquots were stored for
future use. SDS, acetic acid, thiobarbituric acid and water
were then added to the selected aliquots and heated at 958C
for 1 h. The butanol pyridine solution (15:1) was added to
the above reaction mixture and the mixture was vortexed vig-
orously for 2 min followed by centrifugation at 3000 gfor
10 min. The upper organic layer was collected, and optical den-
sity at 532 nm was measured against the control. Control was
prepared in the same way as described above, but water was
taken instead of sample lysate.
4-HNE is considered as a marker for oxidative stress and is a
well-known naturally occurring toxic by-product of lipid per-
oxidation. It was measured in a ninety-six-well format where
samples (about 10 mg/ml) were allowed to adsorb in the
wells for 2 h at 378C. 4-HNE protein adducts were then
Chemopreventive effects of dietary cardamom 987
British Journal of Nutrition
incubated with anti-HNE-His antibody, which was further
incubated with horseradish peroxidase-conjugated secondary
antibody. Initially, a standard curve was prepared with
known concentrations of HNE-bovine serum albumin stan-
dards. 4-HNE contents in the treated/untreated skin tissues
from the different groups of mice were measured as
above and quantified using the HNE-bovine serum albumin
standard curve.
Activity of GST was measured from 200 mg of the treated/
untreated skin tissue lysates by documenting the increase in
the absorbance of 1-chloro-2-4-dinitrobenzene (CDNB) as
the substrate at 340 nm
(14)
. Specific activity of the enzyme is
expressed as the formation of CDNB glutathione (CDNB
GSH) conjugate/min per mg protein. In brief, tissues were
homogenised and centrifuged at 35 000 gfor 2 h at 48C. The
supernatant was collected and incubated with 100 ml CDNB
in distilled water at 308C for 5 min. Then the reaction was
initiated by the addition of 100 ml GSH and optical density
was taken at 340 nm. Control was prepared as described
above and distilled water was taken instead of tissue
homogenate.
Activity of GPx was measured from 100 mg of the treated/
untreated skin tissue lysates
(15)
. For the assay, NADPH
and GR were mixed with the sample and the decrease in
the absorbance of NADPH at 340 nm was immediately
measured in the presence of H
2
O
2
. GPx activity is expressed
as nmol of NADPH utilised/min per mg protein using
the molar extinction coefficient of NADPH at 340 nm
(6·22/nMper cm).
CAT activity was also measured spectrophotometrically
from 100 mg of the skin tissue lysates
(16)
. Tissues were hom-
ogenised in phosphate buffer and centrifuged at 2500 gfor
10 min. The supernatant was used for the enzyme activity
assay in the presence of H
2
O
2
and measured spectrophotome-
trically at 240 nm. The results are expressed as units/mg pro-
tein. The unit is defined as follows: one unit of CAT is the
amount of enzyme that liberates half of the peroxide oxygen
from H
2
O
2
in 100 s at 258C.
SOD activity was measured from 200 mg of the skin tissue
lysates from the different groups of mice. Extraction and puri-
fication of SOD was performed according to the published
protocol
(17)
. SOD was quantified as an inhibition of pyrogallol
auto-oxidation in Tris– HCl buffer (50 mM, pH 7·5), which was
measured spectrophotometrically at 420 nm, and the results
are expressed as units/mg protein. The unit is defined as fol-
lows: one unit of enzyme activity is expressed as the amount
of enzyme necessary for inhibiting the reaction by 50 %.
GSH activity was measured with the cytosolic fraction from
200 mg of skin tissues obtained from the different groups of
mice
(18)
. The assay was designed to measure a decrease in
dithiobis(2-nitro)-benzoic acid reduced by SH groups and
expressed as nmol/mg protein. First, a standard curve was
prepared by measuring the absorbance of different known
concentrations of GSH. The GSH enzyme activities from the
treated/untreated skin samples were determined from the
GSH standard curve.
GR is a flavoprotein catalysing the NADPH-dependent
reduction of glutathione disulphide to GSH. GR activity was
measured from 200 mg of the skin tissue lysates obtained
from the different groups of mice
(19)
. The oxidation of
NADPH was measured spectrophotometrically at 340 nm.
Enzyme activity is expressed as nmol NADPH consumed/
min per mg protein based on the extinction coefficient of
NADPH, which is 6·22/nMper cm.
Statistical analysis
All the experiments were performed at least three times and
were analysed and expressed as means and standard devi-
ation. The data obtained from the ‘untreated’, ‘acetone’,
‘DMBA’ and ‘DMBA þCARD’ groups were statistically ana-
lysed by one-way ANOVA via Tukey’s test, which is a pairwise
comparison of the mean responses to the different treatment
groups using Sigmastat statistical software (version 3)
for Windows; Systat Software Inc.). P,0·05 was considered
significant for all experiments.
Results
Morphological evidences of papillomas following the
7,12-dimethylbenz[a]anthracene and cardamom
treatments
Female Swiss albino mice were grouped and treated as
described in Table 1. The ‘untreated’, acetone- and ‘DMBA’-
treated groups served as the normal control, vehicle control
and carcinogen control, respectively. The effect of cardamom
was determined in carcinogen-treated and cardamom-ingested
mice designated as (DMBA þCARD). At the end of the 12th
week, after the first topical DMBA application on mouse
skin, the total incidences of papilloma growth were found to
be reduced significantly both in number and sizes in the
‘DMBA þCARD’-treated group with respect to the ‘DMBA’
group. No papillomas were observed in the ‘untreated’ and
‘acetone’ groups at any time point. A representative snapshot
of mice from each group is shown in Fig. S1 of the sup-
plementary material (available online at http://www.journals.
cambridge.org/bjn). Cardamom-treated mouse skin did not
show any papilloma growth on the skin at any time point
(Fig. 1(b)).
Effects of aqueous suspension of cardamom on the body
weight and papilloma growth
A normal growth pattern in body weights was observed in
the ‘untreated’ and ‘acetone’-treated groups throughout our
experimental time points (012 weeks). The body weights
of the ‘DMBA’ group followed a much lower slope in
growth pattern when compared with the other groups of
mice (Fig. 1(a)). Growth pattern of the ‘DMBA þCARD’
group falls in between the growth rate of the ‘DMBA’ and
‘acetone’/‘untreated’ groups. Papillomas started to appear on
the DMBA-treated skin from the 5th week onwards and con-
tinued to grow until the end of the 12th week. Of these
mice, 92 % had papilloma growth due to the DMBA treatment
on the shaved skin. Of the mice that received the aqueous
I. Das et al.988
British Journal of Nutrition
suspension of cardamom by oral administration after the first
DMBA application, 29 % showed papilloma growth from the
7th week instead of the 5th week and showed regression in
papilloma growth when compared with the DMBA group
(Fig. 1(b)).
Histology of the skin following induction of carcinogenesis
and the cardamom treatment
The histology of the skin from the ‘DMBA’ group demon-
strated marked differences when compared with the ‘acetone’
group as described previously
(12,20)
. Severe hyperkeratosis
and acanthosis were observed in DMBA-treated mouse skin,
demonstrating progression towards skin carcinogenesis after
8 and 12 weeks (see Fig. S2(B and E) of the supplementary
material, available online at http://www.journals.cambridge.
org/bjn). ‘Acetone’-treated mice demonstrated similar his-
tology to that of the untreated mouse skin of the same age
as shown previously
(12)
(see Fig. S2(A and D) of the sup-
plementary material, available online at http://www.journals.
cambridge.org/bjn). Histology of the affected skins from the
‘DMBA þCARD’ group demonstrated mild hyperkeratosis
and moderate acanthosis of the epidermis, which revealed a
near-normal morphology of the skin after 8 and 12 weeks
(see Fig. S2(C and F) of the supplementary material, available
online at http://www.journals.cambridge.org/bjn).
Effect of the aqueous suspension of cardamom on lipid
peroxidation
Lipid peroxidation is the unstable indicator of oxidative stress
in cells that decompose to form more complex and reactive
compounds such as MDA and 4-HNE. LPO are generated in
the aerobic cells due to oxidative stress and the deleterious
interaction of ROS with lipid molecules. These free radicals
are produced in the body as normal consequences of bio-
chemical processes and increased exposure to xenobiotics.
Here we wanted to see whether aqueous suspension of carda-
mom has any effect on the DMBA-induced generation of LPO
in the treated skins from the different groups of mice. We
killed the different groups of mice at the end of 2, 8 and 12
weeks from the first dose of DMBA treatment. LPO values
obtained from the 2nd and 8th week are given in Table S1
(available online at http://www.journals.cambridge.org/bjn)
and the LPO data obtained after 12th week are shown in
Fig. 2. The DMBA treatment induced the formation of LPO
in the mouse skin, which was significantly higher than that
of the ‘untreated’ and ‘acetone’-treated mouse skins. At the
end of the 12 weeks, the generation of MDA and the
production of 4-HNE were down-regulated to 5·25-fold
(P,0·001; Fig. 2(a)) and 2·75 fold (P,0·001; Fig. 2(b)),
respectively, in the ‘DMBA þCARD’ group when compared
with ‘DMBA’-treated mice.
Effect of the aqueous suspension of cardamom on host
cellular defences
Detoxification enzymes and antioxidants are the key players
that maintain redox homeostasis of the cells and help in
metabolism of the xenobiotics as a part of cellular defence
system. Thus, we investigated the effects of cardamom
0·4
100
80
60
40
20
0
*
*
0·3
0·2
0·1
TBARS
(nmol/mg protein)
(a) (b)
4-HNE
(µmol/mg protein)
0·0
Untreated
Acetone
DMBA
DMBA+CARD
Untreated
Acetone
DMBA
DMBA+CARD
Fig. 2. Effects of ingestion of aqueous cardamom and 7,12-dimethylbenz
[a]anthracene (DMBA) treatment on the generation of lipid peroxides (LPO)
in treated skin tissue lysates after 12 weeks from first DMBA application. Mal-
ondialdehyde (MDA) and 4-hydroxynonenal (4-HNE) are the known toxic by-
products of LPO. Swiss albino mice were grouped and treated as given in
Table 1. Treated/untreated skin tissue lysates from all the groups of mice
were subjected to the (a) MDA assay and (b) 4-HNE assay to determine the
levels of LPO among the samples. In both the cases, the DMBA þCARD
group showed significantly reduced LPO (* P,0·001) when compared with
DMBA-treated mice only. Values are means, with standard deviations rep-
resented by vertical bars. TBARS, thiobarbituric acid-reactive substance.
CARD, cardamom ingested.
After 12 weeks
(b)
No. of papilloma
bearing mice
Incidences of
papilloma (%)
Groups
Untreated 0 (out of 24)
0 (out of 24)
Mean SD
1·72
0·14
6·1
1·2
92
29
22 (out of 24)
7 (out of 24)
0 (out of 24)
Acetone
DMBA
DMBA+
CARD
CARD
No. of papilloma per mice
35
30
25
20
15
00246810 12
Time (weeks)
Body weight (g)
(a)
Fig. 1. Effects of topical application of 7,12-dimethylbenz[a]anthracene
(DMBA) and ingestion of cardamom on body weight and papilloma growth.
Swiss albino mice were grouped and treated as given in Table 1.
(a) DMBA-treated mice (DMBA, ) demonstrated a significantly lower
growth rate than that of DMBA-treated and cardamom-ingested mice
(DMBA þCARD, ). The normal control (untreated, ) and acetone
control (acetone, ) groups of mice showed a normal growth pattern.
(B) Table describing the total incidences of papilloma growth on the skin in
the experimental mice groups. Of the DMBA group, 92 % had papilloma
growth on their treated skin but the percentage dropped down to 29 % for the
DMBA þCARD group. The untreated, acetone-treated and CARD groups
showed no papillomatous growth during the experimental period.
Chemopreventive effects of dietary cardamom 989
British Journal of Nutrition
ingestion against the development of skin papillomas initiated
by DMBA on the activities of GST, GPx, GSH, GR, SOD and
CAT. The treated/untreated skin tissue lysates obtained from
the different groups of mice were used for the activity
assays. Cardamom ingestion, following the DMBA treatments,
elevated the activity levels of GST by 13·1-fold, combined GPx
by 8·62-fold, GSH by 8·17-fold, GR by 16·45-fold, combined
SOD by 22·13-fold and CAT by 13·14-fold when compared
with DMBA-treated mice only. The increase in enzyme activi-
ties exceeded the basal levels of activities set by either
‘untreated’ or ‘acetone’-treated mice in each case (Fig. 3).
Cardamom rescues 7,12-dimethylbenz[a]anthracene-
induced down-regulation of nuclear factor erythroid-2-
related factor 2 and kelch-like ECH-associated protein 1 in
mouse skin
Nrf2 is a basic region-leucine zipper transcription factor that
regulates the induction of phase II detoxification enzymes
during stress condition
(21)
. Keap1 is a cytoplasmic anchor for
Nrf2 that tethers Nrf2 in the cytoplasm under basal unstressed
conditions. Keap1 prevents Nrf2 from translocating to the
nucleus, where it would bind to the antioxidant response
element and activate gene transcription
(22)
. It has been
reported that Nrf2 is released from Keap1 under oxidative
stress, resulting in an enhanced nuclear accumulation and
transcriptional induction of target genes (phase II detoxification
enzymes) that ensure cell survival
(23)
. Immunohistochemistry
(IHC) with Nrf2 and Keap1 on the untreated/treated skins
from the different groups of mice is shown in Fig. 4(a– c)
(Nrf2) and Fig. 4(df) (Keap1) (one representative area from
each group is shown). Skin tissue slides were stained with
either Nrf2 or Keap1 antibody to evaluate the protein
expression; the average expression of Nrf2 and Keap1
from each group of mice was quantified using the IHC picto-
graphs, and analysed through Metamorph imaging software
(Molecular Devices). The quantifications of the expressed
proteins are represented as bar diagrams in Fig. 4(g) (Nrf2)
and Fig. 4(h) (Keap1). It appeared that cardamom ingestion
partially rescued the loss of expression of both Nrf2 and
Keap1 due to the DMBA treatments.
Cardamom restores the 7,12-dimethylbenz[a]anthracene-
initiated activation of NF-
k
B
NF-kB is a transcription factor that has a crucial role in cell
proliferation, inflammation, immunity and apoptosis
(24)
.
During oxidative stress or UV radiation, activation of NF-kB
mainly occurs via IkB kinase-mediated phosphorylation of
inhibitory molecules, such as IkBa. Once activated, IkB
kinases phosphorylate IkBaon Ser32 and Ser36 that lead
towards ubiquitination and degradation of IkBavia the
ubiquitin proteasome pathway
(25)
. During stress conditions,
NF-kB transcription factor subunit (p65) translocates into the
10
8
6
4
2
0
NADPH (nmol/min
per mg protein)
**
Acetone
Untreated
DMBA
DMBA+
CARD
160 20 60
40
20
0
15
10
5
0
120
80
40
0
GSH-CDNB (nmol/min
per mg protein)
NADPH (nmol/min
per mg protein)
GSH (nmol/mg protein)
(a) (b) (c)
(d) (e) (f)
**
**
*
Acetone
Untreated
DMBA
DMBA+
CARD
Acetone
Untreated
DMBA
DMBA+
CARD
Acetone
Untreated
DMBA
DMBA+
CARD
*
Acetone
Untreated
DMBA
DMBA+
CARD
30
25
20
15
10
5
0
30
25
20
15
10
5
0
Protein (units/mg)
Protein (units/mg)
*
Acetone
Untreated
DMBA
DMBA+
CARD
Fig. 3. Effects of ingestion of aqueous cardamom and 7,12-dimethylbenz[a]anthracene (DMBA) treatment on the activities of the detoxification enzymes in the
treated/untreated skin tissue lysates obtained from the different groups of mice (Table 1) after 12 weeks from first DMBA application. The activities of (a)
glutathione-S-transferase, (b) glutathione peroxidase, (c) reduced glutathione, (d) glutathione reductase, (e) superoxide dismutase and (f) catalase were signifi-
cantly increased in the ‘DMBA þCARD’ group compared with the ‘DMBA’ group. The ‘untreated’ and ‘acetone’ groups demonstrate basal enzyme activities in
each case. Values are means, with standard deviations represented by vertical bars. Mean value was significantly different from that of the DMBA group:
*P,0·001, ** P,0·005. CARD, cardamom ingested.
I. Das et al.990
British Journal of Nutrition
(f)
1·2
0·8
Fold change (expression)
compared to normal
Fold change (expression)
compared to normal
(h)(g)
NRF2 Keap1
**
0·4
0·0
1·2
0·8
0·4
0·0
(c)
(e)(b)
(d)(a)
Untreated
Acetone
DMBA
DMBA+CARD
Untreated
Acetone
DMBA
DMBA+CARD
Fig. 4. Cardamom stimulates the expression of nuclear factor erythroid-2-related factor 2 (Nrf2) and kelch-like ECH-associated protein 1 (Keap1). Skin tissues
from the different groups of mice (Table 1) were excised and tissue slides were prepared for immunohistochemistry (IHC). IHC with Nrf2 antibody was performed
on the skin tissues obtained from (a) acetone-treated, (b) DMBA-treated and (c) DMBA þCARD-treated mice. Similarly, Keap1 antibody was also used to stain
the skin tissues obtained from (d) acetone-treated, (e) DMBA-treated and (f) DMBA þCARD-treated mice. The magnifications of the pictographs are 200 £.
Quantification of the expressions of (g) Nrf2 and (h) Keap1 from several mouse skin tissues was evaluated using metamorph imaging software (Molecular
Devices). Values are means, with standard deviations represented by vertical bars. * Mean value was significantly different from that of the DMBA group
(P,0·001). The arrowhead indicates loss of the expression; the arrows indicate gain in the expression. CARD, cardamom ingested. (A colour version of this figure
can be found online at http://www.journals.cambridge.org/bjn).
Chemopreventive effects of dietary cardamom 991
British Journal of Nutrition
(f)
NF-κB-p65 IκBα
(c)
(e)(b)
(d)(a)
1·2
0·8
Fold change (expression)
compared to normal
Fold change (expression)
compared to normal
(h)(g)
**
0·4
0·0
1·2
1·5
0·9
0·6
0·0
0·3
Untreated
Acetone
DMBA
DMBA+CARD
Untreated
Acetone
DMBA
DMBA+CARD
Fig. 5. Cardamom modulates the NF-kB signalling pathway. Different groups of mice were treated as described in Table 1. Skin tissues were excised and slides
were prepared. Immunohistochemistry with NF-kB-p65 antibody was performed on the skin tissues obtained from (a) acetone-treated, (b) DMBA-treated and
(c) DMBA þCARD-treated mice. Similarly, IkBaantibody was also used to stain the skin tissues obtained from (d) acetone-treated, (e) DMBA-treated and
(f) DMBA þCARD-treated mice. The magnifications of the pictographs are 200 £. Quantification of the expressions of (g) NF-kB-p65 and (h) IkBaon skin tissues
from several mice was evaluated using Metamorph Software (Molecular Devices). Values are means, with standard deviations represented by vertical bars.
* Mean values were significantly different (P,0·05). (h) *Mean value was significantly different form that of the DMBA group (P,0·001). The arrowhead indicates
loss of the expression; the arrows indicate gain in the expression. CARD, cardamom ingested. (A colour version of this figure can be found online at
http://www.journals.cambridge.org/bjn).
I. Das et al.992
British Journal of Nutrition
nucleus, where it binds to several regulatory proteins and
regulates gene transcription. So, nuclear import is a key
event of NF-kB activation
(26)
. Thus, deregulation of NF-kB
and IkBaphosphorylation is crucial in inflammation and
cancer
(3)
, and it has become a popular target for therapeutic
applications. IHC with NF-kB-p65 and IkBaon the treated/
untreated skins from the different groups of mice is shown
in Fig. 5(ac) (NF-kB-p65) and Fig. 5(d– f) (IkBa) (one repre-
sentative area from each group is shown) to understand the
impact of cardamom ingestion on the DMBA-treated mice in
regulating NF-kB signalling. Several mouse skins from the
different groups of mice were stained; the average staining
intensities from each group of mice were quantified using
IHC and analysed through Metamorph imaging software
(Molecular Devices) and are represented as bar diagrams
(Fig. 5(g), NF-kB-p65; Fig. 5(h), IkBa).
Effect of cardamom on 7,12-dimethylbenz[a]anthracene-
induced cyclo-oxygenase-2 expression
Cyclo-oxygenase-2 (COX-2; an isoform of COX), an important
inducible protein expressed during several carcinogenesis
processes, is associated with PG synthesis, mitogenesis, apop-
tosis, angiogenesis and metastasis through various biological
pathways
(27)
. COX-2 has been shown to express both in
malignant epithelial cells and in neovasculature that continu-
ously feed the tumour cells. Thus, COX-2 is considered as
one of the markers of cancer progression and targeted inhi-
bition of COX-2 is a promising approach to prevent skin
cancer. Elevated level of COX-2 expression has been observed
with NMSC
(28)
. The level of COX-2 expression in skin tissues
was increased due to the DMBA treatment when compared
with ‘untreated’ and ‘acetone’-treated mice, which corrobo-
rates with the lines of evidence that tumour progression is
associated with increased COX-2 expression (Fig. 6). COX-2
expression in the skin tissue obtained from the ‘DMBA þ
CARD’ group was reduced by 37 % when compared with the
DMBA-treated mouse skin only, demonstrating inhibition of
neoplasia on the mouse skin by the aqueous ingestion of car-
damom (Fig. 6).
Discussion
NMSC is the most frequent skin cancer in the USA with an
incredible impact on physical condition and morbidity
(1)
.
Although solar radiation is the leading cause of human skin
cancer, it is also acknowledged that significant levels of chemi-
cal agents present in today’s environment also contribute to
growing incidences of cutaneous neoplasia in humans. In
the area of cancer prevention, the use of natural phytopro-
ducts and their constituents as potential chemopreventive
agents remains a hot topic of intense research. In the present
study, we have demonstrated the chemopreventive action of
cardamom, which is a spice mostly used in Asian and Scandi-
navian cuisines, on DMBA-induced skin carcinogenesis in
female Swiss albino mice. Phytochemicals that can metabolise
or inhibit the formation of free radicals in vivo may be
considered as potential anti-carcinogenic agents for cancer
chemoprevention. In recent years, phytochemicals present in
the common human diet have gained considerable attention
as photoprotective and chemopreventive agents against skin
cancers
(29)
. These phytochemicals belong to numerous classes
that include phytoalexins, phenols, polyphenols, anthocyani-
dins flavonoids, isoflavonoids and carotenoids. The chemo-
preventive approach appears to have realistic implications in
inhibiting skin cancer in individuals because of their ability
to modify dietary intake and change in the lifestyle.
There are several dietary phytochemicals that demonstrate
an immense effect on chemically induced skin carcinogenesis.
Oral administration of epigallocatechin gallate, which is a
polyphenol, blocked the growth of UV-induced skin tumours
in mice, by decreasing H
2
O
2
-induced ROS and reducing indu-
cible NO synthase expression in the UV-irradiated skin
(30)
.
Pretreatment with genistein, an isoflavone, to hairless mice
before UV exposure considerably blocked the UV-induced
H
2
O
2
and MDA formation in the skin, as well as minimised
the generation of 8-hydroxy-2’-deoxyguanosine in the epider-
mis
(31)
. In SENCAR mice, topical application of genistein
before UVB radiation reduced c-fos and c-jun expression
(32)
that are linked with cancer progression. Resveratrol is a poly-
phenol phytoalexin, and topical application of resveratrol
before UVB irradiation to SKH-1 hairless mice resulted in
considerable inhibition of UV-induced skin oedema as well as
Fold change
(b)
(a)
39
81
kDa
COX-2
72 kDa
Actin
43 kDa
COX-2
37 %
*
1·5
1·0
0·5
0·0
Untreated
Acetone
DMBA
DMBA+CARD
Untreated
Acetone
DMBA
DMBA+CARD
Fig. 6. Effects of ingestion of aqueous cardamom and 7,12-dimethylbenz
[a]anthracene (DMBA) treatment on cyclo-oxygenase-2 (COX-2) expression.
Swiss albino mice were grouped and treated as given in Table 1. COX-2 is a
marker of carcinogenesis. (a) DMBA þCARD group showed reduced
expressions of COX-2 when compared with the DMBA group as determined
by Western blot. (b) Densitometry was performed with at least three different
immunoblotting experiments. Percentage decrease in expression is indicated
by the arrows. Values are means, with standard deviations represented by
vertical bars. * Mean value was significantly different from that of the DMBA
group (P,0·01). CARD, cardamom ingested.
Chemopreventive effects of dietary cardamom 993
British Journal of Nutrition
inhibition of the formation of H
2
O
2
(33)
. Resveratrol treatment to
mouse skin also significantly inhibited the induction of COX-2
and lipid peroxidation, as well as repressed UVB-mediated acti-
vation of the NF-kB pathway
(34)
. Silymarin, which is a polyphe-
nol flavonoid, promotes the reduction of UVA-caused oxidative
stress by reducing GSH depletion, blocking ROS and LPO pro-
duction, as well as deactivating NF-kB activation in irradiated
cells. Moreover, topical application of silymarin protects
SKH-1 hairless mice against tumour initiation either with UVB
or DMBA, stimulates CAT activity and inhibits COX-2
expression
(35)
. Topical application of another well-known
phytochemical, curcumin, also inhibited COX-2 expression by
suppressing p38 and c-Jun N-terminal kinase activities in
HaCaT cells
(36)
. Gingerol, on the other hand, repressed the
UVB-induced intracellular ROS levels, inhibited the activation
of caspase-3, -8, -9 and TNF receptor superfamily, member 6
(Fas) expression, down-regulated COX-2 expression and NF-
kB activation
(37)
. Moreover, delphinidin, a dietary anthocyani-
din, protected cells against UVB-mediated decrease in cell viabi-
lity, as well as down-regulated the levels of lipid peroxidation
and blocked DNA damage. Application of lycopene, which is
found in tomatoes, inhibited UVB-induced ornithine decarbox-
ylase and proliferating cell nuclear antigen inhibition, down-
regulated myeloperoxidase activities and significantly reduced
bifold skin thickness
(38)
. Thus, several individual components
show excellent promise to protect cells against the propagation
of skin carcinogenesis. Researchers are now interested in
the combinatorial effects of several phytochemicals against
chemically induced skin carcinogenesis. Recently, Kowalczyk
et al.
(39)
demonstrated the inhibitory effect of combined
phytochemicals on DMBA-induced murine skin tumorigenesis
and obtained a significantly higher efficacy than individual
compounds.
DMBA is a well-known carcinogen and often used with
croton oil or 12-O-tetradecanoylphorbol-13-acetate (TPA) to
generate experimental papillomas on the skin. TPA and
croton oil have been used for ages to initiate the two-stage
skin carcinogenesis in mice
(39 – 43)
. The use of DMBA with
TPA or croton oil generates ROS, promotes DNA damage,
modulates p53 and COX-2 function, deregulates the cell
cycle and also alters apoptosis in the skin
(12,20,44,45)
. Thus,
our system of generating experimental skin papilloma in
mice by croton oil and DMBA somewhat mimics NMSC in
humans. ROS also induces peroxidation of PUFA and causes
lipid peroxidation in the cellular membrane. Increased lipid
peroxidation has been found to be triggered due to amplified
arachidonic acid metabolism via increased activities of cyclo-
oxygenases, such as COX-1 and COX-2, to produce PG
(46)
.
MDA and 4HNE, the toxic by-products of lipid peroxidation
formed during PG biosynthesis, are mutagenic and genotoxic
that can promote cancer growth. Oral administration of aqu-
eous cardamom to DMBA-treated mice showed significantly
inhibited lipid peroxidation (Fig. 2), as well as demonstrated
a decrease in the expression of COX-2 (Fig. 6), indicating
beneficial functions of cardamom.
Cardamom extracts were also tested on the activities of
phase II detoxification enzymes and antioxidation enzymes.
GST and GPx play a vital role in inducing the detoxification
of foreign compounds in the body to non-toxic and easily
soluble components that can be easily excreted from the
system. On the other hand, GR catalyses the conversion of
glutathione disulphide to GSH for a fresh round of detoxifica-
tion action. The antioxidation enzymes, particularly CAT and
SOD, also provide protection to cells against ROS by detoxify-
ing them on the site where they are produced. SOD and CAT
are regarded as the first line of antioxidant defence that pro-
tect cells against oxidative stress. SOD neutralises the O2
2
anion to H
2
O
2
and O
2
, whereas CAT neutralises H
2
O
2
to O
2
and water
(12)
. Hence, the significant enhancement of the
level of activities of GST, GPx, GSH, GR, SOD and CAT in
the ‘DMBA þCARD’ group (Fig. 3) may be beneficial for inhi-
biting the incidences of papilloma generation due to DMBA
application on mouse skin. Cardamom ingestion by DMBA-
treated mice even elevated the levels of the enzyme activities
beyond the basal levels of the ‘untreated’ and ‘acetone’-treated
groups. Thus, it indicates that intake of whole cardamom
every day in the normal diet has an enormous potential to
boost the cellular defence system of the body that will be
more appropriate to combat different stress conditions.
To understand the mechanism, we have investigated the
molecular function of cardamom on Nrf2 and NF-kB transcrip-
tion factors. The present data indicate that there is a loss of
expression of Nrf2 (Fig. 4(b and g)) as well as Keap1
(Fig. 4(e and h)) in the ‘DMBA’-treated skin when compared
with the ‘acetone’-treated skins (Fig. 4(a), Nrf2; Fig. 4(d),
Keap1). ‘Acetone’-treated mouse skin expresses Nrf2 in the
cytoplasmic region, which closely resembles the expression
of Nrf2 on the skin of ‘untreated’ mice (compare Fig. 4(a)
and Fig. S3(A) of the supplementary material, available
online at http://www.journals.cambridge.org/bjn). Interest-
ingly, cardamom ingestion rescues the nuclear expression of
Nrf2 in the mouse skin of the ‘DMBA þCARD’ group, which
suggests that the cardamom treatment might trigger an acti-
vation of Nrf2 despite the deleterious effects of DMBA, to
maintain cellular homeostasis during oxidative stress (Fig. 4(c
and g)). Nrf2 transcription factor is well known for leading the
induction of phase II detoxification enzymes during oxidative
stress, and we have already demonstrated an induction of sev-
eral phase II detoxification enzymes due to the cardamom
treatments in ‘DMBA þCARD’ mice (Fig. 3). Moreover,
Keap1 is highly expressed in the cytoplasm of the ‘untreated’
and ‘acetone’-treated mouse skin (Fig. 4(d) and Fig. S3(B) of
the supplementary material, available online at http://www.
journals.cambridge.org/bjn). Loss of Keap1 in the ‘DMBA’-
treated skin is restored by cardamom intake, suggesting a
reversion of DMBA-associated malfunction in the skin
(Fig. 4(f and h)). Although an association between Keap1
and Nrf2 is required by the resting cells, our experimental
observations with these two proteins may not be linked to
each other in the present scenario. Keap1 has often been
found to be expressed in low levels and mutated in patients
with cancer
(47)
. Low expression of Keap1 is probably due to
the epigenetic silencing via Keap1 promoter methylation
(48)
.
Furthermore, nuclear expression of NF-kB-p65 has
been observed in 3040 % of the epidermal cells in the
‘DMBA’-treated skin (Fig. 5(b and g)) when compared
I. Das et al.994
British Journal of Nutrition
with the ‘untreated’ and ‘acetone’-treated skins (see Fig. S3(C)
of the supplementary material, available online at http://www.
journals.cambridge.org/bjn and Fig. 5(a and g)). This obser-
vation suggests an activation of the NF-kB signalling cascade
in carcinogen-treated mice, by facilitating the release of NF-
kB-p65 protein from its cytoplasmic inhibitor protein, IkBa.
Additionally, IHC with IkBadisplayed down-regulation of
IkBaexpression in the skins of DMBA-treated mice,
suggesting that the ability to sequester NF-kB-p65 protein in
the cytoplasm was lost (Fig. 5(e and g)). The cardamom treat-
ment restored the expression of NF-kB-p65 protein in the
cytoplasm regardless of the deleterious effects of DMBA on
the skin (Fig. 5(c and g)). It is possibly due to simultaneous
up-regulation of the inhibitory protein IkBain the cytoplasm
(Fig. 5(f and h)) that resembles the resting phase in ‘untreated’
or ‘acetone’-treated mice (see Fig. S3(D) of the supplementary
material, available online at http://www.journals.cambridge.
org/bjn and Fig. 5(d and h)). We observed subtle differences
in the expression levels in NF-kB-p65 or IkBabetween the
‘untreated’ and ‘acetone’-treated groups that may be due to
the variation in the animal pool. There are multiple lines of
evidence supporting the hypothesis that the induction of
COX-2 is regulated by NF-kB
(49)
. Activation of NF-kB occurred
due to DMBA application on the mouse skin, and thus there
might be an increase in COX-2 expression. Additionally,
both SOD and COX-2 expressions are partly regulated by
NF-kB. So it is likely that cardamom modulates the NF-kB
signalling and induces the expression of SOD to inhibit the
translocation of NF-kB-p65 to the nucleus and concurrently
block NF-kB activation.
In general, NF-kB activation is often linked with increased
survival of the tumour cells and resistance to chemotherapy.
Several anti-carcinogenic compounds that could act as poten-
tial inhibitors of NF-kB activity are in the process of develop-
ment for an anticancer therapy
(50)
. However, activation of NF-
kB can also sensitise cells to apoptosis
(51)
. For example, a
combination of both UV radiation and adriamycin treatment
can activate the NF-kB signalling pathway, which in turn inhi-
bits the expression of a couple of anti-apoptotic genes. Induc-
tion of anti-apoptotic proteins by activating NF-kB might be
one of the many possible ways that is required for the devel-
opment of skin carcinogenesis
(52)
. Activation of NF-kB has
been convincingly shown to promote melanoma pro-
gression
(53,54)
. Furthermore, in dysplastic nevi and melanoma
lesions, NF-kB activity was found to be highly up-regulated
when compared with the normal human nevi or melano-
cytes
(54,55)
. Thus, expression of any proteins or agent/s that
could potentially deactivate NF-kB would be of high interest
in the therapy against skin carcinogenesis. For example, in
keratinocytes, expression of mutant JUN leads to the down-
regulation of NF-kB and that eventually suppresses the
tumour phenotype
(56)
. In contrast, it has also been reported
that mice with down-regulated NF-kB function display fea-
tures of squamous cell carcinoma
(57)
. Similar effects have
been shown by using lupeol, an anticarcinogenic compound,
on TPA-induced skin papillomatogenesis in CD1 mice. Lupeol
treatment helps in the reduction of skin oedema/hyperplasia,
and it is associated with the decrease in the expression/activity
of COX-2, NO synthase and epidermal ornithine decarboxy-
lase. Additionally, a decrease in the activation of phosphatidy-
linositol 3-kinase (PI3K) and NF-kB signalling, as well as a
decrease in the degradation of IkBa, was observed in
lupeol-treated mice
(58)
. Another group also showed that skin
papillomas generated in DMBA/TPA in LACA mice were
associated with increased expression of NF-kB and activator
protein 1 (AP1). Inhibition of papillomatosis by Azadirachta
indica (a leaf extract) was accompanied by the induction of
signal transduces and activator of transcription 1/AP1 and
simultaneously knocked down the activity of NF-kB
(59)
.
In the present study, we have also shown the histology
of mouse skin obtained from different groups of mice.
Histology of the skin from the ‘DMBA þCARD’-treated
group showed a near-normal phenotype similar to either the
‘acetone’ or ‘untreated’ group (no squamous cell carcinoma
characteristics), which was remarkably different from the
‘DMBA’-treated group (Fig. S2 of the supplementary material,
available online at http://www.journals.cambridge.org/bjn).
Extrapolating these facts, we can state that cardamom might
also have an impact on the function and expression of TNF-
a. NF-kB activation also facilitates COX-2 expression. We
have showed that cardamom ingestion in DMBA-treated
mice shows down-regulation of COX-2, and could potentially
also down-regulate NO synthase and epidermal ornithine
decarboxylase. Moreover, cardamom might also modulate
the PI3K and AP1 signalling pathways to restore the normal
cellular homeostasis of the papilloma-bearing cells.
It is well known that squamous cell carcinoma, which are
responsible for the majority of NMSC-related deaths, result
from the accumulation of genetic alterations. Several molecu-
lar events occur in skin epidermal cells following UV exposure
such DNA damage, generation of ROS, induction of p53 and
p53-regulated proteins, cell-cycle arrest, DNA repair and
apoptosis. Natural phytoproducts, such as cardamom, with
antitumorigenic properties have an immense potential to
maintain cellular homeostasis when continuously taken as
dietary supplements. There are several active components of
cardamom and not all the components are examined separ-
ately. Several reports have demonstrated that most of the com-
ponents of cardamom act as antioxidants such as limonene,
cineole, linalool, pinene and borneol
(9)
. Cardamom contains
Cu and Mn that are required for the activation of SOD
enzyme, which in turn helps in the decrease in the process
of lipid peroxidation. So in conclusion, generation of
DMBA-induced skin papillomatogenesis most probably
mimics UV-induced NMSC in humans, and the present data
suggest that whole cardamom as a nutritional diet plays an
anti-carcinogenic role in preventing the progression of skin
carcinogenesis in mice. Further investigations with individual
components are required to establish the chemopreventive
role of cardamom against NMSC.
Acknowledgements
I. D. wishes to thank the Director, Jaydeep Biswas, and all the
other staff members of the Department of Cancer Chemopre-
vention, CNCI. T. S. thanks the Lombardi Comprehensive
Chemopreventive effects of dietary cardamom 995
British Journal of Nutrition
Cancer Center, Histopathology core and acknowledges the
support from the pilot grants from the American Cancer
Society (IRG no. 97-152-16-2) and the Fisher Center for Famil-
ial Cancer Center, Georgetown University (Washington, DC,
USA). The authors’ contributions are as follows: I. D. designed
and performed the experiments (mouse set-up, tissue prep-
aration, biochemical and immunohistochemical assays) as
well as assisted in organising the manuscript; A. A. performed
the experiments (immunoblotting with the tissue lysates) and
reviewed the writing of the manuscript. D. L. B., S. S., E. W.
and E. P. were involved in tissue handing, slide preparation,
haematoxylin and eosin and IHC staining of the skin slides
in the USA. A. S. and S. B. provided space and reagents in
India. T. S. supervised the research, designed the experiments,
provided the reagents, space and scholarly wrote the manu-
script. The authors declare that they have no conflict of
interest.
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Chemopreventive effects of dietary cardamom 997
British Journal of Nutrition
... E. cardamomum EO is used mainly for treating fevers, digestive complaints, and respiratory diseases (Gochev et al., 2012); it embraces aromatic compounds with stimulant, astringent, diuretic, carminative, anti-inflammatory, and antioxidant activities (Asghar et al., 2017). These pharmaceutical activities have been attributed to the presence of oxygenated monoterpenes (1,8-cineole, α-terpinyl acetate, and geraniol) and phenyl propanoids (eugenol) (Das et al., 2012;Tangjitjaroenkun et al., 2020). Meanwhile, C. verum extract is used to treat coughing, sore throats, arthritis, and microbial infections (Sharifan et al., 2016). ...
... The standard (Trolox) neutralized generated DPPH radicals at IC50 value of 0.75 ± 0.05 µg/mL. The herein reported antibacterial and antioxidant activities of E. cardamomum EO could be attributed to the presence of high content of oxygenated monoterpenes, mainly 1,8-cineole, α-terpinyl acetate (Das et al., 2012;Elguindy et al., 2016;Tangjitjaroenkun et al., 2020). Its EO was reported to inhibit the growth of Gram-positive and negative bacteria with MIC (0.4-0.8%, v/v) (Gochev et al., 2012). ...
... Such variation in effective concentration might imply the role of extractant on the EO activity. Worthy to note, E. carda-momum EO up-regulated the antioxidant and detoxification enzymes, such as glutathione-S-transferase, glutathione peroxidase, glutathione reductase, superoxide dismutase, and catalase (Das et al., 2012;Elguindy et al., 2016). ...
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Context: Elettaria cardamomum (L.) Maton grains and Cinnamomum verum J. Presl barks are immensely consumed by Jordanian population as food flavoring and remedies in treating different disorders without awareness of their effect or dose toxicity. Aims: To evaluate the antibacterial, antioxidant and cytotoxic effects of essential oils (EOs) hydro-distilled from E. cardamomum grains and C. verum barks. Methods: Hydro-distillation was carried out using Clevenger apparatus; the EOs were analyzed using GC/MS, and their constituents were quantified using GC/FID. The antibacterial activity was determined by agar diffusion test and micro-broth dilution assay, while the antioxidant activity was evaluated by DPPH and ABTS scavenging assays. The cytotoxic activity was evaluated against the MDA-MB-231 breast cancer cell line. Results: E. cardamomum EO was most effective against Bacillus subtilis with MIC 3.75 µL/mL and exhibited antioxidant activity in DPPH assay with IC50 0.057 ±0.013 µL/mL (0.076 ±0.017 µL EO/µg Trolox®) but was ineffective in the ABTS test up to 5 µL/mL; 86.22% of its EO constituent are oxygenated monoterpenes with α-terpinyl acetate as the major component. Whilst C. verum EO was active against both tested Gram-positive and negative bacterial strains with MIC 1.25-5.00 µL/mL but was devoid of significant antioxidant activity up to 5 µL/mL; phenylpropanoids constituted 69.48% of its EO with E-cinnamaldehyde as the major component. Both EOs showed cytotoxic activity against the breast cancer cell line withIC50 0.14-0.46 µL/mL. Conclusions: The current results unveil the potential application of C. verum and E. cardamomum in complementary and alternative medicine as biosafe remedies besides their usage as flavoring and seasoning sources.
... It is called elakkaay in Telugu and elam in Tamil. 15,[28][29][30][31][32] Therapeutic activity of cardamom is shown in Table 2. ...
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... Qiblawi et al. (2012) reported that mice when treated orally with 0.5 mg of cardamom powder in suspension continuously for 15 days resulted in a significant decrease in the level of lipid peroxidation in the liver and acts as a chemo-preventive agent against two-stage skin cancer. Das et al. (2012) reported cardamom has been commonly used in cuisines for flavour and has numerous health benefits, such as improving digestion and stimulating metabolism and having antitumorigenic effects and the effectiveness of nutritional cardamom against 7,12-dimethylbenz[a]anthracene (DMBA)induced skin papillomatogenesis in Swiss albino mice that closely resembles human NMSC. evaluated, that extracts of cardamom which has significantly improved the cytotoxic activity of natural killer cells, indicating their potential as anti-cancer effects. ...
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This reference book is based on the history, cultivation, processing, breeding, protection, phytochemistry, and pharmacological importance of cardamom. Cardamom is scientifically known as Elettaria cardamomum (L.) Maton and is a member of the Zingiberaceae family. It's a shade-loving plant that grows well at an altitude between 600 and 1400 metres above sea level, with annual rainfall ranging from 1300–4000 mm and temperatures ranging from 10 to 35°C. Cardamom is highly cross-pollinated and depends on honeybees for pollination. Cardamom is classified into three types based on the nature of the panicles, namely, Malabar (prostrate panicle), Mysore (erect panicle) and Vazhukka (semi-erect panicle), a natural hybrid between Mysore and Malabar varieties. In India, currently, six research institutions, namely, Cardamom Research Station (CRS), Pampadumpara (Kerala Agricultural University, Kerala), ICAR-Indian Institute of Spices Research (IISR), Kozhikode, Kerala; Cardamom Research Centre, Appangala, Karnataka; Indian Cardamom Research Institute (ICRI), Myladumpara, Kerala and its Regional Research Station, Sakleshpur, Karnataka and Regional Research Station, Mudigere (University of Agricultural and Horticultural Sciences) are engaged in research for the improvement of cardamom farming. These research institutions have begun doing routine surveys to exploit desirable genes using a variety of traditional and modern crop improvement techniques. Also, these research centres/institutes are holding enormous genetic wealth of different accessions. Recently, many researchers have used various biotechnological approaches to conduct studies on micropropagation, assess the diversity in germplasm collections, and elucidate the biotic and abiotic stress tolerance mechanisms in cardamom. Fungal, bacterial and viral diseases pose severe threats to the successful cultivation of this crop. Major pests of cardamom include thrips, shoot and capsule borer, root grub, and whiteflies. Injudicious pesticide applications to manage pests and diseases rises the residue levels in the cured product, limiting its export value. Bio-pesticides control pest in an eco-friendly manner and are considered as the best alternatives to synthetic pesticides. It includes the effective utilization of microbials (bacteria, fungi, virus, and nematodes), macrobials (predators, parasitoids, and parasites), botanicals, organic amendments, semiochemicals, endophytes, and reduced risk pesticides in managing pest and diseases. Cardamom is grown in the throughout tropical mountains mainly for its capsules and its essential oil. Cardamom capsules/seeds accumulate essential oil and other bioactive metabolites, which contribute to their distinctive aroma and are used in the functional food, pharmaceutical, and nutraceutical industries. More than 100 secondary metabolites have been identified from cardamom essential oil. The essential oil of cardamom capsules possesses predominantly monoterpene constituents, such as 1,8-cineole, α-pinene, α-terpineol, linalool, linalyl acetate and nerolidol and the ester constituent α-terpinyl acetate all of which have therapeutic benefits including antioxidant, anticancer, antidiabetic, anti-inflammatory, antifungal, antiviral and gastroprotective activities. Cardamom capsules contain substantial concentration of flavonoids like catechin, myricetin, kaempferol and quercetin. Lutein is said to be the most abundant carotenoid in small cardamom. According to recent investigations, cardamom phenolic constituents’ flavonoids, alkaloids, terpenoids, and anthocyanins are being used to treat cardiovascular, pulmonary, kidney, and lung disorders. Cardamom capsules are a nutraceutical and functional food that can protect humans from several chronic diseases when taken daily. Cardamom oil is a new potential natural source for food, aroma, cosmetics, and pharmaceuticals. Since the 4th century BC, Indian Ayurvedic physicians, as well as Greek and Roman doctors, have used small cardamom capsules to treat bronchitis, asthma, and constipation, as well as colds, coughs, diuretics, carminatives, teeth and gum infections, urinary and kidney disorders, congestion of the lungs, pulmonary tuberculosis, irritation of the eyelids, cataract, nausea, and diarrhoea. Cardamom was used to treat constipation, stomach aches, bladder infections, and dysentery in children in traditional Chinese medicine. Cardamom is also used in Ayurvedic medicine to cure food sickness. Cardamom oils are being employed in the production of plant-based hand lotions and soaps. Digestive problems can be treated with powdered cardamom capsules mixed with pulverised cloves, ginger, and caraway. In addition, using cardamom capsules helps to relieve inflammation and headaches. This reference book entitled “Cardamom [Elettaria cardamomum (L.) Maton]: Production, Breeding, Management, Phytochemistry and Health Benefits” is comprises of twelve chapters contributed by different authors and provide complete information about this wonderful herb. Its occurrence, history, cultivation, post-harvest processing, botany, crop improvement, biotechnology, protection, ethnopharmacological uses, phytochemistry and pharmacological activities are well described with supporting references. The book contains latest information pertaining to cardamom and its cultivation. The information provided in this book will be very useful for students, academicians, researchers, and scientists, as well as others interested/involved nutraceutical and pharmaceutical industries.
... It is a common ingredient used in cooking across India and various parts of Europe [15]. The potential of cardamom as a chemopreventive agent has been reported against two-stage skin cancer model in mice [27,28]. Recently, Elettaria cardamomumi showed a significant immunosuppressive activity [29]. ...
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... The use of cardamom for medicinal purposes is not well established. In vitro and in vivo studies showed antimicrobial ( 58) , cytotoxic, anticarcinogenic properties ( 59,60) , and gastroprotective effect ( 61) of cardamom. In addition, a human study showed that cardamom at the dose of 3 g/ day reduced fatty liver (62). ...
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Stomachic mixture is used widely for the treatment of gastrointestinal conditions. However, the evidence for its efficacy and safety is scarce. The review of pharmacology and toxicity of stomachic mixture constituents provides information for patients and healthcare providers in deciding the use of this drug. Stomachic mixture products registered in Thailand as shown in the Thai Food and Drug Administration database in 2018 contained sodium bicarbonate as their main active ingredient of the stomachic mixture. The herbal components in the stomachic recipe registered to Thai FDA were volatile oils, anthraquinone glycosides, bitter substances, and spicy substances. The amount of each ingredient in the stomachic mixture, when the mixture was used as recommended, was lower than toxic doses of the component. However, the sodium amount in the stomachic mixture could be high for patients requiring sodium restriction. Data regarding the use of stomachic mixture in pregnant and lactating women were insufficient. Therefore, the stomachic mixture should be avoided in patients requiring sodium restriction, pregnant women, and breastfeeding women. Side effects of stomachic mixture on the liver, kidney, heart, gastrointestinal tract, and central nervous system and hypoglycemia are possible.
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Full-text available
Background Food additives act as preventive measures and promote a healthy immune response against pathogenic diseases. There are several functional food additives with antiviral potential that are part of our daily food supplements, which can be exploited to improve the immunity of the human being during the pandemic of Covid-19. Scope and Approach For the development of this literature, an extensive database search using the scientific databases and Google Scholar, as well as commercial search engines such as Google, Google Patent and Patent Scope to search for commercial and patentable applications. Key Finding Food additives such as Phyllanthus emblica, Long pepper, Cinnamon, Turmeric, Cardamom, Ginger, Garlic, Holy basil, and liquorice are used in traditional cultures as preventative treatments. The phytocompounds extracted from these food additives are immune modulators against various pathogenic inflammations. Enhancing the immune response and boosting health are the benefits of these food additives. Conclusion The phytocompounds extracted from food additives such as Phyllanthus emblica, Long pepper, Cinnamon, Turmeric, Cardamom, Ginger, Garlic, Holy basil, and liquorice are immune modulators against various pathogenic inflammations. The research literature and reputable sources online confirm that functional food additives in a regular diet may help cure Covid-19 disease. It is necessary to conduct scientific research to determine the effectiveness of food additives. Proposes the Future Direction The majority of diseases are caused by metabolic disorders. It is clear that the diet plays a major role in controlling the inflammation associated with diseases and metabolic disorders. There are still a lack of phytochemical screenings and their interaction with metabolism. This effort will help the science community to think outside of the box of medicine. Keywords: Covid-19, Functional Food Additives, Immune Response, SARS-CoV-2
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Background & aims Cardamom known as “queen of spice” seems to be an anti-diabetic agent due to its poly phenolic content. Since, recent studies reported controversial results related to its effect on metabolic factors, present meta-analysis examined the effect of cardamom supplementation on glycemic indices and weight profile of randomized controlled clinical trials (RCTs). Methods A wide search was done on biomedical electronic databases including Scopus, PubMed, Cochrane, EMBASE and Iranian databases, for all relevant literature published up to May 2021. Our search strategy included: [HbA1C, Blood Sugar, glycemic index, glucose tolerance test, insulin, insulin resistance, insulin sensitivity, body weight, BMI, body composition, waist circumferences] added to searched queries based on scientific Mesh terms. The included papers required to be RCTs that reported the effect of cardamom on glycemic and weight indices. We excluded studies with: a) non-randomized or non-controlled trials, b) animal studies, c) not available full text articles d) duplicate citations and e) not available full text articles. The risk of bias was assessed based on the Cochrane Risk of Bias tool. The effects of cardamom supplementation were assessed using standardized mean difference (SMD) statistics. The SMD of metabolic risk factors were pooled together using random effect meta-analysis method. Results Totally, six publications enrolling 410 participants was included in present meta-analysis. Daily 3 g supplementation of cardamom from 8 weeks to 3 months showed no significant effect on BMI (WMD: 0.07; 95% CI: [-0.12, 0.27]; P:0.5), weight (WMD: 0.01; 95% CI: [-0.22, 0.21]; P:0.95) and WC (WMD: 0.09; 95% CI: [-0.34, 0.17]; P:0.63), FBS (WMD: 0.10; 95% CI: [ −0.32, 0.12]; P:0.37), insulin (WMD: 0.83; 95% CI: [-2.07, 0.40]; P:0.19) and QUICKI (WMD: 1.14; 95% CI: [-1.11, 3.39]; P:0.32). However, significant effect occurred on HOMA-IR (WMD: 0.40; 95% CI: [-0.65, −0.15]; P:0.00), and HbA1C (WMD: 0.48; 95% CI: [-0.80, −0.16]; P:0.00). Conclusion Final findings suggest ameliorative effect of cardamom on metabolism of glucose.
Article
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Article
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Article
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Article
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