1. No category

International Journal of Pharmaceutical Applications
ISSN 0976-2639.Online ISSN 2278 – 6023
Vol 4, Issue 1, 2013, pp 29-37
http://www.bipublication.com
EFFECT OF CITRUS RETICULATA ESSENTIAL OIL ON ABERRANT
CRYPT FOCI (ACF) DEVELOPMENT IN 1, 2-DIMETYLHYDRAZINE
INDUCED COLON CARCINOGENESIS RATS
Burali Sanganna1, 2*and A.R. Kulkarni3
1
Karpagam University, Coimbatore, Tamilnadu,India
2
RIPER, Anantapur, AP.
3
SETs College of Pharmacy, Dharwad 580 002, India
Corresponding author: E-mail:[email protected]
[Received10/13/2012, Accepted-17/03/2013]
ABSTRACT:
The 1, 2-dimetylhydrazine (DMH) induced rats are associated the risk of colorectal cancer due to formation of
methyldiazonium ions along with free radicals that induce oxidative DNA damaged in the liver and colon. In colon
carcinogenesis end point was formation of aberrant crypt foci (ACF) which could be prevented by variety of drugs
including those which were obtained from the natural origin. The present study was designed to screen the anti cancer
activity of one of the potential natural chemopreventive agent from commonly available citrus reticulata Blanco
essential oil in DMH induced rats. By evaluating the effect of citrus reticulata essential oil on rats body weight
changes, depicting multiple plaque lesions (MPLs), ACF and histopathological changes on colon part. The result
showed that a linear growth in the body weight of rats during sixteen weeks but no significant change in the body
weight compare to normal and other induced groups of rats. The colon cancer was 100% incidence in DMH treated rats
but in the dietary citrus reticulata essential oil significantly inhibited the incidence of colon cancer and number of ACF
63.35%. The histopathological changes in DMH groups were also significantly improved in citrus reticulata essential
oil groups. Overall our result of citrus reticulata essential oil may produce a synergistic colon cancer preventive effect
and may be possible chemopreventive agents against colon cancer.
Keywords: Aberrant crypt foci, Multiple Plaque Lesions, 1, 2, dimethylhydrazine, citrus reticulata Blanco essential
oil, Capacitabine.
INTRODUCTION:
Colon cancer is the second leading cause of cancer
related deaths in USA [1]. Cancer is a major public
health problem in the United States and many other
parts of the world [2]. Recently, it was estimated
that 12 million cases of cancer were diagnosed,
with 7 million deaths worldwide of these numbers
are expected to double by 2030 (27 million cases
with 17 million deaths) [3]. The overall cure rate of
colorectal cancer has not improved dramatically in
the last decade, 5-year survival remaining at
approximately 60% and also mortality rate remains
high [4]. Advances in automated micro- and
EFFECT OF CITRUS RETICULATA ESSENTIAL OIL ON ABERRANT CRYPT FOCI (ACF) DEVELOPMENT
macroscopic image acquisition and digital image
analysis have created new opportunities in the field
of prognostic assessment but one area in
experimental pathology, animal models for colon
cancer, has not taken advantage of these
opportunities [5]. Colon cancer arises due to
diverse genetic and epigenetic changes in the
colonic epithelium. In addition to genetic
predisposition, diet is an also important risk factor
for colon cancer. Evidence suggests that an
association between high fat intake is an increased
risk of colorectal cancer [6]. Colon cancer
incidence has been increasing in recent years in
India. More than 11 million people are diagnosed
with cancer every year [7].
The animal models are designed to test the agents
across tumor type and these were targeted to the
most common cancers like lung cancer, breast
cancer, colon cancer and prostate cancer [8]. The
animal model mimics the mode of human exposure
to environment carcinogen. The 1, 2Dimethylhydrazine (DMH) is a potent colon
procarcinogen that is metabolically activated to the
active carcinogen in the liver, produces free
radicals that induce oxidative DNA damage in the
liver and colon [6]. Colonic adenoma is a particular
type of lesion that is prone to develop into
adenocarcinoma, a malignant tumor that constitutes
the majority of colorectal cancer cases. These
colonic adenomas are generally present in the form
of polyps. The main characteristic of polyps is a
considerably thick mucosa with a typical size of 1
mm. Aberrant crypt foci (ACF), a colon carcinoma
precursor in humans and rats, are a feasible tool to
distinguish between the polyp types serve as a
sensitive, realistic and rapidly appearing in
histopathological intraepithelial neoplasia. ACF
grow via a mechanism of crypt formation and ACF
with more crypts indicate a more advanced growth
stage [5].
In recent years the public has shown an increased
interest in the use of herbal medicines in compared
to synthetic drugs. This is based on the belief that
natural products are intrinsically less dangerous and
can be obtained at a lower cost. However, most
natural products contain a complex mixture of
active components. After identification of the active
Burali Sanganna and A.R. Kulkarni
component can lead to the synthesis of more potent
analogues that can be readily formulated into more
traditional dosage forms [9].
Natural dietary antioxidants compounds protect
against damages caused by reactive oxidants [10].
Currently, there is much biomedical interest in
citrus fruits because consumption of them appears
to be associated with lower risk of colorectal,
esophageal, gastric, and stomach cancers, and
stroke. So the presence active components are
beneficial effects and responsible for the anticancer
activity [11]. The work focused on essential oil of
citrus reticulata Blanco, Essential oils are vegetable
products whose constituents are basically complex
mixture of terpenic hydrocarbons and oxygenated
derivatives such as aldehydes, alcohols and esters.
These are accumulated mainly in secretory cavities
scattered throughout the fruit peels and leaf.
Among many sources, citrus fruits peels are the
most familiar and rich source of essential oils [12].
Essential or Tangerine oil is obtained from the fruit
peel of Citrus reticulata Blanco, family Rutaceae. It
is traditionally used as an antiseptic, antispasmodic,
stomachic, and sedative, diuretic and to improve
circulation [9]. In literature found that essential oil
extracted from citrus reticulata possesses a variety
of bioactivities, such as relaxing isolated tracheal
and intestinal smooth muscle, protecting drug
induced asthma in guinea pig and playing
antiallergic effect by inhibiting the release of
allergy and mediators [13]. Inhibitory effects of
citrus extracts on the experimental pulmonary
fibrosis [14]. Suppressive effects of nitric oxide
(NO) production and inducible nitric oxide
synthase (iNOS) expression by Citrus reticulata
extract in RAW 264.7 macrophage cells [15].
Determining the efficacy of Citrus reticulata
essential oil during the post-initiation stage, at
which point the premalignant lesions developed, is
very important with regards clinical use of these
agents in the secondary prevention of colon cancer
among patients with polyps.
In contrast, a number of studies have suggested that
high consumption of fruits and vegetables
decreases the risk of colon cancer [16]. The present
study was systematically design to evaluate the
potential chemopreventive property of Citrus
30
EFFECT OF CITRUS RETICULATA ESSENTIAL OIL ON ABERRANT CRYPT FOCI (ACF) DEVELOPMENT
reticulata essential oil by using chemically induced
colon carcinogenesis of wistar rats. This study also
involves state of the art animal model to elucidate
its probable mechanism of industrial guidelines of
FDA, USA for pre-clinical evaluation of anticancer drugs [17].
MATERIAL AND METHODS:
Plant material:
In the present study, the Citrus reticulata Blanko
were collected from local market. The fruits were
authenticated by Prof. S. A. Kappali, H.O.D of
Botany. B.V.V.S, Science College, Bagalkot
Karnataka, India, studies includes organoleptic
tests, and
macroscopic
and microscopic
observations were done. Then voucher specimen is
deposited at Science College and number is BSC
/BOT/30. Soon after authentication, the fruits peels
were used for extraction of essential oil.
Preparation of Extracts:
Citrus reticulata Blanco is commonly known as
mandarin fruit. The fresh fully matured ripe fruits
had been washed in potable water and cut into
equal portions to remove the peels. The fruit albedo
layers were peeled off carefully and discarded. The
peel oils were isolated by hydro-distillation in
Clevenger’s apparatus [18]. The Essential oil was
collected by hydro-distillation for 6–8 h. The yield
of oil was recorded (0.5 ml/100 g peel) and stored
in air-tight sealed glass vials covered with
aluminum foil at 4ċ for further Pharmacological
use.
Animals and Diet:
Healthy young Albino wistar rats of either sex of 4
weeks of age were purchased from Shree
Nijalingappa medical college Bagalkot, Karnataka,
India. After 1 week of acclimation, animals were
randomly distributed into control and experimental
groups. All animals were housed in a plastic cage
with a wire mesh top and a hygienic bed of husk in
a specific pathogen-free conditions room. The
animal room was controlled at 23 ±1C, 50 ± 10%
humidity and a 12 h light/dark cycle till the end of
the experimental. Necessary approval were
obtained from IAEC (Reg. No.878/ac/05/CPCSEA)
for the use of experimental animals in the present
studies. .
Burali Sanganna and A.R. Kulkarni
Carcinogen administration:
The experimental animals were divided into four
groups. The animals in groups II, III & IV received
sub- cutaneous injections of DMH at a dose of 20
mg/kg body weight once a week for the first four
consecutive weeks to induce colorectal cancer.
Prior to subcutaneous injection, DMH was
dissolved in 1mM EDTA the pH adjusted to 6.5
with 1mM NaOH to ensure the pH and stability of
the chemical and was used immediately after
preparation. Initial body weights of all animals in
this study protocol were ensured to be between 80
and 120g. The animal weights were recorded once a
week throughout the experimental period and prior
to sacrifice [19].
Experimental design:
All animal were divided in to four groups of six rats
in each groups.
Group I served as Normal control was given normal
saline & diet ad labium. Group –II, III& IV
received DMH (20mg/kg) subcutaneous once a
week for first four weeks, in addition group- III and
IV received standard drug of capacitabine and
Citrus reticulata essential oil at the dose of
(200mg/kg) respectively.
Body weight changes:
The body weight of the control, DMH,
Capecitabine and Citrus reticulata essential oil
treated rats groups was record throughout the study.
The rats were weighed at the beginning of the
experiment subsequently once a week and finally
before sacrifice [20].
Microscopic evaluation of tumors/polyps:
At the end of 16 weeks DMH treated rats were
sacrificed, their colons were excised blotted dry, cut
open longitudinally and the inner surface was
examined for visible macroscopic lesions.
Tumors/polyps were easily discernible in the
inflamed section of the colon. Colon sections of
equal length and tumors were examined grossly for
the location, number and size [7].
Analysis of tumor incidence, average tumor
number [21]
Tumor incidence, average tumor numbers were
calculated using the following formulas:
31
EFFECT OF CITRUS RETICULATA ESSENTIAL OIL ON ABERRANT CRYPT FOCI (ACF) DEVELOPMENT
Tumor incidence% = Number of tumor-bearing
rats/Number of tested rats x100%
Average
tumor
number=Total
number/Number of tested rats
tumor
Average tumor number of tumor-bearing rats =
Total tumor number/ Number of tumor-bearing rats
Tumor inhibitory rate % = (positive control- test
group/positive control) x100%:
Control group
DMH group
mucosal side up on a microscopic slide, and viewed
under a light microscope. The total number of ACF
in the entire colon was determined in every 2 cm
section of the colon, starting from the distal (taken
as 0 cm) to the proximal end of the colons.
Aberrant crypts were distinguished from the
surrounding normal crypts by their increased size,
increased distance from lamina to basal surfaces of
cells, and easily discernible pericryptal zone. The
variables used to assess the aberrant crypts were
occurrence and multiplicity. Aberrant crypt
multiplicity was determined as the number of crypts
in each focus and categorized as containing up to
four or more aberrant crypts/focus [23]. The result
was shown in the Table No 03.
Histology:
For histological examination of lesions in the
intermediate stage of colon carcinogenesis, four rats
were killed at week 16. Lesions, identified by
transillumination and surface examination of
formalin-fixed whole-mount preparations under the
inverse light microscope, were dissected, embedded
in paraffin wax, cut in parallel with the mucosal
surface and stained with haematoxylin and eosin
[24].
Capecitabine group
STATISTICAL ANALYSIS:
The statistical significance of the data has been
determined using one-way analysis of variance
(ANOVA) and significant difference among
treatment groups were evaluated by Bonferroni's
Multiple Comparison Test. The results were
considered statistically significant at P < 0.05. All
statistical analyses were made using Graph pad
prism 5.01 software.
Citrus reticulata group
RESULT:
Fig. 1. Showing Multiple Plaque Lesions (MPLs) in rat
Colons.
Determination of aberrant crypt foci (ACF):
Aberrant crypt foci are considered the ‘gold
standard’ of colon carcinogenesis biomarkers
[22].Topographic analysis of the colonic mucosa
according to Bird was done after a minimum of 24h fixation. Colons were stained with 0.2%
methylene blue solution for 5 to 10 min, placed
Burali Sanganna and A.R. Kulkarni
Effect of Citrus reticulata essential oil extract on
1, 2 dimethylhydrazine induced rats
body
weight changes:
The results in the animal body weight showed a
linear growth during the 16 week treatment
schedule, with a net weight gain at the end of the
study in comparison to their initial weight. Table 01
shows No significant change in the body weight
between the Control and the different treated
32
EFFECT OF CITRUS RETICULATA ESSENTIAL OIL ON ABERRANT CRYPT FOCI (ACF) DEVELOPMENT
groups was observed which shows that the
carcinogenic events did not alter the metabolic
status of the animals in a significant way and the
normal growth pattern achieved.
Table 2 Chemopreventive response of Citrus reticulate
Essential oil in terms of MPLs (Multiple Plaque Lesions)
in DMH induced colon carcinogenesis Rats.
Groups
Table.01 Effect of Citrus reticulata Essential oil on
Total
No. of
ras
Total
No. of
MPLs
in
colon
No. of rats
with MPL/
Total No. of
rats
MPL
incidenc
e%
MPL
burden
MPL
Multipli
city
06
No
MPL
00/06
00
NIL
NIL
06
12
06/06
100
2.0
2.0
06
03
02/06
33.33
0.5
1.5
06
05
03/06
50.00
0.8
1.66
Body Weight (g) in DMH induced colon carcinogenesis
Rats.
Groups
Initial Body Weight (g)
Final Body
Weight (g)
Normal
81.17±1.537
214.2±1.537
DMH
79.17±2.386
175.0±1.291
Capecitabine
Citrus
reticulata
Essential oil
82.83±2.088
190.0±3.651
82.50±2.141
185.8±2.007
Control
DMH
Effect of Citrus reticulata Essential oil extract on
body weight in 1, 2- dimethylhydrazine induced
colon carcinogenesis rats was calculated from first
day of study and every week regularly. The body
weight of each animal was recorded one hour prior
to administration of drug extract on recording day.
The results are depicted as mean ± S.D and one
way ANOVA analysed by Bonferroni's Multiple
Comparison Test. for coming to conclusion.
Effect of Citrus reticulata essential oil extract on
1, 2 dimethylhydrazine induced rats depicting
Multiple Plaque Lesions (MPLs):
Multiple plaque lesions (MPLs) were recognized as
either raised or non-raised lesions with identifiable
tissue growth in carcinogen treated animals, often
appearing singly or in multiple forms throughout
the length of the colon Fig 1 shows gross anatomy
of the mucosal surface of colon depicting the
occurrence of multiple plaque lesions (MPLs) of (a)
Control, (b) DMH, (c) DMH + Capecitabine and
(d) DMH+ Citrus reticulata essential oil. MPLs
incidences were observed in almost all the DMH
treated rats (100%). Standard treated group had less
MPL as compared to the DMH group (33.33%) and
minimum number of MPL noted in the Citrus
reticulata essential oil group (50%). No MPL was
identified in the control animals groups. (Table 2).
Burali Sanganna and A.R. Kulkarni
Capecit
abine
Citrus
reticulat
a
Essentia
l oils
Calculation of MPLs (Multiple Plaque Lesions).
MPL incidence= the percentage of animals having
MPLs.
MPL burden= the total number of MPLs
counted/total number of rats.
MPL multiplicity= the total number of MPLs
counted/number of MPL bearing rats.
Effect of Citrus reticulata Essential oil extract on
ACF (Aberrant crypts foci in 1, 2
dimethylhydrazine induced rats:
Aberrant crypts (ACs) were identified as the crypts
that were 2 to 3 times larger than the surrounding
normal crypts. ACFs are early appearing lesions
recognized on the colonic lumen surface in the
DMH treated rats. These ACFs are characterized by
crypts with altered luminal epithelia and being
larger than the adjacent normal crypts22. ACF
frequencies100 % (P < 0:001) were significantly
higher in the DMH treated rats. Control groups
there were no ACF in the colons of rats due to
injected with Normal saline solution and
Capecitabine groups levels significantly reduced
the number of ACF by 79.63% (P < 0:001) as
compared to the DMH group. ACFs were also
reduced with the simultaneous treatment of Citrus
reticulata Essential oil and DMH by 63.35% (P <
0:001) significantly. The crypt multiplicity (number
of aberrant crypts/focus) was not affected
significantly with each other groups but the
numbers of ACF consisting of 1 crypt level were
significantly decreased in groups treated with
33
EFFECT OF CITRUS RETICULATA ESSENTIAL OIL ON ABERRANT CRYPT FOCI (ACF) DEVELOPMENT
Capecitabine and Citrus reticulata Essensial oil
extract compare to DMH groups [25].
reticulate essential oil 200 mg/kg b.wt. (group 4)
showed mild hyperplasia and mild degree of
dysplastic changes in the mucosal glands (400x).
Groups
No.of
Animals
Normal
04
1 crypt
Nil
2 crypt
Nil
3 crypt
Nil
DMH
06
25.8± 5.2 ***
21.33± 3.44
5.5± 2.8 ***
8.33± 1.5 ***
Capecitabine
Citrus reticulata
Essential oils
06
06
Number of aberrant crypts per ACF
Total no. ACF
% inhibition
≥ 4 crypt
Nil
Nil
Nil
20.83± 6.76
22.5± 6.253
90.5 ± 5.01***
100%
5.333± 1.63
4 ± 2.36
4± 1.41
18.83± 4.26***
8.333± 0.81
8.667 ± 3.50
7.66 ± 1.63
33.17± 1.32***
79.63%
63.35%
Table.03 Effect of Citrus reticulate Essential oil on
aberrant crypts foci and Number of crypts in DMH
induced colon carcinogenesis Rats.
Effect of Citrus reticulata Essential oil extract on
aberrant crypts foci in 1, 2 dimethylhydrazine
induced colon carcinogenesis rats was calculated
from Last day of study. The aberrant crypts foci of
each animal were recorded. The results are depicted
as mean ± S.D. of six rats from each group and one
way ANOVA analyzed by Bonferroni's Multiple
Comparison test for coming to conclusion.
***P<0.001. All treatment groups compared to
DMH group was considered as significant.
% Inhibition =
a) Normal
group
Effect of Citrus reticulata Essential oil extract on
Histopathological
changes
in
1,
2
dimethylhydrazine induced rats:
Fig.2. represents the photomicrographs of the colon
of Normal and experimental rats. Histological
examination of the colon section a) normal (group
1) rats showed normal architecture with mucosal
and submucosal layers (100x). b) DMH alone
treated rat colon (group2) showed proliferating
mucosal glands with severe dysplasia and
hyperplasia
of
crypt
cells
representing
transformation to carcinoma (400x). The colon of
carcinogen rats treated with Capecitabine (200
mg/kg b.wt.) (group 3) showed glands with
dysplasia within mucin vacuoles and normal limits
surrounded by lymphoid aggregates (100x).The
colon of carcinogen rats treated with Citrus
Burali Sanganna and A.R. Kulkarni
b) DMH group
c) Capecitabine
group
34
EFFECT OF CITRUS RETICULATA ESSENTIAL OIL ON ABERRANT CRYPT FOCI (ACF) DEVELOPMENT
d) Citrus reticulata Essential oil group
Fig 2. Micrographs showing H & E stained
histopathological representation of DMH-induced
tumorigenesis in the rat colon. (a) Colon of control
rat with normal mucosal and submucosal layers
(20X). (b) Colon of DMH-treated rat showing
adenocarcinoma with papillary pattern and
dysplastic zone (10X). (c) Colon of DMH-treated
rat with Capecitabine showing carcinoma glands
filled with mucin and lined with pleomorphic cells
and densely packed inflammatory cell infiltrations
(40X). (d) Colon of DMH treated with Citrus
reticulata essential oil rat showing normal mucosal
folds and scattered inflammatory cell infiltrations
(10X).
DISSCUSION:
Colorectal cancer is one of the most common
malignancies in today. The interest in research is
motivated by the current need of new substances of
natural
origin,
which
have
demonstrate
effectiveness in the cancer fields of application and
low degree of toxicity for humans [26]. Hence, this
study was carried out, with an objective to evaluate
chemoprevetive activity of Citrus reticulata
essential oils.
A wide variety of dietary plants including grains,
legumes, fruits, vegetables, tea, wine, etc. contain
polyphenols [27]. The Plant-based non-nutrients
called photochemical are emerging as an important
dietary factor. Citrus fruits contain hundreds of
photochemical and increasing interest in the
possible substances contribute to optimal health and
may protect against some of the common chronic
diseases such as cancer, cardiovascular disease,
degenerative eye, cognitive conditions, and general
Burali Sanganna and A.R. Kulkarni
damage caused by ageing [28]. Medicinal uses
proposed by sellers of essential oils vary from skin
treatments to remedies for cancer. Essential oils
exhibit various and variable antimicrobial activities,
including antifungal, antiviral, antibacterial,
insecticidal,
and
antioxidant
properties.
Monoterpenes have shown prevention of
mammary, lung, skin, liver and forestomach
cancers in rat models, but not examine on colon
cancer by DMH induced rat models. The Citrus
reticulata essential oil has anticancer and
antioxidant activities due to its content of γterpinene, α-terpinene [29].
Naringin and hesperidin have many biological
activities such as antioxidant, antimutagenic effect,
analgesic, anti-inflammatory etc [30]. They are
active constituents of Citrus reticulata Blanco.
Preneoplastic lesions such as ACF, which occurs in
30-45 days after carcinogen administration or even
earlier, have been extensively, used as endpoint in
short term carcinogenesis and chemopreventive
studies [22]. In our present study, DMH treatment
led to increase in the grossly visible multiple plaque
lesions (MPLs) and aberrant crypt foci (ACF) after
a period of sixteen weeks. Citrus reticulata
essential oils treated animals were showing a
significantly decreased in number of MPLs and
ACF.
Histopathological observations of the colon clearly
implies that supplementation with Citrus reticulata
essential oils, under the experimental conditions
greatly inhibits colon carcinogenesis by altering the
efficacy of DMH in initiating the neoplastic
changes. Proliferating mucosal glands with severe
dysplastic changes that represent transforming
carcinoma were observed in the colonic tissue
sections of DMH alone administered rats. The
ability Citrus reticulata essential oils (200 mg/kg
b.wt.) were restore the histological changes in
DMH group, due to anti-carcinogenic potential
effect for the presences of phenolic acids present in
essential oils. The reactive phenolic group
helping to scavenge reactive oxygen species that
initiate lipidperoxidation and preventing cell
proliferation by increase the susceptibility and
decrease the resistance of tumor cells to free radical
attack leading to decreased cell proliferation [10].
35
EFFECT OF CITRUS RETICULATA ESSENTIAL OIL ON ABERRANT CRYPT FOCI (ACF) DEVELOPMENT
Citrus
fruit
contains
other
possible
chemopreventive agents, include d-limonene [31],
auraptene [32], limonin etc. Some of these can
modify activities and expression of the detoxifying
enzymes and CYPs [33], and inhibition of the
enzyme topoisomerase II in cancer cells and
normalization of tumor cells by flavonoids
[28].There are many reports in the literature of the
biological activities of Citrus reticulata essential
oils [29], but there are only few studies on the
antioxidant and anticancer activities of the essential
oils. So take it as a challenge in the discovery of
the new substance for colon cancer.
CONCLSION:
In conclusion, the present study shows that
essential oils of Citrus reticulata have potentially
good sources for anticancer drugs. The overall
results obtained from Citrus reticulata essential oils
was more effective in animal model of 1, 2- DMH
induced colon cancer. The presence of
monoterpines,
Flavonoids,
and
Phenolic
compounds of Citrus reticulata essential oils show
a anti proliferative activity. So colon cancer patient
can use as oral agents of citrus juice daily in the
early morning instead of tea and coffee
significantly reverse the development of adenoma
and also interfere with the multi-step progression
from adenoma to carcinoma and reducing the
mortality of colon cancer incidence.
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