ijapr - international journal of advances in pharmaceutical research

M.S Latha et al. / International Journal of Advances in Pharmaceutical Research
the process, which is followed by regeneration,
growth and clonal proliferation, eventually leading to
cancer (5). There is extensive evidence that the free
radicals participate in NDEA induced
hepatocarcinogenesis (6). Enzymes such as GST, GR
and GPx reduce oxidative stress in the normal tissues.
Extensive liver damage causes decrease in levels of
these enzymes suggesting a high oxidative stress in
the tissues, which may be one of the key factors in
the etiology of cancer (7). They have been proved to
cause numerous cellular anomalies, including but not
limited to protein damage, deactivation of enzymatic
activity, alteration of DNA and lipid peroxidation of
membranes (8). Several herbal drugs like Abrus
precatorius, Scutia myrtina, Funaria indica etc. have
been evaluated for its potential as liver protectant
against NDEA induced hepatocellular carcinoma in
rats (9- 11).
Chemoprevention, which is referred to as the use of
nontoxic natural or synthetic chemicals to intervene
in multistage carcinogenesis, has emerged as a
promising and pragmatic medical approach to reduce
the risk of cancer. Numerous components of plants,
collectively termed “phytochemicals” have been
reported to possess substantial chemopreventive
properties. For many years cancer chemotherapy has
been dominated by potent drugs that either interrupt
the synthesis of DNA or destroy its structure once it
has formed. Unfortunately, their toxicity is not
limited to cancer cells and normal cells are also
harmed (12).
Use of plants and plant derivatives considered as
alternative medicine for many ailments are on the
increase on a global perspective. Woodfordia
fruticosa is a traditional medicinal plant belongs to
the family Lythraceae is used against a wide variety
of diseases. All parts of the plant possess valuable
medicinal properties such as anti inflammatory,
antitumor, hepatoprotective and free radical
scavenging activity but flowers are of maximum
demand. Dried flowers are used as tonic in disorders
of mucous membrane, hemorrhoids and in
derangement of liver (13). Phenolics, particularly
hydrolysable tannins and flavonoids were identified
as major components in Woodfordia fruticosa
flowers. In view of these the present study was
undertaken to evaluate the chemopreventive effect of
Woodfordia fruticosa flower extract on NDEA
induced hepatocellular carcinoma in experimental
rats.
2. MATERIALS AND METHODS
2.1 Chemicals
N-nitrosodiethylamine (NDEA), silymarin,
proliferating cell nuclear antigen (PCNA), cyclin D1,
anti-mouse IgG horse radish peroxidase, streptavidin
horse radish peroxidase conjugate and
diaminobenzidine were purchased from Sigma
Chemical Co., St. Louis, MO, USA. Assay kits for
serum aspartate aminotransferase (AST), alanine
aminotransferase (ALT) and gamma glutamyl
transferase (GGT) were purchased from Agappe
Diagnostics, India. All other chemicals were of
analytical grade.
2.2 Collection of plant material and preparation of
plant extracts
Woodfordia fruticosa flowers were collected from
natural habitat (Kollam, Kerala, India) during
November - January. Plant material was identified by
Dr. V.T Antony, S.B College. A voucher specimen
(Acc. No. 7566) is deposited at the herbarium of the
Department of Botany, S.B College, Changanassery,
Kottayam, Kerala. Flowers were shade-dried and
powdered and 50 g of dried powder was soxhlet
extracted with 400 mL of methanol for 48 h. The
extract was concentrated under reduced pressure
using a rotary evaporator and was kept under
refrigeration. The yield of methanolic extract of
Woodfordia fruticosa (MEWF) was 12.5 % (w/w).
The concentrate was suspended in 5% Tween 80 for
the present studies.
2.3 Animals and diets
Male Wistar rats weighing 150-160 gm were used in
this study. The animals were housed in
polypropylene cages and had free access to standard
pellet diet (Sai Durga Feeds, Bangalore, India) and
drinking water. The animals were maintained at a
controlled condition of temperature of 26–28 o C with
a 12 h light: 12 h dark cycle. Animal studies were
followed according to Institute Animal Ethics
Committee regulations approved by Committee for
the Purpose of Control and Supervision of
Experiments on Animals (Reg. No. B 2442009/4) and
conducted humanely.
2.4 Induction of hepatocellular carcinoma
Hepatocellular carcinoma (HCC) was induced by oral
administration of 0.02% NDEA (2ml, 5 days/week
for 20 weeks (12). Silymarin at an oral dose of 100
mg/kg body weight was used as standard control in
the experiment (14-15). Two different doses of
MEWF (100mg/kg and 200 mg/kg) suspended in 5%
Tween 80 were prepared for oral administration to
the animals. It is reported that the extract of W.
fruticosa flowers are safe up to the dose of
2000mg/kg, p.o (13).
2.5 Experimental design
IJAPR / Dec. 2012/ Vol. 3 /Issue. 12 / 1322 – 1330 1323