Hepatoprotective wrightia tinctoria against ccl4 induced toxicity

1. 178
________________________________
* Corresponding author:
Mumtaz P
Devaki Amma Memorial College of Pharmacy, Chelembra-673634, Kerala, India.
E-mail address: mumthazemil@gmail.com
Available Online at: www.ijrpp.com Print ISSN: 2278 - 2648
Online ISSN: 2278 - 2656
(Research article)
Exploration of Hepatoprotective activity on leaf extract of
Wrightia tinctoria against CCl4 induced toxicity
*Mumtaz P, Periyasamy Selvam.
Devaki Amma Memorial College of Pharmacy, Chelembra-673634, Kerala, India.
________________________________________________________________________
ABSTRACT
The chloroform extract, isolated compounds isatin and rutin of Wrightia tinctoria were studied for
hepatoprotective activity against human liver derived Chang liver cells induced by carbon tetrachloride (CCl4).
The screening of hepatoprotective activity was based on the protection of human liver derived Chang liver cells
against CCl4 induced damage determined by estimating mitochondrial synthesis using tetrazolium assay. The
CCl4 exposed Chang liver cells showed a percentage viability of 43%. These exposed cells, when treated with
different concentrations of chloroform extract of Wrightia tinctoria, showed a dose-dependent increase in
percentage viability and the results were highly significant (P < 0.001, when compared to CCl4 intoxicated
group). The percentage viability ranged between 64 to 77 % at 100–150µg/ml concentration of Wrightia
tinctoria chloroform extract and isolated compounds isatin and rutin. The above result proved the
hepatoprotective activity of Wrightia tinctoria.
Keywords: Wrightia tinctoria, Hepatoprotective, Carbon tetrachloride.
______________________________________________________________________________________________
INTRODUCTION
Liver is one of the vital organs in human body and
the chief site for extreme metabolism and
excretion. It is involved with almost all the
biochemical pathways to increase, fight against
disease, nutrient supply, energy provision and
reproduction. The major functions of the liver are
to metabolize, detoxification of carbohydrates,
proteins and vitamins. Liver diseases are a serious
health problem. In the absence of reliable liver
protective drugs in allopathic medical practices,
herbs play a role in the management of various
liver disorders. Numerous medicinal plants and
their formulations are used for liver disorders in
ethano medical practices and in traditional system
of medicine in India. However, we do not have
satisfactory remedy for serious liver disease, most
of the herbal drugs speed up the natural healing
process of liver. So the search for effective
hepatoprotective drug continues.
Wrightia tinctoria is an important medicinal plant
used in the Indian system of medicine for the
treatment of variety of diseases1
and it reported to
possess analgesic2
, antifertility3
, cytotoxic4
,
hemostasis5
and anti ulcer activity6
. Previously, we
demonstrated the anti-HCV activity of chloroform
extract of Wrightia tinctoria (CWT) in human liver
cells7
. Review of literature revealed that
hepatoprotective activity of Wrightia tinctoria is
relatively less explored. The present work is to
study the hepatoprotective activity of chloroform
extract of Wrightia tinctoria against CCl4 induced
toxicity using Chang liver cells.
EXPERIMENTAL
Extraction
Wrightia tinctoria leaves were collected from
Tenkasi, Tamilnadu, India. The fresh leaves were
shade dried at room temperature for certain days
and then powdered. The 500gm of dried leaves
International Journal of
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powder was subjected to hot continuous extraction
in Soxhlet extractor with chloroform for 72 hours.
It was then filtered to get the crude extracts,
compounds isatin and rutin isolated from ethanolic
extract by using column chromatography. which
was then evaporated to dryness under vacuum and
the dried extracts were used for hepatoprotective
activity.
Preparation of suspensions
Wrightia tinctoria chloroform extracts were
dissolved in DMSO and the volume was made up
to 10ml with MEM (Minimum Essential Medium)
to obtain a stock solution of 1mg/ml concentration
and stored at -20°C prior to use. Further dilutions
were made to obtain different concentrations
ranging from 50–250µg/ml with respective media
and used for in vitro investigations. A suspension
of the standard powder was also prepared in a
similar manner.
Hepatoprotective effect in Chang liver cells
The screening of hepatoprotective activity was
based on the protection of human liver derived
Chang liver cells against CCl4 induced damage
determined by estimating mitochondrial synthesis
using tetrazolium assay. Chang liver cells were
routinely grown and sub-cultured as monolayer in
DMEM supplemented with 10% newborn calf
serum. The experiments in this investigation were
conducted with cells that had been initially batch
cultured for 10 days. At this stage, the cells were
harvested and plated at approximately 30,000
cells/well in 96 well microtitre plates (Nunclon)
and left to rest for 24 h at 37°C in a humidified
atmosphere of 5% CO2
8
. The cells were then
exposed to toxicant (medium containing 15mM
CCl4) along with/without various concentrations of
Chloroform extract of Wrightia tinctoria (CWT)
and isolated compounds isatin, rutin or the medium
alone (as normal). At the end of the period,
cytotoxicity was assessed by estimating the
viability of Chang liver cancer cells by MTT
reduction assay. After 1 h incubation, the test
solution from each well was removed by aspiration
and replaced with 50µl of MTT prepared in MEM
without phenol red (MEM-PR). The plates were
gently shaken and incubated for 3 h at 37 °C in a
humidified 5% CO2 atmosphere. The supernatant
was removed and 50µl of propanol was added and
the plates were gently shaken to solubilize the
formed formosan. The absorbance was measured
using a microplate reader at 540nm.
RESULTS AND DISCUSSION
Hepatoprotective effects in Chang liver cells
Liver injuries induced by CCl4 are the best
characterized system of xenobiotic-induced
hepatotoxicity and commonly used models for the
screening of anti-hepatotoxicity and or
hepatoprotective activities of drug9,10
. Since the
changes associated with CCl4 induced liver damage
are similar to that of acute viral hepatitis11
, CCl4
mediated hepatotoxicity was chosen as the
experimental model. It has been established that
CCl4 is accumulated in hepatic parenchyma cells
and metabolically activated by cytochrome P450
dependent monooxygenases to form a
trichloromethyl radical (CCl3). The CCl3 radical
alkylates cellular proteins and other
macromolecules with simultaneous attack on
polyunsaturated fatty acids, in presence of oxygen,
to produce lipid peroxides, leading to liver
damage12
. Thus, antioxidant or free radical
generation inhibition is important in protection
against CCl4 induced liver lesions13,14
. Hepatotoxic
compounds such as CCl4 are known to cause
marked elevation in serum enzymes and bilirubin
levels. It causes marked decrease in TP levels.
Silymarin is used as standard hepatoprotective
compound since it is reported to have a protective
effect on the plasma membrane of hepatocytes15
.
The present study had been admitted to
demonstrate the role of hepatoprotective activity of
crude chloroform extract, isolated compounds
isatin and rutin of Wrightia tinctoria. To our
knowledge, this is the first study which reveals the
hepatoprotective effect of Wrightia tinctoria
against CCl4 induced toxicity in Chang liver cells.
Cells which are exposed only with toxicant CCl4
showed a percentage viability of 43%. Cells which
are pretreated with extracts showed an increase in
percentage viability and the results were significant
(P < 0.01, when compared to CCl4 intoxicated
cells). The percentage viability ranged from 64 to
77 % cell viability, when pretreated with the
extracts. The above study confirms the
hepatoprotective activity of the extracts in vitro
against CCl4 induced toxicity and results are shown
in Table No. 1 and 2. The increase in percentage
viability of the chang liver cells treated with
Wrightia tinctoria and isolated compounds was
significant (P < 0.01, when compared to standard )
and equally active to that produced by the standard
at the same concentration. The above result proves
the hepatoprotective activity of Wrightia tinctoria

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Further studies for isolation of active constituents
and in vivo models for hepato protective activity
have to be investigated.
Tables 1: CTC50 values of Wrightia Tinctoria using Chang liver cells
Extract
CTC 50 in (µg/ml)
MTT assay against Chang Liver cells
CWT 223.83 ± 4.54
WT ISA 183.45 ± 5.29
Rutin 192.22 ± 5.46
*Average of six independent determinations, values are mean ± S.E.M.
Table 2: Hepatoprotective effect of Wrightia Tinctoria on CCl4 induced toxicity in Chang liver cells
*Average of six independent determinations, values are mean ± S.E.M. + = P < 0.001.
When compared to untreated cells. ++ = P < 0.001, when compared to CCl4 intoxicated cells.
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Treatment Concentration % Viability of cells*
Control (untreated cells) ___ 100
CCl4 15 Mm 43.8 ± 2.58+
CWT 150 µg/ml
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73.08 ± 2.31++
64.99 ± 2.83++
Silymarin (standard) 150 µg/ml
100 µg/ml
85.29 ± 0.65 ++
86.16 ± 0.98 ++

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