Evaluation of Hepatoprotective action of Andrographis paniculata in

Pallav Kaushik Deshpande and Ragini Gothalwal, Evaluation of Hepatoprotective action of Andrographis paniculata in
in-vitro cultured Hepatocytes, Int.J.Curr.Biotechnol., 2015, 3(4):7-10.
International Journal of Current
Biotechnology
ISSN: 2321 - 8371
Journal Homepage : http://ijcb.mainspringer.com
Evaluation of Hepatoprotective action of Andrographis paniculata in in-vitro cultured Hepatocytes
Pallav Kaushik Deshpande* and Ragini Gothalwal
Department of Biotechnology, Barkatullah University, Bhopal - 462 026.
A R T I C L E
I N F O
Article History:
Received 20 March 2015
Received in revised form 13 April 2015
Accepted 25 April 2015
Available online 30 April 2015
Key words:
Andrographis
paniculata,
hepatoprotective,
hepatocytes, Agarose, RPMI-1640.
A B S T R A C T
Liver plays a vital role in metabolism and detoxification. Impairment due to injury or
infection leads to deterioration of function may imparts many implications on one’s
health. Till date treatment for liver diseases by morden medicine is a challenge. Only
phytoconstituents are known remedies for the liver management while allopathic
medicine has little to offer for the hepatic ailments. Andrographis paniculata is well
known plant in Bengal by the name ‘kalmegh’ Kalamegha, meaning “dark cloud”. In
present study single cell suspension of hepatocytes was isolated from adult wister
mice through mechanical shearing, and cultured in culture petriplates in RPMI1640
with 2% agarose to provide 2D frame, supplemented with 5% FBS and streptomycin
(100µg ml-1). Aqueous extract and alcoholic extract of Andrographis paniculata were
selected, Liv52 was selected as control drug. Hepatotoxicity was induced by 15mM
CCl4. Morphological investigation of cultured cells and micro culture tetrazolium
assay (MTA Assay), confirms that hepatoprotective ability at higher dose. Dose
dependent increase in viability was recorded in hepatocytes exposed to CCl4. These
finding prove the efficiency of hepatoprotective activity of Andrographis paniculata
in vitro culture by normalizing biochemical actions altered by CCl4 intoxication.
Introduction
Andrographis paniculata, commonly known as
Siriyanangai in Tamil, belongs to the family Acanthaceae
is widely used in the Indian traditional system of medicine.
The major component of A. paniculata is
andrographolide is a bitter, colorless, and crystalline in
appearance, is called diterpene lactone (Siripong et al.,
1992). Recent research has revealed that Andrographis
paniculata has a surprisingly broad category of
pharmacological activity and some of them are
enormously beneficial, such as antiinflammatory,
antidiarrhoeal,antivial, antimalarial (Shen et al., 2002),
hepatoprotective, cardiovascular, anticancer, and
immune-stimulatory activities (Calabrese et al., 2000).
Andrographis paniculata is used to treat poisonous
bites, diabetes and respiratory tract infection. The plant
possesses anti-inflammatory, antipyretic, antiviral,
immune stimulatory, anticancer, anti-hyperglycemic and
antioxidant properties (Vijayakumar et al., 2007). Many
of the plants are rich in secondary metabolites and are
potent source of drugs. On the other hand, male
reproductive toxicity and cytotoxicity of the plant
Andrographis has also been reported.
Materials and Methods
Plant extract and drug selected for study
Andrographis paniculata was selected for the present
study .Aquaous extract and alcoholic extract were
prepared by adopting methods (Trease and Evans, 1978,
Kokatte, 2006) and selected as a test samples, Liv52
(Himalaya product) was selected as control drug.
Hepatotoxicity was induced by 15mM CCl4 which was
selected after CTC50 determination.
Stock solution preparation
For hepatoprotective and hepatotoxicity activity in
present study stock solution of extract and drug at
various dilutions were prepared with DMSO for better
uptake of molecule and non toxic property.
Cell preparation
In present study single cell suspension of hepatocytes
was prepared from liver of adult Wister mice through
mechanical shearing (Ursal et al., 2014), and cultured in
culture petriplates in RPMI1640 with 2% agarose to
provide 2D frame, supplememted with 5% FBS and
streptomycin (100µg) in CO2 incubator with 5% CO2 at
350C or 24 hrs, then cells were subjected to CCl4 treatment
for toxicity.
*Corresponding author.
Email address: [email protected]
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Int.J.Curr.Biotechnol.
Volume 3; Issue 4; Apr, 2015
Table-1: Protective effect of given extracts on CCl4 induced toxicity on cultured liver cells
Cells and treatment
Control
Aqueous extract
Alcoholic extract
Liv52
CCl4
Conc. in (µg ml-1)
800
250
230
15mM
% viability of cells
100
65.75±1.94
75.73±1.84
78.83±1.94
32.21±1.02
Figure-1: Hepatocytes treated with Liv52
Figure-2: Hepatocytes treated with alcoholic extract of Andrographis paniculata
Figure-3: Hepatocytes treated with aqueous extract of Andrographis paniculata
Volume 3; Issue 4; Apr, 2015
Int.J.Curr.Biotechnol.
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Figure-4: Hepatocytes treated with CCl4 large no of nonviable cells due to stress
Determination of Mitochondrial Synthesis by Micro
culture Tetrazolium (MTA Assay).
The ability of the cells to survive a toxic treatment has
been the basis of most cytotoxicity assays. This assay is
based on the assumption that dead cells or their products
do not reduce tetrazolium. The assay depends both on
the number of cells present and on the mitochondrial
activity per cell. The cleavage of MTA to a blue formazan
derivative by living cells in clearly a very effective
principle on which the assay is based (Freshney, 2000)
In vitro hepatoprotective activity against CCl4 induced
toxicity
Below the CTC50 value two dose levels were selected for
each extract and used for further studies.In present study
modified method of Vijayan et al., 2002 monolayer was
washed once and treated with 100µl of different drug
concentrations for 24 hrs. After 24 hrs of pretreatment
with the extracts, the cells were challenged with CCl4 (15
mM) where 100µl of different drug concentration and
100µl of CCl4 was added. The plates were then incubated
at 370C for further 24 hours in 5% CO2 atmosphere.
Microscopic examination was carried out and
observations were recorded every 24 hours. After 72
hours, the drug solutions in the wells were discarded
and 50µl of MTA in RPMI 1640 was added to each well.
The supernatant was removed and 50µl of propanol was
added to solubilize the formed formazan.
Morphological analysis
Cells were examined microscopically for any
morphological change induced by treatment of drug and
CCl4.
Results and Discussion
Liver injuries induced by CCL4 are the best characterized
system of xenobiotic- induced hepatotoxicity and
commonly used models for the screening of antihepatotoxic and or hepatoprotective activities of drugs
(Clawson 1989, Lin et al., 2002). Percentage of viable cells
exposed to extract CCl4, and Liv52 were given in table1. It
was suggested that, CCl 4 get accumulated and
metabolically activated in hepatic parenchyma cells which
is cytochrome P450 dependent monooxygenases to form
trichloromethyl radical(CCl3). The CCl3 radical acts on
cellular proteins to alkylates and other macromolecules
simultaneous attack on polyunsaturated fatty acids, in
the presence of oxygen ,to produce lipid peroxides,
resulted in liver damage. Thus it was suggested by
9
Bishayee et al., 1995, that antioxidant or free radical
generation inhibition is important in protection against
CCl4 induced liver damage. Which was supported by
Muthu et al., (2008) CCl4 induced a significant rise in
aspartate amino transferase (AST), alanine amino
transferase (ALT), alkaline phosphatase (ALP), total
bilirubin, gamma glutamate transpeptidase (GGTP), lipid
peroxidase (LPO) with a reduction of total protein,
superoxide dismutase (SOD), catalase, glutathione
peroxidase (GPx) and glutathione S-transferase (GST).
Fig:1-3 represents the hepatocytes culture after exposure
to drug and extract treatments, Fig-4 represents the
damaged hepatocytes after CCl4 toxication.
Conclusion
Morphological investigation of cultured cells and MTT
assay, proves that alcoholic extract shows similar results
as Liv52 (250µgml-1, 230 µgml-1 respectively), while
aqueous extract shows hepatoprotective ability at higher
dose (800µgml-1). Dose dependent increase in viability
was recorded in hepatocytes exposed to CCl 4.These
finding prove the efficiency of hepatoprotective activity
of Andrographis paniculata in vitro culture by
normalizing biochemical actions altered by CCl 4
intoxication.
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