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Enicostemma littorale Blume - A potentialhypolipidemic plant
Natural Product Radiance Vol 3(6) November-December 2004
Keywords: Enicostemma littorale, Chhotachirayta, pDimethylaminoazobenzene (p-DAB), Hepatotoxicity, Lipid profile, Lipid
peroxidation, y-Glutamyl transpeptidase.
IPC code; Int. d. 7_ A61K 35n8, A61P 1/16, A61P 3/06
Restoration of liver function by the application of Enicostemma littoraleBlume (aerial part) powder in p-DAB (p-dimethylaminoazobenzene) induced
animals was evaluated by analyzing the serum total cholesterol, triglycerides,
HDL-cholesterol, serum y-glutamyl transpeptidase and liver malondialdehyde
levels. Administration of the chosen plant reduces the hyperlipidemia at significant
level and also reduces the lipid peroxidation and significantly increased HDL
cholesterol level in serum. When comparing the results with the standard
antilipidemia agent namely, vitamin E, the results obtained using the plant is highly
comparable.
(p-DAB) are reported to induce damage
in hepatic systems (WHO, 1975). p-DAB,
a colouring agent commonly used in foods
is reported to be a powerful liver
carcinogen (Mukai & Goldstein, 1976;
Weinberg, 1996; Fabiana et al, 2001).The electrophilic nature of the metabolites
of p-DAB has the capacity to covalently
bind with RNA, DNA and tissue protein
(Labuc & Blunck, 1979; Ohnishi et al,2001). In addition, reactive oxygen
species such as superoxide, hydroxyl
radicals and hydrogen peroxide (Halliwell
& Gutteridge, 1989; Sies, 1991; Grisham,
1992; Moslen, 1994; Halliwell, 1996) are
released due to the induction of p-DAB,
R GopaP, A GnanamanF*, R Udayakumar and S Sadulla1
ICHORD, Central Leather Research Institute, Adyar, Chennai-20, Tamil Nadu
2Department of Biochemistry,]. ]. College of Arts and Science, Pudukkottai-622404, Tamil Nadu
* Correspondent author, E-mail: [email protected] -which further stimulate the damage of liver
by the metabolized products of lipid
peroxidation, consequently leading to DNA
aberrations (Biswas & Khuda-Bukhsh,
2002). Shamberger et al (1974) reported
that hydroxyl radicals are highly reactive
and are the basis for the generation of lipid
- peroxide products such as
malondialdehyde, which are considered
to be a cause for carcinogenesis.
Several plant products have been
shown to exert a protective role against
the formation of free radicals and playing
a beneficial role in maintaining disease
condition (Ajitha & Rajnarayana, 2001).
Abstract
liver is one of the most important
organs for the metabolism of various
chemicals and is responsible for the major
detoxification. Liver damage/hepato
carcinoma, in general associated with the
external agents comes under the
classification as carcinogens (Raghuram
et al, 2003). About 80-90% of cancer
related diseases and the hepatic problems
are mainly due to continuous exposure or
contamination of chemical carcinogens in
the food chain (WHO, 1987). Chemical
carcinogens like N-nitrosodiethylamine
(NDEA) and p-dimethylaminoazobenzene
Introduction
, .
Statistical analysis
and serum was separated as per
conventional methods. Liversamples were
separated and was homogenized in 10%
saline. The homogenate was centrifugedat 15,000 x g for 30 minutes at 4°C and
the supernatant was used for lipid
peroxidation analysis. The serum total
cholesterol (TC) was estimated by the
method of Zak's (1957); triglycerides
(TG) was estimated by method of Van
Handel & Zilversmit (1957). High density
lipoprotein cholesterol (HDL-c) was
estimated by the method of Burstein et al(1970). Low-density lipoproteincholesterol (WL-c) was calculated from
the above measurement by using the
equation, LDLC= TC - (HDLC +TG),
suggested by of Friendwald et al (1972).
The y-glutamyl transpeptidase
activity was estimated by the method of
Orloswski & Meister (1965). Lipid
peroxidative effectwas evaluated through
measuring the hepatic content of
thiobarbituric acid reacting substances
(TBARS),expressed as malondialdehyde(MDA) equivalents by the method ofNiehaus & Samuelsson (1968).
Results were statistically
evaluated using one-way analysis of
variance (ANOVA) for repeated
observation. Values of p< 0.05 were
considered to be significant.
Results and DiscussionBiochemical estimations
Animals were divided into four
groups each group containing six animals.
Group I served as normal animals, which
received the vehicle 3% DMSO (5m1Jkg
body wt). Group II animals with
hepatocellular damage induced by
p-dimethylaminoazobenzene (Img/kg
body wt) in 3% DMSO was injectedintraperitoneally (i.p.) once in every 3
days for 15 days. Group 1lI animals with
hepatocellular damage were induced as
per group II and followed by last dosage
were treated with the plant (1g1kg bodywt) in crude form along with physiological
saline twice a day for 15 days by gavage
method. Group IV animals with
hepatocellular damage induced as pergroup II, after the last dose, vitamin E
(30mglkg bodywt) was givenorally in thesuspended form with sunflower oil.
Administration was done for twice a day
for 15 days.
Plant material and chemicals
Experimental Work
Enicostemma littorale Blume purchased from an authorized firm at
(Family - Gentianaceae) a glabrous Bangalore and were housed in spacious
herb commonly used as a bitter tonic and polypropylene cages and maintained
substitute for Swertia chirayita under standard conditions of temperature
(Roxb. ex Flem.) Karst., is also called 27 ±2°C, relative humidity of 60 ± 5%,
as Chhotachirayta. It is also reported 12 hours of lighVdark cycle, and fed with
to possess antitumor (Dash pellet diet manufactured by Pranav Agro
et aI, 2000), antiarthritic (Sahu et aI, Industries Limited, Shangli, Maharashtra
2000), hypoglycaemic (Raviet aI, 2000) under the trade name "Amrutrat and miceand antimalarial activities (Katewa & feed" and had free access to sterilized
Arora, 2001). The anticancer activitYof water.methanolic extract of the plant has been-
evaluated against Dalton's ascetic Experimental designlymphoma in Swissalbino mice (Kavimani
& Manisenthlkumar, 2000). In the present
study authors have evaluated the
hypolipidemic and antilipid peroxidativeactivities of the plant against p-DAB
induced hepatotoxicity in animals.
The plant, E. littorale was
collected from Chennai, TamilNadu in themonth of December and was identified
and authenticated by Dr T. Anandan;Research Officer, Central Research
Institute for Siddha Medicine, Chennai.
The aerial parts were air-dried and ground
well to fine powder, and used as drug in
the crude form along with physiologicalsaline.
p-DAB was purchased from Sigma
Chemical Company (St Louis, Mo, USA).
Allother reagents used were of analyticalgrade.
Animals
Wistar male albino rats having
body weight between 150-200g were
Table 1 emphases the effect of
The animals were fasted for aerial part of E. littorale on total
12 hours after the last dose of the drug cholesterol, triglyceride, HDL-c, WL-c,treatment and were sacrificed by cervical y-Glutamyl. transpeptidase (y-GT) and
decapitation. Blood was collected in tubes Lipid peroxidation levels of p- DAB
402 - Natural Product Radiance Vol 3(6) November-December 2004
induced hepatotoxicity in animals. In the
p-DAB induced animals, the level of total
cholesterol (87.59 ± 5.57 mg/dl),
triglycerides (153.47 ± 6.94 mg/dl), IDLe (103.23 ± 3.45 mg/dl) and y-GT (8.26
± 1.05 IUIL)were significantlyincreased
and a significantdecrease in HDL-c(10.28
± 1.62mg/dl) in serum was observed
compared to the normal group animals.
Administration of aerial part of the plantsignificantly reduces the level of total
cholesterol (46.79 ± 6.22 mg/dl),
triglycerides (125.73 ± 76.54 mg/dl),
IDL-c (93.59 ± 2.92 mg/dl), y- GT(4.90± 0.48 IUIL)and increases HDL-c(12.25
± 1.65 mg/dl) content in serum samples
analyzed. Malondialdehyde content in theliver was enhanced in the p-DAB induced
hepatotoxicity (168.80 ± 4.70 n moVg)
A·•" ,.
when compared to normal animals. The
plant significantly reduced the
malondialdehyde content in the liver
(90.58 ± 3.18 n mol/g). VitaminEtreated
animals also expressed the similar
reduction of total cholesterol,
triglycerides, LDL-c, y-GT and lipid
peroxides in liver and an increase in
HDL-ccontent in serum samples.
Hepatotoxicity is associated with
biochemical, histological and functional
changes and progressive increase in the
chance of liver damage and death of an
organ. Liverdamage due to increased level
of lipid profile and lipid peroxidation
appears to be an important determinativemechanism of hepatotoxicity studies(Satturwar et al, 2003; Sharma et al,2003). The extract contains several
alkaloids, tlavanoids and reducing sugars
and exhibited hypolipidemia and
antioxidant properties in the present study
similar to the studies carried out byMuraUet al (2002) and Kavimani &
Manisenthlkumar (2000). Animals that
received a subcutaneous implantation ofthe p-DAB displayed cancerous
hyperlipidemia characterized byincreased
serum cholesterol (hyperscholesterolemia) and triglyceride
(hypertriglyceridemia) levels (Irikura
et al, 1985; Kawasaki et al, 1998;Komatsu et al, 1998). The hyper
cholesterolemia in the hepatoma-bearing
animals showed a highly atherogenic
lipoprotein profile, that is, a notable
increase in the cholesterol and significantdecrease in the HDLfraction (Irikura et
Table 1: Hypolipidemic activity of Enicostemma littorale Blume
S.No ExperimentTotalTriglyceridesHDL-LDL-y- GlntamylLipid
design
Cholesterol(mg/dl)CholesterolCholesteroltranspeptidase Peroxides(mg/dl)
(mg/dl)(mg/dl)(lUlL)(n molTBARS/g)
1
GroupI 33.57 ± 3.10561.61 ± 4.7416.78 ± 1.0247.48 ± 2.251.11 ± 0.2379.01 ± 2.43Normal animals2
GroupII 87.59± 5.57153.47 ± 6.9410.28 ± 1.62103.25 ± 3.458.26 ± 1.05168.80 ± 4.70DABinduced animals3
Groupill46.79± 6.22 •125.73 ± 6.97 •12.25 ± 1.62'93. 59 ± 2.92'4.90 ± 0.48'90.58 ± 3.18 •DABinduced + E. littoraledrug treated4
GroupIV41.25 ± 4.12 •111.75± 5.15•14.62 ± 1.25'87.4 ± 3.62'3.25 ± 0.25'85.45 ± 2.25'
DABinduced + VitaminE treated
Valuesare expressedas mean ± SD.GroupIII & IV are comparedto GroupII ('p<O.OOl).
Natural Product Radiance Vol 3(6) November-December 2004
References
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