Evaluation of acute and subchronic toxicity of Legenaria siceraria aerial part

Abstract

Lagenaria siceraria is traditionally being used in many countries in the treatment of various diseases including diabetes. The aim of the present study was to evaluate the safety of the methanol extract of L. siceraria aerial parts (MELS) through acute and subchronic toxicity study in mice. For acute toxicity study 500-2000 mg/kg MELS were administered orally and obvious toxic symptoms and mortality was studied upto 72 h. In subchronic study, effect of multiple weekly dosing of 400 mg/kg (one-fifth of the maximum tolerated dose) of MELS was investigated in mice for six weeks and the evaluation was done by the studies of hematological parameters, biochemical estimations of hepatorenal parameters, antioxidant status, and histological observations of the tissue. The extract was found to be well tolerated upto 2g/kg in acute toxicity study. In subchronic toxicity study it showed no significant alteration on any of the parameters, however an improvement in the lipid profile was observed in the treated group of animals. Hence the results suggest that methanol extract of L.siceraria aerial parts is quite safe and can be used in the treatment of the chronic diseases like diabetes without any toxicity.

Full text

Mazumder et al., IJPSR, 2011; Vol. 2(6): 1507-1512 ISSN: 0975-8232
Available online on www.ijpsr.com 1507
IJPSR (2011), Vol. 2, Issue 6 (Research Article)
Received on 11 February, 2011; received in revised form 21 April, 2011; accepted 28 April, 2011
EVALUATION OF ACUTE AND SUBCHRONIC TOXICITY OF LAGENARIA SICERARIA AERIAL PARTS
P. Saha
1, 2
, U. K. Mazumder*
1
, P. K. Haldar
1
, A. Islam
1
and R. B. Suresh Kumar
1
Department of Pharmaceutical Technology, Jadavpur University
1
, Kolkata, West Bengal, India
Guru Nanak Institute of Pharmaceutical science and Technology
2
, 157/F, Nilgunj Road, Panihati, Kolkata, West
Bengal, India
ABSTRACT
Lagenaria siceraria is traditionally being used in many countries in the treatment
of various diseases including diabetes. The aim of the present study was to
evaluate the safety of the methanol extract of L. siceraria aerial parts (MELS)
through acute and subchronic toxicity study in mice. For acute toxicity study 500-
2000 mg/kg MELS were administered orally and obvious toxic symptoms and
mortality was studied upto 72 h. In subchronic study, effect of multiple weekly
dosing of 400 mg/kg (one-fifth of the maximum tolerated dose) of MELS was
investigated in mice for six weeks and the evaluation was done by the studies of
hematological parameters, biochemical estimations of hepatorenal parameters,
antioxidant status, and histological observations of the tissue. The extract was
found to be well tolerated upto 2g/kg in acute toxicity study. In subchronic
toxicity study it showed no significant alteration on any of the parameters,
however an improvement in the lipid profile was observed in the treated group
of animals. Hence the results suggest that methanol extract of L.siceraria aerial
parts is quite safe and can be used in the treatment of the chronic diseases like
diabetes without any toxicity.
INTRODUCTION: Nature has been a source of
medicinal agents from the ancient times and medicinal
plants, especially have formed the basis of the wide
variety of traditional medicines used in various
countries worldwide. Present estimates indicate that
about eighty percent of the world’s population relies
on traditional medicine for health care delivery
1, 2
. The
exclusive use of herbal drugs for the management of
variety of ailments continues due to easy access,
better compatibility and for economic reasons. Studies
of medicinal plants using scientific approaches showed
that various biological components of medicinal plants
exhibit a variety of properties and can be used to treat
various ailments. However, a number of studies have
reported the toxic effects of herbal medicines
3, 4
.
Hence a systemic scientific study of a medicinal plant
should include a thorough toxicity study before the
recommendation for its use in the treatment of any
disease or disorder.
Lagenaria siceraria (Mol.) Standley, commonly known
as bottle-gourd (in English), belongs to cucurbitaceae
family. It is a climbing or trailing herb, with bottle or
dumb-bell shaped fruits. Both of its aerial parts and
fruits are commonly consumed as vegetable. The plant
is widely available in India, China, European countries,
Brazil and Hawaiian island and traditionally is used as
medicine for its cardiotonic, general tonic, and diuretic
properties
5
. Further, antihepatotoxic, analgesic and
anti-inflammatory, hypolipidemic, antihyperglycemic,
immunomodulatory and antioxidant activities of its
fruit extract have been evaluated
6-10
. Lagenaria
siceraria fruits are good source of vitamin B complex,
Keywords:
L.siceraria,
Cucurbitaceae,
Subchronic toxicity,
Hematological parameters,
hepatorenal,
Antioxidant status
Correspondence to Author:
P. Saha
Department of Pharmaceutical
Technology, Jadavpur University,
Kolkata, West Bengal, India

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ascorbic acid, fibers, proteins, cucurbitacins, saponins,
fucosterols and compesterols, polyphenolics, flavones-
C-glycoside
8, 10-13
. Methanol extract of its leaves
showed the presence of sterols, polyphenolics,
flavonoids, saponins, proteins and carbohydrates
14
. A
novel protein, Lagenin has also been isolated from its
seeds and it possesses antitumor, immunoprotective
and antiproliferative properties
15
. Despite of the
popular use, exploring various medicinal importances
of the various parts of the plant, there is no report on
the toxicity study of its aerial parts. The present
investigation was therefore carried out to study the
acute and subchronic toxicity of the methanol extract
of L. siceraria aerial parts (MELS) in mice.
MATERIALS AND METHODS:
Plant material: The aerial parts of L.siceraria was
collected in November 2008, from Madanpur, West
Bengal, India and identified by the Botanical Survey of
India, Howrah, India. A voucher specimen (P/LS/1/08)
was retained in our laboratory for further reference.
Preparation of plant extract: The aerial parts were
dried under shade and powdered in a mechanical
grinder. The powdered material was extracted with
methanol using soxhlet apparatus. This extract was
filtered and concentrated in vacuo in a Buchi
evaporator, R-114 and kept in a vacuum dessicator
until use. The yield was 18.13% w/w with respect to
dried powder. Aqueous suspension of MELS was
prepared using 2 % (v/v) Tween-80 and used for oral
administration.
Animals: Healthy Swiss albino mice (20 ± 2 g) were
used for the present study. They were maintained at
standard laboratory conditions and fed with
commercial pellet diet (Hindustan Lever, Kolkata,
India) and water ad libitum. The animals were
acclimatized to laboratory condition for one week
before commencement of experiment. The
experiments were performed based on animal ethics
guidelines of University Animals Ethics Committee.
Phytochemical analysis: Preliminary phytochemical
screening of the extract was carried out using standard
methods
16
.
Acute toxicity study: Healthy Swiss albino mice (20 ± 2
g) of either sex, starved overnight, were divided into
five groups (n=6). Group I-IV animals were orally fed
with MELS in increasing dose levels of 0.5, 1.0, 1.5 and
2.0 g/kg, while group V (untreated) served as control.
The animals were observed continuously for first 2 h
for any gross change in behavioral, neurological and
autonomic profiles or any other symptoms of toxicity
and mortality if any, and intermittently for the next 6 h
and then again at 24 h , 48 h and 72 h for any lethality
or death. One-fifth of the maximum safe dose of the
extract tested for acute toxicity was selected for the
subchronic toxicity experiment
17
.
Subchronic toxicity study: Sixteen mice were randomly
divided into two groups of eight mice in each. Group I
(normal control) animals received 2% Tween 80
solution (0.5 ml, p.o.) and Group II animals received
MELS (400mg/kg, p.o., ie., one-fifth of the maximum
tolerated dose) every 72 h for six weeks
18, 19
. During
the experimental period, the animals were weighed
every three days and food and water intake were
monitored daily. At the end of the experiment, after 24
h of the last dose and 18 h fasting, animals were
sacrificed and blood was collected intracardially and
taken into heparinized tube for hematological studies
and non-hepainized centrifuge tube for biochemical
estimations. Liver tissue was collected from the
animals for the evaluation of in vivo antioxidant status
and part of the liver tissue was taken for the
histological studies.
Hematological studies: RBC, WBC counts using of
Neubauer hemocytometer and estimation of
hemoglobin using Sahli’s Hemoglobinometer were
carried out by standard procedures from the blood
obtained intracardially
20, 21
.
Biochemical estimation: The effect of MELS treatment
on the biochemical parameters of the experimental
mice were evaluated by the estimation of serum
biochemical enzymes such as serum glutamic
oxaloacetic transaminase (SGOT) and serum glutamic
pyruvic transaminase (SGPT) activities by the method
of Reitman and Frankel
22
, alkaline phosphatase (ALP)
activities by Kind and King method
23
, total bilirubin
24
,
total protein
25
, urea
26
, uric acid
27
, creatinine
28
,
glucose, total cholesterol, triglyceride, HDL and LDL

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cholesterol
29, 30
. All the analysis was performed by
standard enzymetic methods using commercially
available kit from Span Diagnostics Ltd.
In vivo antioxidant assay: The antioxidant assay was
performed with the liver tissues of the experimental
animals and evaluation of the antioxidant status was
carried out by measuring the level of lipid peroxidation
31
and the amount of enzymatic (Catalase: CAT) and
nonenzymatic antioxidant system (reduced
glutathione: GSH) by the methods of Luck
32
and
Ellman
33
respectively.
Histological studies: After sacrificing the mice, parts of
liver tissues were collected for the histological studies.
The tissues were washed in normal saline and fixed
immediately in 10% formalin for a period of at least 24
h, dehydrated with alcohol, and embedded in paraffin,
cut into 4-5µm thick sections and stained with
hematoxylin- eosin dye for photomicroscopic
observation.
Statistical analysis: Values were presented as mean ±
S.E.M. Data were statistically evaluated by one-way
analysis of variance (ANOVA) followed by post hoc
Dunnett’s test using SPSS software. P<0.01 were
considered as statistically significant.
RESULTS: Preliminary phytochemical screening of
MELS revealed the presence of polyphenolics,
flavonoids, glycosides, triterpenoids, saponin and
carbohydrates. In acute toxicity study, MELS did not
show any mortality or toxic effect upto the dose of 2
g/kg during the observational period of 72 h. It did not
produce any significant changes in behavior, breathing,
cutaneous effects, sensory nervous system responses,
and gastrointestinal effects in male and female mice.
These results showed that in single dose, there are no
adverse effects of MELS, indicating that the medium
lethal dose (LD
50
) is higher than 2000 mg/kg for both
male and female mice. Accordingly one-fifth of the
maximum tolerated dose ie, 400 mg/kg was
considered as the high dose of MELS and used for the
subchronic toxicity study in the present investigation.
In sub-chronic toxicity study, MELS administration did
not show any significant effect on water and food
intake and body weight of the treated animals (data
not shown).
Effect of MELS on hematological parameters has been
presented in Table 1. RBC and WBC count remained
unaltered in MELS treated animals, hemoglobin
content was slightly decreased in group II mice;
however, it was within normal range.
The normal levels of hepatic biomarker enzymes
(SGPT, SGOT and ALP), total bilirubin and protein in
serum and the unaltered values of renal biochemical
parameters (urea, uric acid and creatinine), as shown
in Table 2, indicate that subchronic treatment with
MELS does not posses any significant adverse effect on
hepato-renal functioning of the animals.
TABLE 1: EFFECT OF METHANOL EXTRACT OF LAGENARIA SICERARIA AERIAL PARTS (MELS) ON HEMATOLOGICAL PARAMETERS OF
CONTROL AND TREATED MICE
Groups
Hemoglobin (g %)
RBC (million/cu.mm)
WBC (thousand/cu.mm)
Normal Control (2% Tween 80 )
13.03±1.00
6.70±0.65
3.47±0.29
MELS (400 mg/kg)
12.20±1.65
6.45±0.45
4.03±0.94
Values are mean ± SEM, (n=8), * p<0.01 for MELS treated group vs. normal control group
TABLE 2: EFFECT OF METHANOL EXTRACT OF LAGENARIA SICERARIA AERIAL PARTS (MELS) ON BIOCHEMICAL PARAMETERS FOR
HEPATORENAL FUNCTIONS IN CONTROL AND TREATED MICE
Groups
Hepatic Biochemical parameter
Renal Biochemical parameter
SGOT (IU/dl)
SGPT (IU/dl)
SALP (IU/dl)
Total Bilirubin
(mg/dl)
Total Protein
(g/dl)
Urea
(mg/dl)
Uric acid
(mg/dl)
Creatinine
(mg/dl)
Normal Control
(2% Tween 80)
52.52±1.68
45.25±4.02
88.26±1.48
1.10±0.13
7.25±0.46
40.16±2.50
6.06±0.65
0.90±0.29
MELS
(400 mg/kg)
60.30±2.02
54.92±3.58
89.05±2.90
0.99±0.22
7.77±0.52
45.08±5.05
7.15±0.81
1.31±0.76
Values are mean ± SEM, (n=8), * p<0.01 for MELS treated group vs normal control group

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Table 3 explores the lipid profile and blood sugar level
of normal and MELS treated animals after the six week
experimental period. The results revealed that the
extract does not adversely alter the lipid profile and
blood sugar level of the animals after subchronic
treatment, however there was a tendency of the
increase in HDL cholesterol with subsequent decrease
in LDL cholesterol level after treatment.
No significant difference in case of endogenous
antioxidant status among the normal control animals
and extract treated mice were observed (Table 4).
TABLE 3: EFFECT OF METHANOL EXTRACT OF LAGENARIA SICERARIA AERIAL PARTS (MELS) ON LIPID PROFILE AND GLUCOSE LEVEL IN
CONTROL AND TREATED MICE
Groups
Lipid profile (mg/dl)
Glucose (mg/dl)
Triglyceride
Total cholesterol
HDL
LDL
Normal Control (2% Tween 80)
90.66±3.26
120.45±2.41
76.60±1.06
26.98±3.05
82.25±3.04
MELS (400 mg/kg)
100.09±4.98
123.00±4.71
79.99±1.00
23.98±2.66
80.65±2.66
Values are mean ± SEM, (n=8), * p<0.01 for MELS treated group vs. normal control group
TABLE 4: EFFECT OF METHANOL EXTRACT OF LAGENARIA SICERARIA AERIAL PARTS (MELS) ON ANTIOXIDANT SYSTEM OF CONTROL
AND TREATED MICE
Groups
LPO (nM/mg wet tissue)
GSH (g/mg wet tissue)
CAT (M of H
2
O
2
decomposed/min/mg wet tissue)
Normal Control (2% Tween 80)
98.266.50
34.923.45
71.906.66
MELS (400 mg/kg)
100.302.55
40.021.88
77.803.66
Values are mean ± SEM, (n=8), * p<0.01 for MELS treated group vs. normal control group
Histological observation of the liver tissue of both
normal control mice as well as extract treated mice
(Fig. 1A and 1B) showed normal cellular architecture
with prominent central vein.
FIG. 1A: PHOTOMICROGRAPH OF LIVER SECTION OF NORMAL
MICE
FIG. 1B: PHOTOMICROGRAPH OF LIVER SECTION OF MICE
TREATED WITH MELS 400mg/kg
FIG. 1: HISTOLOGICAL OBSERVATION OF THE LIVER TISSUES OF
NORMAL MICE AND TREATED MICE WITH METHANOL EXTRACT
OF LAGENARIA SICERARIA AERIAL PARTS (MELS)
DISCUSSION: Herbal medicines have received a great
attention as alternatives to synthetic pharmaceutical
products in recent times, leading to the increase in
their demand
34
. Experimental screening method is
therefore important to ascertain the safety and
efficacy of these herbal drugs.
The lack of mortality or toxicity at oral treatment of
over 2000 mg extract/kg body weight obtained
suggests that the methanol extract of L. siceraria aerial
parts is practically nontoxic at single dose. However in
case of subsequent use in the treatment of the chronic
diseases like diabetes whether it will be safe that can
be clear from its sub chronic toxicity study.
The effect on hemoglobin concentration and RBC
count indicated the unlikelihood of the extract to
induce anaemia. Insignificant change in WBC count
was probably due to normal response to foreign bodies
or stress associated with the chronic toxicity studies
35,
36
. Increase in the level of SGPT, SGOT and ALP reflects
the structural and functional dysfunction of
hepatocellular membrane or cell rupture, and thereby
indicates liver damage. Bilirubin is formed from
degeneration of hemoglobin by the action of
reticuloendothelial systems throughout the body.
Increased bilirubin level reflects the depth of jaundice

Mazumder et al., IJPSR, 2011; Vol. 2(6): 748-753 ISSN: 0975-8232
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19, 37
. The normal value of the hepatic biochemical
parameters reveals the safety profile of the extract on
liver function even on its chronic use. The normal
values of the renal biochemical parameters, including
urea, uric acid and creatinine suggest that the extract
does not produce any sort of disturbance in the kidney
function, as has been found in case of various plant
extracts
18
and hence is safe on its chronic use in
various diseases.
Although, the extract possesses antidiabetic property,
however it does not affect adversely normal blood
glucose level. The tendency to improve the lipid profile
in the present study indicates its hypolipidemic
potential, which may be beneficial in further studies.
The endogenous antioxidant status after the chronic
use of the extract was found to be quite equivalent to
that of the normal mice. Free radicals generated either
exogenously or endogenously in our body have been
implicated in causation of several diseases such as liver
cirrhosis, inflammation, atherosclerosis, diabetes,
cancer, neurodegenerative diseases and so forth. The
link between free radicals and diseases has led to
considerable research into nontoxic drug that
possesses antioxidant property and can scavenge the
free radicals.
Thus, in present investigation the improved
antioxidant status in the extract treated animals
indicates that it may be beneficial in face of the
oxidative stress in case of various diseases and
disorders
6, 38
. Histological observations correlate the
other results showing the normal cellular architectures
in the treated group of animals, without any necrosis
or fatty infiltration, which can substantiate the safety
profile of the extract clearly. The present study thus,
provides evidence for the total safety profile of the
methanol extract of the aerial parts of L. siceraria,
suggesting its safe use in single dose treatment as well
as for long term use for the treatment of various
chronic diseases, without producing any toxic effects.
Hence further phytopharmacological studies on the
basis of its ethnobotanical use can help to explore and
establish the bioactive constituents which can be used
safely for the treatment of various diseases and
disorders in future.
ACKNOWLEDGEMENT: Necessary support and
cooperation from Dr. Abhijit Sen Gupta, Director-cum-
Principal & Prof. Dipankar Chakraborty, Registrar, Guru
Nanak Institute of Pharmaceutical Sciences and
Technology, Kolkata are hereby gratefully
acknowledged.
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