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Research

Patil SJ et al., 2010: Pre-Clinical Toxicity Studies of Orally Administered Petroleum Ether Extract of
Citrus medica seeds on the Reproductive Organs of Female Albino Mice.

ISSN 0976 4852

2010, IJCRR, All Rights Reserved.

International Journal of Contemporary Research and Review

Pre-Clinical Toxicity Studies of Orally Administered Petroleum Ether Extract of Citrus
medica seeds on the Reproductive Organs of Female Albino Mice.
Patil SJ1*, Patil SB2
1

Toxicology Laboratory, Bioenergetics and Environmental Sciences Division, National Institute of Animal Nutrition and Physiology,
Adugodi, Bangalore-560030, Karnataka, INDIA
2
Reproductive Biology Laboratory, Department of Zoology, Gulbarga University, Gulbarga-585106, Karnataka, INDIA.

Abstract
In present investigation is an attempt to determine the possible toxicity of orally administered petroleum ether extracts of Citrus medica seeds in the reproductive system of female albino Swiss mice (Mus musculus) at the dose level of 200mg and 400mg/kg body
weight for 15 days. The results revealed that the (administered of petroleum ether extract of Citrus medica seeds to female mice observed) changes in the gain/loss of body weight, organ weight (Liver, Kidney, Adrenal, Ovary and Uterus) morphological and behavioural studies. The detection of haematotoxicity, haemoglobin concentration, total RBC, WBC counts and total differential count
were examined. The biochemical (Blood Sugar, Serum Cholesterol, Total Protein and Blood Urea Nitrogen) and histological changes
in the liver, ovary and uterus were also studied. All the parameters were found to be within normal range. The data suggests that petroleum ether extract of C. medica seeds at both the dose level administration for 15 days non effective and not shown any significant
toxicity on liver as well as reproductive organs.
Key words: Citrus medica; Ovary; Uterus; Liver; Adrenal, Rat; Toxicity
Introduction
Traditional use of meditational plants to
treat different sorts of diseases is widespread throughout the world, especially in
Asian countries. The female reproductive
system and its related endocrine system
can be very sensitive to the exposure/treatment of phytochemical, chemical and physical agents which may cause
adverse effects on gamete production and
cycle normality1-2.
Citrus medica (Rutaceae) is an indigenous
small tree or shrub. Its seeds are as in the
orange, but smaller upto 12-15 seeded in a
one fruit. The seeds are indigestible,
heavy, heating to the body, stimulus, toxic, good for piles and in biliousness, cure
inflammations and Kapha (Ayuravedic)3-4.
Because of its popular use, the toxicity of
petroleum ether extract of Citrus medica
seeds has also been evaluated for various
organs, such as the liver, kidney, adrenal,
ovary and uterus. Without evaluation of
the toxicity of C. medica in relation to the
reproductive system and fertility, these
results should not be considered sufficient
to suggest that general use of C. medica
seeds were safe. Thus, the aim of present

study was to investigate this in adult female mice.

Materials and methods
Collection of Seeds
The fresh seeds of Citrus medica were
collected from Hyderabad Karnataka
areas of northern region of Karnataka,
during fruiting season i.e., in the month of
July to October and authenticated at the
herbarium, Department of Botany, Gulbarga University, Gulbarga, Karnataka,
India. Collected seed material was immediately sprayed with ethanol to cause the
enzymatic degradation of secondary metabolites. The seeds were shade dried,
chopped into small fragments and powdered inside the laboratory within 10-15
days at room temperature (28-30C).
Soxhlet Extraction of Seed Constituents
The shade dried, powdered 100gm seed
material was soxhleted with petroleum
ether (b.p. 60-80C) in a soxhlet extractor
for 48 hours. The extracts was concentrated to dryness in a flash evaporator
(Buchi) under reduced pressure and controlled temperature (50-60C) to obtain

the crude extract. Remaining trace of the

solvent if any was further removed by
placing the crude extract in vaccum over
night. The yield is 15gm and yellow oily
coloured extract was obtained. The extract
was stored in refrigerator at 4 C until
used for experiment.
Animals
Colony bred Swiss female mice (Wistar
strain Mus musculus) of either sex having
good health and normal behaviour weighing 25 to 35gm were selected for toxicity
analysis. The rats were housed in poly
propylene cages measuring 12x10x8,
under well ventilated animal house conditions (Ambient temperature: 28-31C,
photoperiod: 12h natural light and 12h
darkness, relative humidity: 50*Corresponding Author: Dr. Sharangouda J. Patil,
Bioenergetics and Environmental Sciences Division,
National Institute of Animal Nutrition and Physiology, Adugodi, Bangalore-560030, Karnataka, INDIA.
E-Mail ID: shajapatil@gmail.com
Received15 June 2010; Revised 20 June 2010;
Accepted 3 July 2010
International Journal of Contemporary Research
and Review

August, 2010|Volume 01|Issue 03|

Available Online at www.ijcrrmds.info.

Research

Patil SJ et al., 2010: Pre-Clinical Toxicity Studies of Orally Administered Petroleum Ether Extract of
Citrus medica seeds on the Reproductive Organs of Female Albino Mice.

ISSN 0976 4852

2010, IJCRR, All Rights Reserved.

Fig. 1: treated with vehicle showing normal wellorganized hepatic cords with parenchymatous hepatocytes and blood vessels (x
100).

Fig. 3: treated with 0.1% Tween-80 showing

normal folliculogenesis (x 400).

Fig. 2: treated with petroleum ether extract of Citrus medica seeds showing normal histological organization of similar to that of
control (x 100).

Fig. 4: treated with petroleum ether extract of

Citrus medica seeds showing degenerative changes in follicles and
stroma (x 400).

CV: Central Vein; A: Antrum; HS: Hepatic Sinusoids; O: Oocyte; ILV: Inter Lobular Vein; N: Nucleus; GL: Granulosa Layer; HF:
Healthy Follicle; ZP: Zona Pellucida; GF: Graafian Follicle; CO: Cumulus Oophorus; CR: Corona Radiata; TI: Theca Interna; GC:
Granulosa Cells

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Research

Patil SJ et al., 2010: Pre-Clinical Toxicity Studies of Orally Administered Petroleum Ether Extract of
Citrus medica seeds on the Reproductive Organs of Female Albino Mice.

ISSN 0976 4852

2010, IJCRR, All Rights Reserved.

Fig. 5: treated with vehicle showing normal

endometrium with endometrial glands
and luminal epithelial cells (x 100).

Fig. 6: treated with petroleum ether extract of

Citrus medica seeds showing hypertrophied endometrium and luminal epithe-

Fig. 7: treated with 0.1% Tween-80 showing

normal cortex and medulla (x 100).

Fig. 8: treated with petroleum ether extract of

Citrus medica seeds showing hypertrophied cortex and medulla (x 100).

L: Lumen; C: Cortex/Capsule; LE: Luminal Epithelium; M: Medulla; EM: Endometrium; ZR: Zona Reticularis; USG: Uterine Secretory Gland; ZF: Zona Fasciculata; ZG: Zona Glomerulosa

55%). The rats were given pelleted feed

(Hindustan Unilever Limited, India) and
water ad libitum. The experimental protocol was approved by the Animal Ethical
Committee in accordance with the Guidelines for care and use of Laboratory Animals prepared by the Institutional animal
Ethics committee (NIH publication No:
85-88, 1985).
Experimental Design
Toxic effects of the extracts of the plant
Citrus medica seeds was studied order to

know the toxic constituents present in the

extract.
In any screening programme, the toxicity
tests on mice are usually performed first.
In the measurement of acute and chronic
toxicity5 of graded doses (200 and
400mg/kg body weight) of petroleum
ether extract of C. medica seeds obtained
as described were prepared in required
dose in Tween-80 (1%) and suspended in
distilled water. The mice as mentioned
earlier were taken and divided into three

groups each group containing six animals.

The solution of extracts obtained as above
was administered to mice orally by means
of intragastric catheter.
Group-I: control, received 0.21ml Tween80 (1%) orally.
Group II: received 200mg/kg body weight
of petroleum ether extract in 0.2ml
Tween-80(1%) orally.

August, 2010|Volume 01|Issue 03|

Table: 1: Effect of petroleum ether extract of Citrus medica seeds on toxicity of Swiss albino mice.

No. of animals sur- Organ weight (mg/100g body weight)

vived
Liver
Kidney
(%)

Group

Treatment

Dose (mg/kg body

wt.)

Initial body
weight (g)

Final body weight

(g)

Adrenal

Ovary

Uterus

Control

Tween-80 (1%)

27.20.86

29.00.31

6/6 (100%)

5.930.33

0.790.05

0.020.005

0.100.012

384.2056.87

200

27.60.92

28.80.58

6/6 (100%)

5.760.37

0.820.04

0.020.004

0.130.006

384.2061.85

27.00.92

28.60.51

6/6 (100%)

5.120.26

0.840.06

0.030.004

0.110.354

386.2042.34

II
Petroleum ether extract
III

400

MS.E. = Mean Standard Error; Duration: 15 days; Six animals were maintained in each group

Table: 2: Haematological study of petroleum ether extract of C. medica seeds for toxicity of normal Swiss albino mice.
Haematological parameters

Dose
Group

Treatment

(mg/kg
body wt.)

Total differential count (%)

Control

Tween-80 (1%)

Haemoglobin (mg/
100 ml)
10.330.110

II

Petroleum ether
extract

200

10.100.157

5.130.068

5.490.296**

32.631.281***

67.251.221*

0.000

0.130.125

0.000

400

10.480.080

5.640.141

4.400.282

23.881.837*

76.01.732

0.000

0.000

0.000

III

RBC (106/mm)

WBC (10/mm)

Neutrophils

Lymphocytes

Monocytes

Eosinophils

Basophils

5.330.103

4.160.380

19.750.818

78.750.881

0.000

0.250.164

0.000

MS.E. = Mean Standard Error; Duration: 15 days; Six animals were maintained in each group; *P<0.05; **P<0.01: ***P<0.001 when compared with control

Table: 3: Serum biochemical studies of petroleum ether extract of C. medica seeds for toxicity in Swiss albino mice.
Group
I
II
III

Treatment
Control

Petroleum ether extract

Biochemical parameters

Dose
(mg/kg body wt.)

Blood Sugar (mg/100ml)

Serum Cholesterol (mg/100ml)

Total Protein (mg/100ml)

Blood Urea Nitrogen (mg/100ml)

Tween-80 (1%)

111.364.363

63.011.882

6.390.052

17.761.015

200

112.452.928

62.011.054

5.850.098

16.930.541

400

114.085.493

63.081.772

5.080.132

17.690.422

MS.E. = Mean Standard Error; Duration: 15 days; Six animals were maintained in each group

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Research

Patil SJ et al., 2010: Pre-Clinical Toxicity Studies of Orally Administered Petroleum Ether Extract of
Citrus medica seeds on the Reproductive Organs of Female Albino Mice.

ISSN 0976 4852

Group III: received 400mg/kg body

weight of petroleum ether extract in 0.2ml
Tween-80(1%) orally.
The treatments were given every morning
between 9:00 AM to 10:00AM for 15
days. The weight of each animal was monitored daily during experimental period
and also a week after quitting the experiment.
The death due to high toxicity of the constituents present in the extract and
changes in the gain/loss of weight were
observed. The morphological and behavioural changes in the animal due to toxic
effect of the constituents of the extracts
were also observed. After 15th day all the
animals were sacrificed by cervical dislocation. The blood was collected for the
detection of haematotoxicity, haemoglobin concentration, total RBC, WBC
counts and total differential counts were
examined6. The liver, kidney, adrenals,
ovary and uterus were dissected out immediately and separated out from the adherent connective tissue and a weighed to
the nearest mg on an electronic balance.
The biochemical and histological changes
in the liver, ovary, uterus and adrenals
were also studied.
Statistical Analysis:
The data were statistically analyzed and
expressed as mean S.E. Statistical analysis of the variance between control and
experimental values was done using Students-t test using SPSS package7.
Results
General symptoms, body weight & organ weight (Table: 1)
Over the study duration of 15 days, in
mice did not show any external manifestations of toxic symptom, behavioural patterns like intake of food and water and
morality even after the completion of
crude extract treatment. It did not produce
any statistically significant changes in the
body weight. There was no gross change
in their appearance, colour and size. It
was found that, there was no statistically
significant difference in organ weight of
both treated and control groups of both

the doses of petroleum ether extract of

Citrus medica seeds administration.
Haemoglobin studies (Table: 2)
Haemoglobin Content: Haemoglobin
content of the mice treated with petroleum
ether extract of C. medica seeds at 200mg
and 400mg/kg body weight dose level
remained constant when compared to control.
RBC and WBC Count: the extract did
not vary the number of RBC and WBC
significantly at both the dose level, similarly there was no significant difference in
WBC count at 400mg/kg body weight,
whereas, the 200mg/kg body weight
showed slight increase in the number of
WBC significantly (P<0.01) due to the
administration of petroleum ether, when
compared to control.
Total Differential Leukocyte count:
Oral administration of petroleum ether
extract at both the dose level significantly
increased (P<0.05 to P<0.001) to the
number of neutrophills and slightly decreased the number of (P<0.05) lymphocytes. Non significant changes were seen
in the number of monocytes, eosinophills
and basophills.
Serum Biochemical studies (Table: 3)
The treatment of petroleum ether extract
at 200mg and 400mg/kg body weight did
not shown significant alteration in the
blood sugar and serum cholesterol level.
The total protein content was significantly
reduced and urea nitrogen content of
blood was also non significant variation
when compared to control.
Histophathological studies (Figure: 1-8)
Histophathological studies of liver, adrenal, ovary and uterus did not display any
major deformation in the petroleum ether
extract of C. medica seeds administration.
Liver major drug metabolic site has
shown normal histology, it indicates that
the extract was non hepatotoxic, as the
arrangement of cells could be very clearly
observed without any cell destruction/cell
proliferation. Histological studies of
adrenal glands showed hypertrophy but no
significant toxic effect with both the doses
of petroleum ether extract. Reproductive
organs like ovarian histology slightly degenerated and uterine histology shown
hypertrophy of endometrium and luminal
epithelium and did not shown major toxicity due to petroleum ether extract of C.

2010, IJCRR, All Rights Reserved.

medica seeds administration when compared to control.

Discussion
In the study on mice, petroleum ether extract of Citrus medica seeds at 200 and
400 mg/kg body weight for 15 days administration could be considered safe,
although safety studies in animal models
could be often be extrapolated to humans8. It does not necessarily follow that
such products would be completely safe
in humans; therefore, actual controlled
clinical trials on human subjects would be
more definitive evidence on safety.
In this study, a scientific assessment of a
highly patronized herbal preparation marketed and used in all the parts of world
was undertaken. The results of this study
show some level of efficacy and safety,
which are related to some of the claimed
indications of the phytomedicine. However, no abnormalities were observed in the
external morphologies, internal organs
weights like liver, adrenals, ovary and
uterus due to the administration of both
the doses of petroleum ether extract of C.
medica seeds, which was in agreement
with previous reports concerning other
Petroleum ether
phytoconstituents9-10.
extract of C. medica seeds administration
to the female rats did not alter the general
body metabolism of the test animals as
revealed by the data obtained after examination of the clinical toxicity studies i.e.,
haematological and serum biochemical
profiles as the values of all the parameters
studied were within the normal range.
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Soufir JC, Radique C, Dantec MC,

Garner D, Jegou B. Gossypolinduced modifications in the microenvironment of rat epiidymal spermatozoa. Journal of Reproduction
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Kimmel GL, Clegg E, Crisp TM.

Reproductive toxicity testing: a risk
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RJ, editor, Reproductive Toxicology,
2nd Ed., New York: Revan Press,
1995, 75-98.

3.

Kirtikar KR, Basu AD. In: Indian

Medicinal Plants. Vol. I, Lalit Mohan Basu, Allahabad, 1935, 53,
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Patil SJ et al., 2010: Pre-Clinical Toxicity Studies of Orally Administered Petroleum Ether Extract of
Citrus medica seeds on the Reproductive Organs of Female Albino Mice.

ISSN 0976 4852

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10. sessment of fertility and reproductive toxicity in adult female mice after long term exposure to Pueraria
mirifica herb. Journal of Reproduction and Devlopment 2007; 53: 9951005.
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