Vom Journal of Veterinary Science Vol. 10,2015: 65 - 74
PHYTOCHEMICAL, ELEMENTAL AND ACUTE TOXIC EFFECTS OF METHANOL EXTRACT OF
ONION (ALLIUM CEPA) BULBS IN WISTA ALBINO RATS EXPERIMENTALY PRE-EXPOSED AND
RESTED FOR A WEEK
*1
1
2
J, A. OYEWUSI,.2A. B. SABA, 1J. O. OLUKUNLE
College of Veterinary Medicine, Federal University of Agriculture, Abeokuta, Nigeria
Faculty of Veterinary Medicine, Department of Veterinary Physiology and Pharmacology,
University of Ibadan, Nigeria
*Corresponding author: E-mail: [email protected]: 08036676864
SUMMARY
The acute toxic effects following oral pre-exposure, rested for a week and intraperitoneal
administration of methanol extract of onion (Allium cepa L.) in wista albino ratswas studied.
Phytochemical and Elemental analysis of the onion extract was also studied. Eighteen rats
were randomly distributed into 6 groups of 3 rats per group. 300mg/kg, 600mg/kg,
1200mg/kg, 2400mg/kg and 4800mg/kg was administered orally to each rat in groups 1, 2,
3, 4 and 5 respectively. Group 6 (the control group), received no extract. The same dosage
of the same extract was administeredintraperitoneally to the same groups of rats after a
weekof rest was observed from the oral doses. The extract of Allium cepa was screened for
the presence of phytochemical compounds using standard methods as described by
Sofowora, 1993.The elemental analysis of the plant was done using atomic
spectrophotometer.There was no mortality among any of the groups of rats during both
oral and intraperitoneal toxicity tests. The rats significantly (p<0.05) increased in weight
within the one week of rest. However, some behavioural abnormalities such as anorexia,
depression, and unsteady gait were observed. Respiratory depression and muscular rigidity
were equally observed. All of these abnormal observations resolved within 24 hours.
Phytochemical analysis revealed presence of Alkaloid,Flavonoid, Tannin, Saponins,
Glycosides, Cadenolide, Phlobatanins, Oxalate, Phytate and Trypsin-inhibitor. Elemental
analysis revealed presence of P, Na, K, Mg, Ca and Fe.In conclusion,methanolic extract of
onion (Allium cepa) is generally safe but safer when administered orally.
Key words: Elemental, Onion, Phytochemicals, Rats, Safety margin, Toxicity.
INTRODUCTION
onion (Allium cepa L.) possess some
medicinal (health) benefits. Such benefits
include: hypoglyceamic, antidiabetic,
cardiovascular
risk
reduction,
hypolipideamic, cancer risk reduction and
antimicrobial effects.
The onion (Allium cepa) is a bulbous herb
belonging to the family Allicea and is
commercially cultivated worldwide (WHO,
1999). It is the most cultivated specie of
the genus Allium (Eric, 2010). Numerous
research publications have shown that
65
Oyewusi et al: Phytochemical, Elemental and Acute Toxic Effects of Methanol Extract of Onion
Generally, it is believed that onion is
potentially safe when consumed in
various forms by humans. However, apart
from been toxic to dogs and cats, some
handling and consumption side effects
such as allergy, anaphylactic reaction,
intestinal gas and heart burns have been
noticed in humans (Anita, 2011). Some of
these
unpleasant
occurrences
or
observations may be due the presence of
one or more of the phytochemical
contents of the plant under study.
This work was designed to investigate
phytochemical, elemental contents and
the acute toxic effects of methanol extract
of onion (Allium cepa) following oral and
intraperitonium administrations of graded
doses in wista albino rats.
MATERIALS AND METHODS
collection of plant and preparation of
extract
were transferred to the experimental
animal unit of the College of Veterinary
Medicine, Federal University of Abeokuta,
Ogun State, Nigeria.
EXPERIMENTAL PROCEDURE
Eighteen wista albino rats of both sexes
were randomly divided into 6 groups of 3
rats per group. The rats were fed on
standard growers ration obtained from
Vital Feed Nig. LTD. They were supplied
with clean water and feed adlibitum. The
groups of rats were treated with graded
doses of the methanolic onion bulbs
extract. Groups 1, 2, 3, 4 and 5 were
treated orally with 300mg/kg, 600mg/kg,
1200mg/kg, 2400mg/kg and 4800mg/kg
body weight respectively. Group 6 rats
served as control and were not given the
extract. The rats were observed for signs
of toxicity such as behavioral changes and
deathfor 48 hours.
Fresh onion bulbs were purchased from a
local onion market in Abeokuta, Ogun
state, Nigeria. The dry coverings of the
onion bulbs were clean peeled off the
bulbs. The peeled bulbs were weighed on
a laboratory bench weighing scale. The
peeled and weighed onion bulbs were
macerated into tiny pieces with a sharp
knife. Two kg of macerated onion bulbs
was soaked in 3.1 liters of Kermel
methanol for 72hours. The soaked onion
was filtered and the resultant extract
solution was concentrated on a water
bath by evaporation at 65oC.The brownish
black (honey-like) pasty substance left
behind was kept in the refrigerator (40C)
as onion methanolic extract until it is
needed for the study.
The same groups of rats were allowed to
rest for 1 week during which they were
placed under close observation. Each rat
in the six groups was weighed again so as
to determine the new dosage of the
extract that will be given each rat. Fresh
solution of the onion methanolic extract
was prepared. Rats in groups 1, 2, 3, 4 and
5 were treated intraperitoneally (Ip) with
300mg/kg,
600mg/kg,
1200mg/kg,
2400mg/kg and 4800mg/kg body weight
respectively. Group 6 rats served as
control and were not given the extract.
The rats were observed for signs of
toxicity such as behavioral changes and
deathfor72 hours in the first instance and
then for 14 days. The Arithmetic Method
of Karbar (Dede and Dogara, 2004) was
used for the calculation of the LD50.
ANIMALS
Eighteen wista albino rats weighing
between 130g and 203g body weight
were obtained from a reputable breeder
in Abeokuta, Ogun state, Nigeria. They
Phytochemical Analysis
The extract of Allium cepa was screened
for the presence of chemical compounds
as(secondary metabolites) described by
Odebiyiand Sofowora, 1978; Sofowora,
Vom Journal of Veterinary Science Vol. 10,2015: 65 - 74
1993. The phytochemical analysis was
carried out at the College of Natural
Sciences,
Federal
University
of
Agriculture, Abeokuta.
also form just gradually throughout the
layer.
Test for Saponins
0.5g of the onion extract was placed in a
test tube, 5cm3 of water was added and
thenshaken vigorously. A persistent froth
that lasted for at least 15 minutes was
taken as preliminary evidence for the
presence of saponins. Few drops of Olive
oil were added to 0.5g of the extract and
shaken vigorously. Formation of soluble
emulsion in the extract indicated the
presence of Saponins (Sofowora and
Odebiyi, 1978)
Test for Tannins
2.0cm3 of the onion extract was diluted
with distilled water in a test tube. 2 – 3
drops of 5% ferric chloride solution was
added. A green black or blue – black
colouration indicated the presence of
tannins.
Test for flavonoids
0.5g of the onion extract was shaken with
petroleum ether to remove the fatty
materials (lipid layer). The defatted
residue was dissolved in 20cm3 of 80%
ethanol and filtered. 3cm3 of the filtrate
was mixed with 4cm3 of 1% potassium
hydroxide in a test tube. A dark yellow
colour indicated the presence of
flavonoids.
Test for phlobatannins
About 2 ml of the onion extract was
added to 2 ml of 1% HCl and the mixture
was boiled. Deposition of a red precipitate
was taken as an evidence for the presence
of phlobatannins.
Elemental Analysis
The elemental analysis of the plant was
done using atomic spectrophotometer
(210 VGP BUCK, scientific, UK) at the
Central Laboratory, Biotechnology Center
of the Federal University of Agriculture,
Abeokuta, Nigeria.
Test for Alkaloids
10.0cm3 of the onion extract was placed
in 2 separate test tubes. 2 -3 drops of
Dragendoff’s and Mayer’s reagents were
separately added into the test tubes. An
orange red precipitate/turbidity with
Dragendoff’s or white precipitate with
Mayer’s would denote the presence of
alkaloids.
Statistical Analysis
Data generated from this study were
presented as the(mean ±SD). The
difference between the means inthe
treated groups and in the untreated
groups werecompared by the one way
analysis of variance(ANOVA) using the
Prism Graphpad Statisticsoftware (Prism
5).
Test for cardiac glycosides / Cadenolides
(Keller Killiani’s test)
5cm3 of the onion extract was mixed with
2cm3 of glacial acetic acid containing one
drop of ferric chloride (FeCl3) solution,
followed by the addition of 1cm3
concentrated
sulphuricacid
(H2SO4).
Brown ring was formed at the interface
which indicated the presence of de-oxy
sugar of cardenoloides. A violet ring may
appear beneath the brown ring, while in
the acetic acid layer, a greenish ring may
RESULTS
The average weight of the rats in each
group significantly increased (p<0.05)
within the one week interval between the
oral toxicity test and the intraperitoneal
toxicity test (Table 1& figure 1).
67
Oyewusi et al: Phytochemical, Elemental and Acute Toxic Effects of Methanol Extract of Onion
There was no mortality and there was no
observable sign of toxicity in any of the
rats in groups 1, 2 and 3 during the oral
toxicity test. In groups 4 and 5
(2400mg/kg and 4800mg/kg), 2/3 and 3/3
of the rats lost appetite within the first 1
hour post oral administration of the onion
methanolic extract (Table 2).
All the rats in the control group and all
the rats in groups 1, 2 and 3 started eating
immediately they were served. No rat in
any of the 5 treated groups died or
manifests any sign of toxicity within the
one week of rest preceding the
intraperitonium toxicity test.
In the intraperitoneal treatment with
onion methanolic extract, no mortality
was recorded in any of the treated and
the control groups throughout the period
of the experiment (Table 3). However,
various degrees of dose dependent
behavioural abnormalities were observed
and recorded in each of the treated
groups.
Behavioural abnormalities such as loss of
appetite (anorexia), depression, unsteady
gait, clustering, folding and sleeping. In
addition to the behavioral changes,
respiratory distress, muscular rigidity and
apparent partial paralysis were observed
(Table 3). The result of the Determination
of acute toxicity (LD50) of Allium cepa
(Onion) in Rats using Karber Method is
seen in table 4.The Arithmetic Method of
Karbar (Dede and Dogara, 2004) was used
for the calculation. The results of the
phytochemical and elemental analyses are
as presented in Table 5.
TABLE 1: Weight (g) Change in Rats during 1 Week Rest before i/p Administration.
DAYS OF GROUP 1
EXPOSU
RE
GROUP 2
GROUP 3
GROUP 4
GROUP 5
WEEK 1
138.33±32.
63a
129.17±19.
09a
138.33±15.
28a
129.17±19.
09a
111.83±3.5 148.3±22.
5a
95a
WEEK 2
167.67±31.
51b
151.83±18.
91b
160.83±16.
07b
155.00±10.
10b
138.50±3.7 158.5±20.
8b
43b
Values with different superscriptions differ significantly (p<0.05)
GROUP 6
Vom Journal of Veterinary Science Vol. 10,2015: 65 - 74
Figure 1
Table 2: Occurrence of Toxicological Signs Following Oral Administration of Methanolic
Extract of Allium cepa
Grou N Grou
p
p
dosa
ge
Anorex
ial
Depressi Unstea
on
dy gait
Clusteri Sleepi
ng
ng
Respirat
ory
distress
Partial
paraly
sis
Muscu Deat
lar
h
rigidit
y
1
3 300
-
-
-
-
-
-
-
-
0
2
3 600
-
-
-
-
-
-
-
-
0
3
3 1200
-
-
-
-
-
-
-
-
0
4
3 2400
2/3 for 1hr.
-
-
-
-
-
-
0
5
3 4800
3/3 for 1hr.
-
-
-
-
-
-
0
6
3 contr
ol
-
-
-
-
-
-
-
0
-
Numerator – total number affected. Denominator – total number in the group
69
Oyewusi et al: Phytochemical, Elemental and Acute Toxic Effects of Methanol Extract of Onion
Table 3: Occurrence of Toxicological Signs Following Intraperitonium (i/p) Administration of
Methanolic Extract of Allium cepa
Gro
up
N Gro Anore
up
xial
dosa
ge
1
3 300
2
Depres
sion
Unste
ady
gait
Cluste
ring
Sleep
ing
Respira Parti
tory
al
distress paral
ysis
Musc
ular
rigidit
y
Dea
th
3/3
3/3
for 3
hours
-
3/3
3/3
-
-
-
0
3 600
3/3
3/3
for 3
hours
-
3/3
3/3
-
-
-
0
3
3 120
0
3/3
3/3
for 3
hours
-
3/3
3/3
-
-
-
0
4
3 240
0
3/3
3/3
for 7
hours
3/3
-
-
3/3
-
-
0
5
3 480
0
3/3
3/3
for 10
hours
3/3
3/3
-
3/3
3/3hi
nd
limbs
3/3
0
6
3 cont
r
-
-
-
-
-
-
-
0
-
Numerator – total number affected. Denominator – total number in the group
Vom Journal of Veterinary Science Vol. 10,2015: 65 - 74
Table 4. Determination of acute toxicity (LD50) of Allium cepa (Onion) in Rats using Karber
Method
Dose (mg/kg)
Dose
(Dd)
difference No. of Death Mean
(Nd)
(Md)
death Dose difference –
Mean death (Dd Md).
300
0
0
0
0
600
300
0
0
0
1200
600
0
0
0
2400
1200
0
0
0
4800
2400
0
0
0
Control
0
0
0
0
Md = 0, ∑(Md x Dd) = 0, n = 3.
LD50 = Highest Dose - ∑(Md x Dd)/n.
Therefore, LD50 = 4800 – 0 = 4800 mg/kg.
The highest doserepresent the dose that will kill 100%. In this case it is more than 4800
mg/kg.
Table 5. Showing the results of Phytochemical, Elemental and Proximate analysis of raw
onion bulb
Phytochemical Analysis
Chemical
Alkaloid
Flavonoid
Tannin
Saponins
Glycosides
Steroid
Cadenolide
Phlobatanins
Anthraquinone
Phenol
Oxalate
Phytate
Trypsin-inhibitor
Elemental Analysis
Element
Value (mg/g)
P
0.231
Na
0.146
K
2.102
Mg
0.094
Ca
0.312
Mn
Nil
Pb
Nil
Cd
Nil
Zn
Nil
Fe
0.006
Cu
Nil
Co
Nil
Value (%)
0.96
1.12
1.45
1.07
0.56
Nil
0.12
0.76
Nil
Nil
1.23
0.34
0.19
71
Oyewusi et al: Phytochemical, Elemental and Acute Toxic Effects of Methanol Extract of Onion
DISCUSSION
The result of the oral acute toxicity test
with onion methanolic extract shows that
onion has a very wide safety margin when
administered orally even at as high as
4800mg/kg.The results LD50 calculation
using Arithmetic Method of Karbar (Dede
and Dogara, 2004) indicated that the
methanolic extract of Allium cepa had
LD50>4800 mg/kg body weight.
This finding is supported by Clarke &
Clarke (1977), any substance whose
LD50is above 1000mg/kg body weight is
considered
safe.
Similarly,
the
Organization for Economic Cooperation
and Development (OECD Paris, France)
recommended
that
any
chemical
substance with LD50 more than
3000mg/kg is considered safe. The OECD
(Walum, 1998) also recommended the
chemical labeling and classification of
acute systemic toxicity based on oral LD50
values as follow: <5mg/kg (very toxic), >5<50mg/kg
(toxic),
>50-<500mg/kg
(harmful)
and
>500-<2000kg
(no
labelling). Also, according to the toxicity
scale of Hodge &Sterner, any compound
with an oral LD50 of between 5005000mg/kg body weight should be
considered practically nontoxic (Hodge &
Sterner Scale, 2005).
There was equally no mortality during
thei/p toxicity test. Again, corroborating
the safety margin of the plant as indicated
above. The fact that all the rats increased
in weight within a week between the two
tests showed that there was no residual
effect of the oral treatment that was
given previously. The dose dependent
behavioral abnormalities could be due to
the effect of one or more of the active
principles (phytochemicals) of the plant
which increases in concentration with
increase in dosage of the methanol
extract of onion across the groups.
This study of methanol extract of Onion
(Allioncepa) revealed the presence of
alkaloid flavonoid, tannin, saponin,
glycosides, cadenolide, phlobatannin,
oxalate, phytate and trypsin-inhibitor.
Phytochemicals are biologically active
compounds plants that when ingested,
have the potential to prevent or delay the
onset of disease (Guhr & Lachance, 1997).
Each of these detected phytochemical
compounds is known to have some
beneficial importance in industrial and
medicinal sciences (Aiyelaagbe & Paul,
2009). Published records abound on the
medicinal and industrial benefits of many
of the detected phytochemicals in Allium
cepaL.Alkaloids are produced by a large
variety of organisms, including bacteria,
fungi, plants, and animals, and are part of
the group of natural products (also called
secondary metabolites). Many alkaloids
can be purified from crude extracts by
acid-base extraction. Many alkaloids are
toxic to other organisms. They often have
pharmacological effects and are used as
medications, as recreational drugs, or in
entheogenic rituals (HSW, 2009).
One of the richest sources of flavonoids in
human diet is onion (Allium cepaL.,
Liliaceae) (Lachmanet al, 2003).Flavonoids
and their polymers constitute a large class
of food constituents, many of which alter
metabolic processes and have a positive
impact on health. They generally consist
of two aromatic rings, each containing at
least one hydroxyl, which are connected
through a three-carbon “bridge” and
become part of a six-member heterocyclic
ring
(Gary,
2003).
Polyphenolic
compounds, especially flavonoids are
effective antioxidants due to their
capability to scavenge free radicals of
fatty acids and oxygen. Flavonoids have
been referred to as nature’s biological
response modifiers because of strong
Vom Journal of Veterinary Science Vol. 10,2015: 65 - 74
experimental evidence of their inherent
ability to modify the body’s reaction to
allergies, virus and carcinogens. They
show anti-allergic, anti-inflammatory,
anti-microbial and anti-cancer activity
(Aiyelaagbe&Osamudiamen, 2009).
for
use
(Wikipedia, 2013).Cardiac
glycosides are known to work by inhibiting
the Na+/K+ pump. This causes an increase
in the level of sodium ions in the
myocytes, which then lead to a rise in the
level of calcium ions. This inhibition
increases the amount of Ca2+ ions
available for contraction of the heart
muscle, which improves cardiac output
and reduces distention of the heart; thus,
they are used in the treatment of
congestive heart failure and cardiac
arrhythmia. They are also, used to
strengthen a weakened heart and allow it
to function more efficiently, though the
dosage must be controlled carefully, since
the therapeutic dose is close to the toxic
dose (Denwick, 2002).
Tannin is also defined as any phenolic
compound of sufficiently high molecular
weight containing sufficient hydroxyls and
other suitable groups (i.e. carboxyls) to
form effectively strong complexes with
protein and other macromolecules under
the particular environmental conditions
being studied (Horvath, 1981). The tannin
compounds are widely distributed in
many species of plants, where they play a
role in protection from predation, and
perhaps also as pesticides, and in plant
growth regulation (Ferrell & Thorington,
2006). Flavonoids and tannins, has been
severally fingered as anti-inflammatory
and analgesic active principles in
medicinal plants (Terashima et al., 2002;
Musa et al, 2008)
In view of these documented benefits of
the phytochemicals detected in Onion
(Allium cepaL) and the results of the acute
toxicity tests carried out in this work, it is
therefore concluded that methanol
extract of onion (Allium cepaL) is generally
safe but safer when administered orally.
However, there is need for further studies
on the plant to investigate the cause(s) of
the
dose
dependent
behavioral
abnormalities observed.
Saponins occur widely in plant species and
exhibit a range of biological properties,
both beneficial and deleterious (Price et
al, 1987)Saponin is used as a mild
detergent
and
in
intracellular
histochemistrytrolaemia, hyperglycaemia,
antioxidant,
anti-cancer,
antiinflammatory and weight loss etc. It is also
known to have anti-fungal properties
(Aiyelaagbe&Osamudiamen, 2009).
References
1.
Aiyelaagbe OO & Paul MO (2009).
Phytochemical Screening for Active Compounds in
Mangiferaindica Leaves from Ibadan, Oyo State.
Plant Sciences Research2 (1) 11-13
In chemistry, a glycosideis a molecule in
which sugar is bound to another
functional group via a glycosidic bond.
Glycosides play numerous important roles
in living organisms. Many plants store
chemicals in the form of inactive
glycosides. These can be activated by
enzyme
hydrolysis
(Brito-Arias,
2007)which causes the sugar part to be
broken off making the chemical available
2.
Anita C (2011) Side Effects of Eating
Onions. The livestrong foundation. livestrong.com.
3.
Brito-Arias M (2007). Synthesis and
Characterization of Glycosides. Springer. ISBN 9780-387-26251-2.
In:
Glycosides:Wikipedia,
Wikimedia Foundation, Inc., 2013
4.
Clarke EGC & Clarke ML (1977).
Veterinary Toxicology 1st Edition Bailiere Tindall
Ltd, London Pp10
73
Oyewusi et al: Phytochemical, Elemental and Acute Toxic Effects of Methanol Extract of Onion
5.
Denwick, PM (2002). Natural Products: A
Biosynthetic Approach. 2nd Edition, John Wiley
and Sons, Ltd., England, pp 241-243.
6.
Dede, E.B. and F.M. Dogara, 2004. The
acute toxicological effects of gammalin 20 on the
lung and pancreas of guinea pig. J. Applied Sci.
Environ. Manage. 80: 33-35.
7.
Eric B (2010). Garlic and other Alliums:
The Lore and the Science (Cambridge: Royal
Society of Chemistry, 2010).
8.
Ferrell
EK
&Thorington
RW
(2006). Squirrels: the animal answer guide.
Baltimore: Johns Hopkins University Press.
Pp91.ISBN0-8018-8402-0.
9.
Gary RB (2003). Overview of Dietary
Flavonoids: Nomenclature, Occurrence and Intake.
The Journal of Nutrition © 2003. The American
Society for Nutritional Sciences October 1, 2003
vol. 133 (10) Pp 3248-3254
10.
Guhr G &997). Role of phytochemicals in
chronic disease prevention. In: Nutraceuticals:
designer foods III garlic, soy, and licorice. Food and
Nutrition Press. Trumbull, Conn. Pp 311–364.
11.
Hodge and Sterner Scale (2005). Toxicity
Classes. In Canadian Centre for Occupational
Health and Safety. Copy right © 1997-2010.
http://www.ccohs.ca/oshanswers/ld50.html
04/02/2010.
12.
Horvath PJ (1981). The nutritional and
ecological significance of acer-tannins and related
polyphenols. M.S. Thesis. Cornell University,
Ithaca, NY, USA
13.
Houghton M (2009). SAPONINS. The
American Heritage Medical Dictionary of the
English Language, 2009 by Houghton Mifflin
Company.
14.
HSW (HowStuffWorks) (2009) “Alkaloid”
<http://science.howstuffworks.com/alkaloidinfo.htm.
15.
Lachman (2003) In: Gamal A. Mohamed
(2013) Alliuocide A: a new antioxidant flavonoid
from Allium cepaL. Phytopharmacology, 4(2), Pp
220-227 © 220 2013. In: Foresights Publishing U.K.
16.
Musa YM, Haruna AK, Ilyas M, Yaro AH,
Ahmadu AA, Usman H. (2008). Phytochemical,
Analgesic and Anti-Inflammatory Effects of The
Ethylacetate
Extract
Of
The
Leaves
OfPseudocedrellakotschyii. African Journal of
Traditional, Complementary and Alternative
medicines (AJTCAM). 5(1) 92-96
17.
Odebiyi O. O.&Sofowora EA (1978).
Phytochemical screening of Nigerian medicinal
plants II. Lloydia, 41(3) Pp 234-246.
18.
Sofowora A (1993). Screening Plants for
Bioactive Agents. In: Medicinal Plants and
Traditional Medicine in Africa. 2nd edition.
Spectrum Books Ltd., Sunshine House, Ibadan. Pp.
81-93, 134-156.
19.
Terashima K, Takaya Y, Niwa M. (2002).
Powerful antioxidative agents based on garcinoic
acid from garcinia kola. Bioorganic and Medicinal
Chemistry.10(5) Pp 1619 – 25.
20.
Walum E. 1998. Acute oral toxicity.
Environ. Health Perspect. 106: 497– 503.
21.
WHO. Monographs on selected medicinal
plants. Vol. 1. Geneva: World Health Organization;
1999. Pp 5–15.