Acute Toxicity Study and Phytochemical Screening of

Selected Herbal Aqueous Extract in Broiler Chickens

S.R. Hashemi, I. Zulkifli, M. Hair Bejo, A. Farida and M.N. Somchit

ABSTRACT

In order to collect ethnobotanical information about growth and health promoter plants
as feed additive in broiler chickens, five medicinal plants Euphorbia hirta, Solanum
torvum, Zingiber officinale, Curcuma longa and Zingiber zerumbet used by traditional
medical practitioners for the treatment of several ailments of microbial and non-
microbial origins were investigated for phytochemical screening and acute toxicity
study. A total of 30 female broiler chicks were obtained. At 21 days of age, the chicks
were allocated at random into six groups. Five chickens were assigned at random to
each treatment in five replicates and kept in 30 cages (one chickens per cage) till five
weeks of age. Five groups were administered a single oral dose of 2,000 mg kg -1 b.wt.
while 5 mL distilled water was given to the control group of birds as placebo.
Phytochemical screening study showed that plant contained volatile oils, tannins,
alkaloids, saponins, flavonoids. Alkaloids and steroids were only found in the aqueous
extract of Euphorbia hirta. Tissues were harvested and processed for photomicrographic
examinations. Macro and microscopic observations indicated no alteration in liver and
kidneys of the treated birds with 2000 mg kg -1 of selected herbal plants extract. In the
hematological study, a highly significant decrease was observed in AST, ALT, ALP level of
broiler group receiving the aqueous extract of E. hirta 14 after of administration. Acute
toxicity study indicated that water suspensions of selected herbal aqueous extract are
not toxic when administered by the oral route to experimental birds at 2000 mg kg -1
b.wt. In conclusion, the results obtained in the present study are in agreement to a
certain degree with the traditional uses of the plants estimated as prophylaxis against
various diseases and promote of health.

INTRODUCTION

Antimicrobials have been used as feed supplement for more than 50 years in poultry feed
to enhance the growth performance and to prevent diseases in poultry. Most of the
antibiotic growth promoters take action by modifying the intestinal flora, which are
associated with poor health and reduced performance of animals. However, in recent years
great concern has arisen about the use of antibiotics as growth and health promoters in
poultry feed due to emergence of multiple drug resistant bacteria and antibiotic residue
effects (Wray and Davies, 2000). There is evidence to suggest that herbs, spices and
various plant extracts have appetizing and digestion-stimulating properties and
antimicrobial effects (Alçiçek et al., 2004; Zhang et al., 2005). Plant origin like herbs,
spices and various plant extracts are considered to be natural products that consumers
would have received an increased attention and as possible alternatives to antibiotic
growth promoter in improving broiler performance (Hernandez et al., 2004). Knowledge of
the chemical constituents of herbal plants not only for the discovery of therapeutic agents,
but also for new sources of economic materials is desirable. However, it is important to
establish the chemical constituent and safety of herbal plants before they are useful on
supplement in poultry diet.

Solanum torvum Swartz (Solanaceae) distributed widely in Southeast of Asia. To date,

several alkaloid, steroidal glycosides and long chain hydrocarbons and steroids have been
previously isolated from S. torvum (Arthan et al., 2002). Zingiberaceae is one of the
largest families of the plant kingdom. Traditionally, the rhizome of Zingiber zerumbet are
employed as medicine in relieving stomachache, macerated in alcohol which is regarded as
tonic (Sharifah Sakinah et al., 2007). Zingiber officinale Roscoe (family, Zingiberaceae),
known commonly as ginger. In Ayurveda, ginger is considered as valuable medicine
because of its action as rubefacient, antiasthmatic and stimulant to the gastrointestinal
tract (Bhandari et al., 1998). Curcuma longa L., is belongs to the Zingiberaceae family. The
powdered rhizome of C. longa is considered to be stimulating, carminative, purifying,
antiinflammatory and anthelmintic. Externally, the rhizome mixed with alum lime and
saltpetre is applied as a paste to wounds, bruises, inflamed joints and sprains (Araujo and
Leon, 2001; Khattak et al., 2005).

Euphorbia hirta belongs to the family Euphorbiaceae. The plant contains relatively
abundant white latex. The latex is capable of causing dermatitis. The plant has been used
in traditional medicine for the treatment of cough, coryza, asthma, bronchial affections
(Adedapo et al., 2005; Hore et al., 2006).

As no information exists in the alternative, the objective of the present study was to
investigate the safety of Euphorbia hirta, Solanum torvum, Zingiber officinale, Curcuma
longa and Zingiber zerumbet aqueous extract as a dietary additive in poultry diets.

MATERIALS AND METHODS

Plant materials: Whole plant of Euphorbia hirta, edible fruit of Solanum torvum and
rhizomes of Zingiber officinale, Curcuma longa and Zingiber zerumbet were tested for
phytochemical screening and acute toxicity study. Fresh Euphorbia hirta was collected in
April 2007 from the agriculture gardens in Universiti Putra Malaysia and other plants were
obtained from a local market. The plant specimens were identified and authenticated by
the Institute of Bioscience, Universiti Putra Malaysia.

Preparation of the extract: Fresh parts of the plant were cleaned, cut into bits and
rinsed with distilled water. The plants were air dried at room temperature for 24 h, then
oven-dried at temperature of 50°C till they attained a constant weight. The dried plants
were then powdered. In order to obtain the plants extracts, 100 g of each dried plant
material powder was placed in a flask and distilled water was added (1:10 w/v). The flasks
were incubated in a shaking-water bath at 50°C for 48 h and the obtained extracts were
filtered using cotton wool and finally filter papers (Whatman 7 No. 1). The flow-through t
were stored in a deep freezer at -80°C overnight and then subjected to freeze drying
(Jouan LP3, France) at -50°C; 0.2 mbar for 48 h to obtain water-free extracts. Extracts
were then weighed and stored at -20°C till further use.

Phytochemical screening: A qualitative phytochemical test was carried out to detect the
presence of volatile oils, alkaloids, tannins, saponins, flavonoids, glycosides, steroids,
terpenoids and phenols utilizing standard methods of analysis (Sofowora, 1993; Evans,
1996; Haughton and Raman, 1998). The intensity of the coloration determines the
abundance of the compound present. Qualitative phytochemical analysis of the powder of
the five plants were determined as follows: for tannins one g plant grinded, then sample
was boiled in 20 mL ethanol 70% for 2 min on a hot plate. The mixture was filtered and a
portion of the filtrate diluted with sterile distilled water in a ratio of 1:4 and 3 drop of 10%
ferric chloride solution added. Blue-black precipitate indicated the presence of tannins. For
phenol 2 mL of extract was added to 2 mL of ferric chloride solution (FeCl 3); a deep bluish
green solution is formed with presence of phenols. The test for alkaloids was carried out by
subjecting 5 g ground plant material extracted with 10 mL ammoniacal chloroform and 5
mL chloroform. After filtration, the solution was shaken with 10 drops aqueous sulphuric
acid 0.5 M. Creamish precipitate indicated the presence of respective alkaloids. For
steroids Liebermann-Burchard reaction was applied. Two hundred milligram plant material
boiled in 10 mL chloroform and the mixture was filtered; a 2 mL filtrate was added to 2 mL
acetic anhydride and concentrated H2SO4. Blue-green ring indicated the presence of
steroids and red color indicated the presence of terpenoids. The alcoholic extract (15 mL,
corresponding to 3 g of plant material) was treated with a few drops of concentrated HCl
and magnesium Ribbon (0.5 g). Pink-tomato red color indicated the presence of flavonoids.
Froth test for saponins was used. The test for saponin was carried out by subjecting 5 g of
the plant powder extracted with 15 mL methanol. After evaporation, residue was shaken
vigorously with ethyl ether and 5 mL HCl 2N. Precipitate indicated the presence of saponin.
For detection of volatile oils, 1 g fresh plant sample was boiled in 10 mL petroleum ether,
filtered and then 2.0 mL of extract solution was shaken with 0.1 mL dilute sodium
hydroxide and a small quantity of dilute hydrochloric acid. A white precipitate indicated the
presence of volatile oils (Dahiru et al., 2006). The extract was also tested for free
glycoside. Fehling`s solution (A and B) was added to the extract and the solution was
heated on a hot plate and brick-red precipitate indicated the presence of glycosides.

Acute toxicity study: Acute toxicity test was performed according to the World Health
Organization (WHO) guideline (WHO, 2000) and the Organization of Economic Co-operation
and Development (OECD) guideline for testing of chemicals (OECD, 2001). A total of 30
one-day-old commercial female broiler chicks (Cobb 500) were obtained from a local
hatchery. Female broilers were selected because they are generally more sensitive than
males to toxicity study (OECD, 2002). Upon arrival, the chicks were individually wing-
tagged, weighed and assigned in battery cages with wire floors till 21 days. For evaluation
of high doses of aqueous extract of the plant, at 21 days of age, the chickens were
weighed (850±21 g) and randomly divided into 6 groups; each group was further
subdivided into 5 replicates with one chick per cage. One chick was designated as an
experimental unit. Five groups were administered a single oral dose of 2,000 mg kg -1 b.wt.
with aqueous extract of selected plants while distilled water was given to the control group
of broiler as placebo. Birds were fed NRC-type (National Research Council, 1994)
experimental diets from Days 1 to 35 (Table 1). The birds were fed starter diet from 1 to
21 days of age and finisher diet from 22 to 35 days of age. Feed and water were provided
ad libitum. Light was provided for 24 h day-1. All chickens were observed at the first,
second fourth, six hour and once daily thereafter over 14 days [Center for Drug Evaluation
and Research (CDER)] for clinical signs of toxicity.

Histopathological studies: On day 35, all the birds were killed and their livers and kidneys were
removed. Tissues were fixed in 10% neutral-buffered formalin for 24 h, before being processed for
routine paraffin embedding. Tissues were dehydrated with serial ethanol cycles (70% to absolute),
followed by clarification in xylol and then embedded in paraffin. Embedding was carried out with a
paraffin embedding station (EG 1160; Leica). Duplicate slides of each block were obtained.
 Tabl Ingredients and nutrient
e 1: composition of diets

1
Supplied per kilogram of
diet: Vitamin A, 1,500 IU;
Cholecalciferol, 200 IU;
Vitamin E, 10 IU;
Riboflavin, 3.5 mg;
Pantothenic acid, 10 mg;
Niacin, 30 mg; Cobalamin,
10 μg; Choline chloride,
1,000 mg; Biotin, 0.15 mg;
Folic acid, 0.5 mg; Thiamine
1.5 mg; Pyridoxine 3.0 mg;
Iron, 80 mg; Zinc, 40 mg;
Manganese, 60 mg; Iodine,
0.18 mg; Copper, 8 mg;
Selenium, 0.15 mg.
2
Based on NRC (1994) feed
composition table

Slices of 5 μm were produced with a rotation microtome (RM 2155; Leica). Deparaffination
was performed with the following protocol: Xylol 4 min; 100% EtOH 2 min; 90% EtOH 2
min; 70% EtOH 2 min; 60%. Afterwards slices were stained with Mayer hematoxylin and
eosin and mounted with mounting medium (DPX). Sections were then screened under the
light microscope (Leica DM LB2, Leica Microsystems) at low (x5) and high (x40)
magnifications for histopathological study.

Blood sample: Blood samples (4.0 mL) were obtained from each bird for hematological
studies on 0, 7 and 14 days after administration. Plasma was separated by centrifuged at
3000 g for 10 min and stored at -20°C until analysis. The levels of total protein, albumin,
globuline, urea, creatinine, alkaline phosphatase (ALP), serum glutamic-oxaloacetic
transaminase (SGOT or AST) and serum glutamic-pyruvic transaminase (SGPT or ALT)
were measured by specific commercial kits (Roche Diagnostica, Basel, Switzerland) using
an autoanalyzer (HITACHI 902 automatic autoanalyzer).

Statistical analysis: Results were expressed as Mean±Standard error of mean (SEM).

Data were analyzed by one-way ANOVA using the general linear models (GLM) procedure
of SAS (SAS7 Institute, 2005). Multiple means comparisons were accomplished using a
Duncan test. p-values less than 0.05 were considered to be significant.

RESULTS

The results of the phytochemical screening of the investigated aqueous extracts showed
the presence of different types of active constituents like volatile oils, tannins, phenolic,
saponins, terpenoids, steroids, flavonoid and glycosides in Table 2. However, alkaloids and
steroids were only found in the aqueous extract of Euphorbia hirta in this study.

Bird treated with herbal extract at 2000 mg kg-1 b.wt. did not show any immediate
behavioral changes. The birds moved and drank normally immediately after administration.
After 1 h, all the birds showed signs of inappetence for 24 h after the treatment. There was
no mortality till 14 day after administration (Table 3).

Macro and microscopic observations indicated no alteration in the livers and kidneys of the
treated birds with 2000 mg kg-1 of selected herbal plants extract (Fig. 1, 2).

The clinical blood chemistry examination was performed in the female broiler and the
results are shown in Table 4. Serum clinical chemistry showed only a few consistent
changes. The data indicate significant increases in the levels of ALT and AST treated with
the aqueous extract of Zingiber zerumbet at the dose of 2000 mg kg-1 b.wt. after 7 and 14
after administration as compared to the control groups. On the other hand, a highly
significant decrease in AST, ALT and ALP levels among the groups receiving the aqueous
extract of Euphorbia hirta after 14 day of administration was observed. The highest
elevation in serum ALP was observed in Curcuma longa group during the experimental
period. Total protein, albumin, globulin, creatinine and urea levels were not different
among treatment groups 7 days of administration. Following 14 days of administration,
birds given Zingiber zerumbet had the highest blood urea level. Lowest albumin and
creatinine levels were noted for those given Curcuma longa extract.

Ta
bl Phytochemical screening of
e selected herbal plants
2:
-: Absent of phytochemical groups,
+: Present of phytochemical
groups

T Toxicity signs
a observed in broiler
b (first 24 h) that
l received oral single
e dose (2000 mg kg-1)
3 of the aqueous extract
: of herbal plants

-: Absent of
phytochemical groups,
+: Present of
phytochemical groups
Fi Liver histology of untreated
g. and treated broiler with
1: aqueous extract of selected
herbal plants at 2000 mg kg-1
b.wt., C: Control, Eh:
Euphorbia hirta, Zo:
Zingiber officinale, Zz:
Zingiber zerumbet, Cl:
Curcuma longa, St: Solanum
torvum

T
Clinical blood chemistry
a
values of female broilers
bl
administered with aqueous
e
extract of herbal plants1,2
4:
Means within a column with
different letter(s) differ
significantly (p<0.05),
Values are expressed as
Mean±SEM, n = 5, 1: Groups
were administrated orally
with a single dose aqueous
extract at 2000 mg kg-1 b.wt.,
2
: Significantly different
from control, p<0.05
 Fig. Kidney histology of untreated and treated broiler
2: with aqueous extract of selected herbal plants at
2000 mg kg-1 b.wt., C: Control, Eh: Euphorbia
hirta, Zo: Zingiber officinale, Zz: Zingiber
zerumbet, Cl: Curcuma longa, St: Solanum
torvum

The effects of high dose aqueous extract of selected herbal plants on feed intake, body
weight gain feed conversion in broilers are shown in Table 5. Following 7 days of
administration body weight gains of birds that were administered with Zingiber zerumbet
and Zingiber officinale were significantly greater than those of the control groups (p<0.05).
The greatest body weight was observed in the Zingiber zerumbet group at 7 and 14 days
after administration of high dose of aqueous extract. Birds given Zingiber zerumbet had
higher feed consumption throughout the experimental period. At 35 days of age, the
poorest (2.62) feed conversion ratio values were observed in the Zingiber officinale group
(p<0.05) on the other hand, comparatively better (1.84) FCR values were observed in
Solanum torvum group.

Ta The effect of high dose aqueous

bl extract of selected herbal plants
e on feed intake, body weight gain
5: feed conversion in broilers
 Means within a column with
different letter(s) differ significantly
(p<0.05), All data are expressed as
Means±SEM, Broilers were treated
with aqueous extract of selected
herbal plants at 2000 mg kg-1 b.wt.

DISCUSSION

The results of phytochemical screening provide an empirical basis for the use of these
plants in traditional medicinal practices. The biological or therapeutic activities of
medicinal plants are closely related to their chemicals compounds. The phytochemical
screening revealed the presence of alkaloids and steroid in the aqueous extract of
Euphorbia hirta which explains the reason of using Euphorbia hirta in traditional remedies
for treating infectious diseases such as respiratory disease. Phytochemicals have been
reported to have medicinal uses. Specially, saponin has been reported to have
antimicrobial effects and could serve as precursors of steroidal substances with a wide
range of physiological activities (Ojo et al., 2006). Alkaloids, commonly isolated from the
plants, are commonly found to have antimicrobial properties (Omulokoli et al., 1997).
Berberine is an important representative of the alkaloid group. It is potentially effective
against trypanosomas and plasmodia (Freiburghaus et al., 1996; Omulokoli et al., 1997).
Nicholas et al. (2001) screened a variety of marine extracts and identified novel alkaloid
compounds with the ability to inhibit the mycobacterial (Nicholas et al., 2001). The
mechanism of action of highly aromatic planar quaternary alkaloids such as berberine and
harmane (Hopp et al., 1976) is attributed to their ability to intercalate with DNA (Kumar et
al., 2007).

The toxicological evaluation of a plant extract seeks to determine its possible collateral
effects, to ensure the safety of use. Some phytochemicals are able to interfere in the
toxicity of herbal plants. This toxicity can be inherent of the herbal plant products or it
happens during the process of extract preparation (Barros et al., 2005). Alldredge (1993)
attributed reduced feed intake in animals fed tannin-containing diets to strong astringent
property of tannins and induction of internal malaise in mammals, which may contribute to
reduced feed intake in poultry. Increase in feed intake among the Zingiber zerumbet and
Zingiber officinale groups could be due to zingerone`s ability to stimulate catecholamine
secretion from the adrenal medulla (Diepvens et al., 2007). In a review of peripheral and
central control of food intake, Denbow et al. (1989) detailed the effect of catecholamines
and pancreatic polypeptides exert on feed intake regulation, especially in appetite control
centers of the brain. On the other hand, in traditional medicine by Zingiber officinale
(ginger) and Zingiber zerumbet are believed to have weight-loss properties, describing it
as a root that stimulates digestion and speeds up the body processes. Therefore,
catecholamine secretion may be explain for the increased feed intake and feed
conversion ratio during experimental period.
Alterations in blood parameters may be due to changes in cellular integrity, membrane
permeability of cells or even due to exposure to toxic chemicals, (Choudhari and
Deshmukh, 2007). In living systems, liver is considered to be highly sensitive to toxic
agents. The study of different enzyme activities such as AST (SGOT), ALT (SGPT), ALP
(SALP) and total protein have been found to be of great value in the assessment of clinical
and experimental liver damage. Necrosis or membrane damage releases the enzyme into
circulation; therefore, it can be measured in serum. High levels of AST indicate liver
damage, such as that due to viral hepatitis, cardiac infarction and muscle injury. ALT
catalysis the conversion of alanine to pyruvate and glutamate and is released in a similar
manner. Therefore, ALT is more specific to the liver and is thus a better parameter for
detecting liver injury. The rise in the SGOT is usually accompanied by an elevation in the
levels of SGPT, which plays a vital role in the conversion of amino acids to keto acids
(Hoffbrand and Pettit, 1997; Dash et al., 2007). Serum ALP level on the other hand, is
related to the function of hepatic cell. Increase in serum level of ALP is due to increased
synthesis of the enzyme, in presence of increasing biliary pressure (Ojo et al., 2006).

It should be noted that the toxicity dose of selected plants in this study is not clear. The
dose of selected herbal plants used was high, particularly, if the dose is converted to a
corresponding dose in humans (each dose of 2000 mg kg-1 administered in chickens in the
present study would correspond to 140 g in a 70 kg person). No significant toxicity has
been reported following either acute or chronic administration of Zingiber zerumbet,
Zingiber officinale, Solanum torvum and Curcuma longa extracts at standard doses. These
findings are consistent with those Faizah et al. (2002) who found that the extracts of
Zingiber zerumbet were devoid of any mortality or behavioral changes when rats were
given up to 500 mg kg-1 i.p in rats (Faizah et al., 2002). Curcumin is the principal phenolic
yellow pigment in Curcuma longa was given to rats, pigs and monkeys of both sexes at a
dose of 300 mg kg-1 b.wt. No pathological, behavioural abnormalities or lethality was
observed. No adverse effects were observed on both growth and the level of erythrocytes,
leucocytes, blood constituents such as haemoglobin, total serum protein and ALP. Human
clinical trials also indicate that curcumin has no toxicity when administered at doses of 1-8
and 10 g day-1 (Chattopadhyay et al., 2004). In a recent report, up to 12 g of curcumin
was administered in humans without significant toxicity (Poylin et al., 2008). There has not
been any documentation on Solanum torvum toxicity (Israf et al., 2004).

This finding is in agreement with Ogueke et al. (2007) which showed that Euphorbia hirta
ethanol extract was hematologically not toxic to rats. However, the findings of the current
study do not support the earlier research by Adedapo et al. (2005) that showed that some
chromatographic fractions of Euphorbia hirta have potentially deleterious effects on the
serum chemistry of rats. Several aspects might explain this difference. First, it might be
related to the ability of birds to tolerate high doses of plant extract as herbivorous animals
to compare with rat as omnivorous animals. Second, it could be due extraction methods.
There is evidence that the principal active ingredients of the plant must be more of lipid
soluble or non polar, since organic solvent such hexane and ethanol, are organic solvent
which must have easily extracted the lipid soluble phytochemicals (Cowan, 1999). It has
been reported that plant extracts in organic solvent provided more consistent antimicrobial
activity compared to those extracted in water (Parekh et al., 2005). Finally, it is well known
that the same taxon growing in different areas may have widely differing chemical
components (Eloff, 1999). The efficacy of medicinal herbs is affected by different
environmental factors. Temperature, rainfall, day length and soil characteristics are
some of the factors which affect the potency of the medicinal plants. A plant may grow
well in different situations, but fail to produce the same constituents (Dubey et al., 2004).

Acute toxicity study indicated that water suspensions of selected herbal aqueous extract
are not toxic when administered by the oral route to experimental birds at 2000 mg kg-1 b.
wt. In conclusion, the results obtained in the present study are in agreement to a certain
degree with the traditional uses of the plants estimated. Additionally, based on the results
of the liver enzymes study, apart from the traditional uses, Euphorbia hirta may also act as
a prophylactic agent to prevent liver diseases. The present results could be a good basis for
selection of plant species for further investigation in the potential discovery of new natural
bioactive compounds. In addition, these plants could represent striking feed additive
agents, which provide prophylaxis against various diseases, growth and health promoter.

ACKNOWLEDGMENTS

This study was supported by a grant (05-01-04-SF0182) from the Ministry of Science,
Technology and Innovation, Malaysia. The authors gratefully acknowledge S. Zulkepli, M.
Ebrahimi and M. Tabatabaei for their technical assistance.
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