Evaluation of Safety of NaturalivTM Liquid and Zist® Liquid on Acute Oral Exposure in Rats
J. Joshua Allan,
G. Pavan Kumar,
The objective of the current study was to assess the acute oral toxicity of some herbal veterinary preparations in female albino Wistar rats as per the OECD guidelines for testing of chemicals, Acute oral toxicity-Fixed dose procedure (Test guideline 420). The investigational substances, NaturalivTM liquid and Zist® liquid are used in poultry for hepatoprotective and antioxidant properties, respectively. NaturalivTM liquid was tested at 5000 mg kg-1 b.wt., whereas Zist® liquid was evaluated in sequential manner to one animal each at 2000 and 5000 mg kg-1 b.wt. in the sighting study, followed by four animals at 5000 mg kg-1 b.wt. in the main study. The treated animals were observed for mortality (twice daily), adverse clinical signs (once daily), changes in weekly body weight gain and gross necropsy findings. All the treated animals survived throughout the study period. Treatment with NaturalivTM liquid did not reveal any abnormal clinical signs at the tested dose level while Zist® liquid treated rats exhibited few clinical signs for a transient period on the day of dosing. The overall percent body weight gain in rats was found to be normal during the 14 day observation period. The terminal sacrifice revealed no gross pathological lesions that can be attributable to treatment. Based on this, it can be stated that NaturalivTM liquid and Zist® liquid, on single oral administration to female Wistar rats, were found to be safe up to 5000 mg kg-1 b.wt.
Received: March 03, 2010;
Accepted: May 10, 2010;
Published: June 19, 2010
The global poultry industry forms one of the most dynamic aspects of world
agribusiness trade. Despite the fast growth of commercial chicken production
during 1990s, it is perceived that, in the first decade of the 21st century,
the world may experience continued increase in chicken meat and egg production
but not at the same rapid pace (Aho, 2002). The present
poultry industry faces a variety of managemental and economical barriers. To
optimize production levels for combating financial crises and also to protect
consumers safety, various strategies are explored.
The use of herbal feed supplements for poultry is popular worldwide. Herbal
preparations composed of single or multiple plant ingredients are used in poultry
for various indications (Ranade and Desai, 2005; Waghmare
et al., 2006; Ramnath et al., 2008;
Jadhav et al., 2009). Many of the herbal supplements
are based on the earlier compilations of various traditional medicine systems
and are used for medicinal and non medicinal properties (Okitoi
et al., 2007). As observed in current animal healthcare systems,
the cost spent for commercial medicines at times outstrip the value of animals,
especially in case of smaller stocks like poultry (Lans
et al., 2007). Since, the conventional therapeutic agents being expensive
and the availability of professional veterinary services are inadequate particularly
in the developing countries, use of herbs and herbal preparations seems to be
popular alternative largely due to the accessibility of cost effective animal
healthcare services from familiar, local traditional healers.
On the other side, certain limitations are also reported in the published literature
regarding the use of botanical preparations; one of the critical claims being
lack of scientifically validated safety information of the herbal formulations.
Attempts to collate toxicity data on poultry herbal supplements revealed not
only the scanty information available in this aspect but also the fact that
the scientific investigations were not carried out uniformly throughout the
world (Oyagbemi et al., 2008).
With the advancement of standardised procedures for toxicity characterization
of test substances, safety evaluation of veterinary formulations by approved
regulatory protocols is being warranted for universal validation and acceptance.
Various guidelines were made available for assessment of safety of test compounds
by different international, federal and national agencies (WHO,
1993; OECD, 1993; FDA, 1993;
EMEA, 1994). Studies conducted in compliance to the regulatory
guidelines are considered imperative and as essential prerequisite for registration
of products internationally.
Based on the above considerations, the acute oral toxicity studies of two polyherbal
formulations viz., NaturalivTM liquid and Zist® liquid
were performed in accordance with the OECD guidelines for testing of chemicals,
test guideline 420, acute oral toxicity-fixed dose procedure (OECD,
2001a). NaturalivTM liquid, a combination of selected medicinal
herbs, is recommended for broiler and layer birds for optimizing liver functions,
utilization of nutrients, better body weight gain, feed conversion and livability.
Zist® liquid, also an herbal supplement, possesses adaptogenic,
antistress and immunomodulating activities. The liquid formulation, in poultry
birds, helps to adjust and increase the threshold against stressful conditions,
regularizes plasma cortisol levels and improves weight gain, feed conversion
and livability. Zist® also optimizes egg production and hatchability
in breeder birds. Generally herbal formulations are regarded as safe. However,
considering the lack of adequate, scientifically validated safety data for veterinary
herbal formulations in general and also taking into account the presence of
several herbal ingredients in the liquid formulations, testing at an additional
upper dose level of 5000 mg kg-1 was considered since the outcomes
of such a test will provide a direct relevance for protecting target species
health (OECD, 2001a). The findings of study will be useful
for selection of doses for repeated dose toxicity studies and may also provide
preliminary information on the target organ toxicity on acute exposure, if any.
The outcome of the study observations categorizes the test substances and safety
of use can be ascertained.
MATERIALS AND METHODS
The investigational substances viz., NaturalivTM liquid and Zist®
liquid are polyherbal preparations used in poultry for hepatoprotective and
antioxidant properties were developed by M/s Natural Remedies Pvt. Ltd., Bangalore,
NaturalivTM liquid consists majorly of aqueous extracts of Andrographis
paniculata, Terminalia chebula, Eclipta alba, Boerhaavia
diffusa, Mangifera indica and Terminalia arjuna. The plant
materials used were analyzed for respective marker compounds by High Performance
Liquid Chromatography (HPLC) method (Indian Pharmacopoeia,
Zist® liquid contains majorly aqueous extracts of Emblica
officinalis, Withania somnifera and Ocimum sanctum. The plant
materials used were analyzed for respective marker compounds by HPLC method
(Indian Pharmacopoeia, 2007).
Standardization of NaturalivTM Liquid and Zist®
The crude powders obtained from the plant materials, after verifying the
content of marker compounds, were mixed in appropriate proportions and extracted
to prepare NaturalivTM liquid and Zist® liquid. The
Thin Layer Chromatography (TLC) profile of product was compared with the reference
material using High Performance Thin Layer Chromatography (HPTLC). The product
and respective reference standard measuring 20 mL was dried separately on a
water bath to dryness. The residue was extracted with 50 mL of methanol on a
water bath for 30 min and then filtered after cooling. The filtrate was concentrated
to 25 mL. Equal volumes (15 μL) of sample and reference standard were spotted
on Silica gel 60 F254 plate of 0.2 mm thickness as bands. The plate
was developed in a mobile phase consisting of chloroform:methanol:acetic acid
(90:10:2). The dried plate was scanned at 254 and 366 nm. The plate was sprayed
with anisaldehyde sulphuric acid reagent and dried in oven at 100°C. The
fingerprint of the product sample was compared with reference standard. The
reference standard was prepared in the laboratory by mixing the herbs as per
the approved formulation and extracting with water.
Female rats of albino Wistar strain (8 to 12 weeks) were chosen for the
study. The animals were received from Central Animal Facility, R and D Centre,
Natural Remedies Pvt. Ltd., Bangalore, India. The animals were housed in individual
polypropylene cages provided with clean bedding of paddy husk. The rats were
maintained under standard housing conditions (Temperature: 25±2°C,
relative humidity between 30 and 70%, with optimal air changes per hour and
12:12 h dark and light cycle) with one week acclimatization period before treatment
with test substances. The animals were provided with standard pelleted rodent
feed (M/s Gold Mohur Foods and Feeds Ltd., Bangalore, India) and UV treated
water ad libitum. The project was conducted during 2008 in the R and
D Centre, Natural Remedies Pvt. Ltd., Bangalore-560 100, India.
The study was conducted in accordance with the OECD guidelines for testing
of chemicals, test guideline 420, acute oral toxicity-fixed dose procedure.
Healthy adult female rats were randomly allotted to the cages and each animal
was identified by individual cage card number and picric acid marking on fur.
The females were nulliparous and non-pregnant. NaturalivTM liquid
was tested in five animals (one animal for sighting study and four animals for
main study) at 5000 mg kg-1. Zist® liquid was evaluated
in one animal each at the dose levels of 2000 and 5000 mg kg-1 b.wt.
in the sighting study and in four animals at 5000 mg kg-1 b.wt. in
main study. The rats were deprived of feed overnight before and 3 h after the
administration of the test substances. Water was not withheld during this period.
The herbal formulations were administered by oral gavage to rats using an intubation
needle of appropriate size fitted on to a syringe so as to deliver the dose
levels of 2000 or 5000 mg kg-1 b.wt. NaturalivTM liquid
was diluted with demineralised water to obtain 500 mg mL-1 strength
to administer the dose of 5000 mg kg-1 body weight (10 mL kg-1
b.wt.) whereas Zist® liquid was administered as such at 20 and
50 mL kg-1 b.wt. in two equally divided doses at 3 h apart so as
to deliver the dose levels of 2000 and 5000 mg kg-1 b.wt. of plant
Cage Side Observations
The treated animals were observed for mortality and clinical signs to record
the onset, duration and reversal (if any) of toxic effects at various time intervals
(i.e., 10 min, 30 min, 1 h, 2 h, 4 h and 6 h after the administration of the
formulations). The procedure was continued thereafter for 14 days for observation
of mortality (twice daily) and abnormal clinical signs (once daily). The routine
cage side observations included changes in skin and fur, eyes and mucous membrane
and also functions of respiratory, autonomic and central nervous systems and
somatomotor activity and behaviour pattern. Clinical observations of tremors,
convulsions, salivation, diarrhoea, lethargy, sleep and coma, if any, were also
given particular attention.
Body weight of individual animals was recorded on the day of dosing and
at termination on day 15 and also at weekly intervals. Weekly changes in body
weight gain were calculated and recorded.
Gross Pathology and Histopathology
All the rats in the study, dying during the observation period, sacrificed
moribund for humane reasons or sacrificed terminally were subjected to a complete
necropsy and examined for the post mortem findings. Histopathology of organs
and tissues was considered only in case of evidence of any gross pathological
The herbal formulations were analyzed for respective marker compounds. The
marker compounds in NaturalivTM liquid were andrographolide (Andrographis
paniculata), chebulagic acid and chebulinic acid (Terminalia chebula),
wedelolactone (Eclipta alba), boeravinone B (Boerhaavia diffusa),
mangiferin (Mangifera indica) and arjungenin (Terminalia arjuna).
The marker compounds of Zist® liquid include gallic acid (Emblica
officinalis), withanolide A and withaferin A (Withania somnifera)
and eugenol (Ocimum santum) (Table 1). The HPTLC profile
of NaturalivTM liquid and Zist® liquid were found
to be similar to that of respective reference standards (Fig.
1a, b, 2a, b).
Animals treated up to the dose level of 5000 mg kg-1 b.wt. survived
till end of the study period and did not show any toxic clinical signs following
dosing and during the observation period of 14 days, post treatment. The animal
in the sighting study showed reduced body weight gain during the second week
of 14 day observation period as compared to first week. However, the overall
body weight gain was found to be normal at the end of 14 day study period (Table
||HPTLC finger print pattern of standard mix (a) and sample
(b) of Naturaliv™ liquid
||HPTLC finger print pattern of standard mix (a) and sample
(b) of Zist® liquid
||List of major plants with proportions used in formulations
of NaturalivTM liquid and Zist® liquid with respective
||Effect of herbal preparations on body weight and percent body
weight gain in rats (sighting study)
||Effect of herbal preparations on body weight and percent body
weight gain in rats (main study)
Similarly, animals of the main study showed decreased body weight gain during
the second week when compared to first week. But, the overall body weight gain
was found to be normal at the end of 14 day observation period (Table
3). Macroscopic examination of animals sacrificed at termination revealed
Zist® liquid treated rats survived throughout the study period
and did not exhibit any major adverse clinical signs immediately following dosing
and during the observation period of 14 days except for the animal treated at
the dose level of 5000 mg kg-1 in the sighting study exhibited abdominal
twitches, shivering and vocalization at 1 h 5 min post treatment for 20 sec
and rapid jaw movements and grinding of teeth at 2 h post treatment for 45 sec
while two animals in the main study at 5000 mg kg-1 showed rapid
jaw movements and grinding of teeth at 10 min post treatment for 10 sec only
on the day of dosing. In the sighting study, though the weight gain of female
rat was found to be less in second week of experimental period as compared to
first week, the overall weight gain was found to be normal at the end of 14
day observation period (Table 2). Likewise, the rats of main
study exhibited reduced body weight gain during the 2nd week of 14 day observation
period in comparison to 1st week. But, the overall body weight gain was found
to be normal at the end of two weeks of study period (Table 3).
On necropsy, no gross pathological changes were observed in any of the treated
From the long-term traditional use, herbs and herbal formulations are generally
regarded as safe (Bhattacharjee, 1998). However, scientific
research on a number of medicinal plants has documented general and systemic
adverse effects on acute/chronic exposure (Francis, 2000).
Establishing safety profiles of herbal extracts/formulations is now gaining
importance in lieu of regulatory needs across the world (Schilter
et al., 2003; Igboasoiyi et al., 2007;
Abukakar et al., 2008; Younis
and Adam, 2008).
Safety characterization of any substance involves study of effects in biological
systems either in target species or in suitable laboratory animals for specific
period through different routes of exposure (EMEA, 1994).
In toxicological screening programme of a test substance, acute oral toxicity
testing is considered to be the preliminary step (OECD, 1987;
EPA, 1998; Walum, 1998; FDA,
2000). The objectives of acute toxicity study are to provide information
on the biologic activity of a substance and gain insight into its mechanism
of action (Walum, 1998). Furthermore, it also helps
in establishing a dosage regimen for subchronic and other safety studies (OECD,
Different approaches are recommended by various regulatory authorities for
acute oral toxicity testing (EMEA, 1987; EPA,
1998; OECD, 2001a, b, 2008).
Published reviews and revised guidelines indicate that the current approaches
seem to emphasize more on characterization of toxic nature of a test substance
based on the specific data requirements rather than solely depending on death
as the end point. Also, decreasing the number of animals used for testing forms
one of the important considerations of selection of appropriate testing method
(OECD, 2001c). All the treated rats survived the complete
14 day study period indicating the relatively safe nature of test formulations.
Treatment with Naturaliv® liquid did not exhibit any adverse
clinical signs immediately following dosing and during the 14 day observation
period where as symptoms such as abdominal twitches, shivering and vocalization,
rapid jaw movements and grinding of teeth were observed shortly after administration
with Zist® liquid. As mentioned elsewhere, the reported clinical
signs were noticed only in few treated animals and the incidence was only once
in each animal which could largely be due to sudden exposure of excessive amount
of xenobiotic material. Also, the fact that the symptoms were noticed for transient
period of time only on the day of test substance administration and did not
recur at any point during the 14 day observation period indicates the transient
and harmless nature of side effects.
In toxicological studies, observation on changes in body weight or growth rate
is considered to be a critical endpoint (EPA, 1998; OECD,
2001a; Wang et al., 2009). Any substance
that causes adverse effects on weight gain or growth rate of the experimental
animals are considered having some toxic potential (Rodriguez-Burford
et al., 1999). From earlier research works, it can be observed that
body weight of treated animals, depending on the nature of test substances,
was reported to be progressively decreased with the time course after exposure,
or remained transiently affected before returning to normalcy (Joshua
et al., 2008). The findings of the current study indicated that the
experimental animals have shown increase in overall body weight gain during
the 14 day observation period though the percent gain was comparably less during
second week of study period in some animals compared to first week.
Gross necropsy findings provide evidences of treatment related target organ
toxicity. Substances with toxic characteristics result in pathological lesions
or changes in organ weights. Therefore, it is commonly recommended by the regulatory
guidelines that all the treated animals (including those that die during the
study or are removed from the study for animal welfare reasons or sacrificed
at the end of the study) should be subjected to postmortem examination and for
individual animals all gross pathological changes should be recorded (EMEA,
1987; OECD, 2001a, b, 2008).
In the present study, no gross pathological changes were observed in any of
the treated rats that can be attributable to treatment.
A critical review on the safety studies on medicinal plants of the tested herbal
preparations indicates the relatively safe nature of the herbal ingredients
on acute exposure. In an acute oral toxicity study, female rats were treated
with extract of Andrographis paniculata and observed for signs of toxicity
for 14 days. The extract did not show any treatment-related toxic effects in
rats up to the dose level of 5000 mg kg-1. Also, the percent weight
gain in all the treated animals during the 14 day study period was found to
be normal (Chandrasekaran et al., 2009). The
aqueous extract of fruits of Terminalia chebula was reported to exhibit
high margin of safety since the median lethal dose was found to be above 3 g
kg-1 and there was no mortality (Murali et
al., 2007). Ethanolic extract of Eclipta alba did not show any
signs of toxicity and the minimum lethal dose was found to be more than 2 g
kg-1 in mice on oral administration (Singh et
al., 1993). The juice of leaves of Boerhaavia diffusa and aqueous
decoction of Mangifera indica on oral administration did not cause any
toxic effects in mice up to 5000 mg kg-1 (Hiruma-Lima
et al., 2000; Severi et al., 2009).
An acute oral toxicity evaluation reported that Ocimum sanctum leaf extract
was not toxic up to 15 g kg-1 in Sprague-Dawley rats (Khumphan
and Lawso, 2002). In another study, the ethanolic extract of Ocimum sanctum
leaves did not cause any mortality or toxicity up to the dose level of 4 g kg-1
in albino rats (Udupa et al., 2006). Hence, the
available, published literature on the safety characteristics of plant ingredients
authenticate the contribution of the medicinal plants to the overall safety
of NaturalivTM liquid and Zist® liquid.
In conclusion, the findings of the present study indicate that NaturalivTM liquid and Zist® liquid after oral administration as a single dose to female albino Wistar rats were found to be safe up to 5000 mg kg-1 b.wt. Based on this, the herbal preparations can be categorized as unclassified according to Globally Harmonised System (GHS) and can be considered as safe.
The authors are thankful to Sri. R.K. Agarwal, Chairman, M/s Natural Remedies Pvt. Ltd., Bangalore, India for his constant encouragement and support in completing this work successfully.
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