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Prenatal developmental toxicity evaluation of Withania somnifera root extract

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Prenatal developmental toxicity evaluation of Withania

somnifera root extract in Wistar rats
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P. C. Prabu & S. Panchapakesan
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Central Animal Facility, SASTRA University, Thanjavur, Tamil Nadu, India
Published online: 01 Jul 2015.

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Drug Chem Toxicol, 2015; 38(1): 50–56

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RESEARCH ARTICLE

Prenatal developmental toxicity evaluation of Withania somnifera root

extract in Wistar rats
P. C. Prabu and S. Panchapakesan

Central Animal Facility, SASTRA University, Thanjavur, Tamil Nadu, India

Abstract Keywords
Context: Withania somnifera (L) Dunal (Solanaceae) is an important traditional herbal medicine Alternative medicine, ayurveda, herbal, Indian
used for thousands of years and is considered as the Indian ginseng. Reports on the effect of ginseng, safety, Siddha, teratology
Withania somnifera root (WSR) extract on the developing foetus of pregnant rats including
Downloaded by [Panchapakesan S] at 01:44 07 August 2015

mortality, structural abnormalities, changes in growth and effects on dams are not available. History
Objective: The present study was performed to evaluate the prenatal developmental toxicity
potential of WSR extract in rats. Materials and methods: WSR extract was given orally to Received 17 October 2013
pregnant rats during the period of major organogenesis and histogenesis (days 5 to 19 of Revised 11 February 2014
gestation) at the dose levels of 500, 1000 and 2000 mg/kg/day. Clinical observations including Accepted 18 February 2014
mortality, moribundity, behavioural changes, signs of overt toxicity, body weight, gross Published online 19 March 2014
pathological changes of dams and foetal analyses including external malformations, skeletal
and soft tissue malformations were evaluated. Results: No evidence of maternal or foetal toxicity
was observed. WSR extract caused no changes (p50.05) in body weight of parental
females, number of corpora lutea, implantations, viable foetuses, external, skeletal and
visceral malformations. Discussion and conclusion: Under the conditions of the study, the
no-observed-effect level (NOEL) of WSR extract for maternal and developmental toxicity was
concluded to be at least 2000 mg/kg/day.

Introduction
Withania somnifera (L) Dunal (Solanaceae), called as
Ashwagandha (smell of horse) in Sanskrit, is an Ayurvedic
herb that holds a place similar to that of Ginseng in Chinese
medicine. Ashwagandha is used in ayurveda for the treatment
of pthisis (wasting disesases), weakness, inflammation, male
impotence, etc. The anti-oxidant, anti-inflammatory, immune-
modulating and antistress properties of the whole plant
and individual constituents have been widely published
(Mishra et al., 2000). Ashwagandha is also reported to
possess antitumor, haematopoietic, anxiolytic, anti-aging and
antidepressive properties and to exert effects on the various
receptors of the neurotransmitters of central nervous system
(Pattipati et al., 2003). Its active principles namely, sitoindo-
sides VII-X and withaferin A (WA) increase the concentra-
tions of superoxide dismutase and catalase (Bhatnagar et al.,
2005; Gupta et al., 2003). For therapeutic purposes, the roots
Address of correspondence: Dr. S. Panchapakesan, M.V.Sc., Ph.D.,
Professor & Coordinator, Central Animal Facility, SASTRA University,
Thanjavur – 613 402, Tamil Nadu, India. Tel: +91 4362-264101-108
Extn. 680. Fax: +91 4362-264120. E-mail: panjuvet@yahoo.co.in;
panchapakesan@sastra.edu
are considered to be the most active part and are categorised
as rasayanas. The roots promote health by improving defence
against infections, slowing down the ageing process and
revitalising the body during debility (Weiner & Weiner,
1994). As detailed and systematic reports on the different
toxicological aspects of Withania somnifera root extract
(WSR) were not available, we conducted a sub-acute oral
toxicity study in Wistar rats as per the Organization for
Economic Cooperation and Development (OECD) guidelines
407 and concluded the no observed effect level (NOEL) of the
hydroalcoholic extract of W. somnifera to be 2000 mg/kg/day
of body weight (Prabu et al., 2013). The study demonstrated
the non-toxic nature of WSR extract in adult non-
pregnant rats.
Ashwagandha has been considered as an adaptogen that
works on a non-specific basis to normalize the physiology
by acting on the hypothalamic-pituitary-adrenal axis and
the neuro-endocrine system and to invigorate the body by
rejuvenating the reproductive organs. It has been in use for a
very long time even during pregnancy (Sharma et al., 1985). It
has been claimed to strengthen the pregnant individual and
stabilize the foetus. In contrary, Saleh Al-Qura’n (2005) have
reported that higher doses of Withania somnifera might have
abortifacient properties and have classified it under toxic
plants that cause abortion and sterility. To elucidate precisely,
the exact effect of oral WSR extract on mammalian devel-
opment, the present study was conducted in pregnant Wistar
 DOI: 10.3109/01480545.2014.900073
rats following the OECD guidelines 414 for the assessment
of prenatal developmental toxicity (OECD, 2001).
Materials and methods
Plant material
The plant material collected in Neemuch, India (June, 2008),
was certified by Prof. P. Jayaraman, Plant Anatomy Research
Centre, National Institute of Herbal Science, Chennai.
A specimen was deposited (Specimen No.: PM0906) at the
Archives, Central Animal Facility, SASTRA University.
Extract
The hydroalcoholic extract of Withania somnifera roots
certified to contain not more than 10 ppm of heavy metals
was procured from Ellees Aromatics Ltd. (Batch No:
NACOP/WSD-19/08-09) along with the dried plant material
for identification. Air dried roots of Withania somnifera
were ground into a coarse powder and the same was extracted
with 80% methanol in an extractor. The extract was then
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distilled and dried in a vacuum rotary drier, the dried
material was pulverized and sifted to obtain the fine extract.
The yield of dried extract was 10%, expressed as a percentage
weight of the dried roots. The extract was stored in air
tight containers until the time of use. A representative
sample of the extract (Test substance No.: TSS0906) was
deposited at the Archives, Central Animal Facility, SASTRA
University.
Characterisation of WSR extract
Withanolides (ergostane type steroids) are specific for the
family Solanaceae, especially for the genus Withania and
are considered as marker compounds (Bruneton, 1999) for
characterisation. Among the various withanolides, withaferin
A, that has been studied extensively (Ganzera et al., 2003)
was used as the marker compound in the present study.
Quantitative estimation of withaferin A was performed by
high performance thin layer chromatography (HPTLC) tech-
nique following the method described by Khajuria et al.
(2004) with slight modifications using Withaferin A (CAS
No:5119-48-2, Natural remedies, India) as the standard.
A CAMAG HPTLC system with TLC sample applicator
(Linomat 5), TLC scanner 3 (winCATS version 1.3.4) and
winCATS Planar Chromatography Manager software was
used for the estimation. CAMAG Reprostar 3 system was
used for photo documentation (530 nm). Toluene: ethyl
acetate: formic acid (5:5:1) was used as mobile phase
with a dosage flow of 150 nl/s at multilevel calibration
mode. Aluminum-backed HPTLC plates (20 cm  20 cm with
0.2 mm layer of silica gel 60 F254), prewashed with methanol,
were used. Using automated TLC sampler, 10 ml each of three
aliquots of WSR samples and varying concentrations of
standard withaferin A solutions prepared in methanol were
applied in duplicate on the HPTLC plate to prepare the
six-point linear calibration curve. For all these analysis,
experimental parameters were kept identical. The developed
plate was scanned in the remission absorbance mode at
530 nm with a slit width of 6.00  0.45 mm, microscanning
speed of 20 mm and data resolution of 100 mm per step.
Teratological evaluation of Withania somnifera root extract 51
The analytical grade chemicals used in the study were
purchased from Merck, India or Sigma, USA.
Animal care and management
The study was conducted at the Central Animal Facility,
SASTRA University (Registration. No: 817/04/ac/CPCSEA)
after the approval (IAEC approval No.: 80/SASTRA/IAEC/
RPP) by the Institutional Animal Ethics Committee (IAEC),
SASTRA University. The rats were provided due care
conforming to the Committee for the Purpose of Control
and Supervision of Experiments on Animals (CPCSEA)
guidelines for laboratory animal use and care, Government of
India. Wistar rats procured from the Central Animal Facility
were housed in autoclaved polypropylene cages with sterile
paddy husk as the bedding material. A temperature of
22 ± 3  C, relative humidity of 30–70%, a light: dark cycle
of 12 h: 12 h and 12–15 air changes per hour were maintained
in the experimentation room throughout the study. The rats
were fed with standard rodent feed ad libitum (M/s Provimi
Animal Nutrition India private limited) and RO (reverse
osmosis membrane processed) water ad libitum.
Prenatal developmental toxicity study
Healthy rats, not subjected to any previous experimental
procedures, were initially acclimatized to the room conditions
for 7 days. Young adult nulliparous female rats were used. 100
female rats were mated with 100 male rats in the ratio of 1:1.
Mating of siblings was avoided and individual housing was
provided to the breeding rats. The male rats were separated
and vaginal smears were prepared daily by using a 10 ml
pipette and 0.85% normal saline for aspiration (Marcondes
et al., 2008). Day 0 of gestation was considered to be the day
on which a vaginal plug or spermatozoa was observed. Mated
females were distributed in an unbiased way into control (G1)
and treatment groups (G2, G3 and G4) and were treated as
follows:
No. of Day of sacrifice
Group No. female rats Dose of pregnant females
Group I 25 Control 20th day
Group II 25 500 mg/kg/day 20th day
(Low dose)
Group III 25 1000 mg/kg/day 20th day
(Intermediate dose)
Group IV 25 2000 mg/kg/day 20th day
(High dose)
In the repeated dose oral toxicity study conducted earlier at
our facility (Prabu et al., 2013), WSR extract was given at
500, 1000 and 2000 mg/kg/day for 28 days and was non-toxic
up to the highest dose of 2000 mg/kg/day tested. Considering
the results of that study, WSR extract was administered @
500, 1000 and 2000 mg/kg/day to the pregnant rats daily from
day 5 (day of implantation) to day 19 (a day before necropsy)
of gestation. WSR extract freshly dissolved in distilled water
was administered orally by intubation, approximately at the
same time daily as a single dose. The rats were dosed
according to their most recent body weight. The maximum
volume of liquid did not exceed 1 ml/100 g of body weight
and the variability in volume was minimised by changing the
 52 P. C. Prabu & S. Panchapakesan
concentrations to provide a constant volume to rats of
different groups. The control rats received the maximum
volume of the vehicle and were handled in a manner identical
to those of the test group.
Observations of the dams
Clinical observations including death, moribundity, behav-
ioural changes and signs of toxicity were observed at least
once a day. The rats were weighed on day 0 and every 3 days
during gestation and on the day of sacrifice.
Post-mortem examination
On the 20th day of gestation, anaesthesia was induced with
50 mg/kg of thiopentone (ThiosolÔ sodium) and laparotomy
was performed. The uterine horns were exposed, the foetuses
and placentas were collected and then, the rats were
euthanised by exsanguination. The dams were examined
macroscopically for the presence of any structural abnorm-
alities/pathological changes. The uteri including the cervix
were collected and their weights were measured. The number
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of corpora lutea per pregnant rat, number of implantations/
live foetuses per pregnant rat, body weight of the foetus and
their sex were determined. The runts (normally developed
new borns which were significantly smaller than the rest
of the litter i.e., pups whose body weights were more than
two standard deviations below the mean pup weight of the
respective litter) were evaluated for their viability by foetal
movement, and their response to touch demonstrated that they
were not in the process of being resorbed. % Pre-implantation
loss was derived as follows:
(# corpora lutea -- # implantations)
 100
(# corpora lutea)
The embryonal mortality after implantation i.e. % Post-
implementation loss was derived as follows:
(# implantations -- # of live fetuses)
 100
(# implantation)
Analysis of external malformations
The foetuses were weighed and then the head, limbs, body,
tail and anal perforation were examined for the presence of
any external malformations (Wilson, 1965).
Analysis of visceral and skeletal malformations
Half of the foetuses meant for the evaluation of visceral
malformations were preserved in Bouin’s fluid for a week,
then in 80% isopropyl alcohol for 2 days and in 90% isopropyl
alcohol until analysis. For the detection of visceral malfor-
mations, serial sectioning of the foetus was performed using
standard procedures (Wilson, 1965) under a Stereo-zoom
microscope (NikonÕ Microscope, SMZ 1000). The remaining
half of the foetus meant for the evaluation of the skeletal
malformations after evisceration were dehydrated in industrial
methylated spirit for one to two weeks until they became stiff
and pale beige in colour. Industrial methylated spirit (IMS)
was changed every 2 days.
For maceration, the de-skinned fetuses were placed in 1%
KOH for one to two days until the bones of the fetuses were
Drug Chem Toxicol, 2015; 38(1): 50–56
clearly seen through the flesh. Staining was combined with
the last stages of maceration by adding the alcoholic
Alizarin Red S stain solution to the container containing
KOH. The containers were examined regularly for staining
intensity. The foetuses were then transferred to benzyl/
glycerol/alcohol (1:2:2) solution for 2 days, then, to benzyl/
glycerol (1:3) solution for 2 days and to benzyl/glycerol (3:1)
for four days. The fetuses were finally transferred and stored
in pure benzyl alcohol until analysis. The alterations in
the foetuses were categorised as malformations or variations
(Stadler et al., 1983).
Statistical evaluation
The quantitative data derived are tabulated as mean ± stan-
dard deviation of the mean. All the data pertaining to
the litter were analysed on per litter basis. All the quantita-
tive data were evaluated for homogeneity using Bartlett’s
test. When the variance was homogeneous, the measure-
ments were evaluated by one-way ANOVA and post-hoc
analysis was performed using Tukey test. In the absence of
homogeneity of data, the measurements were evaluated by
Kruskal–Wallis test and when significant differences were
observed, the Dunnett test was performed. Chi-Square test
was used to evaluate the data of malformations per litter and
variations per litter, when the difference in the absolute
number of foetuses were greater than three. A probability
level lower than 5% (p50.05) was considered significant.
Statistical analyses were performed using Graph Pad Prism
5 software.
Results
Chemical standardisation of WSR extract
Analysis of the WSR extract by HPTLC confirmed the
presence of 0.043 ± 0.004 g of withaferin A per 100 g of the
root extract which was in agreement to the previously
reported concentrations ranging from 0.003 to 0.066 g per
100 g of root (Ganzera et al., 2003).
(a) Maternal toxic response data
The conception rate ranged from 91% to 96% with 21–24
pregnant rats/group and all the pregnant females survived until
the day of sacrifice. No toxic signs were found in any of the
groups of pregnant females. The maternal data and the
developmental endpoints for the litters collected at necropsy
are given in Tables 1 and 2. Maternal body weight was greater
(p50.05) in group IV as compared to those of control on day
20. Gravid uterine weight (p50.05) was greater in the group
IV as compared to that of the control. The empty uterine weight
and cervix weight did not show any significant difference
among the groups. At necropsy, no treatment related gross
pathological changes were observed in the dams. No other
signs/symptoms/changes of maternal toxicity attributable to
WSR extract were observed during the study.
(b) Developmental endpoints for litters with implants
No differences (p50.05) were found in the corpora lutea
per pregnant female, implantations per pregnant female, mean
early resorptions per pregnant rat, i.e. a conceptus in which
gross evidence of organogenesis is absent, % pre-implantation
loss and % post-implantation loss.
 DOI: 10.3109/01480545.2014.900073 Teratological evaluation of Withania somnifera root extract 53

(c) Developmental endpoints for litters with live foetuses
No differences (p50.05) were found in the total numbers
of live foetus per pregnant female and male foetal body
weight. There was a significant decrease in the mean female
foetal body weight of the medium and high dose groups which
correlated with the insignificant increase in mean live female
foetuses in the treated groups.
(d) Gross external morphology
The quantitative data of gross external morphology of
the foetuses along with the visceral and skeletal alterations
including the individual malformations/variations are pre-
sented in Table 3. Gross external malformations were absent
and the foetuses from different groups appeared normal.
Table 1. Effect of WSR extract over the body weight and feed
consumption of the pregnant female rats.
Interval Control Low dose Medium dose
Body weight (g)
Day 0 210.35 ± 8.78 210.20 ± 9.52 210.30 ± 6.51
Day 4 225.57 ± 8.39 223.91 ± 11.05 220.67 ± 8.23
Downloaded by [Panchapakesan S] at 01:44 07 August 2015
The external variations observed were the presence of three
runts in group I, a runt each in groups II and IV, a foetus in
group I with kinked tail and a foetus with limb hyperextension
in group III. These external variations were found to be
distributed in the control as well as the treatment groups and
were of the spontaneous type observed in Wistar rats.
(e) Visceral examinations
No significant increases in the frequency of visceral
malformations were observed in the treatment groups as
compared to those of the control (Figure 1A–F). A foetus had
domed head i.e. hydrocephalus characterized by the presence
of mild dilation of the lateral ventricles in group I.
(f) Skeletal examinations
Treatment-related skeletal malformations/variations
were absent in the groups II, III and IV. Common skeletal
variations (incomplete ossification of the skull and dumpbell
shaped misshapen centrae) were observed in groups I, II
High dose
and III (except in high dose group) at similar incidences
(Figure 2A–B). The presence of the bifid centra of the
210.51 ± 4.43
218.02 ± 3.50 thoracic vertebrae and the absence of the associated fusion

Day 7 239.22 ± 8.01 236.37 ± 8.72 229.42 ± 8.21 234.10 ± 7.91 of ribs, right arches and right aspects of the centra
Day 10 246.85 ± 8.94 245.44 ± 9.81 238.73 ± 8.83 241.52 ± 6.99 confirmed that it was an variation and not the ‘‘interrelated
Day 13 255.17 ± 9.15 253.82 ± 10.98 253.00 ± 9.71 250.01 ± 6.67
Day 17 266.04 ± 9.54 263.15 ± 11.81 264.09 ± 9.71 261.58 ± 13.05 vertebral -rib-malformation’’.
Day 20 280.60 ± 8.65 281.66 ± 11.72 295.62 ± 17.85 301.66 ± 11.44* The results indicated that the administration of WSR
Feed intake per day (g) extract did not result in an increase in the incidence of skeletal
Day 0 22.01 ± 2.82 20.08 ± 3.42 19.92 ± 2.75 20.58 ± 2.66
Day 4 31.35 ± 4.03 30.24 ± 0.95 32.14 ± 3.06 29.76 ± 2.57 and visceral malformations/variations in Wistar rats.
Day 7 32.49 ± 4.06 29.52 ± 3.07 33.06 ± 3.57 31.31 ± 3.24
Day 10 33.47 ± 3.82 32.22 ± 2.49 33.07 ± 2.73 30.26 ± 2.91 Discussion
Day 13 36.17 ± 3.36 34.74 ± 2.15 35.89 ± 3.66 34.45 ± 1.94
Day 17 37.81 ± 1.96 35.29 ± 2.13 36.96 ± 3.52 36.80 ± 2.26 Research on the evaluation of herbal preparations for their
Day 20 39.80 ± 1.64 37.65 ± 1.68 38.46 ± 2.40 41.44 ± 1.22 positive effects on health and other functions of the body in
Mean values of the treatment groups bearing * vary significantly from animals and humans are gaining importance (Allan et al.,
the control (p50.05). 2012). Despite the availability of numerous reports on the

Table 2. Effect of WSR extract over the pregnancy data of the Wistar rats.

Group I Group II Group III Group IV

Parameter (0 mg/kg) (500 mg/kg) (1000 mg/kg) (2000 mg/kg)
Dams entering study (sperm positive) 24 23 24 24
% Successful pregnancy (viable) 91.66 91.30 95.83 91.66
Viable litters (with at least 1 live implant) 24 21 24 24
Gravid uterus weight (g) 33.16 ± 6.99 31.29 ± 13.01 31.19 ± 11.50 43.24 ± 11.52*
Empty uterus weight (g) 2.69 ± 0.69 2.87 ± 0.90 2.88 ± 1.17 3.53 ± 0.62
Cervix weight (g) 0.31 ± 0.14 0.30 ± 0.13 0.23 ± 0.11 0.33 ± 0.14
Mean Corpora lutea per pregnant rat 10.80 ± 1.55 10.44 ± 1.88 11.00 ± 1.41 11.78 ± 1.30
Mean Implantation sites per pregnant rat 7.60 ± 3.20 9.00 ± 3.54 7.80 ± 2.78 11.00 ± 1.63
Mean early resorptions per pregnant rat 0.30 ± 0.48 0.33 ± 0.71 0.50 ± 0.85 0.60 ± 1.26
Mean Live foetuses per rat 7.30 ± 3.13 8.63 ± 3.54 7.29 ± 3.06 10.40 ± 2.01
Mean Live female foetus per rat
Left uterine horn 1.60 ± 1.07 2.40 ± 1.43 1.22 ± 1.09 2.20 ± 1.14
Right uterine horn 1.90 ± 1.29 1.70 ± 1.77 2.60 ± 1.84 2.50 ± 1.51
Total 3.50 ± 1.84 4.10 ± 2.42 3.70 ± 2.45 4.70 ± 2.11
Mean live male foetus per rat
Left uterine horn 1.50 ± 1.18 2.40 ± 1.35 1.80 ± 1.23 3.90 ± 1.79*
Right uterine horn 2.40 ± 1.43 2.22 ± 1.30 1.80 ± 1.03 1.80 ± 1.03
Total 3.90 ± 1.85 4.40 ± 2.12 3.60 ± 1.84 5.70 ± 1.64
Mean foetal body wt. per litter – Females (g)
Left uterine horn 4.27 ± 0.88 3.29 ± 1.12 2.23 ± 1.12* 3.10 ± 1.25
Right uterine horn 3.90 ± 0.85 3.78 ± 0.52 2.91 ± 0.99 3.05 ± 1.20
Total 4.08 ± 0.85 3.50 ± 0.92 2.62 ± 1.06* 3.07 ± 1.19*
Mean foetal body wt. per litter – Males (g)
Left uterine horn 4.29 ± 1.10 4.02 ± 0.91 3.00 ± 1.29 3.19 ± 1.37
Right uterine horn 4.13 ± 1.03 3.97 ± 0.60 3.38 ± 0.72 3.24 ± 1.28
Total 4.21 ± 1.03 4.00 ± 0.77 3.21 ± 1.00 3.21 ± 1.00
% Pre-implantation loss 29.68 ± 27.25 13.30 ± 29.48 29.35 ± 25.82 3.21 ± 6.76
% Post-implantation loss 5.08 ± 10.57 16.44 ± 32.44 6.59 ± 17.85 5.38 ± 11.48

Mean values of the treatment groups bearing * vary significantly from the control (p50.05).
 54 P. C. Prabu & S. Panchapakesan Drug Chem Toxicol, 2015; 38(1): 50–56

Table 3. Effect of administration of WSR extract over the foetal data of pregnant rats treated with WSR extract.

Parameter CONTROL LOW MEDIUM HIGH

No. of litters examined 24 21 24 24
i. External observations
No. of foetus examined 176 179 175 249
External malformations
No. of foetus with malformations 0 0 0 0
External variations
No. of foetus with external variations 4 (2.27%) 2 (1.12%) 1 (0.57%) 1 (0.40%)
No. of litters with external variations 2 (8.33%) 1 (4.76%) 1 (4.17%) 1 (4.17%)
Tail – kinked 1 0 0 0
Runts 3 1 0 1
Limb hyperextension 0 0 1 0
ii. Soft tissue observations
No. of foetus examined 88 90 88 125
No. of foetus with soft tissue malformations 1 (1.14%) 0 (0) 0 (0) 0 (0)
No. of litters with soft tissue malformations 1 (4.17%) 0 (0) 0 (0) 0 (0)
Hydrocephalus 1 0 0 0
iii. Skeletal malformations
No. of foetus examined 88 89 87 124
No. of foetus with skeletal malformations 1 (1.14) 1 (1.12%) 1 (1.15%) 0 (0)
No. of litters with skeletal malformations 1 (4.17%) 1 (4.76%) 1 (4.76%) 0 (0)
Incomplete ossifications – Skull, Minimal 1 0 1 0
Dumpbell cartilage, Thoracic Centrum 0 1 0 0
Downloaded by [Panchapakesan S] at 01:44 07 August 2015

Mean values of the treatment groups bearing * vary significantly from the control (p50.05).

Figure 1. External and visceral examination

of treated and non-treated foetuses. (A) A
runt with less than 1.5 cm crown – rump
distance along with a normal foetus from the
same litter in group IV. Lateral view. (B) 3
runts of less than 1.5 cm crown – rump
distance in the control group along with a
normal foetus from the same litter. Lateral
view. (C) Control foetus showing the pres-
ence of a kinked tail (arrow). Ventral view.
(D) Control rat foetus with a domed head,
characteristic of hydrocephalus. Lateral view.
(E) Section of rat foetal head of the foetus
shown in figure D with moderate dilation of
lateral ventricles (arrow). Bouin’s fixation.
(F) Section of the foetal head of the control
rat with normal lateral ventricles (arrow).
Bouin’s fixation.

efficacy of the herbal preparations during different disease in usage of herbal preparations for a variety of health aspects,
conditions, the information available on the toxicity profile of systematic studies on the different aspects of toxicity need to
them following internationally accepted guidelines is not be carried out. Pharmacological effects of Withania somnifera
adequate (Joshua et al., 2008). In anticipation of the increase have been well established in published literature. Several
 DOI: 10.3109/01480545.2014.900073
Figure 2. Skeletal examination of treated and
non-treated foetuses. (A) Rat foetus with
dumbbell shaped thoracic centrum (arrow).
Dorsal view. Alizarin red S  8. (B) Rat
foetus with normal thoracic vertebrae and
ribs, Dorsal view. Alizarin red S  8.
studies have reported the effects of Ashwagandha on repro-
ductive hormones and other sexual functions. Abdel-Magied
and co-workers (2001) showed that the administration of
Withania somnifera decreased the serum FSH levels and
increased the serum LH level in rats. Withania somnifera also
significantly enhanced the testosterone and luteinizing hor-
mones levels and lowered the FSH levels in the serum of men
(Ahmad et al., 2009). Such endocrine disruptors, i.e. those
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substances which can interfere with the endocrine (or
hormone system) can cause adverse developmental, repro-
ductive and neurological effects.
Withania somnifera has been used for several years in
different age groups of men and women, even during
pregnancy (Sharma et al., 1985). Prior to this study, reports
were only available on the acute and sub-acute toxicity
(Prabu et al, 2013) and reproductive toxicity of WSR extract
(Sharma et al., 1985). No reports were available regarding the
action of Ashwagandha on the developing foetus. The paucity
of such information for those preparations like Withania
somnifera that are administered during pregnancy, necessi-
tates the need for the conduct of prenatal developmental
toxicology studies to assess the risks and safety associated
with their use during pregnancy. Contrary to the popular
belief that herbal preparations are safe, the use of medicinal
plants during pregnancy may cause various changes in the
embryo-foetal growth resulting in abortive (embryo-lethality)
or embryo-foeto-toxic changes (Almeida & Lemonica, 2000;
Lyra et al., 2005).
Withaferin A content has been reported to be present at
levels ranging from 1.5 to 2.8 mg/tab in different formulations
of Withania somnifera as evaluated by high performance
liquid chromatography (HPLC) (Mathur & Velpandian,
2009). WSR extract used in the present study contained
0.043 ± 0.004 g of withaferin A per 100 g of extract, i.e.
withaferin A content of the extract used was several times
greater than those levels present in marketed formulations.
Further, WSR powder is given at therapeutic dose levels of
3–6 gm per individual per day in practice as per the Ayurvedic
pharmacopeia. Considering the 10% yield of dried extract as a
percentage weight of the dried roots and assuming an adult
body weight as 70 kg, the dose of the extract was determined
to be 4.2–8.4 mg/kg body weight per day. Using the rat
conversion factor, the dose was arrived at 26.0–52.0 mg/kg
and the toxicity study was conducted at doses 10, 20 and
40 times greater than of therapeutic dose levels, ie. at 500,
1000 and 2000 mg/kg body weight per day. The evaluation of
the effect on the foetus is generally performed by adminis-
tering the preparation/compound to pregnant rats from the day
Teratological evaluation of Withania somnifera root extract 55
of implantation to a day prior to parturition (Rogers &
Kavlock, 2001). The dosing was done from implantation to
hard palate closure period to derive information on the effect
of WSR extract over the adult female reproductive functions
and the development of the embryo through major organ
formation. The toxic effect during this period of organogen-
esis is the one which may most likely result in structural
malformations (Wilson, 1965). The intensity of cellular
multiplication and migration, remodeling and development
of tissue/organs (Brent, 1993) increases during the phase of
organogenesis. The outcome of gestation is dependent over
the interactions between the complex dynamic processes of
pregnancy and the perturbations in its environment. The
kind of deformity caused is dependent on the phase of
development of the embryo and the affinity of the toxic agent
towards the particular type of embryonal tissue/organ (Chang
et al., 2002). However, no indications of developmental
toxicity of WSR extract were observed after the oral
administration during the stage of major organogenesis
including gastrulation for cell differentiation and histogenesis
up to 2000 mg/kg/day.
The weight of the uterus is considered to be an indicator of
the possible estrogenicity of the compound. Though there was
an increase in the weight of the uterus as well as the body
weight of the pregnant rats in the high dose treated group, it
was considered to be due to the non-significant increase in the
mean live foetus per pregnant female. No significant differ-
ences were observed in the empty uterine weight among the
different groups. Significant decreases in foetal weight as a
result of treatment is suggestive of possible toxicity (Lyra
et al., 2005). The pregnant rats fed orally with WSR extract
did not show any significant change in the foetal/maternal
body weights. Pre-implantation loss is a measure of the
ovulation, fertilisation, implantation and the uterine receptiv-
ity of the animal. Since WSR extract was not administered
during the pre-implantation period of the rats, the insignifi-
cant decrease in the pre-implantation loss in the high dose
group was not attributed to be the effect of WSR extract.
No relation between the incidence of malformations/
variations and the administration of WSR extract was
observed as evidenced by external, visceral and skeletal
analyses. The incidence of misshapen centrum among the
thoracic vertebrae, hydrocephalus, kinked tail and runts were
within normal limits and are considered to be spontaneous
malformations/variations observed at low incidences in
Wistar rats. The absence of any significant malformations/
variations in the groups treated during this major phase of
organogenesis including gastrulation for cell differentiation
 56 P. C. Prabu & S. Panchapakesan
indicated the non-toxic nature of WSR extract over the
growing foetus.
Conclusion
The present investigation demonstrated that the NOEL for
prenatal developmental toxicity of the hydroalcoholic extract
of Withania somnifera root is at least 2000 mg/kg/day body
weight. The study supports the evidence of safety of Withania
somnifera root extract.
Acknowledgements
The authors gratefully acknowledge the support and facilities
provided by the management of SASTRA University.
Declaration of interest
The authors report no declarations of interest.
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