Journal of Ethnopharmacology 140 (2012) 151–160

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Journal of Ethnopharmacology
journal homepage: www.elsevier.com/locate/jethpharm

Safety evaluation of an Ayurvedic medicine, Arogyavardhini vati on brain, liver

and kidney in rats
Gajendra Kumar a , Amita Srivastava a , S.K. Sharma b , Y.K. Gupta a,∗
a
Heavy Metal Toxicology Laboratory, Department of Pharmacology, All India Institute of Medical Sciences, New Delhi 110029, India
b
Department of AYUSH, Ministry of Health and Family Welfare, Govt. of India, Red Cross Building, New Delhi 110001, India

a r t i c l e i n f o a b s t r a c t

Article history: Ethnopharmacological relevance: Arogyavardhini vati, an Ayurvedic polyherbal formulation has been used
Received 28 July 2011 for liver and skin disorders in the Ayurvedic system of medicine. However, toxicity due to the presence
Received in revised form of heavy metals in this traditional medicine is a matter of concern.
15 December 2011
Aim of the study: To evaluate the safety of Arogyavardhini vati on brain, liver and kidney in rats.
Accepted 5 January 2012
Materials and methods: Arogyavardhini vati at doses of 50, 250 and 500 mg/kg (1, 5 and 10 times of
Available online 14 January 2012
human equivalent dose respectively), mercury chloride (1 mg/kg) and normal saline were administered
orally to male Wistar rats for 28 days. Behavioral parameters were assessed on day 1, 7th, 14th and
Keywords:
Safety
28th using Morris water maze, passive avoidance, elevated plus maze and rota rod. Biochemical parame-
Arogyavardhini vati ters (acetyl-cholinesterase activity, malondialdehyde, reduced glutathione), histopathology and mercury
Brain level in brain, liver, kidney were assessed at the end of the experiment.
Liver Results: There was no significant change in behavioral parameters, acetyl-cholinesterase activity, liver
Kidney function (ALT, AST, ALP and bilirubin) and kidney (serum urea and creatinine) function tests at all doses
Oxidative stress of Arogyavardhini vati (50, 250 and 500 mg/kg) as compared to normal control. However, significant
Mercury change was observed in mercury chloride treated group. Mercury chloride treated group as well as Aro-
gyavardhini vati treated groups (50, 250 and 500 mg/kg) showed increased levels of mercury in brain,
liver and kidney as compared to normal control. Histopathological results showed significant cytoar-
chitectural changes in brain, liver and kidney architecture in mercury chloride treated group. Whereas,
normal cytoarchitecture was observed at all doses of Arogyavardhini vati.
Conclusion: The finding of the present study suggests that Arogyavardhini vati in the doses equivalent up
to 10 times of the human dose administered to rats for 28 days does not have appreciable toxicological
effects on brain, liver and kidney.
© 2012 Elsevier Ireland Ltd. All rights reserved.

1. Introduction
Ayurveda is a widely practiced system of traditional medicine
in India. It recommends the use of plant based medicines as well as
mineral based medicines including sulfur (S), mercury (Hg), arsenic
(As), lead (Pb), copper sulfate (CuSO4 ) and gold (Au) for treating
assorted disease conditions. This specialty of Ayurveda practice of
mineral, metallic compounds and herbal medicines use is known
as rasa shastra (Shastri, 1979; Satpute, 2003; Saper et al., 2008).
The use of these metals in Ayurvedic medicines is done after a rig-
orous process of purification (shodhana) and converting the metal
into compounds (marana). However, recently some metallic prepa-
rations used in Indian system of medicine are suspected to be
harmful, causing hepatic, renal and neurotoxicity and many other
∗ Corresponding author. Tel.: +91 11 26593282; fax: +91 11 26588662.
E-mail address: yk.ykgupta@gmail.com (Y.K. Gupta).
side effects (Upadhyay et al., 2008). Ayurvedic experts have esti-
mated that 35–40% of the approximately 6000 medicines in the
Ayurvedic formulary intently contain at least one metal. Therefore,
heavy metals content in metal-based Ayurvedic preparation can
still be thousand folds higher (Gogtay et al., 2002; Saper et al., 2008;
Kapoor, 2010). Hg is one of the main components of many tradi-
tional Ayurvedic medicines. Recent reports of high concentration
of heavy metals in Ayurvedic or herbal preparations have raised
much concern and controversy. Hg content found in traditional
medicines raised safety issues amongst the public (Kang-Yum and
Oransky, 1992; Lynch and Braithwaite, 2005; Cooper et al., 2007).
Therefore, it is imperative to know the safety profile of Arogyavard-
hini vati which is Hg based Ayurvedic polyherbal formulation used
for centuries.
Mercury is a well-known toxic heavy metal (ATSDR, 1999).
The major target organs of Hg are brain, liver and kidney.
There are plethora of studies which have shown association of
heavy metal exposure with cognitive impairment and psychiatric

0378-8741/$ – see front matter © 2012 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.jep.2012.01.004
152 G. Kumar et al. / Journal of Ethnopharmacology 140 (2012) 151–160
manifestations (Von Berg and Greenwood, 1991; Laksmanna et al.,
1993). Hg exposed human populations and laboratory animals
have demonstrated neurological abnormalities including cognitive
impairment, and behavioral disturbance (Steuerwald et al., 2000;
Cordier et al., 2002). The accumulation and biotransformation of
Hg occurs in liver. Hepatic damages, ultra structural histopatho-
logical changes have been observed as direct effect of Hg exposure
(El-Shenawy and Hassan, 2008) and indirect effects are due to
oxidative stress (Farina et al., 2004; Sharma et al., 2007). Inorganic
Hg has known to be highly toxic to the proximal tubules (Rodin
and Crowson, 1962; Gritzka and Trump, 1968). High doses of mer-
cury chloride (HgCl2 ) are directly toxic to renal tubular cells but
chronic low-dose exposure induces an immunological glomerular
disease. Some of its functions are so sensitive that abnormalities
start appearing depending upon the nature and the degree of initial
damage occurred (Remuzzi and Bertani, 1998). However, little is
known about the disposition and toxicity of Ayurvedic formulations
containing Hg used in traditional medicines.
Arogyavardhini vati is a traditional Ayurvedic formulation used
for centuries with claimed efficacy and safety in treatment of jaun-
dice and other liver and skin disorders. It consists of Picrorrhiza
kurroa, Haritaki (Terminalia chebula), Bibhitaka (Terminalia bel-
lerica), Amalaki (Emblica officinalis), Silajatu-suddha (Asphaltum),
Pura (Guggulu), suddha (Oleo raisin Commiphora mukul), Citra
(Eranda – Ricinus communis), Tikta (Katuka), Nimba vrksa dalambha
(Neem leaves – Azadirachta indica), (nimba) – svarasa (Neem
leaves juice) and metal including rasa suddh (purified Mercury),
Gandhaka-suddha (purified Sulfur), Lauha-bhasma (iron com-
pound), abhra (abhraka)-bhasma (Mica), Sulva (tamra)-bhasma
(copper compound).
A double-blind trial for treatment of acute viral hepatitis was
conducted with Arogyavardhini by Antarkar et al. (1980) which had
showed significant hepatoprotective effects by Arogyavardhini. In
spite of this, safety issues are often raised because of Hg content.
Therefore, the present study was designed with an aim to evaluate
the safety of Arogyavardhini vati in experimental rats on brain, liver
and kidney.
2. Materials and methods
2.1. Experimental animal
Male Wistar rats (150–200 g) were used in the present study.
The animals were obtained from the Central Animal Facility, All
India Institute of Medical Sciences, New Delhi and stock bred in the
departmental animal house. The rats were group housed in poly-
acrylic cages (38 cm × 23 cm × 10 cm) with not more than 4 animals
per cage and maintained under standard laboratory conditions with
natural dark and light cycle. They were allowed free access to stan-
dard dry diet (Ashirwad, Punjab, India) and tap water ad libitum.
However, 12 h before the behavioral testing, the rats were deprived
of food as this is known to enhance their motivation to perform
the test. All experimental procedures described were reviewed and
approved by the Institutional Animal Ethics Committee, All India
Institute of Medical Sciences, New Delhi India (497/IAEC/09).
2.2. Drugs preparation and duration of treatment
Arogyavardhini vati (Maharshi Ayurveda Pharmaceutical Lim-
ited, New Delhi, India) tablet is, suspended in distilled water. HgCl2
(Merck, USA) solution was made using distilled water. Rats were
randomly divided into five groups consisting of 6 rats each, i.e. nor-
mal control, positive control (HgCl2 , 1 mg/kg) and Arogyavardhini
vati (50, 250 and 500 mg/kg) treated groups. The dose of mercury
chloride was selected to match the level of Hg in the top dose of
Arogyavardhini vati and this dose is also known to have deleterious
effect on brain, liver, and kidney. The doses of Arogyavardhini vati
(50, 250 and 500 mg/kg) in rat was calculated by extrapolating the
equivalent human dose (1, 5 and 10 times) and was administered
orally between 10 and 11 a.m. daily for 28 days, in a volume not
exceeding 1 ml/100 g rat weight. Neurobehavioral tests of rats were
performed on day 1, 7th, 14th, 28th. At end of the experiment,
blood samples were withdrawn from retro-orbital sinus and serum
was separated for biochemical estimation (liver and kidney func-
tions test). Animals were then euthanized and brain, liver, kidney
were removed to determine the levels of malondialdehyde (MDA),
reduced glutathione (GSH), acetyl-cholinesterase (AChE) activity
and mercury. A portion of brain, liver, kidney was kept in 10%
formalin for histopathological evaluation.
2.3. Neurobehavioral activity (cognition and motor coordination)
2.3.1. One trial passive avoidance task
Passive avoidance (Ugo Basile) was used to evaluate the memory
retention deficit according to the method described by Reeta et al.
(2009). In acquisition trial, rats were placed in a lighted chamber
and a guillotine door separating the light and dark chambers was
opened. Initial latency (IL) to enter the dark chamber was recorded.
Immediately after the rat enters the dark chamber, the guillotine
door was closed and an electric foot shock (0.2 mA) was delivered to
the floor grids for 3 s. The rat was removed from the dark chamber
5 s later and returned to its home cage. After 24 h, retention latency
(RL) time was noted in the same way as in the acquisition trial, but
foot shock was not delivered.
2.3.2. Morris water maze
Morris water maze was used to evaluate the learning and mem-
ory. The Morris water maze consisted of a large circular pool filled
with water (1.8 m in diameter, 0.6 m in height) and a platform
(10 cm in diameter) submerged 1 cm below the water’s surface. An
automated tracking system (Video tracking system, Stoelting, USA)
analyzed the total path lengths. Rats were given four acquisition
sessions with an inter-trial interval of 10 min. Once a rat located
the platform, it was allowed to remain there for 10 s before being
removed from the tank. If a rat failed to locate the platform within
120 s, it was manually guided to it (Morris, 1984).
2.3.3. Elevated plus maze
Elevated plus maze was used to evaluate the memory retention
deficit according to the method described by Reeta et al. (2009).
Rats were placed at one end of an open arm, facing away from the
central square. Time taken by the rat to move from open arm to
closed arms was recorded and marked as initial transfer latency
(ITL). The animals were allowed to explore the maze for 30 s after
recording initial transfer latency. Retention transfer latency (RTL)
was recorded by placing the rats similarly on the open arm at spec-
ified intervals.
2.3.4. Rota rod
Rota rod was used to evaluate the muscle coordination of rats.
Rats were conditioned to the accelerating rod (Ugo Basile). Each
animal received a training session on the rota rod at constant speed
of 8 rpm and was tested until it achieved the criteria of remaining on
the rotating spindle for 60 s. Each rat received a single base line trial
on the accelerating rota rod in which the spindle speed increased
from 4 to 40 rpm over a period of 5 min. After administration of
drugs for 28 days, each rat received a test trial (Gupta et al., 2005).
2.4. Biochemical estimation and histopathology
At end of the behavioral experiments, blood was withdrawn
by puncturing the retro-orbital sinus for biochemical estimation.
 G. Kumar et al. / Journal of Ethnopharmacology 140 (2012) 151–160
Serum was separated to measure liver and kidney functions test
parameters. Animals were then decapitated under ether anesthe-
sia and brain, liver, kidney was quickly removed. Half of the tissues
were cleaned with ice cold saline and stored at −80 ◦ C to determine
the level of MDA, GSH, AChE activity and Hg and remaining tissue
was kept in 10% formalin for histopathological evaluation.
2.4.1. Acetylcholinesterase (AChE) activity in brain
AChE activity was done in the rat’s brain according to the
method of Ellman et al. (1961). Enzyme activity was expressed as
change in optical density/min/mg protein. Protein estimation was
carried out by the method of Lowry et al. (1951).
2.4.2. Measurements of MDA and GSH levels in brain, liver and
kidney
Malondialdehyde, an indicator of lipid peroxidation was deter-
mined by the method of Ohkawa et al. (1979). The concentration
was expressed in nmol/g-wet tissue. Glutathione was measured
according to the method of Ellman (1959) and concentration was
expressed as mg/g-wet tissue.
2.4.3. Determination of liver and kidney function test
To assess the state of the liver and kidney, serum aspartate
aminotransferase (AST), alanine aminotransferase (ALT), alkaline
phosphatase (ALP), bilirubin, urea and craetinine levels were
estimated by using semi auto analyzer (Mini techno, USA). Concen-
tration of the enzymes was evaluated according to the instruction
of manufacturer of assay kits (Logitech India Pvt. Ltd, Delhi, India).
2.4.4. Mercury estimation in brain, liver and kidney tissues
Mercury level was estimated in tissue by inductively coupled
plasma–atomic emission spectrophotometer (ICP-AES, JY 2000-2,
France). Brain, liver and kidney tissues were digested by cold vapors
digestion procedure according to the method of Jacobs et al. (1960).
Hg levels were expressed in ␮g/g-wet tissue.
2.4.5. Histopathological study
Tissue specimens from brain, liver, and kidney of all experimen-
tal rats were collected at the end of the study and fixed in 10%
formalin, processed by conventional method, embedded in paraf-
fin, sectioned at 4–5 ␮m and stained by Haematoxylin and Eosin
(Bancroft et al., 1991). Tissues were examined under a light micro-
scope (Nikon, Japan). Histopathological evaluation was carried out
by Department of Pathology, AIIMS, blinded to the groups.
2.5. Statistical analysis
Data are expressed as mean ± SEM (Standard Error of the Mean).
A one-way analysis of variance (ANOVA), followed by Tukey test
was used for statistical analysis. SPSS (version 16) statistical soft-
ware was used for the analysis of data and p < 0.05 was taken as the
level of significance.
3. Results
3.1. Effect of Arogyavardhini vati on passive avoidance task
(step-through paradigm)
There was no significant change in latencies of Arogyavardhini
vati treated groups (50, 250 and 500 mg/kg) as compared to normal
control on day 1, 2, 7, 14 and 28. However, there was significant
decrease in mean latency of HgCl2 treated group on day 28th as
compared to normal control group (p < 0.001), though no change in
mean latency on day 1, 2nd, 7th and 14th was observed (Fig. 1).
153
3.2. Effect of Arogyavardhini vati on Morris water maze
Arogyavardhini vati treated groups (50, 250 and 500 mg/kg) did
not show significant change in total distance traveled during the
acquisition trials and probe trial to reach the platform on day 1, 4,
7, 14 and 28 as compared to normal control group. However, there
was significant increase in distance traveled during probe trial on
day 28th to reach the platform of HgCl2 treated group as compared
to normal control (p < 0.001) (Fig. 2).
3.3. Effect of Arogyavardhini vati on elevated plus maze
There was no significant change in latencies of Arogyavardhini
vati treated groups (50, 250 and 500 mg/kg) as compared to normal
control on day 1, 2, 7, 14 and 28. However, there was significant
(p < 0.001) increase in mean retention transfer latencies on day 28th
of HgCl2 treated group as compared to normal control group which
indicate impairment of cognitive function (Fig. 3).
3.4. Effect of Arogyavardhini vati on rota rod
Arogyavardhini vati (50, 250 and 500 mg/kg) treated groups did
not show significant change in the time spent on the spindle of the
rota rod as compared to normal control group on day 1, 7, 14 and 28.
However, there was significant decrease in time spent on spindle of
HgCl2 treated group on day 28th compared to control group which
showed motor incoordination (p < 0.001) (Fig. 4).
3.5. Effect of Arogyavardhini vati on AChE activity in frontal
cortex and hippocampus
There was no significant difference in AChE activity of Aro-
gyavardhini vati treated groups (50, 250 and 500 mg/kg) as
compared to normal control group in frontal cortex as well as in
hippocampus. However, there was significant (p < 0.001) decrease
in AChE activity in frontal cortex as well as in hippocampus of HgCl2
treated group as compared to normal control group (Fig. 5).
3.6. Effect of Arogyavardhini vati on MDA and GSH levels in
brain, liver and kidney
There was no significant difference in MDA level at all doses
of Arogyavardhini vati treated groups (50, 250 and 500 mg/kg) as
compared to normal control group in brain, liver and kidney. GSH
level at all doses of Arogyavardhini vati treated groups (50, 250
and 500 mg/kg) were akin compared to normal control group in
brain, liver and kidney. However, a significantly increased MDA
and decreased GSH levels were observed in the brain, liver and kid-
ney of HgCl2 treated group as compared to normal control group
(p < 0.001) (Table 1).
3.7. Effect of Arogyavardhini vati on mercury level in brain, liver
and kidney
There was significant (p < 0.001) increase in Hg levels with
increasing doses of Arogyavardhini vati treated groups (50, 250 and
500 mg/kg) and HgCl2 treated group as compared to normal control
group in brain, liver and kidney (Table 2).
3.8. Effect of Arogyavardhini vati on liver and kidney function
test parameters
There was no significant change in the serum ALT, AST, ALP,
bilirubin, urea and creatinine levels of Arogyavardhini vati treated
groups (50, 250 and 500 mg/kg) as compared the normal control
154 G. Kumar et al. / Journal of Ethnopharmacology 140 (2012) 151–160

Fig. 1. Effect of Arogyavardhini vati on step through latency in rat. Values are expressed as mean ± SEM. ***p < 0.001, as compared to normal control.

Fig. 2. Effect of Arogyavardhini vati on Morris water maze in rat. Values are expressed as mean ± SEM. ***p < 0.001, as compared to normal control.
 G. Kumar et al. / Journal of Ethnopharmacology 140 (2012) 151–160 155

Fig. 3. Effect of Arogyavardhini vati on elevated plus maze in rat. Values are expressed as mean ± SEM. ***p < 0.001, as compared to normal control.

Fig. 4. Effect of Arogyavardhini vati on rota rod in rat. Values are expressed as mean ± SEM. ***p < 0.001, as compared to normal control.

Table 1
Effect of Arogyavardhini vati on MDA & GSH of rat’s brain, liver and kidney tissue.

Treatment groups MDA (nmol/g wet-tissue) GSH (mg/g wet-tissue)

Brain Liver Kidney Brain Liver Kidney

Normal control 84.59 ± 4.31 64.66 ± 1.48 180.29 ± 3.48 2.23 ± 0.06 3.31 ± 0.21 4.02 ± 0.09
Mercury chloride (1 mg/kg) 122.40 ± 16.99*** 176.15 ± 6.79*** 242.49 ± 1.64*** 1.07 ± 0.15*** 1.71 ± 0.18*** 1.64 ± 0.21***
Arogyavardhini vati (50 mg/kg) 79.21 ± 3.22 67.50 ± 1.90 183.87 ± 3.22 2.31 ± 0.04 3.17 ± 0.25 3.99 ± 0.04
Arogyavardhini vati (250 mg/kg) 78.49 ± 5.25 67.73 ± 2.28 186.74 ± 5.26 2.25 ± 0.02 3.15 ± 0.07 3.85 ± 0.03
Arogyavardhini vati (500 mg/kg) 70.97 ± 3.75 69.11 ± 1.80 191.04 ± 3.75 2.27 ± 0.02 3.06 ± 0.29 3.64 ± 0.02

All values are expressed as mean ± SEM. Statistical analysis by one-way ANOVA followed by Tukey multiple comparisons.
***
p < 0.001, as compared to normal control.
156 G. Kumar et al. / Journal of Ethnopharmacology 140 (2012) 151–160

Fig. 5. Effect of Arogyavardhini vati on rat frontal cortex and hippocampus acetylcholinesterase. Values are expressed as mean ± SEM. ***p < 0.001, as compared to normal
control (Hippocampus); ### p < 0.001, as compared to normal control (frontal cortex).

Table 2
Effect of Arogyavardhini vati on mercury level of rat’s brain, kidney and liver.

Treatment groups Mercury level (␮g/g wet-tissue)

Brain Liver Kidney

Normal control 1.84 ± 0.17 13.40 ± 1.03 12.49 ± 1.34

Mercury chloride (1 mg/kg) 151.03 ± 14.13*** 775.02 ± 34.07*** 948.78 ± 19.70***
Arogyavardhini vati (50 mg/kg) 7.11 ± 1.12*** 21.01 ± 2.98*** 23.68 ± 3.78***
Arogyavardhini vati (250 mg/kg) 9.36 ± 1.36*** 26.50 ± 1.89*** 30.34 ± 5.4***
Arogyavardhini vati (500 mg/kg) 13.34 ± 1.90*** 27.83 ± 3.21*** 34.31 ± 2.64***

All values are expressed as mean ± SEM. Statistical analysis by one-way ANOVA followed by Tukey multiple comparisons.
***
p < 0.001, as compared to normal control.

Table 3
Effect of Arogyavardhini vati on rat’s liver and kidney functions test.

Treatment groups Liver functions tests Kidney function tests

ALT (IU/l) AST (IU/l) ALP (IU/l) Bilirubin (mg/dl) Urea (mg/dl) Creatinine (mg/dl)

Normal control 129.48 ± 1.39 106.50 ± 1.74 41.92 ± 0.53 0.13 ± 0.01 16.32 ± 0.78 0.43 ± 0.03
Mercury chloride (1 mg/kg) 223.78 ± 12.01*** 191.64 ± 7.38*** 104.20 ± 6.46*** 1.09 ± 0.09*** 42.98 ± 3.07*** 1.19 ± 0.09***
Arogyavardhini vati (50 mg/kg) 127.75 ± 1.82 105.08 ± 1.88 42.52 ± 0.86 0.16 ± 0.02 17.70 ± 1.76 0.45 ± 0.04
Arogyavardhini vati (250 mg/kg) 126.25 ± 2.29 104.10 ± 1.83 40.95 ± 0.79 0.14 ± 0.01 16.55 ± 1.58 0.42 ± 0.04
Arogyavardhini vati (500 mg/kg) 127.67 ± 1.56 103.68 ± 1.25 39.75 ± 0.77 0.11 ± 0.01 14.80 ± 1.28 0.40 ± 0.03

All values are expressed as mean ± SEM. Statistical analysis by one-way ANOVA followed by Tukey multiple comparisons.
***
p < 0.001, as compared to normal control.

group, while significant (p < 0.001) change was observed in HgCl2
treated group (Table 3).
3.9. Effect of Arogyavardhini vati on brain, liver and kidney
histology
The brain, liver and kidney of normal control and Arogyavard-
hini vati treated groups (50, 250 and 500 mg/kg) showed normal
cytoarchitecture. However, HgCl2 treated group showed expected
toxicities, i.e. necrosis of neurons in brain, inflammed periportal
zone in liver and disruption of epithelium in proximal convoluted
tubules in kidney (Figs. 6–8).
4. Discussion and conclusion
Ayurvedic medicines are herbal based traditional medicines
(Gogtay et al., 2002; Chopra and Doiphode, 2002). The heavy met-
als in Ayurvedic products are being added intentionally according
to Ayurvedic formulary. The presence of heavy metals is not con-
taminants but is unavoidable in any herbometallic formulation.
Ayurvedic experts have estimated that 35–40% of the approxi-
mately 600 medicines mentioned in the Ayurvedic formulary may
contain at least one metal (Gogtay et al., 2002). The metals in
Ayurvedic formulations may not be present in elemental form
because during the process of sodhna, they may not retain their
original physiochemical form. Therefore, the mere presence of a
chemical compound of metallic origin has nothing to do with the
 G. Kumar et al. / Journal of Ethnopharmacology 140 (2012) 151–160
Fig. 6. Effect of Arogyavardhini vati on rat’s brain histology. Light micrograph of (a) control rat brain showing normal architecture (H&E ×10). (b) 1 mg/kg/day, HgCl2 treated
rat showing pyknosis of neurons, congestion of blood vessels and necrosis of Purkinje cells of the cerebellum (H&E ×20). Arogyavardhini vati treated rat (c) 50, (d) 250, (e)
500 mg/kg/day, showing no histopathological changes (H&E ×10).
toxicity of the finished product after reaction with several organic
and inorganic materials of herbal origin, which is finally responsi-
ble for pharmacological effect (Gogtay et al., 2002; Chauhan, 2008;
Garnier and Poupon, 2006; Dash, 1986).
The US Environmental Protection Agency (EPA, 2001) has
adopted a reference dose (RfD) for methyl Hg of 0.1 ␮g/kg body
weight/day. Arogyavardhini vati formulation used in the present
study contains total Hg of 1841.36 ␮g/g. The calculated total
ingested Hg at doses of 50, 250 and 500 mg/kg were 92.07, 460.35
and 920.70 ␮g/kg, respectively. The dose of HgCl2 in the study is
1 mg/kg which is approximately the same as the high dose of Aro-
gyavardhini vati. Thus, in therapeutic dose of Arogyavardhini vati
(500 mg/day), Hg ingested per day was many fold higher than the
reference dose. It was interesting to note that even this high con-
centration of Hg in Arogyavardhini vati for 28 days did not cause
significant toxicity on liver, kidney and brain. The nonoccurrence
of toxicity could be due to the fact that Ayurvedic detoxification
process (Sodhana) might have contributed in some modification
of metal property resulting abolition of toxicity, yet retaining its
pharmacological property.
Hg exposure causes neurotoxicity, hepatotoxicity and nephro-
toxicity have been reported in several studies (Rodin and Crowson,
1962; Gritzka and Trump, 1968; Von Berg and Greenwood, 1991;
El-Shenawy and Hassan, 2008). Cognitive impairment has also been
reported in mercury treated rats (Baraldi et al., 2002; Coluccia et al.,
2007). ACh level has found to be decreased in Alzheimer’s dis-
ease (Frasco et al., 2005). In the present study, though the effect
of Arogyavardhini vati on the ChAT activity has not been evaluated
157
but its involvement in HgCl2 induced cognitive impairment can-
not be ruled out. There are several studies which shows decreased
ChAT and AChE activity in brain tissues after Hg exposure (Dwivedi
et al., 1980; Frasco et al., 2005). However, Arogyavardhini vati
(50, 250 and 500 mg/kg), administered orally for 28 days did not
affect the AChE activity in frontal cortex and hippocampus, cog-
nitive and motor function of rat as compared to normal control
group. There are plethora of experimental studies on Hg based
Ayurvedic formulations which have shown safety at recommended
dose and duration, i.e. Samirpannag Rasa (Ravi and Jha, 1995), Rasa
Karpur (Rao and Joshi, 1991) and Rasa Sindoor (Kumar and Joshi,
1992).
Oxidative stress is an important mechanism for tissue injury
in Hg poisoning (Halliwell and Gutteridge, 1984; Ichikawa et al.,
1994; Ward et al., 1994). The toxicity of HgCl2 is attributed to the
high affinity of Hg(II) to thiol groups owing to depletion of cellu-
lar GSH (Augusti et al., 2007) and production of free radicals are
involved in Hg-induced tissue injury (Stohs and Bagchi, 1995; Nath
et al., 1996; Zalups, 2000; Jagadeesan and Sankarsami, 2007). In our
study, Arogyavardhini vati (50, 250 and 500 mg/kg) did not affect
the MDA and GSH levels in brain, liver and kidney. This indicates
that Arogyavardhini vati does not cause oxidative stress.
Hg level in tissue has been widely used as a biomarker for mon-
itoring the extent of mercurial exposure (Grandjean et al., 1999).
There is evidence that chronic exposure to low concentration of Hg
causes tissue injury (Sug et al., 1997). In the present study, there was
significant increased Hg level in brain, liver and kidney at all doses
of Arogyavardhini vati (50, 250 and 500 mg/kg) as compared to the
158 G. Kumar et al. / Journal of Ethnopharmacology 140 (2012) 151–160

Fig. 7. Effect of Arogyavardhini vati on rat’s liver histology. Light micrograph of (a) control rat liver showing normal architecture (H&E ×10). (b) 1 mg/kg/day, HgCl2 treated
rat showing pyknosis, vascular, degenerative and necrotic changes in the liver (H&E ×20). Arogyavardhini vati treated rat (c) 50, (d) 250, (e) 500 mg/kg/day, showing no
histopathological changes (H&E ×10).

Fig. 8. Effect of chronic administration of Arogyavardhini vati on rat’s kidney histology. (a) The kidneys of normal control show normal histological architecture. (b) In
mercury chloride (HgCl2 ) treated group, congestion of blood vessels and epithelium disruption in proximal convoluted tubules was observed. Arogyavardhini vati treated
rat (c) 50, (d) 250, (e) 500 mg/kg/day, showing no histopathological changes (H&E ×10).
 G. Kumar et al. / Journal of Ethnopharmacology 140 (2012) 151–160
normal control group. The presence of Hg indicates the mercury as
one of the ingredient of Arogyavardhini vati.
There are numerous studies which have shown significant ele-
vations in serum ALT, AST, ALP, bilirubin due to mercury exposure
(Kumar et al., 2005; Jagadeesan and Sankarsami, 2007; El-Shenawy
and Hassan, 2008). This elevation of liver enzymes serves as a
marker to evaluate functional status of the liver. In our study, no sig-
nificant change in serum ALT, AST, ALP or bilirubin was observed at
all the three doses of Arogyavardhini vati (50, 250 and 500 mg/kg).
In our study, no statistically significant change in serum urea
and creatinine level was observed at 50, 250 and 500 mg/kg of
Arogyavardhini vati in rats. The results of the present study show
that Arogyavardhini vati administered orally for 28 days, did not
affect kidney functions in rats. A study has shown that HgCl2 treat-
ment causes a significant increase in serum creatinine and serum
urea nitrogen indicating an impaired renal function (Siddiqi and
Alhomida, 2005).
Inorganic Hg has also been shown to accumulate in the renal cor-
tex and affect the morphology and function of the proximal tubules
(Greaves, 2000). A study by Jadhav et al. (2007) has also observed
dose-dependent vascular, degenerative and necrotic changes in the
brain and liver of male rats exposed to mercury via drinking water
(Jadhav et al., 2007). In the present study, normal architecture of
brain, liver and kidney was seen at all doses of Arogyavardhini vati
(50, 250 and 500 mg/kg).
In conclusion, the findings of the present study suggest that
Arogyavardhini vati in the doses equivalent up to 10 times of the
human dose administered to rats for 28 days does not have appre-
ciable toxicological effects on brain, liver and kidney. There were
no significant changes in biochemical parameters or histopathol-
ogy (brain, liver, and kidney) at all doses of Arogyavardhini vati.
This was despite dose-related increases in the level of Hg being
present in tissues. Further confirmations of lack of effects after
chronic administration are needed as this is more relevant for some
of the neurological effects seen with Hg ingestion. However, the
results from the present study and clinical use of Arogyavardhini
vati over centuries in traditional Indian System of Medicine sup-
port the notion that Arogyavardhini vati at the recommended dose
and duration is safe for human use.
Acknowledgements
We thank Dr. A.K. Dinda, Additional Professor, Department of
Pathology, All India Institute of Medical Sciences, New Delhi for
his important suggestions and expertise with histopathology. The
financial support by Central Council for Research in Ayurveda and
Siddha (CCRAS), Department of AYUSH, Ministry of Health and
Family Welfare, Government of India for this research work is duly
acknowledged (F. No. Z31014/04/2009/EMR-CCRAS).
References
Antarkar, D.S., Vaidya, A.B., Doshi, J.C., Athavale, A.V., Vinchoo, K.S., Natekar, M.R.,
Tathed, P.S., Ramesh, V., Kale, N., 1980. A double-blind clinical trial of Arogya-
wardhani—an ayurvedic drug—in acute viral hepatitis. Indian Journal of Medical
Research 72, 588–593.
ATSDR, 1999. Toxicological Profile for Mercury (update). Agency for Toxic Sub-
stances and Disease Registry, Atlanta, USA, p. 485.
Augusti, P.R., Conterato, G.M.M., Somacal, S., Einsfeld, L., Ramos, A.T., Hosomi, F.Y.M.,
2007. Effect of lycopene on nephrotoxicity induced by mercurychloride in rats.
Basic & Clinical Pharmacology & Toxicology 100, 398–402.
Bancroft, J.D., Stevens, A., Turner, D.R., 1991. Theory and Practice of Histological
Techniques. Churchill Livingstone, New York, p. 111.
Baraldi, M., Zanoli, P., Tascedda, F., Blom, J.M., Brunello, N., 2002. Cognitive deficits
and changes in gene expression of NMDA receptors after prenatal methylmer-
cury exposure. Environment Health Perspectives 110, 855–858.
Chauhan, P., 2008. Ayurvedic Metallic Medicines are not fatal. Available from:
http://www.wellsphere.com/healthy-living-article/8220-bhasmas-8221-are-
medicinal 8211ayurvedic-metallic-medicines-are-not-fatal/939018.
159
Chopra, A., Doiphode, V.V., 2002. Ayurvedic medicine; core concept, therapeutic
principles, and current relevance. Medical Clinics of North America 86, 75–89.
Coluccia, A., Borracci, P., Giustino, A., Sakamoto, M., Carratu, M.R., 2007. Effects of low
dose methylmercury administration during the postnatal brain growth spurt in
rats. Neurotoxicology & Teratology 29, 282–287.
Cooper, K., Noller, B., Connell, D., Yu, J., Sadler, R., Olszowy, H., Golding, G., Tinggi,
U., Moore, M.R., Myers, S., 2007. Public health risks from heavy metals and
metalloids. Journal of Toxicology & Environmental Health A 70, 1694–1699.
Cordier, S., Garel, M., Mandereau, L., Morcel, H., Doineau, P., Gosme-Seguret, S., 2002.
Neurodevelopmental investigations among methylmercury-exposed children
in French Guiana. Environmental Research 89, 1–11.
Dash, V.B., 1986. Alchemy and Metallic Medicines in Ayurveda. Concept Publications,
New Delhi, pp. 1–5 and 48–92.
Dwivedi, C., Raghunathan, R., Joshi, B.C., Foster, H.W., 1980. Effect of mercury com-
pounds on cholineacetyl transferase. Research Communicational in Chemical
Pathology & Pharmacology 30, 381–384.
Ellman, G.L., Courtney, K.D., Andres, V., Featherstone, R.M., 1961. A new and rapid
colorimetric determination of acetylcholinesterase activity. Biochemical Phar-
macology 7, 88–95.
Ellman, G.L., 1959. Tissue sulfhydryl groups. Archives of Biochemistry & Biophysics
82, 70–77.
El-Shenawy, S.M.A., Hassan, N.S., 2008. Comparative evaluation of the protective
effect of selenium and garlic against liver and kidney damage induced by mer-
cury chloride in the rats. Pharmacological Reports 60, 199–208.
Farina, M., Soares, F.A., Zeni, G., Souza, D.O., Rocha, J.B., 2004. Additive prooxidative
effect of methyl mercury and ebselen in liver from suckling rat pups. Toxicology
Letters 146, 227–235.
Frasco, M.F., Fournier, D., Carvalho, F., Guilhermino, L., 2005. Do metals inhibit acetyl-
cholinesterase (AChE)? Implementation of assay conditions for the use of AChE
activity as a biomarker of metal toxicity. Biomarkers 10, 360–375.
Garnier, R., Poupon, J., 2006. Lead poisoning from traditional Indian medicines.
Presse Medicale 35, 1177–1180.
Gogtay, N.J., Bhatt, H.A., Dalvi, S.S., Kshirsagar, N.A., 2002. The use and safety of
non-allopathic Indian medicines. Drug Safety 25, 1005–1019.
Grandjean, P., Budtz-Jorgensen, E., White, R.F., 1999. Methylmercury exposure
biomarkers as indicators of neurotoxicity in children aged 7 years. American
Journal of Epidemiology 150, 301–305.
Greaves, P., 2000. Histopathology of Preclinical Toxicity Studies: Interpretation and
Relevance in Drug Safety Evaluation, 2nd ed. Elsevier Science, Amsterdam.
Gritzka, T.L., Trump, B.F., 1968. Renal tubular lesions caused by mercurychloride:
electron microscopic observations: degeneration of the pars recta. American
Journal of Pathology 52, 1225–1277.
Gupta, Y.K., Briyal, S., Sharma, U., Jagannathan, N.R., Gulati, A., 2005. Effect of
endothelin antagonist (TAK-044) on cerebral ischemic volume, oxidative stress
markers and neurobehavioral parameters in the middle cerebral artery occlu-
sion model of stroke in rats. Life Sciences 77, 15–27.
Halliwell, B., Gutteridge, J.M.C., 1984. Oxygen toxicity, oxygen radicals, transition
metals and disease. Biochemical Journal 219, 1–14.
Ichikawa, I., Kiyama, S., Yoshioka, T., 1994. Renal antioxidant enzymes: their regu-
lation and function. Kidney International 45, 1–9.
Jacobs, M.B., Yamaguchi, S., Goldwater, L.G., Gilbert, H., 1960. Determination
of mercury in blood. American Industrial Hygiene Association Journal 21,
475–480.
Jadhav, S.H., Sarkar, S.N., Aggarwal, M., Tripathi, H.C., 2007. Induction of oxidative
stress in erythrocytes of male rats subchronically exposed to a mixture of eight
metals found as groundwater contaminants in different parts of India. Archives
of Environmental Contamination and Toxicology 52, 145–151.
Jagadeesan, G., Sankarsami, P.S., 2007. Hepatoprotective effects of taurine against
mercury induced toxicity in rats. Journal of Environmental Biology 28, 753–756.
Kang-Yum, E., Oransky, S.H., 1992. Chinese patent medicine as a potential source of
mercury poisoning. Veterinary Human Toxicology 34, 34–58.
Kapoor, R.C., 2010. Some observations on the metal-based preparations in the Indian
system of medicine. Indian Journal of Traditional Knowledge 9, 562–575.
Kumar, A., Joshi, D., 1992. Study on the role of Gandhak jaran in relation to mercury
and its preparation Rasa Sindur. MD thesis Department of Rasa Shastra, IMS,
BHU, Varanasi.
Kumar, M., Sharma, M.K., Kumar, A., 2005. Spirulina fusiformis: a food supplement
against mercury induced hepatic toxicity. Journal of Health Sciences 5, 424–430.
Laksmanna, M.K., Desiraju, T., Raju, T.R., 1993. Mercurychloride induced alterations
of levels of noradrenaline, dopamine, serotonin and acetylcholinesterase activ-
ity in different regions of rat brain during postnatal development. Archives of
Toxicology 167, 422–427.
Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurement
with Folin-phenol reagent. Journal of Biological Chemistry 193, 265–275.
Lynch, E., Braithwaite, R., 2005. A review of the clinical and toxicological aspects of
‘traditional’ (herbal) medicines adulterated with heavy metals. Expert Opinion
on Drug Safety 4, 769–778.
Morris, R., 1984. Developments of a water–maze procedure for studying spatial
learning in the rat. Journal of Neuroscience Methods 11, 47–60.
Nath, K.A., Croatt, A.J., Likely, S., 1996. Renal oxidant injury and oxidant response
induced by mercury. Kidney International 50, 1032–1043.
Ohkawa, H., Ohishi, N., Yagi, K., 1979. Assay for lipid peroxides in animal’s tissues
by thiobarbituric acid reaction. Analytical Biochemistry 95, 351–358.
Rao, P., Joshi, D., 1991. Study of Rasa Karpura (standardization evaluation of toxicity
and antibacterial activity. MD thesis, Department of Rasa Shastra, IMS, BHU,
Varanasi.
160 G. Kumar et al. / Journal of Ethnopharmacology 140 (2012) 151–160
Ravi, P., Jha, C.B., 1995. Study of Samirpannag Rasa (with special reference to its phar-
maceutical chemical and experimental study. MD thesis, IMS, BHU, Varanasi.
Reeta, K.H., Mehla, J., Gupta, Y.K., 2009. Curcumin is protective against phenytoin-
induced cognitive impairment and oxidative stress in rats. Brain Research 1301,
52–60.
Remuzzi, G., Bertani, T., 1998. Pathphysiology of progressive nephropathies. New
England Journal of Medicine 339, 1448–1456.
Rodin, A.E., Crowson, C.N., 1962. Mercury nephrotoxicitv in the rat and factors influ-
encing the localization of tubular lesions. American Journal of Pathology 41,
297–313.
Saper, R.B., Phillips, R.S., Sehgal, A., Khouri, N., Davis, R.B., Paquin, J., et al., 2008. Lead,
mercury, and arsenic in US- and Indian-manufactured Ayurvedic medicines sold
via the Internet. Journal of American Medical Association 300, 915–923.
Satpute, A.D., 2003. Rasa Ratna Samuchaya of Vagbhatta, trans., Chaukhamba San-
skrit Pratishtana, Varanasi, India.
Sharma, M.K., Sharma, A., Kumar, A., Kumar, M., 2007. Spirulina fusiformis provides
protection against mercurychloride induced oxidative stress in Swiss albino
mice. Food & Chemical Toxicology 45, 2412–2419.
Shastri K., 1979. Rasa Tarangini of Sadananda Sharma, trans., Motilal Banarsidas,
India.
Siddiqi, N.J., Alhomida, A.S., 2005. Effect of mercurychloride on various hydroxypro-
line fractions in rat serum. Molecular & Cellular Biochemistry 271, 159–165.
Steuerwald, U., Weihe, P., Jorgensen, P.J., Bjerve, K., Brock, J., Heinzow, B., 2000.
Maternal seafood diet, methylmercury exposure and neonatal neurologic func-
tion. Journal of Pediatrics 136, 599–605.
Stohs, S.J., Bagchi, D., 1995. Oxidative mechanisms in the toxicity of metal ions. Free
Radical Biology & Medicine 18, 321–336.
Sug, O., Datar, S., Koch, C.J., 1997. Mercuric compounds inhibit human monocyte
function by inducing apoptosis: evidence for formation of reactive oxygen
species, development of mitochondrial membrane permeability transition and
loss of reductive reserve. Toxicology 124, 211–224.
Upadhyay, P.S., Kumar, M., Singh, B.M., 2008. An experimental study on Shvasa-
kasa-chintamani-rasa. Journal of Research & Education in Indian Medicine 14,
21–26.
US Environmental Protection Agency (USEPA), 2001. Water Quality Criterion for
the Protection of Human Health: Methylmercury. EPA Offices of Sciences and
Technology, Washington.
Von Berg, R., Greenwood, M.R., 1991. Mercury: Metal and Their Compound in Envi-
ronment. VCH Publisher, New York, pp. 1045.
Ward, R.J., Abiaka, C., Peters, T.J., 1994. Inflammation and tissue injury: the world of
free radicals. Journal of Nephrology 7, 89–95.
Zalups, R.K., 2000. Molecular interactions with mercury in the kidney. Pharmaco-
logical Reviews 52, 113–114.