Evaluation of safety profile of black shilajit after 91 days repeated administration in rats

Abstract

To evaluate the safety of shilajit by 91 days repeated administration in different dose levels in rats.
In this study the albino rats were divided into four groups. Group I received vehicle and group II, III and IV received 500, 2 500 and 5 000 mg/kg of shilajit, respectively. Finally animals were sacrificed and subjected to histopathology and iron was estimated by flame atomic absorption spectroscopy and graphite furnace.
The result showed that there were no significant changes in iron level of treated groups when compared with control except liver (5 000 mg/kg) and histological slides of all organs revealed normal except negligible changes in liver and intestine with the highest dose of shilajit. The weight of all organs was normal when compared with control.
The result suggests that black shilajit, an Ayurvedic formulation, is safe for long term use as a dietary supplement for a number of disorders like iron deficiency anaemia.

Full text

210
Document heading
Evaluation of safety profile of black shilajit after 91 days repeated
administration in rats
Velmurugan C
1
*
, Vivek B
2
, Wilson E
3
, Bharathi T
4
, Sundaram T
5
1
Department of Pharmacology, Sri Krishna Chaithanya College of Pharmacy, Nimnapalli Road, Madanapalle, Andhra Pradesh
2
Department of Pharmacology, Sri K.V College of Pharmacy, M.G Road, Chickballapur, Karnataka
3
SRF, National Institute of Indian Medical Heritage, Hyderabad, India
4
Department of Pharmaceutical Biotechnology, Sri Krishna Chaithanya College of Pharmacy, Nimnapalli Road, Madanapalle, Andhra Pradesh
5
Department of Pharmaceutics, Sri Krishna Chaithanya College of pharmacy, Nimmanapalli Road, Madanapalle, Andhra Pradesh
Asian Pacific Journal of Tropical Biomedicine (2012)210-214
Asian Pacific Journal of Tropical Biomedicine
journal homepage:www.elsevier.com/locate/apjtb
*Corresponding author: Velmurugan C, Assistant Professor, Department of
Pharmacology, Sri Krishna Chaithanya College of Pharmacy, Nimmanapalle Road,
Gangannagaripalle, Madanapalee-517325, Chittoor District, Andhra Pradesh, India.
E-mail: velu0906@gmail.com
Foundation Project: This work was financially supported by Shanmugha Arts, Science,
Technology and Research Academy University (grant No. 23/SASTRA/IAEC/RPP).
1. Introduction
Shilajit is considered as one of the wonder medicines
of
Ayurveda. Neither a plant nor animal substance, it is a
mineral pitch that oozes from the rocks of the
Himalayas,
as they become warm in the summer months.
Shilajit
(asphaltum) is widely used in the preparation of Ayurvedic
medicines and is regarded as one of the most important
ingredients in
Ayurvedic system of medicine. It is used as
a
n adaptogen
[1]
. Shilajit, also known as shilajit, mumijo,
and momia, is used in the
Ayurveda, the traditional Indian
system of medicine
[2]
. The complement system is involved
in many disease syndromes that have been traditionally
treated with extracts of shilajit and other humic substances
containing high levels of
Fe (e.g., arthritis and asthma)
[3,4]
and they are also used for anti-oxidant, hypoglycemic
and
iron deficiency anemia
[5,6]
. In the Charak Samhita,
shilajit is described as a product of four minerals i.e. gold,
silver, copper and iron, whereas in the
Susruta Samhita
it included two more minerals, i.e. lead and zinc in its
composition
[7]
. Iron has long been considered important
for the body. Lauha bhasma
(calcined iron) has been used
in ancient
Indian medicine. Iron was used for weakness,
which is common in anaemi
a. In 1713 iron was shown to be
present in blood
[8]
. The most obvious clinical result of iron
deficiency is anaemia, and the only indication for therapy
with iron is to provide material for haemoglobin synthesis
[9]
.
The bioavailability of iron in the colon was increased by
prebiotic and synbiotic diets
[10]
, it is mainly stored in liver
and spleen in the form of ferritin which is used to meet
the daily re
quirements not provided by the diet. Nearly all
cells contain ferritin.
The ferritin will release the Fe
+++
to
circulating blood to meet their deficiency, and aggregated
ferritin was referred to as haemosiderin
[9]
. Iron deficiency
is the most common cause of anemia, or a low red blood
cell count.
Lack of iron slows production of the iron-
containing hemoglobin, which limits the rate of red blood
cell
production. Similarly, iron deficiency causes a reduced
concentration of myoglobin in the muscles, commonly
leading to weakness and poor physical endurance.
The
ARTICLE INFO ABSTRACT
Article history:
Received 19 August 2011
Received in revised form 10 September 2011
Accepted 2 October 2011
Available online 28 March 2012
Keywords:
Shilajit
Graphite furnace
Histopathology
Flame atomic absorption spectroscopy
Objective:
To evaluate the safety of shilajit by 91 days repeated administration in different
dose levels in rats.
Methods:
In this study the albino rats were divided into four groups.
Group I received vehicle and group II, III and IV received 500, 2 500 and 5 000 mg/kg of shilajit,
respectively.
Finally animals were sacrificed and subjected to histopathology and iron was
estimated by flame atomic absorption spect
roscopy and graphite furnace.
Results:
The result
showed that there were no significant changes in iron level of treated groups when compared
with control except liver
(5 000 mg/kg) and histological slides of all organs revealed normal except
negligible changes in liver and intestine with the highest dose of shilajit.
The weight of all organs
was normal when compared with control.
Conclusions:
The result suggests that black shilajit, an
Ayurvedic formulation, is safe for long term use as a dietary supplement for a number of disorders
like iron deficiency anaemia.
Contents lists available at ScienceDirect

Velmurugan C et al./Asian Pacific Journal of Tropical Biomedicine (2012)210-214
211
reduced oxygen-carrying capacity of the blood that occurs
with iron-deficiency anemia further aggravates myoglobin
deficiency-related symptoms
[11]
. The decrease of iron leads
to Trypanosoma cruzi infection in host
[12]
, causes defects in
the cognition and learning processes in humans
[13]
, produces
significant alterations in the metabolism of dopamine
and other neurotransmitter in brain
[14]
and reduces
毭
-aminobutyric acid (GABA) and glutamic acid content of the
brain.
On rehabilitation with the iron-supplemented diet for
1 week, these decreased enzyme activities in brain attained
the corresponding control values
[15]
. The iron preparation
used for disorders in an over dose can cause toxicity to some
vital organs.
The Astanga Hrdayam reported that there are
six types of shilajit but it is found that the shilajit coming
out
of iron is the best
[16]
and also there was no scientific
proof for long term safety of black shilajit.
So the present
study was aimed to evaluate the safety of shilajit by
91 days
repeated administration in different dose levels.
2. Materials and methods
2.1. Acute toxicity
Twenty four (12 male and 12 female) albino mice, weighing
approximately
(18-25 g) were used for the study. The acute
toxicity study was carried out as per guidelines for
Ayurveda
and
Siddha drugs. Healthy mice of either sex were selected
and grouped into four comprising six animals in each group.
The animals were fasted for four hours prior to the study
with free access to water.
The test drug was suspended in
the suitable medium
(reverse osmosis water) and given in
graded doses up to maximum dose level.
One group received
vehicle only, which served as untreated
control. The animals
were observed for any toxic manifestations and mortality for
72 hours. Thereafter, animals were observed for 14 days.
2.2. Chronic toxicity study/ 91 days study
The Wistar rats of 4-6 weeks were selected for the present
study.
Body weights of the rats for chronic toxicity study
were taken after the initial stabilization period.
They were
ranging between
(130-140 g) for males and (120-130 g) for
females.
A total of twenty four Wistar rats (12 males
+
12
females
) were randomized and equally divided into four
groups comprising six animals in each group.
The animals
in all the groups were provided with pellet diet depending
upon the body weights and their food consumption profile.
The animals of group II, III and IV were administered with
shilajit at the respective doses of
500, 2 500 and 5 000 mg/kg
once dail
y for 91 days, whereas animals in group I received
vehicle
(reverse osmosis water). After 91 days administration,
blood was collected from retro orbital and serum was
separated.
Finally animals were sacrificed, and different
organs were isolated and weighed.
The isolated organs
were subjected to histopathology studies and the iron was
estimated by flame atomic absorption spectrometry
(AAS)
and the
serum iron by graphite furnace. The experimental
protocol was reviewed and approved by
Institutional Animal
Ethical Committee (No: 23/SASTRA/IAEC/RPP).
2.3. Metal analysis
2.3.1. Sample collection
The samples were cleaned and dried under shade. The
dried samples were then grinded and powdered in an agate
pestle and mortar.
Samples were labeled and stored in pre-
cleaned polyethylene bottles for further analysis.
2.3.2. Reagents and apparatus
All the reagents such as HNO
3
, and H
2
O
2
were purchased
from
MERCK. Millipore water was used for all analytical
work and all the digestion vessel, polyethylene bottles
(sample container), micro pipette tips and others were
washed with
10
%
HCl, rinsed with de-ionized water before
preparing standards, reagents and samples.
2.3.3. Digestion of samples (sample preparation)
A Multiwave 3000 micro oven system (from Anton paar,
USA) with 16 position teflon vessels with capping was
being used for digestion process.
The digestion vessels
were provided with a controlled pressure, temperature and
release valve.
Before using, all teflon vessels were soaked
with
10
%
HNO
3
. The system was initially programmed by
giving gradual rise of
20
%
, 40
%
, and 50
%
power for 5, 15 and
20 minutes, respectively for the due warming up. The powder
samples were used without any further tr
eatment for sample
preparation.
0.25 g of sample was weighed into the teflon
vessels followed by digestion mixture of
HNO
3
, and H
2
O
2
in
the ratio of
3:1, according to the nature of samples which
were being applied.
The resulting solution after microwave digestion was
filtered through
Whatman No. 40 filter paper (if necessary)
and diluted to
25 mL with Millipore water. A sample blank
containing
only acid mixture was prepared at the same time.
The method of standard addition was generally adapted to
calibrate the instrument before going for the observation of
the samples.
2.3.4. Calibration of instruments
More than three working standard solution of elements to
be determined were prepared, covering the concentration
range as recommended by the manufacturer of the
instrument for the elements to be determined.
Before the
analysis of samples, the instruments were calibrated with
prepared working standard solution.
The calibration curves
were obtained for concentration and absorbance data were
statistically analyzed.
Calibration of the instrument was
repeated periodically during operations and blanks were
carried with each set of
10 samples or aspirating any one of
the prepared working standard for every
10 samples to check
the instrument drift and to validate analytical procedures
and performance.
Reagent blank readings were taken and
necessary correction was made during the calculation of
concentration of various elements.
Standard Certified Reference (SRM) of National Institute of
Standard and Technology (NIST) was used for day-to-day for
the evaluation of methods of analysis or test and for long-
term
quality assurance of measurements.

Velmurugan C et al./Asian Pacific Journal of Tropical Biomedicine (2012)210-214
212
2.3.5. Fe analysis by flame AAS/graphite furnace
After calibrating the instrument with prepared working
standard, the digests liquid sample’s solution was subjected
to analysis of
Fe in organs by flame AAS and Fe in serum
by graphite furnace with specific instrumental conditions
as given by instruments manufacturer.
The solution was
introduced into flame, and the reading was recored,
using the mean of the three reading and the quantified
con
centration of the metals in the given samples against the
standard calibration curve obtained from concentration vs.
absorbance of the prepared known concentration on the day
of the analysis.
2.4. Statistical analysis
The statistical analysis was performed by one-way
analysis of variance
(ANOVA) by Dunnett’s test. A different
level at P<
0.05 was considered to be statistically significant.
3. Results
3.1. Acute toxicity
The test drug and vehicle were administered to mice as
per the guidelines, under close observation for
4 hours and
followed by daily observation for
14 days. There was neither
behavioral changes nor mortality and morbidity in all the
groups of animals.
So, the test drug was proved suitable for
further evaluation.
Figure 1.
Photomicrograph of heart (control).
Figure 2.
Photomicrograph of lungs (control).
Figure 3.
Photomicrograph of liver (control).
3.2. Cage side observation
There was no abnormality in the feces color, consistency
and amount, hair coat, respiration character and rate
convulsions, biting, static limb position, abnormal gait,
ataxic gait, head position and rearing activity during the
experimental period.
3.3. Metal estimation
The estimation of metal concentration was given in Table 1,
which showed no significant difference of iron
content in all
Table 1
Estimated iron content of shilajit in various organs and serum (mean
暲
SEM) (n=6).
Groups Heart Liver Spleen Kidney Serum
Group I 5.66暲0.32 8.36暲0.29 6.23暲0.29 5.58暲0.32 256.60暲4.17
Group II 6.79暲0.69 9.08暲0.46 5.86暲0.95 5.41暲0.23 327.17暲8.65
Group III 5.89暲0.34 9.44暲0.38 6.10暲0.40 5.66暲0.32 342.35暲6.33
Group IV 6.21暲0.55 9.87暲0.17* 6.94暲0.39 6.26暲0.25 367.70暲3.45
*: P<0.05 comparing with the control group.
Table 2
Effect of shilajit on organ weights (g) in rats (mean
暲
SEM) (n=6).
Groups Heart Lungs Liver Kidney Spleen Stomach Intestine Testes Ovaries
Group I 0.50暲0.04
a
0.73暲0.05
a
5.95暲0.45
a
1.18暲0.13
a
0.18暲0.02
a
1.32暲0.06
a
11.53暲0.61
a
2.43暲0.17
a
0.06暲0.01
a
Group II 0.65暲0.08
a
1.00暲0.13
a
6.18暲0.84
a
1.32暲0.20
a
0.30暲0.08
a
1.28暲0.19
a
12.72暲1.55
a
2.30暲0.10
a
0.05暲0.01
a
Group III 0.77暲0.10
a
0.98暲0.10
a
6.00暲0.43
a
1.45暲0.16
a
0.30暲0.04
a
1.57暲0.17
a
12.50暲0.68
a
1.93暲0.22
a
0.08暲0.01
a
Group IV 0.87暲0.13
a
0.98暲0.07
a
6.27暲0.73
a
1.28暲0.10
a
0.27暲0.02
a
1.20暲0.09
a
12.33暲0.85
a
2.23暲0.13
a
0.06暲0.01
a
Means bearing same superscripts do not differ significantly (P<0.05).

Velmurugan C et al./Asian Pacific Journal of Tropical Biomedicine (2012)210-214
213
groups of animals, and in all the organs except liver. Metal
estimation
(iron) values of rats treated with shilajit (group IV)
showed significant difference in liver when compared with
group
I, but no significant changes were observed in group II
and group
III.
Figure 4.
Photomicrograph of liver-treated with shilajit (the highest
dose) showing congestion and degeneration of hepatocytes.
Figure 5.
Photomicrograph of spleen (control).
3.4. Organ weights
There was no adverse effect of shilajit on any vital organs
of the rats, which received the drug for
91 days. There was
no indication of atrophy or hypertrophy of any organ of rats
in all treated groups.
It was represented in Table 2.
Figure 6.
Photomicrograph of stomach (control).
3.5. Histopathological studies
Figure 7.
Photomicrograph of intestine (control).
Figure 8.
Photomicrograph of intestine-treated with shilagit (the
highest dose) showing fusion of villi.
Figure 9.
Photomcrograph of kidney (control).
Figure 10.
Photomicrograph of testis (control).

Velmurugan C et al./Asian Pacific Journal of Tropical Biomedicine (2012)210-214
214
The histological changes in the main organs were shown
in
Figure 1-11. On histological studies, the highest dose
of test drug caused changes in liver and intestine showing
congestion and degeneration of hepotocytes, and fusion of
villi
(Figure 4, 8) and all other organs revealed normal. In
group
I, group II and group III, all the organs were normal
and no specific abnormalities were seen.
When compared
with control, the highest dose showed microscopic changes.
Figure 11.
Photomicrograph of ovary (control).
4. Discussion
The overload iron can cause undesirable side effect or
toxicity.
The excess iron is distributed throughout the body
and slowly accumulates in several organs, including heart,
liver, spleen, and skin.
Ferritin is a unique iron storage
protein containing
24 storage proteins. When excess dietary
iron is absorbed, the body produces more ferritin.
Ferritin
is gr
eatly abundant in the heart and liver, therefore there
is a large amount in these organs, and iron rushes to these
organs for storage
[17,18]
. Iron storage is the most common
cause of death among patients with hemochromatosis, sickle
cell disease, thalassemia, and heart disease
[19]
.
The shilajit contains iron and other minerals, and is widely
used for various disorders in long term therapy like anaemia
an
d diabetes. So it may cause toxicity, but the present study
data suggest that long term use of shilajit did not show any
toxicity and the iron content of shilajit almost has the same
level in all the dose level and histopathological studies show
the normal histology of all the organs except the intestine
and liver, which did not have significant change in the
histological architecture.
Hence, it can be considered being
normal.
Again the organ weight shows no significant in all
organs when compared to control and there was no mortality
observed in all treated groups up to the end of study, which
will further support that the shilajit was safe for long term
use.
Conflict of interest statement
We declare that we have no conflict of interest.
References
[1] Agarwal SP, Khanna R, Karmarkar R, Anwer MK, Khar RK.
Shilajit: a review. Phytother Res 2007;
21
: 401-405.
[
2] Winston D, Steven M. Adaptogens: herbs for strength, stamina,
and stress relief.
Vermont: Healing Arts Press Rochester; 2007, p.
202-204.
[
3] Mizuno M. A review of current knowledge of the complement
system and the therapeutic opportunities in inflammatory
arthritis. Curr Med Chem
2006;
13
: 1707-1717.
[
4] Wills-Karp M. Complement activation pathways: a bridge
between innate and adaptive immune responses in asthma. Proc
Am Thorac Soc
2007;
4
: 247-251.
[
5] Ghosh R, Kadam PP, Kadam VJ. Antioxidant and hypolipidemic
activity of
Kumbhajatu in hypercholesterolemic rats. Int J
Ayurveda Res
2010;
1
(3): 159-162.
[
6] Velmurugan C, Vivek B, Shekar SB, Sudha SP, Sundaram T.
Shilajit in management of iron deficiency anaemia. J Pharm
Biomed Sci
2010;
1
(1): 1-2.
[
7] Ghosal S, Singh SK, Kumar Y, Srivatsava R. Antiulcerogenic
activity of fulvic acids and
4-metoxy-6-carbomethyl biphenyl
isolated from shilajit. Phytother Res
1988;
2
: 187-191.
[
8] Tripathi KD. Essentials of medical pharmacology. 6th ed. New
Delhi: Jaypee Brothers Medical Publishers; 2008, p. 581.
[
9] Rang HP, Dale MM, Ritter JM, Moore PK. Pharmacology. 5th ed.
Elsevier Publication; 2007, p. 348.
[
10] Perez CD. Effect of probiotic, prebiotic and symbiotic follow-up
infant formulas on iron bio availability in rats. Food Sci Technol
Int
2007;
13
: 69-77.
[
11] Kraemer WJ. Exercise physiology: integrating theory and
application.
1st ed. New york: Lipincott; 2011.
[
12] Marilda JA, Maria LP, Helen RM, Vanja MV, Marta DL, Maria TB,
et al.
Treatment with the iron chelator desferrioxamine reduces
parasitemia and mortality in experimentally infected mice. Exp
Parasitol
2007;
117
: 43-50.
[
13] Carter CR, Joseph L, Matthew J, Armony-Sivan R, Neil C,
Angelilli ML, et al. Iron deficiency anemia and cognitive function
in infancy. Pediatrics
2010;
126
(2): 427-434.
[
14] Lozoff B. Early iron deficiency has brain and behavior effects
consistent with dopaminergic dysfunction. J Nutr
2011;
141
:
740-746.
[
15] Yehuda S, Mostofsky D. Iron deficiency and overload: from basic
biology to clinical medicine.
New York: Humana Press; 2009, p.
151.
[
16] Murthy KRS. Astanga hrdayam. 5th ed. Varnasi: Krishnadas
Academy; 2001.
[
17] Mozzarelli A, Bettati S. Chemistry and biochemistry of oxygen
therapeutics: from transfusion to artificial blood.
1st ed. United
Kingdom: John Wiley & Sonsltd; 2011, p. 403.
[
18] Prasad MNV. Trace elements as contaminants and nutrients:
consequences in ecosystems and and human health.
United
Kingdom: Wiley; 2008, p. 485-486.
[
19] Nelson JE, Mugford VR, Kilcourse E, Wang RS, Kowdley
KV. Relationship between gene expression of duodenal iron
transporters and iron stores in hemochromatosis subjects. Am J
Physiol Gastrointest Liver Physiol
2010;
298
: 57-62.