Clinical evaluation of purified Shilajit on testosterone levels in healthy volunteers

Abstract

Purified Shilajit, an Ayurvedic rasayana, was evaluated in healthy volunteers of age between 45 and 55 years for its effect on male androgenic hormone viz. testosterone in a randomised, double-blind, placebo-controlled clinical study at a dose of 250 mg twice a day. Treatment with Shilajit for consecutive 90 days revealed that it has significantly (P < 0.05) increased total testosterone, free testosterone and dehydroepiandrosterone (DHEAS) compared with placebo. Gonadotropic hormones (LH and FSH) levels were well maintained.

Full text

ORIGINAL ARTICLE
Clinical evaluation of purified Shilajit on testosterone
levels in healthy volunteers
S. Pandit, S. Biswas, U. Jana, R. K. De, S. C. Mukhopadhyay & T. K. Biswas
Research Unit, Department of Health and Family Welfare, J. B. Roy State Ayurvedic Medical College and Hospital, Government of West Bengal,
The West Bengal University of Health Sciences, Kolkata, India
Keywords
Clinical—dehydroepiandrosterone—Shilajit—
testosterone
Correspondence
Tuhin Kanti Biswas, Research Unit, J. B. Roy
State Ayurvedic Medical College and Hospi-
tal, 170-172, Raja Dinendra Street, Kolkata
700004, India
Tel.: +91-9433173272;
E-mail: biswastuhin@rediffmail.com
Accepted: July 27, 2015
doi: 10.1111/and.12482
Summary
Purified Shilajit, an Ayurvedic rasayana, was evaluated in healthy volunteers of
age between 45 and 55 years for its effect on male androgenic hormone viz.
testosterone in a randomised, double-blind, placebo-controlled clinical study at
a dose of 250 mg twice a day. Treatment with Shilajit for consecutive 90 days
revealed that it has significantly (P<0.05) increased total testosterone, free
testosterone and dehydroepiandrosterone (DHEAS) compared with placebo.
Gonadotropic hormones (LH and FSH) levels were well maintained.
Introduction
Purified Shilajit (PS) is used in Ayurveda, indigenous sys-
tem of Indian medicine, as a remedy for several diseases,
particularly chronic diseases. Shilajit is a pale-brown to
blackish-brown exudate that oozes from sedimentary
rocks worldwide, largely in the Himalayas. Common peo-
ple describe it from their knowledge as pahar-ki-pasina
(sweat of mountains), paharki-khoon (mountain blood),
shilaras (rock juice), asphalt, bitumen, etc. Shilajit is said
to carry the healing power of these great mountains
(David & Vasant, 2001). It is an important drug of the
ancient Ayurvedic materia medica and it is to this day
used extensively by Ayurvedic physicians for a variety of
diseases. Early Ayurvedic writings from the Charaka Sam-
hita (Sharma, 1998) describe Shilajit as a cure for all dis-
eases as well as a Rasayana (rejuvenator) that promises to
increase longevity. It is composed of rock humus, rock
minerals and organic substances that have been com-
pressed by layers of rock mixed with marine organisms
and microbial metabolites (Ghosal, 1994).
Traditional uses of Shilajit primarily focus not only on
diabetes and diseases of the urinary tract, but also on
oedema, tumours, muscle wasting, epilepsy and even
insanity. Modern indications extend to all systems of the
human body with a significant number of additions in the
reproductive and nervous system. Clinical research confirms
many of the properties for which Shilajit has been used
(Talbert, 2004). In Ayurveda, Shilajit is employed for the
management of male reproductive disorders, and in par-
ticular, under the parlance of Vrisya (an aphrodisiac with
special reference to spermatogenesis) (Sharma, 1998).
Several toxicological studies, both acute and sub-
chronic, have already been performed by many scientists
with Shilajit throughout the world. Oral LD50 was found
to be >2000 mg kg
1
(Acharya et al., 1988; Ghosal et al.,
1989), and Shilajit was proved to be safe at doses of 0.2–
1.0 g per kg body weight when used chronically (Kelgin-
baev et al., 1973; Anisimov & Shakirzyanova, 1982; For-
tan & Acharya, 1984; Al-Hamaidi & Umar, 2003).
Clinical evaluation of spermatogenic activity of processed
shilajit in oligospermia (Biswas et al., 2009) revealed that
there was no alteration on objective features related to
any systemic toxicities such as serum urea, uric acid,
serum bilirubin, total protein, serum globulin, SGPT,
SGOT and alkaline phosphatase (Biswas et al., 2009).
Besides, it was also observed that there was a significant
(P<0.001) improvement in spermia (+37.6%), total
sperm count (+61.4%), motility (12.4–17.4% after differ-
ent time intervals), normal sperm count (+18.9%) and
total testosterone (+23.5%) with concomitant decrease in
pus and epithelial cell count compared with baseline
value in 28 patients of oligospermia after 90 days of treat-
ment with PS at a dose of 100 mg twice daily. An
unpublished human safety study of purified Shilajit from
Natreon, Inc., New Brunswick, NJ, USA by the present
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Andrologia 2016, 48, 570–575
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authors has demonstrated the safety of this product at
250 mg twice a day dosing (Jana, Utpalendu, Biswas,
Tuhin; J.B.Roy Ayurvedic Medical College and Hospital,
Kolkata, India, Project No.: JBR/Res/01/2007), and an
unpublished animal study in rats with 100 mg/kg b.w.
(equivalent to human dose of 850 mg) by Natreon
showed a significant increase in testosterone levels. Thus,
a dose of 250 mg twice daily was selected for this study.
Materials and methods
Preparation and analysis of the drug
Purified Shilajit (PrimaVie
TM
, Kolkata, West Bengal,
India), a patented (US 6,440,436, 6,969,612, 6,558,712, EP
1 387 614) standardised extract of native Shilajit from
Natreon, Inc., was used for the study. Shilajit was stan-
dardised to contain not less than 60% w/w of total bioac-
tives, which include not less than 50% w/w of fulvic acids
(FAs), not less than 0.3% w/w of dibenzo a-pyrones
(DBPs) and not less than 10% w/w of dibenzo-a-pyrone
chromoproteins (DCPs), as quantified by HPLC using
external standards (isolated from Shilajit extract by low
pressure (Lobar) chromatography). An HPLC chro-
matogram of this product is shown in Fig. 1.
Selection of healthy volunteers
The clinical trial was conducted between December 2012
and March 2014 at J. B. Roy State Ayurvedic Medical
College and Hospital, Kolkata, India, after obtaining
necessary permission from Institutional Ethics Commit-
tee (IEC) of J. B. Roy State Ayurvedic Medical College
and Hospital, Kolkata, India. A schematic of the study
design is shown in Fig. 2. Healthy volunteers aged
between 45 and 55 years, irrelevant of religion, income
status and occupation, were selected for the present pur-
pose, and the distribution of patients was done by the
method of double-blind randomised techniques. Initially,
145 volunteers were selected on the basis of primary
assessment eligibility and 49 among them were excluded
for various reasons (Fig. 2). A total of 96 volunteers
were enrolled in the present trial and randomly divided
into two equal groups as PS treated and placebo treated,
each with 48 subjects. In the course of the study, 21
subjects discontinued for various reasons and 38 subjects
in PS-treated group and 37 subjects in the placebo
group completed the study (Fig. 2). Mean age of the
volunteers was 49.44 years in the test drug group and
48.89 years in the placebo group, and thus, there is no
bias due to the difference in mean ages. Volunteers were
included in the study after taking their consent in trilin-
gual (English, Bengali and Hindi) using prescribed for-
mat of WHO-Helsinki rules. History of volunteers was
taken according to the standard protocol mentioning
name, age, sex, religion, address, occupation, income
status, history of past illness, family history, personal
history, marital history, general examination, systemic
examination, laboratory investigation, etc.
Dosage regimen
Both the groups received respective drugs in the dose of
250 mg/capsule orally, twice daily after major meals, for a
0.36
0.30
0.25
0.20
0.15
AU
0.10
0.06
0.00
2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00
Minutes
20.00 22.00 24.00 26.00 28.00 30.00
Fig. 1 HPLC chromatogram of Shilajit (PrimaVie
TM
) by RP-C 18 column. Fulvic acids (FAs) t
R
: 2.0–3.0 min; Dibenzochromo proteins (DCPs) t
R
:
3.0–5.8 min; 3,8 -(OH)
2
–Dibenzo-a-pyrone t
R
: 9.07 min; 3-OH- Dibenzo-a-pyrone t
R
: 26.02 min. HPLC analysis was performed under ambient
conditions, with Waters HPLC equipment comprising Waters 515 pumps, Waters photodiode array detector (PDA) model 2996, Waters pump
controller module and EMPOWER software (version 1) Natreon Inc., New Brunswick, New Jersey, USA. Samples (20 ll) were injected by means of a
Rheodyne injector fitted with a 20-ll loop. Compounds were separated on a C18 reverse-phase (Lichrocart
â
column (250 mm 94 mm, i.d., 5-
lm particle; column no 331303, Darmstadt, Germany) using the mobile phase Water : Acetonitrile : o-phosphoric Acid [67 : 32 : 01 v/v/v] with a
flow rate of 0.8 ml/min. The UV absorbance of eluent was monitored at a wavelength of 240 nm.
©2015 The Authors. Andrologia Published by Blackwell Verlag GmbH 571
Andrologia 2016, 48, 570–575
S. Pandit et al. Clinical evaluation of Shilajit on testosterone

total duration of 90 days. Distribution of treatment is as
follows:
Group –I: PS 250 mg BID (38 subjects)
Group –II: Placebo 250 mg (microcrystalline cellulose
124 mg + lactose 124 mg + magnesium
stearate 2 mg) BID (37 subjects)
Both the PS and placebo capsules were white opaque,
Size 1, and looked identical.
Inclusion/exclusion criteria
Eligibility was based on the following inclusion criteria:
Clinically examined normal healthy volunteers, devoid of
any chronic, organic or dreadful diseases, irrespective of
religion and income status with age between 45 and
55 years, not taking any supplements or vitamins and
male subjects. The exclusion criteria were volunteers of
age below 45 years and above 55 years, any chronic,
organic or dreadful diseases, concomitant serous disor-
ders, other drug treatment being received simultaneously
on long basis which may affect the study results, any
immunosuppressive drugs being received, poor nutri-
tional status, subjects likely to fail compliance with the
trial protocol, alcoholism, smoking, using antipsychotic
drugs and steroids/male contraceptives.
Observation criteria
Screening and diagnosis
Ageing men often present with a variety of vague nonspeci-
fic symptoms that may be associated with testosterone defi-
ciency (Nandy et al., 2008). Subjects were, therefore,
screened on the basis of the St Louis University ADAM
Selection assessment for eligibilty
Excluded (n= 49)
Not meeting inclusion criteria (n = 28)
Refuse to sign consent form (n = 09)
Other reason (n =12)
Enrolment for treatment allocation (n = 96)
0 day : Testosterone,
Free testosterone, FSH,
LH, DHES
PS 250 mg kg–1 BD (n = 48) Comparator (n = 48)
30 day : Testosterone,
Free testosterone, FSH,
LH, DHES
60 day : Testosterone,
Free testosterone, FSH,
LH, DHES
90 day : Testosterone,
Free testosterone, FSH,
LH, DHES
Protocol completed
PS (n = 38)
Comparator (n = 37)
Discontinued (n = 21)
Protocol violation (n = 03)
Follow up (n =14)
Physician’s decision (n = 04)
Fig. 2 Schematics of study design.
572 ©2015 The Authors. Andrologia Published by Blackwell Verlag GmbH
Andrologia 2016, 48, 570–575
Clinical evaluation of Shilajit on testosterone S. Pandit et al.

questionnaire (Table 1) (Morales et al., 2000). Besides the
ADAM questionnaire, normalcy of the subjects was estab-
lished after receiving acceptable ranges of different haema-
tological and biochemical parameters such as fasting
glucose, serum urea, creatinine, ALT, AST, Hb%, total
RBC, total and differential counts of WBC, RBC/WBC
morphology, ESR and routine stool examination including
occult blood test. Biochemical investigations were per-
formed by means of auto-analyser (Beckman Coulter, CA,
USA), and haematological investigations were done by
means of automated cell counter (Medonic CA 530 16;
Oden (Merck, Germany). Routine stool examination was
carried out by means of conventional methods.
Estimation of testosterone, free testosterone, LH, FSH and
DHEAs
Both total and free testosterones, LH, FSH and DHEAs
were estimated from fasting blood of each volunteer on
days 0 (baseline), 30, 60 and 90. LH and FSH were esti-
mated by means of ADVIA Centaur XP Immunoassay
Systems (SIEMENS, Berlin, USA) with ADVIA Centaur
LH ready pack primary reagent; testosterone and DHEAs
were estimated by means of Access Immunoassay System
(Beckman Coulter) with Access Testosterone Reagent
Pack and Access DHEAs Reagent Pack respectively. Free
testosterone was estimated by means of AccuBind ELISA
Microwells Test system (Monobind Inc., Lake Forest, CA,
USA) with AccuBind free testosterone reagent pack.
Statistical analyses
Statistical analysis of data of the two treatment groups
collected at different intervals of study was performed by
paired Student’s ‘t-’test using SPSS11.5 version software
(Chicago, USA).
Results
Subject screening
All subjects responded ‘yes’ to questions 1 and 7 or
any three other questions according to the ADAM
questionnaire were primarily selected. Besides, they also
fulfil all inclusion criteria. Fasting glucose, renal investi-
gations like serum urea and creatinine, hepatic investi-
gations like ALT and AST, haematological parameters
and stool investigations were found within normal
range during primary screening for the inclusion of the
volunteers.
Treatment efficacy
A total of 21 subjects discontinued the study, and any
data from these subjects were not included in the calcula-
tions as these subjects discontinued at different time
points of the study and for various reasons.
Testosterone and free testosterone estimation
It was observed that in PS-treated group, there was an
increase in testosterone levels (ng ml
1
) on days 30
(6.82%), 60 (3.09%) and 90 (20.45%) with respect to day
‘0’. The increment of testosterone levels on day ‘90’ was
significant (P<0.05) with respect to the values of day
‘0’. In placebo-treated group, there was a significant
(P<0.05) decreasing trend of testosterone level. The level
of testosterone in PS-treated group on day 90 was found
to be significantly (P<0.05) better than the values of
placebo-treated group in same day (Table 2). The level of
free testosterone (pg ml
1
) in PS-treated group on day
90 (19.14%) was significantly better (P<0.05) than
0 day value and maintain parity with the testosterone
level. Free testosterone level of PS-treated group on day
90 was also found to be significantly higher (P<0.05)
with the values of placebo-treated group on same day
(Table 2).
LH and FSH estimation
LH (mIU ml
1
) and FSH (mIU ml
1
) are inter-related
hormones, which have role in synthesis and release of
testosterone. In the present research work, it was observed
that there was maintenance of LH level in PS-treated
group, while FSH level significantly increased (P<0.004)
in PS-treated group on days 30, 60 and 90 with respect
to baseline. The result of FSH was significantly better in
PS-treated group than placebo group on day 90
(Table 2).
Table 1 The St Louis University ADAM questionnaire for screening of
testosterone deficiency
Sl Questionnaires Comments
1 Do you have a decrease in libido (sex drive)? Yes / No
2 Do you have a lack of energy? Yes / No
3 Do you have a decrease in strength
and/or and endurance?
Yes / No
4 Have you lost height? Yes / No
5 Have you noticed a decreased ‘enjoyment of life’? Yes / No
6 Are you sad and/or grumpy? Yes / No
7 Are your erections less strong? Yes / No
8 Have you noticed a recent deterioration
in your ability to play sports?
Yes / No
9 Are you falling asleep after dinner? Yes / No
10 Has there been a recent deterioration in
your work performance?
Yes / No
Test is considered positive if answers are ‘Yes’ to question 1, question
7, or any 3 other questions.
©2015 The Authors. Andrologia Published by Blackwell Verlag GmbH 573
Andrologia 2016, 48, 570–575
S. Pandit et al. Clinical evaluation of Shilajit on testosterone

DHEAs estimation
DHEAs, the precursor of testosterone, showed interesting
results with PS, where the level of DHEAs (lgdl
1
) grad-
ually increased on day 30 (9.14%), 60 (9.59%) and 90
(31.35%) with respect to values on day ‘0’. Change of
DHEAs in placebo group was irregular. However, the
level of increase in DHEA in PS-treated group on day 90
was found to be significantly higher (P<0.05) than base-
line value of PS-treated group and 90-day value of pla-
cebo (Table 2).
Discussion
Testosterone is an anabolic steroid synthesised primarily
by the Leydig cells in the testes in males, the ovaries in
females and adrenal glands in both sexes. It is synthesised
from cholesterol, with androstenedione, androstenediol,
dehydroepiandrosterone sulphate (DHEAs), progesterone
and pregnenolone acting as some of the intermediate sub-
strates. Testosterone production is regulated by hormonal
secretions from the hypothalamus and the pituitary gland
in the brain via hypothalamus–pituitary–testicular axis.
The process begins as the hypothalamus secretes gonado-
tropin-releasing hormone (GnRH) in generative pulses.
In response to these steady intermittent bursts of GnRH,
the pituitary gland releases luteinising hormone (LH) and
follicle-stimulating hormone (FSH), which act directly on
the testes. FSH activates the Sertoli cells that produce
sperm (spermatogenesis). LH stimulates the Leydig cells
to secrete testosterone in a daily rhythm characterised by
peak levels in the morning and low levels in the evening.
Once it reaches high levels, testosterone production gen-
erates negative loop feedback to the hypothalamus to
downregulate LH release and diminish further testos-
terone production. In this way, testosterone inhibits its
own secretion (Gingrich, 2010). In addition to hypothala-
mic influence, it has been found that testosterone has
direct negative feedback effects on the anterior pituitary
gland. Like most hormones, testosterone is supplied to
target tissues in the blood where much of it is trans-
ported bound to a specific plasma protein, sex hormone-
binding globulin (SHBG) (Brooks, 1975). Most circulat-
ing testosterone is bound by SHBG and albumin; approx-
imately 2% of total testosterone is free (not bound to
protein). SHBG-bound testosterone is so tightly bound
that it is not biologically active. Both free and albumin-
bound testosterones are biologically active and together
are referred to as the bioavailable fraction (Bhasin et al.,
2010). It is reported that the total, free and bio-available
testosterone varies widely in the age group between 18–69
and 70–89 (Rosner et al., 2007). In the current study,
total and free testosterones were estimated to establish
the efficacy of PS for its role on testosterone stimulation
and secretion property considering the reference range 4–
30 pg ml
1
for free testosterone and 1.75–7.81 ng ml
1
for testosterone (total testosterone). DHEAs, the main
precursor of testosterone, was also estimated to rationalise
the role of PS on testosterone synthesis considering refer-
ence range 70–495 lgdl
1
between the age group of 41–
50 years and 38–313 lgdl
1
between the age group of
51–60 years. The present research work reveals that PS
may be able to increase both total testosterone and free
testosterone with respect to baseline value indicating its
potentiality on testosterone secretion level. The significant
betterment of DHEAs with the treatment of PS signifies
its role on testosterone synthesis. Other two gonadotropic
hormones, viz. LH and FSH, were studied in this present
work, to rationalise the hypothalamo-pituitary–testicular
axis, where both of these hormones were in maintained
levels indicating their initial role of triggering of testos-
terone production. This was followed by downregulation
of LH and FSH on one hand and maintenance of the
hypothalamo-pituitary–testicular axis by means of ele-
vated level of testosterone on 30, 60 and 90 days on the
Table 2 Effect of PS on testosterone and its mediators with respect to placebo control
Parameters
PS (250 mg BID) (n=38) Placebo (250 mg BID) (n=37)
Baseline 30 days 60 days 90 days Baseline 30 days 60 days 90 days
Testosterone
(ng ml
1
)
4.84
b
(1.54) 5.17 (1.33) 4.99 (1.41) 5.83
a,b
(1.67) 5.82
b
(1.58) 4.88
a
(1.74) 4.58
a
(1.44) 4.45
a,b
(1.78)
Free Testosterone
(pg ml
1
)
15.36
b
(7.17) 14.20 (3.97) 14.14 (3.59) 18.30
a,b
(7.72) 19.30
b
(5.75) 15.03
a
(4.22) 14.52
a
(6.19) 12.21
a,b
(5.39)
LH (mIU ml
1
) 6.33 (3.88) 6.65 (3.95) 6.64 (3.81) 6.79 (3.67) 6.49 (3.32) 7.82 (5.71) 5.86 (2.66) 7.45 (5.90)
FSH (mIU ml
1
) 6.94 (3.52) 8.17
a
(4.15) 8.52
a
(4.41) 8.41
a
(4.61) 6.91 (6.35) 8.11 (6.06) 7.46 (5.53) 10.23 (11.77)
DHEA-S (lgdl
1
) 145.09
(53.17)
158.35
(63.56)
159.00
(79.56)
190.57
a,b
(73.24)
139.60
(63.18)
136.04
(68.65)
150.92
(76.08)
138.77
b
(74.17)
P<0.05 considered as significant level in paired and unpaired Student’s t-test.
a
Compare to 0 day value of same group.
b
Compare to placebo group.
574 ©2015 The Authors. Andrologia Published by Blackwell Verlag GmbH
Andrologia 2016, 48, 570–575
Clinical evaluation of Shilajit on testosterone S. Pandit et al.

other hand. All these modus operandi of PS on synthesis
and stimulation of testosterone are found to be better
than placebo-treated group in healthy male volunteers in
age group of 45–55 years, who may undergo andropause
in normal course. Placebo, composed with microcrys-
talline cellulose, lactose and magnesium stearate, has nei-
ther stimulation nor inhibiting role on testosterone
secretion or synthesis.
Processed Shilajit (PS) containing biologically active
component di-benzo-alpha-pyrone (DBP) is earlier
reported to increase the spermatogenic activity in selected
patients of oligospermia (Biswas et al., 2009), and the
current study demonstrates that purified Shilajit increases
the total and free testosterone levels in healthy volunteers.
Conclusion
The present study was conducted to evaluate the efficacy
of PS for testosterone secretion and stimulation effects on
normal healthy volunteers in the age group of 45–
55 years. This effect was clarified by estimation of free
and total testosterone on 0, 30, 60 and 90 days where the
rise of these two androgenic markers was significant.
Testosterone synthesis and secretion was supported by the
maintenance levels of two gonadotropic hormones LH
and FSH as well as elevation of testosterone precursor
DHEAs.
Acknowledgements
We acknowledge Natreon Inc, Premise No: 02-360, Rishi
Tech Park (Ground Floor), Action Area 1D, Newtown,
Kolkata 700 156, for financial support to carry out the
study.
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