Advances in Bioscience and Biotechnology, 2016, 7, 199-204
Published Online April 2016 in SciRes. http://www.scirp.org/journal/abb
How to cite this paper: Raguraman, V. and Subramaniam, J.R. (2016) Withania somnifera Root Extract Enhances Telome-
rase Activity in the Human HeLa Cell Line. Advances in Bioscience and Biotechnology, 7, 199-204.
Withania somnifera Root Extract Enhances
Telomerase Activity in the Human
HeLa Cell Line
Vasantharaja Raguraman, Jamuna R. Subramaniam*
Center for Preclinical and Translational Medicine Research, Central Research Facility,
Sri Ramachandra University, Chennai, India
Received 10 March 2016; accepted 12 April 2016; published 15 April 2016
Copyright © 2016 by authors and Scientific Research Publishing Inc.
This work is licensed under the Creative Commons Attribution International License (CC BY).
Aging is a decelerating unidirectional process of life. Shortening of telomeric DNA, the (TTAGGG)n
hexanucleotide repeats, which form the caps at the chromosome ends, is implicated to determine
the aging process, and more importantly the healthy lifespan itself. Telomerase, a ribonucleo-
protein having reverse transcriptase activity, arrests telomere loss through addition of the
TTAGGG repeats de novo, to the ends of the chromosome. The telomere/telomerase maintenance
is an inevitable necessity to delay aging and for a healthy lifespan. Here, we report the potential of
full-spectrum, high concentration Ashwagandha (Withania somnifera), an Ayurvedic medicinal
herb, root extract to increase telomerase activity. HeLa cells, when treated with various concen-
trations of Ashwagandha root extract, showed an increase in telomerase activity measured with
the established Telomerase Rapid Amplification Protocol (TRAP) assay. Ashwagandha root extract
increased telomerase activity with highest enhancement of ~45% at 10 - 50 µg concentration.
Thus, Ashwagandha root extract has the anti-aging inducing potential.
Ashwagandha, Telomerase, HeLa Cell Line, TRAP Assay
Aging, the relentless decelerating phenomenon, is the main cause for wide range of age-associated diseases like
cancer, ischemic heart disease, stroke, type 2 diabetes, neurodegenerative diseases including Alzheimer’s dis-
ease and others . One of the major factors that accelerate aging and cause degeneration of various systems is
V. Raguraman, J. R. Subramaniam
the shortening of “TELOMERES”, the DNA repeat sequences at the end of linear eukaryotic chromosomes, of 2
- 20 kbp length, which act as caps to conserve chromosome integrity and stability . During the process of
DNA replication, telomeres are shortened by 50 - 100 bp with each cell division. The end replication problem
leads to critically short telomeres and ultimately senescence. Hence, telomeres are implicated as one of the fac-
tors that determine aging and lifespan. The enzyme, telomerase, a ribonucleoprotein having reverse transcriptase
activity, carries out telomere replication. Telomerase synthesizes telomeric DNA sequences through the addition
of TTAGGG repeats at the chromosome ends . But, the telomerase activity is extremely low in somatic cells.
Telomerase is shown to be essential for the healthy normal life. Turning off of the telomerase expression in
the conditional knockout mice leads to degeneration of multiple systems, including immune, digestive and
nervous system. When telomerase expression is turned on later in life, these degenerations can be reversed.
More importantly, nervous system, where neurons are thought to be differentiated and stay that way for a life-
time, manifests remarkable improvement from the degeneration . Telomerase activity and short telomeres are
implicated in aging mechanism -. Enhancing telomerase activity is one way to delay aging. A small mole-
cule telomerase activator, TA-65, isolated from the root of the traditional Chinese medicine, Astragalus mem-
branaceus in mice  can precisely do the same.
Ashwagandha (Withania somnifera Dunal) root extract, consisting of several withanolides, is an extensively
used ayurvedic medicine with multitude of protective effects on humans such as revitalization, stress tolerance
 and anti-inflammation . Further, it enhances longevity in C. elegans . With little or no toxic effect,
Ashwagandha  can be considered as supplement in humans . Though Ashwagandha is suggested to in-
crease longevity, so far no evidence has been provided to reinforce this claim except in C. elegans  which is
usually a harbinger to the positive outcome in humans.
Here, we address the efficacy of a high concentration, full-spectrum Ashwagandha root extract to provide an-
ti-aging effect through enhanced telomerase activity in an in vitro cell culture model.
2. Materials and Methods
2.1. Cell Culture
The human HeLa cell line was used in the present study. The cells were maintained in DMEM (Invitrogen) sup-
plemented with 10% fetal bovine serum in a humidified incubator (37˚C and 5% CO2). Cells (40% - 60% con-
fluency) were treated with various concentrations (10 µg, 50 µg, 100 µg, 500 µg, 5 mg) of Ashwagandha Root
Extract (KSM-66, Shri Kartikeya Pharma, Hyderabad) for 72 hours.
2.2. Preparation of Cell Lysate
Cells were collected and centrifuged at 6000 rpm for 6 min. Pellets of 6 × 105 cells were suspended in ice-cold
NP-40 lysis buffer and incubated 30 min on ice . The cell lysate was aliquoted into separate tubes and stored
at −80˚C until analyzed by TRAP assay.
2.3. Telomerase Assay and Quantification
Detection of telomerase activity in HeLa extracts employed the PCR-based telomeric repeat amplification pro-
tocol (TRAP assay) is a modification of the assay described by . The protein concentration was determined
using Lowry’s method . Briefly, total volume of reaction mixture was 50 µl contained 0.5 µl of dNTP mix
(2.5 mM), 5 µl of 10× TRAP Buffer, 0.15 µg TS [5’-AATCCGTCGAGCAGAGTT-3’] primer, 0.15 µg and
ACX [5’GCGCGGCTTACCCTTACCCTTACCCTAACC-3’] primer, 40 µl RNase-free H2O, 0.5 µl of 5 U/µl
Taq DNA polymerase and 2 µl (1000 cells) of cell lysate. The PCR mixture was incubated at 30 min at room
temperature. The PCR was then started at 94˚C for 90 sec followed by a 40-cycle amplification (94˚C for 20 s,
50˚C for 30 s, and 72˚C for 90s). The amplified PCR product will yield a 6-bp incremental ladder was separated
on 12% acrylamide-TBE gels and visualized by ethidium bromide staining in UV light. The images were then
processed and quantified using Image J software. The density/intensity of the amplified 6 bp repeat periodicity
PCR product observed and quantitated by Image J software is represented as densitometric units.
2.4. Statistical Analysis
Statistical analyses on the percentage of telomerase activity in HeLa cell extracts were done using Sigmaplot
V. Raguraman, J. R. Subramaniam
Aging associated telomere shortening is well documented. More importantly, telomerase, which is needed to
maintain the telomere length through replication, is essential for healthy life. In the terminally differentiated so-
matic tissues, telomerase activity is low. Several factors, including stress aggravate the senescence process
through telomere shortening. Given the importance of telomere/telomerase in aging and health, identification of
a supplement from the natural/plant herb based ancient traditional systems of medicine, which could retain the
telomere length or increase telomerase activity, is a less expensive and faster way with the most important added
benefit of dismal toxicity. We evaluated the Ashwagandha root extract powder (KSM-66) suspended in water to
enhance telomerase activity. Using the human cervical carcinoma cell line, HeLa, reported to have telomerase
activity , we determined Ashwagandha’s telomerase activity enhancing effect following published protocols
. First of all, we determined the optimal total protein concentration of the cell extract needed to assess the
telomerase activity (Figure 1). The telomerase activity was highest between 50 ng and 5 µg of total protein of
cell extract. We selected 2 µg as the ideal protein concentration and all further telomerase assays were carried
out under these conditions.
3.1. Identification of Optimal Concentration of Ashwagandha to
Enhance Telomerase Activity
HeLa cells were exposed to different concentrations of full spectrum Ashwagandha root extract as suspension of
the powder in water for 72 hrs following which the cells were processed and telomerase activity determined.
This resulted in dose-dependent increase in telomerase activity upto 50 µg /ml after which the activity started
decreasing as the preparation is a powder suspension (Figure 2).
3.2. Enhancement of Telomerase Activity
Further, HeLa cells were treated with 10 µg and 50 µg of Ashwagandha root extract based on results arrived
from dose response curve. Cells treated with 10 - 50 µg of Ashwagandha root extract enhanced telomerase ac-
tivity by 45% (Figure 3).
Figure 1. Determination of optimal concentration for the telomerase assay. Different concentra-
tions of HeLa cell lysates were assayed for telomerase activity. The activity is measured by
quantifying the density of amplified 6 bp repeats under each concentration with Image J software
and represented as densitometric units in the Y-axis. X-axis represents the amount of total HeLa
cell lysate as protein concentration.
V. Raguraman, J. R. Subramaniam
Figure 2. Effect of various concentrations of Ashwagandha root extract on telomerase activity
in HeLa cells. The concentrations of Ashwagandh is given in the X-axis. The telomerase ac-
tivity measured by quantifying the density of amplified 6 bp repeats (resolved in the 12%
TBE-acrylamide gel), under each concentration with Image J software and represented as den-
sitometric units in the Y-axis.
Figure 3. Results of Quantitative TRAP assays: (a) Gel picture showing 6-bp ladder of telo-
merase activity resolved in 12% TBE-polyacrylamide gels. NC-Negative control—without
Hela cell lysate; C—with HeLa cell lysate; with and without Ashwgandha treatment; The ar-
rows represent the amplified 6-bp periodicity products due to telomerase activity followed by
PCR amplification; (b) Densitometry analyses of percentage of telomerase activity in control
and ashwagandha treated cells (Cumulative of four independent sets). Statistical significance
*P < 0.05.
As non communicable diseases are in the rise due to aging, strategies to increase healthspan is rigorously eva-
luated using all possible systems and organisms. Generally, a gene or its absence or specific chemicals like Re-
serpine  , Aswagandha , Rapamycin  and Resveratrol  can increase lifespan in various or-
ganisms like yeast, C. elegans, drosophila and mice. In addition, they induce stress tolerance. But the exact me-
chanism of their lifespan extension, especially the contribution of telomere/telomerase is not known. One of the
V. Raguraman, J. R. Subramaniam
important qualities for health span is stable telomere at the ends of the chromosomes in the differentiated so-
matic tissues. The hallmark of aging is associated with telomere; the progressive attrition of telomere in human
beings contributes to mortality in several age-related diseases  . Mouse cells without telomerase activity
have short telomere length , emphasizing the essentiality of telomerase for telomere maintenance. Since ag-
ing promotes degenerative pathologies, screening for new drugs is a necessity to develop highly effective thera-
py for the enhancement of telomerase activity in order to avoid critically short telomere length leading to aging
and age-related diseases. Tomás-Loba et al., (2008)  demonstrated that enhanced telomerase activity in mice
can delay aging and confer cancer resistance. Recently, Bernardes de Jesus et al., (2011), demonstrated TA-65
, the telomerase activator obtained from the Chinese medicine, can elongate short telomeres and increase
health span of mice without increasing cancer incidence. Hence, we evaluated the enhancement of the telome-
rase activity, which is to maintain the telomere by adding the hexanucleotide repeats, by an ancient Ayurvedic
herb Ashwagandha root extract (Figure 1). Indeed, Ashwagandha root extract powder, at a concentration of 10
µg - 50 µg/ml (Figure 2), increased telomerase activity by ~45%, in the Human HeLa cell line upon 72 hrs ex-
posure (Figure 3), as determined by the TRAP assay. Ashwagandha is one of the most widely used ancient
ayurvedic herb and generally non-toxic. Moreover, earlier, we reported that Ashwagandha can increase C. ele-
gans lifespan . Therefore, Ashwagandha deserves to be evaluated as a potential anti-aging ayurvedic herbal
preparation in higher organisms and the potential mechanism needs to be investigated.
Thus, Ashwagandha root extract is able to enhance telomerase activity in the HeLa cell line. Given the excep-
tional necessity of the telomeres at the ends of the chromosomes for the maintenance and integrity of the chro-
mosomes, it will be worthwhile to evaluate Ashwagandha under various adult onset disease conditions. This can
provide a holistic protective effect.
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