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Withania somnifera Root Extract Enhances Telomerase Activity in the Human HeLa Cell Line

April 2016
Advances in Bioscience and Biotechnology 07(04)

DOI:10.4236/abb.2016.74018

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CC BY 4.0

Authors:

Vasantharaja Raguraman

University of Missouri

Jamuna Subramaniam

Sri Ramachandra Institute of Higher Education and Research Porur, Chennai, India

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 ribonucleoprotein 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 concentrations 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.

Determination of optimal concentration for the telomerase assay. Different concentrations 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.

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Advances in Bioscience and Biotechnology, 2016, 7, 199-204

Published Online April 2016 in SciRes. http://www.scirp.org/journal/abb

http://dx.doi.org/10.4236/abb.2016.74018

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.

http://dx.doi.org/10.4236/abb.2016.74018

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

This work is licensed under the Creative Commons Attribution International License (CC BY).

http://creativecommons.org/licenses/by/4.0/

Abstract

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.

Keywords

Ashwagandha, Telomerase, HeLa Cell Line, TRAP Assay

1. Introduction

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 [1]. One of the major factors that accelerate aging and cause degeneration of various systems is

Corresponding author.

V. Raguraman, J. R. Subramaniam

200

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 [2]. 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 [3]. 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 [4]. Telomerase activity and short telomeres are

implicated in aging mechanism [5]-[7]. 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 [8] 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

[9] and anti-inflammation [10]. Further, it enhances longevity in C. elegans [11]. With little or no toxic effect,

Ashwagandha [12] can be considered as supplement in humans [13]. Though Ashwagandha is suggested to in-

crease longevity, so far no evidence has been provided to reinforce this claim except in C. elegans [11] 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 [14]. 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 [14]. The protein concentration was determined

using Lowry’s method [15]. 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

201

(ver.10.0).

3. Results

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 [16], we determined Ashwagandha’s telomerase activity enhancing effect following published protocols

[14]. 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

202

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.

(a) (b)

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.

4. Discussion

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 [17] [18], Aswagandha [11], Rapamycin [19] and Resveratrol [20] 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

203

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 [6] [21]. Mouse cells without telomerase activity

have short telomere length [22], 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) [23] demonstrated that enhanced telomerase activity in mice

can delay aging and confer cancer resistance. Recently, Bernardes de Jesus et al., (2011), demonstrated TA-65

[8], 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 [11]. 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.

5. Conclusion

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.

Acknowledgements

The authors thank the Center for Stem Cell and Regenerative Medicine and the Center for Indian Systems of

Medicine, Sri Ramachandra University for the infrastructure and Shri Kartikeya Pharma, Hyderabad for the fi-

nancial support.

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The Impact of Withania somnifera and carbamazepine on Epileptic Phenotype in Drosophila Temperature-Sensitive Mutants

Article

Apr 2024

Background: Epilepsy, a prevalent neurological disorder affecting individuals across all age groups, often reqquires treatment with antiepileptic drugs (AEDs). While Carbamazepine (CBZ) serves a commonly prescribed first-line medication, its efficacy is limited in 30–40 % of patients. In exploring alternative treatment options. Herbal medicines like Withania somnifera (W. somnifera) offer promising benefits with reduced adverse effects. This study aims to assess the therapeutic impacts of W. somnifera and CBZ on behavioral paralysis, reproductive abilities, and lifespan in a Drosophila paralytic temperature-sensitive mutant (paraST76). Methods: paraST76 flies were exposed to three distinct concentrations of W. somnifera or CBZ added to wheat flour-agar media. Results: The results indicate that high concentration of W. somnifera effectively extended the lifespan of Drosophila paraST76 without compromising fecundity and fertility. Conversely, exposure to 40 μg/ml of CBZ resulted in a shortened lifespan and decreased reproductive ability in the paraST76 mutant. Importantly, our findings demonstrate that neither W. somnifera nor CBZ exerted any notable impact on behavioral paralysis, as per our dataset analysis. Conclusion: This study exhibits that W. somnifera is a safe compound, devoid of any adverse effects, and possesses the capability to prolong the lifespan of Drosophila. Nevertheless, it does not affect the voltage-gated sodium channel of the paraST76 mutant

Medicinal Plants, Antioxidant Potential, and Applications to Aging

Chapter

Feb 2024

Aging is a progressive process in the body where wrinkling, fine lines of the skin, graying and thinning of hair, brittle and pale nails, and fragility in bones are obvious due to the breakdown of collagens, elastin, reduced production of melanin, and hormonal changes. DNA damage, telomerase shortening, alteration in specific genes, increase in the reactive oxygen species level, and mitochondrial dysfunction are the leading causes of aging. These internal factors are aggravated by some external factors like chronic sun exposure specifically UVA and UVB, smoking, alcohol consumption, radiation, pollution, exposure to metal ions and toxic chemicals, and nutrient deficiencies. To correct these signs of aging, antioxidants are widely used to treat and prevent the early signs of aging. Many medicinal plants, which have been used for thousands of years, are found in Rasayana category, a category of the Indian traditional health care system (Ayurveda) advocated for its intriguing antioxidant effects. Some of the medicinal plants utilized in Ayurveda as Rasayana for their therapeutic activity have been well-researched for its antiaging properties. This chapter focuses on some important plant material and isolated compounds that are reported to have ability to slow down the rate of aging and used worldwide. Majority of them exhibited anti-aging effects through their antioxidants potential.

The Critical Need to Promote Research of Aging and Aging-related Diseases to Improve Health and Longevity of the Elderly Population

Article

Full-text available

Feb 2015

Due to the aging of the global population and the derivative increase in aging-related non-communicable diseases and their economic burden, there is an urgent need to promote research on aging and aging-related diseases as a way to improve healthy and productive longevity for the elderly population. To accomplish this goal, we advocate the following policies: 1) Increasing funding for research and development specifically directed to ameliorate degenerative aging processes and to extend healthy and productive lifespan for the population; 2) Providing a set of incentives for commercial, academic, public and governmental organizations to foster engagement in such research and development; and 3) Establishing and expanding coordination and consultation structures, programs and institutions involved in aging-related research, development and education in academia, industry, public policy agencies and at governmental and supra-governmental levels.

Telomerase reactivation reverses tissue degeneration in aged telomerase-deficient mice. Nature, 469, 102-106

Article

Full-text available

Nov 2010
NATURE

An ageing world population has fueled interest in regenerative remedies that may stem declining organ function and maintain fitness. Unanswered is whether elimination of intrinsic instigators driving age-associated degeneration can reverse, as opposed to simply arrest, various afflictions of the aged. Such instigators include progressively damaged genomes. Telomerase deficient mice have served as a model system to study the adverse cellular and organismal consequences of wide- spread endogenous DNA damage signaling activation provokes progressive tissue atrophy, stem cell depletion, organ system failure, and impaired tissue in vivo 1. Telomere loss and uncapping injury responses1. Here, we sought to determine whether entrenched multi-system degeneration in adult mice with severe telomere dysfunction can be halted or possibly reversed by reactivation of endogenous telomerase activity. To this end, we engineered a knock-in allele encoding a 4- hydroxytamoxifen (4-OHT)-inducible telomerase reverse transcriptase-Estrogen Receptor (TERT- ER) under transcriptional control of the endogenous TERT promoter. Homozygous TERT-ER mice display short dysfunctional telomeres and sustain increased DNA damage signaling and classical degenerative phenotypes upon successive generational matings and advancing age. Telomerase reactivation in such late generation TERT-ER mice extends telomeres, reduces DNA damage signaling and associated cellular checkpoint responses, allows resumption of proliferation in quiescent cultures, and eliminates degenerative phenotypes across multiple organs including testes, spleens and intestines. Notably, somatic telomerase reactivation reversed neurodegeneration with restoration of proliferating Sox2+ neural progenitors, DCX+ newborn neurons, and Olig2+ oligodendrocyte populations. Consistent with the integral role of SVZ neural progenitors in generation and maintenance of olfactory bulb interneurons2, this wave of telomerase-dependent neurogenesis resulted in alleviation of hyposmia and recovery of innate olfactory avoidance responses. Accumulating evidence implicating telomere damage as a driver of age-associated organ decline and disease risk1,3 and the dramatic reversal of systemic degenerative phenotypes in adult mice observed here support the development of regenerative strategies designed to restore telomere integrity.

Withania somnifera root extract extends lifespan of Caenorhabditis elegans

Article

Full-text available

Jun 2013

Background: In the ancient Indian herbal medicine system several ayurvedic preparations are claimed to have longevity enhancing effects. But, so far, no clear scientific evidence has been provided. One among them, is the roots of the plant, commonly known as Ashwagandha (Withania somnifera Dunal- WSD), which is supposed to have myriad of beneficial effects including long life. Purpose: Here, we evaluated both the root extract (RE) and its purified ingredients (PI-RE) with a similar composition as in RE obtained from the roots of WSD for lifespan extension in the well established model system, C. elegans. PI-RE could extend the lifespan of C. elegans. Methods: We used wild type C. elegans (N2) or RB918: acr-16 (ok789); andNL2099: rrf-3 (pk1426) mutant worms and analysed their lifespan assay in Ashwagandha extract spreaded on plates containing Bacterial Lawns. Results: Strangely, while there was no effect on the wild type worms, the mutant for the human nicotinic acetylcholine receptor, nAchR, α7 equivalent, acr-16, showed around ~20% lifespan extension when treated with PI-RE. Conclusion: Thus, we are able to show that one of the age old healthy longlife supplements, Ashwagandha does extend lifespan of C. elegans.

Telomerase reactivation reverses tissue degeneration in aged telomerase-deficient mice

Article

Full-text available

Jan 2011
NATURE

An ageing world population has fuelled interest in regenerative remedies that may stem declining organ function and maintain fitness. Unanswered is whether elimination of intrinsic instigators driving age-associated degeneration can reverse, as opposed to simply arrest, various afflictions of the aged. Such instigators include progressively damaged genomes. Telomerase-deficient mice have served as a model system to study the adverse cellular and organismal consequences of wide-spread endogenous DNA damage signalling activation in vivo. Telomere loss and uncapping provokes progressive tissue atrophy, stem cell depletion, organ system failure and impaired tissue injury responses. Here, we sought to determine whether entrenched multi-system degeneration in adult mice with severe telomere dysfunction can be halted or possibly reversed by reactivation of endogenous telomerase activity. To this end, we engineered a knock-in allele encoding a 4-hydroxytamoxifen (4-OHT)-inducible telomerase reverse transcriptase-oestrogen receptor (TERT-ER) under transcriptional control of the endogenous TERT promoter. Homozygous TERT-ER mice have short dysfunctional telomeres and sustain increased DNA damage signalling and classical degenerative phenotypes upon successive generational matings and advancing age. Telomerase reactivation in such late generation TERT-ER mice extends telomeres, reduces DNA damage signalling and associated cellular checkpoint responses, allows resumption of proliferation in quiescent cultures, and eliminates degenerative phenotypes across multiple organs including testes, spleens and intestines. Notably, somatic telomerase reactivation reversed neurodegeneration with restoration of proliferating Sox2(+) neural progenitors, Dcx(+) newborn neurons, and Olig2(+) oligodendrocyte populations. Consistent with the integral role of subventricular zone neural progenitors in generation and maintenance of olfactory bulb interneurons, this wave of telomerase-dependent neurogenesis resulted in alleviation of hyposmia and recovery of innate olfactory avoidance responses. Accumulating evidence implicating telomere damage as a driver of age-associated organ decline and disease risk and the marked reversal of systemic degenerative phenotypes in adult mice observed here support the development of regenerative strategies designed to restore telomere integrity.

Rapamycin fed late in life extends lifespan in genetically heterogenous mice

Article

Full-text available

Aug 2009
NATURE

Inhibition of the TOR signalling pathway by genetic or pharmacological intervention extends lifespan in invertebrates, including yeast, nematodes and fruitflies; however, whether inhibition of mTOR signalling can extend lifespan in a mammalian species was unknown. Here we report that rapamycin, an inhibitor of the mTOR pathway, extends median and maximal lifespan of both male and female mice when fed beginning at 600 days of age. On the basis of age at 90% mortality, rapamycin led to an increase of 14% for females and 9% for males. The effect was seen at three independent test sites in genetically heterogeneous mice, chosen to avoid genotype-specific effects on disease susceptibility. Disease patterns of rapamycin-treated mice did not differ from those of control mice. In a separate study, rapamycin fed to mice beginning at 270 days of age also increased survival in both males and females, based on an interim analysis conducted near the median survival point. Rapamycin may extend lifespan by postponing death from cancer, by retarding mechanisms of ageing, or both. To our knowledge, these are the first results to demonstrate a role for mTOR signalling in the regulation of mammalian lifespan, as well as pharmacological extension of lifespan in both genders. These findings have implications for further development of interventions targeting mTOR for the treatment and prevention of age-related diseases.

Erratum: Sirtuin activators mimic caloric restriction and delay ageing in metazoans (Nature (2004) 430 (686-689))

Article

Sep 2004

Protein measurement with the Folin phenol reagent

Article

Nov 1950

Acute and Sub-Acute Oral Toxicity Assessment of the Hydroalcoholic Extract of Withania somnifera Roots in Wistar Rats

Article

Aug 2013
PHYTOTHER RES

Withania somnifera is a widely used medicinal plant for several disorders. Toxicity studies on Withania somnifera are not available. Acute and sub-acute oral toxicities of Withania somnifera root extract in Wistar rats were evaluated in the present study. In the acute toxicity study, WSR extract was administered to five rats at 2000 mg/kg, once orally and were observed for 14 days. No toxic signs/mortality were observed. In the sub-acute study, WSR extract was administered once daily for 28 days to rats at 500, 1000 and 2000 mg/kg, orally. No toxic signs/mortality were observed. There were no significant changes (P < 0.05) in the body weights, organ weights and haemato-biochemical parameters in any of the dose levels. No treatment related gross/histopathological lesions were observed. The present investigation demonstrated that the no observed adverse effect level was 2000 mg/kg body weight per day of hydroalcoholic extract of W. somnifera in rats and hence may be considered as non-toxic. Copyright © 2012 John Wiley & Sons, Ltd.

Reserpine modulates neurotransmitter release to extend lifespan and alleviate age-dependent Aβ proteotoxicity in Caenorhabditis elegans

Article

Dec 2011

Aging is a debilitating process often associated with chronic diseases such as diabetes, cardiovascular and neurodegenerative diseases like Alzheimer's disease (AD). AD occurs at a very high incidence posing a huge burden to the society. Model organisms such as C. elegans become essential to understand aging or lifespan extension - the etiology, molecular mechanism and identification of new drugs against age associated diseases. The AD model, manifesting Aβ proteotoxicity, in C. elegans is well established and has provided valuable insights. Earlier, we have reported that Reserpine, an FDA-approved antihypertensive drug, increases C. elegans lifespan with a high quality of life and ameliorates Aβ toxicity in C. elegans. But reserpine does not seem to act through the known lifespan extension pathways or inhibition of its known target, vesicular monoamine transporter, VMAT. Reserpine's mode of action and the pathways it activates are not known. Here, we have evaluated the presynaptic neurotransmitter(s) release pathway and identified acetylcholine (ACh) as the crucial player for reserpine's action. The corroborating evidences are: i) lack of lifespan extension in the ACh loss of function (hypomorphic) - synthesis (cha-1) and transport (unc-17) mutants; ii) mitigation of chronic aldicarb effect; iii) lifespan extension in dopamine (cat-2) and dopamine and serotonin (bas-1) biosynthetic mutants; iv) no rescue from exogenous serotonin induced paralysis in the AD model worms; upon reserpine treatment. Thus, modulation of acetylcholine is essential for reserpine's action.

The telomerase activator TA-65 elongates short telomeres and increases health span of adult/old mice without increasing cancer incidence

Article

Mar 2011
AGING CELL

Here, we show that a small-molecule activator of telomerase (TA-65) purified from the root of Astragalus membranaceus is capable of increasing average telomere length and decreasing the percentage of critically short telomeres and of DNA damage in haploinsufficient mouse embryonic fibroblasts (MEFs) that harbor critically short telomeres and a single copy of the telomerase RNA Terc gene (G3 Terc(+/-) MEFs). Importantly, TA-65 does not cause telomere elongation or rescue DNA damage in similarly treated telomerase-deficient G3 Terc(-/-) littermate MEFs. These results indicate that TA-65 treatment results in telomerase-dependent elongation of short telomeres and rescue of associated DNA damage, thus demonstrating that TA-65 mechanism of action is through the telomerase pathway. In addition, we demonstrate that TA-65 is capable of increasing mouse telomerase reverse transcriptase levels in some mouse tissues and elongating critically short telomeres when supplemented as part of a standard diet in mice. Finally, TA-65 dietary supplementation in female mice leads to an improvement of certain health-span indicators including glucose tolerance, osteoporosis and skin fitness, without significantly increasing global cancer incidence.