An Aqueous Extract of Withania somnifera Root Inhibits Amyloid β Fibril Formation In Vitro

* CorrespSchool ofUniversityE‐mail: e.j

PHYTOTHERAPY RESEARCHPhytother. Res. 26: 113–117 (2012)Published online 12 May 2011 in Wiley Online Library(wileyonlinelibrary.com) DOI: 10.1002/ptr.3512

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An Aqueous Extract of Withania somnifera RootInhibits Amyloid β Fibril Formation In Vitro

Suresh Kumar,1,2 Robin J. Harris,3,4 Christopher J. Seal1 and Edward J. Okello1,5*1Faculty of Science, Agriculture and Engineering, School of Agriculture, Food and Rural Development, Newcastle University,Newcastle upon Tyne NE1 7RU, UK2University School of Biotechnology, GGS Indraprastha University, Sector 16C, Dwarka, New Delhi 110075, India3Institute for Cell and Molecular Biosciences, Newcastle University, Newcastle upon Tyne NE1 7RU, UK4Institute of Zoology, University of Mainz, D‐55099 Mainz, Germany5Institute of Neuroscience, Newcastle University, Newcastle upon Tyne NE1 7RU, UK

The ability of an aqueous extract of W. somnifera L. Dunal (Family: Solanaceae) roots to inhibit fibril formationby the amyloid‐β peptide in vitro was investigated. W. somnifera is used extensively in traditional Ayurvedicmedicine as a nerve tonic with reputed memory enhancing properties. Inhibition of fibrillogenesis measured bytransmission electron microscopy and ThT fluorescence assay showed that an aqueous extract of W. somniferastrongly inhibited Aβ fibril formation in a concentration‐dependent manner, when compared with controlsamples. These results suggest that the aqueous extract of W. somnifera root has an ability to inhibit theformation of mature amyloid‐β fibrils in vitro, which are known to lead to amyloid plaque formation in vivo.Copyright © 2011 John Wiley & Sons, Ltd.

Keywords: Alzheimer’s disease; amyloid β; fibrillogenesis; W. somnifera.

INTRODUCTION

Extracellular amyloid plaques have been recognized asone of the main cytopathological features of Alzheimer’sdisease (AD) (Selko, 2002). The principle component ofamyloid plaque extracted from AD patients revealedthe presence of characteristic 40 and 42 amino acidsequences, termed the amyloid‐β (Aβ) peptide (Glennerand Wong, 1984; Hucul et al., 1985; Masters et al., 1985).The imbalance between the production and clearanceof Aβ peptides released proteolytically from themembrane‐bound amyloid precursor protein (APP) inthe brain appears to be directly related to the develop-ment ofAD. If overproduction ofAβ is not followed by aparallel increase in clearance of the peptide, this can leadto sporadic cases of AD (LeVine, 2004; Bates et al.,2009). The Aβ monomer is an unstructured, unfolded~4 kD peptide, rich in hydrophobic residues. It is stillunclear whether Aβ ever occurs in a monomeric statein vivo for extended time periods, but it has beensuggested that the smallest stable form of theAβ peptideexists as a dimer, trimer or a tetramer. Furthermore, Aβmonomers are prone to self‐aggregate and form Aβfibrils, a process generally termed Aβ fibrillogenesis.Due to their characteristic crossed β sheets, amyloidfibrils bind dyes such as Congo red and thioflavin, whichspecifically bind only to amyloid fibrils, staining tech-niques that are commonly used to analyse these fibrilstructures in more detail.The formation of well‐ordered fibrillar protein

deposits consisting of Aβ peptides has been shown to

ondence to: Dr Ed Okello, Medicinal Plant Research Group,Agriculture, Food and Rural Development, Newcastle

, Newcastle Upon Tyne NE1 7RU, [email protected]

© 2011 John Wiley & Sons, Ltd.

play a key role in neurodegeneration and is consideredto be one of the main pathogenic factors associatedwith AD. Currently there is no approved therapeuticagent directed towards the inhibition of theformation ofthese Aβ fibrillar assemblies. One possible approach isthe use of small molecules that specifically andefficiently inhibit the fibrillogenesis process. Severalplant‐based compounds such as polyphenols, curcumin,rosmarinic acid, tannic acid, catechin and quercetinhave demonstrated the ability to inhibit the formationof fibrillar assembly in vitro and the associatedcytotoxic i ty (Porat e t al . , 2006). Based onthese observations, this study measured the inhibitionof fibril formation by an aqueous extract of W. somniferaroot under in vitro conditions. The extract used inthis study has been characterized previously to be richin withanolide derivatives and shown to protect differ-entiated PC12 cells against hydrogen peroxide andβ‐amyloid induced cytotoxicity (Kumar et al., 2010).W. somnifera is used extensively in traditional Ayurvedicmedicine as a nerve tonicwith reputedmemory improvingproperties. The techniques used to measure the fibrilformation were a Thioflavin‐T (ThT) fluorescence assayand transmission electron microscopy (TEM).

MATERIALS AND METHODS

Plant material. Roots of W. somnifera were purchasedfrom a herbalist in India. The species was verified andauthenticated by Dr George Wake, School of Biology,Newcastle University, United Kingdom and specimenvoucher (SK‐WS‐01) of this plant root is deposited in theherbarium of the Medicinal Plant Research Group,Newcastle University, UK.

Received 24 July 2010Revised 09 March 2011

Accepted 22 March 2011

114 S. KUMAR ET AL.

Extract preparation. The extract used was prepared asin Kumar et al. (2010). Briefly: the air‐dried plant rootswere ground to powder consistency using an electricgrinder. Then 1 g of powdered plant material wasinfused in freshly boiled de‐ionized water (1:50w/v)for 25min. The infusions were left to cool to roomtemperature and centrifuged (12000 rpm, 15min). Thesupernatants were re‐centrifuged (12000 rpm, 10min).The supernatants were freeze‐dried. The freeze‐driedaliquots were reconstituted in deionized water prior toassay (1mg/mL stock solution).

Chemicals/reagents. Aβ peptides (Aβ 1–42), uranylacetate cholesterol, Thioflavin T, glycine, PBS werepurchased from Sigma‐Aldrich (Gillingham, Dorset,UK). All reagents were prepared in Milli‐Q water(Millipore (UK) Ltd, Watford, UK).

Transmission electron microscopy. Specimens wereprepared on continuous carbon support film formedon copper grids (300mesh), following glow dischargefor 30 s, and negatively stained with 2% w/v aqueousuranyl acetate (pH 4.5) by a single droplet procedure(Harris, 2007). The carbon support films used in thiswork had the advantage that protein fibrils readilyadsorb and can be washed to remove buffer salts beforenegative staining (Harris, 2002). Before use, the gridswere treated by glow discharge, which renders thecarbon surface hydrophilic, thus allowing satisfactoryspreading of aqueous suspensions (Hayat, 2002).Aliquots (20μL) of 50 µg/mL Aβ peptides were

added to equal volumes of plant extracts (50 µg/mL)and incubated overnight at 37 °C with constant oscilla-tion. Immediately after incubation 5μL quantities weretaken for the preparation of TEM specimens. Speci-mens were studied using a Philips CM100 TEM,routinely at instrumental magnifications of 32000× to130000×, at 100 kV. Digital images were recorded usingan Optronics 1824 × 1824 pixel CCD camera, withAMT40 version 5.42 image capture engine supplied byDeben UK.

Thioflavin‐T (ThT) fluorescence assay. Aβ fibrillogen-esis (peptide aggregation) was measured by a ThTassay, in which the fluorescence intensity reflects thedegree of aggregation (LeVine, 1999). ThT bindsspecifically to Aβ fibrils and this binding produces ashift in emission spectrum, the fluorescent signal beingproportional to the amount of amyloid formed. Aβ wasdissolved in deionized water at a concentration of100 µg/mL. The stock was diluted to 0.75 to 10 µg/mL indeionized water. 10μL of Aβ was added to 10μL ofplant extract, which was then incubated overnight at37 °C with constant oscillation. The following day it wastransferred to a microtitration plate and 80μL of ThT(10 µM) in glycine buffer (pH 9.0) was added to eachwell. Fluorescence was read after 15min at Ex 450 nmand Em 490 nm using a Lab Thermosystem microplatereader (Ascent software) (Naiki et al., 1989).

Statistical analysis. Graphpad prism software was usedto calculate the probability (p) value using the Student’st‐test. Values of p< 0.05 were considered significant.The appropriate p values, determined to be statisticallysignificant, were compared with negative controls.

Copyright © 2011 John Wiley & Sons, Ltd.

RESULTS

Effect of the aqueous extract of W. somnifera on Aβfibril formation

Transmission electron microscopy was used to assessthe inhibitory effect of the aqueous extract of W.somnifera extract on Aβ fibril formation. Aβ (50 µg/mL) peptide incubated at 37 °C for 24 h in the presenceof water alone formed fibrils of considerable lengthwhich also formed numerous networks. The networkswere thick, with fibrils tangled into each other, as shownin the control (Fig. 1A). The fibril formation wassignificantly reduced in a concentration dependentmanner when Aβ (50 µg/mL) peptide was co‐incubatedwith increasing concentrations of the aqueous extract ofW. somnifera (6.25–50 µg/mL) (Fig. 1B–E), respectively.The morphology of the fibril networks appears to beless dense, less entangled and increasingly sparse,indicating an inhibition of Aβ fibril formation.

Effect of W. somnifera root extract in the presence ofcholesterol on Aβ fibril formation

Cholesterol is known to promote Aβ fibrillogenesis,predominantly in the form of protofibrils, duringovernight incubation (Harris, 2002, 2008). Aβ (50 µg/mL) peptide co‐incubated with cholesterol (0.2mg/mL)at 37 °C for 24 h promotes fibril formation (Fig. 2A)(control). Mature double helical fibrils were formed,which were often clustered around the cholesterolmicrocrystal. In the presence of an aqueous extract ofW. somnifera (50 µg/mL) the fibril formation wasinhibited significantly (Fig. 2B). Moreover, the mor-phology of the fibrils formed with test samples appearedshort in length compared with the control, againindicating the antiamyloidogenic activity of the W.somnifera extract.

Standardization of the Thioflavin T assay

The ThT dye is known to associate rapidly with fibrillaramyloid aggregates, giving rise to a new spectralemission maximum at around 482 nm. As shown inFig. 3, a concentration‐dependent response was foundfor ThT fluorescence with an increasing concentrationof Aβ (1.25–10 µg/mL) dissolved in the glycine buffer(pH 9.0) and incubated for 24 h at 37 °C. In subsequentexperiments a 5 µg/mL concentration of Aβ was used incombination with the test compound, as the fluores-cence obtained at this concentration was highlysignificant compared with the control containing noAβ (p< 0.001) (see below).

Effect of the aqueous extract from W. somnifera rootson ThT‐induced fluorescence as a measure of Aβfibrillogenesis

When Aβ (5 µg/mL) was co‐incubated with increasingconcentrations of the aqueous extract of W. somnifera(6.25–50 µg/mL) for 24 h at 37 °C, the ThT fluorescenceshowed a concentration‐dependent reduction, indicating

Phytother. Res. 26: 113–117 (2012)

A B C

D E

Figure 1. TEM images showing representative negatively stained Aβ fibrils formed under five different conditions. (A) Control, showingmature fibril formation by Aβ (50µg/mL) peptide in the presence of water alone on incubation at 37 °C for 24h. (B–E) Fibril formations whenAβ peptide incubated with W. somnifera extract 6.25µg/mL (B), 12.5µg/mL (C), 25µg/mL (D) and 50µg/mL (E). The scale bars indicate100nm.

115WITHANIA SOMNIFERA INHIBITS AMYLOID β FIBRIL FORMATION IN VITRO

that the W. somnifera extract progressively suppressedfibrillogenesis.. As shown in Table 1, the aqueous extractinhibitedAβ aggregation by 50%at 50 µg/mL, comparedwith the control (Aβ only) (p< 0.001). As shown inTable 1 inhibition of Aβ aggregation reached amaximum of approximately 50% at a concentration of25 µg/mL W. somnifera extract, compared with thecontrol (p< 0.001).

A B

Figure 2. TEM images showing negatively stained Aβ fibrilsformed under three different conditions. (A) Control, showingmature fibril formation by Aβ (50µg/mL) peptide in the presence ofcholesterol (0.2mg/mL) but no plant extract, after incubation at37 °C for 24h. (B) Fibril formation when Aβ peptide was incubatedwith W. somnifera (50µg/mL) (B) extract. The scale bars indicate100nm.

DISCUSSION

Alzheimer’s disease (AD) is the most common neuro-degenerative disorder in elderly people, and is char-acterized by the cerebral extracellular deposition ofprotein fibrils formed by Aβ, mainly from the 40‐ or 42‐amino acid Aβ peptide. The series of events eventuallyleading to the transformation of Aβ peptides intoneurotoxic oligomeric, protofibrillar and fibrillar assem-blies is thought by many to be a key pathological eventin AD. Moreover, it was found that Aβ oligomers andfibrils exert a cytotoxic effect compared with solublepeptides, when added directly to neuronal cells(Lorenzo and Yankner, 1994). Furthermore, it has alsobeen reported that after interaction with the nerve cellmembrane, Aβ induces a sequence of events that leadsto the intracellular accumulation of reactive oxygenspecies (ROS) (Sopher et al., 1996). In spite of these


studies, the exact neurotoxic mechanism remainsunclear. These facts do, however, support the relevanceof therapeutic strategies targeting Aβ production,oligomerization and fibril formation, clearance and theprevention of neurotoxicity.

Phytother. Res. 26: 113–117 (2012)

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

B 1.25 2.5 5 10

Concentration of A [µg/mL]

Fluo

resc

ence

inte

nsity

[A

.U]

***

***

**

*

Figure 3. ThT fluorescence of different Aβ concentration (1.25–10µg/mL). B, represents blank (no Aβ). Data are presented asmean±SEM (n=3). ***p<0.001, **p<0.01, *p<0.05 com-pared with a blank containing no Aβ.

Table 1. Inhibition of Aβ aggregation by an aqueous extract ofW. somnifera root

Concentration ofW. somnifera (µg/mL)

Inhibition (%) of Aβfibrillogenesis

6.25 29±0.01a

12.50 40±0.02b

25.00 49±0.02b

50.00 50±0.02b

Each value represents mean±SEM (n=3).ap<0.05, bp<0.01 compared with control.

116 S. KUMAR ET AL.

In this study, inhibition of Aβ fibrillogenesis using aroot extract of W. somnifera plant was investigated. Theaqueous extract of W. somnifera inhibited Aβ fibrilformation under in vitro conditions, demonstratedindependently by TEM and a ThT assay, in aconcentration‐dependent manner (Figs 1, 2). TheTEM study elucidated the morphological changes inthe fibrils formed when Aβ peptides were co‐incubatedwith the root extract, compared with the control (Aβalone). This was independently supported by a reduc-tion in ThT fluorescence, obtained in parallel with theTEM study.Interestingly, it was also noted that even on prior

addition of cholesterol, which is known to potentiatefibril formation (Harris, 2002, 2008), the root extractstill showed inhibition of Aβ fibril formation. However,it is uncertain whether there is a direct interactionbetween one or more components in the root extractthat prevents the cholesterol potentiation, rather thanacting directly on the Aβ to prevent fibrillogenesis.These data indicate that the root extract ofW. somniferahas significant antiamyloidgenic potential.This is the first kind of study on an aqueous extract of

this plant showing strong inhibition of Aβ fibrillogen-esis, which strongly supports the claim thatW. somniferaroot extract has possible potential to attenuate cerebralfunctional deficits, including AD, in geriatric patients. Anumber of studies have reported a direct correlationbetween in vitro and in vivo activities of therapeuticallybeneficial compounds (Alberts et al., 1991; Martínezet al., 1999; Liu et al., 2007). Neoandrographolide, aprinciple diterpene lactone isolated from the medicinalherb, Andrographis paniculata Nees showed similar invitro and in vivo antiinflammatory activity (Liu et al.,2007). The fibrillogenesis inhibition by W. somniferaroot extract demonstrated in this study can becorrelated with the neuroprotective activity of W.somnifera extract demonstrated against Aβ inducedcytotoxicity in cultured PC12 cells (Jayaprakasam et al.,2010; Kumar et al., 2010). In animal studies, W.somnifera extract was demonstrated to promote learn-ing and memory (Dhuley, 2001). W. somnifera alsoreversed the memory loss that was induced by oxidativedamage caused by streptozotocin (Parihar et al., 2004).


It was also demonstrated that pretreatment with rootextract of W. somnifera significantly reduced (80%) thenumber of degenerating cells in the hippocampalsubregion of albino rats, thereby exhibiting neuropro-tective effects of plant preparation (Jain et al., 2001).Apart from these neuroprotective activities, the bene-ficial effect of the extract of W. somnifera in othercentral nervous system (CNS) related disorders werealso reported extensively from this plant (Bhattacharyaet al., 2001; Ahmad et al., 2005; Naidu et al., 2006). Theextract of W. somnifera is a complex mixture of variousphytochemicals. The extract used in this study has beenshown previously to be rich in withanolide derivatives(Kumar et al., 2010). Withaferin A and sitoindosidesVII–X, isolated from W. somnifera, were described fortheir potent antioxidant activity in comparison with themajor free‐radical scavenging enzymes including super-oxide dismutase (SOD), catalase (CAT) and glutathi-one peroxidase (GPX) levels in the frontal cortex andstriatum of the rat brain (Bhattacharya et al., 1997;Choudhary et al., 2005). Withanoside extracted from W.somnifera significantly improved memory deficits inAβ‐injected mice (to induce dendritic and axonalatrophy) and prevented the loss of axons, dendritesand synapses in the cerebral cortex and hippocampus(Kuboyama et al., 2006). These results suggest a knownrelationship between oxidative stress and AD. How-ever, the results presented herein suggest that theantifibrillogenesis property of W. somnifera to be aconsequence of its direct interaction with Aβ. Althoughthe above results of ThT fluorescence and TEM dataconfirm the inhibitory action of W. somnifera on Aβfibrillogenesis, they provide no information on themolecular basis of this phenomenon. The only prelim-inary interpretation that can be made is that thechemical compounds present in the aqueous extract ofW. somnifera roots, could bind to hydrophobic regionsof the Aβ peptide and thus inhibit Aβ fibril formation.

These results support the putative claims that anextract of W. somnifera might represent an alternativetherapeutic option, as the compounds present in theextract might be able to ameliorate cognitive deficiencyand neurodegeneration by inhibiting Aβ aggregation.

CONCLUSION

In conclusion, the results presented in this studydemonstrate the potent inhibition of Aβ fibrillogenesisby an extract W. somnifera root. This study reinforces

Phytother. Res. 26: 113–117 (2012)

117WITHANIA SOMNIFERA INHIBITS AMYLOID β FIBRIL FORMATION IN VITRO

the ethnopharmacological observation thatW. somniferacould have potent neuroprotective effects. It is proposedthat the extract of W. somnifera root could have thepotential to become an effective therapy forADpatients,as well as a primary or secondary preventive agent foryounger healthy individuals and patients with mildcognitive impairment. Although the above results fromTEM and the ThT fluorescence assay indicate theinhibitory action of W. somnifera root extract on Aβfibrillogenesis, they provide no information on the


molecular basis of this phenomenon. Hence, furtherdetailed biochemical studies are required to determinethe inhibitory mechanism. In vivo studies using appro-priate amyloid models are warranted in order to provideevidence of correlation with our in vitro findings.

Conflict of Interest

The authors declare that there is no conflict of interest.

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Phytother. Res. 26: 113–117 (2012)

An Aqueous Extract of Withania somnifera Root Inhibits Amyloid β Fibril Formation In Vitro

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