Withania somnifera chemotypes NMITLI 101R, NMITLI 118R,

NMITLI 128R and withaferin A protect Mastomys coucha from

Brugia malayi infection

S. KUSHWAHA,1 V. K. SONI,1 P. K. SINGH,1 N. BANO,1 A. KUMAR,2 R. S. SANGWAN2 & S. MISRA-BHATTACHARYA1

1Division of Parasitology, Central Drug Research Institute, A Unit of Council of Scientific and Industrial Research, Lucknow, UttarPradesh, India, 2Metabolic and Structural Biology Division, Central Institute of Medicinal and Aromatic Plant, A unit of Council ofScientific and Industrial Research, CIMAP, Lucknow, Uttar Pradesh, India

SUMMARY

Withania somnifera is an ayurvedic Indian medicinal plantwhose immunomodulatory activities have been widely usedas a home remedy for several ailments. We recently observedimmunostimulatory properties in the root extracts of chemo-types NMITLI-101, NMITLI-118, NMITLI-128 and purewithanolide, withaferin A. In the present study, we evaluatedthe potential immunoprophylactic efficacies of these extractsagainst an infective pathogen. Our results show that admin-istration of aqueous ethanol extracts (10 mg ⁄ kg) and with-aferin A (0Æ3 mg ⁄ kg), 7 days before and after challengewith human filarial parasite Brugia malayi, offers differen-tial protection in Mastomys coucha with chemotype 101Roffering best protection (53Æ57%) as compared to otherchemotypes. Our findings also demonstrate that establish-ment of B. malayi larvae was adversely affected by pretreat-ment with withaferin A as evidenced by 63Æ6% reduction inadult worm establishment. Moreover, a large percentage ofthe established female worms (66Æ2%) also showed defectiveembryogenesis. While the filaria-specific immunologicalresponse induced by withaferin A and NMITLI-101 showeda mixed Th1 ⁄ Th2 phenotype, 118R stimulated production ofIFN-c and 128R increased levels of IL-4. Taken together,our findings reveal potential immunoprophylactic propertiesof W. somnifera, and further studies are needed to ascertainthe benefits of this plant against other pathogens as well.

Keywords chemotype, infective larvae, lymphatic filariasis,Th1 ⁄ Th2 cytokine, withaferin A, Withania somnifera

INTRODUCTION

Wuchereria bancrofti, Brugia malayi and Brugia timori aremajor human filarial nematodes responsible for lymph sta-tic disease afflicting more than 130 million people world-wide (1). The disease is the second leading cause ofpermanent and long-term disability estimated at 5Æ5 mil-lion disability-adjusted life years (2). The current drugs forthe treatment of lymphatic filariasis principally target lar-val stages ‘microfilariae’ with little or no effect on adultparasite. Moreover, the recent threat of emergence of resis-tance to mainstay drugs has necessitated the need for thedevelopment of alternative strategies like vaccine search orresisting the infection by prior immune stimulation. Thenatural products have been a rich source of drugs andimmunomodulators, among them, Withania somnifera(WS), a terrestrial plant, has been used in traditional med-icine from the time of Ayurveda for over 3000 years forvarious diseases and disorders (3). WS contain a group ofbiologically active constituents known as withanolides inseveral parts of the plant with more prodigal amounts inleaf and root, represented by several individual chemicalmoieties of the phytochemical groups. Among these,withanone and withanolide A in root and withaferin A inleaf are the major components. Diverse pharmacologicalactivities are reported for withaferin A that includes anti-inflammatory, antigenotoxic, antitumor and antioxidantproperties (4–8). However, no information is available onits anthelmintic activity. We recently demonstrated excel-lent immunostimulatory properties in different chemotypesof WS (9). Therefore, in the present study, we hypothe-sized that immunostimulation prior to pathogen invasionmight provide protection against filarial infection. Our

Correspondence: Shailja Misra-Bhattacharya, Division of Parasi-tology, Central Drug Research Institute, A unit of Council ofScientific and Industrial Research, P.O. Box 173, Lucknow226001, Uttar Pradesh, India(e-mails: shailja_cdri@rediffmail.com; shailjacdri@yahoo.com).Disclosures: None.Received: 4 July 2011Accepted for publication: 13 December 2011

Parasite Immunology, 2012, 34, 199–209 DOI: 10.1111/j.1365-3024.2012.01352.x

� 2011 Blackwell Publishing Ltd 199

findings demonstrate that crude root extracts of chemo-types NMITLI-101 (101R), NMITLI-118 (118R) andNMITLI-128 (128R) as well as withanolide withaferin Aoffer varying degrees of protection in the susceptiblerodent host Mastomys coucha against infection of filarialparasite B. malayi. Additionally, treatment with WSextracts not only adversely affected larval establishment inthe host but also led to defective embryogenesis in femaleworms. Overall, our findings reveal potent immunoprophy-lactic properties of WS and suggest that this may be apromising strategy for evaluating other natural products asimmunoprophylactic agents.

MATERIALS AND METHODS

Source of plant material

A set of discrete chemotypes of Ashwagandha (WS) weredeveloped at Central Institute of Medicinal and AromaticPlants (CIMAP, CSIR, India) as a consequence of large-scale phytochemical screening of accessions of the plantcollected from almost all major wild habitats of the coun-try followed by genetic improvement of the selected linesin the identified core collection (10). Of these, threeselected chemotypes, NMITLI 101, NMITLI 118 andNMITLI 128, were investigated for their immunoprophy-lactic potential in this study. The plants were raised at theexperimental farm of CIMAP, Lucknow, India, followingstandard agronomic practices. Roots of 5-month-oldplants were harvested and used for the preparation ofaqueous ethanol extract.

Extraction and fractionation of WS and withaferin A

The extracts were prepared by extraction of the liquidnitrogen powdered fresh root tissue of each chemotype intwo volumes (g ⁄ mL) of aqueous ethanol (75 : 25, v ⁄ v) for24 h with occasional shaking followed by filtrationthrough two layers of muslin cloth and refiltration of thefiltrate through a filter paper. The filtrate was saved, andresidue was re-extracted two more times as mentioned ear-lier. The three filtrates were pooled, concentrated in aflash evaporator at 50�C followed by complete drying in afreeze dryer. The lyophilized extract was weighed andanalysed by TLC and HPLC and used in the currentstudy. The yield of extracts on fresh weight basis was4Æ28%, 3Æ30% and 4Æ08% and dry weight basis was 16Æ0%,15Æ0% and 18Æ8% for NMITLI 101R, NMITLI 118R andNMITLI 128R, respectively. Withaferin A was isolatedfrom the leaf extract of chemotype NMITLI-118, preparedand processed by silica gel column chromatography andcrystallization cycles essentially as described earlier (10).

Animals

Six-week-old outbred male M. coucha (‘GRA’ Giessenstrain) were used for treatment with WS samples. Theanimals were housed under standard conditions of tem-perature (23 € 1�C), relative humidity (55 € 10%) and12 : 12 h light ⁄ dark cycles at National Laboratory Ani-mal Centre, CDRI, Lucknow, India, and fed standardpellet diet and water ad libitum. In each experiment, eachgroup consisted of four Mastomys, and each experimentwas carried out twice. All the animals, animal handlingand experimental protocols employed in the present studywere duly approved by the Institutional Animal EthicsCommittee (IAEC) bearing IAEC no. 129 ⁄ 08 ⁄ Para ⁄ -IAEC.

Recovery of infective larvae (L3) of Brugia malayi

Infective larvae (L3) of B. malayi were recovered from lab-oratory bred vector mosquitoes (Aedes aegypti) fed ondonor Mastomys 9 € 1 days earlier (11). L3 were isolatedfrom gently crushed mosquitoes by Baermann technique,washed and counted in Ringer’s solution.

Experimental plan

The immunoprophylactic efficacy of three chemotypes101R, 118R and 128R and withanolide, withaferin A, wasinvestigated in Mastomys. To ensure that prophylacticeffects of test samples were attributable to immunostimula-tion and not because of antifilarial effects, experimentswere designed to assess any direct antilarval activity ofsamples.

In vitro antifilarial activity of different chemotypes andwithaferin A on Brugia malayi L3The effects of Withania chemotypes and withaferin A wereobserved in vitro to ensure that the immunoprophylacticactivity was not because of lethal effects of samples onL3. Stock solutions of samples (10 mg ⁄ mL) were preparedin DMSO. L3 were washed several times in antibiotic for-tified RPMI medium and placed in 48-well culture platessuch that each well contained 10 L3 ⁄ mL ⁄well. Parasiteswere exposed to different concentrations of samples rang-ing from 7Æ8 to 62Æ5 lg ⁄ mL for 48 h at 37�C in a CO2

incubator. After exposure, L3 were transferred to freshculture medium for 30 min at 37�C and their motility wasobserved. The adverse effect of test sample on larvalmotility was evaluated by scoring the larval motility andgiven the following grades: 4+, no reduction; 3+, 1–49%reduction; 2+, 50–74%; 1+, 75–99% reduction; and D,100% reduction.

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Treatment with chemotypesThe root extracts of all three different chemotypes 101R,118R and 128R were prepared in water and tested for theirimmunoprophylactic efficacy at an optimal immunostimula-tory dose of 10 mg ⁄ kg, which was administered orally for 14consecutive days. Mastomys were subcutaneously (s.c.) chal-lenged with 100 L3 on day 7 since the start of the treatmentwith different chemotypes. Control animals received distilledwater andwere challengedwith L3 under identical conditions.After 90 days of L3 challenge, microfilarial (Mf) density wasmonitored in 10 lL tail blood smear. The monitoring proce-dure continued every month up to day 180 when the animalswere euthanized to ascertain adult worm burden in varioustissues viz. lungs, heart, testes and lymph nodes (12).

Treatment with withaferin AFor evaluating the immunoprophylactic activities of with-aferin A, animals were placed in three experimental groupsalong with matched controls, and following treatmentschedule was followed.1 Animals in group I were pretreated with withaferin A

for 7 consecutive days, prior to L3 challenge that wasgiven on day 7. After L3 challenge, withaferin A wasagain administered for another 7 days.

2 Animals in group II were pretreated with withaferin Afor 7 consecutive days, prior to L3 challenge on day 8.

3 Animals in group III were simultaneously administeredboth L3 and withaferin A at day 0 followed by withafer-in A administration for next 7 days.Therefore, animals in group I and II were aimed for

evaluating the immunoprophylactic activities of withaferinA, while animals in group III were used for assessing thein vivo anti-L3 activity of withaferin A.

A fine suspension of withaferin A was prepared in 0Æ1%Tween-80 and fed orally to Mastomys at 0Æ3 mg ⁄ kg as perthe treatment schedule. Each animal was challenged with100 L3 in the anterior back region via the s.c route. Theanimals in groups II and III along with their respectivecontrols were euthanized on day 30 to assess parasite bur-den and ensure that the activity was not because of directanti-L3 action of withaferin A. Animals in group I werefollowed for 180 days for the assessment of Mf, after whichthey were euthanized to ascertain adult parasite burdenand immune response. For measuring filaria-specific anti-bodies, small amount of blood was collected at varioustime points from these animals via the retro-orbital plexusroute, and antibody levels were ascertained in the serum.

Blood microfilaraemia

The effect of immunomodulators was evaluated on devel-oping Mf pattern by assessing Mf density in the blood of

both treated and control Mastomys by drawing 10 lL oftail blood between 12:00 and 12:45 hours, that is, duringthe peak Mf circulation period (12). Blood was first col-lected on day 90 post-L3 challenge and continued everymonth till day 180. Per cent reduction in the Mf densityin treated animals over control was assessed to observe theeffect of immunostimulation on resulting Mf.

Adult worm establishment and female worm fecundity

Animals treated with the three chemotypes and thosereceived withaferin A for 14 days were euthanized on day180 post-L3 challenge along with their respective controls.Mastomys treated with withaferin A belonging to group IIand III were euthanized early, that is, on day 30 post-L3challenge, and their heart, lungs, testes and lymph nodeswere excised and teased gently under the stereomicroscopeto recover adult worms. Parasites were examined for theirnumbers, sex ratio, motility and death. All the femaleworms recovered on day 180 were teased individually inphosphate-buffered saline (PBS, pH 7Æ2), their intrauterinecontent (eggs, embryo, Mf) was examined microscopically,and percent sterilization of female worms was computed.Percent change or reduction in worm recovery wasassessed by counting the number of worms recovered andcomparing them with matched controls. (12).

Immunological response of host

Humoral immune response

Adult antigen preparation.

Adult worms (male and female) of B. malayi were recoveredfrom gerbils, freed of host tissue and washed several timesin sterile PBS. Thereafter, they were homogenized usingElvejhm tissue grinder in sterile PBS containing proteaseinhibitors (Sigma, St. Louis, MO, USA) and incubatedovernight (O ⁄ N) at 4�C. Next day, the homogenate was son-icated (Soniprep 150; MSE, London, UK) at 10 Kcs for10 min in ice and centrifuged at 20 000 g for 30 min(Biofuge Stratos centrifuge, Kendro laboratory products,Sollentum, Germany) at 4�C, and supernatant was col-lected. The protein content was estimated in the supernatantby Bradford method (13).

IgG antibody level by enzyme-linked immunosorbantassay (ELISA).

IgG antibody titres in treated and control mice sera weremeasured by indirect ELISA (14). In brief, ELISA plates

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(Nunc, Roskilde, Denmark) were coated with B. malayiadult antigen (1 lg ⁄ mL, prepared in carbonate buffer (pH9Æ6) and incubated overnight at 4�C. Thereafter, plateswere blocked with 1% gelatin. Mouse serum (1 : 400) andrabbit anti-mouse IgG-HRP (1 : 10 000) were used asprimary and secondary antibodies, respectively. Reactionwas developed using orthophenyldiamine (OPD) substrate(Sigma) and H2O2. The reaction was terminated by theaddition of 2Æ5 M H2SO4. The absorbance was read at492 nm using an ELISA reader (Tecan, Mannedorf,Switzerland).

Antibody isotype measurement.

Antibody isotypes were determined by antibody isotypingkit as per manufacturer’s protocol (Sigma). Briefly, afteradult antigen coating and blocking, primary antibody(1 : 100) was added and the plate was incubated at 37�C for1 h, followed by re-incubation for another hour at 37�Cwith 1 : 1000 diluted isotype-specific monoclonal antibodies(goat anti-mouse IgG1, IgG2a and IgG2b) in triplicate.Peroxidase-labelled rabbit anti-goat IgG (1 : 5000) and sub-strate were finally added to start the reaction.

Cellular immune response

Splenocytes.

Single-cell suspension of splenocytes was prepared fromspleens collected from animals of various groups alongwith their respective controls. They were minced inincomplete RPMI medium supplemented with antibiotic–antimycotic mixture (Sigma) and filtered through a70-lmcell strainer (BD Biosciences, San Diego, CA, USA). Theresulting cell suspension was centrifuged, and RBCs werelysed with ice-cold lysis buffer at room temperature (RT).RBC-free cells were washed thrice and suspended incomplete RPMI supplemented with 10% foetal bovineserum.

Peritoneal macrophages.

Macrophages were harvested from the peritoneal cavity ofMastomys as described earlier (15). In brief, 5 mL of ice-cold RPMI containing 5 U ⁄ mL heparin was injected intoperitoneal cavity of Mastomys, and their abdomen wasmassaged gently. The medium was aspirated using a syr-inge, and cells were washed and resuspended in completeRPMI. The viability of cells was determined by trypanblue exclusion, and cell densities were assessed using anhemocytometer.

Reactive oxygen species (ROS) in the peritoneal macro-phages.

Production of intracellular ROS in peritoneal macrophag-es was determined by flow cytometry using 2¢, 7¢-dichloro-fluorescin diacetate (DCF-DA; Sigma)-based fluorometricassay as described earlier (15). Harvested macrophages ofboth treated and control groups were adjusted to aconcentration of 1 · 106 cells ⁄ mL in PBS and incubatedwith DCF-DA at a final concentration of 1 lM for15 min at 37�C. After incubation, cells were washed twicein PBS, and ROS levels were determined by measuringthe fluorescence intensity on FACS Calibur (BD Bio-sciences). Data were analysed by CELLQUEST Software(BD Biosciences) and are presented as mean fluorescenceintensities.

Immunophenotyping of T and B lymphocytes.

Cell surface antigen staining of B and T cells was performedwith fluorochrome-conjugated monoclonal antibodiesdirected against CD4, CD8 and CD19 (BD Biosciences)following manufacturer’s protocol as described earlier (15).Briefly, 1 · 106 splenocytes from treated and control Mas-tomys were blocked with mouse Seroblock FcR antibody at4�C for 10 min. Blocked cells were washed and labelled witheither CD4-FITC-, CD8-PE- or CD-19 FITC-conjugatedprimary antibodies and incubated at 4�C in dark for20 min. Thereafter, cells were washed, and pellet was sus-pended in FACS buffer and acquired on FACS Calibur (BDBiosciences). Flow cytometric data were analysed usingCELLQUEST analysis software.

Intracellular Th1 ⁄ Th2 cytokines.

Intracellular cytokines were measured in RBC-free spleno-cyte suspension following the instructions of the manufac-turer (15). Briefly, splenocytes (2 · 106 ⁄ mL) wereincubated with brefeldin A (10 lg ⁄ mL) for 6 h in CO2

incubator at 37�C and re-incubated with mouse SeroblockFcR antibody at 4�C for another 10 min. Thereafter, cellswere washed and labelled with FITC-conjugated ratanti-mouse CD4 monoclonal antibody. After cell surfacestaining was over, cells were fixed and permeabilized withLeucoperm A and Leucoperm B reagents (Serotec,Oxford, UK) and incubated at RT for another 15 min.The CD4-labelled cells were dispensed equally in twotubes, and PE-labelled rat anti-mouse IL-4 and IFN-cwere added in separate tubes. After incubation at 4�Cfor 30 min, cells were washed and suspended in 250 lL of0Æ5% paraformaldehyde and acquired on FACS Calibur.

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In vitro cytotoxicity assay on test samples

Cytotoxicity assay was performed according to themethod of O’Brien et al. (16) using fluorescent dye resazu-rin to evaluate CC50, the concentration at which 50% ofthe cells under investigation became dead. Briefly,1 · 105 Vero cells ⁄ mL were plated in a 96-well flat-bottomculture plate and incubated over night at 37�C in a CO2

incubator for cells to adhere. Control wells contained onlythe medium. After incubation, serially diluted chemotypeextracts 101R, 118R, 128R and withaferin A were addedto the wells containing cells, and the plate was again incu-bated for another 72 h. After incubation, 10 lL of cellviability dye Alamar blue or resazurin (12Æ5 mg ⁄ 100 mLPBS; Sigma) was added to each well, and plate was incu-bated further for 2–4 h in the dark. Thereafter, fluorimeterreadings were taken at excitation and emission wavelengthsof 536 and 588 nm, respectively, and logistic regressionmodel was used for the calculation of the CC50. Thesedata are presented as percentage cytotoxicity.

Statistical analysis

Data are expressed as mean € SEM. Statistical signifi-cance was calculated by one-way analysis of variance (ANO-

VA) followed by Dunnett’s test using the PRISM GRAPHPAD

software (Graph Pad Software, Inc., La Jolla, CA, USA).P £ 0Æ05 was considered significant, whereas P £ 0Æ01 and£0Æ001 were considered highly significant and very highlysignificant, respectively.

RESULTS

Chemotypes do not show any lethal effect on Brugia ma-layi L3 in vitro

We did not observe any lethal effect of the three differentchemotypes of W. somnifera viz. 101R, 118R and 128R onL3 when they were used at a concentration of 62Æ5 lg ⁄ mLfor a period of 48 h (Table 1).

Withaferin A is lethal for Brugia malayi L3 in vitro butnot in vivo

Experiments performed in vitro showed profound lethaleffect of withaferin A on B. malayi L3 when tested at aminimum concentration of 7Æ8 lg ⁄ mL with in 24 h. Toascertain the in vivo larvicidal effects, we administeredwithaferin A simultaneously with L3 challenge and contin-ued its administration for a period of 7 days (group III).Withaferin A did not show any noticeable lethal effect onL3 in vivo as the recovery of parasites on day 30 post-L3

challenge in both the treated and control groups did notshow any appreciable change. We observed a meagre 4Æ3%reduced parasite load in Mastomys treated with withaferinA (14Æ13 € 1Æ2 in treated Mastomys vs.13Æ45 € 2Æ3 inuntreated controls) (Figure 1).

Withaferin A and chemotype 101R followed by chemo-types 118R and 128R exhibited best immunoprophylacticefficacy in Mastomys

In vivo administration of the three different chemotypesand withaferin A showed an adverse effect on the estab-lishment of L3. When animals were treated for 14 consec-utive days, they showed lower Mf density in comparisonwith untreated controls and Mf counts were significantlyreduced (P < 0Æ01) in all groups with the exception ofchemotype 128R during a 180-day follow-up period. (Fig-ure 2a and Table 2).

We also observed significantly reduced adult worm recov-ery in animals treated with withaferin A followed by thechemotypes 101R and 118R. However, 128R had no signifi-cant effect on worm establishment (Table 2). Adverse effecton intrauterine stages was also noticed in the female wormsrecovered from Mastomys treated with withaferin A(66Æ25 € 3Æ94%), 101R (53Æ47 € 3Æ9%) and 118R (43Æ54 €2Æ86%) when compared with untreated controls that

Table 1 In vitro activity of chemotypes 101R, 118R, 128R andwithaferin A on Brugia malayi L3

Extract ⁄compound

Concentration(lg ⁄ mL)

Activity scoreafter 48 h of treatment

101R 62Æ5 4+31Æ75 4+15Æ8 4+7Æ8 4+

118R 62Æ5 4+31Æ75 4+15Æ8 4+7Æ8 4+

128R 62Æ5 4+31Æ75 4+15Æ8 4+7Æ8 4+

Withaferin Aa 62Æ5 D31Æ75 D15Æ8 D7Æ8 D

Control 62Æ5 4+31Æ75 4+15Æ8 4+7Æ8 4+

4+ = 0% reduction in motility, D = 100% reduction in motility ordeath; aL3 died within 24 h of exposure.

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exhibited only (21Æ21 € 1Æ7%) sterilization. These valueswere found to be statistically significant (Figure 2b).

Owing to observed lethal effects of withaferin A and torule out any possibility of direct larval killing by the com-pound on B. malayi, L3 in vivo animals in group III were

fed withaferin A for 7 days starting from the day of L3challenge. No significant effect on L3 establishment wasnoticed in this group (4Æ3% reduction in adult worm estab-lishment as compared to controls). In addition, it was alsoobserved that a 7-day pretreatment of withaferin A was aseffective as a 14-day treatment in terms of parasite recov-ery on day 30 (59Æ8% reduction in adult worm establish-ment) and extending the treatment for another 7 daysafter L3 challenge did not exert significant effect on estab-lishment of L3 (Figure 1).

Withania chemotypes and pure withaferin A trigger genera-tion of filaria-specific antibodiesTreatment with different Withania chemotypes and with-aferin A led to ample generation of filaria-specific IgGantibody, whose levels were maintained in all treatedgroups until the day of autopsy. The highest level offilaria-specific IgG antibody was recorded in withaferinA-treated group, and among the three chemotypes, 101Rinduced highest IgG production (Figure 3a). Serum IgGisotype levels in withaferin A-, 101R- and 118R-treatedanimals demonstrated a significant increase (P < 0Æ01)in specific IgG1 and IgG2a levels in contrast to128R, which showed only high IgG1 isotype titre(Figure 3b).

Peritoneal macrophages of Mastomys produce significantamount of ROS when treated with WS samplesProfound up-regulation in spontaneous ROS production(P < 0Æ001) was noticed in all the animals treated witheither withaferin A, or chemotypes 101R, 118R and 128R.Moreover, this was found to be statistically significantacross all groups when compared with untreated controls(P < 0Æ001) (Figure 4a).

Figure 1 Total parasite recovery from withaferin A-treated Mas-tomys. In group I, animals were fed with withaferin A for 14 con-secutive days and L3 challenge on day 7 since the start oftreatment. In group II, Mastomys treatment was given for 7 daysand L3 challenge on day 8 since the start treatment. In group III,L3 inoculation and withaferin A treatment were started from day0 (the day of L3 challenge) continuing up to day 7 to observein vivo anti-L3 activity of withaferin A. In group I, animals weresacrificed on day 180 post-challenge, and in groups II and III,animals were scarified on day 30. The parasites were recoveredfrom various tissues viz. heart, lungs, lymph nodes and testis. Barrepresents mean € SEM value, and the statistical significancebetween the mean values of immunized and control groups isindicated as *P £ 0Æ05, **P £ 0Æ01 and ***P £ 0Æ001. P £ 0Æ05was considered significant, whereas P £ 0Æ01 and P £ 0Æ001 wereconsidered highly significant and very highly significant, respec-tively.

(a) (b)

Figure 2 (a) Microfilarial density in WS chemotypes- and withanolide withaferin A (WFN A)-treated Mastomys following Brugia malayiinfective larval challenge was measured from day 90 post-challenge in tail blood. (b) Effect of pretreatment of Mastomys with WS chemo-types and withaferin A (WFN A) on female worm embryogenesis was examined. Significant number of females recovered from withaferinA, 101R and 118R had adverse effect on embryogenesis. Bar represents mean € SEM value, and the statistical significance between themean values of immunized and control groups is indicated as *P £ 0Æ05, **P £ 0Æ01 and ***P £ 0Æ001. P £ 0Æ05 was considered significant,whereas P £ 0Æ01 and P £ 0Æ001 were considered highly significant and very highly significant, respectively.

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(a) (b)

Figure 3 (a) Filaria-specific antibody level in the sera of Mastomys at various time intervals after larval challenge was assessed by ELISA.Highest filaria-specific IgG antibody titre was generated in sera of withaferin A-treated Mastomys followed by 101R, 118R and 128R. (b)Filaria-specific antibody isotype level in Mastomys sera was gauged at the end of observation period on day 180. Increased level of filaria-specific IgG1 isotype was found in all treated groups. High titres of IgG2a antibodies were observed in withaferin A-, 101R- and 118R-treated Mastomys sera. UT-UI-C and UT-IN-C symbolize untreated uninfected control and untreated infected control, respectively. Barrepresents mean € SEM value, and the statistical significance between the mean values of immunized and control groups is indicated as*P £ 0Æ05, **P £ 0Æ01 and ***P £ 0Æ001. P £ 0Æ05 was considered significant, whereas P £ 0Æ01 and P £ 0Æ001 were considered highly signif-icant and very highly significant, respectively.

Table 2 Immunoprophylactic effect of chemotypes and withaferin A in Mastomys on day 180 post-L3 challenge

Animalgroups

Ani-malsused

Adult wormcount ⁄ animal

Adultwormsa

Total adultworm recoverya

% Reduction intotal worm recovery

% Change in microfilarial ⁄ 10 lL blood overcontrol (day 180)

101R 8 $ 3, 6, 2, 8, 6, 6, 2, 4# 1, 1, 2, 3, 2, 5, 1, 2

$ 4Æ6 € 2Æ2***# 2Æ1 € 1Æ3**

6Æ75 € 3Æ1*** )53Æ7 )57Æ56

118R 8 $ 8, 4, 9, 8, 5, 7, 7, 5# 4, 3, 2, 4, 2, 3, 4, 3

$ 6Æ6 € 2Æ1***# 3Æ1 € 0Æ9

9Æ25 € 2Æ7** )33Æ3 )49Æ00

128R 8 $ 8, 6, 8, 10, 9, 8, 11, 6# 4, 4, 4, 3, 3, 4, 4, 4

$ 8Æ1 € 1Æ8# 3Æ9 € 0Æ8

11Æ87 € 2Æ0 )18Æ8 )35Æ00

Withaferin A 8 $ 5, 3, 5, 2, 3, 3, 2, 3# 3, 1, 2, 2, 2, 3, 2, 1

$ 3Æ3 € 1Æ1***# 2 € 0Æ7**

5Æ3 € 1Æ3*** )63Æ6 )68Æ29

Control 8 $ 11, 9, 8, 14, 9, 11, 7, 9# 4, 3, 4, 7, 5, 5, 3, 7

$ 9Æ7 € 2Æ3# 4Æ7 € 1Æ5

14Æ6 € 3Æ4 – –

aValues are expressed as mean € SE; Statistical analysis was carried out by one-way anova followed by Dunnett’s multiple comparison;Statistical significance **P < 0Æ01 (highly significant), ***P < 0Æ001 (very highly significant).

(a) (b)

Figure 4 (a) Reactive oxidative burst (ROS) generation in peritoneal macrophages was measured by DCF-DA. All the treated groups dem-onstrated significant increase in ROS over untreated challenged control Mastomys. (b) Splenocytes from Mastomys were stained withFITC-conjugated anti-mouse CD 19 antibody and analysed on FACS. Significant increase in B-cell population was noticed in case ofwithaferin A-, 101R- and 128R-treated animals but not in 118R. Bar represents mean € SE of geometric means values. Bar representsmean € SEM value, and the statistical significance between the mean values of immunized and control groups is indicated as *P £ 0Æ05,**P £ 0Æ01 and ***P £ 0Æ001. P £ 0Æ05 was considered significant, whereas P £ 0Æ01 and P £ 0Æ001 were considered highly significant andvery highly significant, respectively.

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Withania chemotypes and pure withaferin A differentiallyup-regulate T and B lymphocyte populationsExcept chemotype 118R, treatment with all other chemo-types including pure withaferin A resulted in expansionof CD19+ B cells (P < 0Æ001) (Figure 4b). Among the Tcells, significant up-regulation of CD4+ T-helper cellpopulation (P < 0Æ01 to <0Æ001) was observed in thespleens of all the treated animals (Figure 5a). The CD8+cytotoxic T cells also revealed augmented population(P < 0Æ001) in 101R-, 118R- and withaferin A-treatedanimals with withaferin A administration showing themaximal effect (Figure 5b).

Chemotype 101R and withaferin A generate mixed Th1 ⁄ Th2responseIntracellular content of major pro-inflammatory and anti-inflammatory cytokines revealed a mixed Th1 ⁄ Th2immune response in chemotype 101R- and withaferinA-treated animals as evident by up-regulated IL-4 andIFN-c cytokine levels. While 118R stimulated production

of IFN-c (Th1 biased response), 128R exhibited Th2-biased (IL-4) immune response (Figure 6a,b).

In vitro cytotoxicity

Toxicity results obtained from Vero cells demonstratedCC50 value of �253 lg ⁄ mL, which was found to be simi-lar across all chemotypes, thus demonstrating high safetyindex of the three extracts. Our results also show thatwithaferin A was not cytotoxic even at a concentration of300 lg ⁄ mL, which was the maximum concentration testedin the present study.

DISCUSSION

The activation of immune system has important connota-tions in several infectious diseases like AIDS, tuberculo-sis, leishmaniasis, filariasis and malaria all of which areaccompanied by profound immunosuppression. Therefore,sincere search for finding immune modifiers that would

(a) (b)

Figure 5 CD4+ and CD8+ cells in splenocytes from control and treated Mastomys were assessed by flow cytometry after staining with ratanti-mouse monoclonal antibodies. Bar represents mean € SE of geometric means values. (a) Significant increase in T-helper immuneresponse was observed in treated animals as indicated by increased CD4+ T-cell population. (b) Increased percentage of CD8+ cytotoxic Tcells (Tc) was noticed in treated groups except 128R. Bar represents mean € SEM of geometric means values, and the statistical signifi-cance between the mean values of immunized and control groups is indicated as *P £ 0Æ05, **P £ 0Æ01 and ***P £ 0Æ001. P £ 0Æ05 was con-sidered significant, whereas P £ 0Æ01 and P £ 0Æ001 were considered highly significant and very highly significant, respectively.

(a) (b)

Figure 6 Intracellular Th1 and Th2 cytokines were measured in permeabilized splenocytes cells. (a) Significant increase in the anti-inflam-matory (Th2) cytokine IL-4 was seen in withaferin A followed by 128R and 101R. No significant increase in 118R-treated animals wasnoticed. Bar represents mean € SE. (b) Production of pro-inflammatory cytokine IFN-c (Th1) was found significantly higher in treatedgroups except 128R. Bar represents mean € SEM value, and the statistical significance between the mean values of immunized and controlgroups is indicated as *P £ 0Æ05, **P £ 0Æ01 and ***P £ 0Æ001. P £ 0Æ05 was considered significant, whereas P £ 0Æ01 and P £ 0Æ001 wereconsidered highly significant and very highly significant, respectively.

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have the potential to stimulate necessary immune func-tions and suppress unnecessary functions is truly desired.In recent years, use of plants or plant products as immu-nomodulators has gained momentum for developingnovel therapeutics against various immune disorders (17–22). Being derived from plant, these immunomodulatorsare inexpensive, help in maintaining immune systemhomoeostasis and are safe during prolong treatmentschedules (23). The pharmacological activities, particularlystimulation of immune functions by traditionally usedherbs, have been the focus of alternative medicine sincemany years. Withania somnifera, also known as Ashwa-gandha, is one such herb that has been categorized as‘Rasayana’ in Ayurveda, because of its beneficial proper-ties like augmenting defence against diseases, arrestingageing, revitalizing the body in debilitated condition andincreasing the capability of the individual to resist adverseenvironmental factors (24–27).

Recent observations of immunomodulatory propertiesof different WS root chemotypes and withanolides by ourgroup in mice (9) prompted us to further evaluate theiractivities as immunoprophylactant against infection ofhuman filarial parasite B. malayi. Different chemotypesand withaferin A were administered at a dose of 10 and0Æ3 mg ⁄ kg, respectively, which was earlier found to beoptimal for immunostimulatory actions in mice. Our find-ings illustrate that all the chemotypes possess immunopro-phylactic value and resist development of filarial infectionto varying degrees. Among the three chemotypes, 101Rprovided considerable degree of protection followed by118R, while withaferin A even at a low dose of 0Æ3 mg ⁄ kgoffered better protection than 101R. To ensure that theprotection offered by Withania samples was because ofimmunostimulatory activity, additional experiments werecarried out to observe in vitro lethal effect on L3, becauseWS samples were administered continuously for 14 dayscovering both pre- and post-infection period. While nei-ther chemotype exhibited lethal activity on L3 in vitro,withaferin A killed B. malayi L3 in vitro when used at aconcentration of 7Æ8 lg ⁄ mL. Furthermore, when with-aferin A was administered in vivo to Mastomys for 7 daysstarting from the first dose of L3 inoculation, no notice-able killing of larval stages was observed after a month ofL3 infection (group III), thereby confirming that thereduced parasite load in treated animals was because ofimmunostimulation rather than in vivo antiparasiticactivity of withaferin A. Prior immunostimulation ofanimals with withaferin A for 7 continuous days followedby L3 challenge a day after the end of treatment also ledto significant reduction in adult worm recovery, whichfurther confirmed immunoprophylactic properties of with-aferin A.

Pre-administration of test samples led to reduced Mfload in the blood of treated animals, which could be eitherbecause of lower adult filarial parasite burden or becauseof adverse effects of heightened immune response onembryogenesis of female parasites or Mf release. Likewise,adult worm burden was also lowest in case of pure withaf-erin A although chemotype 101R also checked the estab-lishment of inoculated B. malayi larvae to some extent.Differential level of protection offered by different chemo-types may be explained by differentially regulated immunemechanisms all tuned in to acquire antifilaria-specificresponse after parasite challenge.

The parasite-specific IgG antibody was noticed in allthe treatment groups once the larval progression to adultparasites was complete and maintained till the end ofexperiment, withaferin A and 101R treatment groups pro-duced highest amount of serum IgG titres. The immuneparameters indicated significant up-regulation in CD4+T-cell population demonstrating the ability of WS to stim-ulate major immune regulatory T-helper cells. WithaferinA, being the pure withanolide, was highly effective andshowed promising immune stimulation at a dose that was30 times less than the crude extracts of different chemo-types. In addition, there was also a significant increase incytotoxic T cells in Mastomys treated with chemotypes101R, 118R and withaferin A. The increased pool ofT-helper cell and cytotoxic T cell might be a crucial factorfor partial protection in Mastomys because T-cell hypo-responsiveness is a well-established phenomenon in filarialinfections (28,29). The chemotype 101R- and withaferinA-treated animals were found to develop a mixedTh1 ⁄ Th2 response as evidenced by significantly up-regu-lated cytotoxic CD8+ (Tc) T cells and CD19+ B cells, aswell as Th1 cytokine IFN-c and Th2 cytokine IL-4 levelsalong with increased titres of both IgG1 and IgG2a inserum. The response generated by chemotype 101R couldbe attributed to the presence of withaferin A and its vari-ous derivatives like 17-hydroxy and 27-deoxy withaferin Ain this chemotype (9). While chemotype 118R-treated ani-mals were found to show a Th1-polarized immuneresponse as evident by increased numbers of Tc cells, ele-vated IFN-c and IgG2a antibody levels, chemotype 128Rmounted a Th2-biased immune response as evident bysignificant increase in CD19+ B cells, production of Th2cytokine IL-4 and increase in IgG1 antibody levels with-out significant increase in IgG2a over that of control.

Our findings thus suggest that stimulation of both Th1-and Th2-helper immune response provide better degree ofprotection than either a Th1- or Th2-biased stimulation.Immunosuppression has been earlier reported to beaccompanied with invasion of infective filarial larvaefacilitating their establishment in the vertebrate host (30–

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34). The immunostimulation provided by withaferin Aand different chemotypes during infection mounted suffi-cient resistance or at least neutralized to some extent theimmunosuppression brought about by the invading larvaeinterfering with its establishment, further growth anddevelopment. The administration of WS samples was donebefore and after larval challenge to elicit host immune sys-tem for substantial period of time because filarial parasiteshave a long developmental cycle. Down-regulation ofimmune system has been attributed to the induction ofregulatory T cells and alternatively activated macrophages,which are able to suppress both Th1 and Th2 responses(34,35). In humans, active filarial infection is accompaniedwith a loss of antigen-specific T-cell proliferative responses(33–36) and lowered production of both Th1 (IFN-c) andTh2 (IL-5) effector cytokines, indicating the role of bothT and B cells in imparting resistance to filarial parasiteestablishment (37,38). This might possibly be the reason ofadequate protective immune response imparted by chemo-type 101R and withaferin A through generation of mixedTh1 ⁄ Th2 response. Once the active infection is established inthe host, a Th2-dominant response persists because ofdown-regulation of Th1 response by immunomodulatorycytokines IL-10 and TGF-b (39). This phenomenon was alsoobserved in the current study in case of chemotype 128R-treated animals that were not able to ward off infection.

The primary targets of most immunomodulatory com-pounds are believed to be macrophages, which play a keyrole in the generation of immune response. Increasedrespiratory burst could be correlated with increased killingactivity. Significant ROS generation was observed in mac-rophages isolated from Mastomys fed with chemotypes101R, 118R and withaferin A. Macrophage activation wasfurther assisted by increased IFN-c secretion in chemotype101R-, 118R- and withaferin A-treated groups that mighthave contributed towards protection. Moreover, WS sam-ples administered via oral route were found to be safe as

no animal mortality occurred, and CC50 values in vitrowere also quite high. Thus, our present findings demon-strate that development of infective larvae in an immuno-logically elevated environment created by differentchemotypes and withaferin A hampers their survival andfurther development in the vertebrate host.

We believe that using effective chemotypes or withanolideas adjuvants in vaccines may efficiently enhance the efficacyof vaccine. Traditional vaccines mostly use chemical adju-vants like aluminium or oil, but these have many disadvan-tages, such as strong local stimulation and carcinogenesis,together with complicated preparations or failure toenhance immunogenicity of weak antigens (40). The plant-derived adjuvants have lesser side effects and stimulate dif-ferent branches of the immune system, so they have thepotential to be used in design of new vaccines by elicitingthe desired immune response (41,42). The other uses ofactive extracts ⁄withaferin A could be as antifilarial ⁄ and orantiparasitic drug adjuncts because many antiparasiticdrugs including diethylcarbamazine act through the hostimmune system. Taken together, our findings are highly per-tinent and demonstrate the usefulness of chemotype 101Rand pure withaferin A as immune adjuvants for promisingvaccines or as adjuncts to anti-infective immunopropylaxis.

ACKNOWLEDGEMENTS

The authors acknowledge the financial support providedby Council of Scientific and Industrial Research (CSIR)under CSIR-NMITLI project. The authors also acknow-ledge the help extended by Dr Mrigank Srivastava, CDRIfor editing the manuscript. The technical assistance ren-dered by Mr A.K. Roy and Mr R.N. Lal in experimentalmaintenance of infection is gratefully acknowledged. Wethankfully acknowledge the help extended by SAIF Divi-sion, CDRI, Lucknow for flow cytometer facility. Themanuscript bears CDRI communication no. 8172.

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