Research Article

Macrofilaricidal Activity of the PlantPlumbago indica/rosea In Vitro

Nisha Mathew,n K. P. Paily, Abidha, P. Vanamail, M. Kalyanasundaram, and K. BalaramanVector Control Research Centre (ICMR), Indira Nagar, Pondicherry, India

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ABSTRACT The macrofilaricidal property of the plant, Plumbago indica/rosea, was investigated invitro against Setaria digitata, a filarial parasite of cattle. Adult worms were incubated in a mediumcontaining crude extract at concentrations 0.05, 0.04, 0.02, and 0.01mgmL�1 for various incubationperiods of 30min, 1 h, 2 h, and 6 h, respectively, at 371C and the worm motility was compared with that ofsolvent control worms kept in drug-free medium. Complete inhibition of motility was observed forconcentrations ranging from 0.02 to 0.05mgmL�1, whereas in the control all the worms were active.Bioassay-guided fractionation of the crude extract by silica gel column chromatography resulted in theidentification of a very active fraction. The activity of this fraction against adult worms was furtherconfirmed by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide–formazan colorimetricassay, which gave 450% inhibition for the active fraction at concentrations 0.05, 0.002, 0.005, and0.0006mgmL�1 at 30min, 1 h, 2 h, and 6 h incubation periods, respectively. The physical and chemicaldata obtained from melting point determination, thin-layer chromatography and high-performance liquidchromatography analysis and IR, 1HNMR, 13CNMR, and gas chromatography-mass spectrometry spectralanalysis have indicated the chemical structure of the active molecule as 5-hydroxy-2-methyl-1,4-naphthalenedione (plumbagin). Drug Dev. Res. 56:33–39, 2002. �c 2002 Wiley-Liss, Inc.

Key words: filariasis; antifilarial; Plumbagin; 1,4-naphthalenedione; Setaria digitata

INTRODUCTION

Lymphatic filariasis is a vector-borne parasiticdisease affecting millions of people and is the secondleading cause of permanent and long-term disability inthe world. In India, approximately 45% of its 1-billion-plus population lives in known endemic areas [WorldHealth Organization, 2000] and 48 million are infected[Michael et al., 1996], accounting for 40% of the globallymphatic filariasis burden [Das et al., 2001]. Socio-economic studies showed that the annual loss causedby this disease is close to a billion U.S. dollars, andthere is tremendous hardship in affected individuals[Ramaiah et al., 1997]. However, considering themagnitude of filariasis as a public health problem,relatively few drugs are available for its treatment.While the most widely employed drug in the treatmentof lymphatic filariasis for decades has been diethylcar-bamazine, ivermectin is now recommended in areas of

Africa that are coendemic for onchocerciasis [Ottesenet al., 1997]. Although both of these drugs have markedactivity against microfilariae (mf) in interruptingtransmission of the disease, they are much less effectiveagainst the adult worm [Ottesen et al., 1997; Noroeset al., 1997]. Several benzimidazolyl carbamates,although significantly active against gastrointestinalnematodes, are not sufficiently active against the adult

DDR

Contract grant sponsor: International Foundation forScience (IFS), Stockholm, Sweden; Contract grant number:F/2929-1.

nCorrespondence to: Dr. Nisha Mathew, Vector ControlResearch Centre, Indira Nagar, Pondicherry-605006, India.E-mail: nishamathew@yahoo.com

Received 3 March 2002; Accepted 9 June 2002

Published online in Wiley InterScience (www.interscience.wiley.com) DOI: 10.1002/ddr.10056

DRUG DEVELOPMENT RESEARCH 56:33–39 (2002)

�c 2002 Wiley-Liss, Inc.

filarial worms due to poor absorption and teratogenicityof some of them [Townsend and Wise, 1990; Lazdinsand Kron, 1999]. Adult worms may survive for severalyears in the infected person [Vanamail et al., 1990],producing mf and thereby facilitate transmission of thedisease through the vector mosquitoes to moreindividuals. Hence, elimination of the parasite bymeans of a microfilaricide alone is extremely difficult.This warrants an effective and safe drug targetedagainst the adult filarial worm. In view of this, studieswere carried out to identify a lead for the developmentof potent macrofilaricide from medicinal plants.

Plumbago indica/rosea (Family: Plumbaginaceae)is a perennial shrub found growing throughout India,characterized by reddish petiole and bright red flowers.The root of the plant is known to contain anticancer[Devi et al., 1994, 1998], radiosensitizing [Ganasoun-dari et al., 1997], and antifertility factors [Lal et al.,1983; Sarma and Mahanta, 2000]. A number ofnaphthoquinones such as 3-O-30biddroserone, plum-bagin, 2,3-epoxyplumbagin, plumbagic acid, and flavo-noids have been reported from P. indica/rosea [Dindaet al., 1998]. The active principle responsible for theantibiotic property of Plumbaga zeylanica [Krishnas-wamy and Purushothaman, 1980], antimalarial activityof Nepenthes thoredii [Likhitwitayawuid et al., 1998],leishmanicidal and trypanocidal effects of Pera benensis[Fournet et al., 1992] is reported to be plumbagin.Plumbagin is also reported for its action against thegastrointestinal parasites Haemonchus contortus andAscaris suum [Fetterer and Fleming, 1991]. Here wereport in vitro macrofilaricidal activity of root extract ofP. indica/rosea against Setaria digitata (Nematoda:Filariodea), the cattle filarial parasite.

MATERIALS AND METHODS

Extraction and Fractionation of Compounds fromP. indica/rosea Roots

Roots of P. indica/rosea were collected fromsouthern part of the country and identified taxonomi-cally at the Botany Department, Bharathidasan Gov-ernment College for Women, Pondicherry, India.Herbarium sample was kept for future reference. Theroots of the plant were washed, shade dried, andpowdered. About 100 g of the root powder was used forextraction with 750mL analytical reagent (AR) grademethanol (s.d. fine-chem, Mumbai, India) in a Soxhletapparatus for 6 h. After extraction, the solvent wasremoved in a Rotary vacuum evaporator to get a darkbrown semisolid residue. One portion of the residuewas then weighed and reconstituted in ethyl alcohol(AR) (Hayman, Essex, England) and kept as stocksolution for in vitro evaluation against Setaria adult

worms by following a slightly modified method [Ghoshet al., 1993].

Another portion of the crude extract (3.5 g) waspassed through a silica gel (60–120 mesh, E-Merk,Mumbai, India) column. The column was eluted usinga mixture of laboratory reagent grade petroleum ether(s.d fine-chem) and chloroform (AR) (E-Merck) in aratio of 2:3. Fractions were collected and characterizedby thin layer chromatography (TLC) on silica gel G(E-Merck)–coated (1mm thickness) plates run withpetroleum ether/chloroform (2:3) and visualized in aniodine chamber. Fractions with an identical TLCpattern were pooled, named as fractions 1–3, and thesolvent was completely removed from the pooledfractions. After weighing, the residues were reconsti-tuted in ethyl alcohol and used as purified fractions forin vitro evaluation against the adult worm.

In Vitro Screening for Antifilarial ActivityAgainst S. digitata

Adult S. digitata were obtained from the perito-neal cavity of freshly slaughtered cattle and used forthe experiment within an hour. The worms werewashed repeatedly with normal saline (0.85%) to freethem of any extraneous material. After washing, theworms were transferred immediately to Dulbecco’smodified eagle’s medium (Sigma Chemical Co., St.Louis, MO, U.S.A.) with 0.01% Strepto-penicillin(Sarabhai Chemicals, Baroda, India) and supplementedwith 10% heat-inactivated fetal calf serum (SigmaChemical Co., St. Louis, MO).

Dilutions of the crude extract (30%) of P. indica/rosea were made in ethyl alcohol in such a way that100 mL of which, when distributed to sterile disposablePetri dishes (35-mm diameter and 5-mL capacity)containing 3mL medium would give the required testconcentration. Initial screening was done at concentra-tions ranging from 1 to 10mgmL�1 of the crudeextract. A simultaneous control was kept without thetest solution but with 100mL ethyl alcohol in 3mL ofthe medium. Two worms (one maleþone female) wereintroduced into each Petri dish. Three replicates eachwere set up for both test and control. The worms wereincubated at 371C for 100min in an incubator. After100-min exposure, the number of immobilized wormsin each Petri dish was counted. Immediately aftercounting, the worms were washed twice with freshmedium and transferred to another set of fresh Petridishes containing fresh medium without the testsolution to find out whether any of the immotileworms regained motility in the 1-h, 6-h, and 12-hposttreatment period. If the worms did not revive, thecondition was considered as irreversible and theconcentration lethal. Each experiment was repeated

34 MATHEW ET AL.

three times. Similarly, fractions one to three obtainedfrom column chromatographic purification of the crudeextract were also screened for their efficacy.

Further dilutions of the crude extract were madeto get 0.05, 0.04, 0.02, and 0.01mgmL�1, andexperiments were repeated by monitoring the motilityof worms at 30min, 1 h, 2 h, and finally at 6 h to find outthe activity endpoints with different incubation peri-ods. Similar experiments were also performed forfraction 2, because this fraction was found to beeffective in the preliminary screening, with 0.05, 0.005,0.002, and 0.0006mg mL�1.

MTT-Formazan Colorimetric Assay for Viability ofWorms Exposed to Fraction 2

The effect of fraction 2 of the extract on viabilityof adult female Setaria worms was studied by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazoliumbromide) (Sisco Research Laboratory, India) reductionassay following the method described by Comely et al.[1989]. Because of the scarcity of male worms onlyfemale worms were used for these tests. After theexposure of the worms to various concentrations (0.05,0.005, 0.002, and 0.0006mgmL�1) of fraction 2 atdifferent incubation periods (30min, 1 h, 2 h, and 6 h),the parasites were further incubated for 30minindividually in 0.5mL phosphate buffered saline (pH7.4) containing 0.25mgmL�1 MTT. Each time, 10replicates were used. At the end of the MTTincubation, worms were carefully transferred to amicrotiter plate containing 400mL of spectroscopicgrade dimethyl sulphoxide (DMSO) (s.d.fine-chem)and allowed to be at room temperature for 1 h, withoccasional gentle shaking to extract the color devel-oped. The absorbance of the resulting formazansolution was then determined at 492 nm in anenzyme-linked immunosorbent assay reader (TitertekMultiskan MCC/340, Labsystems, Helsinki, Finland)relative to DMSO blank. High values of absorptioncorrelate with high viability of the worms. Positivecontrol was set up with adult females not treated withthe test solution but exposed to alcohol solvent asdescribed in the above experiment. Adult worms thathad previously been heat killed (561C for 30min) andincubated with MTT served as the negative control.Viability of the worms was estimated as percentageinhibition in formazan formation relative to solventcontrols and heat killed worms [Strote et al., 1998] byfollowing the formula:

%inhibition ðparameterÞ ¼ 100� ½ðT�HÞ=ðC�HÞ�� 100

where T, C, and H are absorbance values obtained forthe formazan produced in treated, control, and heat-

killed worms respectively. Nonparametric statisticalMann-Whitney U test was used to see the significantdifference between the absorbance values obtained fortreated worms against that of the control worms.

Physical and Spectral Measurements of theActive Fraction

The melting point of the purified fraction 2 wasdetermined with a melting point apparatus (TechnoInstruments Inc., India) after recrystallizing from ethylalcohol. TLC was done on silica gel G and columnchromatography with silica gel (60–120mesh). High-performance liquid chromatography (HPLC) analysiswas carried out using a Shimadzu (Kyoto, Japan)LC-3A equipped with SPD-2A spectrophotometric,Phenomenex (Torrance, CA) column Ultracarb C8(150� 4.6), 5-mm particle size, mobile phase acetoni-trile (HPLC grade) (s.d fine-chem)/water (Milli-Q,Molshiem, France) (70/30) with 0.01% orthophospho-ric acid, at 1.2mL/min flow rate, column temperatureat 501C and at UV-270 nm.

Infrared spectrum was recorded in KBr on aShimadzu FTIR-8300. Mass spectrum was recorded ona gas chromatography–mass spectrometry (GC-MS)spectrophotometer (Finnigan Mat, San Jose, CA,8230MS). Nuclear magnetic resonance (NMR) spectrawere recorded in deuterochloroform on a JEOL Ltd.(Tokyo, Japan) GSX-400. Spectra were referenced totetramethylsilane (1H) or solvent (13C) signals.

RESULTS

Extraction and Fractionation

The removal of the solvent under reducedpressure from the crude extract of P. indica/rosea rootsresulted in a dark brown semisolid residue. From 100 gof the root powder, 29.2 g of crude residue wasobtained. Purification of the crude residue (3.5 g) bysubmitting to silica gel column chromatography in amixture of petroleum ether–chloroform (2:3) yieldedmany fractions. These fractions were characterized byTLC. Identical fractions shown by TLC were pooledand stripped off the solvent at reduced pressure,resulted in a colorless semisolid (25mg, 0.71%, Rf 0.84)from fraction 1, a yellow powder (105mg, 3%, Rf 0.4)from fraction 2, and a brown solid from fraction 3(35mg, 1%, Rf 0.22).

In vitro Screening for Antifilarial Activity

The methanolic extract of the root of P. indica/rosea was screened for antifilarial activity against adultsof the cattle filarial worm S. digitata. Concentrations of1–10mgmL�1 of the crude extract caused completeimmobilization of the worms in 100min of exposure at

MACROFILARICIDAL ACTIVITY OF PLUMBAGO INDICA/ROSEA 35

371C, whereas in untreated control, all the worms wereactive. The postexposure incubation in normal medium(without test solution) for 6–12 h did not revive theworms, confirming their death due to the treatment.Experiments were continued further with lower con-centrations, and it was found that a test concentrationas low as 0.02mgmL�1 of the crude extract couldimmobilize all the worms exposed for 100min.

Similarly purified fractions (1–3) were alsoscreened for their antifilarial activity in comparisonwith the crude extract at 0.02 and 0.002mgmL�1.Fractions 1 and 3 did not immobilize the worms at0.02mgmL�1 over an exposure period of 100min,while fraction 2 immobilized all the worms at0.002mgmL�1 over an exposure period of 100min.Thus, bioassay-guided fractionation of the crude extractresulted in the identification of fraction 2 as the mostactive fraction.

The results of the effect of the crude extract andthe fraction 2 on the motility of the worms at differentconcentrations and incubation periods are given inTable 1. Incubation of the adult worms in the mediumcontaining the crude extract as well as fraction 2 at0.05mgmL�1 resulted in 100% immobilization even ata minimum incubation period of 30min, and theworms did not revive even after 12-h postincubationperiod in a drug-free medium. The same result wasobserved at 0.04 and 0.02mgmL�1 of crude extractand in the case of fraction 2 at 0.005 and0.002mgmL�1 , respectively, at 1-h and 2-h incubationperiods. At the lowest concentration of crude extracttested (0.01mgmL�1), only 83.3% of the worms werefound to be immobilized at an incubation period of 6 h,while fraction 2 exhibited immobilization of wormseven at a concentration of 0.0006mgmL�1 over 6 h.Thus, the results indicated that at higher concentra-tions, the inhibition in motility was faster, while atlower concentrations it was relatively slow.

MTT–Formazan Colorimetric Assay

The macrofilaricidal effect of the purified fraction2 was further confirmed by comparing the treatedworms to untreated control and heat-killed worms, interms of MTT–formazan colorimetric assay. The resultsare shown in Table 2. MTT is pale yellow in solutionbut when incubated with living cells is reduced byactive mitochondria to yield dark blue formazan withinthe cells. During the assay, the formazan formed isextracted with DMSO and quantitated colorimetrically.The very low absorbance value (0.102) observed for theheat-killed worms was due to the least production offormazan in dead worms. At 0.05mgmL�1, thepercentage inhibition was found to be 470% at allincubation periods, indicating the immediate effect ofthe drug at higher concentration. At 0.005mgmL�1

and at 0.002mgmL�1, the percentage inhibition waso50% at the 30-min incubation period, while at higherincubation periods it was found to be 460%. Fraction2 exhibited 450% inhibition even at a concentration aslow as 0.0006mgmL�1 at an incubation period of 6 h,and this is an indication of the higher activity of thisfraction. Both worm motility assay and MTT reductionassay confirm the macrofilaricidal activity of the rootextract of P. indica/rosea.

Physical and Spectral Measurements

The physical and chemical data from IR, 1HNMR, 13CNMR and GC-MS spectra of fraction 2are given in Table 3. The yellow crystals melted at73–741C. TLC on silica gel G with petroleum ether/chloroform (2:3) showed a single spot with Rf value 0.4.HPLC analysis again confirmed the purity of thecompound by giving a single peak at 3.27min for thefraction 2, while the crude extract gave multiple peaks.The IR spectrum of fraction 2 in KBr showed bandsspecific for 1,4-quinone system at 1675, 1645, and

TABLE 1. In vitro Macrofilaricidal Activity of Plumbago indica/rosea Extract Against Adult Setaria digitata in Terms of Worm Motility

Sample Test conc. (mgmL�1) Incubation time (end point) (h) % Worm motility inhibition

Test (n=18) Control (n=18)

Crude 0.05 0.5 100 00.04 1 100 00.02 2 100 00.01 6 83.3 0

Fraction 2 0.05 0.5 100 00.005 1 100 00.002 2 100 00.0006 6 100 0

n, Total number of worms.

36 MATHEW ET AL.

TABLE

2.In

vitroMacrofilaricidal

ActivityofPurified

Frac

tion2ofPlumbag

oindica/roseaAga

inst

SetariadigitataAdultFe

males

inTermsofMTTreduction

Sample

andtrea

tmen

tco

ncentration

Durationofincu

bation(h)

Absorban

ceof492nm

per

female(m

ean7s.e.m.n¼10)

%Inhibitionrelative

tosolven

tco

ntrolaan

dheatkilled

bworm

scMan

n-W

hitney

Utest

Zva

lue

Sign

ifica

nce

d

Control-alco

hol

0.5

1.85470.067

FF

Hea

t-killed

worm

s0.5

0.10270.008

F3.781

Po0.001

Frac

tion2

0.5

0.59270.027

72.06

3.780

Po0.001

0.05mgmL�

11

0.50670.029

76.96

3.780

Po0.001

20.44270.045

80.60

3.780

Po0.001

60.35470.037

85.62

3.780

Po0.001

Frac

tion2

0.5

1.04870.054

46.05

3.780

Po0.001

0.005mgmL�

11

0.73770.119

63.78

3.780

Po0.001

20.70070.031

65.88

3.780

Po0.001

60.61470.040

70.70

3.780

Po0.001

Frac

tion2

0.5

1.54070.056

17.92

2.797

P¼0.005

0.0006mgmL�

11

1.48970.060

20.87

2.948

P¼0.003

21.26270.074

33.78

3.628

Po0.001

60.90370.085

54.30

3.780

Po0.001

Frac

tion2

0.5

1.03270.067

46.94

3.780

Po0.001

0.002mgmL�

11

1.00370.055

48.60

3.780

Po0.001

20.90370.038

54.30

3.780

Po0.001

60.61970.041

70.51

3.780

Po0.001

aPositive

control.

bNeg

ativeco

ntrol.

cEffectiveif%

inhibition450%.dPva

luerepresentsthelevelofsign

ificance

whileco

mparingthemea

nva

lueofab

sorban

ceobserved

fortheform

azan

form

edbetwee

ntrea

tedan

dco

ntrolworm

s.‘‘Z’’is

stan

dardized

norm

alva

riate.

MACROFILARICIDAL ACTIVITY OF PLUMBAGO INDICA/ROSEA 37

1610 cm�1. Its structure was further confirmed by GC-MS, which showed a base peak at m/z 188 due to theprotonated molecular ion. The 1H NMR and 13C NMRdata are in agreement with the number and type ofprotons as well as carbon atoms respectively present inthe molecule. Thus, the structure of fraction 2 is 5-hydroxy-2-methyl-1, 4-naphthalenedione, also knownas 5-hydroxy-2-methyl-1, 4-naphthoquinone (plumba-gin) as identified by comparison of the physical andchemical data with those published earlier [Evanset al., 1999].

DISCUSSION

In the present study, a methanolic extract of theroots of P. indica/rosea, a traditionally used medicinalplant in many Ayurvedic drug preparations in India,revealed promising macrofilaricidal activity againstcattle filarial worm S. digitata in the in vitro system.The treated worms were completely immobilized dueto the lethal effect of the extract. Purification of theactive fraction of the extract and MTT reduction assayof the worms treated with the active fraction confirmedits effect on the viability of the worms by acting at thecellular level, as indicated by the reduced level ofmitochondrial enzyme that reduces the MTT toformazan [Comely et al., 1989].

In the case of crude extract, complete inhibitionof motility was observed when worms were incubatedfor 2 h at a concentration as low as 0.02mgmL�1. Thepurified fraction was 10 times more effective than thecrude extract, showing complete inhibition of motilityat a concentration of 0.002mgmL�1 and 2-h incuba-tion period. At the lowest concentration of crudeextract tested (0.01mgmL�1), only 83.3% of the wormswere found to be immobilized at an incubation periodof 6 h, while fraction 2 exhibited immobilization ofworms at 0.0006mgmL�1. Like the motility assay,MTT reduction assay confirmed the activity of fraction

2 when incubated in medium containing0.002mgmL�1 and at 0.0006mgmL�1 of fraction 2for 2 h and 6 h, respectively, where the observedreduction in absorbance values with respect to thecontrol and heat treated worms were 450%.

The chemical structure of this active component(fraction 2) has been identified by spectral analysis andfound to be 5-hydroxy-2-methyl-1,4-naphthalenedione.Earlier, phytochemical studies of P. indica root extractsshowed the presence of a number of naphthoquinones[Dinda et al., 1998] in addition to this molecule. Thebiomedical potential of 5-hydroxy-2-methyl-1,4-naphthalenedione has been studied for its anticancer[Devi et al., 1994, 1998], antibiotic [Krishnaswamy andPurushothaman, 1980], antimalarial [Likhitwitayawuidet al., 1998; Suraveratum et al., 2000], and leishmani-cidal [Fournet et al., 1992], trypanocidal [Salmon-Chemin et al., 2001], and ascaricidal [Fetterer andFleming, 1991] properties. Here we have reported themacrofilaricidal activity of the root extract of P. indica.The active principle responsible for the antifilarialaction has also been identified and characterized for itschemical structure. The identification of macrofilarici-dal activity of 5-hydroxy-2-methyl-1, 4-naphthalene-dione that was so far known for other biologicalactivities provides a new lead for the development of abetter macrofilaricidal agent for combating filariasis,the second most debilitating disease in the world.

ACKNOWLEDGMENTS

This research was supported by the InternationalFoundation for Science, Stockholm, Sweden, through agrant to Dr. Nisha Mathew (grant no. F/2929-1) andshe expresses her sincere gratitude toward the IFS.The authors thank Dr. P.K. Das, Director, VectorControl Research Centre for his encouragement duringthe study. They also thank Dr. R. Ravi, DeputyDirector (SG), Dr. K. D. Ramaiah, Assistant Director

TABLE 3. Physical and Spectral Data for the Purified Fraction 2 of Plumbago indica/rosea

S. no. Parameter Data

1 Melting point 73–741C (Uncorrected)2 Molecular formula C11H8O3

3 Molecular weight 1884 TLC Single spot with Rf value 0.4 (petroleum ether: chloroform 2:3)5 HPLC Single peak at 3.27min (mobile phase: CH3CN/H2O 70/30 at 1.2mL/min,

501C column: Ultracarb 5 C8 (150�4.6mm) UV 270nm6 IR (KBr) spectrum 1,675, 1,645, 1,610 cm�1

7 1H NMR (CDCl3) in � ppm 2.20 (s, 3H), 6.81 (d, 1H), 7.25 (m, 1H), 7.62 (m, 2H), 11.95 (s, H)8 13C NMR in � ppm 190.11, 184.59, 161.03, 149.48, 135.99, 135.30, 131.90, 124.04,

119.15, 114.96, 16.45.9 GC-MS (m/e 70ev) 188(100%), 173(25), 160(23), 131(38), 120(20), 92(21), 63(18)

TLC, thin layer chromatography; HPLC, high-performance liquid chromatography; NMR, nuclear magnetic resonance.

38 MATHEW ET AL.

for critical review of this article and Dr. A. Pragasam,Head of Botany Department, Bharathidasan Govern-ment College for Women, Pondicherry, for taxonomicalidentification of the plant, as well as the Staff, RegionalSophisticated Instrumentation Centre, Indian Instituteof Technology, Chennai, for the spectral analysis. Thetechnical assistance rendered by Ms. R. Anilakumari,Mr. S. Srinivasan, and the Staff of the ParasitologyDivision is gratefully acknowledged.

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