Research Article

Antileishmanial Action of Tephrosia purpurea Linn, Extractand Its Fractions Against Experimental Visceral

LeishmaniasisPreeti Sharma, Subha Rastogi, Sunita Bhatnagar, Janmejya K. Srivastava, Anuradha Dube,Purushottam Y. Guru,n Dinesh K. Kulshrestha, Bishan N. Mehrotra, and Bhola N. Dhawan

Central Drug Research Institute, Lucknow, India

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ABSTRACT Tephrosia purpurea (family: Fabaceae), which is used in traditional remedies for thetreatment of febrile attacks, enlargement and obstruction of liver, spleen, and kidney, was found to havesignificant antileishmanial activity, and has been extensively fractionated to locate the abode of activity. Afraction (F062) obtained from N-butanol extract of T. purpurea showed consistent antileishmanial activityat 50 mg/ kg � 5 days by oral route against Leishmania donovani infection in hamsters. Activity wasfurther confirmed in a secondary model, i.e., Indian langur monkeys (Presbytis entellus). Thus, the fractionF062 from this plant possesses potential to produce significant antileishmanial activity by oral routewithout producing any toxic side effects. Drug. Dev. Res. 60:285–293, 2003. �c 2003 Wiley-Liss, Inc.

Key words: Leishmania donovani, Tephrosia purpurea, Golden hamsters, Presbytis entellus

INTRODUCTION

Visceral leishmaniasis (V.L.) or kala-azar is themost dreaded disease among various forms of leishma-niases complex. In V.L., the parasite Leishmaniadonovani heavily infects liver and spleen and severelyimpairs the physiological function of these organs[Chatterjee and Ghose, 1955; Sengupta et al., 1956].The existing drugs are not always effective, possessmany toxic side effects, and have to be given by theparenteral route. A large number of plant productshave been extensively investigated for activity against avariety of infective disorders, but leishmania hasreceived much less attention. The available data havebeen reviewed recently by Guru et al. [1996]. The rootsof Tephrosia purpurea Linn (family: Fabaceae) in theform of a decoction and taken with pepper powder isbeneficial in febrile attacks, and enlargement andobstruction of liver, spleen, and kidneys [Chopraet al., 1958]. In a preliminary in vitro trial, the crudeextract of T. purpurea (stem and bark) was found to

have significant activity against the experimental L.donovani infection in the macrophage–amastigotesystem. The test material was further exploredextensively in vivo (in hamster model) with a view toisolate the most active fraction and also to extracta pure compound that may be responsible foractivity. The results are being reported in the presentcommunication.

DDR

Contract grant sponsors: Indian Council of MedicalResearch, New Delhi, and the Council of Scientific and IndustrialResearch, New Delhi.

nCorrespondence to: P. Y. Guru, Division of LaboratoryAnimals, Central Drug Research Institute, Chattar Manzil Palace,Post Box No. 173, Lucknow-226001, India.E-mail: pyguru@rediffmail.com

Published online in Wiley InterScience (www.interscience.wiley.com) DOI: 10.1002/ddr.10324

DRUG DEVELOPMENT RESEARCH 60:285–293 (2003)

�c 2003 Wiley-Liss, Inc.

MATERIALS AND METHODS

Host

Male golden hamsters (Mesocricetus auratus; 40–45 g) used in this study were procured from the animalbreeding facility of the Institute. These were fed onstandard rodent pellet diet and water ad libitum.

Young male langur monkeys (Presbytis entellus),(3–5 kg body weight) were trapped in the wild andquarantined for 45 days prior to use. During thatperiod, they were monitored for hemoprotozoan andgastrointestinal parasites for tuberculosis (Mycobacter-ium tuberculosis) by skin test and chest radiograph andfor antibodies against L. donovani promastigote antigenby enzyme-linked immunosorbent assay [Engvall andPerlmann, 1972]. These animals were housed indivi-dually in squeeze-back cages and were fed oncommercially available primate feed supplementedwith seasonal fruits and vegetables. Water was providedad libitum.

Care and management of these animals werecarried out according to ethical guidelines laid down atour institute.

Parasite

The strain of L. donovani (HOM/IN/80/Dd8) wasoriginally obtained from Imperial College, London andhas been maintained in golden hamsters by regularpassage of promastigotes/amastigotes.

Plant Material and Its Preparation

T. purpurea (Hindi name: Sarphonka) belongs tothe family Fabaceae. Its stem and bark was collectedfrom Lucknow and adjacent areas (Uttar Pradesh,India), botanically authenticated, and a voucher speci-men was preserved in the Institute’s herbarium.

Powdered plant material was exhaustively ex-tracted with ethyl alcohol by cold percolation, and theextract was concentrated under reduced pressurebelow 501C to give a viscous mass. The extract wasresolved into n-hexane, chloroform, n-butanol, andwater-soluble fractions. Further fractionation of con-stituents of active n-butanol fraction was carried out bycolumn chromatography over silica gel (Figs. 1 and 2).Thin layer chromatography (TLC) was run on silicagel–coated plates using ethyl acetate saturated withwater plus 10% methanol as the solvent system. Spotswere visualised by spraying 1% ceric sulphate solutionin 1 M sulphuric acid followed by heating the TLCplate at 1101C. High-performance liquid chromato-graphy was carried out on a Linhroacart RP-100column using acetonitrile:water:acetic acid (20:80:0.4)as the eluent at a flow rate of 1 mL/min, and ultravioletdetector wavelength was set at 260 nm. The fractions

obtained from column chromatography were tested forantileishmanial efficacy. For in vitro testing, the stocksolution of plant extract was made in 1 mL of absolutealcohol; later the stock was diluted by sterile RPMI-1640 + 10% fetal calf serum (FCS) to get the requiredconcentration of 100 mg/mL. The ethanolic extract andits fractions were either solubilized in triple distilledwater (TDW) or made into a fine suspension with thehelp of permissible amount of absolute ethyl alcohol forin vivo testing.

Experimental Protocol

In vitro assay (macrophage–amastigote system)This was done according to the method of Neal

and Croft [1984]. Briefly, the macrophages wereisolated from peritoneal cavity of Balb/c mice and 1� 106 cells in RPMI 1640 þ 10% FCS medium werelaid over the coverslip of Leighton tube. These wereincubated at 371C in 5% CO2 atmosphere for 24 h toallow the cells to form a monolayer over the coverslip.Promastigotes (3� 106) were added to the macrophageculture. The promastigotes invade the macrophages,transform to amastigotes, and the system becomesready for screening after another 24 h. The testmaterial (100 mg/mL in medium) was added to theleighten tubes after replacing the previous medium.The test extract drug was replaced after 48 h. Afteranother incubation period of 48 h, the coverslips weretaken out, dried, fixed, stained in Giemsa, mounted,and observed under a 100� oil immersion lens. Thenumber of amastigotes was counted along with 500macrophages. Thereafter, the average number ofamastigotes per 100 macrophage cells was used forcalculation of percentage inhibition using the followingformula:

PI ¼ 100�T�100

c

where PI¼ percentage inhibition; T¼ number ofparasites in treated samples/100 macrophage cells;and C¼ number of parasites in control samples/100macrophage cells.

In vivo assaysHamster model: protocol-I. The plant extractand its fractions prepared as above were screened forantileishmanial activity using a standard procedure[Guru et al., 1989]. Briefly, each hamster wasinoculated intracardially with 107 amastigotes isolatedfrom the spleen of heavily infected hamsters. Twenty to25 days after inoculation; splenic biopsies were carriedout [Stauber et al., 1958] for assessing the status ofinfection spleen dab smear stained with Giemsa.Animals having 10 to 15 amastigotes per 100 spleen

286 SHARMA ET AL.

cell nuclei were selected and used for screeningpurposes. Three to 5 days after the spleen biopsy, agroup of 5 infected hamsters was treated orally witheach fraction for 5 consecutive days. Splenic biopsieswere again performed on day 7 after treatment afterthe last dose administered. This was done to assess thestatus of infection in both treated and untreatedinfected animals. Sodium stibogluconate (Stibanate,Gluconate India, Kolkata) by intraperitoneal route wasused as standard positive control. Percent inhibition intreated animals was calculated by the following formula[Guru et al., 1989]:

PI ¼AT� 100

IT� TI

where PI¼ percent inhibition; AT¼actual number ofamastigotes/100 spleen cell nuclei in treated animals;IT¼ initial number of amastigotes/100 spleen cell

nuclei in treated animals; and TI¼ times increase inuntreated control animals.

The treated animals were kept for another monthto observe the survival time in comparison to sodiumstibogluconate.

Protocol-II. This was designed to assess the efficacywhen the immune system of the host still remainsintact, that is, during the early stage of infection.In brief, male golden hamsters were inoculated with1� 107 amastigotes, i.c. The treatment schedulecommenced from day 3 after infection and continuedfor 5 consecutive days. The efficacy was assessedon days 12 to 17 after treatment by killing the animals.The spleen in each of the animals was removed,weighed, and touch smeared. The L. donovani units(LDU) were calculated by multiplying the numberof amastigotes in 103 cell nuclei to the weightof spleen (in grams). The percent inhibition was

Tephrosia purpurea (Fam: Fabaceae) and its fractions Alcoholic extract (Stem & Bark)

n-hexane Chloroform n-butanol Aqueous(F002) (F003) (F004)* (F005)

n-hexane Chloroform n-butanol Aqueous(F010) (F011) (F012)* (F013)

Chromatogrpahic fractionsF031

F032 F033

F034 F035 F036 F037 F049 F050 F051 F052 F053 F054 F055 F056* F057*

F058 F059

*Active

Fig. 1. Tephrosia purpurea (Fam: Fabaceae) and its fractions. Alcoholic extract (stem and bark).

ANTILEISHMANIAL ACTIVITY OF TEPHROSIA PURPUREA 287

calculated using LDU values by the formula asmentioned for in vitro assay (macrophage–amastigotesystem). Sodium stibogluconate was used as a standarddrug for comparison.

Langur (Presbytis entellus) model. The efficacyof F062 was carried out as per method of Anuradhaet al. [1992]. Briefly, langur monkeys were inoculatedintravenously with 1� 108 amastigotes. Splenic biopsywas done 30 days after inoculation for assessing thestatus of infection by Giemsa-stained spleen touchsmear. The monkeys were treated orally with fractionF062 for 10 consecutive days starting 5 days after thespleen biopsy. The status of infection was reassessed bysplenic biopsy on day 7 after treatment. Control

animals received only the vehicle. Sodium stibogluco-nate at the dose of 60 mg/kg � 10, i.m. was used asstandard positive control. Percent inhibition in treatedanimals was calculated as described for hamster model(Protocol-I).

RESULTS

The ethanol extract of Tephrosia purpurea (100mg/mL) produced 62% inhibition in the macrophage–amastigote system, whereas in the hamster model theactivity was 61% at a dose of 500 mg/kg p.o.� 5 andthe treated animals survived longer than controls.Higher dose (1g/kg� 5, p.o.) proved to be toxic (lethal)to the animals (Table 1). The extract was resolved inton-hexane (F002), chloroform (F003), n-butanol (F004),

n-butanol fraction (F047)

Partitioned between chloroform and water

Chloroform fraction Aqueous phase (F060)

(Extracted withChloroform + Ethanol)

(2:1)

Chloroform + Ethanol Aqueous phase (2:1) fraction (F061)

Extracted withn-butanol

n-butanol fraction (F062) Aqueous fraction(=F056 + F057) (F063)

Chromatography

F064

F065

F066

F067 Chromatography

K074 F068 K073 (Kaempferol-3-O-rhamnoglucoside) (Rutin)

K075 F069 (Biochanin A-rhamnoglucoside)

F070

Fig. 2. n-Butanol fraction (F047). Partitioned between chloroform and water.

288 SHARMA ET AL.

and aqueous (F005) fractions (Fig. 1). Amongthese, the n-butanol fraction (F004) showed activity,but the n-hexane (F002), chloroform (F003)and aqueous (F005) fractions have either less or noactivity.

To confirm the activity, a repeat fractionation ofthe alcoholic extract was carried out. There was againeither less or no activity in corresponding n-hexane(F010), chloroform (F011), and aqueous (F013) frac-tions. These fractions were, therefore, dropped (Table1). The activity in the n-butanol (F012) fraction wasconfirmed. Hence, it was subjected to column chro-matography resulting into 18 fractions (F031–F037 andF049–F059).

The antileishmanial activity was found consoli-dated in two fractions, F056 and F057 (approximately70% inhibition), at a dose level of 50 mg/kg/d � 5, p.o.

(Table 2). In a bid to obtain an active fraction similar toF056 and F057 on a large scale, a process offractionation without recourse to chromatography wasdeveloped. Thus, the n-butanol fraction was parti-tioned between chloroform and water. The aqueousphase was then extracted successively with chloroformplus ethanol (2:1) and n-butanol (Fig. 2). The n-butanolfraction thus obtained was designated F062 andshowed a pattern of spots on TLC similar to that ofF056 and F057.

To characterise the compounds present in F062,it was subjected to column chromatography. Sevenfractions (F064 to F070) were obtained. Of these,two fractionsFF065 and F066Fshowed less activitythan F062. Fraction F067 was subjected to columnchromatography and a purified fraction therebyobtained containing the two flavonoids, kaempferol-

TABLE 1 Antileishmanial Activity Profile of Tephrosia purpurea and Its Fractions Against Leishmania donovani Infection in Hamsters

Plant material Dose (mg/kgx5, p.o.) Animals survived/treated Percent inhibition (mean7SD)

Tephrosia 1000 0/5 Toxic (lethal)purpurea 500 5/5 61.071.1(ethanol extract) 250 5/5 41.771.2

FractionsF002 (n-hexane) 100 5/5 39.171.7F003 (chloroform) 100 5/5 NilF004 (n-butanol) 100 5/5 57.272.2F005 (aqueous) 100 5/5 NilF010 (n-hexane) 100 5/5 31.771.1F011 (chloroform) 100 5/5 24.474.2F012 (n-butanol) 100 5/5 63.273.3F013 (aqueous) 100 5/5 24.075.9

TABLE 2: Antileishmanial Activity of Chromatographic Fractions of F012 (n-butanol) of Tephrosia purpurea Against Leishmania donovaniInfection in Hamsters

Fractions Dose (mg/kgx5, p.o) Animals survived/treated Percent inhibition (mean7SD)

F031 50 5/5 42.372.4F032 50 5/5 57.276.5F033 50 5/5 NilF034 50 4/5 NilF035 50 3/5 NilF036 50 4/5 NilF037 50 5/5 NilF049 50 5/5 12.178.2F050 50 5/5 23.875.0F051 50 5/5 NilF052 50 5/5 42.578.6F053 50 5/5 51.676.2F054 50 5/5 55.377.1F055 50 5/5 NilF056 50 5/5 71.975.3F057 50 5/5 73.376.2F058 50 5/5 45.876.5F059 50 5/5 60.277.3

ANTILEISHMANIAL ACTIVITY OF TEPHROSIA PURPUREA 289

3-O-rhamnoglucoside (K074) and biochanin A-rham-noglucoside (K075), was again subjected to columnchromatography to furnish the two flavonoids in pureform (Fig. 3a and b). Repeat chromatography of F068yielded another flavonoid, which was identified as rutin(K073) (Fig. 3c). The structures of all of the threeflavonoids were confirmed on the basis of theirspectrochemical data, namely, 1H magnetic resonance,mass spectrometry, ultraviolet, and so on, and the datawere compared with those reported in the literature,thus confirming their identity. All of the threeflavonoids were found to be inactive. However, thesecompounds are serving as markers for standardisationof the fraction F062 so as to obtain uniformity frombatch to batch (Fig 4).

Meanwhile, to ascertain whether the loss ofactivity of F062 on chromatography was caused bythe separation of compounds having synergistic action,fractions F065 and F066 were combined in the ratio of1:1 to give a fraction F071, which was evaluated foractivity. The fraction F062 exhibited still better efficacy(around 75%) than F071 at the same dose level, that is,50 mg/kg/d � 5, p.o. After being convinced that theantileishmanial activity was consolidated in F062, thisfraction was further column chromatographed intoseven fractions (F064–F070). Although promisingactivity of 62.9% and 69.2% was noticed in twofractions, F065 and F066, respectively, none couldsurpass the efficacy of their parent fraction F062(Table 3). Fractions F065 and F066 were obtained in apoor yield and therefore, their commercial viability as adrug would not be feasible. On the other hand, theyield of F062 fraction is high because it does notinvolve any chromatographic separation. The threecompounds K073, K074, and K075 also did not showany significant activity. Hence, it appears that theconstituents of fraction F062 are acting synergistically.

Fraction F062, when evaluated at doses startingfrom 3.125 to 100 mg/kg administered orally for 5 daysusing both of the protocols, showed an approximatedose-dependent efficacy, producing maximum inhibi-tion of 74.5 7 4.0% at 50 mg/kg in protocol-I and 90.37 7.1% at 100 mg/kg in protocol-II (Figs. 5 and 6).Thereafter, a plateau in activity was observed. Themedian effective dose (ED50) was significantly lower inearly infection (12.97 mg/kg) as compared to theestablished infection (30.36 mg/kg). The sodiumstibogluconate, when evaluated at log doses, produced94.1 7 4.6% inhibition at 100 mg/kg, i.p. in protocol-Iand 96.1 7 3.5% at the dose of 50 mg/kg i.p. inprotocol-II (Fig. 4). The ED50 was 4.4 and 10.5 mg/kgin early and established infection, respectively.

The fraction F062 was found to be very safeand could be administered to hamsters up to 4,640

mg/kg � 1, p.o. without any mortality. In contrast,sodium stibogluconate at 2,150 mg/kg, i.p. provedinvariably fatal (median lethal dose [LD50]value¼ 1,620 mg/kg).

In langur monkeys, the activity of the fractionF062 was 77.42% at the dose of 100 mg/kg/d � 5 byoral route as compared to the standard drug (sodiumstibogluconate), where the activity was 92.52% at thedose of 60 mg/kg/d � 10 by intramuscular route(Table 4).

DISCUSSION

Drug development is a cumbersome, painstaking,and time-consuming process and needs continuedefforts to reach a conclusive end. The antileishmanials,stibanate, pentamidine, amphotericin B, and the veryrecently emerged drug Miltefosine (Asta Medica, TheNetherlands), the drugs of choice for V.L., have several

OH

OH

O

OH

HO O

O

Glc Rha

OH

O

OH

HO O

O

Glc Rha

OH

HO O

OOCH3

K073 (QUERCETIN -3-0- RHAMNOGLUCOSIDE)

K074 (KAEMPFEROL -3-0- RHAMNOGLUCOSIDE)

K075 (BIOCHANIN A + RHAMNOSE + GLUCOSE)

(a)

(b)

(c)

Fig. 3. Chemical structures of three flavonoids: K073, K074,and K075.

290 SHARMA ET AL.

drawbacks including toxicity [Berman, 1988; WorldHealth Organization, 1990; Olliaro and Bryceson, 1993;Thakur, 1993] and need to be administered parent-

erally. The increasing nonresponsiveness to stibanateand pentamidine is a matter of great concern [Sundaret al., 1997; Jha et al., 1998]. In recent years, India has

Fig. 4. HPLC chromatograph of active fraction of plant 324 (F062). X-axis shows the retention time inminutes and y-axis shows the intensity of the peaks.

TABLE 3: Antileishmanial Activity of F062 Fraction of Tephrosia purpurea and Its Chromatographic Fractions Against Leishmania donovaniInfection in Hamsters

Fractions Dose (mg/kg/day x5, p.o.) No. of animals (replicates) Percent inhibition (mean7SD)

F062 50 12 (4) 74.577.4F064 50 6 (2) 44.375.6F065 50 9 (3) 62.9710.3F066 50 9 (3) 69.277.7F067 50 6 (2) 22.078.3F068 50 6 (2) 47.076.3F069 50 6 (2) 22.672.1F070 50 6 (2) 8.574.4F071 50 6 (2) 38.178.1

ANTILEISHMANIAL ACTIVITY OF TEPHROSIA PURPUREA 291

been the centre for clinical development of newantileishmanial drugs such as lipid formulations ofamphotericin B, new drugs such as parenterallyadministered aminosidine and oral miltefosine.The alkyl phospholipid compound miltefosine is thefirst effective oral compound for V.L. and is underclinical trials [Jha et al., 1999; Sundar et al., 2000,

2001]. Thus, the situation demands concerted efforts tohave a dependable drug from natural resources forkala-azar.

The plant T. purpurea is well known for itstherapeutic use in spleen and liver disorders [Nadkarni,1954]. Because L. donovani infection primarily affectsspleen and liver, it is expected that the plant wouldhave favourable action in alleviating symptoms asso-ciated with V.L. This may be because of its preferentialcollection at liver and spleen. It has been possible toconfirm the activity of T. purpurea in accepted modelsof experimental leishmaniasis and also to concentrate itin fraction FO62. With this rationale, this plant wasselected and investigated for its antileishmanial poten-tial in prepatent and patent infections. This was donewith a view to investigate the efficacy of drug when theimmune status is intact (prepatent) and when it issuppressed because of infection (patent).

The ethanolic extract of the plant when used atearly infection (72 h of challenge) was found to bemuch more effective than when used in establishedinfection (Protocol-I). This is probably because of (1)the parasite load being much less, and (2) the immunesystem being completely intact in early infection. It hasbeen well documented that resultant cure is thecombined effect of drug action and immune status[Sacks et al., 1987; Murray et al., 1993].

Apart from significant antileishmanial activity, aremarkable feature of the animals treated with F062was that increase in their survival was much longer incomparison to sodium stibogluconate–treated animalsand untreated infected controls. Furthermore, theLD50 value of the fraction F062 was much more(44,640 mg/kg) than the preferred first-line antileish-manial drug sodium stibogluconate (1,620 mg/kg).Thus, the therapeutic index of F062 is much higherthan the minimum acceptable level.

Although the fraction could not exceed thereference drug, sodium stibogluconate, in terms ofefficacy, the toxicity associated with prolonged use ofthe latter puts severe restrictions on its use. Thus, anymaterial showing even marginal advantage over existingantileishmanial drugs would call for more detailed

TABLE 4: Antileishmanial Efficacy of F062 Fraction and the Standard Drug Sodium stibo- gluconate in Indian Langur Monkeys(Presbytis entellus)a

Monkeys treated with inhibitionPretreatment parasite counts

(mean with range)Posttreatment parasite countsb

(mean with range) Mean percentb

Fraction F062 of Tephrosia purpurea(100mg/kg�10 days, p.o.)

54.2 (45–67) 12.2 (9–15) 77.4

Sodium stibogluconate (60mg/kg�10 days, i.m.) 47.0 (38–55) 3.5 (2–5) 92.5

aNo. of parasites/100 cell nuclei.bTwo langurs in each group.

Fig. 5.

Fig. 6.

292 SHARMA ET AL.

investigation in the different stages of drug develop-ment. The test material (F062) could emerge as anontoxic and effective oral antileishmanial drug, whichcould be safely used even for a longer duration.

As is evident from the results, only one or twofractions had shown antileishmanial efficacy, and theothers either were devoid of activity as such or hadlimited efficacy. This is not surprising, because thebiological activity is not distributed throughout theextract but is confined to particular fractions. Becauseof this, elaborate fractionation was undertaken toobtain the locus of activity and to isolate andcharacterise the active principle. The exercise hasprovided important clues, which warrants concertedefforts towards development of fraction F062 in theform of an oral leishmanicidal agent. It also showedantileishmanial activity in the in vitro amastigotes–macrophage culture system, which indicates its anti-leishmanial action. In addition to these, the hepato-protective as well as immunostimulatory property hasalso been observed (Central Drug Research Institute,unpublished data). It has not shown any action againstthe promastigote stage of the leishmanial parasite. Thebest models for testing antileishmanial activity are theamastigote–macrophage system (in vitro) and hamstersinfected with leishmanial parasites. The promastigotestage of parasites is an insect stage and as such does notmultiply in the vertebrate host. In spite of thisdrawback, the promastigote stage is used by manyworkers. Hence, activity whether present againstpromastigotes or not is not of much relevance. Evenstandard drugs such as sodium stibogluconate isinactive against this stage.

The F062 fraction, which consists of severalconstituents with three marker compounds, is likelyto be acting at various loci with possibility of synergisticeffect, which is a common factor in drugs used inherbal mode. Furthermore, the plant is easily available.Further studies with the material will be reported later.

ACKNOWLEDGMENTS

Placement of J.K.S. under the Scientist PoolScheme of Government of India is gratefully acknowl-edged. This is CDRI Communication No. 5958.

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