leishmanicidal triterpenes from lantana camara

Leishmanicidal Triterpenes from Lantana camara

by Sabira Begum*a), Anjum Ayuba), Syeda Qamar Zehraa), Bina Shaheen Siddiquia),M. Iqbal Choudharya)b), and Samreena)

a) H. E.J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences,University of Karachi, Karachi 75270, Pakistan

(phone: þ9221-99261701-2; fax: þ9221-99261713; e-mail: [email protected])b) Department of Biochemistry, Faculty of Science, King Abdulaziz University, Jeddah 21412, Saudi

Arabia

Two new natural triterpenes, lantaninilic acid and lantoic acid, along with the known triterpeneslantadene A, and oleanolic, ursolic, betulinic, lantanolic, and camaric acid, were obtained from the aerialparts of Lantana camara through bioassay-guided isolation, monitoring the in vitro antileishmanialactivity against promastigotes of Leishmania major. Oleanolic acid (3), ursolic acid (4), lantadene A (5),and lantanilic acid (7) showed significant leishmanicidal activities with IC50 values of 53.0, 12.4, 20.4, and21.3 mm, respectively. The IC50 value of ursolic acid (4 ; 12.4 mm) was found to be comparable with that ofthe standard drugs, pentamidine (IC50 15.0 mm) and amphotericin B (IC50 0.31 mm). The in vitro activitiesof L. camara and its constituents against promastigotes of Leishmania major are reported here for thefirst time.

Introduction. – Lantana camara Linn. (Verbenaceae) is an invasive weed,originating from tropical America. It was introduced in several areas, especially inthe Australian Pacific region, as an ornamental or hedge shrub. It is used traditionallyfor the treatment of eczema, rheumatism, leprosy, bilious fever, swellings, ulcers,toothache, stomachache, influenza, tumors, anemia, and malaria, and as an antisepticfor wounds. Numerous terpenoids, flavonoids, and steroids have been reported fromthis plant [1– 8]. Diverse biological features, such as antimicrobial, nematicidal,anticancer, insecticidal, analgesic, anti-inflammatory, anticonvulsant, CNS-depressant,antihyperglycaemic, antimalarial, hepatotoxic, and antihypertensive activities, havebeen reported for L. camara [9 – 12].

Leishmania major is the protozoan parasite, responsible for cutaneous leishma-niasis with an annual incidence rate affecting 1.5 million people globally. According tothe World Health Organization (WHO) report, 12 million people are infected byprotozoan parasites, while 350 million people are living in the regions with high risk ofinfection. Drugs containing pentavalent antimony (SbV) are prescribed as the first-linetreatment for the leishmaniasis, but they have numerous side-effects. Some of them arereported to be inherently toxic or became ineffective due to resistance developed byparasites against them [13 – 18]. Increasing cases of leishmaniasis in immune-compromised patients have been reported. Therefore, there is an urgent need fordevelopment of new effective antileishmanial drugs.

The present phytochemical investigation on the aerial parts of L. camara affordedeight compounds, namely lantaninilic acid (1), lantoic acid (2), oleanolic acid (3) [19],

CHEMISTRY & BIODIVERSITY – Vol. 11 (2014) 709

� 2014 Verlag Helvetica Chimica Acta AG, Z�rich

ursolic acid (4) [19], lantadene A (5) [20], betulinic acid (6) [21], lantanilic acid (7)[22], and camarinic acid (8) [23] (Fig. 1). Compounds 1 and 2 are new natural products,although compound 1 was reported earlier as a hydrolysis product of lantanilic acid[24], while 2 was isolated before as its methyl ester [25]. The 13C- and 2D-NMR data of1 and 2 is presented here for the first time. The MeOH extract, its fractions, and all ofthe purified constituents 1– 8 were evaluated for their activity against Leishmaniamajor promastigotes.

Results and Discussion. – Structure Elucidation. HR-EI-MS of 1 provided themolecular formula C30H46O5 (m/z 486.5928). IR Bands were observed at 1090 (C�O),1625 (C¼C), 1710 (acid C¼O), 2855 (CH), and 3452– 2650 cm�1 (br. OH and COOH).On treatment with CH2N2, 1 yielded a methyl ester derivative, 1a (d(MeO) 3.61 (s))suggesting the presence of a COOH group in the natural product. Six tertiary Megroups were inferred from six 3-H singlets at d(H) 0.69, 0.82, 0.89, 0.95, 1.02, and 1.05 inthe 1H-NMR spectrum (Table 1). A double doublet at d(H) 2.92 (dd, J ¼ 14.0, 4.1,H�C(18)) and a triplet at d(H) 5.26 (t, J ¼ 3.4, H�C(12)) were also detected [5]

CHEMISTRY & BIODIVERSITY – Vol. 11 (2014)710

Fig. 1. Structures of compounds 1–8 (for atom numbering, see Fig. 3)

(Table 1). The 1H-NMR data and the signals in the 13C-NMR spectrum at d(C) 122.3(C(12)) and 143.3 (C(13)) indicated that 1 is a D12-b-amyrin type of triterpenoid [8].The 1H-NMR spectrum also showed two double doublets at d(H) 4.18 (dd, J ¼ 8.6, 3.0)and at 3.81 (dd, J ¼ 8.6, 1.0) (d(C) 67.7; CH2; DEPT; HMQC) for the two CH2 H-atomsHa�C(25) and Hb�C(25) which exhibited cross-peaks with each other in COSY-458spectrum (Fig. 2) and a long-range W-coupling with Hb�C(1). A triplet of one H-atomat d(H) 3.70 (t, J ¼ 3.2) (d(C) 74.1); HMQC and DEPT) indicated a OH group atC(22) with b-orientation. In the HMBC spectrum, correlations of the signal ofHa�C(22) with those of C(16), C(17), C(18), and C(28) further supported thisassignment. Cross-peaks between Ha�C(22) and Me(29) in the NOESY spectrum


Table 1. 1H- and 13C-NMR Data, and HMBC Features of 1a). Atom numbering as indicated in Fig. 3 ;d in ppm, J in Hz.

Position d(H) d(C) HMBC (H!C)

1 2.48–2.52 (m, Ha), 1.18–1.22 (m, Hb) 34.6 –2 1.49–1.51 (m, Ha), 1.13 –1.17 (m, Hb) 27.8 –3 – 98.2 –4 – 40.7 –5 1.17–1.22 (m) 50.8 –6 1.45–1.49 (m, Ha), 1.38 –1.42 (m, Hb) 19.6 –7 1.27–1.31 (m) 31.2 –8 – 38.4 –9 1.58–1.62 (m) 41.7 –

10 – 34.7 –11 1.88–1.92 (m, Ha), 1.63 –1.67 (m, Hb) 23.7 –12 5.26 (t, J¼3.4) 122.3 C(9), C(11), C(14), C(18)13 – 143.3 –14 – 42.2 –15 2.04–2.08 (m, Ha), 1.61–1.63 (m, Hb) 27.8 –16 1.66–1.72 (m, Ha), 1.08 –1.12 (m, Hb) 24.5 –17 – 52.2 –18 2.92 (dd, J¼14.0, 4.1) 39.0 C(12), C(13), C(14), C(16), C(17),

C(19), C(28)19 1.59–1.65 (m, Ha), 1.11 –1.15 (m, Hb) 45.8 –20 – 30.1 –21 1.46–1.50 (m, Ha), 1.38 –1.42 (m, Hb) 41.1 –22 3.70 (t, J¼3.2) 74.1 C(16), C(17), C(18), C(20), C(28)23 0.95 (s) 27.2 C(3), C(4), C(5), C(24)24 0.89 (s) 18.2 C(3), C(4), C(5), C(23)25 4.18 (dd, J¼8.6, 3.0, Ha), 3.81

(dd, J¼8.6, 1.0, Hb)67.7 C(10), C(3), C(5), C(10)

26 0.69 (s) 17.1 C(7), C(8), C(9), C(14)27 1.05 (s) 25.1 C(8), C(13), C(14), C(15)28 – 177.8 –29 0.82 (s) 33.6 C(19), C(20), C(21), C(30)30 1.02 (s) 27.1 C(19), C(20), C(21), C(29)

a) Assignments are based on 1H- and 13C-NMR (broad-band decoupled, DEPT), 1H,1H-COSY, 1H,1H-TOCSY, NOESY, J-resolved, HMQC, and HMBC spectra.

indicated the b-configuration of the OH group at C(22). A retro-Diels�Alder fragmention peak at m/z 264.1794 (C16H24O3; Fig. 3) indicated the presence of a COOH group atC(14) or C(17). Correlations of the H�C(18) signal with those of C(17) and C(28) inthe HMBC spectrum established the position of COOH at C(17). 13C-NMR Signal(d(C) 98.2) of the quaternary C(3)-atom pointed to the remaining O-atom as an a-OHgroup at C(3). These data established the structure of 1 as 3,25-epoxy-3a,22b-dihydroxyolean-12-en-28-oic acid.

For compound 2, the molecular-ion peak at m/z 486.1352 in the HR-EI-MSprovided the molecular formula C30H46O5. In the IR spectrum, bands at 1122 (C�O),1700 (acid C¼O), and 3450– 2643 cm�1 (br., OH and COOH) were observed. The1H-NMR spectrum showed six Me signals (Table 2), four as singlets at d(H) 0.74, 0.94,1.01, and 1.05, and two as doublets at d(H) 0.85 (J ¼ 6.0) and 0.91 (J ¼ 6.2). It furthershowed a characteristic CH signal at d(H) 2.37 (d, J ¼ 11.8, H�C(18) and a signal atd(H) 5.30 (t, J ¼ 3.4, H�C(12)) assigned to the olefinic H-atom. These data along with13C-NMR resonances (Table 2) of C(12) (d(C) 126.1) and C(13) (d(C) 137.5) indicatedthat 2 is a D12-a-amyrin pentacyclic triterpenoid. Spectroscopic analysis furtherindicated that compound 2 is the ursane isomer of compound 1 based on followingobservations: a b-oriented ether linkage between C(3) and C(25) (d(H) 4.21 (br. d, J ¼


Fig. 3. Mass fragmentation of compound 1

Fig. 2. Key 1H,1H-COSY (H$H) and NOESY(H H) interactions of compound 1

8.6, Ha�C(25)) and 3.87 (br. d, J ¼ 8.6, Hb�C(25)); d(C) 67.9 (CH2(25); DEPT,HMQC), olefinic C-atom (d(H) 5.30 (t, H�C(12)); d(C) 126.1 (CH(12)); DEPT) and137.5 (C(13))), a b-oriented OH group at C(22) (d(H) 3.82 (t, J ¼ 3.2, Ha�C(22)); d(C)73.2 (CH(22)); DEPT, HMQC (Table 2))), and a COOH group at C(17) indicated bythe IR and 13C-NMR (d(C) 179.0) and corroborated through esterification with CH2N2

to 2a (d(H) 3.61 (s, MeO)) and 13C-NMR data of rings D and E (Table 2). Thesespectral data established the structure of 2 as 3,25-epoxy-3a,22b-dihydroxyurs-12-en-28-oic acid. The above assignments were accomplished by taking recourse to 2D-NMRdata, including NOESY (Fig. 4), 1H,1H-COSY, HMQC, and HMBC (Table 2), and the


Table 2. 1H- and 13C-NMR Data and HMBC Features of 2a). Atom numbering indicated in Fig. 4 ;d in ppm, J in Hz.

Position d(H) d(C) HMBC (H!C)

1 2.12–2.18 (m, Ha), 1.17 –1.19 (m, Hb) 34.9 –2 1.16–1.18 (m, Ha), 0.98–1.02 (m, Hb) 28.1 –3 – 98.7 –4 – 40.3 –5 1.16–1.20 (m) 50.3 –6 1.48–1.52 (m, Ha), 1.44 –1.48 (m, Hb) 19.6 –7 1.34–1.36 (m) 31.3 –8 – 38.3 –9 1.58–1.62 (m) 41.9 –

10 – 34.9 –11 1.98–2.02 (m, Ha), 1.69 –1.73 (m, Hb) 23.8 –12 5.30 (t, J ¼ 3.4) 126.1 C(9), C(18)13 – 137.5 –14 – 42.6 –15 2.10–2.14 (m, Ha), 1.68 –1.72 (m, Hb) 29.4 –16 1.75–1.81 (m, Ha), 1.59 –1.61 (m, Hb) 25.0 –17 – 53.4 –18 2.37 (d, J ¼ 11.5) 48.9 C(12), C(13), C(14), C(16),

C(17), C(19)19 1.31–1.33 (m) 39.3 –20 1.40–1.44 (m) 31.6 –21 1.64–1.68 (m, Ha), 1.51 –1.53 (m, Hb) 38.4 –22 3.82 (t, J ¼ 3.2) 73.2 C(20)23 1.01 (s) 27.2 C(3), C(4), C(5), C(24)24 0.94 (s) 18.3 C(3), C(4), C(5), C(23)25 4.21 (dd, J ¼ 8.6, 3.0, Ha), 3.87

(dd, J ¼ 8.6, 1.0, Hb)67.9 C(1), C(5)

C(1), C(3), C(5)26 0.74 (s) 16.9 C(7), C(8), C(9), C(14)27 1.05 (s) 23.1 C(8), C(13), C(14), C(15)28 – 179.0 –29 0.91 (d, J ¼ 6.2) 17.8 C(19), C(20), C(21)30 0.85 (d, J ¼ 6.0) 20.9 C(18), C(19), C(20)

a) Assignments are based on 1H- and 13C-NMR (broad-band decoupled, DEPT), 1H,1H-COSY, 1H,1H-TOCSY, NOESY, J-resolved, HMQC, and HMBC spectra.

proposed structure was finally corroborated by the observed mass fragmentationpattern (Fig. 5). It is important to note that there are only a few pentacyclictriterpenoids with a OH group at C(22) reported in [26] [27].

Comparison of spectral data of the known compounds 3 – 8 with those reported inthe literature established their structures.

Leishmanicidal Activity. The MeOH extract, its AcOEt-soluble fraction and Et2O-soluble subfraction were found to possess activities with IC50 values 41.90, 22.83, and42.42 mg/ml (Table 3), respectively against L. major promastigotes. Compounds 1– 8isolated from the Et2O-soluble fraction were also evaluated for their activities(Table 4). Compound 4 showed the highest activity with an IC50 value of 12.4 mm, while3, 5, and 7 exhibited significant activities (IC50 values of 52.94, 20.38, and 21.34 mm,resp.). Compounds 2, 6, and 8 exhibited moderate activities (IC50 values in the range of97.0– 101 mm). Compound 1 showed only a weak activity (IC50 164 mm; Table 4). Theresults showed that compound 4 was about four times more potent than the 3. Thesefindings indicated that the leishmanicidal activity may be related to the presence of theursane skeleton present in ursolic acid. Similarly, 2 (ursane type) is more active than 1(oleanane type). Furthermore, significant activities of 5 and 7 may be due to thepresence of an a,b-unsaturated ester side chain at C(22) in oleanane skeleton, whereas8 (ursane analog) with an AcO group at that position showed moderate activity.


Fig. 4. Key 1H,1H-COSY (H$H) and NOESY(H H) interactions of compound 2

Fig. 5. Mass fragmentation of compound 2

Activities of all these compounds are reported here for the first time, except for 3, 4,and 6 [28]. In the present study, activities of 3 and 6 were found to be comparable to thereported values (IC50 40.0 and 88.0 mm, resp.). While the IC50 value of 4 (12.4 mm) wasfound to be less than the reported value (IC50 46.1 mm), it is comparable with that of thestandard drug pentamidine, indicating its potential as a pharmacological lead forfurther research.

Conclusions. – This study resulted in the isolation of two new natural triterpenoids,1 and 2, and six known ones, 3– 8. The crude extract, fractions, and compounds 1– 8were screened for leishmanicidal activity against L. major. It could be concluded thatthe active AcOEt-soluble fraction of L. camara and its pure constituents, oleanolic acid(3), ursolic acid (4), lantadene A (5), and lantanolic acid (7), possess potential asleishmanicidal agents.


Table 3. In vitro Leishmanicidal Activity of MeOH Extract and Its Fractions against L. major

Extracts/Fractionsa) IC50 [mg/ml]�S.D.b)

LC 42.0�0.1LC-AQ >100LC-EAR 23.0�0.2LC-EA-PES >100LC-EA-PEI >100LC-EA-ES 42.4�2LC-EA-EI >100LC-EA-EAS >100LC-EA-EAI >100Amphotericin Bc) 0.30�0.1Pentamidinec) 5.10�0.1

a) LC, Extract; AQ; H2O-soluble fraction; EA, AcOEt fraction; EAR, AcOEt residue; PES, petroleumether-soluble fraction; ES, Et2O-soluble fraction; EI, Et2O-insoluble fraction; EAS, AcOEt-solublefraction. b) Standard deviation. c) Positive control.

Table 4. In vitro Leishmanicidal Activity of Compounds 1–8 against L. major

Compounds IC50 [mm]�S.D.a)

Lantaninilic acid (1) 164�0.8Lantoic acid (2) 97.0�0.02Oleanolic acid (3) 53.0�0.02Ursolic acid (4) 12.4�0.03Lantadene A (5) 20.4�0.1Betulinic acid (6) 101�0.3Lantanilic acid (7) 21.3�0.02Camarinic acid (8) 89.0�0.3Amphotericin Bb) 0.31�0.1Pentamidineb) 15.0�0.1

a) Standard deviation. b) Positive control.

Experimental Part

General. Vacuum liquid chromatography (VLC): silica gel 60 PF254 (SiO2; Merck). Columnchromatography (CC): SiO2 9385 (0.040–0.063 mm, Merck), gradient elution with the solvent mixturesindicated. Prep. TLC: SiO2 60 PF254 (Merck). Anal. TLC: Merck Kieselgel Si F254-precoated aluminumcards (0.2-mm thickness); detection with I2 spray. UV Spectra: Hitachi U-3200 spectrophotometer; lmax

(log e) in nm. IR Spectra: Jasco A-302 spectrophotometer; n in cm�1. 1H- (COSY, NOESY, TOCSY, andJ-resolved) and 13C-NMR: Bruker spectrometers, at 300 and 500 and 75 and 125 MHz, resp. d in ppm rel.to Me4Si as internal standard, J in Hz. EI-MS: Finnigan-MAT 311A mass spectrometer; source at 2508and 70 eV; m/z (rel. %). HR-EI-MS: Jeol JMS-HX-110 mass spectrometer; EI source at 2508 and 70 eV;m/z (rel. %).

Plant Material. Aerial parts of Lantana camara Linn. were collected from the Karachi region inFebruary 2009. The plant was identified by Mr. Abdul Ghafoor, Senior Taxonomist, Department of Botany,University of Karachi, and a voucher specimen (No. 63482 KUH) was deposited with the Herbarium.

Extraction and Isolation. Aerial parts of Lantana camara (10 kg) were chopped and repeatedlyextracted with MeOH at r.t. Solvent was removed under vacuum. The extract (LC) thus obtained showedactivity against Leishmania major promastigotes. It was separated into AcOEt and H2O (LC-AQ)phases. The AcOEt fraction (LC-EA) was divided into acidic and neutral fractions. The residue (LC-EAR) obtained upon removal of the solvent from the neutral fraction showed significant leishmanicidalactivity. It was separated into petroleum ether (PE)-soluble (LC-EA-PES), Et2O-soluble (LC-EA-ES),and AcOEt-soluble (LC-EA-EAS) fractions. All these fractions were tested for their leishmanicidalactivity. The VLC (vacuum liquid chromatography; PE/AcOEt of increasing polarity) of the most activeEt2O-soluble fraction (114 g) was performed to furnish twelve fractions, Frs. I –XII. The major fractionFr. V (6.7 g; PE/AcOEt 9.25 : 0.75 eluate) was submitted to CC (SiO2; (PE/AcOEt of increasing polarity)which afforded ten fractions, Frs. V.1–V.10. Fr. V.7 (538.2 mg; PE/AcOEt 8.5 : 1.5 eluate) was furtherpurified by CC (SiO2; (PE/AcOEt of increasing polarity) to give eleven fractions, Frs. V.7.1–V.7.11.Residue obtained from fraction Fr. V.7.2 (256 mg; PE/AcOEt 9 : 1 eluate) on keeping in CHCl3/MeOH1 : 1 at r.t. overnight afforded 3 (25 mg) as colorless crystalline solid. Mother liquor gave 4 (12 mg) onpurification by prep. TLC (CHCl3/MeOH 9.7 :0.3; 2� ). Fr. V.7.5 (25.2 mg; PE/AcOEt 8.5 : 1.5 eluate)gave 5 (15.2 mg). Fr. V.7.3 (80.1 mg; PE/AcOEt 9.25 : 0.75 eluates) was further subjected to CC (SiO2) togive a further amount of 5 (4.9 mg; CHCl3/MeOH 9.05 : 0.05 eluate) and 6 (21.3 mg; CHCl3/MeOH9.25 : 0.75 eluate). Fr. VII (PE/AcOEt 8.5 : 1.5 eluate) afforded nine fractions, Frs. VII.1–VII.9, uponVLC (PE/AcOEt of increasing polarity). Fr. VII.3 (2.3 g; PE/AcOEt 8.75 : 1.25 eluates) was subjected toVLC (PE/AcOEt of increasing polarity)to afford 13 fractions, Frs. VII.3.–VII.3.13. Fr. VII.3.9 gavecompound 7 through crystallization from CHCl3/MeOH 1 :1 at r.t. Fr. IX (8.3 g; CHCl3/MeOH 9.5 : 0.5eluate) was separated by VLC (PE/AcOEt, followed by CHCl3, MeOH of increasing polarity) to furnisheleven fractions, Frs. IX.1 – IX.11. Fr. IX.6 (PE/AcOEt 7.5 : 2.5 eluates) was purified by CC (SiO2; PE/AcOEt of increasing polarity) to afford seven fractions, Frs. IX.6.1– IX.6.7. Fr. IX.6.5 (PE/AcOEt 7 : 3eluates) furnished 8 (9.1 mg). Fr. IX.10 (162.3 mg; CHCl3/MeOH 9.5 : 0.5 eluate) was subjected to CC(PE/AcOEt and CHCl3/MeOH of increasing polarity) which gave twelve fractions, Frs. IX.10.1–IX.10.12. Fr. IX.10.5 (PE/AcOEt 1 : 1 eluates) was purified by CC (SiO2; PE/AcOEt of increasingpolarity) gave ten fractions, Frs. IX.10.5. –IX.10.5.10. Fr. IX.10.5.3 yielded 1 (10 mg), and Fr. IX.10.5.5furnished 2 (8.5 mg).

(3b,22b)-3,25-Epoxy-3,22-dihydroxyolean-12-en-28-oic Acid (1). Amorphous powder. IR (CHCl3):3451–2649, 2935.6, 2854.9, 1710.1, 1625.4. 1H- and 13C-NMR: Table 1. EI-MS: 486 (3), 468 (100), 424(17), 246 (38), 201 (78), 187 (57), 133 (36), 119 (46), 105 (26). HR-EI-MS: 486.5928 (Mþ , C30H46Oþ

5 ;calc. 486.6861).

Methylation of 1. The methyl ester 1a (2.1 mg) was prepared by the reaction of 1 (2.2 mg) with anEt2O soln. of CH2N2. 1H-NMR (CDCl3): 3.62 (COOMe). EI-MS: 500 (Mþ ).

(3b,22b)-3,25-Epoxy-3,22-dihydroxyurs-12-en-28-oic Acid (2). Amorphous powder. IR (CHCl3):3453–2651, 2937.2, 2856.0, 1710.4, 1626.3. 1H- and 13C-NMR: Table 2. EI-MS: 486 (3), 468 (100), 424(17), 246 (38), 201 (78), 187 (57), 133 (36), 119 (46), 105 (26). HR-EI-MS: 486.6002 (Mþ , C30H46Oþ

5 ;calc. 486.6861).


Methylation of 2. The methyl ester 2a (2.1 mg) was obtained from the reaction of 2 (2.2 mg) with anEt2O soln. of CH2N2. 1H-NMR (CDCl3): 3.60 (COOMe). EI-MS: 500 (Mþ ) .

Determination of Leishmanicidal Activity. Leishmania major promastigotes were grown at 22–258 inRPMI-1640 (Sigma) containing 10% heat-inactivated (568 for 30 min) fetal bovine serum. Promastigoteculture in the logarithmic phase of growth was maintained and the final concentration of parasites wasadjusted to 2�106 parasites ml�1. The test compounds, 1.0 mg each, were dissolved in DMSO (50 ml),and the volume were made up to 1 ml with RPMI-1640. Twenty ml of the test compound were added in tothe first well, which contained 180 ml of media, followed by serially dilution. A total of 100 ml of parasitesuspension was added into each well of the 96-well plates and incubated at 21–228 for 72 h.Amphotericin B and pentamidine were used as positive controls. The experiments were performed induplicates, and the numbers of surviving parasites were counted in a Neubauer chamber. IC50 Values weredetermined by a Windows based EZ-Fit 5 Perrella Scientific Software [29].

One of the authors (A.A.) thankfully acknowledges financial support of Higher EducationCommission (HEC) of Pakistan through Indigenous 5000 Ph.D. fellowship scheme, Batch-IV, 2007. Thisresearch was also supported by the Pakistan Academy of Sciences (PAS) (Ref. 5-9/PAS/2592).

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Received May 7, 2013


leishmanicidal triterpenes from lantana camara

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