In vitro antimalarial activity of medicinal plant extracts ... - Springer Link

Parasitol Res (2011) 108:15–22 DOI 10.1007/s00436-010-2034-4

ORIGINAL PAPER

In vitro antimalarial activity of medicinal plant extracts against Plasmodium falciparum Asokan Bagavan & Abdul Abdul Rahuman & Naveen Kumar Kaushik & Dinkar Sahal

Received: 1 July 2010 / Accepted: 16 August 2010 / Published online: 1 September 2010 # Springer-Verlag 2010

Abstract Malaria is a major global public health problem, and the alarming spread of drug resistance and limited number of effective drugs now available underline how important it is to discover new antimalarial compounds. In the present study, ten plants were extracted with ethyl acetate and methanol and tested for their antimalarial activity against chloroquine (CQ)-sensitive (3D7) and CQresistant (Dd2 and INDO) strains of Plasmodium falciparum in culture using the fluorescence-based SYBR Green assay. Plant extracts showed moderate to good antiparasitic effects. Promising antiplasmodial activity was found in the extracts from two plants, Phyllanthus emblica leaf 50% inhibitory concentration (IC50) 3D7: 7.25 μg/mL (ethyl acetate extract), 3.125 μg/mL (methanol extract), and Syzygium aromaticum flower bud, IC50 3D7:13 μg/mL, (ethyl acetate extract) and 6.25 μg/mL (methanol extract). Moderate activity (30– 75 μg/mL) was found in the ethyl acetate and methanol extracts of Abrus precatorius (seed) and Gloriosa superba

Asokan Bagavan and Naveen Kumar Kaushik contributed equally. A. Bagavan : A. A. Rahuman (*) Unit of Nanotechnology and Bioactive Natural Products, Post Graduate and Research Department of Zoology, C. Abdul Hakeem College, Melvisharam-632 509, Vellore District, Tamil Nadu, India e-mail: [email protected] N. K. Kaushik : D. Sahal Malaria Research Laboratory, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi 110067, India D. Sahal e-mail: [email protected]

(leaf); leaf ethyl acetate extracts of Annona squamosa and flower of Musa paradisiaca. The above mentioned plant extracts were also found to be active against CQ-resistant strains (Dd2 and INDO). Cytotoxicity study with P. emblica leaf and S. aromaticum flower bud, extracts showed good therapeutic indices. These results demonstrate that leaf ethyl acetate and methanol extracts of P. emblica and flower bud extract of S. aromaticum may serve as antimalarial agents even in their crude form. The isolation of compounds from P. emblica and S. aromaticum seems to be of special interest for further antimalarial studies.

Introduction Malaria is the world’s most important tropical disease. It is prevalent in about 100 countries and around 2,400 million people are at risk (Kager 2002). In South East Asia alone, 100 million malaria cases occur every year and 70% of these are reported from India (WHO 2004). The use of chloroquine (CQ) to prevent and treat falciparum malaria has led to the wide-spread appearance of CQ-resistant strains of Plasmodium falciparum throughout the affected regions. The resistance has at the same time increasingly extended to other available antimalarial drugs (Peters 1982). The increasing global spread of drug resistance to most of the available and affordable antimalarial drugs is a major concern and requires innovative strategies to combat the disease. There is an urgent need for new chemotherapeutic compounds, which are easy to administer and store, and which are of low cost. One possible source for such affordable treatments lies in the use of traditional herbal remedies. The use of plants for the treatment of malaria extends over at least three continents, including several countries in Africa, in the Americas, and in Asia (Phillipson

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et al. 1987). Such a situation has heralded the need for alternative antiplasmodial therapy. The therapeutic success of two important plant-derived compounds, quinine and artemisinin, against malaria has inspired researchers to search new antimalarial drugs from plants. Achyranthes aspera is an annual, stiff-erect herb found commonly as a weed throughout India. The leaf ethyl acetate extract showed high larvicidal activity on the tick larvae of Rhipicephalus (Boophilus) microplus (Zahir et al. 2009), and the acetone, chloroform, ethyl acetate, hexane, and methanol leaf extracts were tested against the early fourth instar larvae of Aedes aegypti and Culex quinquefasciatus (Bagavan et al. 2008). The compound abruquinone B was isolated from the aerial parts of Abrus precatorius exhibited antiplasmodial and cytotoxic activities (Limmatvapirat et al. 2004), and the extracts of stem and root gave the best results against the schistosomules of the trematode Schistosoma mansoni and cysticercoids of the cestode Hymenolepis diminuta (Mølgaard et al. 2001). Annona squamosa, commonly known as custard apple is a native of West Indies and is cultivated throughout India, mainly for its edible fruit. The major constituents identified in the members of the Annonaceae are, typically, acetogenins (Zeng et al. 1996; Liu et al. 1999; Bermejo et al. 2005) that exhibited in vitro antimalarial activities (Rakotomanga et al. 2004), which could explain the antiplasmodial activity observed for Annona crassiflora, and the other Annonaceae like Duguetia furfuracea and Xylopia emarginata (de Mesquita et al. 2007). The roots extract of Annona senegalensis showed activity against the chloroquinoresistant strain of P. falciparum (Fall et al. 2003). Centella asiatica is a stoloniferous perennial herb, commonly growing in humid areas in several tropical countries. Its aerial parts are used against fever and to reduce uric acid levels (Devkota and Jha 2008). The Umbelliferae plant family extracts were investigated for their parasitic properties; the hydrodistilled oils from the aerial parts of Bupleurum montanum and B. plantagineum showed antiplasmodial activity (Laouer et al. 2009), and the methanolic extract of Ferula oopoda was investigated for its activity against P. falciparum (Esmaeili et al. 2009). Cynodon dactylon, commonly known as bermudagrass or arugampullu in Tamil Nadu, India, possesses various medicinal properties such as antimicrobial, antiviral activity (Dhar et al. 1968). The antimalarial activity of an ethanol leaf extract of Setaria megaphylla (Poaceae) was studied in vivo in mice infected with Plasmodium berghei berghei during early and established infections (Okokon et al. 2007). The roots and tubers of Gloriosa superba, an ornamental climbing herb native of tropical Asia and Africa, have been used in traditional Indian medicine for the treatment of gout, in diseases of skin, and several other purposes (Finnie and Staden 1994). Choochote et al. (2004)

Parasitol Res (2011) 108:15–22

have reported colchicine-like activity in the hexane, dichloromethane, and methanol fractions of G. superba. The hexane, chloroform, ethyl acetate, acetone, and methanol extracts of Mukia maderaspatensis leaves were found active against Haemaphysalis bispinosa, and fourth instar larvae of Anopheles subpictus and Culex tritaeniorhynchus (Bagavan et al. 2009). The water extract of Momordica foetida (Cucurbitaceae) was well tolerated and it delayed the development of P. berghei infection in C57BL mice (Waako et al. 2005), the crude ethanolic extract of dry Cucurbita maxima seeds showed strong antimalarial activity following oral administration, reducing the levels of parasitemia in P. berghei-infected mice and the same extract of Momordica charantia was ineffective in lowering the parasitemic levels of malarious mice (Amorim et al. 1991). Musa paradisiaca, banana, is a perennial treelike herb widely distributed in moist tropics. Due to enriched food value and versatile medicinal value, banana is one of the most important fruits and vegetable crops of India. M. paradisiaca and Musa acuminata are used in the treatment of leishmaniasis against Leishmania donovani by practitioners of traditional medicine (Gachet et al. 2010). Phyllanthus emblica, a euphorbiaceous plant is widely distributed in subtropical and tropical areas of India. It is used as antimicrobial (Rani and Khullar 2004) and the phenolic compounds including the major components 5– 8 from the fruit juice, 8, 9, and 12 from the branches and leaves, and proanthocyanidin polymers from the roots of P. emblica showed stronger inhibition against B16F10 cell growth than against HeLa and MK-1 cell growth (Zhang et al. 2004).Three new norsesquiterpenoid glycosides, 4′hydroxyphyllaemblicin B (1) and phyllaemblicins E (2) and F (3), were isolated from the roots of P. emblica, together with three known compounds, phyllaemblic acid (4), phyllaemblicin B (5), and phyllaemblicin C (6) evaluated for their antiviral activity toward coxsackie virus B3 (CVB3) by an in vitro cytopathic effect inhibitory assay (Liu et al. 2009). Dapper et al. (2007) reported that the aqueous extract of the leaves and stem of Phyllantus amarus displayed antiplasmodial effects against P. berghei infection using Swiss albino mice as models. The aqueous, methanolic and chloroformic extracts obtained from Phyllanthus niruri have been evaluated for the in vitro antiplasmodial activity against CQ-resistant (FCR-3) and CQ-sensitive (D-10) strains of P. falciparum (Mustofa et al. 2007). The flower bud of Syzygium aromaticum, commonly known as clove, a well-known food flavor for exotic food preparations has been reported to possess antimicrobial, antifungal, and antiviral properties. Besides, clove oil possesses cytotoxic, and anesthetic properties (Chaieb et al. 2007). The compound eugenol present in S. aromaticum is very toxic to Lymnaea acuminata (Kumar and Singh


2006). The extracts of the flower buds of S. aromaticum caused high mortality in Sitaphilius zeamais (Cheng et al. 1994) and were toxic to the larvae of Culex pipiens (El-Hag et al. 1999). The compound Phenylpropanoids isolated from the buds of S. aromaticum suppressed the expression of the umu gene following the induction of SOS response in the Salmonella typhimurium TA100 strain (Miyazawa and Hisama 2003), the flavones, kaempferol, and myricetin isolated from the crude MeOH extract of S. aromaticum demonstrated potent growth-inhibitory activity against the periodontal pathogens Porphyromonas gingivalis and Prevotella intermedia (Cai and Wu 1996) and the triterpene aglycones isolated from the methanol extract showed differentiation-inducing activity, but triterpene glycosides showed little activity against human acute promyelocytic leukemia cell line (HL-60) (Umehara et al. 1992). In the present study, the solvent extracts of different Indian medicinal plants were tested for their activity against CQsensitive (3D7) and CQ-resistant (Dd2 and INDO) strains of P. falciparum in culture using the fluorescence-based SYBR Green assay and the active extracts were tested for in vitro cytotoxicity against HeLa cells using 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.

Materials and methods Collection of plant materials The leaf of A. aspera linn. (Amaranthaceae), A. squamosa L. (Annonaceae), C. asiatica (L.) Urban (syn. Hydrocotyle asiatica L.) (Umbelliferae), C. dactylon (L.) Pers. (Poaceae), G. superba L. (Colchicaceae), M. maderaspatensis (L.) M. Roem (Cucurbitaceae), P. emblica L. (syn. Emblica officinalis Gaertn; Euphorbiaceae), flower of M. paradisiaca L. (Musaceae), flower bud of S. aromaticum L. (Myrtaceae), and seed of A. precatorius L. (Fabaceae) were selected on the basis of aromatic smell, bitter taste, ethnopharmacological, and ethnobotanical literature survey. The plant materials were collected from the tropical region of Javadhu Hills, Tiruvannamalai district (12°36′10N, 078°53′07E, altitude 705 m) and Nilgiri Mountain, Nilgiri district (11° 29′ 0″ N, 76° 20′ 0″ E, altitude of 880 m), Tamil Nadu, South India in January 2010 and the taxonomic identification was made by Dr. C. Hema, Department of Botany, Arignar Anna Government Arts College for Women, Walajapet, Vellore, India. The voucher specimen was numbered and kept in our research laboratory for further reference. Preparation of plant extracts The plant materials (leaf, seed, flower, and flower bud) were air-dried for 7–10 days in the shade at the environ-

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mental temperatures (27–37°Cday time) and the dried leaf (850 g), seed (400 g), flower (550 g), and flower bud (240 g) were powdered mechanically using commercial electrical stainless steel blender and extracted with ethyl acetate (2,200 mL, Qualigens) and methanol (2,800 mL, Qualigens) in a Soxhlet apparatus (boiling point range 60– 80°C) for 8 h separately until exhaustion. The extract was concentrated under reduced pressure 22–26 mmHg at 45°C, and the residue obtained was stored at 4°C. In vitro cultivation of P. falciparum CQ-sensitive strain 3D7 and CQ-resistant strains Dd2 and INDO of P. falciparum were used in in vitro blood stage culture to test the antimalarial efficacy of different plant extracts. The culture was maintained at the Malaria Research Laboratory, International Centre for Genetic Engineering and Biotechnology, New Delhi, India. P. falciparum culture was maintained according to the method described by Trager and Jensen (1976), with minor modifications. P. falciparum 3D7 and Dd2 cultures were maintained in fresh O+ve human erythrocytes suspended at 4% hematocrit in RPMI 1640 (Sigma) containing 0.2% sodium bicarbonate, 0.5% Albumax, 45 μg/L hypoxanthine, and 50 μg/l gentamicin and incubated at 37°C under a gas mixture 5% O2, 5% CO2, and 90% N2. Every day, infected erythrocytes were transferred into fresh complete medium to propagate the culture. For P. falciparum INDO strain in culture medium, albumax was replaced by 10% pooled human serum. Drug dilutions Stock solutions of CQ were prepared in water (milli-Q grade) and artemisinin, and test compounds were in dimethyl sulfoxide (DMSO). All stocks were then diluted with culture medium to achieve the required concentrations (in all cases except CQ, the final solution contained 0.4% DMSO, which was found to be non-toxic to the parasite). Drugs and test compounds were then placed in 96-well flatbottom tissue culture grade plates. Assay for antiplasmodial activity The ethyl acetate and methanol extracts of experimental plants were evaluated for their antimalarial activity against P. falciparum strains 3D7, Dd2, and INDO. For drug screening, SYBR green I-based fluorescence assay was setup as described (Smilkstein et al. 2004). Sorbitol synchronized parasites were incubated under normal culture conditions at 2% hematocrit and 1% parasitemia in the absence or presence of increasing concentrations of plant extracts. CQ and artemisinin were used as positive controls,

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while 0.4% DMSO was used as the negative control. After 48 h of incubation, 100 μl of SYBR Green I solution (0.2 μl of 10,000 X SYBR Green I (Invitrogen)/mL) in lysis buffer {Tris (20 mM; pH 7.5), EDTA (5 mM), saponin (0.008%; w/v), and Triton X-100 (0.08%; v/v)} was added to each well and mixed twice gently with multi-channel pipette and incubated in dark at 37°C for 1 h. Fluorescence was measured with a Victor fluorescence multi-well plate reader (Perkin Elmer) with excitation and emission wavelength bands centered at 485 and 530 nm, respectively. The fluorescence counts were plotted against the drug concentration, and the 50% inhibitory concentration (IC50) was determined by analysis of dose–response curves. Results were validated microscopically by examination of Giemsastained smears of extract treated parasite cultures. Cytotoxic activity on HeLa cells using MTT assay The cytotoxic effects of extracts on host cells was assessed by functional assay as described (Mosmann 1983) using HeLa cells cultured in RPMI containing 10% fetal bovine serum 0.21% sodium bicarbonate (Sigma) and 50 μg/mL gentamycin (complete medium). Briefly, Cells (104 cells/ 200 μl/well) were seeded into 96-well flat-bottom tissue culture plates in complete medium. Drug solutions were added after 24 h of seeding and incubated for 48 h in a humidified atmosphere at 37°C and 5% CO2. DMSO (as positive inhibitor) was added at 10%. Twenty microliters of a stock solution of MTT (5 mg/mL in 1X phosphatebuffered saline) was added to each well, gently mixed and incubated for another 4 h. After spinning the plate at 1,500 rpm for 5 min, supernatant was removed and 100 μl of DMSO (stop agent) was added. Formation of formazon was read on a microtiter plate reader (Versa max tunable multi-well plate reader) at 570 nm. The 50% cytotoxic concentration (TC50) of drug was determined by analysis of dose–response curves. Therapeutic index was calculated as a ratio of TC50 HeLa/IC50 3D7.

Results and discussion In the present study, ten species from ten different families were evaluated for their antimalarial potencies. Out of them, seven have been analysed for the first time against P. falciparum. Botanical names, plant part, recovery, appearances of extracts and solubility in DMSO are described in Table 1. Methanolic extract of P. emblica was found to be more potent (IC50 3D7: 3.1 μg/mL) than the corresponding ethyl acetate extract (IC50 3D7: 7.25 μg/mL). Good potencies were also displayed by methanolic and ethyl acetate extracts S. aromaticum flower bud (IC50 3D7: 6.25 μg/mL and 13 μg/mL, respectively). Methanolic


extracts of A. precatorius (IC50 3D7: 37 μg/mL) and G. superba (IC50 3D7: 42 μg/mL) and ethyl acetate extracts of A. precatorius (IC50 3D7:34 μg/mL) and A. squamosa (IC50 3D7: 33 μg/mL) displayed moderate activity. On the other hand, the methanolic extracts of A. squamosa, (IC50 3D7:100 μg/mL) and ethyl acetate extracts of G. superba (IC50 3D7: 73 μg/mL), M. maderaspatensis (IC50 3D7: 100 μg/mL), and Musa paradisiacal (IC50 3D7: 75 μg/mL) exhibited low activity. Finally, the methanolic and ethyl acetate extracts of A. aspera, C. asiatica, and C. dactylon displayed no activity up to 100 μg/mL. Plant extracts which showed IC50 up to 100 μg/mL were further analysed against CQ-resistant P. falciparum strains Dd2 and INDO (Table 2). Extracts of P. emblica, S. aromaticum, and A. precatorius were found to be as active against CQ-resistant Dd2 and INDO as against CQ-sensitive 3D7 giving resistance indices of 1–1.5. However, the ethyl acetate extracts from P. emblica and S. aromaticum gave resistance indices of 2.06 (Dd2) and 0.76 (INDO), respectively. Extracts of A. squamosa showed amazingly better response against CQ-resistant strains than against 3D7 giving resistance indices in a range of 0.5–0.82. Extracts of G. superba exhibited quite different resistance indices with values >1 for Dd2 and 7 to >32 (Table 3). Similar study was conducted by Gessler et al. (1994) who reported that the in vitro study of crude ethanolic, petroleum ether, ethyl acetate, and water fraction of A. aspera showed antimalarial activity with IC50 median values of 78, 72, 3.0, and >500 μg/mL, respectively, against P. falciparum (K I). The stem ethyl alcohol extract of A. aspera showed the IC50 values of 153 μg/mL against CQ-susceptible strain (3D7) of P. falciparum (Simonsen et al. 2001). Hilou et al. (2006) have reported that the stem bark extracts of Amaranthus spinosus exhibited higher suppressive activity (with ED50 =789.36±7.19 mg/kg) on P. berghei berghei in mice. The methanolic extract of A. squamosa leaves showed high antiplasmodial activity with IC50 values of 2 and 30μg/mL against CQ-sensitive strain 3D7 and CQ-resistant strain Dd2 of P. falciparum, respectively (El Tahir et al. 1999). The extracts from Desmopsis panamensis, Pseudomalmea boyacana, Rollinia exsucca, and Rollinia pittieri (Annonaceae) showed good antiplasmodial activity (IC50 < 10 μg/mL), respectively, against CQ-sensitive (F32) and CQ-resistant (W2) P. falciparum (Osorio et al. 2007). The compounds (+)-3acetylaltholactone, goniotriol, (+)-altholactone, and an aporphine alkaloid; (−)-nordicentrine were isolated from flowers of Goniothalamus laoticus exhibited antiplasmodial


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Table 1 Description of plant and their extract used for antimalarial screening Botanical name/family/(herbarium numbers)/ vernacular names/plant part

Crude extract

Yield (%)a

Appearance of extracts

DMSO solubility at 25mg/ml

Achyranthes aspera linn./Amaranthaceae (CAH/Z/022-07) Naayuruvi/leaf

Ethyl acetate Methanol Ethyl acetate Methanol Ethyl acetate Methanol Ethyl acetate Methanol Ethyl acetate Methanol Ethyl acetate Methanol Ethyl acetate

8.7 5.5 6.5 10.5 7.6 10.8 6.9 8.8 7.5 10 10.5 12.8 9.5

Black paste Black paste Brown thick Brown thick Black paste Black dry flakes Dark black paste Dark brown thick paste Dark green paste Dark black paste Dark green paste Dark brown thick Black paste

Small pellet and layerb Small pellet and layerb Completely soluble Completely soluble Small pellet and layerb Completely soluble Small pellet and layerb Completely soluble Small pellet and layerb Completely soluble Small layer Completely soluble Small layer

Methanol Ethyl acetate Methanol Ethyl acetate Methanol Ethyl acetate Methanol

16.9 7.8 8.7 8.5 11.6 6.2 11.8

Dark green paste Brown paste Brown liquid Dark green paste Black dry flakes Brown paste Brown paste

Small layer Small layer Completely Completely Completely Completely Completely

Abrus precatorius L/Fabaceae/(ZD/AP/119-10) Kundumani/Seed Annona squamosa L./Annonaceae (ZD/AS/045-08) Pangiee/Sita/Leaf Centella asiatica (L.) Urban/Umbelliferae (ZD/CA/036-8) Vallarai/Leaf Cynodon dactylon L/Poaceae/(ZD/CD/098-10) Arugampullu/Leaf Gloriosa superba L./Liliaceae/(ZD/GS/073-08)/ Nabhikkodi/Leaf Mukia maderaspatensis (L.) M. Roem/Cucurbitaceae (ZD/GS/073-09) Musumusukkai/Leaf Musa paradisiaca L./Musaceae/(ZD/MP/176-09) Kadali or banana/Flower Phyllanthus emblica L./Euphorbiaceae (ZD/PE/088-08) Kattunelli/Leaf Syzygium aromaticum L./Myrtaceae/ (ZD/SA/0143-10) Kiraambu/Flower bud

soluble soluble soluble soluble soluble

a

Extract yield percentage is regarding the dry powdered plant

b

Floating layer probably lipid, waxy material. Both pellet and the floating layer were discarded. Only the clear supernatant was used in the assay

activity against P. falciparum (IC50 2.6, 7.9, 2.6, and 0.3μg/ mL), respectively (Lekphrom et al. 2009), and the 80% EtOH extracts of Polyalthia viridis and Goniothalamus marcanii were found to show notable antimalarial activity (IC50: 10.0 and 6.3μg/mL) against the drug-resistant K1 strain of P. falciparum (Ichino et al. 2006). Some plants reported in literature have show good antimalarial response against P. falciparum CQ-sensitive strain D10, i.e., C. asiatica leaves (IC50, 8.3 μg/mL), Tridax procumbens whole plant (IC50, 17 μg/mL), Maytenus senegalensis roots (IC50, 15.5 μg/mL), Flueggea virosa leaves (IC50, 19 μg/ mL), and Acacia nilotica twigs (IC50, 13 μg/mL) (Clarkson et al. 2004). Two natural khellactone, (+)-4′-Decanoyl-ciskhellactone (1) and (+)-3′-decanoyl-cis-khellactone (2) were isolated from the methanolic extract of the rhizomes parts of Agelica purpuraefolia (Umbelliferae) showed notable growth-inhibitory activity against CQ-sensitive strains of P. falciparum with IC50 values from 1.5 and 2.4 μM (Chung et al. 2010). Three triterpenes cucurbitacin B, cucurbitacin D, and 20-epibryonolic acid were isolated from Cogniauxia podolaena (Cucurbitaceae) were assayed for antiplasmodial activity with the IC50 values of 1, 2, and 3 are 1.6, 4, and 2 μg/mL on FcM29, a CQ-resistant strain of P. falciparum (Banzouzi et al. 2008).

Subeki et al. (2005) have reported that the bioassayguided fractionation of boiled aqueous extracts from the whole plant of P. niruri led to the isolation of 1-O-galloyl6-O-luteoyl-alpha-d-glucose (1), with IC50 values of 4.7 μg/mL against Babesia gibsoni and 1.4 μg/mL against P. falciparum in vitro. The whole plant ethyl alcohol and dichloromethane extracts of P. niruri produced more than 60% inhibition of the parasite growth in vitro at a test concentration of 6 μg/mL against P. falciparum (Tona et al. 1999). The ellagic acid as the active constituent isolated from the ethanolic extract of Alchornea cordifolia exhibited mild in vitro activity with IC50 in the range of 0.2–0.5μM against P. falciparum (Banzouzi et al. 2002). A. precatorius pentane extract presented an IC50 value below 20 μg/mL on FcM29-Cameroon (CQ-resistant strain) and a Nigerian CQ-sensitive strain of P. falciparum (Ménan et al. 2006). Limmatvapirat et al. (2004) reported recently the presence of antimalarial and antitubercular isoflavanquinone in the aerial parts of A. precatorius. Songsiang et al. (2009) reported that the compound dalparvone isolated from stems ethyl acetate extract of Dalbergia parviflora (Fabaceae) exhibited moderate antiplasmodial activity with an IC50 value of 8.19 μg/mL against P. falciparum (K1, multi-drug-resistant strain). The compound diprenylated

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Table 2 Antimalarial activity of ethyl acetate and methanol extracts of different plant extracts against Plasmodium falciparum

Botanical name

Achyranthes aspera Abrus precatorius Annona squamosa Centella asiatica Cynodon dactylon Gloriosa superba Mukia maderaspatensis Musa paradisiaca Phyllanthus emblica a Values enclosed in parenthesis represent resistance index ratio of IC50 CQ-resistant strain (Dd2 or Indo/IC50 CQ-sensitive strain 3D7)

(–) not tested

Syzygium aromaticum Chloroquine Artemisinin

Antimalarial potency (IC50 μg/ml)a against P. falciparum

Extract

3D7

Dd2

Indo

Ethyl acetate Methanol Ethyl acetate Methanol Ethyl acetate Methanol Ethyl acetate Methanol Ethyl acetate Methanol Ethyl acetate Methanol

>100 >100 34 37 33 100 >100 >100 >100 >100 73 42

– – 43 40 20 82 – – – – 77 88

– – 35 28 18 72 – – – – 32 40

Ethyl acetate Methanol Ethyl acetate Methanol Ethyl acetate Methanol Ethyl acetate Methanol – –

100 >100 75 >100 7.25 3.125 13 6.25 0.021 0.0045

– – 100 (1.33) – 15 (2.06) 4.8 (1.53) 20 (1.53) 9.5 (1.52) 0.072 (3.42) 0.0046 (1)

flavanone from stem bark ethyl acetate extract Erythrina fusca showed notable antimalarial activity (IC50; 1.6 μg/ mL) (Khaomek et al. 2008), and the emodin and lupeol were isolated from the ethyl acetate fraction of stem bark of Cassia siamea found to be the active principles responsible for the antiplasmodial property with IC50 values of 5 μg/ mL, respectively, against the multi-drug-resistant strain of P. falciparum (Ajaiyeoba et al. 2008). Sichaem et al. (2010) have reported that the compounds isomeric indole alkaloids, naucleaorals A (1) and B (2) were isolated from the roots of Nauclea orientalis were subjected to cytotoxic evaluation against KB (human epidermoid carcinoma), HeLa (human cervical carcinoma)

(1.26) (1.08) (0.6) (0.82)

(1.05) (2.09)

(1.02) (0.7) (0.54) (0.72)

(0.43) (0.95)

– – >100 – 9 (1.24) 5 (1.6) 10 (0.76) 10 (1.6) 0.258 (12.28) 0.0045 (1)

cell lines, and antimalarial activity. Compound 1 showed significant cytotoxicity against HeLa cells with an IC50 value of 4.0 μg/mL, while compound 2 exhibited only very modest cytotoxicity against both cell lines with IC50 values of 7.8 and 9.5 μg/mL, respectively, and both compounds showed little potential as antimalarials due to their inactivity in antimalarial assays (IC50 >10.00 μg/mL). Mustofa et al. (2007) reported that the methanolic, chloroformic and aqueous extracts of P. niruri showed good antiplasmodial activity against chloroquine-resistant (FCR-3) and sensitive (D-10) strains of P. falciparum with IC50 values ranging from 2.3 to 3.9, 132.6 to 200.4, and 2.9 to 4.1 μg/mL, respectively, and the mean cytotoxicity Index

Table 3 Therapeutic indices of plant extracts harboring potent antimalarial activity Botanical name

Extract

Phyllanthus emblica

Ethyl acetate Methanol

Syzygium aromaticum

Ethyl acetate Methanol

Antimalarial potency (IC50 μg/ml) against Plasmodium falciparum 3D7

Cytotoxicity (TC50 μg/ml) against HeLa cell line

7.25 3.125

>100 >100

>14 >32

>100 >100

>7.5 >16

13 6.25

Therapeutic index TC50/IC50


(CI=IC50 against HeLa/IC50 against the FCR-3 strain) of methanolic extract (median CI, 41.3) was lower than that of the aqueous extract after 24-h incubation (CI, 106.8). The results of this study are significant since they report for the first time that the extracts of P. emblica and S. aronoticum antagonize the growth of P. falciparum across both chloroquine sensitive and chloroquine-resistant strains. Thus, isolation of compounds from these plants is crucial and of special interest with respect to the discovery of novel and potent antimalarials. In this context, the highly bioactive P. emblica, which is being grown widely in India, offers an opportunity for developing alternatives to rather expensive drugs. Acknowledgements The authors are grateful to C. Abdul Hakeem College Management, Dr. S. Mohammed Yousuff, Principal, Dr. K. Abdul Subhan, HOD of Zoology Department, for providing the facilities to carry out this work. D.S and N.K.K thank MR4 for providing P. falciparum CQ-resistant strains. This study was funded by University Grants Commission, (F.No.35-71/2008 SR) Government of India, New Delhi.

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