Antimalarial Activity of Traditionally Used Western Ghats Plants from India and Their Interactions with Chloroquine Against Chloroquine-Tolerant Plasmodium berghei

Abstract

An ethnopharmacological investigation was undertaken on Western Ghats plants traditionally used to treat malaria; 50 plants were very carefully selected from total of 372 plants, and 216 extracts were prepared and tested for in vivo antiplasmodial activity alone and in combination with chloroquine (CQ) against CQ-tolerant Plasmodium berghei (strain NK65). In in vivo antiplasmodial activity when plant extract alone is used, 81 extracts (or 37.5%) gave 52.90% significant parasitemia inhibition on day 4 postinfection and 39 extracts (or 18%) gave 41–89% mouse survival on day 9 postinfection. In combination with CQ on day 11 postinfection, 103 extracts (or 47.68%) gave mouse survival rate of 92% and on day 14 gave maximum mouse survival up to 70–79%. The fact that these activities were up to fourfold higher with CQ mostly resulted in longer mouse survival because of significant parasitemia inhibition. Our investigation have confirmed that above 70% of the plant extracts showed moderate to high in vivo antimalarial activity when used alone and in combination with CQ, and most of the extracts showed border line to good synergistic activity.

Introduction

M alaria remains one of the most serious tropical diseases, as it affects about 500 million people and kills more than 2.5 million annually, mostly children in Africa (WHO 2006). 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. History shows that plants have been an important source of medicines against malaria, with two of the major drugs used in malaria treatment, namely quinine and, more recently, artemisinin, both derived from traditional medicine and from plants. These two drugs are now the mainstay of the treatment of severe malaria worldwide. Because of limited availability and/or affordability of pharmaceutical medicines in many tropical countries, the majority of the populations depend on traditional medicinal remedies (WHO 2002, Zirihi et al. 2005), mainly from plants.

The Western Ghats is known for its richest biodiversity and the area is one of the eight biodiversity hotspots identified in the worldwide. The Western Ghats mountain range stretches for about 1600 km from the south most tip of India in Tamil Nadu from 8°N to 21°N latitudes and 173°E to 77°E longitudes to the river Tapti with the state Maharashtra, and the region covers an area of 1,60,000 sq km supporting a population of more than 50 million people. About 30% area is covered with forest and it is the ecologically richest region of India; of the 15,000 plant species recorded so far, 4000 are endemic to the region. The diverse natural wealth of the region supports numerous tribal communities who harvest nearly 150 uncultivated food plants and more than 500 medicinal plants from the wild. Chloroquine (CQ) continues to play a major role for malaria treatment in primary health care system because of its efficacy and low cost. Unfortunately, the resistant strains of Plasmodium falciparum, the most virulent of the four species infecting humans, have emerged in the past decade, making the discovery of new antimalarial agents with a novel mode of action a global health priority. The malarial multidrug resistant parasites led WHO (2006) to recommend combination therapy as first-line treatment to control malaria globally. In an attempt to impede selection of drug resistance, use of monotherapy is being discouraged for most parasitic diseases (WHO 2000). In the case of malaria, for instance, not only are novel combinations being tried, but also attempts are being made to enhance the potency and/or even reverse resistance of conventional drugs such as CQ (Winstanley 2000). There are no documented data on interactions of herbal remedies with conventional drugs such as CQ. In this study, several Western Ghats plants used as traditional remedies for malaria and malaria-like symptoms in India were evaluated for antimalarial activity against CQ rodent parasite Plasmodium berghei in mice, alone or in combination with the conventional antimalarial drug CQ.

Materials and Methods

Antimalarial plant screening

The tribal communities such as Kaani, Gowlis, Siddis, Halakkiokkaligas, and Kunabis inhabiting the Western Ghats region are one of the richest knowledge systems on tribal medicines in India, as their treatments of diseases are almost entirely confirmed to herbal medicines. An ethnopharmacological investigation of Western Ghats medicinal plants traditionally used against malaria and malaria-like symptoms was undertaken starting from the above tribal communities to their traditional healers called Plathis and was enriched by relevant literature survey and interviews. An inventory of 372 plants was developed. Selection was further optimized to narrow down the identification of potentially new antimalarial compounds. Weighted criteria were applied to this collection based on the quality of documented references, relevance and redundancy of the information, the specific use of the plant to treat malaria or malaria-like symptoms, the convergence of remedies and practices in different parts, chemical composition, and biological activity reports; 50 plants were selected and are listed in Table 1.

Table 1.

Selected Western Ghats Plants Traditionally Used to Treat Malaria and Malaria-Like Symptoms

Family	Species	Local name	Collecting place	Part	V.N.
Acanthaceae	Acanthus ilicifolius L.	Harikala	Mahabaleshwar	R	KUPS72
	Andrographis paniculata Nees.	Sriyanagai	Bharathiar University	W	KUPS 80
Apocyanaceae	Alstonia scholaris L.	Alipaven	Ghataprabha	B	KUPS 83
	Catharanthus roseus L. Spraque	Sadasawagan	Kannoor	R	KUPS 87
Apiaceae	Trachyspermum ammi L. Spraque	Ajawa	Sienna Village	Se	KUPS 91
Asteraceae	Eclipta alba L. Roxb.	Garuga	Somavarpet	L	KUPS 94
	Ageratum conyzoides L.	Billykalai	Subramanya village	L	KUPS 97
	Chrysanthemum coronarium L.	Panjupoo	Pushpagiri	L	KUPS 111
	Chromolaena odorata L.	Ottraikalai	Shesha Parvata	L	KUPS 116
	Vernonia amygdalina Del.	Kasepisedi	Kumara Parvata	L	KUPS 117
Aristolochiaceae	Aristolochia tagala L.	Orikodi	Bidalli	R	KUPS 120
Anacardiaceae	Mangifera indica L.	Mankai	Heggademane	B	KUPS 123
Balanitaceae	Balanites aegyptica L. Peplile	Hingan	Bhattaramane	L	KUPS 125
Caricaceae	Carica papaya L.	Pappali	Siribagilu	L	ZEBU 126
Caesulpiniaceae	Tamarindus indica L.	Puliyam	Gundya	F	KUPS 129
Caesalpiniaceae	Caesalpinia pulcherrima	Ratanagandisedi	Hassan	B	KUPS 130
	Cassia occidentalis L.	Kesudo	Sakaleshpur	L	KUPS 133
Cannabaceae	Cannabis sativa var. indica	Ghanja	Yedakumeri	L	KUPS 134
Compositae	Taraxxacum afficinale Web.	Dandelion	Siribagilu	L	KUPS 136
	Spilanthes oleracea L.	Palacrere	Adimali	L	KUPS 138
Convolvulaceae	Evolvulus alsinoides L.	Visnugandhi	Munnar	W	KUPS 141
Cucurbitacea	Telfairia occidentalis Hook.F	Varikai	Munnar	L	KUPS 142
Crassulaceae	Kalanchoe pinnata	Pather Sedi	Munnar	L	KUPS 143
Cyperaceae	Cyperus rotundus L.	Korai	Marayoor	L	KUPS 147
Dillieniaceae	Dillenia indica L.	Chatta	Chinnar	L + F	KUPS 150
Ebenaceae	Diospyros peregrina (Gaertn) Katlati Gurke.	Katlati	Parambikulam	B	KUPS152
Euphorbiaceae	Euphorbia hirta	Palaai	Parambikulam	L	KUPS 153
Euphorbiaceae	Acalypha siamensis	Sarusedi	Mannavan Chola	L	KUPS 171
	Phyllanthus amarus	Nelli	Uttara Kannada	W	KUPS 183
Fabaceae	Butea monosperma (Lamk) Taub.	Khakar	Mittabagilu	L	KUPS 189
				L	ZEBU 192
Gentianceae	Swertia chrayita (Roxb.ex. Fleming) H.Karst.	Balmony	Savanalu	L	KUPS 192
Lecythidaceae	Careya arborea Roxb.	Kumbhi	Savanalu	R + B	KUPS 196
Liliaceae	Aloe barbadensis Mill	Sottrukattralai	Malavanthiyavara	L	KUPS 197
Menispermaceae	Tinospora cordifolia	Thippatheega	Melanthabettu	L	KUPS 215
Molluginaceae	Mollugo cerviana Ser.	Paratakam	Melanthabettu	W	KUPS 233
Myrsinaceae	Embelia riber Burm.F.	Baberang	Melanthabettu	F	KUPS 246
Papaveraceae	Argemone mexicana	Daturi	Neriya	Se	KUPS 261
Piperaceae	Piper nigrum	Kalimiri	Neriya	F	KUPS 268
Ranunculaceae	Aconitum ferox Wall.	Bilva	Amedikallu	R	KUPS 277
Rutaceae	Aegle marmelos	Kuvalum	Maruthamalai	B	KUPS 293
	Citrus sinensis (L) Osbeck	Orngi	Maruthamalai	Se	KUPS 312
Serophulariaceae	Bacopa monnieri (L) Pennel	Brami	Kanuvai	L	KUPS 319
Solanaceae	Datura metel L.	Dutra	Kanuvai	L + R	KUPS 323
Simaroubaceae	Brucea javanica (L) Mess.	Macussas	Kanuvai	Se	KUPS 332
Verbenaceae	Lippia chevalieri M.	Haras	Periyanaikampalayam	L	KUPS 243
Zingiberaceae	Costus speciosus (Koening ex retz) J.E. Smith	Kottam	Amedikallu	Rhi	KUPS 359
	Curcuma longa L.	Manjal	Mittabagilu	Rhi	KUPS 361
	Rauwolfia serpentina	Sarpagandha	Bidalli	R	KUPS 367

R, root; W, whole plant; B, bark; Se, seeds; L, leaves; F, fruits; V.N., voucher number; Rhi, rhizome.

Collection and identification

Plant collection was carried out, starting from mid 2005 from areas spreading out from Kanniyakumari, Kalakad, (TamilNadu) to Palakad through Eravikulam, Idukki (Kerala), Kudremukh, Banavasi, and Udupi (Karnataka). Collections were undertaken from areas endemic for malaria, with variable vegetable covers. Botanic identification was first conducted in the field. The parts of the plant traditionally used were collected and allowed to dry. The specimens were then chopped into small pieces and put on plates in a drying room for 12 h at 40–50°C, until desiccated. Plants were crushed into powder using a hammer mill and stored at room temperature in plastic bags under vacuum with a dehumidifying pellet sachet. The specimen identification was confirmed by the Botanical Survey of India, Tamil Nadu Agricultural University Campus, Coimbatore, India. And by comparison with the Western Ghats flora database system—named “Shayadri-Western Ghats Biodiversity Information System”—developed by the Indian Institute of Science (IISc), Bangalore, India. These voucher herbarium specimens were deposited at the departmental General Library, School of Life Sciences, Bharathiar University, Coimbatore, India.

Extraction

Crude extractions from the plants were performed in the medicinal chemistry laboratory of the School of Life Sciences, Bharathiar University, Coimbatore, India. For each part of the plant, 50 g of powdered dried material was sequentially macerated three times in ethanol 80% for 2 h with constant shaking at room temperature. The filtrates were pooled and evaporated to dryness and the powdered extract was stored in a dry place in dark. Crude extract fractionation was performed with a semiautomatized Soxhlet extractor (Soxhlet Avanti 2055 apparatus). Each crude extract (2 g) was mixed with sand (15 g) and sequentially extracted with 100 mL of cyclohexane (15 min, 180°C) and methanol (60 min, 27°C). The filtrates were taken to dryness under vacuum and the powdered residues were stored at 4°C, and 216 organic extracts were obtained. Extracts were conditioned in 96-tube microplates at a concentration of 10 mg/mL in dimethyl sulphoxide and stored at −20°C.

Biological assays

Experimental mice

For in vivo antimalarial assays of plant extracts a CQ-tolerant P. berghei (strain NK65), a rodent malaria parasite, was used. The blood stage CQ-tolerant-induced parasite was maintained at the parasitic bank of Department of Parasitology, Bharathiar University, Coimbatore, India. Donor mouse for the experimental mice, having 0–15% parasitemia, was sacrificed and bled by cardiac puncture. The parasitemia was adjusted downward using physiological saline, and each of the experimental Swiss albino 7-week-old mice weighing about 32 g was inoculated intraperitoneally with approximately 10⁵ parasitized erythrocytes in volumes of 0.2 mL (Ishih et al. 2003). The infected animals were divided into groups, each group consisting of five animals per cage, and maintained in an animal care facility with commercialized diet and water ad libitum.

In vivo antimalarial assay

For screening of the plant extracts alone, the 4-day suppressive method of Peters et al. (1975) was used. Within 3 h postinoculation of mice with the parasite (i.e., on day 0), treatment of the experimental groups was initiated by oral administration of the test extract, dissolved in an aqueous solution of 0.2% dimethyl sulphoxide (v/v) and diluted to a desired final volume with saline, at a dose of 500 mg/kg body weight and treatment was done twice a day (at 8-h interval) for 4 days, up to day 3 postinfection. A positive control group that received CQ at a dose of 20 mg/kg body weight once a day for 2 days, as well as an untreated control group that received saline only, was also included. All the extracts and combinations were tested in parallel in five independent repetitions. Twenty-four hours after the last treatment (i.e., on day 4 postinfection), parasitemia of individual mouse was determined by microscopic examination of Giemsa-stained thin blood smears prepared from mouse tail blood.

For assessing the in vivo interactions of CQ and the plant extracts, treatment was started on day 4 postinfection based on the method of Ishih et al. (2004). Infected mice were randomized into CQ/plant extract treated groups (CQ 20 mg/kg body weight, once a day for 2 days + plant extract 500 mg/kg body weight, twice a day for 4 days), a CQ-treated positive control group, and an untreated control group, which received water only. For all mice before initial treatment on day 4 postinfection, thin blood smears were prepared, after which CQ dose followed by plant extract dose was administered by oral route. In both studies, in vivo antimalarial activity of the test drugs was assessed by monitoring mouse survival and parasitemia for over a 30-day period.

Data and statistical analysis

Percentage suppression of parasitemia for the plant extracts was calculated as follows: 100 − [(mean parasitemia treated/mean parasitemia control) × 100] (Gessler et al. 1995). For comparison of average parasitemia, one-way analysis of variance and two-tailed Student's t-test were used, with p < 0.05 considered significant.

Ethical approval

Handling of the animals was done in accordance with the Guide for Care and Use of Laboratory Animals, Bharathiar University; this work was approved by the ethical committee for using animals (no. 722/02/a/CPCS EA).

Results

In vivo antiplasmodial activity of plant extract alone

The results of the percentage of parasitemia inhibition for mice on day 4 postinfection and their corresponding survival on day 9 postinfection, relative to controls, are presented in Table 2. From different solvent extractions, 45 cyclohexane extracts showed significant parasitemia inhibitions (p < 0.05), ranging from 40% to 89%, out of which 22 extracts showed most significant parasitemia inhibitions ranging from 52% to 89%, namely Andrographis paniculata Nees. (Acanthaceae), Alstonia scholaris L. (Apocyanaceae), Chromolaena odorata L. (Asteraceae), Vernonia amygdalina Del. (Asteraceae), Mangifera indica L. (Anacardiaceae), Caesalpinia pulcherrima (Caesalpiniaceae), Cannabis sativa var. indica (Cannabaceae), Taraxxacum afficinale Web. (Compositae), Spilanthes oleracea L. (Compositae), Evolvulus alsinoides L. (Convolvulaceae), Kalanchoe pinnata (Crassulaceae), Swertia chrayita (Roxb.ex. Fleming) H.Karst. (Gentianceae), Careya arborea Roxb. (Lecythidaceae), Vanda terrablata (Roxe) Hook.ex.G.Don (Orchidaceae), Piper nigrum (Piperaceae), Citrus sinensis (L) Osbeck (Rutaceae), Datura metel L. (Solanaceae), Costus speciosus (Koening ex retz) J.E.Smith (Zingiberaceae), Dillenia indica L. (Dillieniaceae), Diospyros peregrina (Gaertn) Katlati Gurke. (Ebenaceae), and Euphorbia hirta (Euphorbiaceae).

Table 2.

In Vivo Antiplasmodial Bioassays on Chloroquine-Tolerant Plasmodium berghei with Plant Extracts Alone and in Combination with Chloroquine

				In vivo antiplasmodial activity of Plasmodium berghei NK65
Species		Extraction		Plant extract alone (500 mg/kg)		Plant extract + CQ (500 mg/kg + 20 mg/kg)
Scientific name	V.N.	Solvents	Yield (%)	% Mean (±SE) inhibition on day 4 p.i.	% Survival on day 9 p.i.	% Mean (±SE) inhibition on day 11 p.i.	% Survival on day 14 p.i.
Acanthaceae	KUPS72	CH	3.6	18 ± 3.2	2	29 ± 2.0	7
Acanthus ilicifolius L.		MC	10.3	7 ± 0.1	0	25 ± 1.8	5
		CE	58.7	10 ± 0.1	0	22 ± 1.8	0
		ME	2.8	6 ± 1.5	0	19 ± 1.7	0
Andrographis paniculata Nees.	KUPS80	CH	4.5	89 ± 2.1^a	62	92 ± 3.6^a	69
		MC	15.8	82 ± 1.3^a	53	90 ± 2.7^a	73
		CE	84.3	80 ± 2.2^a	57	91 ± 3.1^a	77
		ME	1.8	84 ± 3.2^a	59	90 ± 3.2^a	79
Apocyanaceae	KUPS83	CH	2.6	86 ± 1.2^a	62	89 ± 3.5^a	62
Alstonia scholaris L.		MC	1.7	87 ± 2.0^a	53	89 ± 3.3^a	68
		CE	8.9	89 ± 3.2^a	60	83 ± 4.1^a	65
		ME	60.3	90 ± 3.1	57	91 ± 4.5	70
Catharanthus roseus L. Spraque	KUPS87	CH	12.3	48 ± 2.7^a	12	63 ± 2.3^a	27
		MC	2.7	31 ± 3.9	10	56 ± 4.2^a	25
		CE	21.6	12 ± 2.7	0	49 ± 3.2^a	20
		ME	57.9	5 ± 0.4	0	35 ± 1.6	20
Apiaceae	KUPS91	CH	19.6	43 ± 1.2^a	15	65 ± 3.5^a	32
Trachyspermum ammi L. Spraque		MC	3.3	40 ± 0.4^a	0	60 ± 3.7^a	30
		CE	23.8	22 ± 2.1	0	57 ± 4.1^a	24
		ME	60.2	0	0	0	0
Asteraceae	KUPS94	CH	6.7	38 ± 1.1^a	30	58 ± 3.1^a	33
Eclipta alba L. Roxb.		MC	3.2	19 ± 0.4	0	49 ± 3.8^a	26
		CE	22.2	0	0	0	0
		ME	84.6	9 ± 2.7	0	45 ± 4.8^a	25
Ageratum conyzoides L.	KUPS97	CH	9.7	22 ± 1.2	7	73 ± 4.0^a	50
		MC	19.0	27 ± 1.5	3	59 ± 3.5^a	42
		CE	20.7	11 ± 2.9	5	48 ± 3.1^a	31
		ME	51.5	0	0	0	0
Chrysanthemum coronarium L.	KUPS111	CH	18.0	42 ± 3.2^a	0	61 ± 3.0^a	22
		MC	5.2	18 ± 0.4	7	37 ± 2.1	20
		CE	20.6	0	0	0	0
		ME	92.3	0	0	0	0
Chromolaena odorata L.	KUPS116	CH	11.9	60 ± 3.1^a	0	77 ± 3.1^a	37
		MC	14	18 ± 2.0	28	55 ± 2.1^a	30
		CE	20.2	4 ± 0.2	23	31 ± 2.0	22
		ME	46.7	0	0	0	0
Vernonia amygdalina Del.	KUPS117	CH	4.8	87 ± 3.6^a	52	90 ± 3.1^a	57
		MC	1.2	82 ± 3.8^a	57	86 ± 3.2^a	63
		CE	25.1	85 ± 2.0^a	48	90 ± 4.0^a	60
		ME	76.9	78 ± 3.1^a	39	82 ± 3.2^a	62
Aristolochiaceae	KUPS120	CH	10.0	42 ± 4.2^a	28	78 ± 2.8^a	53
Aristolochia tagala L.		MC	9.7	28 ± 1.8	33	63 ± 2.5^a	44
		CE	18.3	25 ± 2.1	7	59 ± 2.1^a	37
		ME	78.3	37 ± 3.2	9	45 ± 3.0^a	22
Anacardiaceae	KUPS123	CH	6.0	53 ± 1.8^a	13	63 ± 2.1^a	20
Mangifera indica L.		MC	2.3	47 ± 1.2^a	16	69 ± 3.0^a	27
		CE	17.0	31 ± 2.1	0	57 ± 3.8^a	32
		ME	68.2	46 ± 3.2^a	5	69 ± 3.1^a	30
Balanitaceae	KUPS125	CH	9.8	42 ± 1.8^a	10	63 ± 3.2^a	30
Balanites aegyptica L. Peplile		MC	3.7	37 ± 1.5	0	58 ± 2.1^a	25
		CE	20.1	20 ± 1.8	0	43 ± 4.0^a	22
		ME	65.7	0	0	0	0
Caricaceae	KUPS126	CH	17.6	32 ± 2.1	12	65 ± 2.8^a	33
Carica papaya L.		MC	8.8	41 ± 2.7^a	17	71 ± 3.7^a	39
		CE	21.0	12 ± 2.1	0	37 ± 2.0	21
		ME	60.8	14 ± 1.4	0	39 ± 3.1^a	20
Caesulpiniaceae	KUPS129	CH	7.0	19 ± 1.1	0	33 ± 2.1	20
Tamarindus indica L.		MC	1.6	20 ± 1.3	0	39 ± 3.2^a	20
		CE	18.3	13 ± 1.5	0	35 ± 3.0	20
		ME	85.1	12 ± 2.8	0	27 ± 1.8	0
Caesalpiniaceae	KUPS130	CH	12.9	60 ± 2.6^a	45	75 ± 3.1^a	39
Caesalpinia pulcherrima		MC	1.6	39 ± 1.0^a	12	43 ± 2.7^a	32
		CE	18.6	27 ± 2.1	8	48 ± 2.5^a	30
		ME	85.1	0	0	0	0
Cassia occidentalis L.	KUPS133	CH	1.8	39 ± 1.6^a	33	65 ± 2.5^a	48
		MC	0.6	23 ± 2.0	11	67 ± 1.9^a	50
		CE	10.2	16 ± 1.6	0	43 ± 2.0^a	38
		ME	88.2	20 ± 1.3	7	50 ± 1.5^a	41
Cannabaceae	KUPS134	CH	5.8	62 ± 2.1^a	44	23 ± 2.1	10
Cannabis		MC	1.3	57 ± 2.6^a	41	18 ± 2.5	5
sativa var. indica		CE	7.6	35 ± 2.6	26	12 ± 2.5	0
		ME	72.7	49 ± 2.4^a	33	20 ± 2.1	5
Compositae	KUPS136	CH	12.3	57 ± 2.7^a	43	63 ± 3.0^a	47
Taraxxacum		MC	2.5	48 ± 2.1^a	40	59 ± 3.1^a	42
afficinale Web.		CE	15.9	19 ± 1.2	0	41 ± 3.0^a	20
		ME	72.6	15 ± 1.0	19	35 ± 2.1	20
Spilanthes oleracea L.	KUPS138	CH	11.9	52 ± 3.0^a	37	20 ± 2.8	10
		MC	7.3	63 ± 0.4^a	41	17 ± 1.9	11
		CE	16.4	0	0	0	0
		ME	36.7	0	0	0	0
Convolvulaceae	KUPS141	CH	12.5	52 ± 2.3^a	48	72 ± 2.3^a	52
Evolvulus alsinoides L.		MC	2.5	31 ± 1.9	20	53 ± 2.5^a	39
		CE	16.9	16 ± 2.0	15	38 ± 2.0	20
		ME	73.3	0	0	0	0
Cucurbitacea	KUPS142	CH	11.8	32 ± 2.3	12	53 ± 2.1^a	23
Telfairia occidentalis		MC	9.3	46 ± 3.1^a	23	62 ± 2.5^a	25
Hook.F		CE	11.5	33 ± 1.6	20	57 ± 2.0^a	20
		ME	36.5	0	0	0	0
Crassulaceae	KUPS143	CH	2.2	57 ± 3.2^a	49	63 ± 2.3^a	33
Kalanchoe pinnata		MC	0.4	45 ± 2.9^a	40	67 ± 2.7^a	35
		CE	18.5	32 ± 3.8	18	59 ± 3.1^a	29
		ME	77.2	0	0	0	0
Cyperaceae	KUPS147	CH	16.7	85 ± 3.5^a	58	87 ± 3.1^a	60
Cyperus rotundus L.		MC	14.5	80 ± 3.2^a	55	83 ± 3.5^a	58
		CE	29.0	84 ± 3.5^a	59	87 ± 3.0^a	611
		ME	36.7	87 ± 3.8^a	50	90 ± 4.1^a	63
Dillieniaceae	KUPS150	CH	49.7	51 ± 3.1^a	18	70 ± 3.8^a	53
Dillenia indica L.		MC	0.2	5 ± 1.0	0	32 ± 2.0	20
		CE	25.3	7 ± 0.4	0	0	0
		ME	25.2	11 ± 1.2	0	0	0
Ebenaceae	KUPS152	CH	9.6	78 ± 3.2^a	32	81 ± 2.1^a	41
Diospyros		MC	2.1	73 ± 3.0^a	43	85 ± 3.5^a	47
peregrina (Gaertn)		CE	22.3	64 ± 2.5^a	50	78 ± 2.9^a	40
Katlati Gurke.		ME	78.2	51 ± 3.1^a	36	70 ± 3.2^a	37
Euphorbiaceae	KUPS153	CH	5.2	89 ± 3.5^a	50	87 ± 3.8^a	52
Euphorbia hirta		MC	3.5	81 ± 3.5^a	43	85 ± 3.8^a	57
		CE	26.2	83 ± 3.5^a	43	85 ± 3.8^a	48
		ME	76.3	80 ± 3.5^a	47	87 ± 3.2^a	50
Euphorbiaceae	KUPS171	CH	16.8	42 ± 1.7^a	36	67 ± 2.1^a	37
Acalypha siamensis		MC	7.1	50 ± 2.3^a	41	59 ± 2.3^a	30
		CE	24.0	7 ± 2.1	0	38 ± 2.5	32
		ME	43.3	0	0	0	0
Phyllanthus amarus	KUPS183	CH	5.6	38 ± 2.3	19	50 ± 1.5^a	22
		MC	3.2	5 ± 1.0	0	35 ± 1.8	20
		CE	8.5	6 ± 1.0	0	39 ± 2.5	20
		ME	73.5	0	0	0	0
Fabaceae	KUPS189	CH	13.0	19 ± 1.2	0	32 ± 2.0	20
Butea monosperma		MC	1.2	6 ± 0.4	0	37 ± 2.2	20
(Lamk) Taub.		CE	19.2	0	0	0	0
		ME	74.5	5 ± 0.9	0	30 ± 2.3	20
Gentianceae	KUPS192	CH	4.5	75 ± 2.3^a	39	89 ± 3.8^a	42
Swertia chrayita		MC	2.2	77 ± 3.0^a	52	80 ± 3.1^a	48
(Roxb. ex. Fleming)		CE	9.7	70 ± 3.1^a	43	85 ± 3.3^a	40
H.Karst.		ME	72.5	72 ± 3.2^a	50	87 ± 3.7^a	44
Lecythidaceae	KUPS196	CH	14.3	58 ± 1.8^a	43	70 ± 2.1^a	24
Careya arborea Roxb.		MC	1.2	61 ± 3.2^a	32	78 ± 3.2^a	37
		CE	20.6	50 ± 2.4^a	30	78 ± 3.1^a	40
		ME	72.0	48 ± 1.5^a	37	67 ± 2.7^a	43
Liliaceae	KUPS197	CH	8.3	7 ± 1.2	0	33 ± 2.0	20
Aloe barbadensis Mill		MC	14.9	10 ± 0.9	0	41 ± 2.1^a	20
		CE	7.9	0	0	0	0
		ME	59.7	0	0	0	0
Menispermaceae	KUPS215	CH	17.0	42 ± 2.6^a	18	60 ± 3.2^a	25
Tinospora cordifolia		MC	10.0	21 ± 2.4	11	43 ± 3.5^a	20
		CE	5.7	13 ± 2.8	0	40 ± 3.1^a	20
		ME	63.5	0	0	0	0
Molluginaceae	KUPS233	CH	3.9	35 ± 2.7	12	59 ± 3.0^a	23
Mollugo cerviana Ser.		MC	4.6	24 ± 1.2	10	52 ± 3.8^a	27
		CE	19.3	16 ± 1.8	0	43 ± 3.5^a	20
		ME	59.0	0	0	0	0
Myrsinaceae	KUPS246	CH	3.8	38 ± 1.3	39	59 ± 2.7^a	42
Embelia riber Burm.F.		MC	0.7	19 ± 1.4	23	47 ± 2.8^a	38
		CE	21.3	0	0	0	0
		ME	29.8	7 ± 1.2	0	35 ± 2.0	20
Myrtaceae	KUPS252	CH	5.6	49 ± 2.5^a	28	67 ± 3.1^a	21
Psidium guajava L.		MC	2.7	40 ± 2.0^a	23	59 ± 3.5^a	24
		CE	18.2	12 ± 1.8	20	61 ± 3.7^a	23
		ME	71.4	6 ± 0.8	0	37 ± 2.1	20
Orchidaceae	KUPS260	CH	3.8	52 ± 2.8^a	37	63 ± 3.1^a	39
Vanda terrablata (Roxe)		MC	1.1	57 ± 3.5^a	29	71 ± 3.5^a	20
Hook.ex.G.Don		CE	17.4	13 ± 1.7	0	43 ± 3.1^a	20
		ME	66.0	5 ± 0.8	0	37 ± 2.1	20
Papaveraceae	KUPS261	CH	0.2	49 ± 2.8^a	31	67 ± 1.8^a	23
Argemone mexicana		MC	0.1	12 ± 2.3	18	43 ± 2.8^a	20
		CE	37.5	18 ± 2.4	20	50 ± 2.0^a	20
		ME	95.0	0	0	0	0
Piperaceae	KUPS268	CH	1.5	58 ± 2.1^a	47	67 ± 2.8^a	50
Piper nigrum		MC	4.8	62 ± 2.5^a	55	70 ± 3.2^a	29
		CE	13.6	12 ± 1.0	27	37 ± 2.1	20
		ME	80.1	0	0	0	0
Ranunculaceae	KUPS277	CH	10.5	45 ± 2.3^a	32	63 ± 3.1^a	36
Aconitum ferox Wall.		MC	1.9	18 ± 1.5	27	37 ± 2.0	20
		CE	19.3	0	0	0	0
		ME	80.6	5 ± 0.5	0	28 ± 1.0	0
Rutaceae	KUPS293	CH	4.7	48 ± 1.4^a	18	60 ± 2.3^a	20
Aegle marmelos		MC	1.1	53 ± 2.3^a	27	63 ± 2.0^a	20
		CE	16.4	20 ± 2.7	0	46 ± 2.1^a	22
		ME	66.0	0	0	0	0
Citrus	KUPS312	CH	0.2	64 ± 3.5^a	55	68 ± 2.8^a	33
sinensis (L) Osbeck		MC	0.1	36 ± 3.7	28	53 ± 2.5^a	37
		CE	37.4	8 ± 1.1	0	37 ± 1.7	20
		ME	95	0	0	0	0
Serophulariaceae	KUPS319	CH	4.9	30 ± 1.3	18	52 ± 1.8^a	25
Bacopa monnieri		MC	1.5	25 ± 1.0	21	38 ± 1.8	25
(L) Pennel		CE	12.9	15 ± 2.0	10	35 ± 1.3	27
		ME	83.0	0	0	0	0
Solanaceae	KUPS323	CH	15.1	89 ± 3.6^a	48	90 ± 4.0^a	52
Datura metel L.		MC	2.0	85 ± 3.2^a	45	87 ± 4.1^a	47
		CE	19.7	87 ± 3.6^a	43	90 ± 3.8^a	49
		ME	70.2	82 ± 3.5^a	39	94 ± 4.5^a	42
Simaroubaceae	KUPS332	CH	6.0	38 ± 2.4	22	50 ± 3.5^a	23
Brucea		MC	9.2	13 ± 2.1	20	34 ± 2.6	20
javanica (L) Mess.		CE	9.8	10 ± 1.0	0	32 ± 1.8	20
		ME	75.8	17 ± 2.5	0	20 ± 1.7	0
Verbenaceae	KUPS243	CH	7.2	48 ± 3.4^a	28	61 ± 2.5^a	20
Lippia chevalieri M.		MC	6.0	39 ± 3.7^a	21	59 ± 2.5^a	33
		CE	95.0	20 ± 1.4	0	48 ± 1.9^a	24
		ME	73.6	5 ± 1.3	0	20 ± 1.7	0
Zingiberaceae	KUPS359	CH	5.1	52 ± 2.1^a	38	70 ± 1.8^a	0
Costus speciosus		MC	3.2	17 ± 1.4	23	43 ± 2.0^a	42
(Koening ex. Retz)		CE	19.8	8 ± 1.2	0	37 ± 1.5	30
J.E.Smith		ME	75.2	37 ± 2.0^a	32	62 ± 2.0^a	40
Curcuma longa L.	KUPS361	CH	12.6	33 ± 1.4	23	55 ± 1.8^a	38
		MC	7.4	47 ± 2.1^a	35	49 ± 2.2^a	30
		CE	20.2	25 ± 2.3	24	48 ± 1.7^a	27
		ME	37.8	31 ± 2.1	28	57 ± 2.8^a	20
Rauwolfia	KUPS367	CH	2.3	46 ± 3.1^a	20	51 ± 2.0^a	24
serpentina		MC	1.5	52 ± 3.1^a	20	58 ± 2.3^a	25
		CE	17.2	27 ± 3.1	12	43 ± 2.5^a	19
		ME	83.7	39 ± 2.1^a	15	49 ± 2.5^a	22

Statistically significant (p < 0.05).

CQ, chloroquine; CH, cyclohexane; MC, methylene chloride; CE, crude extract; ME, methanol extract; SE, standard error.

Methylene chloride extracts from 26 plant species showed significant parasitemia inhibitions, out of which, 16 plant species, namely A. paniculata Nees. (Acanthaceae), A. scholaris L. (Apocyanaceae), V. amygdalina Del.(Asteraceae), C. sativa var. indica (Cannabaceae), S. oleracea L. (Compositae), S. chrayita (Roxb.ex. Fleming) H.Karst. (Gentianceae), Cyperus rotundus L. (Cyperaceae), D. peregrina (Gaertn) Katlati Gurke. (Ebenaceae), E. hirta (Euphorbiaceae), Acalypha siamensis (Euphorbiaceae), S. chrayita (Roxb.ex. Fleming) H.Karst. (Gentianceae), C. arborea Roxb. (Lecythidaceae), V. terrablata (Roxe) Hook.ex.G.Don (Orchidaceae), P. nigrum (Piperaceae), Aegle marmelos (Rutaceae), and D. metel L. (Solanaceae), showed most significant parasitemia suppressions ranging from 53% to 87%. In addition, nine crude extracts, namely A. paniculata Nees. (Acanthaceae), A. scholaris L. (Apocyanaceae), V. amygdalina Del. (Asteraceae), M. indica L. (Anacardiaceae), Telfairia occidentalis Hook.F (Cucurbitacea), K. pinnata (Crassulaceae), C. rotundus L. (Cyperaceae), as well as D. peregrina (Gaertn) Katlati Gurke. (Ebenaceae), and 8 out of 12 methanolic extracts, namely A. paniculata Nees. (Acanthaceae), A. scholaris L. (Apocyanaceae), V. amygdalina Del. (Asteraceae), C. rotundus L. (Cyperaceae), D. peregrina (Gaertn) Katlati Gurke. (Ebenaceae), E. hirta (Euphorbiaceae), S. chrayita (Roxb.ex. Fleming) H.Karst. (Gentianceae), D. metel L. (Solanaceae), showed most significant parasitemia inhibitions ranging from 62% to 89% and 68% to 90%, respectively.

In contrast, 7 out of 28 plant extracts, namely Acanthus ilicifolius L. (Acanthaceae), Ageratum conyzoides L. (Asteraceae), Tamarindus indica L. (Caesulpiniaceae), Butea monosperma (Lamk)Taub. (Fabaceae), Aloe barbadensis Mill (Liliaceae), Mollugo cerviana Ser. (Molluginaceae), and Bacopa monnieri (L) Pennel (Serophulariaceae), showed nonsignificant parasitemia inhibitions (p > 0.05) ranging from 7% to 39% and/or no activity at all.

Based on day 9 postinfection, relative to untreated controls, 39 extracts (19.5%) showed significant mouse survival, up to a further 2 weeks in some cases. Fourteen cyclohexane extracts of A. paniculata Nees. (Acanthaceae), A. scholaris L. (Apocyanaceae), V. amygdalina Del. (Asteraceae), C. pulcherrima (Caesalpiniaceae), C. sativa var. indica (Cannabaceae), T. afficinale Web. (Compositae), E. alsinoides L. (Convolvulaceae), K. pinnata (Crassulaceae), E. hirta (Euphorbiaceae), C. rotundus L. (Cyperaceae), C. arborea Roxb. (Lecythidaceae), P. nigrum (Piperaceae), C. sinensis (L) Osbeck (Rutaceae), and D. metel L. (Solanaceae) showed 43–89% mouse survival, up to a further 14–117 days in some cases. Eleven methylene chloride extracts of A. paniculata Nees. (Acanthaceae), A. scholaris L. (Apocyanaceae), V. amygdalina Del. (Asteraceae), C. sativa var. indica (Cannabaceae), T. afficinale Web. (Compositae), C. rotundus L. (Cyperaceae), D. peregrina (Gaertn) Katlati Gurke. (Ebenaceae), E. hirta (Euphorbiaceae), A. siamensis (Euphorbiaceae), S. chrayita (Roxb.ex. Fleming) H.Karst. (Gentianceae), P. nigrum (Piperaceae), and D. metel L. (Solanaceae) showed 41–82% mouse survival. The eight crude and five methanolic extracts of A. paniculata Nees. (Acanthaceae), A. scholaris L. (Apocyanaceae), C. rotundus L. (Cyperaceae), D. peregrina (Gaertn) Katlati Gurke. (Ebenaceae), as well as S. chrayita (Roxb.ex. Fleming) H.Karst. (Gentianceae) showed mouse survival of 43–80% and 47–89%, respectively. A. paniculata Nees. (Acanthaceae), A. scholaris L. (Apocyanaceae), V. amygdalina Del.(Asteraceae), C. rotundus L. (Cyperaceae), E. hirta (Euphorbiaceae) showed 47–62% and 62–79% mouse survival on days 9 and 16 postinfection, respectively, with sustained effect on day 14 postinfection relative to controls.

In vivo antimalarial activity of plant extracts in combination with CQ

In CQ + plant extract combination studies, the parasitemia levels on day 4 postinfection (before initial treatment) were not different among all groups and microscopic examination of smear on day 9 postinfection detected no parasites; however, the recrudescent parasites reappeared by day 11 postinfection, and hence, parasitemia levels at day 11 postinfection were considered the most significant in assessment of chemoinhibition. Table 2 summarizes the parasitemia inhibitions (%) for mice on day 11 postinfection and the corresponding survival on day 14 postinfection when all mice of CQ-treated control group died. Among four different solvent extractions, 45 cyclohexane extracts of A. paniculata Nees. (Acanthaceae), A. scholaris L. (Apocyanaceae), C. odorata L. (Asteraceae), V. amygdalina Del. (Asteraceae), C. rotundus L. (Cyperaceae), D. peregrina (Gaertn) Katlati Gurke. (Ebenaceae), S. chrayita (Roxb.ex. Fleming) H.Karst. (Gentianceae), P. nigrum (Piperaceae), as well as Brucea javanica (L) Mess (Simaroubaceae) in combination with CQ showed significant parasitemia inhibitions ranging from 50% to 92% (p < 0.05) and 28 methylene chloride extracts also showed best activities ranging from 52% to 90%.

The most significant antimalarial activities were exhibited by A. paniculata Nees. (Acanthaceae), A. scholaris L. (Apocyanaceae), V. amygdalina Del. (Asteraceae), Caesalpinia pulcherrima (Caesalpiniaceae), C. sativa var. indica (Cannabaceae), T. afficinale Web. (Compositae), E. alsinoides L. (Convolvulaceae), K. pinnata (Crassulaceae), E. hirta (Euphorbiaceae), C. rotundus L. (Cyperaceae), C. arborea Roxb. (Lecythidaceae), P. nigrum (Piperaceae), C. sinensis (L) Osbeck (Rutaceae), as well as D. metel L. (Solanaceae), with significant parasitemia inhibition activities ranging from 89% to 90% by cyclohexane extracts. The methylene chloride extracts of A. paniculata Nees. (Acanthaceae), A. scholaris L. (Apocyanaceae), D. peregrina (Gaertn) Katlati Gurke. (Ebenaceae), S. chrayita (Roxb.ex. Fleming) H.Karst. (Gentianceae), v. amygdalina del (Asteraceae) as well as D. metal 1 (solanaceae) also exhibited significant plasmodial activities of 90%, 89%, 65%, 85%, 80%, 86%, and 87%, respectively.

Sixteen crude extracts and 14 methanolic extracts of plants (Table 2) also showed significant parasitemia inhibitions on day 11 postinhibition, ranging from 57% to 91% and 53% to 94%, respectively. Different extractions of the five plants A. ilicifolius L. (Acanthaceae), Chrysanthemum coronarium L. (Asteraceae), T. indica L. (Caesulpiniaceae), B. monosperma (Lamk) Taub. (Fabaceae), and A. barbadensis Mill (Liliaceae) in combination with CQ showed nonsignificant parasitemia inhibition (p > 0.05) activities of up to 39% (Table 2).

Based on day 14 postinfection, when 100% mortality of CQ-alone treated control occurred, mice in the group treated with CQ + methanolic extract of A. paniculata Nees. (Acanthaceae) showed the highest mouse survival of 79% at day 14 postinfection and other extracts showed survival rates ranging from 69% to 77% on day 14 postinfection. A. scholaris L. (Apocyanaceae) showed the highest survival of 70% on day 14 postinfection. The methanolic extracts of A. paniculata Nees. (Acanthaceae) and A. scholaris L. (Apocyanaceae) in combination treatment showed longer mouse survival by up to a further 2 weeks (day 25 postinfection) relative to controls. CQ + D. indica L. (Dillieniaceae) extracts also had sustained effect on day 14 postinfection, ranging from 58% to 63% mouse survival. The crude and methanolic extracts of D. indica L. (Dillieniaceae), E. hirta (Euphorbiaceae), and B. javanica (L) Mess. (Simaroubaceae) in combination with CQ on day 14 postinfection showed mouse survival ranging from 39% to 48.33%, which was not statistically significant (p > 0.05).

Discussion

As shown from the results of the in vivo antiplasmodial studies presented in Table 2, 55 cyclohexane extracts, 26 methyl chloride extracts, 9 crude extracts, and 12 methanolic extracts, representing 70% of the all the 50 plant species screened, showed significant parasitemia suppressions (p < 0.05) on day 4 postinfection, ranging from 40% to 89%, which strongly validate the ethanomedical use of the herbs in management of malaria. The plants A. paniculata Nees. (Acanthaceae), A. scholaris L. (Apocyanaceae), C. odorata L. (Asteraceae), V. amygdalina Del. (Asteraceae), M. indica L. (Anacardiaceae), C. pulcherrima (Caesalpiniaceae), S. oleracea L. (Compositae), E. alsinoides L. (Convolvulaceae), K. pinnata (Crassulaceae), C. rotundus L. (Cyperaceae), D. peregrina (Gaertn) Katlati Gurke. (Ebenaceae), E. hirta (Euphorbiaceae), S. chrayita (Roxb.ex. Fleming) H.Karst. (Gentianceae), C. arborea Roxb. (Lecythidaceae), V. terrablata (Roxe) Hook.ex.G.Don (Orchidaceae), P. nigrum (Piperaceae), A. marmelos (Rutaceae), C. sinensis (L) Osbeck (Rutaceae), D. metel L. (Solanaceae), C. speciosus (Koening ex retz) J.E.Smith (Zingiberaceae), and Rauwolfia serpentina (Zingiberacea) had shown very high in vivo chemosuppression activities and also significant in vitro antiplasmodial activities against both CQ-sensitive and CQ-resistant P. falciparum isolates in a previous study (Kuppusamy and Murugan 2006). The organic bark extracts of A. scholaris L. had shown the highest in vitro inhibition of parasite growth with IC₅₀ values as low as 1.0 μg/mL, (Keawpradub et al. 1999), which is consistent with the findings of this study, in which the methanolic extract showed a remarkable in vivo parasitemia suppression of 90%, which can be compared with the antiplasmodial activities of Vernonia lasiopus, which showed the highest in vitro inhibition of parasitic growth, with IC₅₀ values as low as 1.0 μg/mL, and remarkable in vivo parasitemia suppression of 59.3% with mouse survival rates of 60% on day 4 postinfection when using its root bark extracts (Muregi et al. 2007). The presence and/or quantities of bioactive compounds in plants are influenced by survival factors including seasons, environment, plant part used, intraspecies variations, and plant age (Weenen et al. 1990, Dua et al. 2004) and this may explain the discrepancies observed in in vitro and in vivo activities of plant parts used.

The two Asteraceae plants C. odorata L. and V. amygdalina Del showed the highest in vivo parasitemia suppression, ranging from 60% to 90% with mouse survival rates up to 63%. Many Asteraceae plants have been investigated chemically and found to contain several metabolites including triterpenes, oxygenated sesquiterpenes, flavones, and vernolic acid (Oketch-Rabah 1996). Secondary metabolites of the genus Vernonia and artemisinin, isolated from the Chinese herbs Artemisia annua, belongs to the same class of compounds and has been avidly used in the synthesis of semisynthetic antimalarials effective against multidrug-resistant strains of P. falciparum (Trigg 1989, Oketch-Rabah 1996). It is important to investigate Vernonia species further. The organic extracts of C. rotundus, Diospyrus peregrine, S. chrayita, E. hirta, and Datura metal showed remarkable in vivo plasmodial activity up to 90% in this study. Although not much has been reported in literature about the biological activity of these plants, their leaf decoctions are traditionally used to treat malarial fever, veneral diseases, and rheumatism among other ailments in some parts of India and Congo (Tona et al. 2004). The fact that all the organic extracts of these plants showed remarkable antimalarial activity underscores the need for further investigation of these plants. In some cases, remarkable suppression of parasitemia by extracts translated into either a higher or longer mouse survival on day 9 postinfection or a relatively longer survival with 68% mouse survival on day 11 postinfection. Most of the plants maintained similar suppression levels on days 4 and 11 postinfection, suggesting that the bioactive agents in these plants may have a good onset of action and fast acting. This may suggest that the bioactive compounds in these plants may have an enough half-life, because some antimalarial drugs including artemisinin-based derivatives are known to be fast acting and have an enough half-life.

In contrast, Kalanga pinnata, Argemone Mexicana, and Psidium guajava had shown moderate antiplasmodial activity with longer mouse survival on day 9 postinfection and shorter survival on day 11 postinfection, which mean that high percentage of survival on day 9 postinfection does not translate into considerably longer mouse survival. This may suggest that the bioactive compound in the plant may have a slow onset of action and have a short half-life. On the other hand, some extracts had shown very mild or no parasitemia suppression with short or no mouse survival, implying that other than direct antiplasmodial benefits to the hosts, such as acting as reducing fever, calming convulsions and headache, and possibly even immunostimulatory effects (Dahanukar et al. 2000). In combination with CQ, some of the extracts showed up to threefold better chemosuppression as well as longer mouse survival when compared with CQ-alone treated controls, suggesting synergistic interactions of the two drugs. As expected, high chemosuppression in most cases led to high mouse survival at day 14 postinfection and subsequently a longer mouse survival, as in the case of the group treated with CQ-A. scholaris and V. amygdalina. Some plant extracts such as A. conyzoides, Aristolochia tagala, and Cassia occidentalis, which lacked activity when used alone, demonstrated both higher as well as prolonged mouse survival when used in combination with CQ. As noted earlier, this emphasizes the possibility of other pharmacological effects of plants being involved, besides direct antiparasitic effects. On the other hand, some extracts including C. sativa var. indica and S. oleracea, which had significant activity when used alone, showed little or no suppression in combination with CQ. Antagonistic interactions among drugs as well as toxicity cannot be ruled out in such cases, emphasizing the need to avoid simultaneous use of conventional drugs with natural products before the safety and efficacy of such combinations have been authenticated.

Our investigation on Western Ghats plants, which are used as traditional medicines to treat malaria, has confirmed the fact that about 70% of 50 plants screened, which have not been previously studied until this report, showed moderate to high in vivo antimalarial activity when used alone, and most of the extracts showed enhanced CQ activity, which forms a basis for further detailed studies of these plants, including isolation and characterization of the bioactive compounds, with the ultimate objective of finding novel antimalarial compounds that can be used to fight against the drug-resistant malarial parasites.

Footnotes

Acknowledgments

The authors thank Dr. K. Sasikala (Professor and Head) for her warm support and critical evaluation of the manuscript and Dr. Maruthappan for help with solvent provision and crude extract fractionation. Special thanks to Dr. Gunasekaran for technical help. Warm thanks also to Dr. Ramesh for help with botanical determination.

Disclosure Statement

No competing financial interests exist.

References

Dahanukar

, Kulkarni

, Rege

NN.

Pharmacology of medicinal plants and natural products. Ind J Pharmacol, 2000; 32:81–118.

Dua

, Ojhaa

, Royb

, Joshib

et al. Anti-malarial activity of some xanthones isolated from the roots of Andrographis paniculata. J Ethnopharmacol, 2004; 95:247–251.

Gessler

, Tanner

, Chollet

, Nkunya

MHH

, Heinrick

Tanzanian Medicinal plants used traditionally for the treatment of malaria: in vivo antimalarial and in vitro cytotoxic activities. Phy Res, 1995; 9:504–508.

Ishih

, Miyase

, Terada

Comparison of antimalarial activity of the alkaloid fraction of Hydrangea macrophylla var. Otaksa leaves with the hot-water extract in ICR mice infected with Plasmodium yoelii 17 XL. Phyto Res, 2003; 17:633–639.

Keawpradub

, Kirby

, Steele

JCP

, Houghton

PJ.

Antimplasmodial activity of extracts and alkaloids of three Alstonia species from Thailand. Plan Med, 1999; 65:690–694.

Kuppusamy

, Murugan

Antimalarial activity of traditionally used Western Ghats plants from India and their interactions with chloroquine against CQ-resistant Plasmodium falciparum. Presented at the International conference on diversity of insects: Challenging issues in management and conservation: Tamil Nadu, India, 2006; 195–210.

Muregi

, Ishih

, Miyase

, Suzuki

Antimalarial activity of methanolic extracts from plants used in Kenyan ethnomedicine and their interactions with chloroquine (CQ) against a CQ-tolerant rodent parasite, in mice. J Ethnopharmacol, 2007; 111:190–195.

Oketch-Rabah

HA.

Antimalarial and antileishmarial compounds from Kenyan medicinal plants [Ph.D. thesis]

Department of Medicinal Chemistry, The Royal Danish School of Pharmacy: Denmark, 1996; 80–82.

Oudhia

Traditional medicinal knowledge about useful herb Ajwain in Chattisgarh. India Research note, 2003; 23–24.

10.

Peters

, Portus

, Robinson

BL.

The four-day suppressive in vivo antimalarial tests. Ann Trop Med Parasitol, 1975; 69:155–171.

11.

Tona

, Mesia

, Musuamba

, Bruyne

TDE

et al. In vitro antiplasmodial activity of extracts and fractions from seven medicinal plants used in the Democratic Republic of Congo. J Ethnopharmacol, 2004; 93:27–32.

12.

Trigg

PI.

Wagner

, Hikino

, Farnsworth

. Economic and Medicinal Plant Research, 3. London: Academic Press, 1989; 19–55.

13.

Weenen

, Nkunya

MHH

, Bray

, Mwasumbi

et al. Antimalarial activity of Tanzanian medicinal plants. Planta Med, 1990; 56:368–370.

14.

[WHO] World Health Organization. Severe falciparum malaria. Trans R Soc Trop Med Hyg, 2000; 94:36–37.

15.

[WHO] World Health Organization. Centre for Health Development. Traditional medicine: planning for cost-effective traditional health services in the new century-a discussion paper. 2002. www.who.or.jp/tm/research/.

16.

[WHO] World Health Organization. www.who.int/tropics/malaria/en/. 2006 May 18.

17.

Winstanley

PA.

Chemotherapy for falciparum malaria: the armoury, the problems and the prospects. J Parasitol, 2000; 16:146–153.

18.

Zirihi

, Mambu

, Guede-Guina

, Bodo

, Grellier

In vitro antiplasmodial activity and cytotoxicity of 33 West African plants used for treatment of malaria. J Ethnopharmacol, 2005; 98:281S–285S.