Antimalarial activities of medicinal plants traditionally used in the villages of Dharmapuri regions of South India

Journal of Ethnopharmacology 141 (2012) 796–802

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Antimalarial activities of medicinal plants traditionally used in the villages of Dharmapuri regions of South India Chinnaperumal Kamaraj a,1 , Naveen Kumar Kaushik b,1 , Abdul Abdul Rahuman a,∗ , Dinesh Mohanakrishnan b , Asokan Bagavan a , Gandhi Elango a , Abdul Abduz Zahir a , Thirunavukkarasu Santhoshkumar a , Sampath Marimuthu a , Chidambaram Jayaseelan a , Arivarasan Vishnu Kirthi a , Govindasamy Rajakumar a , Kanayairam Velayutham a , Dinkar Sahal b,∗∗ a Unit of Nanotechnology and Bioactive Natural Products, Post Graduate and Research Department of Zoology, C. Abdul Hakeem College, Melvisharam 632509, Vellore District, Tamil Nadu, India b Malaria Research Laboratory, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi 110067, India

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Article history: Received 20 November 2011 Received in revised form 20 January 2012 Accepted 5 March 2012 Available online 13 March 2012 Keywords: Antiplasmodial activities In vitro Plasmodium falciparum culture SYBR green assay Cytotoxicity Traditional medicine Dharmapuri region Chloroquine resistance

a b s t r a c t Ethnopharmacological relevance: An ethnopharmacological investigation of medicinal plants traditionally used to treat diseases associated with fevers in Dharmapuri region of South India was undertaken. Twenty four plants were identified and evaluated for their in vitro activity against Plasmodium falciparum and assessed for cytotoxicity against HeLa cell line. Aim of the study: This antimalarial in vitro study was planned to correlate and validate the traditional usage of medicinal plants against malaria. Materials and methods: An ethnobotanical survey was made in Dharmapuri region, Tamil Nadu, India to identify plants used in traditional medicine against fevers. Selected plants were extracted with ethyl acetate and methanol and evaluated for antimalarial activity against erythrocytic stages of chloroquine (CQ)-sensitive 3D7 and CQ-resistant INDO strains of Plasmodium falciparum in culture using the fluorescence-based SYBR Green I assay. Cytotoxicity was determined against HeLa cells using MTT assay. Results: Promising antiplasmodial activity was found in Aegle marmelos [leaf methanol extract (ME) (IC50 = 7 ␮g/mL] and good activities were found in Lantana camara [leaf ethyl acetate extract (EAE) IC50 = 19 ␮g/mL], Leucas aspera (flower EAE IC50 = 12.5 ␮g/mL), Momordica charantia (leaf EAE IC50 = 17.5 ␮g/mL), Phyllanthus amarus (leaf ME IC50 = 15 ␮g/mL) and Piper nigrum (seed EAE IC50 = 12.5 ␮g/mL). The leaf ME of Aegle marmelos which showed the highest activity against Plasmodium falciparum elicited low cytotoxicity (therapeutic index > 13). Conclusion: These results provide validation for the traditional usage of some medicinal plants against malaria in Dharmapuri region, Tamil Nadu, India. © 2012 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Malaria is one of the most prevalent infections in tropical and sub-tropical regions. It is estimated that half of the world population is still at risk of contracting malaria and an estimated 243 million cases led to nearly 863,000 deaths in 2008, mostly of African children aged below 5 years. In Sub-Saharan regions, 45 countries were endemic for malaria in 2008 (WHO, 2009). Spread of

∗ Corresponding author. Tel.: +91 94423 10155/04172 269009; fax: +91 04172 269487. ∗∗ Corresponding author. Tel.: +91 11 26742357; fax: +91 11 26742316. E-mail addresses: [email protected] (A.A. Rahuman), [email protected] (D. Sahal). 1 These authors contributed equally. 0378-8741/$ – see front matter © 2012 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2012.03.003

drug resistance has played an important role in the occurrence and severity of epidemic diseases in the world (Majori, 2004). As a consequence of resistance to drugs such as quinine, chloroquine, primaquine and mefloquine, the vehemence of malaria has increased in many endemic regions of the world (Schlitzer, 2007). The only hope against drug resistant severe cerebral malaria in the form of artemisinin combination therapy has also been shattered by the recent reports of clinical artemisinin resistance from South East Asia (Dondorp et al., 2009; White, 2010). The highly adaptive character of the malaria parasite accentuates the difficulty of obtaining an antimalarial vaccine along traditional lines. About 500 million new malaria cases reported annually is a challenge (Sahu et al., 2008) that underscores the urgent requirement of new drugs against malaria. In this context, we carried out an ethnopharmacological study in the Malaiyur, Amman Nagar, Periyur, Tamaran Malai and Gollapatti

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villages, located close to the Hogenakkal River, Dharmapuri region, Tamil Nadu, India in order to validate the traditional usage of plants against malaria like fevers. Twenty three plants with the highest ethnopharmacological reputations were dried, extracted with ethyl acetate and methanol and crude extracts were assayed for in vitro antiplasmodial and cytotoxic activities. 2. Materials and methods 2.1. Survey methodology and collection of ethnomedical information An ethnobotanical survey was conducted in 2010 to identify plants used in traditional medicine against diseases associated with malaria like fevers. During this survey, 25 traditional healers (THs) and 20 herborists (who sell dried plant material and advise people on the medicinal use of herbs) were interviewed with standardized questionnaires (Supplementary Appendix A). Herborists were interviewed using a validated questionnaire and species of plants were identified using a combination of local names and voucher specimens. Plants used in the study were collected from the five villages (Malaiyur, Amman Nagar, Periyur, Tamaran Malai and Gollapatti) of Dharmapuri region shown in Supplementary Fig. 1. The traditional healers have been using symptamatology to treat fevers. During our survey, the following details were collected: age and sex of THs, ethnic groups, plants used in their pharmacopoea against fever, nausea, headache and some other symptoms, plant species used in treatment of fever, methods of disease diagnosis, combination regimen of the traditional preparations, availability of raw material, conservation methods or cultivation practices of the medicinal plants, modes of preparation and routes of administration (Table 1 and Supplementary Table 1). 2.2. Identification of plant materials The plant materials were collected from the tropical Dharmapuri region, Tamil Nadu, South India between September and November 2010 and the taxonomic identification was made by Dr. C. Hema, Department of Botany, Arignar Anna Government Arts College for Women, Walajapet, Vellore, India. During raw material collection, sustainable harvesting was practiced in order to protect the habitat. For each medicinal plant collected, its vernacular name, the part used, preparation, administration and posology were obtained. The voucher specimens were deposited in our research laboratory for further reference.

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method described by Trager and Jensen (1976) with minor modifications. Plasmodium falciparum (3D7) 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 ␮g/L hypoxanthine, and 50 ␮g/L gentamicin and incubated at 37 ◦ C under a gas mixture of 5% O2 , 5% CO2 , and 90% N2 . Every day, infected erythrocytes were transferred into fresh complete medium to propagate the culture. For Plasmodium falciparum (INDO strain) in culture medium, albumax was replaced by 10% pooled human serum. 2.5. Drug dilutions Stock solutions of plant extract and artemisinin were prepared in dimethyl sulfoxide (DMSO) while CQ stock solution was in water (Milli-Q grade). 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 nontoxic to the parasite). Drugs and test plant extracts were then placed in 96-well flat bottom tissue culture grade plates. 2.6. In vitro antiplasmodial assays The ethyl acetate and methanol extracts of experimental plants were evaluated for their antimalarial activity against 3D7 and INDO strains of Plasmodium falciparum. For drug screening, SYBR green I-based fluorescence assay was set up 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, while 0.4% DMSO was used as the negative control. After 48 h of incubation, 100 ␮l of SYBR Green I solution (0.2 ␮l of 10,000× 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 cantered 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 Giemsa stained smears of extract treated parasite cultures.

2.3. Preparation of crude plant extracts 2.7. Cytotoxic activity on HeLa cells using MTT assay The collected plants samples were air-dried in the shade at the environmental temperatures (27–37 ◦ C). The samples were powdered mechanically using a commercial electrical stainless steel blender and extracted with ethyl acetate and methanol (Qualigens) in a Soxhlet apparatus (boiling point range of 60–80 ◦ C) for 8 h. The extract was concentrated on a rotary evaporator under a reduced pressure of 22–26 mm Hg at 45 ◦ C and the residue obtained was stored at 4 ◦ C. All extracts obtained were weighed and their yields calculated. 2.4. In vitro cultivation of Plasmodium falciparum CQ-sensitive strain 3D7 and CQ-resistant strain INDO of Plasmodium falciparum were used 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. Plasmodium falciparum culture was maintained according to the

The cytotoxic effects of extracts on host cells were 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 ␮g/mL gentamycin (complete medium). Briefly, cells (104 cells/200 ␮l/well) were seeded into 96well 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 1× phosphate buffered saline) was added to each well, gently mixed and incubated for another 4 h. After spinning the plate at 1500 rpm for 5 min, supernatant was removed and 100 ␮l 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.

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Table 1 Statistical analysis of the interviewee responses. Reported literature

Locations Malaiyur Amman Nagar Periyur Tamaran Malai Gollapatti Total Mean age Experience (mean average) Level of education (%) Number of patients treated

THs (M/F)

10(7/3) 3(1/2) 5(2/3) 3(3/0) 4(3/1) 25 54.8 yearsa 25.6 yearsa 22.36%a 29.8%a

Occupation

Herborists (M/F)

Agriculturea (%)

Othera (%)

78.3 56.4 83.7 43.8 82.3

21.7 43.6 16.3 56.2 17.7

Plant details Collected plants Belonging families of collected plants Most cited plants(using fever)

97 43 23

In vitro activity of cited plants (a) Promising (b) Good (c) Moderate (d) Marginally potent (e) Poor or inactive

1 species 5 species 8 species 5 species 7 species

Main reported families Lamiaceae Euphorbiaceae Cucurbitaceae Acanthaceae Fabaceae Average annual rainfall Average relative humidity Average high temperature Average low temperature Sunlight Average wind storms

8 species 6 species 6 species 5 species 5 species 958.5 mm 86% 28 ◦ C 20 ◦ C 7.44 h/day 10 to 15 km

Parts of plant used Leaf/flower Bark/stem bark/seed/fruit Rhizome/root Latex Whole plant

47% 23% 12% 7% 21%

Preparation methods Extraction/juice/decoction Boiled vegetable Powder/tablets

62% 18% 20%

Mode of administration Decoction/infusion External Other kinds of beverage Other

65% 17% 10% 8%

Response of patient Good Fair Poor

42% 54% 4%

6(4/2) 2(2/0) 5(4/1) 4(1/3) 3(3/0) 20 56.2 yearsa 26.8 yearsa 18.92%a 16.4%a

Occupation Agriculturea (%)

Othera (%)

87.2 49.6 68.4 24.8 86.2

12.8 50.4 31.6 75.2 13.8

THs, traditional healers; M, male; F, female. a Mean percentage.

3. Results 3.1. Ethnomedical data In order to know the plants in traditional usage, an ethnobotanical study was performed in September 2010 in five villages viz., Malaiyur, Amman Nagar, Periyur, Tamaran Malai and Gollapatti, located close to the Hogenakkal river, Dharmapuri region, Tamil Nadu, South India. These villages are inhabited by 17 native communities. During this survey, 25 traditional healers (THs) (9 women and 16 men; mean age: 54.8 years) and 20 herborists

(who sell dried plant material and advise people) were interviewed individually with standardized questionnaires. These traditional healers who belong to different ethnic groups (Irular, Kurumans, Malai Vedan, Malai Pandaram and Vaithiyar), who do not always speak the same language or dialect and who live in different regions treat fevers symptomatically. These THs and herborists cited many plants used in their pharmacopoea against fever, nausea, headache, etc. They revealed the methods used for traditional preparations, availability of raw material, conservation methods or cultivation practices of the medicinal plants, modes of preparation and route of administration (Supplementary Table 1). Their

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Table 2 Antimalarial activities of ethyl acetate and methanol extracts of different plant extracts against Plasmodium falciparum. S. no.

Botanical name

Plant parts

Acacia concinna (Willd.) DC. Var Adhatoda vasica Nees. Aegle marmelos L. Corr.

Seed Leaf Leaf

4 5 6 7 8 9 10 11 12

Andrographis lineata Wallich ex Nees Argemone mexicana L. Cassia auriculata L. Cassia nictitans L. Cocculus hirsutus (L.) Diels Coriandrum sativum L. Cuminum cyminum L. Datura metel L. Diospyros melanoxylon Roxb.

Leaf Leaf Flower Leaf Leaf Seed Seed Leaf Leaf

13

Eclipta prostrata (L.)

Leaf

14 15 16 17

Lantanna camara L. Leucas aspera (Willd.) Link. Momordica charantia L. Nelumbo nucifera Gaertn.

Leaf Flower Leaf Leaf

18

Phyllanthus amarus Schum. & Thonn.

Leaf

19 20

Piper nigrum L. Solanum trilobatum L.

Stem Seed Leaf

21 22 23

Tagetes erecta L. Trachyspermum ammi L. Zingiber zerumbet (L.) Roscoe ex Sm. Chloroquine Artemisinin

1 2 3

Fruit Leaf Seed Rhizome

Solvents

EA ME EA ME ME EA ME EA ME ME EA ME EA ME EA ME EA EA EA EA ME EA ME EA EA EA ME ME ME EA EA

Plasmodium falciparum (IC50 ␮g/mL)a

Cytotoxicity (TC50 ␮g/mL)

Selectivity index (TC50 /IC50 )

3D7

INDO

HeLa

HeLa/3D7

100 ± 3.0 >100 30 ± 0.9 7 ± 0.2 63 ± 1.8 66 ± 1.9 30 ± 0.9 >100 >100 >100 >100 22 ± 0.6 29 ± 0.87 25 ± 0.75 36 ± 1.0 30 ± 0.9 19 ± 0.57 12.5 ± 0.4 17.5 ± 0.5 69 ± 2.0 35 ± 1.0 26 ± 0.78 15 ± 0.45 62.5 ± 1.8 12.5 ± 0.37 >100 100 ± 3.0 >100 >100 25 ± 0.7 42.5 ± 1.2 0.021 0.0045

– – – 6 ± 0.3 (0.86) – – – – – – – – – – – – 20 ± 1.5 (1.05) 20 ± 0.5 (1.6) 9 ± 0.4 (0.51) – – – 26 ± 1.4 (1.73) – 12 ± 0.6 (0.96) – – – – – – 0.258 (12.28) 0.0045 (1)

– – – 90 ± 5.5 – – – – – – – – – – – – 42 ± 2.3 63 ± 1.6 68 ± 3.2 – – – >100 – 87 ± 2.2 – – – – – – – –

– – – 12.8 – – – – – – – – – – – – 2.2 5.04 3.9 – – – >6.6 – 7 – – – – – – – –

–, not tested; numbers in parentheses represent resistance indices (IC50 INDO/IC50 3D7); EA, ethyl acetate; ME, methanol; ND, not done. a Values given represent mean ± standard deviation of three independent observations.

knowledge of traditional plants was appreciable although many of them did not have formal education. As a result of our ethnopharmacological survey a total of 97 medicinal species belonging to 43 families were documented. The survey was conducted during September and November 2010 (low rain season). Interviews took place in the interviewees’ homes and conversation in Tamil, the language of the natives, facilitated transparent and unambiguous communication. The data collected from 45 interviewees in the study are shown in Table 1. Most cited 23 plants samples were dried, extracted with ethyl acetate and methanol and the crude extracts were assayed for in vitro antiplasmodial and cytotoxic activities.

3.2. In vitro anti-plasmodial activities To explore the possible use of traditional plants against malaria, 32 extracts obtained from 23 plants were assayed for antiplasmodial activity against 3D7, the chloroquine-sensitive strain of Plasmodium falciparum. As shown in Table 2, we observed a wide spectrum of activities which can be categorised into (a) promising (IC50 below 10 ␮g/mL, leaf methanol extract of Aegle marmelos), (b) good (IC50 > 10–20 ␮g/mL, leaf ethyl acetate extract of Lantana camara, flower extract of Leucas aspera, leaf extract of Momordica charantia, leaf methanol extract of Phyllanthus amarus and seed extract of Piper nigrum), (c) moderate (IC50 > 20–40 ␮g/mL, leaf ethyl acetate extract of Aegle marmelos, flower methanol extract of Cassia auriculata, leaf methanol extract of Datura metel, leaf ethyl acetate and methanol extracts of Diospyros melanoxylon and Eclipta prostrata, leaf methanol extract of Nelumbo nucifera,

leaf ethyl acetate extract of Phyllanthus amarus and seed ethyl acetate extract of Trachyspermum ammi), (d) marginally potent (IC50 > 40–70 ␮g/mL, leaf methanolic extracts of Andrographis lineata, leaf ethyl acetate extract of Argemone mexicana and Nelumbo nucifera, stem ethyl acetate of Phyllanthus amarus, and rhizome ethyl acetate extract of Zingiber zerumbet) and (e) poor or inactive (IC50 > 70 ␮g/mL, seed ethyl acetate extract of Acacia concinna, leaf methanol extract of Adhatoda vasica, leaf ethyl acetate extract of Cassia nictitans, leaf methanol extract of Cocculus hirsutus, seed methanol extract of Coriandrum sativum, leaf ethyl acetate extract of Cuminum cyminum and leaf, fruit ethyl acetate and methanol extract of Solanum trilobatum). Plant extracts which showed good to promising activity (IC50 below 20 ␮g/mL) (Aegle marmelos, Lantana camara, Leucas aspera, Momordica charantia, Phyllanthus amarus and Piper nigrum) on further analysis against CQ-resistant strain INDO of Plasmodium falciparum exhibited similar potency with IC50s of 6, 20, 20, 9, 26 and 12 ␮g/mL, respectively (Table 2) and showed the resistance indices in the range of 0.51–1.73. Furthermore they were also non-toxic to mammalian HeLa cell line as evident from the therapeutic index values of 2.2–12.8 (Table 2).

4. Discussion Ethnopharmacology is by definition a multidisciplinary science, where success hinges on interaction of ethnobotanists, natural products chemists, pharmacologists, taxonomists, traditional healers and user communities. Interviewing traditional healers for accurate information about herbal recipes, their component herbs

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and their medicinal and other uses constitutes an important activity in ethnopharmacological field investigation. Importantly, plantderived compounds offer an approach to chemotherapy since the use of plant products with specific clinical activity can be a useful starting point for medicinal chemistry. In the present study, we have collected ethnobotanical data on medicinal plants that are used to treat malaria and other diseases. Our ethnopharmacological cues are based on first-hand information collected by personal contact with traditional healers who had sound knowledge of traditional medicine, as well as through personal observations of applications of the herbal remedies administered by traditional healers. In the gathering and recording of information each THs was asked about the plants used for medicinal purposes in their area, and for each plant the following descriptors were recorded: vernacular names, part of plant used, the mode of preparation, the form of administration of the remedies, the diseases and also the symptoms for which they were recommended. The present work based on ethnopharmacology of Indian medicinal plants of the Dharmapuri region of South India was an attempt to identify new drug leads against malaria. Our study has revealed that information gathered from traditional healers was true in relation to the results of the in vitro tests. Khalid et al. (1986) also reported that the leaf and immature bark extracts of Aegle marmelos exhibited in vitro antimalarial activity (IC50 = 48.2 ␮g/mL) against Plasmodium falciparum and a new anthraquinone, 1-methyl-2-(3 -methyl-but2 -enyloxy)-anthraquinone isolated from seeds of Aegle marmelos was found to have significant antifungal activity (Mishra et al., 2010). Dichloromethane and methanol extracts of Lantana camara have been reported to exhibit in vitro antimalarial activity against the 3D7 (IC50 = 5.7 ␮g/mL) and W2 (IC50 = 14.1 ␮g/mL) strains of Plasmodium falciparum (Jonville et al., 2008). The leaves ethanolic extracts of Lantana sp., have been reported to display good activity (IC50 < 17.5 ␮g/mL) against Plasmodium falciparum (Valadeau et al., 2009). Flavonoids (linaroside and lantanoside) isolated from Lantana camara were found to be active against Mycobacterium tuberculosis (Begum et al., 2008). In previous reports, leaf ethyl acetate and methanol extract of Leucas aspera showed good antiplasmodial activity (IC50 = 7.81 and 22.76 ␮g/mL) against chloroquine (CQ)-sensitive (3D7) strain of Plasmodium falciparum which was cultured following the candlejar method (Bagavan et al., 2011a) and chloroform extract of Leucas cephalotes (IC50 =3.96 ␮g/mL) showed good antiplasmodial activity against erythrocytic stages of Plasmodium falciparum was determined using a modified [3H]-hypoxanthine incorporation assay with the chloroquine- and pyrimethamine-resistant K1 strain (Dua et al., 2011). Sadhu et al. (2003) isolated eight lignans and four flavonoids from whole plant of Leucas aspera having prostaglandin inhibitory and antioxidant activities. Earlier authors Mesia et al. (2008) reported the methanol extract of Momordica charantia (whole plant) showed good antiprotozoal activity against Trypanosoma brucei (IC50 = 7 ␮g/mL) and Trypanosoma cruzi (IC50 = 37.0 ␮g/mL) whereas the same was not potent against Plasmodium falciparum (IC50 > 64 ␮g/mL). In contrast, Momordica charantia aerial part of aqueous extract displayed a moderate in vivo activity (LD70 = 904 mg/kg of body wt.) on the ˜ rodent malaria against Plasmodium vinckei petteri (279BY) (Munoz et al., 2000). However, Amorim et al. (1991) reported that the oral administration of crude dried leaves ethanolic extracts of Momordica charantia was ineffective in lowering the parasitaemia level against Plasmodium berghei-infected mice at 500 mg/kg. Ling et al. (2009) isolated three compounds, (19S, 23E)-5beta,19epoxy-19-methoxy-cucurbita-6,23-dien-3beta and 25-diol; (19R, 23E)-5beta,19-epoxy-19-methoxy-cucurbita-6,23-dien-3beta and 25-diol; and 3beta, 7beta,25-trihydroxycucurbita-5,23-dien-19al-3-O-beta-d-glucopyranoside from Momordica charantia having inhibitory effects against pests (Liriomyza sativae).

Boiling water extract of Phyllanthus amarus has been previously reported to be marginally active in vitro and in vivo against Plasmodium falciparum (IC50 80.11 ␮g/mL) and Plasmodium berghei infected mice (ED50 112.78 ± 32.32 mg/kg) (Traore et al., 2008). However our results indicate good antimalarial activity (IC50 = 15 ␮g/mL) for the methanolic extract of leaves of Phyllanthus amarus. This difference in activity may be related to the solvents used in the two studies. The methanol extracts of Phyllanthus niruri have earlier been found to have significant in vitro antiplasmodial activity against chloroquine-resistant (FCR-3) and chloroquinesensitive (D-10) strains of Plasmodium falciparum (IC50 = 2.3–3.9 and 3.2–2.5 ␮g/mL), respectively and also significant in vivo activity against Plasmodium berghei infected mice with an ED50 of 9.1 mg/kg/day (Mustofa Sholikhah and Wahyuono, 2007). Earlier, we have reported promising antiplasmodial activity in the leaf ethyl acetate and methanol extract of Phyllanthus emblica (IC50 = 7.25 and 3.125 ␮g/mL, respectively) (Bagavan et al., 2011b). Our finding of the presence of antimalarial activity in Phyllanthus amarus is supported by previous reports which indicated ellagic acid (formed by hydrolysis of tannins) as the active constituent of an ethanolic extract of the same plant (Dhooghe et al., 2011). Xiang et al. (2011) isolated a polyphenolic compound 1,2,4,6-tetra-O-galloyl␤-d-glucose (1246TGG) from Phyllanthus emblica having in vitro antiviral activities against herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2). Piper nigrum has been used by South Indian THs to treat fevers in general, malaria asthma, cold, intermittent fever, cholera, colic pain and diarrhoea (Simpson and Ogorzaly, 1995). In our observation, the ethyl acetate seed extract of Piper nigrum showed promising in vitro antiplasmodial activity against Plasmodium falciparum 3D7 and INDO strains with IC50 values of 12.5 and 12.0 ␮g/mL, respectively with a low cytotoxicity (TC50 = 87.0 ␮g/mL) and a significant therapeutic index of 7.0. alkaloids piperine, guineensine, pipericide, N-feruloyltyramine and N-isobutyl-2E, and 4E-dodecadienamide have been isolated from Piper nigrum and piperine has been reported as a stimulator of in vitro melanocyte proliferation (Lin et al., 2007). Cassia auriculata (flower), Datura metel (leaf), Diospyros melanoxylon (leaf), Eclipta prostrata (leaf), Nelumbo nucifera (leaf) and Trachyspermum ammi (seed) displayed moderate anti-malarial activities in our study. Previous reports demonstrate the antiplasmodial (Kamaraj et al., 2011) anti-diabetic (Gupta et al., 2010), larvicidal (Kamaraj et al., 2009) and anti-cancer (Prasanna et al., 2009) effects of leaf extracts of Cassia auriculata. Juan-Badaturuge et al. (2011) isolated the antioxidant kaempferol-3-O-rutinoside, kaempferol, quercetin and luteolin from Cassia auriculata. Datura metel has been well known for its use in traditional Indian and Chinese systems of medicine for centuries as a narcotic antispasmodic useful in the treatment of cough, asthma, diarrhoea, and it has also been used as an analgesic, to control fever and kill parasites (Bonde, 2001; Parrotta, 2001; Rajesh Sharma, 2002). The aqueous extracts of Datura metel are known to exhibit moderate larvicidal activity against filariasis and avian malaria vector of Culex quinquefasciatus (Rahuman et al., 2008; Tandon and Sirohi, 2010). Three new withanolide glycosides, daturametelins, daturataturin A and 7,27-dihydroxy-1-oxowitha-2,5,24-trienolide were isolated from the methanol extract of the aerial parts of Datura metel. Withanolide 7,27-dihydroxy-1-oxowitha-2,5,24-trienolide has been reported to have anti human colorectal carcinoma (HCT116) activity (IC50 = 3.2 ␮M) (Ma et al., 2006). Kantamreddi and Wright (2008) reported four Indian Diospyros species viz., Diospyros melanoxylon, Diospyros peregrine, Diospyros sylvatica, and Diospyros tomentosa in which methanolic extracts were found to have significant activity (IC50 = 16.5–92.9 ␮g/mL), against chloroquine-sensitive 3D7 strain of Plasmodium falciparum and similar activities against chloroquine-resistant K1 strains

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(IC50 = 20.5–121.6 ␮g/mL). The compound 4-O-(3 -methylgalloyl) norbergenin isolated from the stem bark of Diospyros sanza-minika was highly active against Plasmodium falciparum (IC50 = 0.6 ␮g/mL), whereas 4-O-galloylnorbergenin (IC50 = 3.9 ␮g/mL), 11-O-phydroxy-benzoyl-norbergenin (IC50 = 4.9 ␮g/mL) and norbergenin and 4-O-syringoylnorbergenin (IC50 ≥ 32 ␮g/mL) were less active (Tangmouo et al., 2010). Eclipta prostrata showed moderate antiplasmodial activity (IC50 = 30 ␮g/mL) in our study. In previous reports Eclipta prostrata has been studied for antileishmanial and larvicidal activites. Dasyscyphin C compound isolated from Eclipta prostrata showed good leishmanicidal activity (Khanna et al., 2009) and the leaf extracts showed good mosquito larvicidal effect (Khanna and Kannabiran, 2007; Elango et al., 2009). Bapna et al. (2007) reported moderate anti-malarial activity at 750 mg/kg/day of methanolic leaf extract of Eclipta alba against Plasmodium berghei ANKA strain in mice. Orobol, isolated from Eclipta prostrata (Tewtrakul et al., 2011) exhibited anti-inflammatory effect. In the present investigation, leaf ethyl acetate extract Nelumbo nucifera showed low activity whereas methanol extract showed moderate activity against chloroquine sensitive (3D7) strain of Plasmodium falciparum (IC50 = 69 and 35 ␮g/mL) when compared with earlier report. Nelumbo nucifera has been used for various medicinal purposes in oriental medicine and two compounds, (R)roemerine and N-methylasimilobine isolated from its leaf exhibited good in vitro antimalarial activity (IC50 = 0.2 ␮g/mL) (Agnihotri et al., 2008). Armepavine, a compound from Nelumbo nucifera, was found to exert immunosuppressive effects on T-lymphocytes in lupus nephritic mice (Weng et al., 2009). Liensinine, negferine, and isoliensinine isolated from the leaves and embryo of Nelumbo nucifera showed potent anti-HIV activity (IC50 : 0.8 ␮g/mL) (Kashiwada et al., 2005). In the present investigation, seed ethyl acetate extract of Trachyspermum ammi has shown moderate in vitro antiplasmodial activity against CQ-sensitive strain of Plasmodium falciparum (3D7), with IC50 values of 25 ␮g/mL. Previous authors also reported the seed cyclohexane, methylene chloride and methanol extract of Trachyspermum ammi to have good in vitro antimalarial activity (IC50 : 8.75–9.23 ␮g/mL) (Chenniappan and Kadarkarai, 2010). A novel compound, (4aS, 5R, 8aS) 5,8a-di-1propyl-octahydronaphthalen-1-(2H)-one isolated from the seed of Trachyspermum ammi showed good antibiofilm and antiadherence activities (Khan et al., 2010) whereas a phenolic monoterpene isolated from the same source was found to have antifilarial activity (Mathew et al., 2008). Leaf methanolic extracts of Andrographis lineata, leaf ethyl acetate extracts of Argemone Mexicana, Nelumbo nucifera, stem of Phyllanthus amarus, and rhizome of Zingiber zerumbet, seed ethyl acetate extract of Acacia concinna, leaf methanol extract of Adhatoda vasica, leaf ethyl acetate extract of Cassia nictitans, leaf methanol extract of Cocculus hirsutus, seed methanol extract of Coriandrum sativum, leaf ethyl acetate extract of Cuminum cyminum, methanol extract of leaf and fruit of Solanum trilobatum displayed comparatively low antimalarial activity. It is evident that medicinal plants are continuously being screened for their pharmacological properties and many interesting results with crude extracts have occasionally led to the isolation and identification of the active principles. However, as a source of new drugs, the medicinal plants are understudied, considering the high percentage of plants not yet screened for their biochemical composition or for their pharmacological properties.

5. Conclusion Our data reflects on the different plants used by traditional healers form Dharmapuri region of South India against different

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diseases including malaria. The different plant species which are used belong to different plant families. The choice of these medicinal plants by traditional healers was found to be legitimate as confirmed by the antiplasmodial screening carried out in the present study. It was heartening to find that some, though not all of the plants mentioned by traditional healers showed promising results against malarial parasites in vitro. This indicates that some of these traditional remedies probably owe their effectiveness at least in part to the presence of a component that can kill the malaria parasite. Acknowledgements The authors are grateful to C. Abdul Hakeem of the College Management; Dr. W. Abdul Hameed, Principal; and Dr. Hameed Abdul Razack, HOD of Zoology Department for providing the facilities to carry out this work. All traditional healers and herborists are highly acknowledged for sharing their indigenous medicinal knowledge on plants and help rendered during the field work. NKK thanks ICMR, New Delhi for Senior Research Fellowship. NKK, DM and DS thank MR4 who generously provided the Chloroquine resistant INDO strain used in the study. Thanks to X. Su who deposited this strain with MR4. This study was funded by University Grants Commission (F. no. 35-71/2008 SR) Government of India, New Delhi. Chinnaperumal Kamaraj gratefully thanks CSIR, New Delhi for Senior Research Fellowship (CSIR Sc.No. 8/524 (0005)/2011EMR-1). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.jep.2012.03.003. References Agnihotri, V.K., ElSohly, H.N., Khan, S.I., Jacob, M.R., Joshi, V.C., Smillie, T., Khan, I.A., Walker, L.A., 2008. Constituents of Nelumbo nucifera leaves and their antimalarial and antifungal activity. Phytochemistry Letters 1, 89–93. Amorim, C.Z., Marques, A.D., Cordeiro, R.S., 1991. Screening of the antimalarial activity of plants of the Cucurbitaceae family. Memórias do Instituto Oswaldo Cruz 2, 177–180. Bagavan, A., Rahuman, A.A., Kamaraj, C., Kaushik, N.K., Mohanakrishnan, D., Sahal, D., 2011a. Antiplasmodial activity of botanical extracts against Plasmodium falciparum. Parasitology Research 108, 1099–1109. Bagavan, A., Rahuman, A.A., Kaushik, N.K., Sahal, D., 2011b. In vitro antimalarial activity of medicinal plant extracts against Plasmodium falciparum. Parasitology Research 108, 15–22. Bapna, S., Adsule, S., Mahendra, S.S., Jadhav, S., Patil, L.S., Deshmukh, R.A., 2007. Antimalarial activity of Eclipta alba against Plasmodium berghei infection in mice. Journal of Communicable Diseases 39, 91–94. Begum, S., Wahab, A., Siddiqui, B.S., 2008. Antimycobacterial activity of flavonoids from Lantana camara Linn. Natural Product Research 22, 467–470. Bonde, K., 2001. The genus Datura: from research subject to powerful hallucinogen. Journal of Ethanobotanical Leaflet 29, 335–336. Chenniappan, K., Kadarkarai, M., 2010. In vitro antimalarial activity of traditionally used Western Ghats plants from India and their interactions with chloroquine against chloroquine-resistant Plasmodium falciparum. Parasitology Research 107, 1351–1364. Dhooghe, L., Meert, H., Cimanga, R.K., Vlietinck, A.J., Pieters, L., Apers, S., 2011. The quantification of ellagic acid in the crude extract of Phyllanthus amarus Schum. & Thonn. (Euphorbiaceae). Phytochemical Analysis 22, 361–366. Dondorp, A.M., Nosten, F., Yi, P., Das, D., Phyo, A.P., Tarning, J., Lwin, K.M., Ariey, F., Hanpithakpong, W., Lee, S.J., Ringwald, P., Silamut, K., Imwong, M., Chotivanich, K., Lim, P., Herdman, T., An, S.S., Yeung, S., Singhasivanon, P., Day, N.P., Lindegardh, N., Socheat, D., White, N.J., 2009. Artemisinin resistance in Plasmodium falciparum malaria. The New England Journal of Medicine 361, 455–467. Dua, V.K., Verma, G., Agarwal, D.D., Kaiser, M., Brun, R., 2011. Antiprotozoal activities of traditional medicinal plants from the Garhwal region of North West Himalaya, India. Journal of Ethnopharmacology 136, 123–128. Elango, G., Rahuman, A.A., Bagavan, A., Kamaraj, C., Zahir, A.A., Venkatesan, C., 2009. Laboratory study on larvicidal activity of indigenous plant extracts against Anopheles subpictus and Culex tritaeniorhynchus. Parasitology Research 104, 1381–1388. Gupta, S., Sharma, S.B., Singh, U.R., Bansal, S.K., Prabhu, K.M., 2010. Elucidation of mechanism of action of Cassia auriculata leaf extract for its antidiabetic activity in streptozotocin-induced diabetic rats. Journal of Medicinal Food 13, 528–534.

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