Journal of Ethnopharmacology 165 (2015) 152–162

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Research Paper

Antiplasmodial activity of medicinal plants from Chhotanagpur plateau, Jharkhand, India Niharika Singh a,1, Naveen Kumar Kaushik a,1, Dinesh Mohanakrishnan a,1, Santosh Kumar Tiwari b, Dinkar Sahal a,n a b

Malaria Research Group, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi 110067, India Department of Genetics, Mahrishi Dayanand University, Rohtak, Haryana 124001, India

art ic l e i nf o

a b s t r a c t

Article history: Received 26 November 2014 Received in revised form 13 February 2015 Accepted 14 February 2015 Available online 24 February 2015

Background: The alarmingly increasing problem of drug resistance in treatment of malaria has led to an urgent need for identifying new anti-malarial drugs for both prophylaxis and chemotherapy. Aim of the study: The present study presents a systematic exploration of the ex vivo blood stage antiplasmodial potential of medicinal plants to corroborate their traditional usage against malaria in Jharkhand, India. Methods: An ethnobotanical survey in and around Ranchi was done to grasp the traditional knowledge of medicinal plants used by local healers for malaria, other fevers and for other medicinal purposes like, antiamoebic, antihelmenthic, antidote to poisons, etc. Following the survey, the selected 22 plant samples were extracted in ethanol for studying ex vivo SYBR Green I fluorescence assay based antiplasmodial activity against both chloroquine-sensitive Pf3D7 and chloroquine resistant PfINDO strains of Plasmodium falciparum grown in human red blood cell cultures. Cytotoxicity was determined against HeLa and L929 cells using MTT assay. Further the most potent extract was chromatographed on reverse phase HPLC towards antiplasmodial activity guided purification of metabolites. Results: Of the 22 plant species assayed, the highest antiplasmodial activity (Pf3D7IC50 r5 mg/ml) was seen in leaf ethanol extracts of Corymbia citriodora (Hook.) K.D.Hill & L.A.S.Johnson, Calotropis procera (Aiton) Dryand. and Annona squamosa L. and bark ethanol extract of Holarrhena pubescens Wall. ex G.Don. Leaf ethanol extract of H. pubescens, bark ethanol extract of Pongamia pinnata (L.) Pierre and whole plant ethanol extract of Parthenium hysterophorus L. showed promising activity (IC50 6–10 mg/ml). Good antiplasmodial activity (IC50: 11–20 mg/ml) was observed in leaf ethanol extract of Bryophyllum pinnatum (Lam.) Oken and whole plant ethanol extract of Catharanthus roseus (L.) G.Don. The extracts of plants showing highest to good antiplasmodial activity exhibited HeLa/Pf3D7 selectivity indices of the order of 20–45. Bioassay guided fractionation of P. hysterophorus led to fivefold enrichment of antiplasmodial activities (IC50
450 ng/ml) in some fractions. Conclusion: These results provide confirmation to the traditional usage of some medicinal plants against malaria in areas around Ranchi, Jharkhand. & 2015 Elsevier Ireland Ltd. All rights reserved.

Keywords: Medicinal plants Chhotanagpur Antiplasmodial activity Resistance index Selectivity index Stage specificity of action Cytocidal action Scientific validation of tribal medicine.

1. Introduction Caused by a protozoan parasite, Malaria which kills worldwide 367,000–755,000 people and causes disease in 124–283 million people annually continues to be one of the most important diseases of the developing world. It has been estimated that nearly 22% population in India is under high transmission risk of 41 case per

n

Corresponding author. E-mail address: [email protected] (D. Sahal). 1 These authors contributed equally.

http://dx.doi.org/10.1016/j.jep.2015.02.038 0378-8741/& 2015 Elsevier Ireland Ltd. All rights reserved.

1000 population with 881,730 confirmed reported cases and 440 deaths in 2013 (WHO, 2014). The most severe form of malaria viz. cerebral malaria is caused by Plasmodium falciparum. Even as a number of chemotherapeutic attempts have been made to facilitate the eradication of malaria, yet none has succeeded largely due to emergence of drug resistant forms that have rendered chloroquine, quinine, primaquine and mefloquine ineffective in the vast majority of malaria-endemic areas (Shah et al., 2011). A remarkable feature about malaria therapy is that the two herbal treatments viz. cinchona bark and qinghao leaves were used to treat malaria effectively for hundreds of years even prior to our basic understanding of malaria. With advances in analytical techniques,

N. Singh et al. / Journal of Ethnopharmacology 165 (2015) 152–162

the active antimalarial molecules viz. quinine in the bark of cinchona trees and Artemsinin in the leaves of qinghao (Artemisia annua) were identified and used as the magic bullets against Malaria. This background seems to suggest that even as we are facing rampant resistance against the quinolines like Chloroquine and resistance against the endoperoxide artemisinin is threatening to spread out from its origin in Cambodia, a concerted effort into traditional herbal remedies as a source of new therapeutics against malaria is the need of the hour. It must be noted that despite recent successes in rational drug design and synthetic chemistry techniques by pharmaceutical companies, natural products and particularly medicinal plants remain an important source of new drugs (Lombardino and Lowe, 2004). Further traditional knowledge and usage of medicinal plant extracts to treat febrile illnesses offer the advantage of increased rate of success in identifying new antimalarial compounds. Also, it is both convenient and economic to have readily available cures in the vicinity of patients afflicted by a disease. WHO (2014) has identified malaria endemic regions in India and specified eastern part of India including Jharkhand and Orissa as falling under high transmission risk of more than 1–10 cases per 1000 population. So we conducted an ethnophamacological survey in the Chhotanagpur plateau of Jharkhand which is well known for its rich floral biodiversity and extensive usage of traditional medicines (Prasad, 1988). In the present study of 22 different plant species belonging to 13 different families selected on the basis of their usage against febrile diseases, seven have been found to offer very good to promising potential against blood stage P. falciparum in human red blood cell culture.

2. Materials and methods

153

malaria, methods of disease diagnosis, availability of raw materials, conservation methods or cultivation practices of the medicinal plants, modes of preparation and routes of administration. The views of traditional healers were compared with the information available in literature of the State Forest Department (Paria and Chattopadhyay, 2000; Prasad, 1988; Singh et al., 2001), for medicinal usage, availability of plants and the regions where they were growing. 2.2. Collection and identification of plant materials The plant materials were collected from the areas in and around Ranchi (Fig. 1, Supporting information Table 1) between March and April 2012 and the taxonomic identification was done by Dr S. K Singh, Department of Botany, A.N College, Magadh University, Patna, India. During raw material collection, sustainable harvesting was practiced in order to protect the habitat. For each collected medicinal plant, information about its vernacular name, the part(s) used, methods of preparation of extracts, administration and posology were obtained. The voucher specimens were deposited in the Department of Botany, A.N. College, Magadh University, Patna for further reference. 2.3. Preparation of crude plant extracts The collected plants samples were air-dried in shade at the environmental temperatures (27–37 1C). The samples (5 g) were powdered mechanically and extracted with ethanol (50 ml  4¼200 ml). Ethanolic extract was concentrated to dryness under reduced pressure at 40 1C using Labconco RapidVap. The residues obtained were weighed and stored at 4 1C.

2.1. Survey methodology and collection of ethnomedical information 2.4. In vitro cultivation of P. falciparum An ethnobotanical survey was conducted in March–April, 2012 in and around Ranchi, Jharkhand (geographically classified as Chhotanagpur plateau) (Fig. 1) to identify plants used in traditional medicine against diseases associated with malaria like fevers. During this survey, traditional healers (THs) and horticulturists were interviewed with standardized questionnaires. During our survey, the following details were collected: age and sex of THs, plant species used as antitoxins and in treatment of fevers like

Chloroquine (CQ) sensitive 3D7 and CQ resistant strain INDO strains of P. falciparum were used for ex vivo blood stage culture to test the antiplasmodial efficacy of different plant extracts. The cultures were 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 previously (Trager and Jensen, 1976) with

Fig. 1. Sites of plant sample collection for the study in Ranchi, Jharkhand.

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minor modifications. Cultures were maintained in fresh O þ human erythrocytes suspended at 4% hematocrit in RPMI 1640 (Invitrogen, USA) containing 0.2% sodium bicarbonate, 0.5% albumax, 45 mg/L hypoxanthine, and 50 mg/L gentamicin and incubated at 37 1C 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. 2.5. Dilutions of drugs and test samples Stock solutions of plant extract and artemisinin (ART) 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% (v/v) DMSO, which was found to be non-toxic to the parasite}. Drugs and test plant extracts were then placed in 96-well flat bottom tissue culture grade plates. 2.6. Ex vivo antiplasmodial assays The ethanol extracts of experimental plants were evaluated for their antiplasmodial activity against 3D7 and INDO strains of P. 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 ART were used as positive controls, while 0.4% (v/v) DMSO was used as the negative control. After 48 h of incubation, 100 ml of SYBR Green I (Invitrogen, USA) solution (0.2 ml of 10,000X SYBR Green I/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 371 C for 1 h. Fluorescence was measured with a Victor fluorescence multi-well plate reader (Perkin-Elmer, Morrisville, NC) 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. 2.7. Cytotoxic activity on HeLa and L929 cells using MTT assay The cytotoxic effects of extracts on host cells were assessed by functional assay as described (Mosmann, 1983) using HeLa and L929 cells cultured in RPMI containing 10% fetal bovine serum, 0.21% sodium bicarbonate (Sigma, USA) and 50 mg/ml gentamicin (complete medium). Briefly, cells (104 cells/200 ml/well) were seeded into 96-well flat-bottom tissue culture plates in complete medium. Drug solutions

Table 1 Antiplasmodial activity, resistance index, cytotoxicity and selectivity index of ethanol extracts of plants collected from Ranchi, Jharkhand, India. S.No.

Name of plant (Voucher number)

1

Albizia procera (Roxb.) Benth.(A6)

2

Alstonia scholaris (L.) R. Br (A3)

3 4

Annona squamosa L. (A7) Azadirachta indica A.Juss. (A5)

5 6 7 8 9 10 11

Bryophyllum pinnatum (Lam.) Oken (K5) Calotropis procera (Aiton) Dryand. (C4) Carum carvi L.(C5) Cascabela thevetia (L.) Lippold (T) Catharanthus roseus (C8) (L.) G.Don Corymbia citriodora (Hook.) K.D.Hill & L.A.S.Johnson (B4) Dalbergia sissoo DC (D6)

12 13

Eclipta prostrata (L.) L. (T2) Holarrhena pubescens Wall. ex G.Don (H5)

14 15

Ipomoea carnea Jacq. (B3) Madhuca longifolia (J.Koenig ex L.) J.F.Macbr. (M6)

16 17

Mimosa pudica L. (M3) Moringa oleifera Lam. (H4)

18

Paederia foetida L. (P5)

19

Pongamia pinnata (L.) Pierre (P4)

20 21

Parthenium hysterophorus L. (P4) Saraca indica L. (S6)

22

Ziziphus jujuba Mill. (M3)

a b c

Part used

Leaves Bark Leaves Bark Leaves Leaves Seeds Seed oil Leaves Leaves Seeds Leaves Whole Leaves Leaves Roots Whole Leaves Bark Leaves Leaves Bark Seeds Seed oil Whole Leaves Bark Fruit Leaves Fruits Leaves Bark Whole Leaves Bark Bark Leaves

Selectivity index TC50 HeLa/IC50 Pf3D7. Numbers in parenthesis denote resistance index (IC50Pf INDO/IC50Pf3D7). Hemolysis.

IC50 (lg/ml)

Selectivity indexa

TC50 (lg/ml)

Pf 3D7

Pf INDO

HeLa

L929

HeLa

L929

23.5 Hemolc 20.5 28 2.1 32 4100 62 14 2.5 27 4100 16 5 4100 20 23 7 4.5 Hemol 55 24.1 92 4100 51 4100 4100 4100 4100 4100 22.8 9.5 6 21 38 25 4100

43(1.82)b Hemol 22.5(1.09) 35 (1.25) 3.3(1.57) – – – 29 (2) 2.9 (1.16) 30 (1.1)

– 8 – – 95 – – – – 100 – – – 4100 – 4100 – 4100 94 38 – – – – – – – – – – – 4100 412.5 4100 – – –

– 11 – – 4 100 – – – – 39 – – – 4 100 – 4 100 – 44 4 100 35 – – – – – – – – – – – 100 4.5 4 100 – – –

– – – – 45 – – – – 40 – – – 4 20 – 45 – 4 14.3 20.8 – – – – – – – – – – – – 4 10.5 42 45 – – –

– – – – 4 30 – – – – 15.6 – – – 4 20 – 45 – 6.2 4 22 – – – – – – – – – – – – 10.5 0.75 45 – – –

37 (2.3) 17 (3.4) – 14.5(0.73) – 8.5 (1.2) 11 (2.4) Hemol – – – – – – – – – – 20.4 (0.9) 22.5 (2.4) 2.1 (0.35) 20 – 16 (0.64) 4100

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155

were added after 24 h of seeding and incubated for 48 h in a humidified atmosphere at 37 1C and 5% CO2. DMSO 10% (v/v) was used as a positive inhibitor. Twenty microliters of a stock solution of MTT (5 mg/mL in 1X 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 ml of DMSO (stop agent) was added. Formation of formazon was read on a microtiter plate reader (Versa max tunable multi-well plate reader, Molecular Devices, Sunnyvale, USA) at 570 nm. The 50% cytotoxic concentration (TC50) of drug was determined by analysis of dose– response curves. Selectivity index (SI) was calculated as a ratio of TC50 mammalian cells/IC50 P. falciparum.

Indian state of Jharkhand with its dense forest and tribal population is special in this regard since local inhabitants of this area have strong faith in traditional medicines. We conducted a survey to grasp the knowledge of medicinal plants from traditional healers and herborists and shortlisted 22 different plants which are suggested to be used against febrile diseases and other ailments (Supporting information Table 1). The medicinal reputation of the selected plants was further confirmed with the available literature (Prasad, 1988; (Paria and Chattopadhyay, 2000; Singh et al., 2001).

2.8. Bioassay guided fractionation and isolation of active Fractions

Ethanolic extracts of plants collected from Ranchi, Jharkhand were assayed for antiplasmodial activity against chloroquine sensitive P. falciparum Pf3D7. As shown in Table 1, a wide range of IC50 values were obtained, based upon which, the plants were classified for their antiplasmodial potential as (a) highly active (IC50 r5 mg/ml, leaf ethanol extracts of Corymbia citriodora (Hook.) K.D.Hill & L.A.S.Johnson, Calotropis procera (Aiton) Dryand., Annona squamosa L. and bark ethanol extract of Holarrhena pubescens Wall. ex G.Don, (b) promisingly active (IC50 5.1–10 mg/ml, leaf ethanol extracts of A. squamosa, H. pubescens, bark ethanol extract of Pongamia pinnata (L.) Pierre, whole plant ethanol extract of P. hysterophorus L., (c) good activity (IC50 10.1–20 mg/ml, leaf ethanol extract of Bryophyllum pinnatum (Lam.) Oken, whole plant ethanol extract of Catharanthus roseus (L.) G.Don, (d) moderate activity (IC50 420.1–40 mg/ml, leaf ethanol extract of Alstonia scholaris (L.) R. Br, Albizia procera (Roxb.) Benth, Saraca indica L., Azadirachta indica A.Juss, P. pinnata (L.) Pierre, bark ethanol extract of A. scholaris (L.) R. Br, Ziziphus jujube Mill., S. indica L, Madhuca longifolia var. latifolia (Roxb.) A.Chev., seed ethanol extract of Carum carvi L., whole plant ethanol extract of Eclipta prostrate (L.) L., (e) Marginal potency (IC50 40.1 –70 mg/ml, leaf ethanol extract of M. longifolia var. latifolia (Roxb.) A.Chev., seed ethanol extract of A. indica A.Juss, whole plant ethanol extract of Mimosa pudica L. and (f) poor or inactive (IC50 70.1 to 4100 mg/ml, leaf ethanol extract of Paederia foetida L., Dalbergia sissoo DC, Cascabela thevetia (L.) Lippold, Moringa oleifera Lam., fruit ethanol extract of P. foetida L., M. oleifera Lam., seed ethanol extract of M. longifolia var. latifolia (Roxb.) A.Chev.,

2.9. Microscopic evaluation of concentration dependent effect of potent fractions and stage specific inhibition assay on growth of P. falciparum Two most potent fractions of P. hysterophorus L. were evaluated for dose dependent and stage specific inhibition of Pf 3D7 growth. Highly synchronized ring stages culture was used to study the dose dependent inhibition. Different concentrations of fractions were transferred to wells containing ring stage P. falciparum culture (2% parasitemia, 2% hematocrit, 100 ml) in tetraplicate, and incubated under standard culture condition for 48 h. Following the drug incubation, a thin smear was made from wells of each respective drug concentration and stained with Giemsa for determining the stage of the culture. The remaining wells in the plate were centrifuged in a swinging bucket rotor. Drug containing medium was removed and cell pellets were spin washed three times with 200 μl of fresh medium, re-suspended in 100 ml of complete medium and further incubated at 37 1C for 24 h. Following the incubation, smears were made from wells of each respective drug concentration, stained with Giemsa and seen under a light microscope to determine an effect on parasite stage transition. Assisted by autocount (Ma et al., 2010) parasitemia was determined by visual inspection of 10–12 fields (
3000 cells). Similarly, stage specificity of the action of test samples was determined by treating highly synchronized ring, trophozoite and schizont stages with respective IC100 (F14 and F19 at 3.125 mg/ml each). Drug pressures were maintained for 48, 24 and 12 h for ring, trophozoite, and schizont stages, respectively, following which drugs were removed by media wash and the cultures were maintained in drug free complete RPMI medium for 48 h. Percentage of stage-specific inhibition of each compound was calculated in comparison to drug free control by microscopic counting of 3000 cells for each stage. The parasites with pycnotic morphology indicated in figure legend were considered as nonviable cells.

3. Results 3.1. Ethnopharmacological relevance of plants India is a rich habitat of traditional medicinal knowledge which continues to be in practice even in the era of modern medicine. The

50 40 30

mVolts

The ethanolic extract of Parthenium hysterophorus L. which showed good antiplasmodial potency and selectivity index (Table 1) was chosen for antiplasmodial activity guided reverse phase HPLC fractionation. Chromatography was performed on C18 Deltapak (19 X 300 mm, 15 m,) column (Waters, USA) using water– methanol gradient (5–95%, 1%/min) at a flow rate of 10 ml/min on a Gilson prep HPLC system. Dual wavelength detections were made at 214 and 254 nm. Fractions were collected, dried, weighed and analyzed for antiplasmodial and cytotoxic activities.

3.2. Antiplasmodial activity of collected plant extracts

20 10 0 1

2

3

4

5 6

7

8 9 10 11 12 13 14 15 1

6 17 18 19 20 21

-10 0

20

40

60

80

100

120

Minutes Fig. 2. Semiprep Reverse phase HPLC Chromatogram of ethanol extract of P. hysterophorus. DMSO supernatant of 100 mg/ml was injected into Deltapak (C18, 19 X 300 mm, 15 m) column and fractionated using Methanol water (10 ml/ min, 1%/min) 5–95% gradient (red line). Profiles in red (lower chromatogram) and blue (upper chromatogram) represent on line absorbencies at 254 nm and 214 nm, respectively. Fractions were collected, dried, weighed and analyzed for antiplasmodial and cytotoxic activity. Filled and empty bars indicate fractions (1–21) collected. The zoom of 60–120 min (marked by dotted box) has been shown just above the chromatogram tracing. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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N. Singh et al. / Journal of Ethnopharmacology 165 (2015) 152–162

Table 2 Antiplasmodial activity, resistance index, cytotoxicity and selectivity index of RPHPLC fractions of P. hysterophorus. Fraction number

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 Total

Recovered weight (mg)

8.0 2.4 2.3 5.6 4.2 1.4 7.5 5.5 5.9 1.7 3.5 4.2 2.8 4.8 4.0 2.3 2.8 5.5 9.3 5.5 2.2 91.4/100

IC50 (lg/ml)

Selectivity index (IC50 mammalian cell/IC50 PfINDO)

IC50 (lg/ml)

Pf3D7

Pf INDO

HeLa

L929

HeLa

L929

450 450 450 9 2 2.4 3.6 4.6 7 8.1 9 3.6 3.8 2.1 2 2.8 2.2 2.6 1.2 17 2.6

450 450 450 1.0 (0.1)a 0.45 (0.2) 0.45 (0.2) 0.45 (0.1) 1.0 (0.2) 1.2 (0.2) 4.5 (0.6) 2.3 (0.3) 0.6 (0.2) 0.9 (0.2) 0.65 (0.3) 0.45 (0.2) 0.5 (0.2) 0.45 (0.2) 1.0 (0.4) 0.61(0.5) 12 (0.7) 0.9 (0.3)

– – – 4 25 4 25 4 25 4 25 4 25 66 46 60 31 34 30 25 48 37 37 26 4 100 73

– – – 4 25 25 25 4 25 4 25 53 43 64 28 26 4 25 4 25 38 35 35 35 19 4 100

– – – 425 455 455 455 425 55 10.2 26 51 37 46 455 96 82 37 42 48 81

– – – 425 455 455 455 425 44 9.5 27 46 28 438 55 76 77 35 57 1.5 4111

–: Not done. a

Numbers in parenthesis denote resistance index (IC50Pf INDO/IC50Pf3D7).

A. indica A.Juss. Hemolysis (80%) was seen in the ethanolic extracts of bark of A. procera (Roxb.) Benth. and Ipomoea carnea Jacq. at 12.5 and 3.125 mg/ml, respectively. Plant extracts with IC50 Pf3D7: o25 mg/ml when tested for antiplasmodial activity against the chloroquine resistant PfINDO strain displayed resistance index values ranging from 0.35–3.4 (Table 1). All those plant extracts which displayed Pf3D7 IC50 o21 mg/ml were tested for their cytotoxic effect against mammalian cell line HeLa and L929. Most of them were found to be non-toxic with selectivity indices 410 (Table 1). 3.3. Bioassay guided fractionation of P. hysterophorus Since the ethanol extract of P. hysterophorus extract showed the highest antiplasmodial potency (IC50: 2.1 mg/ml, Table 1) against CQ resistant strain PfINDO, it was selected for activity guided fractionation by RPHPLC using water–methanol gradient (Fig. 2). Fractions collected as indicated were analyzed for ex vivo antiplasmodial activity. The fractions showed a wide range of antiplasmodial potencies with IC50 values ranging from 1.2 to 17 μg/ml against a chloroquine sensitive strain (3D7) and 0.45–12 mg/ml for chloroquine resistant strain (INDO) of P. falciparum (Table 2). It is worth noting that like the crude extract of P. hysterophorus, which showed a resistance index of 0.35, most chromatographic fractions derived from it were also found to be more effective against CQ resistant INDO strain than against CQ sensitive 3D7 strain giving resistance indices ranging 0.1–0.7. More interestingly fractionation led to significant improvement in selectivity indices (
10 to
111) compared to crude which exhibited a poor SI of 0.75. 3.4. Dose dependence and stage specificity of fractions of P. hysterophorus During the course of activity guided purification of metabolites in the crude extract of P. hysterophorus it was observed that (a) almost all its fractions exhibited promising antiplasmodial potency (Table 2), (b) there was 3-6 fold greater antiplasmodial potency in several fractions compared to the already promising antiplasmodial potency of its crude extract (IC50 6 mg/ml) and (c) like the crude extract, the potency of the fractions was around

three fold higher for the CQ resistant PfINDO strain than the CQ sensitive Pf3D7 strain. Fractionation was found to not only enhance antiplasmodial potency but it also led to significant increase in selectivity index from 0.75 (for crude extract) to more than 111 for some of its fractions. We chose to study fractions 14 and 19 for stage specificity of action since both these fractions scored high in antiplasmodial potency, resistance index, selectivity index and were associated with well resolved chromatographic peaks. Both fractions 14 and 19 showed progressive decline in % parasitemia as a function of increasing concentration from 0.78 mg/ml to 6.25 mg/ml (Fig. 3). Importantly it was observed that while the lower concentrations up to 1.56 mg/ml led to around 50% inhibition of growth, the higher concentration of 3.12 mg/ml led to only little more than the initial parasitemia with arrest of culture at the trophozoite stage. At 6.25 mg/ml the cultures showed the initial parasitemia of
1% with trophozoite as the predominant life cycle stage. The highest concentration of 12.5 mg/ml (IC100) led to complete inhibition of growth. In order to find if growth inhibited cultures were alive or dead, we washed the 14/19 48 h exposed cultures free of “drug” and incubated them in drug free medium for 24 h. Such drug withdrawal experiments with both 14 and 19 showed that the lower concentrations of 0.78–1.56 mg/ml were cytostatic and not cytocidal since following drug withdrawal, the predominantly ring stage cultures gave rise to predominantly trophozoite stage cultures suggesting viability. However concentrations of 3.12 mg/ml and higher of both 14 and 19 were cytocidal since there was no rise in parasitemia following incubation in drug free medium (Fig. 3). Since exposure of ring stage cultures to 14/19 led to arrest of culture at trophozoite stage, we wanted to confirm that the trophozoite stage was the true target for the action of fractions 14/19. In order to confirm this we exposed synchronized trophozoite stage cultures to the action of fractions 14/19 at a concentration of 3.12 mg/ml for 24 h, intercepted the culture by washing and extension of culture for 48 h in drug free medium. As shown in Fig. 4A and B, the cultures remained arrested in trophozoite stage and showed no increment in parasitemia. This provided strong proof of the cytocidal action of 14/19 on the trophozoite stage at drug concentrations which were not inhibitory to the transition of rings into trophozoites. Further to study the effect of

N. Singh et al. / Journal of Ethnopharmacology 165 (2015) 152–162

µg/ml

0h

48h(D+)

W

157

24h(D-) 14

% Parasitemiaat 48h of Ph-14 treatment

12

0

10

%P %R %T %S

8 % 6 0.78

4 2 0 0

1.56

0.78

1.56 3.12 µg/ml

6.25

12.5

Wash removal of drug % Parasitemiaat 72h of Ph-14 treatment

14

3.12

12 10 6.25

%P

% 8

%R

6

%T

4

%S

2 12.5

0 0

µg/ml

0h

48h(D+ )

W

0.78

1.56 3.12 µg/ml

6.25

12.5

24h(D-) % Parasitemiaat 48h of Ph-19 treatment

14 12

0

10

%P

8 0.78

%R

% 6

%T

4

%S

2 0 0

1.56

0.78

1.56 3.12 µg/ml

6.25

12.5

Wash removal of drug 3.12

14

% Parasitemiaat 72h of Ph-19 treatment

12

%P

10 6.25

%

%R

8 6

%T

4

%S

2 12.5

0 0

0.78

1.56 3.12 µg/ml

6.25

12.5

Fig. 3. Dose dependent effect of P. hysterophorus fractions 14 and 19 on the ex vivo growth of Pf3D7 in human red blood cell culture. Left panels: Micrographs of synchronized ring stage Pf3D7 cultures treated with different concentrations (0.78–12.5 mg/ml) of fraction 14 (left panel, A) and fraction 19 (left panel, B) for 48 h (D þ ) followed by removal of drug and incubation in drug free medium for further 24 h (D  ). W represents the transition from D þ to D states of the culture. Right panels: Distribution of P. falciparum life cycle stages in presence of fraction 14 (panel a, right top) and fraction 19 (panel b, right top) at 48 h of growth and distribution of stages following withdrawal of fraction 14 (panel a, right bottom) and fraction 19 (panel b, right bottom) after 24 h in drug free condition (D  ) growth. P total parasitemia, R % ring stage, T % trophozoite, S % Schizonts.

fractions 14 and 19 against egress and invasion, synchronized schizont stage cultures of the parasite were treated with 6.25 mg/ml of each for 12 h followed by removal of drug by washing and incubation in drug free medium for a period of an additional 24 h. It was interesting to see (Fig. 4C) that both 14 and 19 caused delay in egress since against the control where egress followed by invasion and formation of rings was evident at 24 h, the 14 and 19 treated smears showed what appeared to be recently egressed merozoites. More interesting was to see that the schizonts which egressed with a history of delayed egress

gave rise to merozoites that were invasion incompetent resulting in no increment in parasitemia. Thus the fractions 14/19 appeared to target all stages of the life cycle of blood stage malaria parasite.

4. Discussion Malaria is the most devastating parasitic disease affecting the entire developing world causing grave emotional and economic

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24h (D+)

W

48 h (D -)

0h

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Fig. 4. Stage specificity of antiplasmodial action of P. hysterophorus fractions 14 and 19. (A) Micrograph of synchronized trophozoite stage Pf3D7 cultures treated with 3.12 mg/ml (IC100) of fractions 14/19 for 24 h (Dþ ) followed by removal of drug by spin washing and incubation in drug free medium for an additional 24 h (D ). (B) Distribution of malaria parasite life cycle stages in fraction 14/19 treated trophozoite stage culture at 48 h (24 h Dþ and 24 h D ) of growth. (C) Micrograph of synchronized schizont stage Pf3D7 cultures treated with 6.25 mg/ml (2  IC100) with fractions 14/19 for 12 h (Dþ ) followed by removal of drug and incubation in drug free medium for an additional 24 h (D ). (D) Distribution of malaria parasite life cycle stages in fraction 14/19 treated schizont stage culture at 36 h (12 h Dþ and 24 h D ) of growth. P total parasitemia, R % ring stage, T % trophozoite, S % Schizonts.

turmoil and retarding the progress of nations. The scenario is grim since we have neither a credible vaccine against Malaria nor a drug that is not yet tamed through drug resistance by the clever bug that the malaria parasite is. To overcome the problem of resistance against the available antimalarials, a traditional healthcare system can be looked upon as an important source of developing lead molecules for better efficacy and minimum toxicity. In this context, an ethnobotanical survey was carried out in and around Ranchi followed by identification and collection of 22 different plant species (Supporting information Table 1) which have continued to be in traditional use against febrile diseases or are remedies for other ailments. As shown in the present study, the ex vivo red blood cell culture based antiplasmodial screening of these plant extracts has revealed a spectrum of antiplasmodial activities (Table 1). It was interesting to observe for the first time antiplasmodial activity (IC50: 23.5 mg/ml) in the leaf ethanolic extract of A. procera (Roxb.) Benth. A. procera, a tall deciduous tree belonging to Leguminosae is a potential source of natural antioxidants and can be used to prevent diseases associated with free radicals. Phytochemical screening has revealed the presence of saponins, steroids, tannins, glycosides and flavonoids in the extracts (Khatoon et al., 2013). A paste of its leaves is given by the local healers for intestinal worms. In contrast to leaf extract, we found the bark extract of A. procera to be hemolytic which may be because of the presence saponins (Yoshikawa et al., 1998). The finding of potential antiplasmodial activity in ethanol extracts of leaf (IC50 20.5 mg/ml) and bark (IC50 28 mg/ml) of A. scholaris (L.) R. Br (Table 1) was similarly interesting. Since antiquity, A. scholaris commonly known as devil's tree has been used for the treatment of many human ailments. Literature suggests that A. scholaris is useful in treating malaria, abdominal disorders, dyspepsia, leprosy, skin diseases, tumors, chronic and foul ulcers, asthma, bronchitis, helminthiasis, agalactia, and debility. Preclinical studies have shown that it possesses anti-microbial, anti-diarrhoeal, anti-plasmodial, anti-oxidant, anti-inflammatory hepatoprotective, nootropic, anti-stress, antifertility, immunomodulatory, analgesic, anti-ulcer, wound healing,

anti-cancer, chemopreventive, radiation protection, radiation sensitization, and chemosensitization activities. The diverse pharmacological activities associated with A. scholaris are supposed to be due to the presence of alkaloids, flavonoids and phenolic acids (Baliga, 2012; Shankar et al., 2012). A. squamosa, L. (Annonaceae) 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 (Bermejo et al., 2005; Liu et al., 1999; Zeng et al., 1996) that are known to exhibit in vitro antimalarial activities (Rakotomanga et al., 2004). We have earlier reported moderate antiplasmodial activity (IC50
30 mg/ml) in the ethyl acetate and methanol extracts of the leaves of A. squamosa (Asq) (Bagavan et al., 2011). Similarly, the methanolic extracts of the leaves of have been reported to show high to moderate antiplasmodial activity against the chloroquinesensitive strain 3D7 (IC50: 2 mg/ml) and chloroquine resistantstrain Dd2 (IC50: 30 mg/ml) of P. falciparum (El Tahir et al., 1999). In the present study we have observed high antiplasmodial activity in ethanolic extract of Asq against both CQ sensitive 3D7 (IC50: 2.1 mg/ml) and CQ resistant INDO (IC50: 3.3 mg/ml) strains of P. falciparum with resistance index of 1.57 (Table 1). A. indica A.Juss. (Meliaceae) is used popularly among the traditional people as a topical medicine to treat chickenpox. A. indica (Ai) is as a source of pesticide and insecticide because of the presence of bioactive component azadirachtin (Koriem, 2013). Ai is renowned to have various medicinal uses, such as a contraceptive for intravaginal use, for treatment of vaginal infections, treatment of gastric ulcers, cardiovascular disease, malaria, rheumatism and skin disorders, external applications for treatment of septic wounds, ulcers and boils, treatment of allergic skin reactions, asthma, bruises, colic, conjunctivitis, dysmenorrhea, fever, gout, headache, itching due to varicella, kidney stones, leukorrhoea, psoriasis, scabies, sprains, muscular pain, and wounds (Koriem, 2013). Besides, Ai is widely used as a mosquito repellant. Here we have found moderate antiplasmodial activity in

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leaf extract (IC50 32 mg/ml) and seed oil (IC50: 62 mg/ml) of Ai (Table 1). B. pinnatum (Lam.) Oken (Crassulaceae) is a perennial herb native to Madagascar and used in folkloric medicine in tropical Africa, tropical America, India, China, and Australia. In traditional medicine, the leaves of B. pinnatum (Bp) have been used for its antimicrobial and antifungal properties. In general, Bp has been reported to have various medicinal properties like anticancer (Supratman et al., 2001), antimicrobial (Akinsulire et al., 2007), anti-inflammatory and analgesic (Afzal et al., 2012), antineoplastic (Afzal et al., 2013), and as a tocolytic agent to prevent premature labor (Furer et al., 2013). Here we have observed good antiplasmodial activity in leaf ethanol extracts of Bp {IC50 14 mg/ml (Pf3D7) and 29 mg/ml (Pf INDO)} (Table 1). C. procera (Aiton) Dryand. belongs to Apocynaceae, the milkweed family. C. procera (Cp) continues to be in traditional use as purgative and medicine for intermittent fevers. The plant is versatile since it is known to possess diverse activities including analgesic, anti-arthritic, anti-asthmatic, antibacterial, anticonvulsant, antipyretic, contraceptive, antiulcer and wound healing (Kadiyala et al., 2013). Chemical analysis of the plant shows the presence of cardenolides like Calotropin. The in-vitro P. falciparum schizonticidal activity of the ethanolic extracts of different parts of Cp has been found to be moderate to poor (IC50 0.11–1.2 mg/ml) (Sharma and Sharma, 2000). However, in the present study we have observed that the ethanolic leaf extract of Cp has high antiplasmodial activity (IC50: 2.5 mg/ml) with low toxicity (TC50: 10 mg/ml). It is interesting to note that Calotropis gigantea another species of the same genus has also been reported to be effective against P. falciparum 3D7 strain (Wong et al., 2011). C. carvi L. also known as Caraway, meridian fennel, Shahi-Jeera etc. is a biennial plant of family Apiaceae, and is native to western Asia, Europe and Northern Africa. Fruits of C. carvi (Cc) are used in Indian families as part of spices in various cuisines. Traditional healers recommend it for treatment of inflammation, anorexia, febrile diseases etc. The oil of caraway derived from Cc fruits is known to possess insecticidal (Pitasawat et al., 2007), antiplasmodial (Fujisaki et al., 2012) antiobesity (Kazemipoor et al., 2013), antimicrobial (Gilani et al., 2013) and anticancer (Aydin et al., 2013) activities. In the present study, we have found that the ethanolic extract of Caraway seed possesses good antiplasmodial activity (IC50: 27 mg/ml (Pf3D7), 30 mg/ml (PfINDO) with resistance index of 1.1 (Table 1). D. sissoo DC (Leguminosae) possesses immense traditional applications. It is widely used as analgesic, anti-inflammatory, antipyretic, antimicrobial, anti-diarrheal, anti-ulcerogenic, spermicidal, larvicidal and mosquito repellant in the traditional system of medicines. Chemical investigation has resulted in isolation and characterization of various phytoconstituents like dalbergenone, dalbergin and methyl dalbergin and a new 4-phenyl chromene, dalbergichromene (Beldjoudi et al., 2003; Mukerjee et al., 1971). However heartwood flavonoids of Dalbergia louvelii species of genus Dalbergia are found to have antiplasmodial activity (IC50 5.8–8.7 mM) (Beldjoudi et al., 2003). Here it is interesting that we have found for the first time that ethanolic root extract of D. sissoo (Ds) possesses good antiplasmodial activity {IC50 20 mg/ml (Pf3D7),14.5 mg/ml (PfINDO)} with resistance index of 0.73. In contrast the leaf ethanolic extract of Ds was found to be inactive (IC50: 4100 mg/ml) (Table 1). C. citriodora (Hook.) K.D.Hill & L.A.S.Johnson (synonym Eucalyptus citriodora) also called as lemon eucalyptus belongs to family Myrtaceae. Its lemon-scented oil has insecticidal, antifungal, antiseptic and anti-inflammatory properties. Chemical analysis of the plant shows the anti-inflammatory Eucalyptol/Cineole to be the major constituent (Gbenou et al., 2013). Cineole is an effective treatment for rhinosinusitis (Fischer and Dethlefsen, 2013). C. citriodora (Cc) leaf extract is also known to have antibacterial and anti-cancerous properties (Bhagat et

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al., 2012; Rakover et al., 2008). In the present study we have for the first time observed promising antiplasmodial activity {IC50 5 mg/ml (Pf 3D7)} in ethanolic extract of the leaves of Cc. E. prostrata (L.) L. (synonym Eclipta alba) is a small branched creeping and moisture-loving perennial herb, which has been used as a traditional medicine in different countries mainly in tropical and subtropical regions of the world. E. prostrate (Ep), popularly known as “Bhringaraj” in India plays a significant role in the Ayurvedic, and Unani systems of medicine. Well known for its medicinal value, it has been used for its analgesic, antimytotoxic, antihepatotoxic, antibacterial, antioxidant, antihaemorrhagic, antihyperglycemic, and immunomodulatory properties. Broad range of chemical constituents which have been isolated from E. alba include coumestans, alkaloids, thiopenes, flavonoids, polyacetylenes, triterpenes and their glycosides (Sidra et al., 2013). It has been reported that 750 mg/kg/day of methanol extract of E. alba increased the survival time of P. berghei ANKA infected mice (Bapna et al., 2007). In the present study we have observed that ethanol extract of Ep inhibits the growth of P. falciparum {IC50 23 mg/ml(Pf 3D7)} (Table 1) indicating direct action on the parasite. H. pubescens Wall. ex G.Don (synonym Holarrhena antidysenterica) (Apocynaceae) is well known traditionally for curing dysentery and diarrhea. Chemical analysis of H. pubescens (Hp) shows mostly a rich abundance of alkaloids. The principle alkaloid is Conessine, having structural similarity to quinine and quinidine. It is a good anesthetic and antimuscarinic agent. The whole plant chloroform extract of H. antidysenterica has been found to show in vitro antiplasmodial activity (IC50: 5.5 μg/ml) (Verma et al., 2011). In the present study we have found that the ethanolic extract of Hp bark is slightly more potent {IC50 4.5 μg/ml (Pf 3D7)}than the ethanolic extract of Hp leaves {IC50 7 μg/ml(Pf 3D7)}. However, the potency order was reversed in the case of Cq-resistant Pf INDO strain where the leaves ethanol extract (IC50 8.5 μg/ml) was more potent than the bark ethanol extract (IC50 11 μg/ml) (Table 1). M. longifolia var. latifolia (Roxb.) A.Chev. (synonym Madhuca indica) (Mahua) (Sapotaceae) is a forest based tree that is virtually the lifeline of the tribal belt in Central India. M. longifolia (Ml) is culturally the most identified with Indian life in the plains. Besides its tasty fruits, its flowers are also relished for their sweet and delicious taste. But the tree wins in fame due to the liquor distilled from the flowers, which is also used to make vinegar. The cake of Ml is insecticidal and is also used as bait for fishing. This plant is also endowed with antifungal (Sidhu et al., 2009) and antioxidant (Chaudhary et al., 2012) activity. It was interesting for us to observe antiplasmodial activity in bark, leaf and seed of Ml. The bark was found to be most potent {IC50 24.1 μg/ml (Pf 3D7)} followed by leaves {IC50 55 μg/ml (Pf 3D7)} and seeds {(IC50 92 μg/ml (Pf 3D7)}. Seed oil was found not active up to 100 μg/ml (Table 1). M. pudica L. (Leguminosae) commonly called as touch-me-not or Chuimui is used traditionally as antidote for scorpion sting and as an antidiabetic. According to the Unani system of medicine, M. pudica (Mp) root is useful in diseases arising from blood impurities and bile, bilious fevers, piles, jaundice, leprosy etc. (Azmi et al., 2011). Laboratory experiments have demonstrated that Mp has antimicrobial (Ojala et al., 2000), antimyotoxic (Mahanta and Mukherjee, 2001), wound healing, anti-asthmatic (Yang et al., 2011), anticancer (Hullatti et al., 2013) and anti-mumps (Malayan et al., 2013) activities. It is interesting that in the present study we have found Mp to have moderate antiplasmodial activity (IC50: 51 mg/ml) (Table 1). P. pinnata (L.) Pierre (Leguminosae) is a handsome flowering tree with foliage like the Beech. Sanskrit writers call it Karanja and Naktamāla or Naktamālaka, “garland of the night”. In traditional medicine P. pinnata (Pp) has proven useful for skin diseases, ulcers and bleeding wounds. Flavonoids isolated from Pp have exhibited moderate anti-leishmanial activity (IC50: 32 mM) (Kapingu et al., 2006). Ethanolic extract of Pp has also been reported to have

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moderate antiplasmodial activity against CQ sensitive Pf3D7 (IC50: 24–40 mg/ml) (Simonsen et al., 2001). In the present study we have found that the leaf ethanolic extract of Pp is nearly equipotent against both CQ sensitive 3D7 and CQ resistant INDO strain (IC50s: 22.8 and 20.4 mg/ml, respectively). In contrast, the ethanolic bark extract is more potent against the sensitive than the resistant strain (IC50s: 9.5 and 22.5 mg/ml respectively) (Table 1). P. hysterophorus L. is a wild growing weed belonging to family Compositae and is a common invasive species in India. It is looked down upon since it is known to cause respiratory and allergic problems (Bhatia et al., 2014). However, it is also rich in many biological activities which include anti-cancer, antioxidant and antiHIV activities (Kumar et al., 2013). Hydroethanolic (80% ethanol) extract of P. hysterophorus (Ph) has been reported to have weak antiplasmodial activity (IC50: 45.2 mg/ml) (Valdes et al., 2010). However, in the present study, we have found that the whole plant ethanol extract of Ph has promising antiplasmodial activity against both CQ-sensitive {IC50 6 μg/ml (Pf 3D7)} and resistant strain {IC50 2.1 μg/ml (Pf INDO)}, with resistance index of 0.35 (Table 1). In view of its impressive antiplasmodial potency, we subjected the ethanolic extract of Ph to activity guided purification using RPHPLC. A well resolved chromatogram (Fig. 2) together with assessment of antiplasmodial activity of fractions helped us to identify the fractions with promising antiplasmodial activity and low toxicity to two mammalian cell lines (Table 2). Evaluation for stage specificity of action by two of the most potent fractions viz. 14 and 19 revealed that they were inhibitory to the parasite growth in a dose dependent stage specific manner. Thus while 3.125 mg/ml was sufficient to inhibit the growth of trophozoites, the growth inhibition of rings was seen only at 6.25 mg/ml (Figs. 3 and 4A and B). Furthermore schizonts treated with 6.25 mg/ml of fractions 14/19 showed delayed egress resulting in merozoites that were invasion incompetent (Fig. 4C and D). Finally it was interesting to observe in drug exposure followed by drug withdrawal experiments that 14/19 killed all stages of the malaria parasite (Figs. 3 and 4). The molecule in the dominant peak representing fraction 14 is likely to be amphipathic since its RPHPLC retention time is approximately midway between the retention times expected of very polar versus very non polar molecules. Further its 20 fold greater absorbance at 214 nm over the absorbance at 254 nm suggests that the molecule may be aliphatic in nature. Indeed chemical analysis of Ph shows Sesquiterpene lactones to be the major chemical constituent in all parts including pollens and trichomes (Picman et al., 1979). Some of the other chemical constituents found in Ph and reported to have antiprotozoan, antibacterial and antitumor properties are Parthenin/ Hymenin, Coronopilin, Ambrosin, Tetraneurin A and Charminarone. Since a large number of fractions of Ph have been found to exhibit promising antiplasmodial activity (Table 2), it is quite likely that the fractions may have some of the compounds listed above (Pandey, 2009). S. indica L (Ashoka) is one of the most legendary and sacred trees of India. It is an evergreen tree used extensively as a tonic by local people, as a uterine sedative and for women related problems like menorrhagia (scant menses), dysmenorrhea (painful menses, menstrual cramps), and depression, bleeding hemorrhoids and uterine fibroids (Pradhan et al., 2009). Experimentally S. indica (Si) has been found to possess antimutagenic and genoprotective properties (Nag et al., 2013). It has also been reported to possess antiprogestational and antioestrogenic activity against menorrhagia (Sharma et al., 2009). In the present study on the antiplasmodial activity of the ethanolic extracts of Si against Pf 3D7, we have found the leaf extract to be more potent (IC50 21 μg/ml) than the bark extract (IC50 38 μg/ml) (Table 1). C. roseus (L.) G.Don (synonym Vinca rosea) (Apocynaceae) is also known as Nayantara and is widely used by the traditional healers as cure of many ailments. While the hot water extract of dried entire

plant is taken orally by human adults for cancer and menorrhagia, the hot water extract of dried leaves is taken orally for Hodgkin's disease. Pulp of nodes of C. roseus (Cr) mixed with cow dung is used externally for cuts and wounds. The presence of antitumor activity in the leaf extracts has been attributed to the two alkaloids Vincristine and Vinblastine (Gupta et al., 2005; Mangeney et al., 1979). Leaves methanol extract of Cr has been reported to be not active against Plasmodium up to a concentration of 100 mg/ml (Kamaraj et al., 2012). However, in the present study we have found that the ethanolic extract of whole plant possesses good antiplasmodial activity (IC50 16 mg/ml) against chloroquine sensitive strain Pf3D7. Z. jujuba Mill. (synonym Zizyphus mauritiana) (also known as Ber) belongs to Rhamnaceace. Phytochemical investigations have established the genus Ziziphus to be a rich source of cyclopeptide alkaloids (El-Seedi et al., 2007). Cyclopeptide macrocycles of Ziziphus species showed interesting biological properties, including sedative (Han et al., 1989), analgesic (Trevisan et al., 2009), antibacterial (Morel et al., 2002), antifungal (Pandey and Devi, 1990) and antiplasmodial (Suksamrarn et al., 2006) activities. Cyclopeptide alkaloids isolated from the root of Z. mauritiana have been found to exhibit potent antiplasmodial activity (IC50s: 3.7– 10.3 μM). Here, we tested the antiplasmodial activity of ethanolic extracts of Z. jujuba leaves and bark and found that while the leaves extract is inactive (IC50 Pf 3D7: 4100 μg/ml), the bark extract is active {IC50: 25 μg/ml (Pf 3D7), 16 μg/ml(Pf INDO)}. In summary, our study shows that the ethanolic extracts of some of the plants studied exhibit good ex vivo antiplasmodial activity against CQ-resistant and sensitive strains of Pf. Since many of these plants are in consumption by way of food/drinks in many parts of India, they might as nutraceuticals assist in keeping malaria under control. Detailed evaluation may result in generation of therapeutic formulations for malaria or may provide potential molecules for malaria therapy.

Acknowledgments We thank MR4 who generously provided the chloroquineresistant INDO strains used in the study. Thanks to X Su who deposited these strains with MR4, BEI Resources Repository, NIAID, NIH. NS thanks the Summer Research Fellowship programme jointly sponsored by IASC (Bangalore), INSA (New Delhi) and NAS (Allahabad). NKK (45/82/2009-PHA/BMS) and DM (45/90/2011PHA/BMS) thank Indian Council for Medical Research (ICMR), New Delhi, for Senior Research fellowship. We thank the ICGEB, New Delhi for internal funding. We thank Dr Shailendra Kumar Singh, AGM, Environmental Engineering Section, Mecon, Ranchi who has been a source of inspiration for this work.

Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.jep.2015.02.038. References Afzal, M., Gupta, G., Kazmi, I., Rahman, M., Afzal, O., Alam, J., Hakeem, K.R., Pravez, M., Gupta, R., Anwar, F., 2012. Anti-inflammatory and analgesic potential of a novel steroidal derivative from Bryophyllum pinnatum. Fitoterapia 83, 853–858. Afzal, M., Kazmi, I., Anwar, F., 2013. Antineoplastic potential of Bryophyllum pinnatum lam. on chemically induced hepatocarcinogenesis in rats. Pharmacognosy Research 5, 247–253. Akinsulire, O.R., Aibinu, I.E., Adenipekun, T., Adelowotan, T., Odugbemi, T., 2007. In vitro antimicrobial activity of crude extracts from plants Bryophyllum pinnatum and Kalanchoe crenata. African Journal of Traditional, Complementary, and Alternative Medicines: AJTCAM/African Networks on Ethnomedicines 4, 338–344.

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Aydin, E., Turkez, H., Keles, M.S., 2013. Potential anticancer activity of carvone in N2a neuroblastoma cell line. Toxicology and Industrial Health, http://dx.doi. org/10.1177/0748233713484660. Azmi, L., Singh, M.K., Akhtar, A.K., 2011. Pharmacological and biological overview on Mimosa pudica Linn. International Journal of Pharmacy & Life Sciences 2, 1226–1234. Bagavan, A., Rahuman, A.A., Kaushik, N.K., Sahal, D., 2011. In vitro antimalarial activity of medicinal plant extracts against Plasmodium falciparum. Parasitology Research 108, 15–22. Baliga, M.S., 2012. Review of the phytochemical, pharmacological and toxicological properties of Alstonia scholaris Linn. R. Br (Saptaparna). Chinese Journal of Integrative Medicine, http://dx.doi.org/10.1007/s11655-011-0947-0. Bapna, S., Adsule, S., Shirshat Mahendra, S., Jadhav, S., Patil, L.S., Deshmukh, R.A., 2007. Anti-malarial activity of Eclipta alba against Plasmodium berghei infection in mice. The Journal of Communicable Diseases 39, 91–94. Beldjoudi, N., Mambu, L., Labaïed, M., Grellier, P., Ramanitrahasimbola, D., Rasoanaivo, P., Martin, M.T., Frappier, F., 2003. Flavonoids from Dalbergia louvelii and their antiplasmodial activity. Journal of Natural Products 66, 1447–1450. Bermejo, A., Figadere, B., Zafra-Polo, M.C., Barrachina, I., Estornell, E., Cortes, D., 2005. Acetogenins from Annonaceae: recent progress in isolation, synthesis and mechanisms of action. Natural Product Reports 22, 269–303. Bhagat, M., Sharma, V., Saxena, A.K., 2012. Anti-proliferative effect of leaf extracts of Eucalyptus citriodora against human cancer cells in vitro and in vivo. Indian Journal of Biochemistry & Biophysics 49, 451–457. Bhatia, H., Manhas, R.K., Kumar, K., Magotra, R., 2014. Traditional knowledge on poisonous plants of Udhampur district of Jammu and Kashmir, India. Journal of Ethnopharmacology 152, 207–216. Chaudhary, A., Bhandari, A., Pandurangan, A., 2012. Antioxidant potential and total phenolic content of methanolic bark extract of Madhuca indica (koenig) Gmelin. Ancient Science of Life 31, 132–136. El-Seedi, H.R., Zahra, M.H., Goransson, U., Verpoorte, R., 2007. Cyclopeptide alkaloids. Phytochemistry Reviews 6, 143–165. El Tahir, A., Satti, G.M., Khalid, S.A., 1999. Antiplasmodial activity of selected Sudanese medicinal plants with emphasis on Maytenus senegalensis (Lam.) Exell. Journal of Ethnopharmacology 64, 227–233. Fischer, J., Dethlefsen, U., 2013. Efficacy of cineole in patients suffering from acute bronchitis: a placebo-controlled double-blind trial. Cough 9, 25. Fujisaki, R., Kamei, K., Yamamura, M., Nishiya, H., Inouye, S., Takahashi, M., Abe, S., 2012. In vitro and in vivo anti-plasmodial activity of essential oils, including hinokitiol. The Southeast Asian Journal of Tropical Medicine And Public Health 43, 270–279. Furer, K., Raith, M., Brenneisen, R., Mennet, M., Simoes-Wust, A.P., von Mandach, U., Hamburger, M., Potterat, O., 2013. Two new flavonol glycosides and a metabolite profile of Bryophyllum pinnatum, a phytotherapeutic used in obstetrics and gynaecology. Planta Medica 79, 1565–1571. Gbenou, J.D., Ahounou, J.F., Akakpo, H.B., Laleye, A., Yayi, E., Gbaguidi, F., BabaMoussa, L., Darboux, R., Dansou, P., Moudachirou, M., 2013. Phytochemical composition of Cymbopogon citratus and Eucalyptus citriodora essential oils and their anti-inflammatory and analgesic properties on Wistar rats. Molecular Biology Reports 40, 1127–1134. Gilani, S.R., Mahmood, Z., Hussain, M., 2013. Preliminary evaluation of antimicrobial activity of cream formulated with essential oil of Trachyuspermum ammi. Pakistan Journal of Pharmaceutical Sciences 26, 893–896. Gupta, M.M., Singh, D.V., Tripathi, A.K., Pandey, R., Verma, R.K., Singh, S., Shasany, A.K., Khanuja, S.P., 2005. Simultaneous determination of vincristine, vinblastine, catharanthine, and vindoline in leaves of Catharanthus roseus by highperformance liquid chromatography. Journal of Chromatographic Science 43, 450–453. Han, B., Park, M.H., Park, J.H., 1989. Chemical and pharmacological studies on sedative cyclopeptide alkaloids in some Rhamnaceae plants. Pure and Applied Chemistry 61, 443–448. Hullatti, K., Pathade, N., Mandavkar, Y., Godavarthi, A., Biradi, M., 2013. Bioactivityguided isolation of cytotoxic constituents from three medicinal plants. Pharmaceutical Biology 51, 601–606. Kadiyala, M., Ponnusankar, S., Elango, K., 2013. Calotropis gigantiea (L.) R. Br (Apocynaceae): a phytochemical and pharmacological review. Journal of Ethnopharmacology 150, 32–50. Kamaraj, C., Kaushik, N.K., Mohanakrishnan, D., Elango, G., Bagavan, A., Zahir, A.A., Rahuman, A.A., Sahal, D., 2012. Antiplasmodial potential of medicinal plant extracts from Malaiyur and Javadhu hills of South India. Parasitology Research 111, 703–715. Kapingu, M.C., Mbwambo, Z.H., Moshi, M.J., Magadula, J.J., Cos, P., Berghe, D.V., Maes, L., Theunis, M., Apers, S., Pieters, L., Vlietinck, A., 2006. A novel isoflavonoid from Millettia puguensis. Planta Medica 72, 1341–1343. Kazemipoor, M., Radzi, C.W., Hajifaraji, M., Haerian, B.S., Mosaddegh, M.H., Cordell, G.A., 2013. Antiobesity effect of caraway extract on overweight and obese women: a randomized, triple-blind, placebo-controlled clinical trial. Evidence-Based Complementary and Alternative Medicine 2013, 928582. Khatoon, M., Islam, E., Islam, R., Rahman, A.A., Alam, A.H., Khondkar, P., Rashid, M., Parvin, S., 2013. Estimation of total phenol and in vitro antioxidant activity of Albizia procera leaves. BMC Research Notes 6, 121. Koriem, K.M., 2013. Review on pharmacological and toxicologyical effects of oleum azadirachti oil. Asian Pacific Journal of Tropical Biomedicine 3, 834–840.

161

Kumar, S., Chashoo, G., Saxena, A.K., Pandey, A.K., 2013. Parthenium hysterophorus: a probable source of anticancer, antioxidant and anti-HIV agents. BioMed Research International 2013, 810734. Liu, X.X., Alali, F.Q., Pilarinou, E., McLaughlin, J.L., 1999. Two bioactive monotetrahydrofuran acetogenins, annoglacins A and B, from Annona glabra. Phytochemistry 50, 815–821. Lombardino, J.G., Lowe, J.A., 2004. The role of the medicinal chemist in drug discovery—then and now. Nature Reviews Drug Discovery 3, 853–862. Ma, C., Harrison, P., Wang, L., Coppel, R.L., 2010. Automated estimation of parasitaemia of Plasmodium yoelii-infected mice by digital image analysis of Giemsa-stained thin blood smears. Malaria Journal 9, 348. Mahanta, M., Mukherjee, A.K., 2001. Neutralisation of lethality, myotoxicity and toxic enzymes of Naja kaouthia venom by Mimosa pudica root extracts. Journal of Ethnopharmacology 75, 55–60. Malayan, J., Selvaraj, B., Warrier, A., Shanmugam, S., Mathayan, M., Menon, T., 2013. Anti-mumps virus activity by extracts of Mimosa pudica, a unique Indian medicinal plant. Indian Journal of Virology: An Official Organ of Indian Virological Society 24, 166–173. Mangeney, P., Zo Andriamialisoa, R., Langlois, N., Langlois, Y., Potier, P., 1979. Preparation of vinblastine, vincristine, and leurosidine, antitumor alkaloids from Catharanthus species (Apocynaceae). Journal of the American Chemical Society 101, 2243–2245. Morel, A.F., Araujo, C.A., da Silva, U.F., Hoelzel, S.C., Zachia, R., Bastos, N.R., 2002. Antibacterial cyclopeptide alkaloids from the bark of Condalia buxifolia. Phytochemistry 61, 561–566. Mosmann, T., 1983. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. Journal of Immunological Methods 65, 55–63. Mukerjee, S., Saroja, T., Seshadri, T., 1971. Dalbergichromene: a new neoflavonoid from stem-bark and heartwood of Dalbergia sissoo. Tetrahedron 27, 799–803. Nag, D., Ghosh, M., Mukherjee, A., 2013. Antimutagenic and genoprotective effects of Saraca asoca bark extract, Toxicology and Industrial Health, http://dx.doi.org/ 10.1177/0748233713483200. Ojala, T., Remes, S., Haansuu, P., Vuorela, H., Hiltunen, R., Haahtela, K., Vuorela, P., 2000. Antimicrobial activity of some coumarin containing herbal plants growing in Finland. Journal of Ethnopharmacology 73, 299–305. Pandey, D., 2009. Allelochemicals in Parthenium in response to biological activity and the environment. Indian Journal of Weed Science 41, 111–123. Pandey, V., Devi, S., 1990. Biologically active cyclopeptide alkaloids from Rhamnaceae plants. Planta Medica 56, 649–650. Paria, N., Chattopadhyay, S.P., 2000. Flora of Hazaribagh District, Bihar. volume 1. Botanical Survey of India, Kolkata. Picman, A.K., Rodriguez, E., Towers, G.H., 1979. Formation of adducts of parthenin and related sesquiterpene lactones with cysteine and glutathione. ChemicoBiological Interactions 28, 83–89. Pitasawat, B., Champakaew, D., Choochote, W., Jitpakdi, A., Chaithong, U., Kanjanapothi, D., Rattanachanpichai, E., Tippawangkosol, P., Riyong, D., Tuetun, B., Chaiyasit, D., 2007. Aromatic plant-derived essential oil: an alternative larvicide for mosquito control. Fitoterapia 78, 205–210. Pradhan, P., Joseph, L., Gupta, V., Chulet, R., Arya, H., Verma, R., Bajpai, A., 2009. Saraca asoca (Ashoka): a review. Journal of Chemical and Pharmaceutical Research 1, 62–71. Prasad, U.N., 1988. Survey Report on Availability and Resources of Medicinal Plants in the Tribal and other Areas of North & South Chhotanagpur Divisions As well as Tribal Area of Dumka & Other Areas of Gaya, Aurangabad, Nawadah & Munghyr District, Divisional Forest Officer (DFO), Forest Resources Survey Division, Bihar. Published by Forest Department of Bihar. Rakotomanga, M., Razakantoanina, V., Raynaud, S., Loiseau, P.M., Hocquemiller, R., Jaureguiberry, G., 2004. Antiplasmodial activity of acetogenins and inhibitory effect on Plasmodium falciparum adenylate translocase. Journal of Chemotherapy 16, 350–356. Rakover, Y., Ben-Arye, E., Goldstein, L., 2008. The treatment of respiratory ailments with essential oils of some aromatic medicinal plants. Harefuah 147 (783–788), 838. Shah, N.K., Dhillon, G.P., Dash, A.P., Arora, U., Meshnick, S.R., Valecha, N., 2011. Antimalarial drug resistance of Plasmodium falciparum in India: changes over time and space. The Lancet. Infectious Diseases 11, 57–64. Shankar, R., Deb, S., Sharma, B.K., 2012. Antimalarial plants of northeast India: an overview. Journal of Ayurveda and Integrative Medicine 3, 10–16. Sharma, P., Sharma, J.D., 2000. In-vitro schizonticidal screening of Calotropis procera. Fitoterapia 71, 77–79. Sharma, P.K., Ramanchandran, R., Hutin, Y.J., Sharma, R., Gupte, M.D., 2009. A malaria outbreak in Naxalbari, Darjeeling district, West Bengal, India, 2005: weaknesses in disease control, important risk factors. Malaria Journal 8, 288. Sidhu, O.P., Chandra, H., Behl, H.M., 2009. Occurrence of aflatoxins in mahua (Madhuca indica Gmel.) seeds: synergistic effect of plant extracts on inhibition of Aspergillus flavus growth and aflatoxin production. Food and Chemical Toxicology: An International Journal Published for the British Industrial Biological Research Association 47, 774–777. Sidra, S., Hussain, S., Malik, F., 2013. Accentuating the prodigious significance of Eclipta alba – an inestimable medicinal plant. Pakistan Journal of Pharmaceutical Sciences 26, 1259–1266. Simonsen, H.T., Nordskjold, J.B., Smitt, U.W., Nyman, U., Palpu, P., Joshi, P., Varughese, G., 2001. In vitro screening of Indian medicinal plants for antiplasmodial activity. Journal of Ethnopharmacology 74, 195–204.

162

N. Singh et al. / Journal of Ethnopharmacology 165 (2015) 152–162

Singh, N., Mudgal, V., Khanna, K., Srivastava, S., Sahoo, A., Bandopadhyay, S., Aziz, N., Das, M., Bhattacharya, R., Hajra, P., 2001. Flora of Bihar: Analysis. Botanical Survey of India, Kolkata p. 777. Smilkstein, M., Sriwilaijaroen, N., Kelly, J.X., Wilairat, P., Riscoe, M., 2004. Simple and inexpensive fluorescence-based technique for high-throughput antimalarial drug screening. Antimicrobial Agents and Chemotherapy 48, 1803–1806. Suksamrarn, S., Panseeta, P., Kunchanawatta, S., Distaporn, T., Ruktasing, S., Suksamrarn, A., 2006. Ceanothane-and lupane-type triterpenes with antiplasmodial and antimycobacterial activities from Ziziphus cambodiana. Chemical and Pharmaceutical Bulletin 54, 535–537. Supratman, U., Fujita, T., Akiyama, K., Hayashi, H., Murakami, A., Sakai, H., Koshimizu, K., Ohigashi, H., 2001. Anti-tumor promoting activity of bufadienolides from Kalanchoe pinnata and K. daigremontiana x tubiflora. Bioscience, Biotechnology, and Biochemistry 65, 947–949. Trager, W., Jensen, J.B., 1976. Human malaria parasites in continuous culture. Science 193, 673–675. Trevisan, G., Maldaner, G., Velloso, N.A., Sant'Anna Gda, S., Ilha, V., Velho Gewehr Cde, C., Rubin, M.A., Morel, A.F., Ferreira, J., 2009. Antinociceptive effects of 14membered cyclopeptide alkaloids. Journal of Natural Products 72, 608–612.

Valdes, A.F., Martinez, J.M., Lizama, R.S., Gaiten, Y.G., Rodriguez, D.A., Payrol, J.A., 2010. In vitro antimalarial activity and cytotoxicity of some selected cuban medicinal plants. Revista do Instituto de Medicina Tropical de Sao Paulo 52, 197–201. Verma, G., Dua, V.K., Agarwal, D.D., Atul, P.K., 2011. Anti-malarial activity of Holarrhena antidysenterica and Viola canescens, plants traditionally used against malaria in the Garhwal region of north-west Himalaya. Malaria Journal 10, 20. Wong, S.K., Lim, Y.Y., Abdullah, N.R., Nordin, F.J., 2011. Assessment of antiproliferative and antiplasmodial activities of five selected Apocynaceae species. BMC Complementary and Alternative Medicine 11, 3. WHO 2014. Malaria: World Malaria Report 2014. Visiting date 12/02/2015. 〈http:// www.who.int/malaria/publications/world_malaria_report_2014/en/〉. Yang, E.J., Lee, J.S., Yun, C.Y., Ryang, Y.S., Kim, J.B., Kim, I.S., 2011. Suppression of ovalbumin-induced airway inflammatory responses in a mouse model of asthma by Mimosa pudica extract. Phytotherapy Research 25, 59–66. Yoshikawa, K., Satou, Y., Tokunaga, Y., Tanaka, M., Arihara, S., Nigam, S.K., 1998. Four acylated triterpenoid saponins from Albizia procera. Journal of Natural Products 61, 440–445. Zeng, L., Ye, Q., Oberlies, N.H., Shi, G., Gu, Z.M., He, K., McLaughlin, J.L., 1996. Recent advances in Annonaceous acetogenins. Natural Product Reports 13, 275–306.