Biological activity screening

Plant extracts were selected for the screening of antiplasmodial activity through two different strategies: 1) selection of extracts from plants used in traditional medicine for treating malaria or with a reported antiplasmodial activity in the literature and 2) a random selection of available plant extracts. In order to obtain further information on the biological activities of the extracts, all samples were tested againstP. falciparumand for theirin vitro cytotoxicity on human fibroblasts or mice splenocytes. Whenever possible, samples were also screened for activity against other parasites such asTrypanosoma cruzi,T. b. brucei, Leishmania infantum, and L. amazonensis. Even though the aim of this project was the search for antimalarial compounds, the investigation of activity against other parasites was also considered as part of a general investigation approach to detect whether an activity is specific or not. In the same way, the cytotoxicity assay results were used to discard samples which were potentially toxic in an unspecific way.

It is also important to highlight that the investigation of crude extracts in screening tests may lead to false-positive or false-negative results, due to the presence of interfering molecules, especially tannins. These are secondary metabolites widely distributed in the plant kingdom. The reactivity of such polyphenolic compounds to proteins has been associated with bioassay interference, particularly in assays involving enzymes (Wall et al., 1996). As a matter of fact, a pre-purification step is usually recommended to remove tannins from complex plant matrices. Some of the techniques used in this way are the precipitation with skin powder or lead acetate, and polyamide column chromatography (Hostettmann et al., 1998). The disadvantage of such techniques is that they are not specific and may completely remove other polyphenols together with tannins. The complete removal of polyphenols is controversial and some authors claim they may play an important role in specific activities (Phillipson et al., 1998). Accordingly, the extracts considered in the present work were not pre-purified for tannin removal before screening assays. As an alternative approach, major fractions of extracts were co-screened in order to increase chances of finding an activity by concentration of active principles and/or removal of interfering compounds.

A primary sample selection was based on the in-house availability of plant species from the family Asteraceae. The antiparasitic activity and toxicity of their extracts are presented in Table 3.1.

A secondary sample selection included three plant extracts and their fractions from a collaboration project between the University of Geneva (Switzerland) and the Federal University of Paraíba (João Pessoa, Brazil). The decoction of Argemone mexicanaL. was also considered for secondary screening based on ethnopharmacological data and its previously published clinical results (Willcox et al., 2007) in collaboration with Antenna Technologies (Geneva, Switzerland). The antiparasitic activity and toxicity of these samples are shown in Table 3.2.

Considering the results of the primary and the secondary screenings, the following species were selected for phytochemical investigation: Syzygium cumini (L.) Skeels (Myrtaceae), Licania octandra (Hoffmanns. ex Roem. & Schult.) Kuntze (Chrysobalanaceae) andArgemone mexicanaL. (Papaveraceae).

Table 3.1.Antiparasitic activity and cytotoxicity of primary selected MeOH extracts of plants from the family Asteraceae.

Activity

Plant information Anti- a P.falciparum Anti- a T.cruzi Anti- a L.amazonensis a,b Cytotoxicity

Species Organ Country %

Achillea erba-rottaAll. leaves Italy 5.36 NA NA 37.60

Artemisia borealisPall. leaves Italy NA NA NA 34.50

Blumea sessilifloraDecne. aerial parts Indonesia NA NA NA 2.80

Centaurea vallesiaca(DC.) Jord. aerial parts Switzerland NA NA NA 36.30

roots Switzerland NA NA NA 40.50

Eupatorium inulifoliumKunth aerial parts Indonesia NA NA NA 0.00

roots Indonesia NA NA NA 2.10

Hieracium alpinumL. aerial parts Italy NA NA NA 8.30

Melanthera aspera(Jacq.) Steud. ex Small leaves Panama NA NA NA 0.90

stems Panama NA NA NA 3.20

Mikania leiostachyaBenth. twigs Panama NA NA NA 18.50

Rolandra fruticosa(L.) Kuntze aerial parts Panama 18.39 NA NA 0.00

Spilanthes iabadicensisA.H. Moore aerial parts Indonesia NA NA NA 14.00

Vernonia cinerea(L.) Less. aerial parts Indonesia NA NA NA 7.10

Vernonia patensKunth leaves Panama 37.45 NA NA 5.70

flowers Panama 45.88 NA NA 0.30

stems Panama NA NA NA 6.20

NA = not active;^acf Experimental section (V.6.1) for experimental assay description;^btested on mouse splenocytes.

Table 3.2.Antiparasitic activity and cytotoxicity of secondary selected extracts and fractions of plants from different botanical families.

Activity

Plant information Anti- a,b P.falciparum Anti- a,c T.cruzi Anti- a,d T.b.brucei a,e Cytotoxicity

Species Family Organ Extract/

Fraction Country IC50(µg/ml)

Decoction >64.00 39.24 0.57 >64.00

AM 8_A >64.00 <0.25 >64.00 >64.00

AM 8_B >64.00 51.42 10.08 >64.00

AM 8_C >64.00 >64.00 1.32 >64.00

Argemone mexicanaL. Papaveraceae aerial parts

AM 8_D

Mali

1.71 0.25 0.25 24.98

EtOH 80% extract 29.84 >32.00 13.74 >32.00

HF 12.11 32.00 32.00 32.00

AcF 9.86 24.78 16.19 >32.00

BF 18.80 >32.00 13.26 >32.00

Licania octandra(Hoffmanns. ex

Roem. & Schult.) Kuntze Chrysobalanaceae leaves

AqF

Brazil

>32.00 >32.00 >32.00 >32.00

decoction 32.00 34.56 4.31 >64.00

CPC A >64.00 34.56 2.69 >64.00

CPC B 27.07 33.99 5.55 >64.00

CPC C <0.25 34.56 2.05 >64.00

Syzygium cumini(L.) Skeels Myrtaceae stem bark

CPC D

Brazil

19.80 7.50 1.86 26.44

acf Experimental section (IV.6.2) for experimental assay description;

bIC50>16 µg/ml: inactive; IC50between 2 and 16 µg/ml: moderately active; IC50<2 µg/ml: highly active;

cIC50>30 µg/ml: inactive; IC50between 2 and 30 µg/ml: moderately active; IC50<2 µg/ml: highly active;

dIC50>5 µg/ml: inactive; IC50between 1 and 5 µg/ml: moderately active; IC50<1 µg/ml: highly active;

etested on human fibroblasts (MRC-5 cell line); IC50>30 µg/ml: not toxic; IC50between 10 and 30 µg/ml: moderately toxic; IC50<10 µg/ml: highly toxic.