Antiplasmodial activity of Vernonia cinerea Less ( Asteraceae ) , a plant used in traditional medicine in Burkina Faso to treat malaria

Research and development of new antiplasmodial molecules in plant is a very important way for the development of new anti-malarial drugs. In this study, Vernonia cinerea Less (Asteraceae) was selected for its promising antiplasmodial activity because it is traditionally used in Burkina Faso to treat malaria. The aim of this study was to investigate the antiplasmodial activity of this whole plant. Five crude extracts of V. cinerea Less were prepared from the solvents of increasing polarity (CH2Cl2, CH3OH, CH3OH/H2O (1/1), H2O and alkaloids extracts). The method of Ciulei (1982) and thin layer chromatography were used for chemical characterization. The p-LDH technique was used in vitro. Extracts were evaluated in vitro for efficacy against the Plasmodium falciparum strain K1, which is resistant to chloroquine, and 3D7, which is sensitive to chloroquine. The crude extracts of alkaloids showed the IC50=4.25 μg/ml with the strains 3D7 and IC50=2.56 μg/ml with the K1 strains. The CH2Cl2 extracts showed the IC50= 8.42 μg/ml and IC50=5.85 μg/ml on strains 3D7 and K1, respectively. The CH3OH extracts showed the IC50=21.08 μg/ml, CH3OH/H2O extracts gave 41.56 μg/ml and H2O extracts gave 37.17 μg/ml on strains of P. falciparum K1. The present study highlighted the very promising antiplasmodial activity of V. cinerea Less. The most antiplasmodial activity of this plant extracts merit further study about its in vivo antiplasmodial activity in Plasmodium berghei infected mice.


INTRODUCTION
Malaria is a potential fatal parasitic disease that remains a public health concern in tropical countries, thus also in Burkina Faso.Globally, 198 million cases of malaria are registered per year, causing about 438 000 deaths (WHO, 2015).The burden is particularly heavy in Africa where 90% of all fatal cases occur, of which 78% occurs in children under the age of five (WHO, 2014).Efforts for malaria eradication have been made from 2000 to 2013 *Corresponding author.E-mail: somaaboubakar@yahoo.fr Tel: (+226) 20 97 01 02, (+226) 76129189.Fax: (+226) 20 97 04 57.
Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License (WHO, 2014), but this is hampered by the resistance of Plasmodium falciparum to antimalarial drugs available (Dondorp et al., 2012).The history of malaria has shown that plants are a source of new molecules.This is the case of quinine that has been isolated from Chincona and artemisinin that has been isolated from Artemisia annua (Batista et al., 2009;Bero and Joëlle, 2011;Bero et al., 2009;Kaur et al., 2009;Nogueira and Lopes, 2011;Phillipson and Wright, 1991).Over 80% of the world's population use medicinal plants for healing (WHO, 2008(WHO, , 2010)).In Burkina Faso, the majority of the population use medicinal plants as the first therapeutic means (Bero et al., 2009;Traoré et al., 2009) in order to contribute to find new antimalarial molecules having a wide margin of safety and efficiency.In traditional medicine, Vernonia cinerea Less (Asteraceae) has many therapeutic uses.Its vernacular name is "little ironweed (USA)" and the whole plant is used in therapeutic.It is used to treat malaria fever, vomiting, inflammation, infections, diuresis, cancer, abortion and gastrointestinal (Jain and Puri, 1984).The decoction is used to treat cardiac pathologies, wounds, colic and diarrhea (Rivière et al., 2005).In Burkina Faso, the plant is recommended in the care of malaria and for the care of dysentery and wounds in therapeutic use.The dosage used is usually a decoction of 50 g/l (Http://www.jardinsdumonde.org).In the face of resistance to artemisinin which is the core molecule of ACT, which extracted from A. annua (Asteracea), the priority is to research of new active molecules against emerging resistant strains.It was in this context that we chose V. cinerea Less which is a plant of the Asteracea family.The aim of the study was to evaluate the antimalarial activity of extracts from V. cinerea Less (Asteraceae) that is used in traditional medicine in Burkina Faso to treat malaria.

Plant collection
The plant was collected in the Comoé region (West Burkina Faso), GPS 10°38'N, 4°45'W in August 2010 and authenticated by a botanist M. Madou Ouedraogo from the Comoé Regional Forestry Department.After collection, a voucher specimen of plant was deposited in the herbarium of Centre National de Recherche et de Formation sur le Paludisme (CNRFP) in Burkina Faso (Ouagadougou).Then the plant was sprayed and the raw material obtained was sent to the laboratory of pharmacognosy for preparation of crude extracts.

Preparation of plant extracts
Five (5) types of extracts were prepared from the plant powder.We obtained three organic extracts, one aqueous with water and one crude alkaloid.Crude organic extracts were prepared by maceration for 16 h successively with dichloromethane (CH2Cl2), methanol (CH3OH) and water-methanol (CH3OH/H2O) solvents.Plant powder (10 g) was used for these organic extraction methods with 100 ml of each solvent.CH2Cl2 extract was air dried at room temperature.CH3OH and CH3OH/H2O extracts were freeze-dried with lyophilisator (Brand) after total evaporation of solvents.Aqueous extracts were prepared by boiling 10 g of plant powder in 100 ml of purified water for 30 min.After cooling, solutions were filtered on cotton wool and freeze-dried.Crude alkaloid extracts were obtained by alkalization with 28% NH4OH to pH9 of the plant powder and extraction with CH2Cl2 for 24 h.Plant powder (10 g) was used by applying the classical alkaloids extraction method (Sanon et al., 2003).After 16 h of maceration with ammoniac and CH2Cl2, a percolation was made with CH2Cl2 solvent.Then 100 ml of CH2Cl2 layer was concentrated under vacuum and then extracted with a 3% solution of H2SO4 to pH3.The aqueous acid solution was alkalinized again with NH4OH and extracted with CH2Cl2, and a crude alkaloid extracts was obtained by concentration.The yields were calculated using the following: (1)

Phytochemical screening
For characterizing the major chemical groups, non-hydrolyzed extracts and hydrolyzed extracts were prepared.1 g of each lyophilizate was weighed and mixed with 100 ml of distilled water then decanted into a bottle to get non-hydrolyzed extracts.To obtain hydrolyzed extracts, 25 ml of non-hydrolyzed extracts were removed and mixed with 15 ml of 10% HCl, this was heated under reflux for 30 min.After cooling, the mixture was transferred to a separating funnel.The liquid-liquid partition was done by the addition of 3×10 ml of dichloromethane.The organic phase was recovered and filtered and then stored in vials.
Phytochemical screening of plant extracts was made according to Ciulei method (1982).Chemical groups were identified by liquid medium characterization tests of the extract.Triterpenes and sterols were identified with Liebermann-Büchard test.Tannins presence has been highlighted by the reaction of FeCl3 1% test tubes.Saponins were identified with the observation of persistent foam column.Coumarins were detected with NH4OH 10%, UV (366 nm).Emodols and anthracenosids were identified with the Bornträger test.Carotenoids were detected with the H2SO4.

Chromatographic analysis thin layer (TLC)
Five microliters of each of the dichloromethane extracts, nonhydrolyzed extracts and hydrolyzed extracts were deposited on chromatography plates (silica gel G60, Merck).The plates were dried in ambient air and placed in migration vats covered before hand containing appropriate solvent systems.The distance covered of the eluent (solvent front) is predefined at 8 cm from the extracts of the deposition line.At the end of migration, the plates were removed and oven dried, and then the UV (254 or 366 nm) was read and after visualized with a reagent specific to the desired chemical groups.Alkaloids were identified with migration solvent toluene-ethyl acetate-diethylamine (17.5: 5: 2.5) and revealed with Draggendorf test.Triterpenes and sterols were identified with migration solvent n-hexane-ethyl acetate-toluene (6: 2: 4) and revealed with sulfuric acid 3% in ethanol.
Tannins has been highlighted by the solvent migration ethyl acetate-methanol-water (2: 1: 1) and revealed with aqueous solution of ferric chloride to 1%.Coumarins, emodols and anthracenosids were detected with migration solvent n-hexane-ethyl acetate-toluene to 6: 2: 2 and revealed with the KOH solution (1 N).

Strains of P. falciparum
The biological material used was strains of P. falciparum, the species responsible for the majority of malaria cases in Africa.Strains of P. falciparum resistant to chloroquine K1 and sensitive to chloroquine 3D7 were used.The K1 parasites were provided by the laboratory Warhust, London School of Hygiene and Tropical Medicine, London, England, United Kingdom (LSHTM).The 3D7 parasites were provided by the Laboratory Nuguchi Memorial Institute on Medical Research (NMIMR) (Ghana).They were maintained in continuous culture in human blood in the Laboratory of "Centre National de Recherche et de Formation sur le Paludisme" (CNRFP) in Burkina Faso (Ouagadougou).

Continuous culture of parasites in vitro by the method of Trager
The strains in continuous culture were maintained using the technique of Trager and Jensen (1976) and we renewed the culture medium every 24 h.Parasites were thawed and cultured in flasks containing complete culture medium composed of RPMI 1640, Lglutamine 2%, Stamps Hepes 2%, Gentamicin 0.5%, Albumax 5% and Hypoxanthine 0.5%.The flasks containing the culture were aerated with mixed gas composed of 2% O2, 5% CO2, and 93% N2.The flasks were then incubated in the CO2 incubator at 37°C under conditions for maximal growth.Parasitaemia was controlled by making blood smears after the renewal of the culture medium.When parasitaemia reached 6%, a subculture was made using fresh blood without the interference from blood group A+.

In vitro evaluation of antiplasmodial activity
The antiplasmodial activity of extract from V. cinerea Less was evaluated using the technique of Plasmodium Lactate Deshydrogenase (pLDH).Reference products (Dihydroartemisinin and Chloroquine) and extracts were dissolved in dimethylsulfoxide (DMSO) or in distilled water.The starting concentration of the extracts was 10 mg/ml that was further diluted to reach a final concentration of 100 µg/ml.The tests were performed on 96-well plates filled with a fixed volume of parasitized erythrocytes (2% parasitaemia).Samples were serially diluted with complete culture media (RPMI 1640 with albumax) to achieve the required concentration with DMSO concentration < 0.5%.Each extract was applied in a series of duplicate dilutions (final concentrations ranging from 0.78 to 50 μg/ml) on two rows.Dihydroartemisinin was used to validate the malaria test and chloroquine diphosphate salt (Sigma Aldrich) was used to validate the real chloroquine resistance of malaria strain K1.Infected and uninfected erythrocytes A+ were used as positive and negative controls, respectively.
After 72 h of incubation, the plates' counterpart's tests plates were prepared and the various substrates and coenzyme were then added.100 µl MALSTAT (160 ml distilled H2O, 200 μl Triton X100, 2 g of L-Lactate, 0.66 g Trizma base, 66 mg 3-acétylpiridine adenine di-nucleotide (APAD), at pH 9), 25 μl NBT/PES (100 ml of distilled water, 160 mg of NTB and PES 8 mg) and 20 μl of blood from the test plate was dispensed in each well including positive and negative controls.After 10 min of incubation, the plates were read on a spectrophotometer at a wavelength of 650 nm.Data were scored and analyzed using Microsoft Excel 2007.The mean optical density of negative controls was subtracted from that of each product to obtain the percentage of viability. (2) The 50% inhibitory concentrations (IC50) were calculated graphically with the Table Curve 2D v.5.0 software using the percentages of viability or cells proliferation versus log concentration.The IC50 of various extracts obtained were analyzed according to the criteria Deharo (Deharo et al., 2001); good activity IC50<= 5 µg/ml, moderate activity 5<IC50≤10 μg/ml, and inactive IC50>10 µg/ml.

RESULTS AND DISCUSSION
Five types of extracts were prepared.Phytochemical screening of V. cinerea Less revealed the presence of alkaloids, triterpenes and sterols, saponins, tannins, emodols, anthracenosids, coumarins, and carotenoids (Tables 1 and 2).The in vitro antiplasmodial activity of the crude extracts on strains reference K1 and 3D7 was assessed by using five crude extracts of plant prepared.Amongst the 5 extracts tested, alkaloids extracts were identified as having good antimalarial effects (IC 50 < 5 μg/ml), CH 2 Cl 2 with moderate effects (5 μg/ml ≤ IC 50 < 10 μg/ml), and CH 3 OH, CH 3 OH/H 2 O and H 2 O as inactive (IC 50 ≥ 10 μg/ml) (Table 3) according to Deharo et al. (2001).The best antimalarial effects were obtained with alkaloids extracts of plant (Figures 1 and 3).
The crude alkaloids extracts from the whole plant showed good antimalarial effects against the chloroquineresistant strain K1, with IC 50 values 2.56 μg/ml.The moderate antimalarial effects were obtained with dichloromethane extracts against K1, with IC 50 values 5.85 μg/ml (Figures 2 and 4).
In Cambodia, a similar study showed that dichloromethane extracts of V. cinerea had an IC 50 = 18.3 μg/ml with the W2 malaria strain chloroquine-resistant (Hout et al., 2006).Although, our results are different from those of Simonsen et al. (2001) in India on ethanol extracts (82 µg/ml) tested on 3D7.
Based on Deharo's efficiency criteria, results from Cambodia and India are the same as our findings.These differences may be related to many parameters, including the local environment and the collection periods, which contribute to the variation of the chemical components of the plant as shown in a previous study on seasonal effects on bioactive compounds (Aires et al., 2011).
In Burkina Faso, a previous study showed that alkaloids extracts of bark of Terminalia avicennoides had an IC 50 = 2.9 μg/ml with the K1 malaria strain (Sanon et al., 2013).Based on Deharo's efficiency criteria, results from this plant are a same from our findings.
The previous study was conducted with Dicotoma tomentosa (Asteracea) also collected in Burkina Faso, and in a different area.The antiplasmodial activity obtained with this plant (IC 50 = 1.9 ± 0.2 µg/ml) was different from our dichloromethane extracts based on Deharo's efficiency criteria (Jansen et al., 2012).
In summary, our study confirms the pharmacological % viability = OD product − OD negative control OD positive control X 100  (Iwalewa et al., 2003).Another study showed a good antiplasmodial activity of vernolide C and D molecules against W2 (Chea et al., 2006).This activity could be due to the presence of alkaloids (Bruneton, 1993) and triterpenes in the plant which mentioned by Chea et al. (2006).Alkaloids are one of the most important classes of natural products providing drugs since ancient times.The outstanding example is quinine from Cinchona succirubra (Rubiaceae) used for the treatment of malaria for more than three centuries (Kaur et al., 2009).Several plants of the Asteraceae family have been revealed as a good source for antimalarials.The most famous one is A. annua, the Chinese herb from which artemisinin (qinghaosu) was isolated (Liu et al., 1992).The good activity observed from the present investigation with V. cinerea Less which is a member of this family, thus supports the use of this  study have shown that V. cinerea Less has a very promising antiplasmodial activity.The CH 2 Cl 2 extracts and crude alkaloids allowed us to get good IC 50 on plasmodial strains.These results support the traditional use of this plant in traditional medicine for the treatment of malaria.Further studies will be needed, in particular in vivo tests on mice infected with P. berghei to assess antiplasmodial activity.

Figure 1 .
Figure 1.Curve of growth inhibition 3D7 of crude alkaloids.

Figure 3 .
Figure 3. Curve of growth inhibition K1 of crude alkaloids extracts.

Table 1 .
Summary of phytochemical screening of plant extracts was made according toCiulei method (1982).

Table 2 .
Chemical groups showed by chromatographic analysis thin layer.

Table 3 .
In vitro antiplasmodial activity of crude extracts obtained from Vernonia cinerea Less.