Antiprotozoal Activities of Millettia richardiana (Fabaceae) from Madagascar

With at least 60% of the Millettia species (Fabaceae) being in medicinal use, we found it relevant to assess the potential antiprotozoal and antifungal activities of Millettia richardiana. Water and methanol crude extracts of the stem barks from M. richardiana and the six fractions resulting from the fractionation of the methanol extract were tested. The dichloromethane extracted fraction showed the best in vitro antiprotozoal activities (IC50 = 5.8 μg/mL against Plasmodium falciparum, 11.8 μg/mL against Leishmania donovani and 12.8 μg/mL against Trypanosoma brucei brucei) as well as low cytotoxicity on several cell lines. The phytochemical analysis showed this selected fraction to be rich in terpenoids and alkaloids, which could explain its antiparasitic activity. A phytochemical study revealed the presence of lonchocarpenin, betulinic acid, β-amyrin, lupeol, palmitic acid, linoleic acid and stearic acid, among which betulinic acid and lupeol could be the compounds responsible of these antiprotozoal activities. By contrast, neither the crude extracts nor the fractions showed antifungal activity against Candida. These results confirm the importance of the genus Millettia in Malagasy ethnomedicine, its potential use in antiparasitic therapy, and the interest of developing a sustainable exploitation of this plant. Moreover, both molecules betulinic acid and lupeol appeared as very relevant molecules for their antiprotozoal properties.


Introduction
In a previous paper, we listed all the medicinal or toxic species of Millettia from subtropical Africa and Madagascar and showed that about 60% of the species in the genus Millettia are known for their medicinal properties [1]. amygdalina (syphilis). Several researchers, and mostly the team of Rasoanaivo, have begun to validate the pharmacological potential of the Malagasy Millettia species in the laboratory, essentially for their anticancer activity [2][3][4]. All these eight Millettia are endemic species in Madagascar and most are threatened or vulnerable according to IUCN standards [5]. M. lenneoides and M. richardiana are the least endangered species. Nothing has been published on these plants, but as the medicinal potential of M. lenneoides is already known, we choose to assess the biological properties of M. richardiana.
The present study was thus undertaken to assay the antiparasitic potential of Millettia richardiana (Baill.) Du Puy & Labat (Synonyms: M. baronii Drake, Mundulea hysterantha Baker, Mundulea richardiana Baill., Neodunnia atrocyanea R. Vig.), one of the still plentiful species, which had not yet been studied despite its broad distribution. M. richardiana is a small tree (up to 10 m in height) or a shrub of the dry deciduous forests growing on limestone, sand or on dunes in the west of Madagascar, below 300 m altitude, from Toliary to Antsiranana. The young stalks are covered with brown hairs. The leaves are alternate, compound with seven pairs of leaflets; the rachis and veins are also covered with gold-coloured brown hairs. Inflorescences in the axillary position are pseudo-racemes of 2-4 flowers only. The fruits are obovate flattened pods, ending in a bent beak, 9 cm long, covered with yellowish soft hairs. According to the regions, the vernacular names differ: Hararandrato, Hazovola, Lovanjafy, Marary, Mararybotry, Mararytoloha, Manaribôraka, Taintsindambo, Tankiandambo, Taitsindambo, Tsimahamasabary.
We report here the results of the antiprotozoal (against Plasmodium falciparum, Trypanosoma brucei, Leishmania donovani) and fungicidal (against Candida albicans, Candida glabrata and Candida parapsilosis) assays of two crude extracts and six fractions from the stem bark of M. richardiana. Their cytotoxicity was also evaluated on KB, Vero and MRC5 cells. These M. richardiana biological data led to bio-phytochemical correlation between active principles and pharmacological results.

Results and Discussion
Malaria, African trypanosomiasis and leishmaniasis are a group of tropical diseases that are especially endemic in low-income populations in developing regions of Africa, Asia and South America. Together, these three parasitic diseases cause up to an estimated 2 million deaths annually [6]. Otherwise, Candidemia are opportunistic fungal infections causing alarming problems with serious infections, especially in immunologically compromised people [7]. Natural products have played a key role throughout the history of drug discovery as a source of both novel compounds. Many plants are daily used in tropical and sub-tropical areas to treat infectious diseases. In this context, as at least 60% of the Milletia species being known for their medicinal properties, we have thought relevant to assess the potential antiprotozoal and antifungal activities of M. richardiana, even if we found no written record of its ethnobotanical use. The two crude aqueous (traditional) and methanol (MRE1) extracts from M. richardiana showed extraction yields of 10.2% and 8.4%, respectively whereas the 6 fractions from MRE1 had extraction yields ranging from 4.1% to 43.0% (Table 1). The chemical classes present in M. richardiana were identified through the TLC screening and the most represented were saponins, flavonoids, terpenoids, alkaloids, sugars, tannins, phenolics and phenolic acids (Table 1).
Results concerning the antiprotozoal, antifungal and cytotoxicity assays for the extracts and fractions of M. richardiana are reported in Table 2. The aqueous crude extract, following the traditional preparation, showed neither activity whatever the pathogen tested nor any cytotoxicity. No activity was also found with the other crude extract in methanol (MRE1). Both types of extraction did not seem to extract the active compounds. Furthermore, two other fractions MRE3 (ethyl acetate) and MRE4A (butanol), containing terpenoids, alkaloids, saponosids, and flavonoids showed weak activity against Trypanosoma and Leishmania. However MRE4A was the most cytotoxic of the extracts and fractions (61% of cell growth inhibition for KB line). It should be noted that all M. richardiana extracts and fractions tested had IC 50 higher than 10 μg/mL on Candida sp., showing no antifungal activity at pharmacological level, by comparison with the control drug (5-fluorocytosine) active at 0.75 µg/mL.
On the contrary, the DCM fraction MRE2 extracted from MRE1, shows more promising IC 50 values against the protozoan parasites tested compared with the control drugs (IC 50 : 5.8 μg/mL against P. falciparum, 12.5 μg/mL against T. brucei brucei and 11.8 μg/mL against L. donovani). The P. falciparum data are particularly interesting since IC50 values around 5 μg/mL are considered as good in the laboratory efficacy score for in vitro antiplasmodial activity of crude extracts and correspond to the concentration value warranting bioassay-guided fractionation [8]. Moreover, the trypanocidal activity of the MRE2 was comparable to the control drug (IC 50 = 12.5 µg/mL for MRE2 versus 7.4 µg/mL for pentamidine) and leishmanicidal activity was only less than 10 times lower than that of the control drugs (11.8 µg/mL versus 1.4 µg/mL for pentamidine and 1.2 µg/mL for miltefosine). These values are also quite promising since they were obtained from crude extracts (grouping many compounds and so diluting the pharmacological activity) and were compared with control drugs corresponding to pure molecules. The good results on all these three tropical parasites thus support the pharmacological potential of this fraction. Furthermore, MRE2 presented a low cytotoxicity with percentages of inhibition ranging from 15% to 22% at 10 µg/mL. DCM extraction is known to give fractions that are the most active in ethnopharmacological studies [9]. The preliminary phytochemical analysis showed this selected fraction to be rich in terpenoids and alkaloids, which could explain its antiparasitic activity. This DCM fraction MRE2 was then chromatographed over a silica gel column eluting successively with CH 2 Cl 2 and mixtures of CH 2 Cl 2 -CH 3 OH, and yielded seven compounds: lonchocarpenin, betulinic acid, β-amyrin, lupeol, palmitic acid, linoleic acid and stearic acid [10]. β-amyrin, palmitic acid, linoleic acid and stearic acid have no antiprotozoal activity reported. By contrast, lupeol is known to show antiplasmodial (with IC 50 obtained in vitro ranging from 5 to 45 µg/mL) and trypanocidal (IC 50 : from 4 to 19.3 μM) activities [11]. Moreover, betulinic acid exhibited moderate in vitro activities against P. falciparum (IC 50 : 9.89 μM) [12], against L. major (IC 50 : 40 μM) [13] but also against T. brucei [14]. Therefore, lupeol and betulinic acid might both explain the antiprotozoal activity of the fraction MRE2.  The Millettia genus is already largely known for its antiprotozoal activities, in Africa as well as in Asia. In Africa, M. griffoniana had trypanocidal activity due to griffonianone E (IC 50 = 8.6 µg/mL on T. brucei brucei and 6.35 µg/mL on T. b. rhodesiense) and antiplasmodial activity due to the presence of griffonianone E, 7-methoxyebenosin and prenylated isoflavonoids, with IC 50 ranging from 28.3 µg/mL to 36.7 µg/mL [15,16]. M. usaramensis showed also antiplasmodial properties (IC 50 = 21 µg/mL) which its most active compound (IC 50 = 8.7 µg/mL) against P. falciparum, was a chalcone [17]. M. zechiana and M. versicolor presented moderate antiplasmodial activity (IC 50 = 16.1 µg/mL and 33 µg/mL, respectively) and low cytotoxicity [18]. Anti-leishmanial properties (IC 50 = 32 µM for 6,7-dimethoxy-3',4'methylenedioxyisoflavone on L. infantum) and low cytotoxicity (IC 50 = 43 µM on MRC-5 cells) [19] were also reported for M. puguensis.
It should be noted that all the antiprotozoal compounds isolated from these various Millettia spp are phenolic compounds belonging to the groups of isoflavans, isoflavonoids, rotenoids and pterocarpans, whereas the fraction MRE2, according to our preliminary analysis, appears to contain little or no flavonoids and other phenolic compounds. The active molecules that are present in the fraction MRE2 seem to belong principally to terpenoids as suggested by the results of the phytochemical analysis.

Biological Assays
Assays on Leishmania donovani and Trypanosoma brucei brucei were performed at the Antiparasitic Chemotherapy Department of the Faculty of Pharmacy of Châtenay-Malabry University Paris-Sud 11, France. The experiments on Plasmodium falciparum and on Candida spp were performed at the Parasitology-Mycology Department, Toulouse Hospital University, France. The KB, Vero and MRC5 cells were cultured at the Institut de Chimie des Substances Naturelles (ICSN-CNRS), Gif-sur-Yvette, France.

Antiplasmodial Activity
The extracts and fractions were screened for their antiplasmodial activity on FcM29-Cameroon, a chloroquine-resistant strain of Plasmodium falciparum (IC 50 for chloroquine = 445 nM) as previously reported by Desjardins et al. [24] and modified as follows. Extract dilutions were tested 3 to 5 times independently, each dilution in triplicate, in 96-well plates with cultures at a parasitaemia of 1% and a haematocrit of 1%. For each test, the plates of parasite culture were incubated with plant extracts for 48 h and radioactive hypoxanthine was added to the medium 24 h after the beginning of incubation [25]. The control parasite culture (without drug or with 2% DMSO) was referred to as 100% growth. Parasite growth was estimated by [ 3 H]-hypoxanthine incorporation. Concentrations of extracts inhibiting 50% of the parasite growth (IC 50 ) were graphically determined as concentration versus percent inhibition.

Leishmanicidal Activity
The test was performed as previously described by Mbongo et al. [26] and Okpekon et al. [27]. Promastigotes of Leishmania donovani were grown at 27 °C in a 5% CO 2 atmosphere in the dark, in M199 medium containing 10% fetal calf serum (FCS) and supplemented with 40mM HEPES, 100mM adenosine, 0.5 mg hemin/L, and 50 mg gentamycin/ml with concentrations of extract . After 1 h at 27 °C under a 5% CO 2 atmosphere, culture medium containing 1.75 × 10 6 parasites/mL, from a logarithmic phase culture, was added. After a 72 h incubation period, the viability of parasites was evaluated by the tetrazolium-dye (MTT) colorimetric method. The results are expressed as the concentration inhibiting parasite growth by 50% (IC 50 ). Pentamidine isethionate and miltefosine were the reference drugs. These tests were performed three times and each concentration was tested in duplicate (n = 6).

Antitrypanosomal Activity
The antiparasitic activity was assessed using the method described in Loiseau et al. [28]. Bloodstream forms of Trypanosoma brucei brucei were maintained in vitro for 48 h in the dark at 37 °C in a 5% CO 2 atmosphere, in minimum essential medium (Gibco-BRL, Life Technologies, Cergy Pontoise, France) including 25 mM HEPES and Earle's salts, and supplemented with 2 mM L-glutamine, 1 g of additional glucose per liter, 10 mL of minimum essential medium non-essential amino acids (100×; Gibco-BRL) per liter, 0.2 mM 2-mercaptoethanol, 2 mM sodium pyruvate, 0.1 mM hypoxanthine, 0.016 mM thymidine, 15% heat-inactivated horse serum (Gibco-BRL), and 50 mg gentamycin per milliliter. The minimum active concentration (MAC) was defined as the minimum concentration at which no viable parasite was observed microscopically. Pentamidine was the reference drug. These tests were performed three times and each concentration was tested in duplicate (n = 6).

Antifungal Activity
The antifungal activity of the extracts and fractions was evaluated on Candida albicans American Type Culture Collection 90028 (ATCC 90028), C. parapsilopsis (ATCC 22019) and C. glabrata (ATCC 24220). A micro-dilution method adapted from the Clinical and Laboratory Standards Institute (CLSI, formerly NCCLS-M27A) [29] was used. The culture medium used was RPMI 1640 (Sigma, Saint-Quentin Fallavier, France) with 2 mM L-Glutamine and 0.165 M morpholinopropanesulfonic acid (MOPS) buffer (Sigma). Inoculates were prepared by suspending the yeast in a sterile saline solution and adjusting to a final concentration of 2.5 × 10 6 yeast cells/mL. 100 µL of this suspension was added to each well of 96-well plates and 100 µL of various concentrations of drugs added (with the maximum concentration at 10 µg/mL), and the plates incubated for 48 h at 35 °C. The results were determined using a spectrophotometer (Elx 808, Vetra Microplate Reader, Avantec, Illkirch, France) at a wavelength of 550 nm. The MIC (Minimal Inhibitory Concentration) was determined graphically by plotting concentrations of tested drugs versus percentage inhibition. For comparison, the control 5-fluorocytosine was routinely used as a reference in this test.

Cytotoxicity
The cytotoxicity of M. richardiana extracts and fractions was tested against KB (human epidermoid carcinoma), Vero (African green monkey kidney) and MRC5 (Normal Human Fetal Lung Fibroblast) cells. These cells were grown in Dulbecco's modified Eagle's medium supplemented with 25 mM glucose, 10% (v/v) foetal calf serum, 100 UI penicillin, 100 µg/mL streptomycin and 1.5 µg/mL fungizone and kept under 5% CO 2 at 37 °C. Microplates of 96-wells were seeded with 600 KB cells per well in 200 µL medium. After 24 h, extracts dissolved in DMSO, at a concentration of 10 mg/mL, were added to the cells for 72 h at a final concentration of 1% in a fixed volume of DMSO. Controls received an equal volume of DMSO. After two hours of incubation with the MTS reagent (Promega, Madison, WI, USA), the number of viable cells was determined by measuring the optical density of each well at 490 nm with a spectrophotometer. The positive control used was taxotere (Rhône-Poulenc, Sanofi, Paris, France).

Conclusions
Despite absence of biological activity for crude extracts, the fractionation of the methanol extract of the stem bark of M. richardiana yielded a fraction, active against the parasites P. falciparum, L. donovani and T. brucei brucei. This dichloromethane fraction was particularly promising for its antiprotozoal activities especially against L. donovani and T. brucei brucei with results close to those obtained with the control drugs and its low cytotoxicity. These findings support the interest in the genus Millettia as antiparasitic plants in the traditional pharmacopoeia. Some active compounds such as lupeol and betulinic acid here isolated can explain this pharmacological activity. Further investigations by bio-guided fractionations could permit to identify all the active antiparasitic compounds. M. richardiana is currently the object of trials of cultivating with the aim of developing a sustainable exploitation of the plant, without endangering the Malagasy biodiversity.

Author Contributions
M. Rajemiarimiraho participated to phytochemical investigations, chemical characterization and in the draft of the manuscript; J.-T. Banzouzi conceived this study, designed the experiments for the phytochemical part, and wrote the paper; M.-L. Nicolau-Travers carried out the antiplasmodial and antifungal assays; S. Ramos participated to phytochemical investigations; Z. Cheikh-Ali carried out the antileishmanial and antitrypanosomal assays; C. Bories managed the antileishmanial and antitrypanosomal assays; O.L. Rakotonandrasana participated to botanical investigations; S. Rakotonandrasana carried out botanical investigations; P.A. Andrianary participated to phytochemical investigations; F. Benoit-Vical designed the experiments for the pharmacological part, managed the antiplasmodial and antifungal assays and wrote the paper.