Plant selection and collection
In the framework of a random study of Cameroonian plants as sources of anti-malarial agents, the fruits of S. juglandifolia were collected during the rainy season in Mt. Kalla, Yaoundé area, Cameroon in October, 2010. The plant sample was brought to the Laboratory for Phytobiochemistry and Medicinal Plants Studies, University of Yaoundé I, Cameroon for phytochemical and biological studies. Botanical identification was done at the National Herbarium, Yaoundé by Mr. Victor Nana, where a voucher specimen is deposited under the identification number 9176 SRFCam. The genus Sorindeia includes trees, shrubs or lianas that are confined to tropical Africa, including Madagascar, the Comoro Islands and the Mascarene Islands
. Sorindeia juglandifolia is a tree of up to 23 m height of which no specific use and no pharmacological studies have been found. Therefore, the approach adopted in this study differs from the reverse pharmacology approach
, since there is no evidence of plant use against malaria. A related species, Sorindeia mildbraedii, is used to provide chewing sticks for oral hygiene
Plant extraction and structure elucidation
Reagents and materials
Melting points were determined on a Büchi-540 melting point apparatus. UV spectra were determined on a Spectronic Unicam spectrophotometer. IR spectra were determined on a JASCO Fourier Transform IR-420 spectrometer. 1H- and 13C NMR spectra were run on a Bruker spectrometer equipped with 5 mm 1H and 13C probes operating at 500 and 125 MHz, respectively, with TMS as internal standard. Silica gel 230–400 mesh (Merck) and silica gel 70–230 mesh (Merck) were used for flash and column chromatography, while precoated aluminum-backed silica gel 60 F254 sheets were used for TLC. Spots were visualized under UV light (254 and 365 nm) or using MeOH-H2SO4 reagent.
Extraction and fractionation
The ground fruits (2 kg) were extracted at room temperature with methanol (2× 5 L, 48 h each). Solvent was evaporated under reduced pressure to yield 28g of extract. A portion (20g) of the extract was fractionated by flash chromatography over silica gel (70–230 mesh, Merck, 7 × 42 cm), eluting with a gradient of increasing polarity of mixtures of hexane-ethyl acetate 100:0–0:100, resulting in the collection of 35 fractions of 500mL each, which were combined on the basis of TLC analysis to yield 18 subfractions, labelled SJFR1-SJFR18. All these fractions were tested in vitro against the Plasmodium falciparum chloroquine-resistant W2 strain and the P. falciparum cysteine protease falcipain-2.
Fractions SJFR8-10 [Hex:EtOAc (1:1)] and SJFR17-18 [Hex:EtOAc (0:1)] showed the best potencies against falcipain-2 and P. falciparum W2, respectively. Subsequent TLC analysis suggested the combination of the respective fractions into SJFRA and SJFRB, which were submitted to phytochemical investigation.
Fraction SJFRA [7g, composed of subfractions 20–22, obtained with Hex:EtOAc (1:1)] was subjected to column chromatography over silica gel (230–400 mesh, Merck, 5 × 42 cm), eluting with gradient mixtures of hexane-ethyl acetate 30:70–80:20, resulting in 82 subfractions of 200 mL, combined on the basis of TLC analysis into two subfractions, SJFRA1 (1–25) (3.8g), and SJFRA2 (26–82) (0.3g).
Subfraction SJFRA1 showed significant anti-plasmodial activity, and was, therefore, subjected to column chromatography over silica gel (230–400 mesh, Merck, 5 × 42 cm), eluting with gradient mixtures of hexane-ethyl acetate 30:70 – 20:80 to yield SJFRA1-41, identified as 2,3,6-trihydroxy benzoic acid (1, 125mg).
Column chromatography over silica gel (230–400 mesh, Merck, 5 × 42 cm) of SJFRB [6g, composed of subfractions 34–35, obtained with Hex:EtOAc (0:1)] eluting with gradient mixtures of hexane-ethyl acetate 80:20 – 0:100 and EtOAc- MeOH (1:0 to 98:2) yielded SJFRB-41, identified as 2,3,6-trihydroxy methyl benzoate (2, 35mg).
Evaluation of biological activities
The protocol described in this report was approved by the Institutional Review Board (IRB-No: 001/UY11 BTC/ IRBI 2009), Biotechnology Centre, University of Yaoundé 1, Cameroon, and the World Health Organization Research Ethics Review Committee (WHO ERC-A80689).
In vitro screening of Sorindeia juglandifolia extracts against Plasmodium falciparum W2
Plasmodium falciparum strain W2, known to be resistant to chloroquine and other anti-malarials
, was cultured in sealed flasks at 37°C, in a 3% O2, 5% CO2 and 91% N2 atmosphere in RPMI 1640, 25 mM HEPES, pH 7.4, supplemented with heat inactivated 10% human serum and human erythrocytes to achieve a 2% haematocrit. Serum and erythrocytes were purified from blood voluntarily given by adults with no recent contact with malaria, and who have signed an informed consent form. Parasites were synchronized in the ring stage by serial treatment with 5% sorbitol (Sigma)
 and tested at 1% parasitaemia. Stock solutions of plant extracts were prepared as 1 mg/ml in DMSO, diluted as needed for individual experiments, and tested in triplicate as described previously
[7, 8]. The stock solutions were diluted in supplemented RPMI 1640 medium so as to have at most 0.2% DMSO in the final reaction medium. Equal volumes (100μl) of inhibitors and 1% parasitaemia, 4% haematocrit culture were then added and gently mixed thoroughly. Negative controls contained equal concentrations of DMSO. Positive controls contained 1 μM chloroquine phosphate (Sigma). Cultures were incubated at 37°C for 48 h. Parasites at the ring stage were thereafter fixed by replacing the serum medium by an equal volume of 1% formaldehyde in PBS. Aliquots (5 μl) of each culture were then added to round-bottom polystyrene 96 well plates containing 150 μl of 100 mM NH4Cl, 0.1% Triton X- 100 and 1 nM YOYO-1 nuclear dye (Molecular Probes) in PBS, and the parasitaemia of treated and control cultures were compared using a Becton-Dickinson FACSort flow cytometer equipped with AMS-1 loader (Cytek Development) to count nucleated (parasitized) erythrocytes. Data acquisition was performed using Cell Quest software. These data were normalized to percent control activity and 50% inhibitory concentrations (IC50s ) were calculated using Prism 5.0 software (GraphPad) with data fitted by nonlinear regression to the variable slope sigmoidal dose–response formula, y = 100/[1 + 10(logIC50− x)H
where H is the hill coefficient or slope factor
In vitro screening of Sorindeia juglandifolia extracts against Plasmodium falciparum field isolates
Blood sampling and purification of Plasmodium falciparum isolates
Plasmodium falciparum isolates were prepared as described previously
. Blood was collected from outpatients presenting at the Etoug-Ebe Baptist Health Centre in Yaoundé with uncomplicated malaria. Patients older than five years of age with mono infection with P. falciparum and parasite density greater than 6,000 asexual parasites per microliter of blood were considered for inclusion in the study. Those with recent history of self-medication with anti-malarial drugs were excluded. After patients agreed to participate in the study and signed the consent form, 5 mL of blood was collected from them by venipuncture into heparinized tubes. The blood was centrifuged at 3,000 rpm for 15 min and the supernatant discarded. The pellets were suspended in 10 mL of pre-warmed RPMI 1640 medium and centrifuged again twice at 3,000 rpm for 5 min, and the supernatant was then discarded. The volume of the packed-cells was estimated and mixed with an equal amount of RPMI 1640, 25 mM HEPES, pH 7.4, and subsequently used at 4% haematocrit for experiments. Thin smears were evaluated to assess the parasitaemia, the developmental stages of parasites, and the colour, shape and size of red blood cells. Nine such isolates were prepared and subsequently used for experiments.
Evaluation of antiplasmodial activity
The activity of plant products was assessed as previously described
. All the fractions and pure compounds that showed activity against P. falciparum W2 and falcipain-2 were tested against the P. falciparum field isolates. Stock solutions of plant extracts were prepared at 1 mg/ml in DMSO and diluted as needed for individual experiments in 96-well plates using RPMI 1640, to achieve a maximum final DMSO concentration of 0.2%. Chloroquine was used as positive control at 1 μM, and the negative control was an equal concentration of DMSO. To assess the susceptibility of parasites to drugs, they were maintained in RPMI 1640 supplemented with 25 mM HEPES buffer (pH 7.4), 0.05% ampicillin, and 10% AB+ human serum at 37°C, in a candle jar. To achieve this, 200 μl parasite suspensions at 4% haematocrit and 0.1%– 0.3% parasitaemia were added to 50 μl of each concentration of inhibitor in individual wells, each tested in triplicate. Cultures were incubated at 37°C for 48 h in a candle jar. To estimate the effects of inhibitors after 48 h, thin smears from treated and control cultures were fixed in methanol for 30 s, stained using fresh 5% Giemsa solution (Sigma) in phosphate buffer (pH 7.1), and examined by counting the parasitaemia per 100 erythrocytes. IC50s were calculated from curves plotting percent growth versus drug concentration using Prism 5.0 software with data fitted by nonlinear regression to the variable slope sigmoidal dose–response formula y = 100/[1 + 10(logIC50− x)H
, where H is the Hill coefficient or slope factor
Assessment of falcipain-2 inhibition by plant extracts
Falcipain-2 activity was measured as hydrolysis of the fluorogenic substrate benzyloxycarbonyl-Leu-Arg-7-amino-4-methyl-coumarin (Z-Leu-Arg-AMC) using a Fluoroskan II spectrofluorometer, as described previously
. For inhibitor assays, recombinant falcipain-2 prepared as described previously
 was incubated with inhibitors (added from stocks concentrated 100- to 1,000-fold in dimethyl sulfoxide [DMSO]) with 10 mM dithiothreitol, pH 5.5, for 30 min at room temperature before the substrate (50 μM) was added. Equal concentrations of enzyme were used for each experiment. Multiple inhibitor concentrations were studied in triplicate, and the rates of hydrolysis were determined and compared to those of controls containing equal concentrations of DMSO. All values were normalized to the control activity, and IC50s were calculated with the Prism 5.0 program, with data fitted by nonlinear regression.
In vivo assessment of 2,3,6-trihydroxy benzoic acid against Plasmodium berghei
Compound 1 (2,3,6-trihydroxy benzoic acid), obtained from the fractionation of S.juglandifolia extract, was assessed in vivo using the Plasmodium berghei rodent parasite model, according to the procedure described by Okokon et al. with a few modifications. Plasmodium berghei strain B (MRA-406, MR4, ATCC® Manassas Virginia) was obtained from BEI Resources
 and used for the experiments. Female Swiss albino mice aged eight weeks and weighing ~20 g were used for efficacy and toxicity experiments. These animals were bred in the animal house of the Faculty of Medicine, University of Yaoundé I. They were fed a standard laboratory diet (S.P.C. Ltd., Bafoussam, Cameroon) and given clean tap water ad libitum. Animal welfare requirements were strictly considered during the experiments, as described in the protocol approved by the National Ethics Committee (Reg. No.FWAIRB00001954). To start infections, a cryotube of parasites was thawed and the undiluted contents were injected immediately i.p. into naïve recipient mice. Three days after infection, the parasitaemia was evaluated daily in tail vein blood through Giemsa-stained thin smear observation under oil immersion at 100X magnification. Passage of infection into new mice consisted of an i.p. injection of 200 μL of 106 infected erythrocytes/mL sterile PBS. The test compound was dissolved and diluted in Tween 20: Ethanol (7:3 v/v).
Evaluation of the suppressive effect of the compound
Preliminary screening of the compound consisted of a suppressive test. To achieve this, animals were infected i.p. with 200 μL of 106 infected erythrocytes/mL sterile PBS. Four hours after infection, duplicate batches of animals were treated as follows: four batches of six mice each were given test drug orally at respective dosages of 10, 30, 50 and 100 mg/kg, and subsequently once daily for four consecutive days. Negative controls consisted of one batch of 6 infected mice receiving no drug (dose 0), and one batch of six uninfected animals. Positive controls consisted of one batch of infected mice receiving quinine sulfate at 24 mg/kg.
Evaluation of the curative effect of the compound
Curative assays were subsequently conducted using the same setting, with animal receiving drug at daily oral dosages of 10, 30, 50 and 100 mg/kg for 5 consecutive days, starting day 3 (72 hours) after infection. Thin blood smears were made daily from the tail blood of mice and the level of parasitaemia assessed by examination of Giemsa-stained smears using a microscope at 100X magnification. In both cases, activity was expressed as ED50s that were calculated based on percent parasitaemia obtained 24 h after the last drug administration using GraphPad Prism 5.0 software.
Evaluation of the acute toxicity of active extracts
The acute toxicity profile of the active fractions was determined as previously described
[6, 8] and according to the Organization for Economic Cooperation and Development (OECD) protocol briefly described below, with some modifications
. Before the experiments, animals were starved for 12 h in wire mesh bottom cages to prevent coprophagy but allowed free access to water.
Mice were studied in groups of 6 animals (3 males and 3 females). They received the extract solutions in olive oil per os in doses ranging from 1,000 to 7,000 mg/kg. The negative control group received 1ml olive oil. The animals were carefully observed for 2 h, and thereafter for 7 days during which mortality, body weights and gross behavioural changes were recorded daily.