Author’s Accepted Manuscript Antiplasmodial And Cytotoxic Activities Of The Constituents Of Turraea Robusta And Turraea Nilotica Beatrice N. Irungu, Nicholas Adipo, Jennifer A. Orwa, Francis Kimani, Matthias Heydenreich, Jacob O. Midiwo, Per Martin Björemark, Mikael Håkansson, Abiy Yenesew, Máté Erdélyi PII: DOI: Reference: www.elsevier.com S0378-8741(15)30107-0 http://dx.doi.org/10.1016/j.jep.2015.08.039 JEP9704 To appear in: Journal of Ethnopharmacology Received date: 2 July 2015 Revised date: 15 August 2015 Accepted date: 26 August 2015 Cite this article as: Beatrice N. Irungu, Nicholas Adipo, Jennifer A. Orwa, Francis Kimani, Matthias Heydenreich, Jacob O. Midiwo, Per Martin Björemark, Mikael Håkansson, Abiy Yenesew and Máté Erdélyi, Antiplasmodial And Cytotoxic Activities Of The Constituents Of Turraea Robusta And Turraea N i l o t i c a , Journal of Ethnopharmacology, http://dx.doi.org/10.1016/j.jep.2015.08.039 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. 1 Antiplasmodial and cytotoxic activities of the constituents of Turraea robusta and Turraea nilotica Beatrice N. Irungua,b, Nicholas Adipoa, Jennifer A. Orwaa, Francis Kimanic, Matthias Heydenreichd, Jacob O. Midiwoe, Per Martin Björemarkb, Mikael Håkanssonb, Abiy Yenesewe*, Máté Erdélyib,f* a Centre for Traditional Medicine and Drug Research, Kenya Medical Research Institute, P.O. Box 54840-00200, Nairobi-Kenya b Department of Chemistry and Molecular Biology, University of Gothenburg, Gothenburg, SE-412 96 Sweden c Centre for Biotechnology Research and Development, Kenya Medical Research Institute, P.O. Box 54840-00200, Nairobi, Kenya d e Institut für Chemie, Universität Potsdam, P.O. Box 601553, D-14415 Potsdam, Germany Department of Chemistry, University of Nairobi, P.O. Box 30197-00100, Nairobi, Kenya f Swedish NMR Centre, University of Gothenburg, Gothenburg, SE-405 30 Sweden *Corresponding authors Máté Erdélyi, Department of Chemistry and Molecular Biology, University of Gothenburg, Gothenburg, SE-412 96 Sweden Tel: +46766229033 E-mail address: mate@chem.gu.se Abiy Yenesew, Department of Chemistry, University of Nairobi, P.O Box 30197-00100, Nairobi, Kenya Tel: +254 733 832 576 E-mail address: ayenesew@uonbi.ac.ke Abstract Ethnopharmacological relevance: Turraea robusta and Turraea nilotica are African medicinal plants used for the treatment of a wide variety of diseases, including malaria. The genus Turraea is rich in limonoids and other triterpenoids known to possess various biological activities. 2 Materials and methods: From the stem bark of Turraea robusta six compounds, and from various parts of Turraea nilotica eleven compounds were isolated by the use of a combination of chromatographic techniques. The structures of the isolated compounds were elucidated using NMR and MS, whilst the relative configuration of one of the isolated compounds, toonapubesin F, was established by X-ray crystallography. The antiplasmodial activities of the crude extracts and the isolated constituents against the D6 and W2 strains of Plasmodium falciparum were determined using the semiautomated micro dilution technique that measures the ability of the extracts to inhibit the incorporation of (G-3H, where G is guanine) hypoxanthine into the malaria parasite. The cytotoxicity of the crude extracts and their isolated constituents was evaluated against the mammalian cell lines African monkey kidney (vero), mouse breast cancer (4T1) and human larynx carcinoma (HEp2). Results: The extracts showed good to moderate antiplasmodial activities, where the extract of the stem bark of T. robusta was also cytotoxic against the 4T1 and the HEp2 cells (IC50 < 10 g/ml). The compounds isolated from these extracts were characterized as limonoids, protolimonoids and phytosterol glucosides. These compounds showed good to moderate activities with the most active one being azadironolide, IC50 2.4 ± 0.03 M and 1.1 ± 0.01 M against the D6 and W2 strains of Plasmodium falciparum, respectively; all other compounds possessed IC50 14.4 - 40.5 M. None of the compounds showed significant cytotoxicity against vero cells, yet four of them were toxic against the 4T1 and HEp2 cancer cell lines with piscidinol A having IC50 8.0 ± 0.03 and 8.4 ±0.01 M against the 4T1 and HEp2 cells, respectively. Diacetylation of piscidinol A resulted in reduced cytotoxicity. Conclusion: From the medicinal plants Turraea robusta and Turraea nilotica, twelve compounds were isolated and characterized; two of the isolated compounds, namely 11-epi- 3 toonacilin and azadironolide showed good antiplasmodial activity with the highest selectivity indices. Keywords: Turraea robusta; Turraea nilotica; antiplasmodial activity; cytotoxicity; limonoid; toonapubesins F; toonacilin; azadironolide. 1.0 Introduction The genus Turraea (family Meliaceae) consists of circa 70 species, mainly shrubs and small trees, and is widely distributed in Eastern Africa. Several of its species are used in indigenous medicine for the treatment of gastrointestinal disorders (Kokwaro, 2009). For example T. floribunda is used as an emetic and purgative, T. holstii is used to treat diarrhoea and constipation, and T. mombassana against excess bile, malaria and other fevers. T. robusta is utilized to treat malaria, stomach pain, diarrhoea and gastrointestinal discomfort (Gathirwa et al., 2008; Kokwaro, 2009). Its leaves are used as an antidote for poisoning. The roots of Turraea nilotica are also used for the treatment of stomach disorders in traditional medicine (Kokwaro, 2009). The methanolic root bark extract of T. robusta was previously reported to possess antiplasmodial and antimalarial activities, with negligible toxicity (mice oral LD50 > 5000 mg/kg body weight) (Irungu et al., 2007; Gathirwa et al., 2008). Previous phytochemical investigations of the root bark of T. robusta led to the isolation of triterpenoids which included five limonoids (Rajab et al., 1988; Bentley et al., 1992). There are no phytochemical and antiplasmodial reports on the stem bark of T. robusta. The root and stem bark of T. nilotica led to the identification of a limonoid and three protolimonoids (Mulholland and Taylor, 1988; Bentley et al., 1995). These compounds were not evaluated for their antiplasmodial and cytotoxic properties. In our search for plant secondary metabolites with 4 potential cytotoxic and/or antiplasmodial activities, the crude extracts and the isolated secondary metabolites of T. nilotica Kotschy and Peyr, and of T. robusta Gürke were tested against chloroquine resistant (W2) and chloroquine sensitive (D6) strains of Plasmodium falciparum. In addition, their cytotoxicity against three mammalian cell lines, African green monkey kidney (vero), mouse breast cancer (4T1) and human larynx carcinoma (HEp2) was evaluated. Here, the isolation and the characterization of the constituents of the crude extracts as well as their antiplasmodial and cytotoxic activities are reported. 2.0 Materials and Methods The stem bark of Turraea robusta Gürke (BN/2011/1) was collected from Chiromo Campus (01o16’31.34’’S; 036o38’64’’E) of the University of Nairobi in July 2011. The leaves, stem and root bark of Turraea nilotica Kotschy & Peyr. (BN/2012/1) were collected from the Mombasa Diani area (039o38’17.06’’E; 04o1λ’04.72’’S) in February 2012. The plants were authenticated by Mr. Patrick Mutiso of the Herbarium, School of Biological Science, University of Nairobi where voucher specimens were deposited. 2.1. Antiplasmodial assay Continuous in vitro cultures of asexual erythrocytic stages of P. falciparum strains (W2 and D6) were maintained following previously described procedures (Kigondu et al., 2009). A drug assay was carried out following a modification of the semiautomated micro dilution technique that measures the ability of the extracts to inhibit the incorporation of (G-3H) hypoxanthine into the malaria parasite (Gathirwa et al., 2008). Plates were harvested onto glass fibre filters and hypoxanthine (G-3H) uptake determined using a micro-beta trilux liquid scintillation and luminescence counter (Wallac, MicroBeta TriLux) and results recorded as counts per minute (cpm) per well at each drug concentration. Data was 5 transferred into Microsoft Excel 2007, and expressed as percentage of the untreated controls. Results were expressed as the drug concentration required for 50% inhibition of (G-3H) hypoxanthine incorporation into parasite nucleic acid, using a non-linear regression analysis of the dose-response curve. The criterion for scoring activity of compounds described by Batista et al., (2009) was adopted: IC50 < 1 M, highly active; IC50 ≥ 1 and <20 M, active; IC50 ≥ 20-100 M, moderate activity; IC50 > 100 inactive. 2.2. Rapid Cytotoxicity assay colorimetric assay was carried out using 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetra-zolium bromide (MTT) (Mosmann, 1983). This assay is based on the ability of a mitochondrial dehydrogenase enzyme of viable cells to cleave the tetrazolium rings of the pale yellow MTT and thereby form dark blue formazan crystals, which are largely impermeable to cell membranes, resulting in their accumulation within healthy cells. The amount of generated formazan is directly proportional to the number of cells (Mosmann, 1983). In this assay, the mammalian cell lines African monkey kidney (vero), mouse breast cancer (4T1) and human larynx carcinoma (HEp2) were used. Cells were maintained in Eagle’s Minimum Essential Medium (MEM) containing 10% fetal bovine serum (FBS). A cell density of 20 000 cells per well in 100 L serum were seeded on λ6-well plates and incubated for 12 hours at 37 °C and 5% CO2 to attach to the surface. Following 12 hours, the medium was replaced with maintenance medium containing the appropriate drug concentration, 0.14 to 100 g/mL, or vehicle control (≤ 1.0% v/v DMSO). After an incubation of 48 hours, cell viability was measured by addition of 10 µL of MTT reagent (5 mg MTT in 1ml of PBS). The plates were incubated for an additional 4 hours at the same conditions. Next, all media was removed from the plates and 100 µL DMSO was added to dissolve the formazan crystals. The plates were read on a Multiskan EX Labsystems scanning multi-well spectrophotometer at 562 nm, and 620 nm as reference. The results were recorded 6 as optical density (OD) per well at each drug concentration, the data was transferred into the software Microsoft Excel 2007 and expressed as percentage of the untreated controls. Percentage cytotoxicity (PC) as compared to the untreated controls was calculated using the following equation: [ ] (1) where A is the mean OD of the untreated cells and B is the mean OD at each drug concentration. The drug concentration required for 50% inhibition of cell growth, using nonlinear regression analysis of the dose-response curve, was calculated. Cytotoxicity was defined as toxic if IC50 < 100 ug/ml. 2.3. Extraction and Isolation 2.3.1. Turraea robusta The air dried and ground stem bark of T. robusta (1.4 kg) was extracted with MeOH/CH2Cl2 (1:1) at room temperature (2 x 3 L, 48 h each). The filtrate was dried in vacuo using a rotary evaporator to yield dark red oil (144 g). A 79 g portion of the extract was fractionated using silica gel column chromatography (600 g, 5 cm × 30 cm) with gradient elution of petroleum ether (40-60 oC) and increasing proportions of ethyl acetate. A total of 41 eluents, ca. 250 mL each, were collected and combined into 11 fractions, labelled TR1-TR11 on the basis of their TLC profiles. Fraction TR4 (1.2 g) was re-chromatographed over silica gel (10 g, 2 cm × 30 cm) using petroleum ether/acetone (95:5) to yield azadirone (1, 32.4 mg). Fraction TR7 (4.0 g) was fractionated on SephadexR LH-20 (40 g, 2 cm × 30 cm) eluting with methanol and was further purified by PTLC eluting with petroleum ether/chloroform/methanol (16:2:1) to yield 12α-acetoxy-7-deacetylazadirone (2, 7.0 mg) and mzikonone (3, 6.3 mg). Fraction TR8 (3.0 g) was re-chromatographed on silica gel column (30 g, 2 cm × 30 cm) eluting with 7 petroleum ether/ethyl acetate (9:1) and a subfraction containing one major compound was purified on PTLC, eluting with petroleum ether/acetone (7:4) to yield azadironolide (5, 16.5 mg). Fractions TR9-10 were combined (145.2 mg) and were separated on PTLC eluting with petroleum ether/chloroform/methanol (10:2:1) yielding 11-epi-toonacilin (4, 3.0 mg). The purification of fraction TR11 (331.9 mg) on PTLC using petroleum ether/chloroform/methanol (12:2:1) eluent gave turranolide (6, 17.7 mg). 2.3.2. Turraea nilotica The air dried and grounded stem bark of T. nilotica (1.2 kg) was extracted and dried following the procedure described in section 2.3.1. to yield a dark gum (59 g). A 58 g portion of the extract was fractionated by silica gel column chromatography (400 g, 5 cm × 30 cm) using petroleum ether (40-60 °C) and increasing proportions of ethyl acetate as eluent. A total of 50 fractions, ca. 250 mL each, were collected and combined into 20 major fractions, labelled TN1-20, upon TLC analyses. Repeated silica gel column chromatography (10 g, 2 cm × 30 cm) of fraction TN12 (1.35 g) eluting with 9:1 petroleum ether/acetone yielded niloticin (8, 9.3 mg). Fractions TN13-15 (7g) were combined and purified by silica gel column chromatography (100 g, 2 × 30 cm) using petroleum ether and increasing portions of acetone; crystallization of the residue from an acetone/dichloromethane mixture yielded toonapubesin F (11, 20.1 mg). A repeated column chromatographic (30 g, 2 cm × 30 cm) separation of fraction TN16 (3 g) in silica gel followed by crystallization from a methanol/dichloromethane mixture yielded piscidinol A (10, 775.6 mg). Fraction TN18 was crystallized from methanol/dichloromethane (1:9) to yield hispidol B (9, 84.2 mg). The root bark of Turraea nilotica (837 g) was extracted and dried as described in section 2.3.1 above yielding 13 g of a yellowish gum. A portion of the extract (11 g) was fractionated by 8 silica gel column chromatography (88 g, 2 cm × 30 cm) using a petroleum ether:acetone gradient, with increasing polarity. Forty six fractions, ca. 100 mL each, were collected and combined into 20 major fractions, labelled TN21-40, based on their TLC profile. Fraction TN27 (140.7 mg) was purified on PTLC eluting with petroleum ether/acetone (9:1) to yield azadirone (1, 8.3 mg). Fraction TN32 (366 mg) was subjected to RP-HPLC (CH3OH/water) yielding 12α-acetoxy-7-deacetylazadirone (2, 18.5 mg) and mzikonone (3, 4.4 mg). Repeated column chromatographic purification of fraction TN36 (358 mg) followed by PTLC with a petroleum ether/chloroform/methanol (10:2:1) mixture as eluent yielded 1α,3α-diacetyl-7αtigloyvilasinin (7, 11.6 mg). The dried and grounded leaves of T. nilotica (500 g) were extracted as described in section 2.3.1, yielding 30 g of a dark green gum. A 20 g portion of the crude extract was fractionated using silica gel column chromatography (200 g, 5 cm × 30 cm) with petroleum ether/acetone gradient with increasing polarity. A total of fifty eight fractions, ca 100 mL each, were collected and combined into the 12 major fractions and labelled TN41-52, based on TLC profiling. Fraction TN43 was crystallized from acetone giving β-sitosterol (15) and stigmasterol (16) as a mixture (4.7 mg). Fraction TN50 was crystallized from acetone yielding sitosterol 3-O-β-D-glucopyranoside acetate (12) and stigmasterol-3-O-β-Dglucopyranoside acetate (13) as a mixture (9.8 mg). Fraction TN52 was crystallized in acetone to yield sitosterol-3-O-β-D-glucopyranoside (14, 4.7 mg). 2.3.3. Structure Elucidation For structure elucidation 1H, 13 C, gCOSY, gNOESY, gHSQC and gHMBC NMR spectra were acquired on an Bruker Avance III HD 800 MHz, on a Varian VNMR-S 500 or a Varian 400 MR spectrometer. The spectra were processed using the MestReNova (v9.0.0) software. Chemical shifts were referenced indirectly to tetramethylsilane via the residual solvent signal 9 (CHCl3, 1H at 7.26 ppm and 13 C at 77.16 ppm). LC(ESI)MS spectra was acquired on a PE SCIEX API 150EX instrument (Perkin Elmer, Waltham, MA, USA) equipped with a Turbolon spray ion source (30 eV ionization energy) and a Gemini 5 mm C-18 110 Å HPLC column, using water:acetonitrile gradient (80:20 to 20:80). 2.4. X-ray diffraction Crystals of compound 11 was selected and mounted under a stereo microscope on to a glass fibre and transferred to a Rigaku R-AXIS IIc image plate system. Diffracted intensities were measured using graphite-monochromated Mo Kα ( = 0.710 73 Å) radiation from a RU-H3R rotating anode operated at 50 kV and 40 mA. Using the R-AXIS IIc detector, 90 oscillation photos with a rotation angle of 2° were collected and processed using the CrystalClear software package. An empirical absorption correction was applied using the REQAB program under CrystalClear. All structures were solved by direct methods (SIR 97) (Altomare et al., 1999) and refined using full-matrix least-squares calculations on F2 (SHELXL-97) (Sheldrick, 2007) operating in the WinGX program package. Anisotropic thermal displacement parameters were refined for all the non-hydrogen atoms. Hydrogen atoms were included in calculated positions and refined using a riding model. Displacement ellipsoids are drawn with ORTEP-3 for Windows under WinGX. 2.5. Acetylation A 20 mg portion of compound 8 or 10 was dissolved in 1 mL pyridine and then 1 mL acetic anhydride was added. The mixture was stirred overnight, and subsequently methanol was added and the solvent was removed under vacuum. The acetate precipitated by addition of water while stirring briskly, and it was filtered and dried. The procedure yielded niloticin acetate (17, 5 mg) and piscidinol A diacetate (18, 7 mg), respectively. 10 3.0 Results and Discussion 3.1. Antiplasmodial and cytotoxic activities The crude extracts of T. nilotica and T. robusta were evaluated for antiplasmodial activity against the chloroquine-resistant (W2) and chloroquine-sensitive (D6) Plasmodium falciparum strains (Table 1). The stem bark of T. robusta showed high antiplasmodial activity with IC50 2.8 ± 0.02 g/mL and 2.3 ± 0.05 g/mL against the W2 and D6 strains, respectively. The stem and root barks of T. nilotica also displayed considerable antiplasmodial activities (IC50 < 10 g/ml) while its leaves showed only moderate activity. Cytotoxicity of these extracts was evaluated against three mammalian cell lines, namely African green monkey kidney (vero), mouse breast cancer (4T1) and human larynx carcinoma (HEp2) (Table 1). The stem bark extract of T. robusta was cytotoxic against the 4T1 and the HEp2 cells (IC50 < 10 g/ml), whereas the extract of T. nilotica possessed moderate cytotoxicity against the three cell lines studied. Notably, all the extracts had low selectivity index (< 10), defined as the ratio of IC50 vero cells to IC50 P. falciparum (D6). This may indicate that the good to moderate antiplasmodial activity observed for the above extracts may be due to their general cytotoxicity. The fractions obtained from the crude extracts were evaluated for their cytotoxicity against vero cells. Generally, they had lower cytotoxicity than the crude extracts. Except for fraction TN12 (IC50 = 4.34 ± 0.04 g/mL), most fractions showed moderate cytotoxicity. 11 Table 1. Antiplasmodial and cytotoxic activities of selected plant parts IC50 (µg/mL) Plant (part) W2 D6 Vero 4T1 Hep2 SI T. robusta (SB) 2.8±0.0 2.3±0.1 21.9±2.0 5.3±0.6 4.2±1.0 9.5 T. nilotica (SB) 7.3±0.1 6.9±0.0 17.7±1.3 ND 22.4±4.4 2.6 T. nilotica (RB) 9.5±1.0 7.9±1.0 13.7±2.0 18.6±1.2 27.2±3.6 1.7 T. nilotica (L) 59.0±4.0 47.4±3.0 21.5±2.0 39.1±4.0 37.4±1.3 0.5 Chloroquine a 108.0±0.1 a 7.7.0±0.0 43.9±0.5 ND ND 5701 Legend: SB: stem bark, RB: root bark, L: leaves, SI: selectivity index [IC50 vero / IC50 (D6)]; Positive control: podophyllum resin, IC50 (4T1) = 0.47 ± 0.05 µg/mL melarsoprol IC50 (Vero) = 0.76 ± 0.01 µg/mL; a IC50: half maximal inhibitory concentration given in nM for chloroquine. ND: not determined, due to the small sample amount available. From the stem bark of Turraea robusta Guerke six known compounds were isolated: three ring A-D-intact limonoids, namely azadirone (1), 12α-acetoxy-7-deacetylazadirone (2) and mzikonone (3), and the ring B seco limonoid 11-epi-toonacilin (4) (Figure 1). In addition, it gave two other triterpenoids azadironolide (5) and turranolide (6). Compound 5 was isolated as an epimeric mixture, indicated by doubling of some of its NMR signals. Epimeric mixtures of limonoids having a hemiacetal functionality in place of a furan ring were previously reported (Cheplogoi and Mulholland, 2003; McFarland et al., 2004). The secondary metabolite content of T. robusta stem bark was similar to those reported for other Turraea species, which have limonoids as the main constituents. Compounds 2 and 4 were reported from this species for the first time. This is the first report on the occurrence of compound 5 in the genus Turraea. 12 Figure 1. The structures of the limonoids azadirone (1), 12α-acetoxy-7-deacetylazadirone (2), and mzikonone (3), the ring B seco limonoid 11-epi-toonacilin (4), the triterpenoids azadironolide (5), and turranolide (6), isolated from the stem bark of Turraea robusta. From the root bark of Turraea nilotica, four limonoids azadirone (1), acetoxy-7deacetylazadirone (2), mzikonone (3), (Figure 1) and 1α,3α-diacety-7α-tigloyvilasinin (7) (Figure 2) were isolated. From its stem bark, four protolimonoids niloticin (8), hispidol B (9), piscidinol A (10) and toonapubesin F (11) were isolated. A mixture of sitosterol-3-O-β-Dglucopyranoside acetate (12) and stigmasterol-3-O-β-D-glucopyranoside acetate (13), sitosterol-3-O-β-D-glucopyranoside (14) as well as a mixture of β-sitosterol (15) and stigmasterol (16) were isolated from the leaves. Compounds 1, 2, 3, 7 and 9 are reported here from this species for the first time. There is no previous report on compounds 11-14 from genus Turraea. 13 Figure 2. In addition to 1-3 shown in Figure 1, the limonoid 1α,3α-diacety-7α-tigloyvilasinin (7), the protolimonoids niloticin (8), hispidol B (9), piscidinol A (10) and toonapubesin F (11), the phytosterols sitosterol-3-O-β-D-glucopyranoside acetate (12), stigmasterol-3-O-βD-glucopyranoside acetate (13), and sitosterol-3-O-β-D-glucopyranoside (14) were isolated from the root and stem barks of Turraea nilotica. The structure of toonapubesin F (11), first isolated from Toona ciliate var. pubesins (Wang et al., 2011), was further confirmed by an X-ray crystallographic analysis (Figure 4) from a single crystal obtained by slow crystallization from a mixture of dichloromethane and acetone. The atomic coordinates are given in the Supplementary Information. 14 Figure 3. ORTEP picture of toonapubesin F (11). All nonpolar hydrogens have been omitted for clarity. Displacement ellipsoids are drawn at the 50% probability level. As the stereochemistry of the toonapubesin backbone is known (Wang, 2011), the chirogenic centre at C4 is (S). Hence, this data allows identification of the oxidized methyl functionality on C4, i.e. C25 and not C24. The roots, stem and leaves of Turraea nilotica, were found to contain different secondary metabolites. Limonoids were isolated from the root bark, and protolimonoids from the stem bark while phytosterols were isolated from the leaves. Limonoids, protolimnoids and steroids share a tetracyclic triterpenoid skeleton; however, they differ in the oxidation state of their side chain. Based on the previously proposed biosynthetic pathway of limonoids, (Champagne et al., 1992; Tan and Luo, 2011) T. nilotica is likely to follow the phytosterol (leaves) → protolimonoid (stem bark) → limonoid (root bark) biosynthetic pathway. The antiplasmodial potency of some limonoids and other triterpenoids has been previously reported (Maneerat et al., 2008; Mohamad et al., 2009). Therefore, some of the compounds isolated in this work were tested for antiplasmodial activities against the D6 and W2 15 Plasmodium falciparum strains (Table 2). Their activity was scored according to the classification of Batista et al., (2009), where IC50 < 1 M is highly active; 1 ≤ IC50 ≤ 20 M is active, 20 M ≤ IC50 ≤ 100 M is moderately active, and IC50 > 100 is inactive. Of the eight compounds tested, two showed good activity with the most active substance being the epimeric mixture azadironolide (5) (IC50 < 2.4 M), and six showed moderate activities against the W2 and D6 strains (Table 2). The antiplasmodial activity of compound 1 is in agreement with the previous literature data (Chianese et al., 2010). The isolated compounds were also tested for cytotoxicity (Table 2) against the mammalian cell line African green monkey kidney (vero cells). Most of them showed moderate cytotoxicity (IC50 > 20 M) and a low selectivity index [SI = IC50(vero)/IC50 (D6) < 10]. Thus, the observed moderate antiplasmodial activity of these compounds is likely due to general cytotoxicity, rather than due to a specific activity against the Plasmodium parasite. It should, however, be noted that compounds 4 and 5 that are classified as active, were observed to have comparably high selectivity index SI > 10.5 and 11.5, respectively, though significantly lower than chloroquine (SI = 5702). Although the investigated plants are in traditional medicinal use against malaria, and their crude extracts showed promising antimalarial activities, their isolated metabolites did not display considerable activity against P. falciparum. This may be explained by synergetic effects or by the loss of one or some minor yet highly active metabolites during the purification process. For an improved understanding of the traditional medicinal applicability of these plants further studies are necessary. Some limonoids and protolimonoids isolated from other plants were previously reported to possess substantial cytotoxicity (Maneerat et al., 2008), which, along with the high to moderate cytotoxicity of the crude extracts and isolated compounds against ‘normal’ vero 16 cells motivated the evaluation of cytotoxicity of the isolated constituents against the cancerous cell lines 4T1 and HEp2 (Table 2). Table 2 Antiplasmodial and cytotoxic activities (IC50 in M) of compounds isolated from Turraea nilotica and Turraea robusta. Compound Azadirone (1) 12α-Acetoxy-7deacetylazadirone (2) Mzikonone (3) 11-epi-Toonacilin (4) Azadironolide (5) Niloticin (8) Hispidol B (9) Piscidinol A (10) Niloticin acetate (15) Piscidinol A acetate (16) Chloroquine D6 23.4±0.2 W2 29.6±1.0 4T1 14.4±0.0 HEp2 12.8±0.0 Vero > 229.4 SIb >9.8 31.0±0.2 30.2±0.5 104.6±7.1 40.3±2.2 134.1±2.9 4.3 36.6±0.8 40.5±3.7 38.8±0.4 59.3±1.0 139.6±4.7 3.8 c c 17.1±0.2 14.4±0.5 88.6±3.2 68.1±1.3 > 180.5 >10.5 2.4±0.0 48.2±2.3 36.8±2.0 37.6±1.4 1.1±0.0 77.0±5.7 37.2±3.2 36.3±4.4 14.7±0.2 14.5±0.5 21.7±3.2 8.0±0.0 8.5±0.5 6.9±0.6 7.4±0.7 8.4±0.0 27.6±0.6 14.5±0.4 130.0±3.1 41.1±5.8 11.5 0.3 3.5 1.1 68.3±5.3 172.9±4.5 ND 121.9±0.1 >c200.8 ND ND ND 15.2±0.8 >c179.2 108.0±0 ND ND 43.9±0.5 a 7.7±0.02 a 5701 The mean values of at least three independent experiments are reported. ND: not determined, due to the small amount of sample available. a IC50: half maximal inhibitory concentration given in nM for chloroquine. b SI = IC50 (vero)/IC50(D6); c values >100ug/ml not cytotoxic, Positive control: podophyllum resin, IC50 (4T1) = 0.47 ± 0.05 µg/mL, melarsoprol IC50 (Vero) = 0.76 ± 0.01 µg/mL Compounds 1, 5, 8-10 showed high cytotoxicities against the 4T1 and HEp2 cell lines with IC50 < 20 µM. It should be stressed that these compounds showed moderate cytotoxicity against ‘normal’ vero cells, yet high cytotoxicity against cancerous cell lines. Hence these compounds may be promising leads for development of anticancer agents (Diantini et al., 2012). For assessment of the importance of the free hydroxyl groups of compound 8 and 10 on their bioactivities, they were acetylated to give niloticin acetate (17) and piscidinol A diacetate (18), respectively (Figure 4). The acetate derivatives 17 and 18 showed lower cytotoxicity as 17 compared to the parent, non-acetylated compounds 8 and 10, indicating the importance of free hydroxyl group(s) in the side chain. A reduction in antiplasmodial activity of compound 17 in comparison to the parent compound 8 was also observed. O OA c OA c O Ac H O H OH H 17 O 18 Figure 4. The structure of niloticin acetate (17) and piscidinol A diacetate (18). 3.2. The chemotaxonomic significance of Turraea limonoids Various classes of limonoids were reported from the genus Turraea with each species synthesizing more than one class of limonoids. Ring intact limonoids were reported from eight Turraea species, namely T. robusta, T. nilotica, T. cornucopia, T. parvifolia, T. floribunda, T. holstii, T. wakefieldii and T. pubescens (Bentley et al., 1λλ2; Ndung’u et al., 2004; Owino et al., 2008; Yuan et al., 2013). So far, ring A seco limonoids have only been isolated from T. wakefieldii (Ndung’u et al., 2003), whereas ring B seco limonoids were reported in T. floribunda, T. holstii and T. pubescens (Mulholland et al., 1998; McFarland et al., 2004; Yuan et al., 2013). Ring A-B seco limonoids were reported from T. mombassana and T. obtusifolia, while ring C seco limonoids from T. holstii and T. pubescens (Adul et al., 1993; Sarker et al., 1997; Mulholland et al., 1998; Yuan et al., 2013). Interestingly, so far no ring D seco limonoid was reported from the genus Turraea although several examples were isolated from other Meliaceace genera. The presence of compounds 1-4 and 7 in T. robusta and T. nilotica is in line with the fact that limonoids are common constituents of the genus Turraea. Except for compound 1 that was reported from other genera of the Meliaceace 18 family (Zhou et al., 1997), the limonoids disclosed here have not been reported from any other genus. The occurrence of five limonoids in Turraea species is shown in Table 3, among which 3 was isolated from five species. Table 3. Occurrence of some limonoids in Turraea species Limonoid Azadirone (1) 12α-acetoxy-7deacetylazadirone (2) Mzikonone (3) 11-epi-toonacilin (4) 1α,3α-diacety-7αtigloyvilasinin (7) 4.0 Turraea species T. nilotica T. robusta - - - T. robusta T. cornucopia T. pubescens - - T. robusta T. robusta T. cornucopia T. holstii T. parvifolia T. pubescens T. nilotica T. cornucopia T. pubescens - T. nilotica T. parvifolia - - - Conclusion Six compounds were isolated from the stem bark of Turraea robusta. Four of them were limonoids and two of them triterpenoids. Of these six compounds, azadironolide (5) is new to the genus. The secondary metabolites present in the stem bark were also found in the root bark. Compound 3 is a common limonoid in the genus Turraea, and was previously reported from five Turraea species (Cheplogoi and Mulholland, 2003; Owino et al., 2008; Yuan et al., 2013). From the leaves, root and stem bark of Turraea nilotica twelve compounds were isolated. Toonapubesin F (11), sitosterol 3-O-β-D-glucopyranoside acetate (12), stigmasterol3-O-β-D-glucopyranoside acetate (13) and sitosterol-3-O-β-D-glucopyranoside (14) are new to the genus. Different secondary metabolites were observed in the leaves, and thus the roots and the stem were observed to contain limonoids and protolimonoids, whereas the leaves contained phytosterols. Out of the isolated compounds, 11-epi-toonacilin (4) and azadironolide (5) showed good antiplasmodial activity with the highest selectivity indices among the isolated compounds. Azadironolide (5), niloticin (8), hispidol B (9) and piscidol A (10) were cytotoxic to HEp2 19 and 4T1 cells with IC50 < 20 M. The cytotoxicity of the latter constituents against the ‘normal’ vero cell line was comparably low, indicating some degree of selectivity. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/... Acknowledgements This work was supported by IFS (grant nr. F/4575-2), UNICEF/UNDP/WORLD BANK/WHO Special Programme for Research and Training in Tropical Diseases (SPHQ10RTG-6 ID: A90208) and by the Swedish Research Council (grant nr. 2012–6124). References Adul, G.O., Bentley, M.D., Benson, B.W., Huang, F.-Y., Gelbaum, L., Hassanali, A., 1993. Two new prieurianin-class limonoids from Turraea mombasana. Journal of Natural Products 56, 1414–1417. Altomare, A., Burla, M.C., Camalli, M., Cascarano, G.L., Giacovazzo, C., Guagliardi, A., Moliterni, A.G.G., Polidori, G., Spagna, R., 1999. SIR97: a new tool for crystal structure determination and refinement. Journal of Applied Crystallography 32, 115–119. Batista, R., de Jesus Silva Júnior, A., de Oliveira, A.B., 2009. Plant-derived antimalarial agents: New leads and efficient phytomedicines. Part II. Non-alkaloidal natural products. Molecules 14, 3037–3072. Bentley, M.D., Adul, G.O., Alford, A.R., Huang, F.-Y., Gelbaum, L., Hassanali, A., 1995. An insect antifeedant limonoid from Turraea nilotica. Journal of Natural Products 58, 748– 750. Bentley, M.D., Gaul, F., Rajab, M.S., Hassanali, A., 1992. Tetranortriterpenes from Turraea robusta. Journal of Natural Products 55, 84–87. Champagne, D.E.; Koul, O.; Isman, M.B.; Scudder, G.G.; Neil Towers, G.H., 1992. Biological Activity of Limonoids from the Rutales. Phytochemistry. 31, 377–394. Cheplogoi, P.K., Mulholland, D.A., 2003. Limonoids from Turraea parvifolia (Meliaceae). Biochemical Systematics and Ecology 31, 799–803. 20 Chianese, G., Yerbanga, S.R., Lucantoni, L., Habluetzel, A., Basilico, N., Taramelli, D., Taglialatela-Scafati, O., 2010. Antiplasmodial triterpenoids from the fruits of neem, Azadirachta indica. Journal of Natural Products 73, 1448–1452. Diantini, A., Subarnas, A., Lestari, K., Halimah, E., Susilawati, Y., Supriyatna, S., Julaeha, E., Achmad, T.H., Suradji, E.W., Yamazaki, C., Kobayashi, K., Koyama, H., Abdulah, R., 2012. Kaempferol-3-O-rhamnoside isolated from the leaves of Schima wallichii Korth. inhibits MCF-7 breast cancer cell proliferation through activation of the caspase cascade pathway. Oncology Letters 3, 1069–1072. Gathirwa, J.W., Rukunga, G.M., Njagi, E.N.M., Omar, S.A., Mwitari, P.G., Guantai, A.N., Ndiege, I. O., 2008. The in vitro antiplasmodial and in vivo antimalarial efficacy of combinations of some medicinal plants used traditionally for treatment of malaria by the Meru community in Kenya. Journal of Ethnopharmacology 115, 223–231. Irungu, B.N., Rukunga, G.M., Mungai, G.M., Muthaura, C.N., 2007. In vitro antiplasmodial and cytotoxicity activities of 14 medicinal plants from Kenya. South African Journal of Botany 73, 204–207. Kigondu, E.V., Rukunga, G.M., Keriko, J.M., Tonui, W.K., Gathirwa, J.W., Kirira, P.G., Ndiege, I.O., 2009. Antiparasitic activity and cytotoxicity of selected medicinal plants from Kenya. Journal of Ethnopharmacology 123, 504–509. Kokwaro, J.O., 2009. Medicinal Plants of East Africa (3rd ed.). Nairobi: University of Nairobi Press. Maneerat, W., Laphookhieo, S., Koysomboon, S., Chantrapromma, K., 2008. Antimalarial, antimycobacterial and cytotoxic limonoids from Chisocheton siamensis. Phytomedicine. 15, 1130–1134. McFarland, K., Mulholland, D. A., Fraser, L.-A., 2004. Limonoids from Turraea floribunda (Meliaceae). Phytochemistry. 65, 2031–2037. Mohamad, K., Hirasawa, Y., Litaudon, M., Awang, K., Hadi, A. H.A., Takeya, K., Morita, H., 2009. Ceramicines B–D, new antiplasmodial limonoids from Chisocheton ceramicus. Bioorganic Medicinal Chemistry 17, 727–730. Mosmann, T., 1983. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. Journal of Immunolgy Methods 65, 55–63. Mulholland, D.A., Monkhe, T.V., Coombes, P.H., and Rajab, M.S., 1998. Limonoids from Turraea holstii and Turraea floribunda. Phytochemistry 49, 2585–2590. Mulholland, D.A., Taylor, D.A., 1988. Protolimonoids from Turraea nilotica. Phytochemistry 27, 1220–1221. Ndung’u, M., Hassanali, A., Hooper, A.M., Chhabra, S., Miller, T.A., Paul, R.L., Torto, B., 2003. Ring A-seco mosquito larvicidal limonoids from Turraea wakefieldii. Phytochemistry 64, 817–823. 21 Ndung’u, M.W., Kaoneka, B., Hassanali, A., Lwande, W., Hooper, A.M., Tayman, F., Torto, B., 2004. New Mosquito Larvicidal Tetranortriterpenoids from Turraea wakefieldii and Turraea floribunda. Journal of Agriculture and Food Chemistry 52, 5027–5031. Owino, J., Ndung’u, M., Hassanali, A., 2008. Two azadiron limonoids from Turraea cornucopia. Journal Kenya Chemical Society 5, 43–49. Rajab, M. S., Bentley, M. D., Hassanali, A., Chapya, A., 1988. A new limonoid from Turraea robusta. Phytochemistry 27, 2353–2355. Su, R., Kim, M., Kawaguchi, H., Takehiko, Y., Katsumi, G., Tooru, T., Takahashi, S., 1990. Triterpenoids from the fruits of Phellodendron chinense SCHNEID.The Stereostructure of Niloticin. Chemical and Pharmaceutical Bulletin. 38, 1616–1619. Sarker, S.D., Savchenko, T., Whiting, P., Šik, V., Dinan, L., 1997. Two limonoids from Turraea obtusifolia (Meliaceae), prieurianin and rohitukin, antagonise 20hydroxyecdysone action in a Drosophila cell line. Archives of Insect Biochemistry and Physiology 35, 211–217. Sheldrick, G.M., 2007. A short history of SHELX. Acta Crystallography A. 64, 112–122. Tan, Q.-G.; Luo, X.-D., 2011. Meliaceous Limonoids: chemistry and biological activities. Chemical Reviews 111, 7437–7522. Wang, J.-R., Liu, H.-L., Kurtán, T., Mándi, A., Antus, S., Li, J., Guo, Y.-W., 2011. Protolimonoids and norlimonoids from the stem bark of Toona ciliata var. pubescens. Organic & Biomolecular Chemistry 9, 7685–7696. Yuan, C.-M., Tang, G.-H., Zhang, Y., Wang, X.-Y., Cao, M.-M., Guo, F., Hao, X.-J., 2013. Bioactive limonoid and triterpenoid constituents of Turraea pubescens. Journal of Natural Products 76, 1166–1174. Zhou, J.-B., Minami, Y., Yagi, F., Tadera, K., Nakatani, M., 1997. Ring C-seco-limonoids from Melia toosendan. Phytochemistry 46, 911–914. 22 Graphical abstract Azadironolide isolated as an epimeric mixture from Turraea robusta stem bark inhibits growth of Plasmodium falciparum strain (W2) with an IC50 of 1.1 ± 0.01 M HO O O % Inhibition 100 80 60 40 20 H O H OAc 0 0.0 0.2 0.3 0.6 1.3 2.5 5.0 10.0 Concentration (μg/ml)