Constituents of the Roots and Leaves of Ekebergia capensis and Their Potential Antiplasmodial and Cytotoxic Activities

A new triterpenoid, 3-oxo-12β-hydroxy-oleanan-28,13β-olide (1), and six known triterpenoids 2–7 were isolated from the root bark of Ekebergia capensis, an African medicinal plant. A limonoid 8 and two glycoflavonoids 9–10 were found in its leaves. The metabolites were identified by NMR and MS analyses, and their cytotoxicity was evaluated against the mammalian African monkey kidney (vero), mouse breast cancer (4T1), human larynx carcinoma (HEp2) and human breast cancer (MDA-MB-231) cell lines. Out of the isolates, oleanonic acid (2) showed the highest cytotoxicity, i.e., IC50’s of 1.4 and 13.3 µM against the HEp2 and 4T1 cells, respectively. Motivated by the higher cytotoxicity of the crude bark extract as compared to the isolates, the interactions of oleanonic acid (2) with five triterpenoids 3–7 were evaluated on vero cells. In an antiplasmodial assay, seven of the metabolites were observed to possess moderate activity against the D6 and W2 strains of P. falciparum (IC50 27.1–97.1 µM), however with a low selectivity index (IC50(vero)/IC50(P. falciparum-D6) < 10). The observed moderate antiplasmodial activity may be due to general cytotoxicity of the isolated triterpenoids.


Introduction
Ekebergia capensis Sparrm (Meliaceae) is a deciduous tree attaining a height of up to 30 m. It is widely distributed in the central and Nyanza regions of Kenya [1,2], and is also widespread in South Africa, Swaziland, Zimbabwe, Uganda and Ethiopia. The Zulu community in South Africa uses its wood to facilitate childbirth [3]. In Kenya, the Sabaot community uses its leaf macerations internally and externally to treat headache, fever, cough and skin diseases, while the Agĩkũyũ community treats diarrhea with its stem bark [1,4]. Pharmacological studies have indicated antiplasmodial, antiinflammatory, hypotensive, uterotonic and antituberculotic activities of the crude extracts of this plant [3,[5][6][7][8], providing scientific support for their indigenous use. Phytochemical investigations of its stem bark led to isolation of triterpenoids, steroids and flavonoids [3,9,10]. The safe application of E. capensis in traditional medicine requires the presence of metabolites with useful pharmacological properties and low toxicity levels. Here, the isolation, spectroscopic identification, and biological evaluation of the constituents of E. capensis root bark and leaf extracts are reported, with special attention given to the evaluation of the cytotoxicity of the constituents.

Antimalarial Activity and Cytotoxicity
Previous phytochemical studies have revealed the in vitro antiplasmodial potency of some triterpenoids [9,19]. Therefore, the constituents of E. capensis were tested against the chloroquine sensitive (D6), and the chloroquine resistant (W2) strains of Plasmodium falciparum. Seven metabolites showed moderate in vitro antiplasmodial activity against the D6 (IC50 = 27-97.1 µM), whilst four had moderate activity against the W2 (IC50 = 64-82.7 µM, Table 2) strains. Due to the low amount isolated, the bioactivity of 1 was not evaluated. A moderate in vitro antiplasmodial activity of oleanolic acid (4) against the 3D7 strain of P. falciparum has been previously reported [20].
For evaluation of the possible risks associated with the application of E. capensis extracts in traditional medicine, we have studied their cytotoxicity using vero cells ( Table 2). The flavonoid containing leaf extract showed a low, whereas the root bark extract, rich in triterpenoids, a high toxicity towards vero cells. The latter observation is in agreement with the cytotoxicity reported for triterpenoids previously isolated from other plants [11,16].
The substantial toxicity of the root bark extract of E. capensis towards vero cells motivated us to assess the anticancer properties of its metabolites. Indeed, compounds 2, 3, 6 and 7 exhibited even higher toxicity against the 4T1 murine breast cancer cell line than against vero cells (Table 2). This toxicity and the corresponding low selectivity index (IC50(vero)/IC50(D6) < 10) suggest that the observed moderate antiplasmodial activity of the metabolites likely originates from their cytotoxicity. For further evaluation of anticancer potency, we assessed the toxicity of the samples against the HEp2 and MDA-MB-231 human cancer cell lines (Table 2). Of the seven triterpenoids isolated from the root bark, oleanonic acid (2) showed the highest cytotoxicity, 1.4 μM against HEp2 cells. Here, it should be noted that the toxicity of 2 against "normal" vero cells is as low as that of the positive control chloroquine, motivating its further assessment as a possible anticancer agent. It is interesting to note that the root extract was more toxic to vero cells than to HEp2 cells, while the reverse situation (Table 2) was observed for oleanonic acid (2). The high toxicity of the root extract to vero cells may be due to synergistic effect of some of its constituents. Therefore, the interaction of oleanonic acid (2), the most toxic metabolite against HEp2 cells, with triterpenoids 3-7 against vero cells was evaluated. 3-Epi-oleanolic acid (3) markedly antagonized the cytotoxic effects of 2 at all concentrations tested, whereas its stereoisomer (4) showed only slight antagonistic effect ( Table 3). The cytotoxicity of oleanonic acid (2) was antagonized by high concentrations of ekeberin A (5), but at lower relative concentrations 5 enhanced the toxicity of 2. Triterpenoids 6 and 7 showed weak antagonistic effects. Overall, no significant synergistic effect was observed.

Plant Material
The root bark and leaves of Ekebergia capensis were collected from Gakoe forest, Kiambu County, in April, 2013. The plant was authenticated by Mr. Patrick Mutiso and a voucher specimen (BN/1/2013) was deposited at the Herbarium of the School of Biological Sciences, University of Nairobi.

Cytotoxicity Assays
A rapid colorimetric assay was carried out using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) [25,26]. This assay is based on the ability of a mitochondrial dehydrogenase enzyme from 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 [25]. 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 μL were seeded on 96-well plates and incubated for 12 h at 37 °C and 5% CO2 to attach to the surface. Samples of the tested extracts and isolated compounds were added to the cultured cells in rows H-B over a concentration range of 0.14 to 100 μg/mL, whereas wells 1-8 of row A served as untreated controls and wells 9-12 as blank (1% DMSO, v/v). The plates were incubated for 48 h at 37 °C and 5% CO2, followed by an addition of 10 µL MTT viability indicator reagent. The plates were incubated for additional 4 h 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 as optical density (OD) per well at each drug concentration. The data was transferred into the software Microsoft Excel and expressed as percentage of the untreated controls. Percentage cytotoxicity (PC) as compared to the untreated controls was calculated as PC = [A − B/A] × 100, where A is the mean OD of the untreated cells and B is the mean OD at each drug concentration [26]. The drug concentration required for 50% inhibition of cell growth was estimated using nonlinear regression analysis of the dose-response curve.
Cytotoxicity tests on MDA-MB-231 cells were carried out following a previously described procedure [27]. MDA-MB-231 human breast cancer cells were cultured in Dulbecco's modified eagle medium (DMEM) supplemented with 10% (v/v) fetal bovine serum, 2 mM L-glutamine, 100 units/mL penicillin and 100 μg/mL streptomycin at 37 °C in humidified 5% CO2. For cytotoxicity assays, cells were seeded in 96-well plates at optimal cell density (10,000 cells per well) to ensure exponential growth for the duration of the assay. After a 24 h preincubation growth, the medium was replaced with experimental medium containing the appropriate drug concentrations or vehicle controls (0.1% or 1.0% v/v DMSO). After 72 h of incubation, cell viability was measured using Alamar Blue reagent (Invitrogen Ab, Lidingö, Sweden) according to the manufacturer's instructions. Absorbance was measured at 570 nm with 600 nm as a reference wavelength. Results were expressed as the mean ± standard error for six replicates as a percentage of vehicle control (taken as 100%). Experiments were performed independently at least six times. Statistical analyses were performed using a two-tailed Student's t-test. p < 0.05 was considered to be statistically significant.
The interaction of oleanonic acid (2) with other triterpenoids was studied using the fixed concentration ratios oleanonic acid: 'other triterpenoid' 0:1, 1:3, 1:1, 3:1, 1:0. The vero cell cytotoxicity assay was used, as described above, to evaluate the toxicity of the mixtures. To determine whether there was synergy, additive effect or antagonism, the sum of fractional inhibition concentration (∑FIC) was calculated using the formula Ax/Ay + Bx/By = K, where Ax and Bx are the IC50s when the substances are used in combination, and Ay and By are the IC50s, when the substances are used alone. The data was scored with the scale ∑FIC < 1: synergism, 2 > ∑FIC ≥ 1: additive, 4 > ∑FIC ≥ 2: slight antagonism, ∑FIC ≥ 4: marked antagonism [28].

In Vitro Antiplasmodial Assay
Continuous in vitro cultures of asexual erythrocytic stages of Indochinese chloroquine-resistant W2 and Sierra Leonean chloroquine-sensitive D6 strains of P. falciparum were maintained following the modified procedure described by Trager and Jensen [29]. Drug assay was carried out following a modification of the semiautomated microdilution technique, which measures the ability of the extracts to inhibit the incorporation of (G-3 H) hypoxanthine into the malaria parasite [30]. Plates were harvested onto glass fibre filters and (G-3 H) hypoxanthine uptake was determined using a micro-beta trilux liquid scintillation and luminescence counter (Wallac, MicroBeta TriLux) with the results recorded as counts per minute (cpm) per well at each drug concentration. Data was transferred into the software Microsoft Excel and was expressed as the percentage of the untreated controls. The drug concentration required for 50% inhibition of (G-3 H) hypoxanthine incorporation into parasite nucleic acid was calculated with nonlinear regression analysis of the dose-response curve. The criterion described by Batista and co-workers for scoring activity was adopted [31], i.e., IC50 < 1 μM: highly active; 20 μM > IC50 ≥ 1: active; 100 μM > IC50 ≥ 20: moderately active; IC50 > 100 inactive.

Conclusions
Phytochemical analysis indicated that the root bark of E. capensis contains pentacyclic triterpenoids. The major secondary metabolites of the root bark are present in the stem bark as well [9]. The triterpenoid 3-oxo-12β-hydroxy-oleanan-28,13β-olide (1) is a new compound. Most constituents of the root bark showed moderate antiplasmodial activity with low selectivity indices, revealing their limited applicability for antimalarial drug development. The triterpenoids 3-7 showed comparable cytotoxicity towards "normal" (vero) and tumor cells, whereas oleanonic acid (2) possessed low toxicity against vero cells yet high toxicity (1.4 μM) against the 4T1 and HEp2 cancer cell lines. Its low activity against MDA-MB-231 human breast cancer cells indicates some selectivity of its anticancer activity. No significant synergism on the cytotoxicity of oleanonic acid (2) with other constituents of the root bark was detected. Based on the above observations we recommend further evaluation of oleanonic acid (2) on additional normal and cancerous cell lines for careful evaluation of its potency as anticancer lead. Whereas the root bark extract of E. capensis possesses high toxicity against "normal" (2.8 μM, vero) cells, no toxicity for its leaf extract or its constituents was observed. Although in vitro toxicity cannot be directly extrapolated to in vivo toxicity, our observations suggest a low risk of the indigenous application of E. capensis leaf extracts, but a substantial risk associated with the traditional medicinal use of its root extracts.