Assessment of antiproliferative and antiplasmodial activities of five selected Apocynaceae species

Background Studies have shown that the barks and roots of some Apocynaceae species have anticancer and antimalarial properties. In this study, leaf extracts of five selected species of Apocynaceae used in traditional medicine (Alstonia angustiloba, Calotropis gigantea, Dyera costulata, Kopsia fruticosa and Vallaris glabra) were assessed for antiproliferative (APF) and antiplasmodial (APM) activities, and analysed for total alkaloid content (TAC), total phenolic content (TPC) and radical-scavenging activity (RSA). As V. glabra leaf extracts showed wide spectrum APF and APM activities, they were further screened for saponins, tannins, cardenolides and terpenoids. Methods APF and APM activities were assessed using the sulphorhodamine B and lactate dehydrogenase assays, respectively. TAC, TPC and RSA were analysed using Dragendorff precipitation, Folin-Ciocalteu and DPPH assays, respectively. Screening for saponins, tannins, cardenolides and terpenoids were conducted using the frothing, ferric chloride, Kedde and vanillin-H2SO4 tests, respectively. Results Leaf extracts of A. angustiloba, C. gigantea and V. glabra displayed positive APF activity. Dichloromethane (DCM) extract of C. gigantea, and DCM and DCM:MeOH extracts of V. glabra showed strong APF activity against all six human cancer cell lines tested. DCM extract of A. angustiloba was effective against three cancer cell lines. Against MCF-7 and MDA-MB-231 cell lines, DCM extract of C. gigantea was stronger than standard drugs of xanthorrhizol, curcumin and tamoxifen. All five species were effective against K1 strain of Plasmodium falciparum and three species (C. gigantea, D. costulata and K. fruticosa) were effective against 3D7 strain. Against K1 strain, all four extracts of V. glabra displayed effective APM activity. Extracts of D. costulata were effective against 3D7 strain. Selectivity index values of extracts of A. angustiloba, C. gigantea and V. glabra suggested that they are potentially safe for use to treat malaria. Extracts of K. fruticosa had the highest TAC while D. costulata had the highest TPC and RSA. Phytochemical screening of extracts of V. glabra also showed the presence of terpenoids, tannins and saponins. Conclusions Leaf extracts of C. gigantea and V. glabra showed great promise as potential candidates for anticancer drugs as they inhibited the growth of all six cancer cell lines. Against K1 strain of P. falciparum, all four extracts of V. glabra displayed effective APM activity. The wide spectrum APF and APM activities of V. glabra are reported for the first time and this warrants further investigation into its bioactive compounds.


Background
The family Apocynaceae consists of about 250 genera and 2000 species of tropical trees, shrubs and vines [1,2]. A characteristic feature of the family is that almost all species produce milky sap. Leaves are simple, opposite and whorled. Flowers are large and colourful.
In traditional medicine, Apocynaceae species are used to treat gastrointestinal ailments, fever, malaria, pain and diabetes [2]. Of the five species studied, roots and leaves of Calotropis gigantea (L.) Aiton are used to treat skin and liver diseases, leprosy, dysentery, worms, ulcers, tumours and earaches [3]. Its latex has been reported to have wound healing properties [4]. Kopsia fruticosa (Ker.) A. DC. is used to treat sore and syphilis, and has cholinergic effects [5]. Leaves and barks of Dyera costulata Hook have been used for treating fever, inflammation and pain [6]. Stems, leaves and latex of Alstonia angustiloba Miq. have been used for gynaecological problems and skin sores in Indonesia [7]. Leaves are externally applied to treat headache in Malaysia [8]. Vallaris glabra Kuntze is well known in Thailand because the scent of its flowers is similar to that of pandan leaves and aromatic rice [9]. Its use in traditional medicine has not been reported, and its bioactivity and phytochemistry have yet to be studied. Species of Apocynaceae have also been reported to possess anticancer properties [2,10]. Species having cytotoxic activity include those of Allamanda [11], Alstonia [12,13], Cerbera [14,15], Nerium [16,17], Plumeria [18] and Tabernaemontana [19]. Species of Apocynaceae, notably those of Alstonia, are also known to have antimalarial properties [20][21][22].
Prompted by the anticancer and antimalarial properties of Apocynaceae, leaf extracts of five selected species used in traditional medicine were assessed for antiproliferative (APF) activity against six human cancer cell lines and for antiplasmodial (APM) activity against two strains of Plasmodium falciparum. Their extracts were also analysed for total alkaloid content, total phenolic content and radical-scavenging activity. Having wide spectrum APF and APM activities, leaf extracts of V. glabra were further screened for saponins, tannins, cardenolides and terpenoids. Information from the screening will serve as a useful guide to further work on isolating compounds with APF and APM activities.

Extraction of leaves
For crude extraction, fresh leaves of each species (40 g) were cut into small pieces and freeze-dried overnight. Dried samples were blended and extracted with 250 ml of methanol (MeOH) three times for 1 h each time. Samples were filtered and the solvent was removed using a rotary evaporator (Eyela). The dried crude extracts were stored at -20°C for further analysis. For sequential extraction, fresh leaves of each species (40 g) were freeze-dried, ground and extracted successively with hexane (HEX), dichloromethane (DCM), DCM: MeOH (1:1) and MeOH (HmbG Chemicals). For each solvent, the suspension of ground leaves in 250-300 ml of solvent was shaken for 1 h on the orbital shaker. After filtering, the samples were extracted two more times for each solvent. Solvents were removed with a rotary evaporator to obtain the dried extracts, which were stored at -20°C for further analysis.

Antiproliferative activity
Antiproliferative (APF) activity of extracts (25 μg/ml) was initially screened for growth inhibitory activity against three human cancer cell lines (MCF-7, MDA-MB-231 and HeLa) using the sulphorhodamine B (SRB) assay [23]. Growth inhibitory activity with less than 50% cell growth was considered positive while that with more than 50% cell growth was considered negative. Extracts with positive growth inhibition were further tested against six human cancer lines (MCF-7, MDA-MB-231, HeLa, HT-29, SKOV-3 and HepG2) using six different extract concentrations. Human cancer cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). The cells were seeded 24 h prior to treatment in 96-well plates at densities of 10,000-20,000 cells/well. Each cell line was designated one plate. Initial cell population of each cell lines prior to addition of extracts was determined by fixing with trichloroacetic acid (TCA) (Sigma). Extracts were dissolved in dimethyl sulphoxide (DMSO) (Sigma) and serially diluted from 8-25 μg/ml. Control cultures were treated with the same volume of DMSO. The concentration of DMSO was kept within 1% to avoid any interference with cell viability. After the addition of extracts, the plates were incubated for 48 h. After incubation, the cells were fixed with 50 μl of cold 50% TCA and incubated for 1 h at 4°C. The plates were then washed with tap water and air dried. Cells were stained with 100 μl of 0.4% SRB solution (Sigma) diluted with 1% acetic acid followed by incubation for 10 min at room temperature. Unbound dye was removed by washing with 1% acetic acid. Bound stain was then solubilised with 200 μl of 10 mM trizma base (Sigma). Absorbance of each well at 505 nm was obtained using a microplate reader. Dose-response curves were constructed to obtain GI 50 or concentration of extract that causes growth inhibition (GI) of cells by 50%. GI 50 was calculated using the formula [(T z -T i )/(T c -T i )] × 100 = 50 where T z is the absorbance of cells treated with extracts or drugs at the end of incubation, T i is the absorbance of cells prior to treatment with extracts or drugs and T c is the absorbance of untreated cells at the end of incubation [24]. IC 50 or inhibition concentration at which there is a 50% reduction in cells was obtained using the formula T z /T c × 100 = 50. Activity is considered to be effective when GI 50 value ≤20 μg/ml [25].

Antiplasmodial activity
Antiplasmodial (APM) activity of extracts was assessed in vitro in human blood using the lactate dehydrogenase assay with slight modifications [26]. Chloroquineresistant K1 and chloroquine-sensitive 3D7 strains of Plasmodium falciparum were tested. Standard drugs of artemisinin (Sigma) and mefloquine (Sigma) were used as positive controls. Extracts dissolved in DMSO (10 mg/ml) were diluted with deionised water to 320 μg/ml. The solution was serially diluted two-fold six times to give seven different concentrations. Aliquots of each concentration (10 μl) were transferred into 96-well microtiter plates. Parasitised red blood cell suspensions (1% parasitaemia, 190 μl) were added to each well. Parasitised and non-parasitised red blood cells were used as negative controls. The plates were incubated for 24 h at 37°C in a candle jar and were subsequently chilled at -20°C to lyse the red blood cells. The plates were then allowed to cool to room temperature, and 20 μl of blood suspension was dispensed into a new microtiter plate containing 100 μl MALSTAT™ reagent (Flow Inc.), and 20 μl of nitroblue tetrazolium (Sigma) and phenazine ethosulphate (Sigma) mixture. Absorbance was measured with a plate reader at 630 nm. Percentage inhibition at each concentration was determined and the mean of EC 50 values of parasite viability was calculated using probit analysis. EC 50 or effective concentration is the extract concentration that kills 50% of malaria parasites. Activity is effective if EC 50 value ≤10 μg/ml [27]. The selectivity index (SI) for APM activity was calculated based on the ratio of cytotoxicity (IC 50 ) on HepG2 and MCF-7 cells to APM activity (EC 50 ) on chloroquine-resistant K1 strain.

Analysis of TAC, TPC and RSA Total alkaloid content
Total alkaloid content (TAC) of extracts was determined using the Dragendorff precipitation assay [28]. For each species, extracts (15 mg) were dissolved in 1 ml of distilled water that was acidified to pH 2.0-2.5 with 0.01 M HCl. Analysis was conducted in triplicate. Alkaloids were then precipitated with 0.4 ml of Dragendorff reagent. After washing with 0.5 ml of distilled water to remove traces of the reagent, the precipitate was later treated with 0.4 ml of 1% sodium sulphide, resulting in a brownish-black precipitate. Precipitates formed at each stage were recovered by centrifugation at 14,000 rpm for 1 min. The resulting precipitate was dissolved in 0.2 ml of concentrated nitric acid and diluted to 1 ml with distilled water. Addition of 2.5 ml of 3% thiourea to 0.5 ml aliquots of this solution resulted in a yellow colored complex. Absorbance was measured at 435 nm and TAC was expressed as boldine equivalent in milligram per gram of extract. The calibration equation for boldine (Sigma) was y = 1.068x (R 2 = 0.9959) where y is absorbance and × is mg/ml of boldine. Dragendorff reagent was prepared by dissolving 0.8 g of bismuth nitrate (Sigma) in 40 ml of distilled water and 10 ml of glacial acetic acid. The resulting solution was mixed with 20 ml of 40% potassium iodide.

Total phenolic content
Total phenolic content (TPC) of extracts was determined using the Folin-Ciocalteu (FC) assay [29][30][31]. Extracts (300 μl in triplicate) were introduced into test tubes followed by 1.5 ml of FC reagent (Fluka) at 10 times dilution and 1.2 ml of sodium carbonate (Fluka) at 7.5% w/v. The tubes were allowed to stand for 30 min in the dark before absorbance was measured at 765 nm. TPC was expressed as gallic acid (GA) equivalent in milligram per gram of extract. The calibration equation for GA (Fluka) was y = 0.0111x -0.0148 (R 2 = 0.9998) where y is absorbance and × is mg/ml of GA.

Phytochemical screening
Qualitative phytochemical screening for saponins, tannins, cardenolides and terpenoids from leaf extracts of V. glabra was carried out using standard phytochemical procedures [32,33]. The frothing test was used for saponins, the ferric chloride test for tannins, the Kedde test for cardenolides and the vanillin-H 2 SO 4 test for terpenoids. To the best of our knowledge, this study represents the first report of cytotoxic activity from DCM leaf extract of A. angustiloba and DCM and DCM:MeOH extracts of V. glabra. Earlier studies have reported cytotoxic activity from the root bark of Alstonia macrophylla Wall. ex G. Don [12] and the stem bark of Alstonia scholaris R. Br. [13]. A recent study on Vallaris solanacea (Roth) Kuntz has reported potent cell growth inhibitory activity of cardenolide glycosides isolated from the plant [34]. The finding of strong APF activity from DCM and DCM:MeOH extracts of C. gigantea from this study is supported by an earlier report that DCM leaf extracts of C. gigantea had strong inhibitory activity against KB, BC and NCI-H187 cancer cell lines [35]. Ethanol root extracts of C. gigantea were also reported to be cytotoxic to K562 and SGC-7901 human cell lines [36].

APM activity
Against chloroquine-resistant K1 strain of P. falciparum, leaves of V. glabra were most effective as all four extracts had APM activity with EC 50 less than 10 μg/ml ( Table 2). DCM extract was the strongest with EC 50 of 0.85 μg/ml. Three extracts of A. angustiloba, and two extracts of C. gigantea, D. costulata and K. fruticosa showed APM activity. It should be noted that DCM: MeOH extracts of all five species displayed APM activity with A. angustiloba having the strongest activity (EC 50 of 0.46 μg/ml).
Against chloroquine-sensitive 3D7 strain of P. falciparum, extracts of A. angustiloba and V. glabra showed no activity. Extracts of D. costulata were the exception in that DCM, DCM:MeOH and MeOH extracts showed positive APM activity with EC 50 of 8.31, 2.13 and 3.56 μg/ml, respectively. Generally, extracts were less effective against 3D7 strain. This finding complements an earlier report that alkaloids from the extracts of Alstonia species were effective against chloroquine-resistant K1 strain but not against chloroquine-sensitive T9-96 strain [22]. Previous studies on the APM activity of Apocynaceae were focused on Alstonia species [21,22]. This study is the first to report the APM activity of leaf extracts of D. costulata, C. gigantea, K. fruticosa and V. glabra. A notable finding is the APM activity of V. glabra against K1 strain in all solvent fractions.

Selectivity index
The selectivity index (SI) for APM activity indicates the safety of an extract to be used for antimalarial therapy [37]. The index was calculated based on the ratio of cytotoxicity (IC 50 ) on HepG2 and MCF-7 cells to APM activity (EC 50 ). The SI was calculated for the most potent extracts against K1 strain of P. falciparum with EC 50 ≤1.00 μg/ml. They were DCM:MeOH extracts of A. angustiloba (0.46 μg/ml) and C. gigantea (0.97 μg/ml), and HEX and DCM extracts of V. glabra (1.00 and 0.85 μg/ml), respectively ( Table 2) Recently, a study on the antimalarial and cytotoxic activity of plants in the Democratic Republic of Congo considered SI values of extracts >10 as high [38]. With SI values relatively higher than 10, the extracts of A. angustiloba, C. gigantea and V. glabra are potentially safe for use to treat malaria.

Analysis of TAC, TPC and RSA
Of the five species analysed, MeOH crude and DCM extracts of K. fruticosa had the highest TAC (100 and 129 mg BE/g of extract), respectively (

Phytochemical screening
Phytochemical screening showed the presence of terpenoids in all leaf extracts of V. glabra except MeOH extract while saponins and tannins were present in DCM and DCM:MeOH extracts, and in DCM:MeOH and MeOH extracts, respectively (Table 4). Cardenolides were not detected. Terpenoids are used for the treatment of human diseases such as cancer and malaria, and infectious diseases caused by virus and bacterial [39]. Taxol and artimesinin are renowned terpenoid-based anticancer and antimalarial drugs, respectively. Terpenoids inhibit the growth or induce apoptosis of breast cancer cells such as MCF-7, MDA-MB-231 and T47D [40]. They are among the most important natural antimalarial drugs, which also include quinones and alkaloids [41].
Tannins are water-soluble polyphenols that are present in many plant foods [42]. Literature on the effects of tannins on human health is vast and sometimes conflicting. Incidences of esophageal cancer have been attributed to consumption of tannin-rich foods such as herbal teas, suggesting that tannins might be carcinogenic. However, reports have indicated a negative association between consumption of tea and the incidence of cancer. Teas with high tannin content have been suggested to be anticarcinogenic Table 3 Phytochemical analysis of total alkaloid content, total phenolic content and radical-scavenging activity of leaf extracts of Apocynaceae species and antimutagenic which may be related to their antioxidative property in protecting cellular oxidative damage against lipid peroxidaton and superoxide radicals. The antimicrobial activities of tannins are well documented. Saponins are naturally occurring glycosides with a distinctive foaming characteristic and bitter taste [43,44]. They have a wide range of properties, which include both beneficial and detrimental effects on human health. Saponins affect the immune system in ways that help to protect the human body against cancers, and also lower cholesterol levels. They decrease blood lipids, lower cancer risks and lower blood glucose response.

Conclusions
The DCM extract of C. gigantea, and DCM and DCM: MeOH extracts of V. glabra inhibited the growth of all six human cancer cell lines. Against MCF-7 and MDA-MB-231 human cell lines, DCM leaf extract of C. gigantea had stronger APF activity than standard drugs of xanthorrhizol, curcumin and tamoxifen. With wide spectrum APF activity, leaves of these two species are therefore promising candidates as alternative resources for anticancer drugs. Against K1 strain of P. falciparum, all four extracts of V. glabra displayed effective APM activity. Selectivity index values suggested that extracts of V. glabra are potentially safe for use to treat malaria. The wide spectrum APF and APM activities of V. glabra are reported for the first time. This warrants further investigation into its bioactive compounds.