Bioactivities and chemical constituents of leaves of selected Apocynaceae species in Peninsular Malaysia
thesisposted on 31.05.2017 by Wong, Siu Kuin
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Leaves of ten Apocynaceae species (Allamanda cathartica, Alstonia angustiloba, Calotropis gigantea, Catharanthus roseus, Cerbera odollam, Dyera costulata, Kopsia fruticosa, Nerium oleander, Plumeria obtusa and Vallaris glabra) were assessed for antiproliferative (APF) activity against selected cancer cells using the sulphorhodamine B (SRB) assay. Leaves were sequentially extracted with hexane (Hex), dichloromethane (DCM) and methanol (MeOH). Extracts were also analysed for total alkaloid content (TAC), total phenolic content (TPC), free radical scavenging (FRS) and antityrosinase (AT) activity using the Dragendorff precipitation, Folin-Ciocalteu, 1,1-diphenyl-2-picrylhydrazyl and dopachrome assays, respectively. Five species (Alstonia angustiloba, Calotropis gigantea, Dyera costulata, Kopsia fruticosa and Vallaris glabra) were also assessed for antiplasmodial (APM) activity using the lactate dehydrogenase assay. Leaf extracts of Alstonia angustiloba, Calotropis gigantea, Catharanthus roseus, Nerium oleander, Plumeria obtusa and Vallaris glabra displayed positive APF activity. DCM extract of Calotropis gigantea, and DCM and DCM:MeOH extracts of Vallaris glabra showed strong APF activity against all six human cancer cell lines (MCF-7, MDA-MB-231, HeLa, HT-29, SKOV-3 and HepG2). Against MCF-7 and MDA-MB-231 breast cancer cells, DCM extracts of Calotropis gigantea and Nerium oleander were stronger than or comparable to standard drugs of xanthorrhizol, curcumin and tamoxifen. In term of antiplasmodial (APM) activity, all five species were effective against chloroquine-resistant K1 strain of Plasmodium falciparum. All four solvent extracts of leaves of Vallaris glabra displayed effective APM activity which is stronger than mefloquine. Against chloroquine-sensitive 3D7 strain, only extracts of Dyera costulata, Calotropis gigantea and Kopsia fruticosa showed positive APM activity. Among the ten species studied, leaf extracts of Kopsia fruticosa had the highest TAC while those of Dyera costulata had the highest TPC, FRS and AT activity. There was a strong correlation between TPC and FRS of extracts (R2 = 0.991) but not between TPC and TAC (R2 = 0.193), and not between TAC and FRS (R2 = 0.168). Vallaris glabra was chosen as the main species for further study. Stem and flower extracts displayed effective inhibition against MCF-7 and SKOV-3 cancer cells. Both leaves and flowers possessed broad-spectrum APF activity. From the MeOH leaf extract of Vallaris glabra, three types of caffeoylquinic acids and a flavonol were isolated by column chromatography. They were identified by 1H-NMR, 13C-NMR and ESI-MS analyses to be 3-O-caffeoylquinic acid (3-CQA) or neochlorogenic acid, 4-O-caffeoylquinic acid (4-CQA) or cryptochlorogenic acid and 5-O-caffeoylquinic acid (5-CQA) or chlorogenic acid (CGA). The flavonol was identified as quercetin 3-O-glucoside (QG) or isoquercitrin. From the DCM leaf extract of Vallaris glabra, a fatty acid and a triterpenoid were isolated. They were identified as stearic acid (SA) and ursolic acid (UA), respectively. Caffeoylquinic acid content of fresh leaf extracts of Vallaris glabra, Dyera costulata, Alstonia angustiloba, Nerium oleander, Kopsia fruticosa and Plumeria obtusa were quantitatively analysed using reversed-phase HPLC. Nerium oleander had the highest content of 5-CQA followed by Vallaris glabra which had the highest content of 3-CQA and 4-CQA. The 5-CQA content of Vallaris glabra was twice that of Lonicera japonicaflowers, the commercial source of CGA, while its 3-CQA and 4-CQA content was about 16 times higher. APF activity of the five compounds (3-CQA, 4-CQA, 5-CQA, QG and SA) isolated from leaf extracts of Vallaris glabra were tested against four cancer cell lines. Only SA displayed weak inhibitory activity. Although the yield of UA was too low for analysis of its inhibitory activity, a review of existing literature showed that there is a wealth of references on the potent anticancer properties of UA including those of the cancer cells used in this study. Most publications concluded that UA is a potent chemo-preventive agent for cancer. MDA-MB-231 breast cancer cells treated with DCM leaf extract of Vallaris glabra and stained with fluorescent dye Hoechst 33342, provided evidence that the extract had an apoptotic effect on the MDA-MB-231 cells, based on nuclear morphological changes. Caspase colorimetry showed that the apoptotic effect involved activation of caspase-8, -9 and -3, but not caspase-6.